PS-1 lntrons and the Origin of Genes. Walter Gilbert. By studying the distribution of introns in the genes for ancient conserved proteins, proteins that are homologous between bacteria and eukaryotes, which have no introns in the prokaryotes and have introns in the eukaryotes, we can show that the positions of introns are correlated with the boundaries of modules, compact regions of" polypeptide chain, for specific sizes of *nodules with diameters around 21, 28 and 33 Angstroms. Furthermore, we can show that this correlation is carried mostly by the phase zero introns, those that lie between the codons. The introns that break the codons, phases 1 and 2, do not show any strong correlation. We interpret this to mean that there is a pattern of added introns, occurring equally in all three phases and totalling some 2/3 of the total number of intron positions while there is an excess of phase zero introns (about 1/3 of the total) which is correlated with structure and is a candidate for a set of ancient introns coming from the progenote.

Congress On In Vitro Biology

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Abstracts

JS-1

JS-2 Identification and Characterization of Human Cell Lines Using DNA Hypervariable Probes YVONNE A. REID. Cell Biology Program, ATCC, Rockville, MD 20852. E-mail: [email protected]

Microbial Identification and DNA Fingerprinting. J. TANG. Bacteriology Program, American Type Culture Collection, Rockville, MD 20852. E-mail: [email protected] Traditionally, bacteria are identified by a variety of morphological, biochemical, and physiological tests in order to describe the organisms qualitatively. Other more sophisticated methods, like the lipid and cell wall analysis that classify bacteria on the basis of chemical composition of the cell, help resolve some of the ambiguity in microbial identification. In recent years, nucleic acid-based detection and molecular typing methods brought bacterial identification to another level especially in the field of diagnostic microbiology. A number of DNA fingerprinting methods have been developed that enable one to differentiate an organism to the species and strain level rapidly. These techniques include variations of restrictive fragment length polymorphism, ribotyping, and PCR amplification of special regions or repetitive sequences. Each identification scheme has its uniqueness and limitations. A polyphasic approach, which combines physiological testing, cytochemical analysis, and molecular techniques, will provide a complete and accurate identification of microorganisms.

The possibility of cross-contamination of cell lines is a major concern to cell biologists. Cross-contamination among cell lines has been reported at frequencies as high as 35%. The wide use of animal cells by scientists in disciplines such as cell biology, toxicology and biotechnology has produced an increase in cell culture activity and, consequently, the risk of crosscontamination. The discovery of DNA hypervariable regions has made possible positive identification of human cell lines. These hypervariable regions, distributed throughout the genome, are composed of distinct base pair sequences. Detection of these sequences is used to generate distinct DNA profiles. The amplified products or restriction fragments generated from human DNA are separated in a gel and detected by various methods (chemiluminescence, silver, florescence). The application of DNA profiling techniques such as RFLP and AmpFLP gives scientists the assurance of positive identification of human cell lines.

JS-3 Amplified Fragment Length Polymorphism (AFLP) for Molecular Typing in Plant and Microorganism. IHY-JHU L1N, Agricultural Biotechnology R&D, Life Technologies, Inc., (GIBCO BRL), 9800 Medical Center Drive, Rockville, MD 20850. AFLP, a PCR based DNA fingerprinting technique, involves restriction enzyme digestion, adapter ligation and selective amplification. A comparison of AFLP with other DNA fingerprinting techniques such as RFLP and RAPD, demonstrates the superior reproducibility and higher number of polymorphisms per reaction with AFLP. Similar to RFLP and RAPD, a quantitative analysis of AFLP based on the Similarity Index (Sl) is achieved by analyzing DNA band patterns. A study of peach diversity using AFLP was able to demonstrate the relatedness of different peach varieties based on the SI and dendogram. The consistency of this quantitative analysis was obtained by using 8 different AFLP primer combinations for the study of peach diversity. Moreover, no difference in the AFLP patterns was observed in rice plants germinated from seeds directly compared to those rice plants regenerated from HiGrow rice medium. Similarly, AFLP was used to confLrm the genetic variation in tobacco plants that were regenerated from years of continuous cell suspension or callus cultures. In microorganisms, heterogeneous groups among Agrobacterium biovar 1 and biovar 2 were identified after examination of 16 strains of Agrobacterium biovar 1 and 6 strains of Agrobacterium biovar 2. Moreover, DNA sequences of the 16S ribosomal RNA genes of Agrobacterium biovar 1 strains C58 and LBA4404 showed that the sequence homology between C58 and A. rubi, a different species of Agrobacterium, was higher than the homology between C58 and LBA4404. Moreover, we successfully placed two isolates, Arl-Nw and 57RR, of the plant pathogenic fungus Collectotrichum, into C. trifolii and C. gloeosporioides based on AFLP. These two isolates were difficult to classify based on morphological criteria.

Congress On In Vitro Biology

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Abstracts

Vertebrate- Symposia V-1

V-2 Syndecan-4: a transmembrane regulator of cell-matrix adhesion. JOHN R. COUCHMAN, Robert Longley, Eok-Soo Oh and Anne Woods. University of Alabama at Birmingham, Department of Cell Biology, 1670 Univ. Blvd., VII 20IC, Birmingham, AL 35294-0019. E-mail: [email protected]

Syndecan-4 is a transmembrane heparan sulfate proteoglycan present in a wide variety of epithelial and mesenchymal cells, omen as a focal adhesion component. It appears to regulate the balance between stable anchorage and migration, and acts as a co-receptor with integfins. Over-expression of wild type syndecan-4 in CHO cells leads to enhanced focal adhesion formation and a decrease in cell locomotion. However, transfection with a form of syndecan-4 having a cytoplasmic deletion of the last eleven amino acids has the opposite effect, being a dominant negative for focal adhesion formation. We have now established that the central region of the cytoplasmic domain of syndecan-4 can interact with, and increase the activity of, protein kinase C (PKC). Moreover, evidence from crosslinking and nuclear magnetic resonance spectroscopy shows that this same region can bind inositol phospholipids. This has two results. First there is a ternary complex formed between PKC, syndecan-4 cytoplasmic domain and the inositol phospholipids. Second, all syndecans have a tendency to dimerize, and this is stabilized by the presence of the phospholipid. PKC activity is omen required in the adhesion process, and in many primary cultures, leads to focal adhesion formation and stable anchorage. This can regulate downstream events through integrin-mediated signaling, such as anchorage-dependent growth, matrix assembly and selective gene expression.

How do Hemidesmosomes Assemble? JONATHAN JONES, Cell and Molecular Biology, Northwestem University Medical School, Chicago, IL 6061 l. E-mail In many epithelial tissues, the hemidesmosome plays an essential role in maintaining epithelial adhesion to the basement membrane. However, the hemidesmosome is more than just a stable "spot" weld since it is also the site of signal transduction, mediated by its cL6[34 integrin component. As befits its multiple functions, the hemidesmosome is a complex junction containing many proteins. The keratin eytoskeleton attaches to its cytoplasmic plaque while its transmembrane elements interact with components of the extracellular matrix. We have studied hemidesmosome assembly using various cell culture models. In summary, our results indicate that hemidesmosome assembly involves recruitment of a6134 integrin heterodimers as well as cytoskeletal elements and cytoskeleton-associated proteins to the cell surface. In our cell culture models, these phenomena appear to be triggered by extracellular matrix, in particular a specific taminin-5 isoform. Our recent data indicate that proteolytic processing of the alpha chain subunit of laminin-5 is necessary for laminin-5-induced nucleation of hemidesmosome formation. In cultured cells, laminin-5 alpha chain processing can be mediated by plasmin which apparently cleaves the alpha chain within its globular C-terminal domain. Finally, as a consequence of cell interaction with laminin-5, there is an induction of both phosphorylation and dephosphorylation of subunits of a6134 integrin. Such events are apparently necessary for subsequent cytoskeleton anchorage to the hemidesmosome cytoplasmic plaque.

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V-3

Development of Media Free of Animal and Human Components. STEPHEN F. GORFIEN, Ph.D., Cell Culture Research and Development, Life Technologies, Inc., Grand Island, NY 14072

Matrix Regulation of Intestinal Epithelial Phenotype. M.D. BASSON. Departments of Surgery, Yale University and CT VA Health Care System, 333 Cedar St, POB 208062, New Haven, CT 06520-8062. E-mail: Intestinal epithelial cell interactions with the extracellular matrix seem likely to regulate diverse aspects of the intestinal epithelial phenotype, including proliferation, polarity, cell shape, motility, and the expression of various conventional differentiation markers such as the digestive enzymes of the apical microvillous brush border. Using the well-differentiated human Caco-2 intestinal epithelial cell line as a model, we have demonstrated alterations in the intestinal epithelial phenotype in response to different purified matrix substrates as well as during such specialized cell-matrix interactions as cell motility and the response to strain. These appear driven by integrin-mediated intracellular signals. Indeed, the ligation of individual integrin subunits by functional antibodies results in both acute intracellular signalling events similar to those initiated by cell-matrix adhesion and chronic alterations in cell phenotype akin to those which occur during repetitive mechanical deformation of the matrix at frequencies and amplitudes consistent with peristalsis and villous motility.

Congress On In Vitro Biology

Many serum-free media contain components that have been derived from animal blood products or tissues. Some of these media components are commercially available in recombinant form, while others are still only obtained from animal sources. The recent emphasis has been towards development of chemically-defined nutrient formulations in which the biochemical composition and structure of all components are known. Most chemically-defined media are also protein-free, although it is possible to have a defined formulation that contains a recombinant form of a hormone (insulin) or growth factor (EGF, FGF, etc.). Recombinant transferrin is not currently commercially available, but transferrin has successfully been replaced by inorganic iron carriers in several applications. In some cases, it may not be feasible or desirable to replace a protein component of a formulation with a recombinant form or a non-protein constituent. However, a change in the source of the protein (i.e., from bovine to porcine or from animalderived to plant-dedved) may be warranted. Some of the choices available in serum-free, protein-free and defined nutrient media will be highlighted using examples from our work in mammalian cell culture systems. Strategies for customizing formulations to fit end-user needs will be discussed.

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Abstracts

V-5

V-6

Applications of Plant Protein Hydrolysates in Cell Culture Media. VIJAI. K. PASUPULETI.Quest International, P.O. Box 630, Norwich, New York 13815. E-mail:

Elimination of Animal and Human Derived Components from Commercial Manufacturing Processes. S. R. ADAMSON, Mammalian & Microbial Cell Sciences, Genetics Institute, Andover, MA 01810. E-mail:

Traditionally the cell culture media utilizes fetal calf serum as a main ingredient and minerals, vitamins, amino acids, hormones, etc. are used as important media constituents. Several reasons have prompted the industry to move towards serum free media and as a result all the media manufacturers have developed a variety of proprietary formulations without fetal calf serum. The focus of my presentation is towards the development and applications of plant protein hydrolysates in cell culture media.

Concems for virus and prion contamination of raw materials used in cell culture manufacturing processes employed to produce parenteral proteins have prompted efforts to eliminate components derived from animal and human tissue. A historical perspective starting from the early 80's will be presented, focusing on choices made in industry of animal derived components (e.g., human versus bovine). Consideration will be given to specific risks from viral and prion based infectious agents associated with the use of these components. In more recent years technology has emerged which allows the growth of mammalian cells in essentially chemically defined medium. The presentation will include approaches to medium development for two commercial processes currently in manufacturing at Genetics Institute and some recommendations for future developments with a focus on regulatory initiatives developing throughout the world.

Plant protein hydrolysates offers several advantages, to mention a few; Animal free origin, BSE free, batch to batch consistency, controlled enzyme hydrolysis to give a broad range of molecular weight peptides, amino acids including glutamine, protein free, cost effective and so on. t will be discussing the development and manufacturing of plant protein hydrolysates on a commercial scale. Further I will be highlighting the applications of plant protein hydrolysates with different cell lines for cell growth and the production of monoclonal antibodies. Plant protein hydrolysateshave the potential to supplement/replace fetal calf serum, individual amino acids and provide pH and temperature stable glutamine.

V-7

V-8 Research Use of Human Biological Materials: An Update from the National Bioethics Advisory Commission. ERIC M. MESL1N. National Bioethics Advisory Commission, Rockville, MD 20892-7508.

The Development o f F i s h Cell Cultures for Study o f Rickettsia and Rickettsial-like Organisms. J.L. FRYER. Department o f Microbiology, Center for Salmon Disease Research, Oregon State University. Corvallis, OR 97331. E-mail: Cultured fish cells currently have application in many fields o f biological research, and teleost cell lines have been initiated from a broad range o f tissues and organs Cell lines reported in the literature represent 74 species from 34 families o f fishes. Fish cells are readily grown in vitro using standard techniques for the culture of mammalian cells. These cell lines are most frequently used for the culture, diagnosis and study o f viruses in fish. There are approximately 80 viruses that have been isolated from fish. Fish cell lines have also been helpful in the detection o f emerging pathogens o f fish. Examples are the highly fastidious intracellular Gram negative bacteria observed in fish and shellfish. Piscirickettsia salmonis is the first o f these previously unrecognized rickettsial pathogens o f fish to be grown and characterized. Since the isolation o f P . salmonis in 1989, the impact o f rickettsial pathogens in fish has become increasingly apparent. Growing awareness o f the emergence o f these fastidious intracellular organisms has led to the discovery o f rickettsial diseases among diverse species o f fish from different geographic locations and aquatic environments.

When President Clinton established the National Bioethics Advisory Commission in 1995, his Executive Order provided NBAC's initial research agenda. NBAC was asked to direct its attention to consideration of protection of the rights and welfare of human research subjects; and issues in the management and use of genetic information, including but not limited to, human gone patenting. In response to its charge, NBAC formed a subcommittee to address issues in the management and use of genetic information. The subcommittee met for the first time in December 1996 to set priorities for 1997. The use of human biological materials in genetic research was chosen as the first topic because the issue is well-defined, clearly important, and a matter of considerable current interest to professional organizations, government agencies, and the research community. Many important advances made in medical science would have been impossible were it not for the analysis of collected and stored human tissues. However, the increasingly common practice of performing genetic analyses on these tissues is triggering a renewed interest in examining how these samples have been (and will be) collected, stored, used, and shared, and how the results of the research performed will be disseminated. A matter of concern is how to handle consent for the use of tissue samples collected in the past for diagnostic or research purposes, especially when the research purposes for which the samples might be used was not foreseen. This presentation will discuss the current status of the Commission's report.

Congress On In Vitro Biology

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Abstracts

V-9

V-10

Cell Cultures from the Japanese Pufferfish (Fugu): Potential for Molecular Genetics. D. W. Barnes. Division of Cell, Developmental and Molecular Biology/Genetics, American Type Culture Collection, University Blvd., Manassas Va 20110 USA The Japanese pufferfish (genus Fugu) possesses a highly compact genome and has become a focus of interest for sequencing and mapping the genomes of vertebrates. In addition, Fugu is an aquacultured marine species of economic importance, prized as a gourmet food, and subject to commercially significant epidemics of microbial/viral origin. Although genomic libraries have been derived and used to study the molecular biology of Fugu, living biological material is difficult to obtain for laboratories distant from the Asian Pacific. We established cell cultures from kusafugu, Fugu niphobles, and torafugu, F. rubripes. Cultures derived from F. niphobles fry and F. rubripes eye have been passaged more than one year, representing approximately 180 population doublings. Proliferating cultures were also initiated from F. rubripes brain, liver, fin, spleen, kidney, swim bladder and muscle. F. rubripes eye-derived cells possessed a chromosome number in the diploid range; F. niphobles fry cells were slightly hyperploid. Flow cytometry confirmed that the relative amounts of DNA present in cultured ceils from both Fugu species were similar to that in blood cells of F. rubripes, and approximately 15% of that diploid human ceils. Telomerase activity was easily detectable in lysates ofF. niphobles and F. rubripes cell cultures, consistent with the notion that the cells are capable of indefinite proliferation. These cultures have uses in studies of regulation of genome size and gene expression, assignment of genomic sequences to specific chromosomes and fish pathology/virology.

V-11

Nucleospora salmonis: A microsporidan parasite of salmonid fish - development of new diagnostic approaches. R.HEDRICK, J. Barlow, T. McDowell, S. Gresoviac, and M. Creorgiadis. Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA 95616. E-mail: Nucleospora salmonis, formerly Enterocytozoon salmonis, is an important parasite of salmonid fish. This microsporidian parasite has been associated with losses of salmon in North and South America and similar parasites have been detected in at least two marine fish species. The unusual infection of the host cell nucleus distinguishes this parasite from all others in the phylum Microspora. Detection of spores of the parasite in stained tissue smears or sections has been the principal means for detection. Unfortunately, sporogenesis often occurs late in infection or at very low levels in fish suffering subclinical infections. We have developed a nested PCR test based on the 16S-like rRNA gene that effectively detects prespore and spore stages of the parasite in tissues from fish. The test has been effective in detecting the parasite in several species of salmonids from both North and South America. Sequence data on parasites from these different regions indicates both similarities and differences that may represent either movement of the pathogen with fish or contamination from indigenous sources.

V-12 Use of Established Fish Cell Lines in the Isolation and Characterization of Aquatic Animal Viruses. J. R. WINTON. Western Fisheries Research Center, Seattle, WA 98115. E-mall:[email protected]

Beginning in the late 1950s, mammalian cell culture techniques were adapted for use in the establishment of fish cell lines. An early goal of this work was to isolate and further Characterize viral pathogens that were causing severe losses among salmon, trout and other commercially important species reared in hatcheries. The development of fish cell lines immediately led to the isolation and characterization of the first viral pathogens of fish. A recent review listed more than 150 cell lines from fish and new ones are continually being reported as new species of fish, molluscs, and crustaceans are cultured and new disease problems are encountered. This presentation will review the use of fish cell lines for the isolation and characterization of a wide range of viruses from aquatic animals. Also discussed, will be the application of fish cell culture methods in the development of new diagnostic techniques for aquatic animal viruses that include DNA probes and the polymerase chain reaction. Finally, the application of fish cell cultures in the creation of novel strategies for control of virus diseases including attenuated and genetically-engineered vaccines will be reviewed.

CongressOn In VitroBiology

Mycoplasma: A Fresh Look at an Old Nemesis. J.W. HARBELL t and J. Luczak ~ qnstitute for In Vitro Sciences, Inc. Gaithersburg, MD 20878 ([email protected]) 2MA Bioservices, Inc. Rockville, MD 20850 ([email protected]) Occult mycoplasma infection of cultured cell stocks continues to plague the basic research and biotechnology communities. Contamination rates in excess of 15% have been found in mammalian cell cultures tested, with some countries reporting much higher rates. Infection rates in cells prepared for biopharmaceutical production have been shown to be as high as 4% despite increased control of contamination "portals of entry" associated with GMP manufacturing. Although the mycoplasma species involved have been predominantly of bovine, porcine and human origins, it is generally believed that most new infections now come from other contaminated cell cultures maintained within the laboratory. The routine use of antibiotics, which suppress but do not eliminate the infection, often mask overt signs of infection. Infected lines are generally unacceptable for use. Even in the absence of overt cytopathic changes, mycoplasma can have a profound impact on the cells' basic biochemistry, cytokine and growth factor response patterns, membrane integrity and virus production. In some reports, infections have been cured with antibiotics although success has been dependent on the infecting strain and has been difficult to measure. Prevention is the advisable approach. Successful prevention of contamination involves sound sterile technique, quarantine of new cultures, and an ongoing surveillance program. A number of detection methods are available including broth & agar culture, DNA fluorochrome staining (Hoechst stain), PCR and others commercial techniques. Each has certain strengths and weaknesses. Routine testing programs combined with quarantine of new cell lines entering the laboratory have provided the greatest success in preventing this nemesis of cell culture from gaining a foothold.

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"9"-13

V-14 Adventitious Agent Considerations in Animal-Derived Raw Materials L.J. SCHIFF. Genzyme Transgenics, Two Taft Court, Rockville, MD 20850:

Adventitious agent contamination is common to all biotechnology products derived from cell culture. If present in the final product, serious clinical consequences could result. The main portals for introducing adventitious agents include: 1) the source cell line, commonly called the cell substrate, 2) the use of contaminated raw materials isolated from animal tissue or blood, such as animal serum components, 3) monoclonal antibodies used in the manufacture of other products, 4) excipients used in formulating the final product, and 5) breakdown ofcGMP. This presentation will focus on animaland human-derived biological materials and ancillary products used in production, and ways to minimize transmission of adventitious agents. Biological raw materials used for production can potentially introduce contaminants such as mycoplasma, viruses, and the transmissible spongiform encephalopathies (TSE). Raw materials have to be screened for freedom of mycoplasma and viral agents by the manufacturers of these cell culture additives and/or by the manufacture of the biological product. The characteristics of the TSE agents do not permit easy detection and, therefore, a risk assessment approach must be used. The use of murine antibodies in affinity chromatography also has the potential for introducing retroviruses into the product. For safety testing, these antibodies should be tested the same manner as antibodies intended for in vitro administration. Human plasma products widely used as stabilizers and excipients have been shown to transmit viruses to recipients. These products require the same quality of testing as a final biological product.

V-15

Adventitious Viral Agents and Viral Clearance Issues in Cell Culture-Based Biotechnology. MARSHALL DINOWITZ, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080. E-mail: [email protected] An essential factor in the manufacture of pharmaceutical and biotechnological products is the incorporation of appropriate and adequate barriers to adventitious and endogenous viral agents. Raw materials testing, appropriate in-process testing and virus clearance validation are all necessary to provide an effective barrier to these agents. This presentation will focus on the multifaceted approach used to enhance viral safety with an emphasis on the strategies for virus inactivation and removal validation. Utilizing guidance provided by the International Conference on Harmonization (ICH) document on virus safety and the FDA Points to Consider, as well as other guidance documents, the role and expectations for virus clearance by the manufacturing process will be discussed. The rationale and procedures appropriate for demonstrating the robustness of the process for removing and inactivating a spectrum of viruses, including endogenous viruses, will be presented. Study design considerations and strategies for proper management and control of such studies will be highlighted including the incorporation of the necessary study controls for process scale-down, sample handling, storage and analysis as well as considerations of utilization of proper expertise, safety and statistical validity.

V-16 Exogenous Animal Viruses: The Risk of Using Baboons as Transplant Donors. J.S. ALLAN. Depart.~ merit of Virology and Immunology, Southwest Found-ation for Biomedical Research, San Antonio, TX 78228. E-mail: .

Baboons are being proposed as donors of bone marrow cells to treat AIDS patients and as heart donors for organ replacement. Baboons are genetically closely related to humans and as such the viral agents harbored in baboons are more likely to be, infectious and possibly pathogenic in humans. Of most concern are common viruses that comprise the normal viral flora in baboons and which are persistent latent infections. Two important examples are the herpesviruses and the retroviruses. Baboons harbor at least 3 herpesviruses (baboon CMV, herpes papio, SA8) and 4 retroviruses (simian foamy virus, simian T cell lymphotropic virus, baboon endogenous virus, and simian endogenous retrovirus). There are also likely to be several yet to be discovered viral infections in baboons. Simian foamy viruses are commonly found in healthy baboons and are highly cytopathic in vitro. Assays have been developed to detect SFV in the peripheral blood ceils of naturally infected monkeys. Recently, we have shown that human liver transplant recipients harbored SFV in several tissues at autopsy. Most: evidence suggests that microchimerism was established and baboon cells continue to circulate in transplant patients. These ceils may also harbor both endogenous and exogenous infections that might later be transmitted to humans. While there are several steps that can be taken to reduce the overall risk of infectious diseases in humans, the current paradigm indicates that viruses with long clinical latencies are difficult to detect and even more difficult to contain once they are established in the population. Issues related to the baboon resource and its future prospects for use in xenotransplantation will be considered.

Congress On In Vitro Biology

Endogenous Animal Viruses: Evidence that Endogenous Porcine Retrovimses Can Infect Human Cells. C. PATIENCE, Y. Takeuchi & RA. Weiss, Section of Virology, Institute of Cancer Research, London, SW3 6JB. E-mail: Xenotransplantation of primate and pig organs is viewed as a means to alleviate the shortage of human donor organs Although baboon to human xenotransplants have been attempted, most attention is being focussed upon the pig as a suitable donor of cells, tissues and vascularized organs due to a variety of reasons. The risks of infectious disease transmission from graft to recipient, and conceivably on to the new host population, has until recently remained a topic of active debate but relatively little research Although the known infectious pathogens of the donor species can be monitored and largely eliminated through the breeding of specific pathogen-free herds, unknown infections may exist which are asymptomatic in their natural host, but which could cause disease in the xenografted recipient. The normal germ line DNA of many vertebrate species contains sequences related to infectious exogenous retroviruses. Such endogenous retroviruses might cause disease if transmitted as a zoonosis, even when they are not normally pathogenic in their natural host species. Pig cell lines in culture have for many years be known to release retrovirus particles, but these were not previously thought to infect human cells. A re-examination of the host range of pig retroviruses was considered appropriate in the light of porcine xenotransplantation. The identification of an endogenous C-type retrovirus from cells of the domestic pig that is capable of infection, and serial transfer to replicate in a range of human cell lines will be described.

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V-17

V-18

Detection of Animal Retroviruses in Human Cell Culture. B.J. POTTS and K.G. Field. Retrovirology Department, Tektagen, Inc. Malvern, PA 19355 e-mail: The potential for retrovirus infection of animal cellular and tissue based products that are intended for human use presents a major challenge for virus testing. Animal retroviruses which may show the greatest risk to humans include; porcine endogenous retrovirus (PERV), bovine BLV and BIV, simian SIV, Foamyviruses (SFV), STLV and Type D viruses (SMRV), ovine VISNA, caprine CAEV and equine EIAV. Whether these viruses can replicate and produce progeny in human cell culture is a key question. These animal retroviruses were inoculated into primary human peripheral blood mononuclear cells (huPBMCs) enriched for monocytes/ macrophages. Positive controls included animal cell lines known to support replication of these animal viruses and HIV-I, HIV-2, HTLV-I and HTLV-II in the PBMC culture. Viral infections were done either by inoculation of cell free virus (SIV, PERV, BIV, SMRV, SFV, VISNA, CAEV and EIAV) or by co-cultivation with gamma irradiated (10,000 rads) virus infected cells (STLV, BLV, four PERV cell lines). Virus replication was detected by either a Mg ++ or Mn ++ reverse transcriptase assay (RT), an antigen capture ELISA (p19 for HTLV, BLV and STLV) and by transformation. All of these viruses were detected by these methods when grown in their respective animal cell line. The animal viruses that produced positive RT signal in cell free supernate after 30 days in culture included SIV and SFV Types 1,2 and 5. Animal viruses that infected and transformed huPBMCs included STLV and BLV. All of the remaining viruses (BIV, SMRV, VISNA, CAEV, EIAV and PERV) did not replicate in the huPBMCs as detected by a virion associated Mg ++ or Mn ++ dependent RT assay.

XENOTRANSPLANTS AND HUMAN INFECTION: REGULATORY CONSIDERATIONS CAROLYN A. WILSON, Ph.D., Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, FDA, Bethesda, MD 20892. E-mail: [email protected] The field of xenotransplantation, the treatment of human disease with cells of non-human derivation, is being investigated for a number of clinical indications. While xenotransplantation raises a number of scientific issues, such as the need to overcome immune rejection, the primary regulatory focus has been on the inherent risk of infectious disease to the xenograft recipient and the potential for spread to the public. Xenotransplantation clinical protocols provide the novel setting where large number of cells are implanted in a usually mamunesuppressed recipient resulting in long-term chronic cell to cell contact: an environment ideal for the processes of adaptation and selection to occur which may result in an infectious agent capable of efficiently infecting human ceils and of human pathogenicity. Of particular concern, are those agents with long latency periods, where human to human transmission of a previously unknown virus may occur prior to manifesting its pathogenic phenotype. The "Public Health Service Guideline on Infectious Disease Issues in Xenotransplantation" was drafted to address these public health issues. The Guideline provides recommendations for use of characterized herds of source animals routinely screened for infectious agents of concern combined with screening of xenografts when feasible. In addition, specific recommendations for patient follow-up are also described. The PHS intends that these in-process con~'ols for the production of xenografts will minimize the opportunity for known or novel cross-species infections to occur.

Congress On In Vitro Biology

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Abstracts

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T-2

Overview of Culture Models and Standardization. R. OSBORNE. The Procter & Gamble Company, Human & Environmental Safety Division, Miami Valley Laboratories, Cincinnati~ OH 45253-8707. Human skin and corneal models are proving useful in toxicology for investigation of mechanisms of epithelial response to chemical irritants and for prediction of human irritation to chemicals or products. Air-interfaced stratified epithelial cultures allow for topical application of test substances in their native form, modeling topical exposures in vivo. It is possible to detect in vitro cellular and tissue changes that are early steps in the process of inflammation in vivo, including cytotoxicity, release of inflammatory mediators and cytokines, and disruption of barrier function. Five stages of standardization of human epithelial cultures are proposed: (1) standardization of manufacturing procedures (e.g., source of tissue, sterility and adventitious agent testing, reproducibility); (2) determination of the structure of the in vitro construct relative to the in vivo tissue (e.g., cell types, threedimensional architecture, tissue-specific markers); (3) determination of the physiology of the in vitro construct relative to the in vivo tissue (e.g., barrier function, percutaneous absorption); (4) determination of the mechanisms of response to chemical irritants relative to in vivo (e.g., cytotoxicity, mediator and cytokine production, wound healing); and (5) validation of in vitro test results from cultures to in vivo irritant responses. It is proposed that consideration of these standardization factors will lead to mechanistically-sound in vitro tests for safety assessments, and also will make it possible to calibrate in vitro responses across different culture models.

In Vitro Techniques to Assess Ocular irritation. S.S. MATSUMOTO, M.E. Stern, D.C. Rupp and R.N. Decker. Allergan, Inc., Irvine, CA 92713. The high degree of innervation of the cornea and conjunctiva requires that eyecare products be comfortable as well as safe and effective. In pre-clinioal screening of eyecare drugs and formulations, the potential for ocular irritation (i.e., a sensation of burning or stinging) can be assessed by mechanistically-based studies using cultured cells. An overview of the physiological mechanisms of ocular irritation will be followed by some examples of cell culture methods that are based on these mechanisms. The ability of therapeutic chemicals to disrupt the ocular surface can be assessed with epithelial cell monolayer cultures possessing intercellular tight junctions. The integrity of the epithelial barrier can be measured by the fluorescein permeability or by the electrical impedance of the monolayer. The applied chemicals can induce the ocular surface to produce inflammatory mediators, such as eicosanoids, and this can be measured in corneal epithelial cell cultures. The direct nerve stimulation effect of applied chemicals or the indirect effect of induced inflammatory mediators can be measured with cultured sensory nerve cells using fluorescent probes. Finally, the status of in vitro ocular irritancy testing for eyecare products will be discussed.

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No Abstract Submitted

Comparison o f l n Vitro and In Vivo Human Skin Response to Consumer Products and Ingredients with a Range of Irritancy Potential. M.K. ROBINSON, M.A., Perkins, and R. Osborne; The Procter & Gamble Co., Miami Valley Laboratories, Cincinnati, OH. Human skin equivalent cultures are being investigated as a preclinicalskin irritationscreen to aid in the design of safe and efficient

human studies. Our approach has been to directly compare m vffro to in vivo human skin responses using historic or concurrent skin response data for products and ingredients including surfactants, cosmetics, antiperspirants and deodorants (AP/DO). The in vivo data consisted of visual scores (i.e., erythema and edema) from skin patch tests and diary accounts of skin irritation from product use studies. For the in vitro studies we evaluated cornified, air-int~rfaeed human skin cultures (EpidermTM) using methods to parallel human clinical protocols by topical dosing of neat or diluted test substances to the stratum comeum surface of the skin cultures. In the in vitro studies we evaluated endpoints that have been shown previously to be relevant to human skin irritation in vivo, including the MTI" metabolism assay of cell viability, enzyme release (lactate dehydrogenase and nspartate aminotransferase) and inflammatory cytokine expression (Interleukin-tc0. For surfactants, dose-response curves of M T r cell viability data clearly distinguished strongly irritating from milder surfactants, and rank ordered irritancy potential in a manner similar to in vivo 3-patch test results. For AP/DO products, all the m vffro endpoints correlated well with consumer reported irritation (r=0.78-0.94), with IL-hz release showing the greatest capacity to distinguish irritancy over a broad range. IL-Ia also showed the best prediction of human skin scores f~om 14-day cumulative irritancy tests of cosmetic products. These results confirm the potential value of comified human skin cultures as an m v~tro preclinical screen for prediction of human skin irritation responses, and identify the need to customize in vitro endpoints for irritancy prediction for different product classes.

Congress On In Vitro Biology

8-A

Abstracts

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T-6

No Abstract Submitted

Developing Strategies for Enhancing the Transport of Polypeptides: Enhancing Permeability and Stability. Patdck J. Sinko, Rutgers University College of Pharmacy 160 Frelinghuysan Road, Piscataway NJ 08854. EMail: [email protected] Little clinical success has been achieved in the mucosal delivery of peptide-based therapeutics. The low oral bioavailability of peptides results from several factors including high preabsorptive clearance due to metabolism, high cellular efflux or poor absorptive permeability and lumenal clearance. We have used a biopharmaceuticel approach to develop an oral salmon calcitonin delivery strategy that utilizes in vitro and in vivo techniques. The use of in vitro techniques atlow for the rapid evaluation of a peptide delivery strategy. The validity of this technique is assessed by comparing the results to in v/vo data.

T-7

T-8 Pharmacokinetic Modeling Approaches for Predicting In Vwo Absorption of Poorly Absorbed Drugs.Glen D. Leesman, NaviCyte, Inc., 851 East Glendale, Sparks NV 89431 • [email protected]

Historical Perspective on the Development of Human Liver Tissue Models for Research Applications. C. E. GREEN. Metabolism and Pharmacokinetics Department, SRI International, Menlo E-mail: [email protected]

The prediction of oral absorption of poorly absorbed drugs is a function of interactions between the drug, the formulation and the characteristics of the gastrointestinal tract (GIT). Physiochemical properties of the drug include dose, solubility, dissolution rate, particle size and permeability in the various sections of the GIT. The major physiologic variables in the GIT which affect absorption are permeability, pH, gastric emptying time, intestinal transit time of both soluble and insoluble drug and water flux.

The availability of functionally viable human tissues and technical advances in in vitro assays using these preparations have led to general acceptance of data obtained in vitro with human liver cells. For example, human and animal liver specimens are being used with increasing frequency to determine the metabolite profile, rate of metabolism, and the specific enzymes involved, both early in the development process for new drugs and later to elucidate mechanisms of action. Approximately 15 years ago research with human liver specimens was conducted only by laboratories that had direct access to human specimens, either excess samples from pathology, autopsy specimens or organ donor livers. Problems with tissue quality and availability were common. Today most laboratories that want to work with human liver cell and tissue models have access to these preparations. Several different notfor-profit organ procurement and commerical organizations provide fresh and frozen liver specimens, microsomal fractions, primary human hepatocyte cultures, liver slices, and human liver cell lines that maintain liver specific functions. Difficulties in working with human liver models still exist and are related to the large degree ofinterindividual variability in the human population and the to the frequent need for fresh tissue. Current research on cryopreservation of human liver cells and stabilization of differentiated functions are aimed at solving these problems and further expanding the usefulness of these models.

Those drugs which are poorly absorbed fall into one of the following two classes in the Biopharmaceutical Classification System (BCS). These two classes are i)low solubility/high permeability and ii) high solubility/low permeability. This presentation focuses on predicting the rate of ab~cn-gaion of poorly absorbed drugs using the IDEATM pharmacokinetic modeling software. The importance of in vitro correlates and predictors in determining the input function will be discussed in detail.

Congress On In Vitro Biology

9-A

Abstracts

T-9

T-10

Use of Human Liver Slices as ~ In Vitro ~ e ~ r c h Tool. D.C. BODE. ~tcmatio~al I~zstitute for the Adv~cemc~t of Madici~e, Extort, PA 19341. Email: The precision-cut liver slice is an i~ vitro model of hepatic physiology and pathophysiology with multiple applications, notably in drug metabolism a~d toxicolo87. The major advantage of slices vs. isolated hepatocytes is the fact that slices con~i~z all of the various cell types found i~ the liver,

Human Hepatocyte Supply : A Practical Look at the Advantages and Limitations of This Valuable Emerging Research Tool. JESS STENGEL, BioWhittaker's Clonetics Hepatocyte Laboratory, Walkerswille, MD 21793 E-mail: <[email protected]> Human hepatocytes are an extremely valuable tool for studying drug development. Although hepatocytes can be cryopreserved and recovered with basic functions intact, the percentage of isolations that perform well after cryopreservation is quite low. Now that fleshly isolated human hepatocytes are available on a frequent basis commercially, it has become much easier for researchers to obtain cells on a schedule which satisfies their research needs and progress in hepatocyte research is accelerating. The US FDA has recommended in vitro studies to examine metabolism and toxicity of drug candidates. The hepatocytes which are supplied commercially are well suited for these studies as they have been shown to express active drug metabolizing enzymes and are able to metabolize compounds such as 7-EC. Hepatocytes can be obtained in a number of multi-well formats, such as 96-well plates, particularly useful for high-throughput screening assays, as well as in T-flasks and in suspension cultures shipped to arrive within hours of isolation. Plated format cells are available with a number of extra-cellular matrices, such as rat tail collagen, Matrigel or gelatin. Clonetics also offers ~mian hepatocytes t~om cynomolgus and rhesus. Hepatocytes from both species can obtained freshly isolated and/or eryopreserved. Hnman hepatocytes offer unique advantages. Because of the differences in metabolism between different species, it is the human hepatocyte which is the ideal cell for drug research. Researchers now have expanding opportunities to use the most relevant model for development of human-use drugs.

with the native extracellularm a ~ and eytoarchit~ture i~tact. Thus, toxicologic ~ d metabolic fuzdi~gs o b t ~ e A i~ vitro with slicesmay be more representative of i~ vivo results t h ~ f m ~ g s obtained with hepatocytes. G~ the other ha~d, isolated huraa~ hepat~ytes are more suitable for m vitro c~jme i~ductio~ studies tha~ slices, gece~tly, cor~siderable progresshas ~ made i~ terms of cryopreservatio~ of both preparation.

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T-12

The Importance of High Throughput Pharmacokinetic Screening in Drug Discovery. Patrick J. Sinko, Rutgers University College of Pharmacy 160 Frelinghuysen Road, Piscataway NJ 08854. E-Mail: [email protected]

The Use of a Human Liver Cell System in ADME/PK Studies. JAMES H. KELLY, Andrea L. Spiering, Norman L. Sussman. Amphioxus Cell Technologies, Inc., Houston, TX 77082-2646, emaih [email protected] The widespread implementation of high throughput methods for drug discovery has shifted the rate limiting step in drug development. Compounds that score positively in the initial rounds of screening now pile up at the second phase of development, creating a new bottleneck as decisions are made on which compounds to advance or reject. A variety of studies are performed, generally using rodents, to examine absorption, distribution, metabolism, excretion and pharmacokinetics (ADME/PK). New cell culture-based methods are being examined as a means of converting at least some of these studies into a high throughput format. Unlike first round screens, these studies require intact cell systems. Since the liver is frequently the major site of drug metabolism, there is a need for a highly characterized system to examine human liver metabolism of new drug candidates. Primary hepatoeytes are generally a poor choice for high throughput methods for a variety of reasons, primarily related to their inability to multiply in culture. We have developed a human liver cell line which has been selected for adult liver specific function and which retains all of the major liver metabolic pathways. These cells are packaged as a unified system, ACTIVTox, including cell culture medium tailored to the particular study. For example, cells can be obtained preinduced with general inducers of drug metabolism, such as phenobarbital, or cultured in completely hormone free medium. In addition, since ACTIVTox is based on a cell line, standard cell culture quality control/quality assurance mechanisms such as cell banking can be applied. Finally, cells can be cultured in small hollow fiber devices such that long term drug interaction and secondary metabolite studies can be easily envisioned. While primary human hepatocytes will remain the standard of comparison for all in vitro studies of liver function, their unpredictable availability limits their usefulness in studies requiting large numbers of replicate cultures. ACTIVTox is uniquely positioned to fill this need. Congress On In Vitro Biology

Selecting new~erapeutic agents has historically been an expensive and inefficient process. Although most efforts focus on discovering a =hit" or new therapeutic agent, it is also important from a resource perspective to identify and eliminate drug candidates that are likely to fail in the clinic. Drug candidates that fail late in the development cycle waste signifcant resources and delay the time market for new chemical entities. However, a new paradigm is emerging that is costeffective, efficient and offers higher throughput HTPkS TM High ThrP_n2hputPharmaeokinatic Screening. HTPkSTM is a set of hardware and cell-based tools that enable efficient drug selection by allowing the simultaneous assessment of the absorption, metabolism and toxicological properties of potential therapeutic agents, This information can then be fed back to discovery chemists to produce highly focused template libraries. High Throughput pharmacokinatic strategies and approaches wilt be discussed.

I O-A

Abstracts

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T-14

Assessing Compound Liability By High Through-Put Screens: Practical Considerations to Converting In Vitro Induction, Mechanistic and Cytotoxicity BioAssays of Primary Cultures to High Density Plate Formats. L. Vemetti', S. Roberts 2, D. Burns t, and B. Beutel'. Department of Pharmaceutical Discovery' and Drug Safety2, Abbott Laboratories, Abbott Park, I160064

A Novel Microtiter-based DNA Damage Assay. R.E. Davidson and D. A. PEREIRA. Precendidate Technology, Pfizer Central Research, Groton, CT 06340. E-mail:

The utilization of high throughput screening ( HTS ) has revolutionized the way that the pharmaceutical industry discovers and develops drugs. A key to the success of HTS technology has been the adoption of miniaturized and formatted assays. The benefits of the former are the significant reductions in the amount of compound and reagents needed due to reduced assay volumes. The use of fixed formats facilitates automation and hence increases capacity. Because nonmicrotiter bacteriabased assays employ traditional microbiological methods, they require relatively large quantities of compound and are more difficult to automate. We have applied an HTS strategy, using microtiter plate formats, in an attempt to develop a miniaturized DNA damage assay. In the process we have discovered a novel " n o n m u t a t i o n a l " phenomenon that appears to correlate with DNA damage in S a l m o n e l l a t y p h i m u r i u m .

Standard in vitro assays to assess cytochrome P-450 induction, protein and DNA synthesis, mitochondrial function and cell membrane integrity were evaluated as potential high-throughput assay screens. These assays are typically used in the 6 or 24 well plate formats. Miniaturization to the 96 well microtiter plate was first developed and then the assays were evaluated for higher density format plates such as 384 or 864 well microtiter plates. Problems encountered with miniaturization included edge effect and other plate biases, rapid dehydration of small liquid volumes, increases in statistical scatter with the smaller buffer volumes and lower whole cell numbers, decreasing signal to noise ratios as well as lack of instrument sensitivity with certain end-point measurements and the increase in the variation of the biological response between isolations of ceils. Solutions to some of these problems will be discussed. For certain assays such as mitochondrial function and plasma membrane integrity acceptable results were achieved with 3000 cells in a 2-5 I.tL volume. Other assays such as protein and DNA synthesis and/n vitro induction required higher cell density and assay volume.

T-15 Automated Systems for Biological Assays in High Throughput Screening. J. Wegrzyn, K. Houck, M. Stamper, and W. JANZEN. Sphinx Pharmaceuticals, a Division of Eli Lilly and Company, 4615 University Drive, Durham, NC 27707. E-mail: [email protected] The automation of biological assays presents a particularly challenging problem. Although a broad spectrum of devices exist, many factors must be considered when selecting automation. Some critical requirements to determining cost versus benefit are throughput, flexibility, and lifecycle. The following four examples will be used to illustrate these dimensions: 1. Workstation automation of the Ames II assay in 384 plates (low throughput, low flexibility, long lifecycle). 2. Modular workstation automation of assays in the Sphinx HTS facility (high throughput, high flexibility, short lifecycle). 3. Full automation of SPA assays employing the Beckman ORCA arm (high throughout, low flexibility, moderate lifecycle). 4. Automation of the production of fermentation sample extracts for screening (high throughput, low flexibility, long lifecycle). Each of these represents a different set of needs which, in turn, required a different solution for optimal performance.

Congress On In Vitro Biology

T-16 In vitro Testing of Embryotoxicity Using the Embryonic Stem

Cell Test (EST): an Intedaboratory Compadson. H. SPIELMANN; G. Scholz, S. Bremer, H.G. Holzhuetter, ZEBET (National Center for Documenlation & EvaJualion of Alternatives to Animal Experiments) at the BgW, D-12277 Berlin, Germany, e-mail: Embryonic stem (ES) cells of the mouse differentiate under appropriate conditions into the major embryonic tissues. In contrast to all of the existing in vitro embryotoxicity tests these cells allowed to develop an in vitro embryotoxicity test without taking embryonic cells or tissues from pregnant animals. The development of such an assay was recently described (In Vitro Toxicol. 10:119-127, 1997). This assay, the ES Cell Test (EST), uses two permanent mouse cell lines: ES cells to represent embryonic tissue, and fibroblasts (3T3 cells) to represent differentiated tissue. The EST takes advantage of the possibility to maintain ES cells in the undifferentiated stage in the presence of the cytokine leukemia inhibiting factor (LIF). Upon withdrawal of LIF ES cells will form embryoid bodies (EB) under appropriate conditions and differentiate into the major embryonic tissues. In addition, ES cells are more sensitive to toxic agents than differentiated cells. Therefore, the inhibition of differentiation of ES cells into cardiac myoblasts (identified by contraction) and cytotoxicity of ES cells and 3T3 cells (MTT-test) were used as endpoints in the EST for predicting the embryotoxic potential of chemicals. 16 test chemicals were correctly classified in the EST into three groups of embryotoxic potential: non-, weak/moderate and strong embryotoxic. The EST is currently evaluated within a European prevalidation projekt of three in vitro embryotoxicity tests. In this study, chemicals were tested in two laboratories: ZEBET at the BgW (Bedin, Germany) and ECVAM at the JRC (Ispra, Italy). Here we describe the results obtained from testing of a set of test chemicals representing the three groups of embryotoxic potential in the two laboratories (ZEBET and ECVAM). An independent biostatistical evaluation showed that the EST is highly reproducible and performance is stable in different laboratories.

11-A

Abstracts

T-17

T-18 Endocrine Disruptor Screening Strategies. W.R. K E L C E and L.E. Gray, Jr. Reproductive Toxicology Division, National Health and Environmental Effects Research Lab, US EPA, Research Triangle Park, N C 27711. E-mall: .

The United States Congress recently mandated via the F o o d Quality Protection Act (Public L a w 104-170) and the Safe Drinking Water Act (Public L a w 104-182) that chemicals be screened for estrogen and other h o r m o n e activities as deemed appropriate by the E P A Administrator. In response to this congressional mandate, screens to assess estrogen, androgen and thyroid hormone agonist and antagonist activity are being developed. Specific in vitro screening assays include cell-free steroid hormone receptor binding and steroidogenic enzyme activity, whole cell receptor binding (includes potential for test chemical metabolism), hormone-dependent cell proliferation, transcriptional activation (reporter gene assays to distinguish agonists f r o m antagonists) and mechanism specific assays such as r e c e p t o r - D N A binding (electrophoretic mobility band-shii~ and promoter interference assays). Since disposition and test chemical metabolism can alter the effects o f a chemical in vivo, both in vitro and in vivo approaches are used. Short-term, hormone specific in vivo assays (such as delayed pul:/ertal maturation assays for (anti) estrogens and (anti) androgens) are sensitive and non-invasive assays to assess adverse endocrine effects in whole animals. The technical merits and limitations o f these screening strategies will be stressed using specific examples derived from empirical efforts to develop and/or use these technical assays. (This abstract does not reflect EPA policy).

( ),ngre~ On In Vitro Biology

Development and Characterization of n 3-Day Automation Compatible Caco-2 Assay System DARWIN ASA, Ph.D., Becton-Dieklnson Lnbware, Bedford, MA 01730 E-maih [email protected]

Caco-2 cells are being increasingly used as an in vitro model system for screening drug candidates for their intestinal absorption potential. The traditional method of culturing Caco-2 cells to obtain a differentiated phenotyp¢ requires up to 3 weeks culture and involves many labor intensive steps. Recently. the BIOCOAT~ Caco-2 Assay System ( which provides a monolayer of Caco-2 cells ready for compound permeability screening in 3 days) has been reported to be a more productive and efficient s'.'2,stemto use with Cac¢~2 cells to generate compound permeability data 1,2,-L To further charucteriz¢ the BIOCOAT system and compara it's performance to the traditional 21-day Caco-2 system, several measures of Caco--2 cell differentiation were tested. Caco-2 cells cultured in either the 3-day BIOCOAT or 21-day culture system were tested for barrier fimction, brush border enzyme activity, transporterfunction, and morphologicalcharacteristics indicative of cell differentiation. Also, the BIOCOAT 3-day Caco-2 system was adapted from using individual cell culture inserts to using an automationcompatible MultiwellTM Insert format suitable for use with commonlaboratory automationequipment. Our results indicate that Caco-2 cells cultured in the BIOCOAT 3-day system possessesmany of the cell differentiation features found in the traditional 21 day Caco-2 system . Many of the biological and morphological markers indicative of Caco-2 cell differentiation compare favorably between the 3-day B1OCOAT and 21-day Caco-2 culturesystems. Therefore, the B1OCOAT Caco-2 system provides a more convenient and productive alternative to the 21-day Caco-2 system to generate compound permeability data, while retaining many of the important properties of the 21day Caco-2 system .By developing and characterizing a more efficient system for Caco-2 culture, we believe that the possibilitiesfor the use of Caco-2 cells as an in vitro model for intestinal compound permeability will be greatly enhanced. 1. Chong,S., et.al., PharmRes. (1997), Vol. 14, pp 1835-1837. 2. Asa, D. and LaRocca, P. Poster presentation at 4th International Conference on Drug Absorption,Edinburgh,(1997). 3. Woods, W. and Asa, D. Poster presentation at AAPS Annual Meeting, Boston, (1997).

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Abstracts

1-2

I-I Molecular Determinants of Baculovirus Virion Occlusion and Occlusion Body Shape. B. A. FEDERICI, J. E. Eason, R. H. Hic¢, and L .r. Johnson. Interdepartmental Graduate Prob..am in Genetics, University of California, Riversld¢, CA 92521. E-mall: Baculovlrus occlusion body assembly results in the selective occlusion of virions into large polyhedra containing many virions per polyhedron in the nuclear polyhedrosis viruses (NPVs), or small granules containing one virion per granule in the granulosis viruses (GVs). Despite this selectivity and structural differences between polyhedra and granules, the occlusion body matrix proteins, polyhedrin and granulin, share high amino acid sequence similarity. Mutants of NPVs suggest virion occlusion and occlusion body shape are determined partly by the tertiary structure of polyhedrin and its interaction with the virion envelope, This process was studied in vitro in Tn5 cells by substituting Trichoplusia ni GV granulin for Autographa californica MNPV polyhedrin, TnGV granulin synthesized by the AcMNPV produced very large (2-5 gm) cuboidal polyhedra in the cytoplasm and nucleus of infected cells, but only a few per cell. Aside from their atypically large size, these polyhedra were aberrant in that they lacked beveled edges, containe6 fractures, and occluded few virions. Placing a nuclear localization signal (KRKK) in granulin directed more granulin to the nucleus, and resulted in more structurally uniform polyhedra in which no virions were observed. Although polyhedra formed by granulin were aberrant, they appeared to interact normally with AcMNPV pl0, fibrous structures, and the polyhedral calyx. These results provide additional evidence that specific interactions occur between polyhedrin and the virion surface, and that these interactions facilitate normal virion occlusion and polyhedron assembly in w i l d - t ~ AcMNPV.

1-3

No Abstract Submitted

The role of viral ubiquitin in baculovirus infection LINDA A. GUARINO, Department of Entomology, Texas A&M University, College Station, TX 778432128 E-mail The baculovirus Autographa californica nuclear polyhedrosis virus potentially encodes more than 150 proteins, one of which has high homology to the eukaryotic protein ubiquitin. Ubiquitin is highly conserved and varies by only three amino acids among mammals, yeast and plants. The viral protein, however, differs from animal ubiquitin at 18 amino acid residues, although all of the residues known to be essential for ubiquitin function have been conserved. These observations raise interesting questions with respect to why the viral protein has diverged from eukaryotic ubiquitin and why the virus encodes its own ubiquitin gene when host cells contain abundant amounts of ubiquitin. One answer may be that ubiquitin is a major structural protein of the budded form of AcNPV virions. It is present in the viral envelope and is anchored to membranes by phospholipid. This type of modification has not been found in other systems, suggesting that enzymes responsible for the acylation may be unique to insects. To study the role of ubiquitin in infection, we constructed a recombinant virus with a frameshift mutation within the coding sequence of the viral ubiquitin gene. This mutant was viable, indicating that viral ubiquitin is not essential for growth in tissue culture. However, the yields of infectious budded virus were decreased, and viral particles contained only host ubiquitin anchored to the membranes. Pulse chase analyses also revealed differences in the pattern of protein synthesis. Together, these results suggest that viral ubiquitin is a nonessential protein that confers a growth advantage to wildtype particles.

1-4 Functional Analysis of Baculovirus Enhancins in the Infection Process of Insect Cells. R.R. GRANADOS, P. Wang and J. Zhong. Boyce Thompson Institute, Comell University, Ithaca, NY 14853. E-mail: Baculovirus enhancins 'are viral encoded proteins which have been reported to increase the efficiency of virus infection to insect larval hosts and cultured cell lines. Earlier work by Tanada and co-workers (1959-1989) reported that enhancins functioned by mediating the binding and fusion events of viruses to insect ceils under in vivo and in vitro conditions. Granados and co-workers (1988-1997) presented a second hypothesis which proposed that enhancins increased virus infections in insect larvae by their proteolytic action on the midintestine peritrophic matrix, disintegrating this protective barrier and facilitating the passage of virus from the midgut lumen to the intestinal epithelial cells. In 1997 we characterized the binding and fusion of Autographa californica nucleopolyhedrovirus (AcMNPV) to cultured insect ceils (Sf21) in order to examine if enhancin could affect these early infection events. Under our conditions using 35S and R-18 labelled virus we were not able to demonstrate that enhancin could increase AcMNPV binding and fusion activity on Sf 21 cells. We recently developed a primary midgut cell culture assay system for analyzing the binding and fusion events of baculoviruses to midgut cells. Under these conditions we were also not able to demonstrate that binding and fusion of R-18 labelled AcMNPV to midgut cells could be increased by enhancin. The role of enhancin in affecting in vitro infection of cultured insect cells is yet to be confirmed.

CongressOn In VitroBiology

13-A

Abstracts

P-1

P-2

No Abstract Submitted

Development of Fungus Resistant Plants. KENNETH BARTON. Monsanto Life Sciences Company, 700 Chesterfield Village Parkway, St. Louis, MO 63198. E-mail: [email protected]. The rapid advancement ofbiotechnology is catalyzing transition in agricultural crop protection practices to minimize grower inputs, while enhancing protection delivered at the time of seed planting. Novel agricultural chemicals are now delivered in ultra-low use rates, and increasingly are available for "on the seed" application. Genetic disease resistance traits are also rapidly being developed through use of molecular breeding and transgenic disease resistance technology. Fungal protection for crops is advancing particularly fast, with transgenic crops now in field trials to assess efficacy and durability of a variety of disease resistance genes. The use of genes encoding antifungal proteins affords near-term opportunities for increasing protection against major pathogens such as Verticillium (early die) and Phytophthora (late blight) in potato, and Fusarium (headscab) in wheat. Issues of utilizing these protective mechanisms include resistance durability, stacking of multiple transgenic traits, and development of combined packages of seed-delivered agricultural chemicals and genetics. Within several years, crops expressing the new transgenes will be available to growers to reduce the cost of crop protection while maximizing field productivity.

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P-4

Nematode Resistance Genes in Tomato. V.M. WlLLIAMSON, S.B. Milligan, J. Bodeau, J. Yaghoobi, and I. Kaloshian. Center for Engireering Plants f o r Resistance Against Pathogens, University of California, Davis~ CA 95616. E-mail: [email protected] Root-knot nematodes (Meloidogyne spp) are endoparasitic roundworms that cause economic damage to many crops. Many modern tomato varieties carry a dominant locus, Mi, that confers resistance to root-knot nematodes. In resistant plants, there is no development of the feeding site. Instead, a localized tissue necrosis or hypersensitive response occurs at or near the site where feeding would normally be initiated. To clone M.i, molecular markers closely linked to the gene were identified by analysis of recombinants. These markers were used to isolate tomato DNA spanning Mi as Bacterial Artificial Chromosome clones. 52 kb of contiguous DNA was sequenced and three open reading frames with similarity to cloned plant disease resistance genes were identified. Susceptible tomato transformed with one of the genes, Mi-1.2, were resistant to nematodes and progeny segregated for resistance. Mi-1.2 has highest similarity to Prf, a tomato gene required f o r resistance to Pseudomonas syringae. Prf and M i - l . 2 share structural motifs including a potential leucine zipper, a nucleotide binding site and a leucine-rich repeat region that are characteristic of a family of plant proteins including several that are required for resistance to bacteria, fungi, and, now, nematodes. How a single gene can mediate resistance to a nematode has long been a question of interest. The cloned gene will allow us to explore the mechanism of resistance, and, possibly, to transfer resistance to other crop species that are damaged by nematodes but for which no natural resistance is currently available.

Genetic Engineering for Abiotic Stress Tolerance. HENRY T. NGUYEN, Jingxian Zhang, and Natalya K Klueva. Dept. of Plant & Soil Sci., Texas Tech Univ., Lubbock, TX79409. Email: Drought, low temperature, and high salinity are limiting factors for plant growth and crop productivity. In their quest to feed the ever-increasing world population, agricultural scientists have to contend with these adverse environmental factors. If crops can be redesigned to better cope with abiotic stress, agricultural production can be increased dramatically. Recent advances in understanding crop abiotic stress resistance mechanisms and an advent of molecular technology allow us to address these issues much more efficiently than in the past. This paper will review the current status of research on mechanisms that plant use to adapt to abiotic stress and summarize functions of stress-induced genes and their products. Emphasis will be placed on the most significant achievements of genetic engineering approach for improving plant abiotic stress resistance. Improved resistance to drought, salinity, and low temperature has been observed in transgenic plants which express genes regulating osmolytes, dehydrins, antioxidant enzymes, and membrane composition. Although the results are not always consistent, these studies collectively foretell a scenario where biotechnology would arm our future crops with new tactics to survive in hostile environments. Further experiments are needed to determine if the achieved increases in stress tolerance are applicable in agriculture.

Congress On In Vitro Biology

14-A

Abstracts

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P-5

Redirecting the synthesis of storage lipids in oilseed crops. V.C. KNAUF. Calgene LLC, 1920 Fifth Street, Davis, CA 95616. E-mail: [email protected]

Plants as Biofactories for the Production of Biodegradable Plastics: Multigene Pathways for the Biosynthesis of Polyhydroxyalkanoic acids.

Ken Gmy~, Nancy Biest Taylor, Susan Colburn, Katie Houmiel, Debbie Mahadeo, Tim Mitsky, Steve Padgette, Steve Reiser, Damian Rodriguez, Steve Slater, Rob Stock, Tien Tran, Greg Thorne, and Henry Valentin Polyhydroxyalkanoates (PHAs) are a class of naturally produced polyesters that have both thermoplastic properties similar to polyethylene or polypropylene, and inherent biodegradability. Poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV), or BiopoW~ is commercially available and is produced through fermentation of Ralstonia eutropha using glucose and propionic acid as the carbon sources. While the process to produce PHBV is quite efficient - up to 90% of the cell dry weight can be polymer - it is costly relative to that associated with the manufacturing of commodity-based plastics. An attractive alternative to fermentation-based production of PHAs is the biosynthesis of this material in transgenic plants. The advantages to this route include, but are not limited to, decreased costs, and production from a renewable resource. One of the challenges to the plant approach, however, is to metabolically engineer target plants so as 'to generate 'the appropriate intermediates for PIqA production. These intermediates must originate from common carbon metabolite pools found in the plant. For PHA copolymers such as PHBV, novel pathways must be designed that take into consideration the following: the pool of carbon available during different plant developmental stages; the specific plant tissue targeted for production, such as seed or leaf; and the targeted subcellular location for biosynthesis. As an example, propionyl-CoA is an essential intermediate required for the biosynthesis of the C5 component of PHBV, but is normally in very low abundance in the plant cell. We have proposed utilizing threonine as the carbon source for propionyl-CoA, and have designed a 2-step route to produce this metabolite at levels sufficient for copolymer production. Metabolic studies of transgenic and wild-type plants aimed at determining 2-ketoacid, and free amino acid levels demonstrate the effects the transgenes have on the carbon flow. This work as well as other strategies for PHA copolymer production in plants will be addressed.

The kinds of fatty acids found in the storage lipid reserve triacylglycerols and the relative content of triacylglycerol in seeds are highly heritable traits. The kinds of fatty acids found in seed oils are often the same as those found in plant structural lipids. However, the ratios of these fatty acids in seed triacylglycerols can vary and hence palm oil is different than olive oil is different than soybean oil. These ratios for a given seed oil is also higbly heritable. Other seed triacylglycerols may contain unusual fatty acids; e.g., castor beans contain high levels of a hydroxylated fatty acid, ricinoleate. Many examples now exist for the genetic manipulation of the heritability of fatty acid composition of triacylglycerols in the major oilseed crops of canola and soybean. Not only have ratios of fatty acids been altered, but in some cases, wholly novel vegetable oils have been created. Practical applications range from better food functionality to healthier food oils to vegetable oil feedstocks for industrial uses. Specific examples of genetically engineered oils with enhanced levels of capric, stearic, ricinoleic, and nervonic fatty acids each demonstrate the superficial simplicity of making dramatic changes in fatty acid composition in agronomically viable crop plants. Each example, however, also demonstrates that re-creating in canola the capric-, stearic-, ricinoleic-, or nervonic-rich oils of Cuphea hookeriana, Garcinia mangostana, Ricinus communis, and Lunaria annua, respectively, may not be so straightforward.

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P-7

Meiotic Prophase Chromosome Behavior in Maize. W. Z. CANDE (1), H. Bass (2), A. Franklin (1), L. Harper (1), J. Sedat, J.(3), Agard, D.(3), RieraLizarazu, O.(4), Rines, H.(4), Philips, R.(4).(1) Dept. of Molecular and Cell Biology, Univ. of California, Berkeley, (2) Dept. Biol. Sci., Florida State U., TaUahassee, (3) Dept. of Biochemistry and Biophysics, University of California, San Francisco, (4) Dept. of Agronomy and Plant Genetics, University of Minnesota, St. Paul. E-mall: Zcande @uclink4.berkeley.edu

Quantification of Metabolic Fluxes for Metabolic Engineering in Plants. J.V. SHANKS, Depts. of Bioengineering and Chemical Engineering, Rice University, Houston TX 77251-1892. E-mail: Metabolic engineering to plants is hindered by a lack of quantitative information on fluxes in the metabolic pathways. Analysis of fluxes in metabolic pathways in response to an environmental or genetic manipulation can help identify ratelimiting steps. Since total productivity is the goal for enhanced production of a valuable product, the product of the biomass productivity times the metabolite specific yield should be optimized. This implies a systems analysis of both primary and secondary metabolic pathways. This talk will provide an overview of in situ measurements using 3~p and ~3C NMR spectroscopy to monitor primary metabolism and physiology and a strategy using HPLC photodiode array analysis, TLC, and MS to obtain temporal concentrations of secondary metabolites. The model system is hairy root cultures of Catharanthus roseus and indole alkaloid pathways.

Congress On In Vitro Biology

We are investigating the chromosomal rearrangements that occur during pairing and synapsis of homologous chromosomes of maize. Using a polyacrylamide gel embedding technique in combination with fluorescence in situ hybridization (FISH), we are conducting a three dimensional (3-D) analysis of meiotic telomere behavior. Images are collected using a computerized epifluorescence microscope and the data stacks are processed by 3-D deconvohition. We demonstrate that telomeres undergo a dramatic rearrangement from a random distribution at leptotene to a clustered distribution on the nuclear envelope at zygotene, revealing the presence of a meiotic prophase telomere motility system. To analyze the kinetics of whole chromosome pairing in 3-D we are using an oat-based chromosome addition line in which a single maize homologous chromosome pair has been introduced. By selectively staining the maize chromosomes with maize FISH probes we can follow the progression of pairing relative to telomere clustering. We are using immunocytochemistry in 3D to describe the redistribution of enzymes involved in meiotic recombination during initiation of pairing. During leptotene, tad51 is present in the nucleus but has a diffuse distribution. At the end of leptotene just as the telomeres are beginning to cluster, rad51 foci begin to appear on the chromatin, The foci are at a maximum early in zygotene (400-600/nucleus), but as pairing proceeds their number decreases until in pachytene only a few (10-20) persist.

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P-9

P-10 Maize Sperm Cell Analysis Using a B Chromosome Specific Probe for In Situ Hybridization. P. KEIM, M. Rusche, L. Shi, T. Zhu, H.L. Mogensen, M Rougier, A. Chaboud, C. Dumas. Northern Arizona University, USA; Ecole Normale Superieure de Lyon, France. E-mail:

The two sperm cells of common origin within the pollen tube of flowering plants are each involved in a fertilization event. It has long been recognized that preferential fusion of one sperm with the egg can occur in B chromosome-containing lines of maize. If the second pollen mitosis begins with a single B chromosome, nondisjunction will result in one sperm possessing two B chromosomes and the other containing no B chromosomes. The B chromosome-containing sperm most often fertilizes the egg, whereas the sperm nucleus with no B chromosomes fuses with the polar nuclei. In this study, we used a B chromosome-specific DNA sequence (pZmBs) and in situ hybridization to identify and track the B chromosome-containing sperm cell within mature pollen and pollen tubes. Using genetic line TB-10L18, our results indicate that nondisjunction of the B centromere occurs at an average frequency of 56.6%, based on four plants and 1306 pollen grains analyzed. This is consistent with the results of genetic studies using the same B-A translocation. In addition, our results suggest that B chromosome nondisjunction can occur during the first microspore division. Spatial distribution of the B chromosome-specific probe appears to be largely confined to one tip of the sperm nucleus, and a DNA fragment found outside the pollen nuclei often hybridizes to the B chromosome-specific probe. Within pollen tubes, the position in which the B chromosome-containing sperm travels (leading or trailing) in relation to the sperm cell lacking B chromosomes appears to be random.

P-11

No Abstract Submitted

Molecular cytogenetic analysis of the structure and evolution of plant genomes. J S (PAT) HESLOPHARRISON. John Innes Centre, Norwich, NR4 7UH, England. E-mail: The range of approaches to analysis of the structure and changes in plant genomes is expanding rapidly. The analyses give valuable and complementary data which are applicable to quantifying, understanding and sometimes modifying the types of change seen during in vitro culture. Molecular cytogenetic techniques include DNA:DNA in situ hybridization in combination with chromosomal, DNA, RNA and immunocytochemical analysis of plant genomes. Molecular cytogenetics has many applications to in vitro biology, where the capability to provide an overview of the structure of the plant genome and its repetitive DNA is particularly valuable. The accelerated evolution seen in tissue culture can also provide pointers for what is happening in whole plants over longer (evolutionary) and shorter (plant breeding) timescales. Different repetitive DNA sequence families may show wide variation in sequence and abundance even between closely related plants. Introduction ofgenomes, chromosomes or chromosome segments from one species to another, by either sexual or protoplast fusion methods, is an important breeding method and enables study of interactions between genomes. Often, the chromosomal composition of the resultant plants can be analysed in detail by using labelled genomic DNA to paint chromosomes from one of the progenitors. Tandemly repeated DNA sequences may be used as probes to identify individual chromosomes and to characterize chromosome rearrangements that often happen during culture. The activation of retroelement sequences may have consequences for somaclonal variation and the stability of both cultures and regenerant plants, and the work to be discussed has implications for understanding the wide range of induced and inducible mutations that can occur.

P-12 Cell Division Control and the Cell Death Programm in Plants. Marco Takita, Chris Lamb and PETER DOERNER. Plant Biology Laboratory, The Salk Institute, La Jolla, CA 92037. E-mail: Programmed cell death is instrumental for pathogen containment during plant defense, but is also crucial for vascular development and the sculpting of leaf organs. Birth and death of cells are tightly coupled in animals, where in addition to its role in development, apoptosis is a powerful mechanism to remove cells that proliferate at inappropriate times. Although plant cells, particularly within the meristems responsible for organogenesis are long-lived, very little is known about their apparently very successful mechanisms of genetic surveillance and removal of cells gone awry, as neoplasias are infrequent. We are examining the relationship of cell death and cell division during pathogen or stress-signaling induced apoptosis as well as following treatment with promutagens. Present data indicates that in contrast to animal systems, cells in any cell cycle phase are equally likely to die following such treatments and that the activity of CDK-cyclin complexes is not required to activate or mediate the death program during pathogen defense. Thus, death during defense is not likely to be initiated through illegitimate activation of proliferation, but rather through distinct signals.

Congress On In Vitro Biolog7

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P-14

P-13 Soluble Signals Controlling Programmed Cell Death. aR.I. PENNELL, ap_j. Meijer, bT.A. Valentine, CL.S. Forsberg and bp.F. McCabe. aplant Biology Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, bDepartment of Biology, University College London, Gower Street, London WC1E 6BT, United Kingdom, cComplex Carbohydrate Research Center, University of Georgia, 220 Riverbend Road, Athens, GA 30602. Programmed cell death (PCD) occurs in plants during development and defense. We show that culture of carrot single cells at a density of <104 cells ml -l activates a cell death process involving condensation and shrinkage of the cytoplasm and nucleus and fragmentation of the DNA. Similarly, certain embryogenic cells ('state C' cells) can condense and shrink when sorted with the monoclonal antibody (Mab) JIM8 into JIM8(-) cell populations. Cell death triggered by modest abiotic stress treatments also causes cell condensation and shrinkage and the formation of DNA fragments, but the same abiotic stresses at high levels cause rapid necrosis with cell swelling and lysis. These common morphological features of cells dying at low cell density, in cultures of sorted cells, and following modest abiotic stress treatments reveal the existence of PCD pathways in plants. The addition of a cell-free, cell-conditioned growth medium allows ceils at low cell density to remain alive, and the addition of a growth medium conditioned by a culture of JIM8(+) cells allows state C cells in JIM8(-) cell cultures to remain alive and develop into embryos. These experiments show that cell-derived signal molecules suppress PCD that is otherwise induced by default. Manipulation of signal pathway intermediates that regulate PCD in animals shows that Ca2+ and protein phosphorylation events are PCD pathway intermediates in plants.

Congress On In Vitro Biolog7

Cell Death, Cell Arrest and Cell Protection Determine Floral Organ Fate in Maize. H. LAPARRA, A. Calder6n-Urrea, A. DeLong, D. Li, S. Gtinther, Y. Yoshioka and S. L. Dellaporta. Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520-8104. E-mail: helene.laparra @yale.edu Maize is a monoecious plant, containing unisexual flowers separated on terminal, staminate and axiUary pistillate inflorescences. The sex determination process occurs through selective abortion of inappropriate organs in immature bisexual floral meristems. In the tassel, all pistil primordia abort leading to the development of staminate florets. In the ear, only pistil from primary florets (El) develop to a mature stage; stamens are arrested and secondary pistils abort. The passage from a bisexual to a unisexual stage is controlled by hormonal and environmental factors, and by sex determination genes. From genetic and molecular studies, we will present the following model: TS2 (TASSELSEED2) induces cell death signaling in pistils leading to their abortion. TS2 expression, at the mRNA level, is found in all pistils. In ears, SK (SILKLESS) protects the E l pistil from the TS2mediated cell death process, permitting their sexual maturation. In tasselseedl mutant plants, no TS2 mRNA is detected, indicating that TS 1 is a positive regulator of TS2 expression. We will describe and discuss the process of TS2-mediated cell death, stamen cell arrest, and SKmediated cell protection.

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W-2 The Agricultural Biotechnologyfor Sustainable ProductivityProject: A Model for Technology Development and Transfer. C.L. IVES. Institute of InternationalAgriculture,MichiganState University,East Lansing, M148824. E-mail:.

The Agricultural Biotechnologyfor Sustainable Productivity (ABSP) project represents an integrated approach to agricultural biotechnologyresearch and development programs by establishinglinkages between developingCountry public and privatesector institutionsand the US privatesector,where much of the technologylies, as well as the US public sector.ABSPis funded by the US Agency for International Development(USAID) and is implementedthrough a consortiumof public and privatesector institutionsin the US and developing countries, with Michigan State University(MSU) as the lead institution. The project has Collaborations in Costa Rica, Egypt, MorocCo, Indonesia and Kenya. Researchprimarilyfocuseson the applicationof plant biotechnologyto the production constraintsin food crops (banana, cucurbits, maize, pineapple, potato, sweetpotato, tomato). It involves Contractual partnerships with the private sector in the US with DNA Plant Technology, Garst Seed Co., and Pioneer Hi-Bred and informal linkages with the BiorechnologyIndustry Organization and Monsanto Co. In developing countries, private sector Collaborationshave been developedin Costa Rica and Indonesia.Additionally, public sector institutions in the US and developingcountries are part of the consortium. In addition to research activities, the project assists developing countries in capacity development, both human and institutional, in those areas which are critical for the sustainable use and management of biotechnology-derivedproducts, pardcolady in the areas of biosafety and intellectual property rights. The ABSP project representsa unique model for internationaldevelopmentassistancein agriculturalbiotechnology.

W-3

The Western Australian State Agricultural Biotechnology Centre: A Role In Technology Transfer to Developing Countries. S.J. WYLIE and M.G.K. Jones, State Agricultural Biotechnology Centre, Murdoch University, Perth 6150, Australia. E-mail: wylie @central.murdoch.edu.au The Western Australian State Agricultural Biotechnology Centre (SABC) was established in 1994 to improve agricultural productivity in Western Australia through biotechnology. The Centre provides facilities for researchers from around the world who work with plants, animals, fish and fungi. The plant biotechnology group has close ties with agricultural training institutions in a number of developing countries in the region, and provides training for post-graduate research students and visiting scholars from those institutions. Since 1994 there have been 25 researchers from developing countries who have received training in plant biotechnology at the SABC. Countries involved include China, Thailand, Malaysia, Indonesia, Singapore, India, Srilanka, Kenya, Nigeria, Mauritius, and Iran. The major research projects under way at present include characterization of locally significant plant viruses, in particular bean yellow mosaic potyvirus, cucumber mosaic cucumovirus and subterranean clover mottle sobemovirus; developing constructs for resistance to viruses and fungi in legumes; optimising in vitro culture and transformation methods for narrow-leafed lupin (L. angustifolius) and yellow lupin (L. luteus); nematode / host interactions; molecular mapping for quality and disease resistance traits in lupins, wheat and barley; expression of antibody fragments in plants for protection against pathogenic fungi, and molecular diagnostics. Significant advances include an in vitro transformation and regeneration system for yellow lupin, the introduction of BYMV-resistance genes into narrow-leafed lupin, yellow lupin and subterranean clover, the identification of a molecular marker for starch quality in noodle wheat, and a molecular map o f narrow-leafed lupin. Some of these projects will be discussed in detail, with possibilties for training.

W-4 Agdcultural Biotechnology and the Developing World Opportunities and Challenges from the Perspective of a Commemial Company. JUDITH A. CHAMBERS, Director for International Government Affairs, Monsanto Company, Washington, DC, E-mail:

Understanding the patent process and its implications. W Murray Spruill. The Bell Seltzer Intellectual Property Group of Alston & Bird, 3605 Glenwood Avenue, Suite 310, Raleigh, NC 37622

Growing population pressures, the need to conserve the earth's natural resource base and changing global dynamics with respect to trade have led to the evolution of new partnerships in international agricultural research and development. Monsanto, as a leading company in the area of agricultural biotechnology, has initiated a number of novel technology transfer efforts in agricultural biotechnology which are designed to address agricultural production constraints in less developed countries and to increase the economic status of small or resource poor farmers. A common feature of these programs is the application of Monsanto's state of the art technologies to crop varieties important in tropical agriculture to ultimately increase yields in an environmentally sustainable way. Human capacity development and an increased awareness on the part of developing country scientists and policymakers for the corporate perspective on intellectual property rights and biosafety are secondary outcomes of these initiatives. Monsanto's interests in sustainable development, microcredit and biodiversity conservation are important factors in the company's participation in these initiatives. Several case studies will be examined in detail, including a =flagship" program on the development of a virusresistant sweet potato for Africa. Various international conventions currently under discussion will also be examined for their likely impact on these types of initiatives.

The implications of intellectual property in research have expanded as patents have issued covering various aspects of the scientific process. These aspects include starting materials, methods, as well as final products, even including living organisms. As long as the claimed invention meets the basic criteria for patentability and falls into one of the statutory classes of invention, patent protection can be claimed. Today patents issue on most any subject matter as the patent drafter has found a way to claim any subject matter so that it falls into one of the statutory classes. Issued patents can have financial as well as design implications on research planning. The holder o f a patent has the right to exclude others from making, using, and selling the claimed invention. While patent rights are important, few researchers are comfortable with negotiating through the patent process. We will explore patentable subject matter and the patent process and provide recent trends in patent law.

Congress On In Vitro Biology

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W-6

W-5 INTELLECTUAL PROPERTY RIGHTS: KEEPING RECORDS AND DETERMINING INVENTORSHIP. Lisa A. Haile, Fish & Richardson, San Diego, CA 92037. E-mail: [email protected] A laboratory notebook is a vital record of events leading to a patentable invention. The recorded information can establish dates of conception and reduction to practice of a technology as well as the inventorship of a patent claiming the technology. The United States laws implementing the general agreement on tariffs and trade (GATT) came into force on June 8, 1995. As a result, as of January 1, 1996, it has been possible for foreign inventors to establish a date of invention in a U.S. patent application by reference to knowledge or use of the invention in a foreign country which is a member of the World Trade Organization (WTO), or activity with respect to it in a foreign country which is a member of the awarded to the first person to invent, not the WTO. Since United States patents are first applicant, an applicant's ability to obtain a patent for his invention may often depend upon how well he documents inventive activity with respect to his invention. This applies, not only to the situation in which an inventor becomes involve in an interference, but also when he wishes to avoid or "swear behind" a reference applied against an application by proving a prior date of invention. Under the new law, it therefore becomes essential for inventors to maintain an accurate and welldocumented laboratory notebook.

W-7

No Abstract Submitted

Congress On In Vitro Biology

Intellectual Property Technology Transfer

Rights: Disclosure and University

Spencer Lemons, North Carolina State University, Office of Technology Transfer and Industry Research, 1 Holladay Hall, Raleigh NC 27695-7003. < [email protected] > For a university scientist, knowing when to disclose/discuss a new technology is especially challenging. To develop a new technology, the scientist/inventor often needs to discuss it with industry and others to raise the necessary funds, but once it's disclosed, patent fights may he lost or the idea may be "developed" by others. Unfortunately, there are no easy answers. We will discuss these matters and review the relevant intellectual property issues. In addition, we will discuss how a typical university technology transfer office operates and how this effects what you do next. Further, we will review some of the ways in which you may realistically present or protect your ideas without hampering your intellectual property rights or your research efforts.

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No Abstract Submitted

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W-10

Genetic Engineering o f Ascochyta Blight Resistance in Chickpeas and Mechanisms o f Technology Transfer. A. de Kathen 1, H. Kieseker j, H.J.Jacobsen 1, B. van Dorrestein 2 and M. B A U M 2. ~Dept. o f Molecular Genetics, University o f Hannover, Herrenhaeuser Str. 2, D-30419, Hannover, Germany. 2The International Center for Agricultural Research in the Dry Areas (ICARDA), P.O. Box 5466, Aleppo, Syria, E-mail: M.Baum~cmaet.com. In recent years ICARDA has integrated a number o f biotechniques into its conventional breeding programs such as the use o f molecular markers for marker-assisted selection, production o f doubled-haploid lines as well as using ovule- and embryo-rescue techniques for interspecific hybridization. In a research project between ICARDA and the University o f Harmover, Germany, transgenie chickpeas will be produced with increased resistance against Ascochyta blight pathogen (Ascochyta rabieO, a devastating disease limiting chickpea production in the Mediterranean region. Two companies (ZENECA and BAYER) agreed in providing antifungal genes for this purpose and the University o f Hannover develops a transformation protocol and generates transgenic prototypes. For kabuli- type chickpea, a protoplast-system suitable for transformation and somatic hybridization has been developed as well as a protocols for high-efficient regeneration applicable for Agrobacterium-mediated DNA transfer. The project can be also be used by the International Center to develop the necessary infrastructure to carry out future genetic engineering projects tackling issues such as intellectual property rights, biosafety and human resource development.

W-11

In Vitro Regeneration of Chickpeas and Technology Transfer the CLIMMEthiopian Experience. BEJIGA GELETU(a), Natalie F. Fletcher(b), Joanne E. Barton(b), and John Hamblin(b). (a)Alemaya University of Agriculture, Debre Zeit Agricultural Research Centre, PO. Box 32, Debre Zelt, Ethiopia. (b)Centre for Legumes in Mediterranean Agriculture (CLIMA), University of Western Australia, Nedlands, WA 6907

AUSTRALIA. A protocol for gene transfer to lupins (Lupintts aogustifolius), developed in Australia, is being adapted for the r~tsfer of genes to a range of legumes including chickpeas (Citer arientinum) at the Centre for Legumes in Mediterranean Agriculture (CLIMA). To ensure that suitable new lines are introduced into the breeding program advanced breeding lines and lines dose to release are used in development of the gene transfer systems. Several desi chickpea lines have been evaluated under Australian conditions. ICC14880 (Heera) and ICCV88202 (Sona) gave better yields than the popular variety Tyson and have recently been released. Reports of varietal differences in shoot and root formation in culture for chickpeas led to the investigation of the~e elite lines and the kabuli chickpea Kaniva in culture. Differences recorded for root and shoot formation in the three vasieties tested suggest that the gene transfer procedure is genotype dependent. Varietal differences could have significant implications for the production of uniform numbers of transformants from each variety. The results suggest that Kaniva and ICC14880 would be more suited to gene transfer studies. This study is of significance not only to the development ofa gene transfer protocol for chickpeas but also because it provided several other opportunities, These included collaboration and the transfer of technology and skills. Gene rechnulogy provides an opportunity for the introduction of genes to assist in the management of weeds, pests and disease in legume crops world wide. While it is recognized that this technology cannot yet be used in Ethiopia due to lack of facilities, CLIMA can contribute to the increased awareness of the techniques involved in the development of the technology. A technology transfer package which could deliver some of the benefits of gene transfer technology to agricultural systems in this region would assist the development of this region.

W-12 Experimental Designs and Data Analysis for In Vitro Research. M.E. COMPTON, School o f Agriculture, University o f Wisconsin-Platteville, Platteville, WI 53818. E-mail: .

Statistics is an important tool for biological research because it provides an objective, nonbiased way to evaluate experimental treatments. In statistics, we use the mathematical probability that an event can occur to determine if treatment effects are real. Statistical methods available to the biological researcher vary from simple to extremely complex. Therefore, it is important for the biologist to carefully select a statistical procedure that is easy to interpret and facilitates clear presentation o f experimental results. In this presentation I will discuss several factors that the in vitro biologist should consider before beginning an experiment, some examples o f experimental designs that can be employed, the basics o f randomization and replication and types o f data analysis appropriate for various data.

CongressOn In Vitro Biology

Comparing Treatment Means Correctly and Appropriately. CARL MIZE, ForestryDepartment, Iowa State University,239 BesseyHall Ames, IA 50011-1021. E-mail: . After doing an analysis of variance on their data, researchers are often interested in being able to say which treatment meansare different.There are a number of techniques that can be used to look for differences.The correct technique depends upon the types of treatments that were used and the objectivesof the research. I will discussfour techniques that can be used to compare means: ranking treatment means, multiple comparison procedures, fitting responsemodels,and using contractsto makeplanned comparisons.For a singlefactor experimentwith art objectiveof identifyingthe treatmentswith, say, the three highestresponses,rankingthe meansand selectingthe treatments with the three highest means is the appropriate analysis. Multiple comparison procedures, Duncan's multiple range test being one of the best known, are all similar in operation, somewhatdifferent in distinguishingbetweentreatments, and have relatively little use. In single factor experimentswith quantitative treatments, such as the amount of a hormone per liter, fitting a regressionline to the treatment meansis a powerfultechniquefor evaluatingtreatment means. Planned contrasts can be used in single factor and muhifactor experiments. These contrastsallowa researcherto developa meaningfulinterpretationof the experiment, much more meaningful than a technique such as Duncan's multiple range test.

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W-14

No Abstract Submitted

Application of Imaging Tools to Plant Cell Culture: Relationship between Plant Cell Aggregation and Flavonoid Production. M.-F. PIPPIN, J.F. Reid and M.A.L. Smith. Nat. Resources Env. Sci. Dept. and Agric. Eng. Dept. Univ. of I11. Urbana-Champaign, Urbana, IL 61801. email: [email protected] Due to the lack of reliable on-line sensors for direct measurements of substrate and biomass concentration, plant cell bioprocess monitoring and control rely on variable measurements linked to the culture metabolic activities (pH, oxygen uptake and carbon dioxide evolution) as indirect gauges of the culture status. Full confidence in monitoring of process state can only be gained by direct measurement of biomass (for specific growth rate) and degree of cellular aggregation, which is strongly correlated with production of many important secondary metabolite products. Image analysis tools were developed to measure biomass concentration, aggregate size and distribution, and pigmentation from anthocyanin producing cell suspension cultures of ohelo (Vacciniumpahalae). The ex-situ imaging system used can image cell aggregates from 301am to 2mm in diameter. The image analysis algorithm is based on extracted geometric features (e.g. breadth for aggregate categorization), morphological methods (for biomass volume estimates), and hue, saturation and intensity characteristics of the colored cell samples (for pigmentation). The status of the imaging technology will soon allow the implementation of in-situ imaging probes.

W-15

W-16 Do We Really Need Special Bioreactors for the Development of Efficient Plant Cell Bioprocesses? J. ARCHAMBAULT, R. D. Williams, M. Perrier, M. Jolicoeur and C. Chavarie. BIOPRO Research Center, I~cole Polytechnique de Montr6al, Montr6al, Canada, H3C 3A7. e-mail:[email protected]

Various authors have published results showing that plant cell cultures can be grown in microbial type bioreactors, thus suggesting the use of existing fermentation facilities and conventional equipment for industrial production of plant products using this cell culture technology. However, production results from plant cell cultures carried out in these bioreactors are generally lower than those obtained in uncontrolled flask cultures. Furthermore, in view of the sensitive nature and high sterility requirements of this culture process, the suitability of conventional fermentation facilities may not be a valid incentive fostering the industrial use of plant cell based bioprocesses. Our research group has successfully grown a variety of plant cell species in suspension, immobilized, hairy root, and somatic embryo cultures in flasks and laboratory scale bioreactors up to 20-L. Various plant cell based bioprocesses are under study for optimization and eventual industrial use, including production of secondary metabolites and somatic embryos. Our experience has shown us that specifically designed and operated bioreactors may be more appropriate for plant cell cultures. And in fact, if our ultimate objective is maximum performance of plant cell based bioprocesses, then our first goal must be to develop culture systems, and not just novel bioreactor configurations, that best suit the specific growth and production requirements of the biological system under study.

Congress On In Vitro Biology

Preliminary Experiments on Douglas-Fir Somatic Embryo Yield and Quality from Stirred Bioreactors. R.TIMMIS1, H.A. Surerus-Lopez1, B.M. Barton1 and R.S. Cherry2. lWeyerhaeuser Company, PO Box 2999, Tacoma, WA 984772999. E-maih'[email protected]'. 2Idaho National Engineering and Environmental Laboratory, PO Box 1625, Idaho Falls, ID 83415-2203. E-maih '[email protected]'. A bioreactor comprising four independently controlled 1.5 liter vessels was constructed at the INEEL as a research tool for improvement of somatic embryogenesis. Start-up experiments were carried out at Weyerhaeuser on several lines of Douglas-fir (Pseudotsugamenziesii (Mirb) Franco) embryogenic culture with the initial goal of obtaining embryo yield and quality equivalent to that from 1 liter Erlenmeyer flasks on a shaker. Electrolytic control of pH was attempted, based on the work of Thompson and Gerson (1985), and proved effective for periods of a few days. Low speed, winged, magnetically driven stirrers were effective in mixing without evidence of cell damage, and produced early-stage embryo structures that in some cases appeared better developed than those from shaker flasks. Yields of embryos after plating from three genetic lines did not differ significantly between flasks and bioreactor vessels, although embryos from the latter tended to be fewer in number and larger. In making yield comparisons, care was required to allow for differences in concentration of structures per ml of plated (settled cell) volume, which could arise between bioreactor vessels and shaker flasks as a consequence of structure size and methods of settling. In an experiment where embryos were germinated, those from bioreactor vessels exhibited a doubled percentage germination compared with those from shaker flasks, suggesting possible benefits of reduced shear stress or changed oxygen availability.

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W-18 Large scale photoautotrophic micropropagation and its advantages against microbial contamination. Chieri KUBOTA, Faculty o f Horticulture, Chiba University, Matsudo, Chiba 271-0092, Japan. E-mail:

Gas Exchange in Bioreactors for Culture of Differentiated Plant Tissues. P.J. WEATHERS(a), Y.J. Kim(b) and B.E. Wyslouzil(b). Biology/Biotechnology(a) and Chemical Engineering(b) Depts., Worcester PolyTechnic Institute, Worcester, MA 01609. E-mail: .

Photoautotrophic (sugar-flee) micropropagation has been examined for many different plant species. One o f the listed advantages o f photoautotrophic micropropagation (Kozai, 1991) is the low risk o f contamination, which facilitates the use o f large culture vessels (e.g., a 2.6-liter vessel by Kubota and Kozai, 1992; a 12.8-liter vessel by Heo and Kozai, 1997; a 100liter vessel by Kirdmanee et al., 1995), and thus, contributes to the reduction in production cost. In the preliminary experiments for controlling contamination in photoautotrophic micropropagation, the addition o f AgNO3 to the media at a relatively low concentration (e.g., 5 mg per liter) reduced the fungal growth rate in the liquid culture medium. The strategic introduction o f microbes such as beneficial bacteria will also be discussed as a potential method for producing quality micropropagated transplants and/or controlling plant morphology under photoautotrophic culture conditions.

The culture of differentiated plant tissues, such as roots and plantlets, is stronglyaffectedby four main factorsin all bioreactors:moisture, temperature, soluble nutrients, and gases. Light is also a critical factor required for some cultures, e.g. plantlets.K~suringefficientgas exchangeis a particular challenge, especially in densely packed reactors containing >50% biomass by volume. Nutrient mist technology, or aeroponics, is one approach to alleviate this problem. Ultrasonicmist reactorsproduce droplets in the 5-10 micron range that readilypenetrateand sustain the growth of denseroot beds. Innovationsin the design of nutrient mist reactors, including the use of acoustic windows, have made fabricationsimple and cost effective.The result is a new tool for studying the biological response of differentiated tissues in culture and especiallyfor addressingthe issueof gas exchange.Various mist reactor designs will be described and typical results of ongoing biological studies will be presented.

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Congress On In Vitro Biology

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W-21

In Vitro Propagation and Differentiation of Multipotential Neural Progenitor Cells From Human Fetal Brain: Development and Applications. Z. ZHANG and I. Sarel. BioWhittaker, Inc., Walkersville, MD 21793. E-mail:

Hematopoietic Stem Cells. MARY C. TSAO. BioWhittaker, Inc., 8830 Biggs Ford Rd., Walkersville, MD 21793. E-mail: Human bone marrow tissue offers a wealth of multilinage stem and progenitor cells for applied research that can lead to clinical cell therapies. Using these ceils for bone marrow transplantation with high-dose chemotherapy has enhanced the treatment of solid tumor cancers. The rapid discoveries of new cytokines, new antibiotics to minimize infection in cytopenic patients, and monoclonal antibodies to pbenotypically isolate stem and progenitor cells has heightened the exponential growth in bone marrow transplantation. A limited number of these multipotent stem and progenitor cells can also be isolated from either umbilicalcord and peripheral blood mobilized by the addition of several cytokines. Bone marrow tissue cells can be separated into both hematopoietic and nonhematopoietic stem cell populations. The nonhematopoietic stem cells can differentiate into osteoblast, chondrocyte, adipocytes, dendritic, and myoblast from multipotential stem and progenitor mesenchymal cells. The hematopoietic stem and pluripotent progenitor cells can repopulate the myeloablativepatient with lymphocytes, platelets, monocytes, granulocytes, and red ceils. Bone marrow tissue, therefore, is an ideal model system to study the cellular and molecular requirements for the differentiation of multilinage cells. New discoveries are continuously being generated for the different hematopoietic and nonhematopoietic stem cells isolated from these tissues. The focus of my presentation will be on the isolation and expansion of human hematopoietic stem and pluripotent progenitor cells in a serum-free system.

It has become obvious that progenitor ceils from mammalian embryonic brain would substantially facilitate drug discovery, cell therapy and basic research by providing a readily renewable source of mature neurons and glia. Until recently, most of the progenitor cells were isolated from fetal rodent central nervous system (CNS). The progenitor cells we have isolated recently from the fetal human brain resemble in many respects those from the embryonic rat brain. In both primary and secondary expanded cultures, the cells form spherical aggregates of undifferentiated cells in suspension and are immunoreactive for the progenitor marker nestin. When plated on an adhesive substrate, these cells can be induced to differentiate into both neurons and glia as confirmed by the expression of respective neuronal and glial markers. The proliferation potential of the progenitor cells was demonstrated by both an increase in nucleic acid synthesis and incorporation ofbromodeoxyuridine into the nuclei. The undifferentiated neural progenitor cells were capable of survival and neuronal differentiation when transplanted into a rat model of Parkinson's disease, and partially ameliorated lesion-induced behavioral deficit in these animals. Therefore, the neural progenitor cells derived from the developing human CNS can be used for such purposes as clinical and laboratory transplantation, and pharmaceutical and toxicological research.

W-24

W-23

Agrobacterium-mediated Plant Transformation

Low-Toxicity, Lipid-Mediated Transfection of Normal Human Cells. P.L. Pingerelli * and S.KARMIOL 2 . ~Stratagene Cloning Systems, La Jolla CA, 92037; 2BioWhittaker, Inc., Walkersville, MD, 21793. E-mail: . The ability to transfect cells provides for a powerful method of evaluating gene analysis and recombinant protein expression. The unfortunate aspect of this technology is the variable responses observed with different cell types. This condition requires the determination of the concentrations of the various components that make-up the transfection reagent. Kits containing normal human cells with optimized transfection reagents would be beneficial. This approach has been taken with a lipid-mediatedtransfection vehicle, LipoTAXI from Stratagene Cloning Systems. Four normal human Clonetics cell strains (BioWhittaker, Inc), small airway epithelial cells, prostate epithelial cells, skeletal muscle cells and mammary epithelial cells have been optimized with respect to DNA concentration and LipoTAXI volume. The control plasmid used to evaluate the transfection optimizations encodes the beta-galactosidase enzyme controlled by the cytomegalovirus (CMV) promoter. Protein was determined to assess the relative cytotoxicity: decreased protein is associated with cytotoxicity and increased protein with proliferation. In addition, a comparison of this technology with other comparable technologies was made demonstrating consistent effectiveness. The use of such optimized systems would significantly reduce or eliminate investigator trail and error of the transfection process.

CongressOn In Vitro Biology

with High Molecular Weight DNA. C. M. HAMILTON. Plant Science Center, Biotechnology Building, Cornell University, Ithaca, NY 14853. E-mail: [email protected] A bacterial artificial chromosome (BAC) type vector that also functions as a binary vector for Agrobacteriummediated plant transformation is described. The binaryBAC (BIBAC) vector is useful for the construction of large insert genomic DNA libraries and for plant transformation with high molecular weight DNA. Once individual library BIBAC clones of interest are identified, the BIBAC plasmid DNA is readily isolated and introduced into a suitable strain of Agrobacterium for plant transformation experiments. The BIBAC is capable of transferring at least 150 kb of DNA, intact, into plants (tobacco and tomato plants). The efficiency and integrity of high molecular weight DNA transfer is affected by the presence of an additional helper virulence p l a s n a a [~-10 copies/cell) that carries VirG, or VirG and VirE. Several plant genomic DNA libraries have been constructed in BIBAC vectors that have average insert sizes comparable to those of traditional BAC vectors. The development and use of plant genomic DNA BIBAC libraries is discussed. The development of BIBAC technology should streamline plant breeding efforts, reduce the time risk involved to identify plant genes and creates a number of new possibilities for plant biotechnology.

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W-25

W-26

Cell Micromanipulation With Laser Scissors and Tweezers, Michael W. Betas, Beckman Laser Institute, University of California, Irvine, CA 92612. Email Microscope-based laser systems can be used to ablate targeted subeellular regions and move cells and their components about at will. The intense, monochromatic, short-pulse lasers are focused to diffr cfion-limited spots using high magnification microscope objectives. Using various physical mechanisms, single photon or multiphoton processes are initiated causing selective molecular and structural alteration of the target. These "laser scissors" are used to alter specific molecular components of chromosomes and other mitotic organelles i.e., centrosomes and kinetchores to study structure and function (cell motility, gene activity, and gent localization). Cell populations can be cloned from a single that has had a specific genetic site inactivated. Selected chromosome fragments arc generated, collected, and subjected to PCR and sequencing; DNA insert sizes vary from 100 to 1200 bp. These laser microbeams are used to "optoporate" cell membranes so foreign DNA can enter the cell with resulting integration into the cell genome at frequencies as high as I%. Fully differentiated rice planflets have been generated from single "optoporated" cells, with stable incorporation and expression of the inserted genes. Besides pulsed laser scissors, continuous wave near infrared lasers are used as "optical tweezers" to induce forces on cells and cell organelles. Optical tweezers are used to study a variety of cell motility problems from manipulation of molecular motors to movement of individual organdies (chromosomes) and whole cells. Using laser tweezers to hold a cell or subcellular structure, laser scissors can be used to cut and/or alter specific molecular components. These two noninvasive optical tools provide the cell biologist with new, exciting methods to manipulate cell structure and function without disrupting other cell structures and functions. The role of these optical tools in biotechnology has yet to be defined.

Biolistic Delivery of Large DNA to Plants: Purified DNA vs. Biological Projectiles. J.R. KIKKERT, Cornell University, New York State Agricultural Experiment Station, Department of Horticultural Sciences, Geneva, NY 14456-0462 USA. E-mail: [email protected] The ability to transform plants with large DNA fragments (>100 kb) would aid map-based gene cloning, and enable transformation with very large genes/gene clusters,or important genes that have been mapped but not cloned. Purified YAC DNA (50-150 kb) has been delivered into plants using conventional biolistics (1-2). However, the delivery of larger YACs may be limited by DNA shear which can occur during DNA purification, coating of tungsten/gold particles, or upon impact with the plant cells. Additionally, the isolation of large quantities of purified high molecular weight DNA is difficult and time consuming. As an alternative, our laboratory has investigated the use of biological projectiles for plant genetic transformation (3, 4). The intact phage particles or yeast and bacterial cells carry and protect DNA cargos as they are bombarded into plant cells. Plant transformation using biological projectiles was initially optimized using small DNA constructs containing plant marker and reporter genes. E. coli was the most effective projectile tested. When maize cells were bombarded with E. coli, rates of transient gene expression (2,000 per plate) and stable transformation (50 per plate) were only 2 to 3 fold lower than when purified plasmid was used. Rates in tobacco were lower, and when E. coli that contained a 150-kb BAC (C. Hamilton, Cornell Univ.) was used stable transformants were not recovered. The fate of the E. coli chromosome was tested by bombardment of maize cells with an E. coli strain that had the GUS and bar genes inserted into the chromosome. A small number of stable transformants were obtained, but the size of the insert has not been determined. References: (1) J VanEck et al. (1995) Pit Cell Rep 14:299, (2) G Adam et al. (1997) The Plant J 11:1349, (3) J Rasmussen et al. (1994) Plt Cell Rep 13:212, (4) I Kikkert et al. (1998) In Vitro Cell Dev Biol, submitted.

W-27

W-28 A General Approach for Developing a Commercial Micropropation System. B.H. MCCOWN and D.D. McCown, Department of Horticulture, University of Wisconsin-Madison, Madison, WI 53706 and Knight Hollow Nursery, Inc., 3333 Atom Road, Middleton, WI 53562. E-mail:

Five distinct steps can be recognized in the establishment of a plant in a commercial micropropagation system, especially if the most utilized approach (shoot culture) is the focus. Failure at any one step can make the total system commercially infeasible. When considering a plant without extensive previous history of microculture, the first step involves an analysis of the potential market (economic reality) as well as the plant's general growth habit (biological reality). The general growth habit of the plant can provide valuable predictive information as to the potential ease of microculture. For example, plants showing indeterminant herbaceous growth (e.g. Chrysanthemum, Solanum, Dieffenbachia) or continuous woody seasonal growth (e.g. Betula, Ulmus, Thuja) are generally much more amenable to microcultrue than those that are determinant herbaceous (e.g. Panix, Paeonia) or episodic woody organisms (e.g. Quercus, Pinus). At times, an episodic habit can be overcome in microculture (Syringa, Rhododendron). The next four steps involve the actual manipulation and microculture of the plant and include the initiation, stabilization, optimization, and production phases. The most intensive analytical step is usually the optimization phase where hormonal response curves, replication, repetition through multiple subcultures, and evaluation of productivity and product quality are involved.

Congress On In Vitro Biology

Establishment and maintenance of contaminant-free perennial plants in vitro. R.M. SKIRVIN, M.A. NORTON, AND S. MOTOIKE. University of Illinois, Department of Natural Resources and Environmental Sciences, 258 ERML, 1201 W. Gregory, Urbana IL 61801 E-mail Perennial plant tissue cultures are established by disinfecting field or greenhouse-grown plant parts and transferring them to sterile medium in vitro. Typically shoots are harvested from field or greenhouse-grown plants placed in water either to force growth from dormant branches or to maintain them until ready for explanting In spite of extreme care 90 to 100% contamination rates in newly established in vitro cultures are not unusual. We have performed many experiments to reduce contamination rates in our laboratory. Some of these procedures include in vitro methods such as minimizing the amount of time a stem cutting is maintained in water prior to explanting; adjusting medium pH to a more acid condition; and use of pH neutralized bleach to sterilize instruments during subculture. Other methods to reduce contamination include parent plant pretreatments such as establishing field grown plants in a greenhouse where inoculum levels can be better controlled; trellising vining plants to get them offth¢ soil; avoiding wetting foliage; and selecting vigorous ¢xplants that are not in contact with soil Some of these methods will be discussed in detail using specific examples of plants we have established in culture.

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Abstracts

W-29

W-30 Challenges Associated with Micropropagation of

Micropropagation systems for woody plants of the Chihuahuan desert. W.A. MACKAY, Texas A&M University Research and Extension Center, 17360 Coit Rd., Dallas, TX 75252-6599. E-mail:[email protected].

Zephyranthes and Hippeastrum sp (Amaryllidaceae). R.H. SMITH, J. Burrows and K. Kurten. Department of Soil & Crop Sciences, Texas A&M University, College Station, TX 77843. Email: Conventional propagation of amaryllis, Hippeastrum sp. hybrids by bulb offsetts is slow, seasonal, and variable; additionally, some amaryllis hybrids do not produce many offsets. From seed, it takes approximately 2 years to flower. Zephyranthes also known as zephyr, rain or fairy lily are small bulbous herbs usually with a solitary flower with a potential for native landscape uses. Alkaloids from Zephyranthes may have medicinal value. Micropropagation of these bulbs has challenges related to contamination of stage I cultures as well as genotype differences in culture media requirements. There are literature reports on in vitro propagation of both genera, however, the application of these reports to new cultivars leave unanswered questions regarding surface disinfestation, explant, nutrient media, and multiplication rates. Surface disinfestation of container grown Hippeastrum spp. hybrid, San Antonio Rose bulbs has resulted in contamination rates of 92 to 100% in spite of various treatments some of which killed the explant. Twin scale explants of San Antonio Rose bulbs responded on a Murashige and Skoog salt medium with 2 mg/l NAA. Transfer to soil is not a problem. Aseptically germinated seed ofZephyranthes sp. served as the source of clean bulb tissue. Results of micropropagation will be presented.

CongressOn In VitroBiology

There are many Chihuahuan desert species that have potential as landscape plants in the arid communities of the southwestern United States. Within these plant populations there are superior genotypes that offer even greater interest for the landscape. However, it is difficult to clonally propagate many of these species using conventional techniques and the seed-derived populations often do not breed true. Therefore, selection of superior genotypes in wild populations coupled with clonal propagation through tissue culture may offer an attractive option. Although it is relatively easy to achieve disinfestation of explants from desert plants, due to a general lack of surface contamination by fungi and bacteria, the timing of collection of explants can be critical because of the brief, periodic flushes of growth. Additionally, there may be years where, due to the harsh environment, the amount of suitable explant material is limited. Various strategies used to develop micropropagation protocols for selected desert trees and shrubs will be discussed.

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Abstracts

V-1001

V-1002

Responses of Human Uterine Smooth Muscle Primary Cultures to in vitro Conditions Are Menstrual Phrase Dependent. J. ANDERSEN, T.Y. Peresleni, J.J.Wu, E. Geimonen, Department of Ob/Gyn, School of Medicine, SIJNY Stony Brook, NY 11794-8091. Email: [email protected] A system to culture primary uterine smooth muscle cells from human myometrial tissue has been developed,The cultures have been used to study women's health issues such as factors causing preterm labor. Connexin43 is the primary gap junction protein found in uterine smooth muscle at parturition. The responses of the cultures to estrogen and progesterone have been characterized using transient expression assays and using analysis of the effects of the hormones on connexin43 (cx43) gene expression. Primary cultures derived from women in follicular phase have little or no response to estrogen while primary cultures from women in luteal phase demonstrate estrogen receptor-mediated transcriptional activation during transient expression assays. No differences in the response were found in cultures taken from women in follicular or luteal phases of the menstrual cycle to progestin. However, the interaction of progestins with protein kinase A (PKA) and protein kinase C (PKC) signal transduction cascades appears to be dependent on the menstrual phase. Progestin inhibits the PKC-induction of cx43 expression in follicular-derived primary cultures and inhibits the PKA-induction of cx43 expression in luteal-derived primary cultures. In summary, the results demonstrate the need for caution and for consideration of cell type and origin before forming general conclusions about biological mechanisms.

V-1003 Biological and Genetic Characterization of a Novel Human Pheochromocytoma Cell Line R. PFRAGNER1, A. Behmel 2, D.P. Smith3, B.A.J. Ponder3, G.Wimsberger4, T.Henn5, B Niederle 6. Departments of ~General and Experimental Pathology, 2Medical Biology and Human Genetics, 4Internal Medicine, University of Graz, SCCRI St.Anna Children's Hospital, Vienna, ODepanment of Surgery, University of Vienna, AUSTRIA, 3CRC Human Cancer Genetics Research Group, Addenbrooke's Hospital, University of Cambridge, U.K. E-mail: [email protected], at Pheochromocytomas - catecbolamine-19roducing tumors derived from adrenal medullary chromaffin cells - occur sporadically or as, part of dominan.t cancer syndromes like MEN2A, 2B and omers. Like_.f.an~.ilialtumors some s.t~oraaic tumors showed RETmutations. We have established anit characterized a continuous cell line from a sporadic human adrenal l~heochromoc.yt.oma, KNA. The tumor cells retainea essential phenotypical and immunological properties, such as neuroendocrine granules and positive reactions to chromogranines and related pep.tide, NSE and VIP-antibodies. Nerve g!'.owth factor induce~l a dosedependent formation ofneurite:like,processes. , ..... ~nromosomes were aip.mi.d (~o,.,~.x~,.n=50)to nyj~oaipioia .[~3,~,,x.x., n=15). In hypocliploic1metaplaases most trequentlv #19, 17, 21, and 22 were rmssing. Chromosome arms l p ahd 4q snoweo al~parenuv consistent interstitial deletions, oq, 8o. 13a and 220 shbwcd clonal deletions. These were partially confirmed by CGH-studies. DNA-sequence analyses showed a heterozv.~ous vhriant TGC (cysteine) to TGG (tryptophan..).in codon 6-1"1 in exon 10 of tlae RET proto-onco~ene, l n e cvsteine 611 tryptophan variant in.the .KN.A cell fine is very likely ko be a mutation associated witia the development of the pheochromocyl;.oma from which the cell line was derived. In which case it is the first report of this mutation in a sporadic Lumor, including pheochromocvtoma. t)ur new continuous cell line KNA reoresents a useful model for in vitro-studies on differentiation and progression of neoplastic cells at cellular and molecular level.

Congress On In Vitro Biology

Use of an In Vitro System to Define the Differentiation-Dependent Life Cycle of Human Papillomavirus. C. MEYERS, T.J. Mayer, D. Huber, and M.A. Ozbun. Dept. of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, PA 17033.. Email: . Investigation of the life cycle of the important human pathogen human papillomavirus (HPV) have in the past been hindered by the lack of a tissue culture system in which the virus could replicate. Our laboratory using an organotypic (raft) epithelial culture system to grow full thickness neoplastic tissue using a cell line derived from a cervical neoplasia biopsy was the first to demonstrate the complete HPV life cycle in vitro. This in vitro culture system has allowed us to characterized and define the temporal expression patterns of HPV transcripts during the complete differentiation-dependent life cycle of the virus. We have also characterized 5 viral promoters, determined their temporal usage, and their dependency on differentiation. Furthermore, we have begun to investigate the spatial and temporal expression pattern of viral proteins in the host tissue. This organotypic (raft) culture system has permitted the study of a number of the differentiation-specific aspects of HPV, however, these investigations have been limited to a single virus type. Recently, we have artificially introduced linearized HPV genomic DNA into primary keratinocytes by electroporation, followed by clonal expansion and induction of epithelial stratification and differentiation in organotypic culture. This has resulted in the in vitro synthesis of abundant infectious virions. Six different HPV types have now been replicated using this system. We expect that any naturally occurring HPV type or mutant for which cloned DNA is available can be propagated in this system.

V-1004 In Vitro Growth and Differentiation of Guinea Pig Marrow, Spleen, and Thymus Stromal Cells and BloodBorn Fibroblasts. S.A.KUZNETSOV, N.S.Fedarko, M.T.Collins, M.H.Mankani, and P.Gehron Robey. Craniofacial and Skeletal Diseases Branch, National Institute of Dental Research, NIH, Bethesda, MD 20892. E-mail: . Stromal cells of various hematopoietic and lymphoid organs feature similar fibroblastic morphology but distinctive differentiation capacities. While bone marrow stromal cells (BMSCs) form a bone and bone marrow organ when transplanted in vivo, spleen and thymus stromal cells (respectively, SSCs and TSCs) do not. It is also possible to culture fibroblastic cells of blood origin (blood-born fibroblasts, BBFs), which have been incompletely characterized. To find in vitro characteristics which might correlate with in vivo differentiation potential, we studied long-term cultures of guinea pig BMSCs, SSCs, TSCs, and BBFs in osteogenic medium (ctMEM, 2% FBS, 0.5% ITS+, 10-*M dexamethasone, 10-SM ascorbic acid phosphate, 5 mM Na-13glycerophosphate). The four populations had different growth rates and distinctive growth patterns. BMSCs formed homogenous layers stable for at least 6 weeks, with negligible alizarinpositive deposits; they had lower maximum cell density and higher collagen content than any other population. SSC multilayers retracted, forming nodules which became alizarin-positive and condensed with time. TSC cultures underwent repeated cycles of retraction, detachment, and regrowth. BBFs developed dense, stable multilayers in which long, sprouting cells became increasingly discernible. All populations were positive for acid phosphatase, partially positive for et-naphthyl acetate esterase, and negative for alkaline phosphatase, except for occasional cells in early BMSC and TSC cultures. Thus, profound differences between the four cell populations have been found. However, features usually attributed to osteogenesis in vitro, such as alkaline phosphatase activity and alizarin-positive nodules, did not correlate with in vivo osteogenic potential.

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Vo1005

V-1006 Establishment and Characterization of Immortalized Human Fetal Brain Cells in Culture for Neural Transplant Studies. K. N. Prasad 1, J. E. Prasad 1, F. G. La Rosa 1, A.R. Hovland 1, E.D. Clarkson 2, C.R. Freed 2. Depts. of Radiology 1 and Medicine 2, University of Colorado School of Medicine, Denver, CO 80262.

There are many limitations associated with the use of human fetal tissue transplantation for the treatment of Parkinson's diseases (PD). Therefore, the establishment of a homogeneous population of immortalized human fetal neurons for use in neural transplant studies is a logical progression. Initially, we established a line of immortalized rat dopamine neurons in vitro. These cells were tested by grafting into the striata of 6hydroxy-dopamine (6-OHDA) lesioned rats (the animal model of PD). The results were an improvement in neurological deficits without any evidence of rejection or tumor production. We have also established a nonclonal, immortalized human cell line from human fetal brain cells. These cells were stably transfected with the plasmid, pSV5neo, which contains the polyoma virus large T-antigen (LTa) gene. All cells in culture express the LTa protein as determined by immunostaining with a primary LTa antibody. The cells also express tyrosine hydroxylase, the dopamine transporter protein, and neurofilament proteins. The cells grow in MCDB medium containing 10 % fetal bovine serum, penicillin and streptomycin. However, estrogen, I~-epidermal growth factor and insulin are-necessary for optimal growth. These immortalized human cells have been tested by transplantation into the striata of naive rats. No tumor formation or rejection at 30 days post transplantation has been detected. In addition, the grafted cells can be recultured in vitro. These cells have been patented. (Supported by NS35348 and NS 29982)

V-1007 A Comparison of the Effects of Powdered and Liquid Fetal Bovine Serum on Normal Human Cell Growth, Metabolism and Urokinase Formation. T. E. Robey and J, M. RYAN. Dept. 456, Abbott Labs, North Chicago, IL. 60064 The attachment, growth, function and survival of many cultured mammalian cells is dependent on the presence of serum in the growth medium. Serum is supplied as a liquid and usually stored frozen to preserve quality. A spray dry process for serum preparation has been developed (HyClone Labs, Inc.) that may offer stability, shipping and storage advantages. We have compared the performance of powdered FBS (pFBS) and liquid FBS (IFBS) in supporting normal diploid human neonatal kidney (HNK) cell growth, carbohydrate metabolism and urokinase formation. The IFBS and pFBS used in these studies were derived from the same serum production lot. pFBS was reconstituted in water at 65 gm/L and added to Medium 199 at a final concentration of 10%. IFBS was used at a 10% final concentration. Cells grown in pFBS had a higher cloning efficiency in pFBS than IFBS (23% vs 15%). The growth rates (doubling time = 19.8 hrs. (pEBS) and 18.8 hrs. (1FBS)) and maximum cell densities (0.6x 105/cm2) were equivalent in both media. There was no difference in the rate of glucose utilization or lactate production in cultures maintained in medium containing pFBS or IFBS. The replicative life spans of cells grown in 1FBS (17 pd) and pFBS (15 pd) were similar. Cells grown ih either media produced equivalent amounts ofurokinase, a functional marker for HNK cells. These results show that medium containing serum supplied as a powder supports HNK cell growth, carbohydrate metabolism and urokinase production equivalent to medium containing serum supplied in liquid form.

Congress On In Vitro Biolog7

Chemotherapy-lnduced Alopecia In Vitro. Lingna Li, Eugene Baranov and Robert M. HOFFMAN, Ph.D. AntiCancer, Inc., 7917 Ostrow Street, San Diego, CA 92111. E-mail: . Chemotherapy-induced alopecia (CIA) or hair loss is a major problem in clinical oncology. It is particularlydevastatingto women and is becoming more prevalent as alopecia-inducingchemotherapeutic agents, such as the taxanes, are becoming more frequently used in breast and ovarian cancer. The pharmacological toxicity of chemotherapy on the hair follicleis the disruption of the hair cycle resulting in CIA. No pharmacologicalprinciple inhibits CIA in a reliable, cost-efficient, unharmful and long lasting manner. One major reason for this is the paucity of relevant in vitro discoverymodels for studying the cyclic activity for the hair follicle and for screening effective anti-CIA agents. We have developed a new model of CIA in histocultured C57B16 mouse skin based on the full thickness skin histoculture technology we have previously developed. The results obtained thus far demonstrate: 1) the hair cycle can be perturbed in skin histocuhure; 2) the hair cycle can be positioned and quantified by a morphometric index based on hair cycle quantitative morphology; 3) doxorubicin has a large effect on the hair cycle of histocultured mouse skin measured by the morphometric index; 4) the skin histoculture system can be used for discoveryof anti-chemotherapy-inducedalopecia.

V-1008

Efficient Cultivation of Cells Using Powdered Serum. JOSEPH C. F. CAMIRE, Nathan W. Briggs, Bill B. Barnett. HyCIone Laboratories, Logan, UT 84321. email: [email protected] A process to produce powdered fetal bovine serum for cell culture has been developed. Cultivation of cells in media supplemented with powdered fetal bovine serum showed performance characteristics similar to controls. Studies were performed using AIF, Balb/3T3, BHK-21, CHO-K1, CRFK, MDCK, MRC-5, Sp2/0-AG14, and Vero cells. All nine cell lines were successfully carried beyond 10 passages in basal medium supplemented with powdered fetal bovine serum. Hybridoma and Vero ceil lines cultivated in media containing liquid serum or powdered serum from corresponding serum lots showed equivalent growth. Cell densities for the hybridoma cell line reached 1.8 x 106 cells/mL and for the Vero cell line 1.05 x 10s celis/cm2.

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V-1009

V-1010 Apoptosis Induced by Iron Deprivation in Cultured Tumor Cells. J. KOVAR 1,2, T. Valenta *, K. Spevakova 1, L.L. Stunz 2, K. KriegerbeckovaI and J.D. Kemp 2. lInstitute of Molecular Genetics, 14220 Prague, Czech Republic, 2University of Iowa College of Medicine, Iowa City, IA 52242. E-mail: .

Iron deprivation induced death of some cultured tumor cells (38C13, Jiyoye) while other cells (EL4, HeLa) were resistant. Three distinct iron-depriving conditions were used: (1) Monoclonal antibodies against transferrin receptor, (2) iron chelator deferoxamine, and (3) defined iron-free culture medium. DNA fragmentation analysis by agarose gel electrophoresis, flow cytometric analysis aider propidium iodide staining, and electron microscopy revealed typical apoptosisassociated changes in mouse B cell lymphoma 38C13. The addition of ferric citrate prevented apoptosis under all three irondepriving conditions. Two possible mechanisms of apoptosis induction by iron deprivation were considered: (1) Impaired DNA replication, resulting from insufficient function of iron-containing ribonucleotide reductase, led to apoptosis via p53 activation. Apoptosis induction via p53 activation could involve p53 effect on Bcl-2 and/or Bax expression. (2) Decreased mitochondrial activity, resulting from affected function of iron-containing enzymes of the respiratory chain and potentially associated with decreased Bcl-2 expression, led to apoptosis via changed activity of Bcl-2/Bax system. Both hypothetical pathways of apoptosis induction by iron deprivation were tested.

Injectable Hydrogels Containing Autologous Chondrocytes as Engineered Tissue Bulking Agents. K. BORLAND, T. Zhou, J. Birkhead, D. Deane and D. Omstead. Reprogenesis, Inc., 21 Erie St., Cambridqe, MA 02139 An alginate hydrogel has been developed that can be delivered via injection to effect a bulking function in tissues. Autologous chondrocytes, isolated and expanded from an auricular cartilage biopsy, are incorporated into the gel. In vivo, these dedifferentiated cells revert to a differentiated chondrocyte phenotype and resume synthesis of cartilage matrix components which serves to remodel the gel into a permanent cartilaginous tissue construct. This chondrocyte-alginate gel suspension is being tested in clinical trials for two urological indications, vesicoureteral reflux and stress urinary incontinence. Alginate, an extract of brown seaweed, is cross-linked by mixing with two calcium sources of disparate solubility. The resulting gel is injectable via cystoscope, divides the tissue plane without extravisation, and is compatible with chondrocytes and host tissue. Volume is retained beyond one year, and the strength modulus of alginate-chondrocyte gel implants is demonstrated to increase over time in vivo. Chondrocyte expansion from a primary 50 mg biopsy isolate (recovery 15.0 x 103 cells/mg +0.8), is a two step culture process which produces a therapeutic complement of cells in approximately 42 days (3.5 day generation interval). Transformation of gel to cartilage over time is demonstrated by histochemical stains of human chondrocytes implanted in immunodeficient mice.

V-1012

V-1011 Coordinated Movement of Neighboring Cells in Confluent Monolayers. C.R. KEESE, M. Linton, C. Lo, and I. Giaever. Applied BioPhysics, Inc. and Rensselaer Polytechnic Institute, Troy, NY 12180 Confluent monolayers of normal fibroblastic and epithelial cells organize into regular morphological patterns indicating that neighboring cells interact and communicate with each other. A more striking example of such cell-cell interactions are morphological oscillations of MDCK cells reported here. These oscillations were discovered using a biosensor referred to as Electric Cell-substrate Impedance Sensing (ECIS). In this method, cells are seeded out on a small electrode immersed in ordinary tissue culture medium. By measuring the changes in impedance of the electrode as a function of time, many important properties of the cells on the electrode can be inferred such as motion, morphology changes, and cell membrane capacitance. The oscillations of MDCK cells were I i i observed with confluent cell layers, Random movement 3.0 where a few hundred u) Oscillatory cells located on the Behavior electrode displayed n. 2 . 0 similar behavior in unison. The figure shows data after 1.0 inoculation of MDCK I I I cells at time zero. 10 20 30 Time (hours) Another type of cell interaction can be observed by culturing cells on two closely spaced electrodes. The impedance fluctuations are measured independently on each electrode and compared to detect coordinated movements in the two cell populations.

Congress On In Vitro Biology

Tools to Simplify G-ene Expression. K. COLVIN, S. Calvin, L. Jacobsen and N.Balgobin. Boehringer Mannheim Corporation Indianapolis, IN 46256. E-mail:[email protected] As the sequencing of the human genome progresses, the ability to express and study the effects of genes is becoming essential. Researchers are faced with selecting expression vectors, screening recombinant clones, identifying a transfection reagent and monitoring expressed proteins. Simplified methods are needed that can be readily used. Epitope tagging is a technique in which DNA is "tagged" by a genetic sequence that once translated is readily recognized by an antibody to that protein. Thus proteins can be localized, identified, and purified by an "epitope tagging antibody". Several products from Boehringer Mannheim Corp. make this valuable technology easy to apply. These products include: Product: Epitope tag expression vectors Screening kits i Fug eneTM 6 Transfection Reagent Purified antibodies to epitopes Western Blotting Reagents Labeled antibodies Immunoprecipation kits

Use: Add peptide sequence to eDNA of interest Identif7 clones rapidly High transfection efficacy with minimal cytotoxieity Localize, identify or isolate expressed proteins Identifff expressed proteins Localize expressed proteins Isolate expressed proteins

Typical results will be presented.

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V-1013

V-1014

Direct Delivery of Competent Genes to Cells and Tissues. R. HELLER m, M. Jaroszeski t, L. Heller2, C. Pottingert, L. Lucas I and R. Gilberd. Univ of South Florida, Depts. 1Surgery and 2Chem. Engineering, Tampa, FL 33612. A key issue in the development of effective gene therapy protocols is the establishment of appropriate delivery methods. A common problem of gene therapy is inefficient gene delivery and insufficient expression. Electroporation is a physical phenomena that temporarily permeabilizes cell membranes which allows nonpemaeant molecules to gain intracellular access. Electroporation has been used successfully to increase the effectiveness of chemotherapy in treating several types of cancer (electrochemotherapy; ECT). Although successful, this treatment is localized. Therefore, research is focused on combining ECT with an immunotherapy protocol to obtain a systemic response. Recently, work has been initiated to examine the use of electroporation to deliver cytokine genes to melanoma tumors. Delivery of plasmid DNA was tested using the green fluorescent protein (GFP) reporter gene. Mice receiving GFP and electroporation showed a twofold increase of GFP expression compared to those receiving gene alone. Effectiveness of treating tumors was examined by treating established melanoma tumors with various combinations of IL-2, Neomycin and eleetroporation. Tumors that received bleomycin or IL-2 gene without pulses did not completely respond. The group receiving Neomycin and pulses had a 50% response rate while those receiving Neomycin, IL-2 gene and electric pulses showed a 67% response. Efforts are now focused on perfecting the delivery procedure. Future directions include looking at effects of different cytokine genes as well as testing the treatment in a metastatic model.

V-lO15

Gender Determination by Amelogenin Allelic Discrimination. P.K. Bender, M.A. Hansen, and J.C. Beck. CorieU Cell Repositories, Coriell Institute for Medical Research, Camden, NJ 08103. E-mail: The Coriell Cell Repositories routinely determine the gender of cell cultures as part of its quality control program. Because of the large number of cultures processed a simple, rapid, and accurate method for gender determination was needed. The method tested is based on allelic discrimination of the amelogenin genes. One amelogenin allele is located on the X-chromosome, while the other allele is located on the Y-chromosome. Although these two alleles are highly conserved and share regions of sequence identity, there are regions of sequence diversity. Primers made to conserved sequences serve to amplify both alleles in PCR assays. Allele-specific probes are designed from the regions of sequence diversity and labeled with different reporter fluorochromes in addition to the standard quencher. These probes are detected in a TaqMan assay with the PE Applied Biosystems model 7200 fluorescent plate reader. Fluorescence from each reporter indicate presence of the corresponding 'allele, DNA from monochromosomal rodent cell hybrids containing either the human Ychromosome or the human X-chromosome were used as standards for the X and Y amelogenin sequences. The rodent genomic DNA (hamster or mouse) did not interfere with the TaqMan assay. In our tests the assay correctly identifies male and female DNA samples. Results with cell lines that are polyploid for the X and/or Y chromosomes were not obscured by the differences in the relative copy number. Furthermore, the Y amelogenin sequence is detected in a sample of female DNA when seeded with as little as 0.5% male DNA. The assay has proved simple, rapid, and accurate for gender determination, and is now routinely used by the repository. (Supported by NIH contract NO 1-GM4-2102)

V-1016 Automated Detection of Fluorescent Signals Using the FishFinder Image Analysis Software System. Wen-Jeng Ko, DELIA R. BETHELL and David W. Sammons. BioSeparations, Inc., Tucson, AZ 85719. E-mail:

Fluorescent labels are used as reporter molecules for a number of different applications. Fluorescent In Situ Hybridization (FISH) techniques are used to detect and identify specific chromosomes and chromosome regions. Gene expression products are identified in whole cells through the use of green fluorescent protein (GFP). Using two model systems, the FishFinder Image Analysis Software System has been used to locate and capture specific fluorescent images while scanning a slide, return to the specific slide location and allow visual confirmation, either immediately or at a later time. One model system is for detection of small fluorescent spots, such as those found with FISH of specific chromosomes. Male blood was spiked into female peripheral blood at al level of 0.1% v/v. Lymphocytes were separated, centrifuged onto slides and labeled by FISH using X and Y chromosome probes. A set of parameters defining the intensity and size of a fluorescent signal of interest was selected. The slide was automatically scanned. Fields containing images matching the parameter settings were captured. At completion of the scan, captured fields were reviewed and verified for the presence of the Y chromosome. The second model system was for the detection of fluorescent cells. Slides were prepared from peripheral blood and cell nuclei were labeled with DAPI. Parameters were set for the detection of whole cells. Slides were scanned and verified as described above. Final reports were prepared including tables summarizing the number of chromosomes or nuclei captured and an image gallery of captured fields.

Congress On In Vitro Biology

Microgravity Suppression of Epstein-Barr Virus Reactivation. J.P. LONG, S. Pierson, & J.H. Hughes. Department of Medical Microbiology & Immunology, The Ohio State University, Columbus, OH 43210 E-mail: [email protected] Rotating-wall bioreactors (RW-bioreactors) allow for the growth of cells in simulated microgravity. Conventional nonadherent cell culture studies use static growth conditions; however, B-cells in vivo exist in various vectorial states and quasi-gravity environments. Therefore, we investigated the disruption of Epstein-Barr Virus (EBV) latency in B-cells cultured in RW-bioreactors. Lytic viral gene expression was measured by RT-PCR and lytic viral protein expression by indirect immtmofluorescence. Specifically, the message for the lytic switch protein was reduced 10-fold in simulated microgravity, while lytic early and late viral proteins were reduced up to 7-fold, P <0.05. Suppression of latency disruption was dependent upon microgravity and not motion, gas exchange, cell density or culture surface to volume relationships. In summary, the cultivation of lymphoblastoid cells in simulated microgravity resulted in significant suppression of the activation of latent EBV. RW-bioreactors, by virtue of providing a simple culture environment triggering marked differences in viral activation, f~ruish a model whereby both host and viral factors involved in regulating the maintenance of latency can be examined. Furthermore, RWbioreactors render an alternative strategy for conducting functional studies with nonadherent immune cells.

29-A

Abstracts

V-1017

V-1018

Use of Rotating Bioreaetors for Development of an In Vitro Blood-brain Barrier Cell Culture Model. A.E. SROUFE, G.L. Sanford, S. Hooker, D. Ellerson and K. Dutt. Morehouse School of Medicine, Atlanta, GA 30310. E-mail: gsroa~link.msm.edu The blood-brain barrier (BBB) protects the brain from transient changes in blood composition and maintains homeostasis in the brain parenchyma. In mammals, the barrier function of the BBB interface resides at the level of capillary endothelial cells whose specialized properties are the result of induction signals from surrounding astrocytes. The permeability of this barrier is affected by developmental, physiological and pathological processes. Therefore, it is importaint to understand how it is induced, maintained and potentially modulated. Several two-dimensional in vitro models of the BBB exist, however, there are few models in which a three-dimensional environment is allowed. The rotating bioreactor, developed by NASA, allows co-location of cells of different size and density, augments cell-to-cell contact, and enhances three-dimensional spatial freedom. In this study, normal human astrocytes (NHAs) and bovine aortic endothelial ceils (BAECs) were co-cultured on Cytodex-3 microcarrier beads in the rotating bioreactor system. Cells were continuously cultured for six days at 8 rpm and subsequently rinsed in PBS, fixed and utilized for immunolabelling and electron microscopy (EM). Samples were immunopositive for glial fibrillary acidic protein and factor VIII, identifying astrocytes and endothelial cells, respectively. Scanning EM revealed two distinct cell types in contact on the surface of the beads. Thus, it appears that NHAs and BAECs can be successfully co-cultured in the rotating bioreactor system rendering a novel three-dimensional model for the BBB. (Supported by NASA NCCW 0083 and NIH/RCMI RR03034).

V-1019 Differential Effect of Collagen and Agar Gel on the Developmental Pathway of a Cloned Fetal Respiratory Epithelial Cell Line M3E3/C3. M. EMURA1, A. Ochiai2, G. Singh3 and P.-G. Germann4. qnst. of exp. Path., Hannover Medical School, Hannover, Germany; zPath. Div., Nat. Cancer Cntr. Res. inst., Tokyo, Japan; 3Dep. of Path., Univ. of Pittsburgh, Pittsburgh, USA; 41nstof Tox. & Path., Byk Gulden, Hamburg, Germany. E-mail: .

Cultivation of tissue specific stem coils and artificial development of therefrom derived tissues, in particular such complex tissues as the lung may still be a dream in biotechnology. We were almost incidentally able to isolate a clone of undifferentiated respiratory epithelial cells from a fetal Syrian hamster on day 15 of gestation. This cell line frozen stocked at 20-23 passages has shown a multipotontial responsiveness to different culture factors incuding collagen and agar contact supporting gel by developing morphological and biochemical characteristics of vadous respiratory cell typos. Under 0.35% bactoagar overlay the cells grown on a plastic undeday differentiated on a pathway towards type II pneumocytes within 8 days of cultivation. Phosphatidylcholine content increased to more than 60 i.tg/106 cells as compared with about 45 pg/f06 coils in cultures under a conventional condition. Tritiated choline incorporation into cellular phospholipid fraction was also 2.6 times higher with agar oveday than under conventional condition. Immunocytochemicel reaction to an antisurfactant (hamster) antiserum and was only appreciably positive in most of the cells grown under agar overlay. In contrast, the cells grown on a collagen gel undeday for 6 days and longer with concomitant presence of 8-24 p.g/rnlof vitamin A developed morphological and biochemical characteristics belonging to a Clara cell (nondliated bronchiolar epithelial cell) type. In these cells (Clara cell specific) CC10 antigenicty was posilk,o and the frequency of these positive cells was dependent on the vitamin A cooncontration.The results indicate that collagen and agar appear to possess completely different properties in directing the pathway of cell differentiation.

Congress On In Vitro Biology

Animal Derived Component Free Media. G.W. REESE, J.C.F. Camire, P.K. Balls and B.B. Barnett., HyClone Laboratories, Inc., Logan, Utah 84321. E-mail:

Recent concern over adventitious agents in tissue culture media has prompted the development of animal derived component free (ADCFTM) media. Replacements and alternative sources must be found for a wide array of components including: serum, transferrin, albumin, amino acids, growth factors, lipids, and cholesterol. Important parameters in formulating ADCF TM media include the capacity to maintain viable, healthy cells that retain their morphological and physiological characteristics while producing the desired products. Alternative sources of materials include plant derived lipids, oils, proteins and peptides, recombinant growth factors, synthetic fatty acids and trace elements. When selecting components, care must be taken to insure that ingredients of an ADCF TM medium have not been manufactured using arfimal derived components. For example, plant peptides should not be processed using animal derived enzymes, and growth factors must not be stabilized with serum albumin. In this report, we identify common media components that must be avoided in ADCF TM media and describe the cultivation of CHO, fibroblast and insect cell lines in ADCFTM media.

V-1020 Cultivation of Human Skin Cells in Microcapsules. Claudia SCHNEIDER, H. H~ibner and R. Buchholz, Technical University of Berlin, Seestr. 13, 13353 Berlin, Germany. The use of autologous cultured epidermal cell sheets has become a standard method for coverage of extensively burned patients. The disadvantages of these sheets grafts are: the relatively long time needed to grow the grafts, technical difficulties in applying the fragile grafts resulting in an uncertain take, and high costs. The aim of our reaseach is the development of a novel kind of skin grafts in form of encapsulated keratinocytes. In this manner the production of skin grafts will be possible in shorter time and with lower costs. The cultivation of encapsulated cells has two significant advantages. First to remark is the higher growth rate of cells in microcapsules compared to normaly cultured cells, which makes skin grafts available in much shorter time. This is an important aspect by treatment of severe burned patients. The second important advantage lies in the protection of the sensitiv skin cells from mechanical stresses which permits a simple handling during cultivation and transplantation processes. These points result in lower costs and improving take rate. The new technique also aims to improve the cosmetic and functional results of the regenerated skin. Our institute is working on encapsulation systems for several years. The choosen polymer for encapsulation used here consists of components of human cells. It is medically authorized and totally biodegradable. We would like to thank Prof. Dr. med. H. Mau and Dr. med. A. Bettermarm just as the FNK of the Technical University of Berlin.

30-A

Abstracts

V-1021

V-1022 IDENTIFICATION OF A TUMOR CELL LINE FOR THE ISOLATION OF P53 MUTANT PROTEIN AND ITS UTILITY IN ASSAYS FOR P53 MUTANT PROTEINS AND THEIR AUTOANTIBODIES. P. Zhang, B. W. Lyons, L. L. Wu, J. T. Wu. Dept. Path. and ARUP, Univ, Lit. Sch. Med., Salt Lake City, UT 84108 E-mail:

Expression of p53 suppressor proteins in patients with tumor is recognized to be associated with higher risk for metastasis. In fact, both p53 mutant proteins and their autoantibodies are detected in the human serum; autoantibodies against p53 mutant proteins have also been shown to appear at an earlier stage during the progression of the malignant disease. Conceivably establishing assays for detecting serum p53 mutant proteins or their autoantibodies would help assess the prognosis and better manage patients with malignant disease. In order to find a reliable source for p53 mutant proteins we have searched nine tumor cell lines including RD, NB-4, MCF-7, COLO 320 HSR, LNCaP, I-I292, T-47D, HFF, and SK-BR-3 cell line using an ELISA kit from Oneogene Science for p53 mutant proteins. We found that the RD cells (Rhabdomyosarcoma, embryonal) contained the highest concentration of p53 mutant protein, higher than any earlier finding reported in ~ e literature. Up to 2 lag of p53 mutant proteins per 1 x 10 ceils could be extracted using buffer containing I% Triton X-100. Apparently, p53 mutant proteins are not very stable, attempt to purify them have met with little success. However, the p53 protein is useful as a calibrator for p53 mutant protein assay when it was extracted in the presence of sulfhydryl reagent and partially purified on Pharmacia FPLC through Mono Q column. The product was also applicable as antigen in the autoantibody assay for capturing autoantibodies from the patients' sera.

V-1023

EXTRACTION OF C-erbB-2 ONCOPROTEINS FROM SK-BR-3 CELLS: DEVELOPMENT OF ASSAYS TO ASSESS THE AGGRESSIVENESS OF BREAST TUMORS. J. T. Wu, P. Zhang, B. W. Lyons, A. Toney, L. L. Wu. Dept. Path. and ARUP, Univ. Ut. Sch. Med., Salt Lake City, UT 84108 Email: Measurement of soluble c-erbB-2 oncoprotein (19185) in breast tumor cytosol is routinely performed to assess the aggressiveness of the tumor. In addition, the ectodomain (p120) of p185 is detectable in serum from patients with various carcinomas. To develop various assays for c-erbB-2 oncoproteins we found that p185 can be extracted from SKBR-3 cells, a breast tumor cell line, in significant amounts with a buffer containing 1% Triton X-100. Extraction of p185 from the cell was almost instantaneous. A total of 17,000 units of p185 could be obtained from 4 x 106 cells. After a simple gel filtration chromatography, p185 and p120 could be separated from each other and from the majority of other cell proteins. Large amounts of p120 can also be obtained by simple extraction of the cells in the absence of detergent. Based on the determination of the slope of the calibration curves, p185 and p120 apparently have similar affinity for the assay antibodies and both were found useful as calibrators for these two assays, namely an assay for both p120 and p185 and an assay specific for p185. The difference between these two assays mainly relate to the antibody used to coat the solid phase. Both procedures and detecting systems are almost identical. Therefore, isolation of c-erbB-2 oncoproteins from SK-BR-3 cells not only allowed the development of assays using commercially available antibodies but permits the future development of an assay specific for serum p120.

V-1024 ISOLATION OF FREE PSA FROM LNCAP TUMOR CELL LINE USING HOLLOW FIBER BIOREACTOR: DEVELOPMENT OF AN ASSAY FOR SCREENING PROSTATE CANCER. B. W. Lyons, T. Haven, G. H. Liu, L. L. Wu, J. T. Wu. Dept. Path. and ARUP, Univ. Ut. Sch. IVied., Salt Lake City, UT 84108 E-mail:

Measurement of free PSA (fPSA) and a calculation of %fPSA is now a routine procedure during prostate cancer screening to differentiate benign prostate hyperplasia (BPH) from cancer. We have established a procedure for the production of milligram quantities of free PSA (fPSA) from LNCaP ceils derived from a haman carcinoma of the prostate for use in the fPSA assay. By growing LNCaP ceils in a serum-free medium in the presence of a synthetic androgen (R1881) and taking advantage of the special design of the CP-IO0 Hollow Fiber Bioreactor, relatively pure fPSA could be harvested from the cell culture medium. Since the fPSA was continuously released supematants were collected weekly. A total of 2.47 mg of fPSA was produced after 20 weeks. This ~ S A could be readily purified further by onestep chromatography on Sephacryl S-200 which removed most other proteins from the fPSA preparation. The isoelectric point (pI) of the major fPSA isoforms from the LNCaP cell culture were higher (6.8 and 6.6) than tPSA from seminal fluid (6.4 and 6.1). Thus the LNCaP cell line is a reliable source for large quantities of pure fPSA both for the preparation of assay calibrators and controls and for studying the difference in fPSA between benign prostate disease and prostate cancer.

Congress On In Vitro Biology

DEVELOPMENT OF BINDING, BLOCKING AND MODULATIING ANTI-ACETYLCHOLINE (AChR) AUTOANTIBODIES USING SOLUBLE RECEPTOR FROM A RHABDOMYOSARCOMA (RD) CELL LINE. B. W. Lyons, M. Astill, L. L. Wu, J. T. Wu Dept. Path. & ARUP, Univ. Lit. Sch. Med., SLC, UT 84108 Email: Measurement of binding, blocking and modulating antiAChR autoantibodies are important in the diagnosis and management of patients with myasthenia gravis. In the past, soluble AChR from amputated human leg muscle and fresh muscle biopsies were required for the binding, blocking and modulating antibody assays. We found that RD cells expresse muscle-type AChR and could be utilized for establishing these assays. For the binding assay, soluble AChR was extracted from RD cells using buffer containing 1% Triton X-100 and various protease inhibitors. The soluble AChR is used to quantify serum binding antibodies, after specifically labeled with [125I]ct-bungarotoxin (or 125IctButx), by forming a complex with the sample antibodies. For the blocking antibody assay, live RD ceils were plated into shell vials at 6 x 104 cells/vial. After incubation with the serum sample, ~25Ic~Butx was added. The decrease of J251c~Butx binding to the cells indicates the amount of blocking antibody present in the sample. For the modulatin~ antibody assays the live RD ceils were plated at 7.5 x 10 ceils per cm 2 in multiwell plates. After labeling the cells specifically with mlaButx (12 wells) the extent of modulation was determined as the percentage of AChR internalized with and without modulating antibodies after incubation with the sample. The discovery of RD cells makes it possible for clinical laboratories to perform all three assays.

31-/t

Abstracts

V-1025

Initial Characterization of Proteins that are Involved in EpitheliaI-Stromal Interactions that May Link Endometrial Cancer and Aging. D.K. TARKA, V. R. Torti, and D. G. Kaufman. Curriculum in Toxicology, Department of Pathology and Lab Medicine, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599. E-mail: .

V-1026

Altered Morphology, Adhesion and Glycosidase Release in U87MG Tumor Cells Differentiated with Retinoic Acid and HMBA. C. CHUNG, Z. Flores, P. Luu, and J.R. Wilson. Biology Department, La Sierra University, Riverside, CA 92515.

Endometrial cancer is typically a disease of older women. It is speculated that age-related changes in stromal cells that directly effect the regulation of the endometrial epithelial cells may increase the risk of cancer formation. Conditioned medium from young stromal cells (YCM) has been shown to inhibit epithelial cell growth in vitro and we believe that this is due to certain constituents, probably proteins, that are present in YCM. To characterize these components, we fractionated the YCM via ultrafiltration into broad molecular weight ranges using filters with nominal cut-offs at 10 kD, 30 kD, and 100 kD. The fractionated medium was used to culture Fetal Endometrial Epithelial Cells (FEEC) in a 7-day assay of colony forming e~ciency of the cells. We found an approximate 50% growth increase with the protein fraction over 100 kD. This result indicates that a dynamic combination of inhibitory and stimulatory effects cause the overall inhibitory effect of the YCM. Identification of these growth factors that control the endometrial epithelialstromal microenvironment could lead to new approaches to therapy or prevention of this form of cancer. Supported by NIH Grants ES07126, ES07017, and CA31733.

The effects of differentiation agents on a brain tumor cell line, U87MG, were studied. AII-Trans retinoic acid (RA) and hexamethylene bisacetamide (HMBA), were used alone or in combinations. Definite morphological changes were noted. When untreated cells become confluent, cells aggregate and form clusters, whereas this cluster formation was greatly reduced in seven-day cultures treated with a combination of IRA and HMBA. In addition, treatment with RA significantly suppressed cell adhesion to the extracellular matrix protein, fibronectin, by 80% relative to untreated control cells. HMBA also reduced adhesion by 24% whereas the cells treated with a combination of IRA and HMBA had adhesion levels reduced by 52%. We also observed that untreated cells release the acid hydrolase, N-acetyl-glucosaminidase (GNase) into the culture media, however treatment of cells with either IRA or HMBA reduced this release by 49% and 34% respectively and in combination the release was reduced to 20% Future studies will investigate the role of cell surface glycoproteins in the differentiation process and in cell-matrix and cell-cell interactions regulated by IRA and HMBA. (Supported by La Sierra University and the Ryckman Undergraduate Research Fund).

V-1027

V-1028

A CelI-ELISA Procedure for the in vitro Quantitation of Myelin Production in Rat Brain Primary Cell Cultures. W.T. BLUE and J.W. Nemie,. West Virginia School Of Osteopathic Medicine, 400 N. Lee St., Lewisburg, WV 24901. E-mail: A cell culture system derived from neonatal rat brain was developed to investigate oligedendrocyte myelination of neuronal axons. Neonatal Slr,tgue-Dawleyrat brains were used to create primary cell cultures in 24well plates using standard tissue culture techniques, lmm*mo.histechemical detection of myelin+and specific cell types, were confirmed using specific rabbit antibodies to myelin basic protein and cell-specific markel~. Staining was accomplished with enzyme-conjugated goat antirabbit antibodies and corresponding substrates. A cell-ELISA assay was developed to quantify myelin production in replicate cultures. For the assay, one row of wells was left cell-free and used for the construction of a standard curve by binding known quantities of purified MBP to the bottom of the wells. Following 30 minutes of binding, the wells were washed with PBS containing 0.05% Tween-20 (PBS-T). The cell monolayers in the remlining wells were fixed with methanol COntaining 1% H20 ~, then washed with 95% ethanol, distilled water, and PBS-T. All wells were then treated with Superblock (Pierce) for a minimum of 30 minutes. Myelin q.Antltafion was accompfished by the addition of rabbit anti-MBP, followed by goat anti-rabbit IgG, conjugated with horseradish peroxidase. Development followed the addition of ophenylenediamine (OPD, Si~,mu). The culture supernatauts were transfered to plastic cuvettes, and their ODo2 determined in a speetropbotometer. Assay results indicated the following: the MBP standard curve was linear and ~.producible, and quantifies of myelin determined in replicate cultures were very similar, Using the cell-ELISA assay, myelin production could be detected much earlier than by imm~mohistochemical stainin~ and observation. In a time-course assay, myelination, and the accumulation of myelin, continued for approximately 40 days in vitro, followed by a leveling off of production and a relatively steady-state maintenance of myelin amounts through day 72. The ability to quantitatively assess the active myelination process should allow for direct investigation of theories of causation of various demyelinafing diseases, such as multiple sclerosis.

Congress On In Vitro Biolog7

Prostanoid Production by Cultured Umbilical Endothelial Cells From the Second and Third Trimester in Normal Women and Women with Severe Gestational Hypertension. C.D. LOX, D. Timm, and S.D. Prien. Dept. Obst.Gynecol. Texas Tech HSC. Lubbock, TX 79430. E-mail [email protected] It is known that the balance of the vasoconstrictor thromboxane and the vasodialator prostacyclin are altered during severe gestational hypertension (pre-eclampsia), a disease of unknown etiology. The altered expression of these two prostanoids has also been observed in cultured human umbilical endothelial cells (HUVEC), when exposed to serum from women with pre-eclampsia. All of these prior studies were on women at or near term. It is not known if the maternal-fetal unit is predisposed or capable of producing the proper milieu for pre-eclampsia during early gestation, hence this study. Endothelial cells were collected from umbilical cords following delivery, starting at 23 weeks of gestation. Following collagenase digestion, the ceils were incubated utilizing standard culture techniques for this cell line. The cells were from women with normal (control) pregnancies or those with clinical pre-eclampsia. These cultured cells were then incubated with media containing either normal serum, serum from women with pre-eclampsia, or sera from women with eclampsia. After 24 hours, the media was analyzed by RIA for content of thromboxane A2 and prostacyclin. The resultant data suggest.that before 30 weeks of gestation, the HUVEC cells are not especmUy responsive to sera from gestation hypertension, even those cells from women with hypertensive disease. After 30 weeks the previously observed responses of this cell line to produce more thromboxane and less prostacyclin was again demonstrated. This suggests that the umbilical vasculature needs a period of time to become responsive to hypertensive stimuli, perhaps around 30 weeks of gestation.

32-A

Abstracts

Vertebrate - Contributed/Poster V-1029

Sessions

V-1030

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Chondrocyte Morphology is Correlated with Chondrogenic Potential. B.B. STONE and B.E. DeFranzo. Genzyme Tissue Repair, Cambridge, MA 02139. e-mail: [email protected] Human chondrocytes dedifferentiate in tissue culture and grow with a variety of morphologies. When these cell populations are transferred to suspension cultures they can be shown to produce a variety of molecular and biochemical markers indicative of chondrogenesis. This report describes the correlation of different cell morphologies with chondrogenic potential. Five strains of human chondrocytes were diluted to one cell per tissue culture well and expanded to produce single colonies. These were photographed, the different morphologies were described, and the cells were harvested. They were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) for the presence of mRNA for aggrecan, a proteoglycan indicative of chondrogenic cells. Seven clones (P of single progenitor cell = 0.52-0.83) produced positive signal for aggrecan mRNA. These aggrecan-positive clones also contained from 2 up to 7 distinct cell morphologies. This indirect statistical approach showed that single progenitor chondrocytes can produce progeny colonies which contain multiple cell morphologies. Therefore these varied morphologies were not a result of the presence of contaminating cell types. A number of these progeny colonies produced aggrecan mRNA, confirming that the parental cell in each case was from a chondrogenic cell type.

To study the mechanisms of morphogenesis in salivary gland regeneration, we established an RSMG-1 cell line derived from the salivary gland of 10-week-old Wistar female rats in serum-free medium. The proliferation of RSMG-1 cells was promoted by HGF, but was inhibited by activin A. In three-dimensional collagen gel culture, RSMG-1 cells formed branching structures. HGF potently induced branching morphegenesis of RSMG-1 cells. Sections of the RSMG cells embedded in collagen gel revealed well-formed lumina in the presence of HGF. This morphology closely resembles that found in vivo, and the cells also expressed activin A and activin receptors. Exogenous activin A at a high concentration reduced this morphogenic effect. These findings suggested that the growth and morphogenesis of salivary gland regeneration is modulated by HGF and actMn A. The RSMG-1 cell line may provide a useful tool for the study of the mechanisms of salivary gland regeneration.

V-1031

V-1032

Effect o f Increased cyclic AMP Concentration on Muscle Protein Synthesis and 13-Adrenergic Receptor Expression in Chicken Skeletal Muscle Cells in Culture. R.B. YOUNG, J.R. Vaughrt, K.Y. Bridge, and *C.K. Smith, II. Marshall Space Flight Center, ES76, Huntsville, AL 35812. *Lilly Research Labs, Greenfield, IN. E-mail: [email protected],go v. Analogues o f epinephrine are known to cause hypertrophy o f skeletal muscle when fed to animals. These compounds prestmmbly exert their physiological action through interaction with the 13-adrenergie receptor. Since the intracellular signal generated by the 13-adrenergic receptor is cyclic AMP (cAMP), experiments were initiated in cell culture to determine if artificial elevation o f cAMP by treatment with forskolin would alter muscle protein metabolism and [3-adrenergic receptor expression. Chicken skeletal muscle cells after 7 days in culture were treated with 0.2-30 I.tM forskolin for a total o f three days. At the end o f the treatment period, both the concentration o f cAMP and the quantity o f myosin heavy chain (MHC) were measured. Concentration o f cAMP in forskolin-treated cells increased up to 10-fotd in a dose dependent manner. In contrast, the quantity o f MHC was increased approximately 50% above control cells at 0.2 laM forskotin, but exhibited a gradual decline at higher levels o f forskolln so that the quantity o f MHC in cells treated with 30 bdCIforskolin was not significantly different from controls. Curiously, the intracellular concentration o f cAMP which elicited the maximum increase in the quantity o f MHC was only 40°,4 higher than cAMP concentration in control cells. (Supported by Lilly Research Laboratories, Greenfield, IN.)

Congress On In Vitro Biolog7

~ . M FURLEd,T. Okamo~, H. I - ~ , J. D. S=o4, M Asast*r~, and S. Sailol. Depalme~ of 1era

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The Effects of g-2-MSH on DAG Activity in Synaptosomesfrom Hypertensive and NormotensiveRats. T.J. ROBINSON and C.A. Hughes. Departmentof Biology,MorganState University,Baltimore, MD 21251. E-maik. The phosphoinosiddesignalingsystemhas been shown to play a role in blood pressure regulation. There has also been reports that phosphoinositide metabolisms is altered in genetically developed hypertension, g-2-MSH peptide, a POMC-derived peptide, administered to conscious rats causes increases in blood pressure, heart rate and cerebralblood flow. However, the cellular and molecular mechanismsthat cause hypertension are not clearly defined. We hypothesizedthat in the brain g-2-MSH akets the levelsof DAG activity during hypertension. In order to test our hypothesis, we use 3Hcyfidine(6 pmole) to measurethe CDP-DAG activityin synaptosomesisolated from the forebrain, midbrain and hindbrain of normal and hypertensiverats. The resultsof our data show that DAG activityis increasedin all of the brain regionsof SHR as comparedto WKY rats. However,when g-2-MSH (0.1 nM to 5 nM) was added, though increasesin DAG activitywas observed in the SHR rats in the forebrain and midbrakt regions, this increase was concentrationdependent.When g-2-MSH (0.1 nM to 5 nM) to synaptosomes isolated from the WKY control, no change was seen in the forebrain and midbrain of these animals. In the midhrain of SHR, a down regulation (0.5 and 1 nM) of DAG activity. [Supported in part by NIH/MBRS grant #GM/S06AI51971-01A1 and NIH/NCRR/RIMI grant #2P20RR11606-02].

33-,4

Abstracts

V-1033

V-1034

Carbonic anhydrases (CAs) catalyze the hydration of carbon dioxide to form bicarbonate ion (HCO~) and proton (H~). Among the seven CA isoen~'mes, the CA IV is expressed in specialized epithelial cells and corresponds to the membrane-associated form. Because of its localization on the surface of plasma membranes, its role in the regulation of extracellular pHs was proposed. Some authors have suggested that it exists in a channellike conformation allowing the secretion of HCO3- ions, in particular in pancreatic duct ceils. In this cellular model. HCO3 ion secretion is regulated by the cAMP-dependent chloride channel CFTR (Cystic Fibrosis "l~ransmembrane conductance Regulator) localized on apical plasma membranes. A failure of its targeting was shown in cystic fibrosis (CF) ceils homoz-,'gous for AF5(I8 mutation and is responsible for important alterations in ionic exchanges, in particular in pulmonary, and pancreatic epithelia. This study was designed to determine whether the targeting of other membrane proteins such as CA IV is altered in CF cells. To this end. two human pancreatic duel cell lines were chosen as study models: the CFPAC-1 line expressing a AF508 CFTR and the Capan-1 line expressing the wild .type form. Immunoblotting and immunocytochemical reactions carried out either on fixed cells permeabilized or not. or on living cells x~ere performed. The antibodies used correspond to a polyclonal antibody (ECL 885) directed against a sequence of the fourth extracellular loop of human CFTR (amino acids: 885-904), and to three polyclonal antibodies directed against three peptidic sequences of human CA IV (amino acids: 1-18, 125-138, 276-294). Immunoc.vtochemical reactions were analyzed by episcopic or confocal fluorescence microscopy. Comparative qualitative and quantitative analysis showed that: i) whatever the cell line_ 100% of cells expressed CFTR and CA IV: it) in Capan-I line, 98'/o of cells displayed CFTR and CA IV targeted to their apical plasma membranes: iii) in CFPAC-I line. CFTR and CA IV were targeted to plasma membranes in only 6% and 4% of polarized cells respectively. In conclusion, a defective targeting of CA IV to apical plasma membranes occurs in human pancreatic duct cells expressing a mutated CFTR (AF508). These data suggest the existence of changes in the transport and/or the nmturation of membrane proteins in CF cells.

The Paradigms of Cell Surface Annexin I1: Subcellular Localization and Biochemical Characteristics in both Cultured Human Fibroblasts and Keratinocytes. Alice S.P. Ma. Kansas City VA Medical Center, KC, MO 64128. We studied extracellular annexin (anx) II in both cultured human fibroblasts and keratinocytes. With a two-step labeling method, anx II was labeled on non-permeabilized cells, followed by cell fixation and then cell permeabilization before f-actin staining. Anx II was detected on the cell surface side where actin bundles ware submembranous. The staining patterns in fibroblasts suggest anx II's translocation to the cell surface to be related to ce~l movement, whereas in keratinocytes, to detachment of the top layer cells. Isolation of anx II by immunoaffinity column chromatography showed that it existed in isoforms with pl 5 . 4 - 8 5 By first separating the cultured cells into intra- and extracellular pools, and using different elution buffers, we ware able to further separate and analyze these isoforms by one and two-dimensional gel electrophoreses. In both cell types, extracellular anx II was characterized by isoforms of acidic pls, 5.4-5.8 and it co-eluted with a 46 kD protein. The same co-elution was also isolated from the intracellular pool, but only extracellular anx II was found to be selectively degraded. Taken together, our findings of extracellular anx II's subcellular topography and biochemical characteristics suggest that a subset of acidic anx II isoforms, when co-localized with submembranous actin, was translocated to the cell surface. Although the mechanism of this is not clear, the process was related to cell detachment during cell movement.

V-1035

V-1036

DEFECTIVE PLASMA MEMBRANE TARGETING OF CARBONIC ANHYDRASE IV IN HUMAN POLARIZED PANCREATIC DUCT CELLS EXPRESSING A MUTATED CFTR tAF508). E. Hollande~, C. Salvador~, S. Cantet ~, L. Alvarez, J Esclassan, N Carter-" and M. Fanjui .1 Laboratoire tie Biologie Celtulaire (dE 1962), Universit~ Paul Sabatier, 38 rue des 36 Pouts, 31400 Toulouse, France - 2 St George's Hospital Medical School Dept of Child Health, London, [.'K

Explant Cultures of Gubcrnaculum Testis. RACHEL I. HUOT and Joseph Ortenberg, Department of Urology, Louisiana State University Medical Center, New Orleans. LA 70112. E-MaiI: . The gubernaculum testis is believed to play a major role in testicular descent but few attempts have been made to grow this tissue in culture. Our laboratory has been interested in the hormonal and neurologic regulation of the testicular descent process. To this end, we have successfully developed explant cultures of gubernacula derived from 4 neonatal (within 3 days of birth) New Zealand White rabbits. Eight gubcrnacula were removed aseptically, rinsed with medium, and allowed to adhere directly to plastic tissue culture flasks (25cm2) either as an intact organ (lx4mm) or as small pieces (lxlmm). Tissue was allowed to attach overnight in lml of medium at 37oC in a humidified atmosphere of 5% CO2 in air. Tissue was randomly assigned to one of three different culture media: an enriched Eagle's Minimal Essential Medium, F12, and RPMI 1640, all supplemented with fetal bovine serum (10%), Lglutamioe (2raM), and antibiotics. RPMI 1640 yielded the best cellular outgrowth in our hands while MEM elicited the poorest growth in terms of cellular proliferation outwards from the tissue. Cells rapidly migrated from the explant as a broad sheet of cells by the following day of culture and the proliferative region attained a diameter of greater than 1.5 cm by. the end of the week. Two cell types predominated: epithelioid and elongated fibroblastlike cells presumably derived from mesenchymal and muscle ceils, respectively, as reported histologicallyas the two gubernacular major cell types by other laboratories. Multilayering of cells was common, especially adjacent to the tissue. Cultures could still actively proliferate at the time of their termination several months later. Gubernacula from a neonatal SpragueDawley rat were also successfully cultivated. In contrast, fibrotic gubernacular tissue from 2 prepubertal boys demonstrated only sparse growth of fibroblastic cells. These failed to proliferate and thus underline the importance of using neonatal gubernacnlar tissue for studies of the gubernaculum Gubernacular cultures should provide a valuable tool for the investigation of gubernacular function.

Congress On In Vitro Biology

Iden~cation of osteocalcin mRNA in nonskeletal human tissues by reverseb'anscriptasepolymerasechain reaction. B. Knoblauch;U. Stahl; A. Battmann; U. Pollex. Department of Pathology,Justus-Liebig-UniversityGiessen,Germany. Bone formation and ossification are generally considered a process limited to the skeleton. Metaplasiaof woven bone can occur in various tissues suggestingthe presence of cells with bone forming capacity in nonskeletal tissues. Osteocatcin is a 5,7 kOa protein known to be a highly specificmarker of osteogenesis.In rat the presenceof osteocalcin m-RNA but not protein was demonstrated for various organs. Total cellular RNA was isolatedfrom ten 10 pm sectionsof fresh frozen tissue samples by RNAzol B-method. Reverse transcription was performed with routinetechniqueusing random priming (Gone Amp, Perkin Elmer). A 294 lop osteocalcinfragmentwas amplifiedby a touch-down protocol. Amplification of the house-keepinggene HPRT was used to prove the quality of RNA e~action and RT-PCR. Specifity of PCR products was demonstrated by restriction enzyme analysis (8amH I). Immuflohistochemis~ was performed on 5 pm sections of formaline-fix~ parafffinembeeded tissue specimens. Monoclonal osteocalcin antibody (Dr. 8aylink, LomaUnda) in a dilutionof 1:800 was incubatedover night at room temperature. Immunostaining was completed using commercially available APAAP-technique (Dako Ltd.). OsteocalcinmRNA was detected in the total RNA from different human tissues includingseveral nonosteoidtissue. Immunohistochemicallywe found a positivereactionwith the monoclonalosteooalcinantibodyonly in human bone. Using the ultrasensitive RT-PCR we were able to detect osteocalcin m-RNA but not -protein in most of the analyzed fissues. Based on this data, we conclude that the presence of osteocalcin m-RNA is not limitedto bone an may reflect an early label of osteogenic potency or a ,leaky" expressionof the gene in non differe~ated stem cells.

34-A

Abstracts

V-1037

V-1038

Apoptosis During the Differentiation of the Retina in Postnatal Mice. C.E. GAGNA and W.C. Lambert. Dept. of Pathology and Laboratory Medicine, UMDNJ-Medical School, Newark, NJ 07103. The purpose of this project was to investigate apoptosis in the postnatal mouse retinal ganglion cells. This was achieved by examining tissue sections, fixed in 10% NBF, for the presence of double-stranded (ds-) DNA, single-stranded (ss-) DNA, and right-handed B-DNA and left-handed Z-DNA, using antibodies: Anti-ds-Z-DNA, anti-ds-B-DNA and anti-ss-DNA antibodies. Some retina were maintained in tissue culture. In the developed mouse retina, apoptosis is a normal and necessary cellular process. We observed the usual morphological and biochemical changes associated with apoptosis of the mouse retina. Cell death occurred primarily during the first 12 days after birth. Using a novel image analyzing system, we quantitated the DNA fragmentation. No difference in data was observed between tissue sections and cultured cells. Our data shows that the DNA within retinal cells undergoes degradation. We observed an increase in ss-DNA and a decrease in ds-B-DNA, along with a decline and/or absence of Z-DNA. The increased fragmentation of ds-B-DNA and the decline and/or absence of Z-DNA would indicate an decrease and/or inhibition of cellular gent function. These results support the conclusion of others that apoptosis occurs within the developing mouse retina. It is speculated that genetically regulated apoptosis acts to fine-tune neuronal networks during the final stages of development.

The Effects of Long Term Storage and Low Temperatures on DNA Structure and Function. C.E. GAGNA and W.C. Lambert. Dept. of Pathology and Laboratory Medicine, UMDNJMedical Schoolr Newark r NJ 07103. DNA and cell banks are maintained at very low storage temperatures. Optimum storage conditions must still be assessed: the possibility of degradation, the resuspension of lyophilized DNA, and the sticking of DNA to storage units. This group has undertaken to ascertain the structural and functional recovery of purified DNA and genes after freezing, for 17 years, using different storage buffers. Double-stranded (ds-) B-DNA, dSZ-DNA and single-stranded (ss-) DNA were analyzed using antibodies (ELISA). DNA structures were also analyzed by using CD UV spectroscopy, electrophoresis, microinjection of DNA into cells maintained via cell culture, and transfeotion studies. Results indicate that the loss of DNA stored at -70°C, -120°C, -196oC and liquid nitrogen using non-freezing solutions, is minimal and biological activity remains intact. Long term storage at +4°C and -20°C allowed for the production of ss-DNA, and biological activity declined. Repeated freezing and thawing had little effect on B-DNA structure-function if stored in nonfrozen solutions, as compared to frozen or lyophilized samples. Z-DNA was more sensitive to repeated freezing and thawing. Data reveals which buffers and storage conditions are best for the long term storage of DNA. This should aid scientists in the storage of DNA and cell lines for future studies.

V-1039

V-1040 In Vitro Effect of Retinoic Acid and Tamoxifen on Extracellular Matrix of Chick Embryo Ovaries: A Structural and Histochemical Study. R.E. AVILA, M.E. Samar, R. Ferraris, L. Olmedo, S. Fabro, F. Esteban, J. A. Pedrosa, M.A. Peinado. Histology and Pathology, School of Medicine, National University of Cordoba, CONICOR, CONICET, Cordoba, (5000) Argentina. E-maih .

Apoptosis in Low Density Cell Culture of Crystalline Lens Anterior Epithelial Cells. C.E. GAGNA and W.C. Lambert. Dept. of Pathology and Laboratory Medicine, UMDNJ-Medical School, Newark, NJ 07103. Our group has examined cell death occurring in low density cell cultures of the bovine lens epithelium. High density dissociated cell cultures of lens epithelial cells survived for many weeks. However, when the epithelial cells were cultured at a low density, they died in an apoptotic manner. Cell death was determined by first fixing the cells in acetic acid/methanol followed by quantitation of immunohistochemistry. We used anti-double-stranded (ds-) Z-DNA, anti-dsB-DNA and anti-single-stranded (ss-) DNA antibodies. High density cell cultures revealed no ss-DNA, and intense ds-B-DNA and Z-DNA immuaoreactivity. Cells undergoing denudeation, in low density culture, exhibited high levels of ss-DNA, decreased ds-B-DNA and either low or no ZDNA intmunoreactivity. Pyknotic cells were only observed in the anterior epithelium. These results suggest that the epithelial cells do not need signals from other cell types to survive and that epithelial cells support one another by secreting survival factors. The data suggests that celt death can occur within the anterior epithelium in the adult bovine lens. Our findings also suggest that pyknosis produces elevated amounts of denatured SS-DNA, which together with the elimination of left-handed Z-DNA may negatively effect normal gene functions of the low density cell cultures.

Congress On In Vitro Biology

It is known that embryo cells have a similar behavior to that of some tumor cells. Taking into account that retinoic acid and tamoxifen are employed in antirumoral therapy, we have analyzed the effect of both substances on extracellular matrix from the right (atrophyed) and left (functional) chick embryo ovaries. Explants of 7 to 19 day-old embryos were separately cultured for 4 days in MEM containing either 10% bovine fetal serum (BFS) (control) or BFS and all-trans-retinoic acid or tamoxifen citrate (SIGMA) respectively. Cultures were processed for uhrastructural (TEM) and histochernical analyses (PAS, PAS/amihse, Alcian blue at pH 2,5 and 1.0, methyhtion/saponification and methenamine/silver, ruthenium red and different lectins bind to peroxidase: WGA, PNA, UEA, DBA, and ConA) The more significant results appears in the 7 day-old ovaries cultured for 4 days with retinoic acid. The medullar extracellular matrix of left ovary and right ovaries showed an increase of collagen fibrils and bundles. In addition, we detected strong PAS and periodate reactions. It also appears an increase in the ruthenium red granulations and PNA labelling reaction. These results showed that retinoic acid produce modifications in the different secretory components of the extracellular matrix. Finally, the active role played by retinoic acid in the differentiation of the chick embryo ovaries may support a preventive function in the development of the neoplasm.

35-A

Abstracts

V-1041

V-1042 Morphological Analysis of Apoptotie Germ and Epithelial Cells from Chick Embryo Ovaries Cultured with Gonadotrophin Hormones. RE. AVILA, ME. Samar, RFerraris, S.Fabro,FEsteban, J A. Pedrosa, MA Peinado Histology and Pathology, School of Medicine,National Univerity of Cordoba, CONICOR, CONICET, Cordoba,(5000) Argentina. E-mail: [email protected]

Apoptosis (programmed cell death) is a normal physiological process that occurs during development. The present study analyzes the occurrence of apoptotic germ and epithelial cells in explants of both chick embryo ovaries (atrophied right and functional left) cultured with luteinizing (LIt) and human chorionic gonadotrophin (hCG) hormones. Explants from 7, 11, 15 and 19 day-old embryos were separately cultured for 4 days in MEM with 10% bovine fetal serum with LH or hCG or without these hormones (control). Then, cultures were processed for their ultrastructural microscopic study (TEM). Morphologically, the apoptotic cells showed chromatin condensation, apoptotic bodies and a strong reduction of their size. Results indicated a less number of germ apoptotic cells in hormone treated ovaries than in control ones. In addition, the first apoptotic cells appeared later in treated hormone explant ovaries (15-day-old embryos) than in control (I 1-day-old embryos), llowever, the types and location of the apoptotic cells was similar in both experimental and control groups; in fact, apoptotic process were detected in medullae germ and epithelial cells from left and right ovaries These results nllow to assume that medullae germ and epithelial programmed cell death from both ovaries is modulate by gonadothophin hormones !during ovary differentiation.

Biomateria],s on the Internet. M. BATI~AGLIA l and R. Zanoniz. IIMS CNR, 00133 Rome, adept, of Chemistry, University of Rome La Sapienza, 00185 Rome, Italy, E-mail: The Internet, a rapidly growing international network of computers that permits exchange of scientific information throughout the world, offers useful resources for educational, research, health care and business institutions involved in the field of biomaterials. Among others, these include: Scientific societies (e.g., "http://www.biomaterials.org/" and "http:// www.gen.emory.edu/MEDWEB/keyword/bioteehnology/soeiet les and associations.html'); Biocompatibility sites (e.g., "http://bittburg,ethz.ch/lsst/bio/default.html" and "http://www. univie.ac.at/uni-zahnklinik/Database.html");Tissue engineering (e.g., "http://www.pittsburgh-tissue.net/" and "http:// www.ukrv.de/ch/rheuma/TissueWeb.html'); Polymers (e.g., "http://iskra.pse.umass.edu/index.html'); Comprehensive biomaterials sites (e.g., "http://www.soealbio. org/matech/index.htm'); Scientific journals (e.g., "http://www.bio. net/BIO-JOURNALS.htmI" and "http:// www.gen.emory.edu/MEDWEB/keyword/electronic t)ublicatio ns. html"); Search engines for finding scientific reformation on the Internet (e.g., "http://www.chem. msu.su/eng/ comparison.html'); Scientific news releases (e.g., "http:// www.eurekalert.org/"); Usenet newsgroups and discussion lists (e.g., "sci.polymers" and "biomat-l'). A detailed list with description of biomaterials resources is available from the authors ('http://pages.inrete. it/mbiomed/index.htm" < [email protected]> < mbattaglia@mclink. it>). Supported by Consiglio Nazionale delle Ricerche Progetto Finalizzato MSTA II.

V-1043 Virology on the Internet. M. BATI'AGLIA. IMS CNR, 00133 Rome, Italy. E-maih The Internet, a rapidly growing international network of computers that allows exchange of scientific information throughout the world, offers resources that are useful for educational, research and health care institutions involved in the field of virology. Among others, these include: Scientific societies (e.g., "http://www.sivb.org/index.html" and "http:// www.asmusa.org/"); Culture collections (e.g., "http://www.atcc.org/", and "http://www.gdb.org/annex/ ecacc/HTML/ecacc.html"); Electron microscopy sites (e.g., "http://cimewww.epfl.ch/EMYP/comp_arc.html" and "http:// momo.mih.unibas.ch/Homepages/bernd f/emlinks.htm'); Molecular and genomic databases (e.g., "http:// www.ncbi, nlm.nih.gov/" and "http://blocks. fhcrc.org"); Comprehensive virology sites (e.g., "http:// www.tulane.edu/'dmsander/garryfavweb.html",'http://fiona.u msmed.edu/- yar/homeb, html" and "http://www.geocities, eom/CapeCanaveral/1819/Virus.html'); Scientific journals (e.g., "http://www.bio.net/BIO-JOURNALS.html" and "http://www.gen.emory.edu/MEDWEB/keyword/electronic_pu blications.html"); Search engines for finding scientific information on the Internet (e.g., "http:// www.chem.msu.su/eng/comparison.html"); Scientific news releases (e.g., "http://www.eurekalert.org/"); Usenet newsgroups and discussion lists (e.g., "bionet.virology" and "biosafty"). A detailed list with description of virology resources is available from the author ('http:// pages, inrete, it/mbiomed/index,htm" < mbatt@biocell,irmkant. rm.cnr.it > < [email protected]>).

Congress On In Vitro Biology

36-A

Abstracts

T-1001

T-1002

Establishment of a New Human Hepatocyte Cell Line Expressing Various Forms of p450.

Insulin alters the Pharmacodynamics of Xenobiotic-indueible CYP Gene Expression in Primary Rat Hepatocyte Cultures. J.S. SIDHU and

M. NAMBA, S. ASAHI*, H. MIYAZAKI and K. FUKAYA. Dept. Cell Biol., Okayama Univ. Med. Sch., Okayama 700, Japan, *Taketa Chem. Ind., Ltd., Osaka 532, Japan. E-mail: [email protected]

Hepatocyteswere obtainedby perfusionwith trypsinsolution

CJ. Omieeinski. Dept. of Environmental Health, University of Washington, Seattle, WA 98195. Email: In this investigation, we examined the effects o f insulin on gene induction responsiveness in primary rat hepatocytes. Cells were

from the liver of a 21-w-old embryo and transferred to cultures. Next day the SV40T and pSV3neo genes were cotransfected into the cells, and the cultures were maintained in the presence of O418. Serum-free culture medium, ASF104, was used after the gene transfection. One month later 13 epithelial colonies were cloned with the stainless cylinder and each clone was expanded to mass cultures to examine cellular characteristics. As a result, one of the clones retained measurable activity of NADPHcytochrome c reductase, 1A2, 2El and 3A4 forms of p450, ornithine carbamoyl transferase, and UDP-flueuronyltransferase. Expression of CYP1A2 was prominently enhanced by phenobarbital, and that of CYP3A4 was enhanced by phenobarbital, rifampicin and dexamethasone. This immortalized cells are now over the 200th passage level and continuously growing. Tumorigenicity was not detected in nude mice when transplanted. To our knowledge, no human hepatocyte cell lines with the above-mentioned drugmetabolizing enzymes have not been established yet. This success may be due to serum-free culture condition from the very early stage of culture and cloning functioning liver cells. This cell line will be useful for toxicology studies and construction of the artificial liver organ.

cultured for 72h either in the presence or absence o f I g M insulin and then exposed to increasing concentrations o f phenobarbital (PB; 0.01-3.5 raM). Culturing in the absence o f insulin produced 1.5-2-fold increases in the induction magnitude of CYP2B 1 and CYP2B2 rnRNA expression resulting from PBexposures, without altering the beg-shaped dose-response curve characteristic o f this agent (Kocarek et aL, 1990). However, for the CYP3A1 gene, insulin removal led to a pronounced shift in both the PB induction magnitude and dose-response relationships o f the induction response, with higher levels o f CYP3A1 expression resulting fi'om exposures to lower concentrations o f inducer. Insulin removal also reduced the time required for maximal induction o f CYP2BI/2 and CYP3A1 gene expression. The insulin effects were not specific for PB induction, as insulin removal similarly enhanced both dexamethason¢- and ~-naphthoflavone-indueible CYP3A1 and CYP1.A1 expression profiles, respectively. In contrast, the level o f albumin mRNA expression was considerably reduced in cells starved o f insulin. We conclude that insulin is an important regulator of inducible and liver selective gene expression in rat hepatocytes. Supported by USPHS Grant GM32281 (CJO).

T-1003

T-1004

Rotating Wall Vessel Culture of Yolk Sac Cells provides a Model System for Studies of Toxicity. T.G. HAMMOND, W.C. Campbell, T.J. Goodwin, and J.H. Kaysen. Tulane Environmental Astrobiology Center, Tulane University School of Medicine, & New Orleans Veteran's Affairs Medical Center, New Orleans LA, 70112, and NASA Johnson Space Center, Houston TX, "17058. E-maih In somc epithelial cell lines, the uptake and degradation of proteins is so pronounced as to be regarded as a specialized function known as "degradative cndocytosis". The endosomal pathway of the visccral yolk sac has highly specialized structures for "degradative endocytosis". The yolk sac epithelial cell line (BN/MSV) was dcrived from yolk sac teratocarcinoma induced by fetectomy and yolk sac infection with Mouse Sarcoma Virus. When grown under conventional conditions in modified Eagles Medium supplemented with 2.5 mM L-gtutamine, 10% fetal calf serum, and an antibiotic cocktail, the cells initially form a monolayer, and at confluence release into the supernatant a few cellular aggregates, often surrounding a fluid filled central cavity, referred to as embryoid bodies. Embryoid bodies are greatly prized as they most accurately reflect the natural yolk sac structure and function for studies of fetal toxicity and associated malformation. There are 1-3 embryoid bodies per 10cm2 flask in adherent culture. The number of embryoid bodies is increased when the cells are grown under nonadherent culture conditions to 6-10 per 10cm2 flask. Rotating wall vessel bioreactors (Syntheeon Inc., Houston, TX), a form of nonadherent culture with the added elements of three-dimensionality and simulated microgravity, substantially induced embryoid body formation in BN/MSV cell culture, such that 99°/, of the culture material was embryoid bodies. Rotating wall vessel culture of BN/MSV cells provides a model system for studies of toxic teratogenesis and fetal loss.

C°ngress On In VitroBiology

Evidence of p53-Dependent Apoptosisin Two Mammalian Cancer Cell Types Treated with TaxoL SAC MAJUMDAR, D.A. Bills and C.A. Bachman. Department of Biology, Lafayette College, Easton, PA 18042. E-mail: . Apoptosis is known to require specificgenetic pathways to be initiated, and is characterizedby the cleavageof DNA into oligonucieosomalfragments,as well as chromatin condensationand nuclear disintegration.The tumor suppressor gene p53 has been shown to play a role in apoptotic activation. In this invesrigaron, enzyme immunoassays and morphological studies were performed to determine whether apoptosis is triggered in murinc erythroieukemiaceils(GM-86 cell line) treated with the anti-cancerdrug taxol. Once the mechanism of cell death was identified as apoptosis, the relative concentration of wild-typep53 activated fragment (XVAFl)in HeLa cellswas immunoassayed to determine if taxol is acting via a p53 dependent apoptotic pathway. Taxol is a diterpenoidchemicalproduct of the western yew tree and is known to be a potent anticancer drug. Exponentiallygrowing ceils were exposedto 0.1,0.5, 1, 5 or 10 v.g/mlof taxol for 24, 48, 72 and 96 hours. Cell Death Detection ELISA and Cellular DNA Fragmentation ELISA assays developed by Boehringer Mannheim showed evidenceof apoptotic death in GM-86 MEL cells especiallyafter 72 and 96 hours of taxol treatment. Taxol was also found to induce chromatincondensation,micronucleiand mukinudei formation. The wild-type p53 activated fragment ('WAFI) EL|SA purchased from Oncogene Science is a "sandwich"enzyme immunoassaythat employs both mouse monoclonaland rabbit polyclonalantibodies.This assayshowed that taxol caused significantlyhigher amounts of WAF1 expressionin HeLa cellsas comparedto untreatedcellsat 72 and 96 hours of treatment, suggesting the p53-dependentapoptotiecelldeath.

37-A

Abstracts

T-1006

T-1005 NewTherapeuticlmplicationsforTetracyciines: Their Role in Protecting Extracellular Matrix Breakdown Mediated by Tumor Cells. Y.GU, E.J. Roemer and S.R. Simon. Department of Biochemistry, State University of New York at Stony Brook, Stony Brook, NY 11794

Atorvastatin and a LongTlived Metabolite are more Myotoxic than Pravastatm in Primary Cultures of Neonatal Rat Muscle Cells. B.A. MASTERS, O.P. Flint, R.E. Gregg, and S.K. Durham. Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543.

Previously we used a complete interstitial extracellular matdx(ECM) to demonstrate that tumor call-mediated matrix degradation was inhibited by the zinc-chelating matrix metalloprotease(MMP) inhibitor 1,10-phenanthroline (OP), suggesting that MMPs contribute to the degradative activity of tumor cells. In this study, we show that 6demethyl,6-decxy,4-de[dimethylamino]tetracycline (CMT-3), a derivativedevoid of antimicrobial activity, can effectively inhibit MMPs released by two different cell lines (COLO 205 human colon carcinoma, and E-10transfected human breast carcinoma line derived from MDA-MB-231), thereby preventing tumor-cell mediated ECM degradation. At 10 pM, CMT-3 reduced levels of MMP-2 in COLO 205 serum-free conditioned medium (SFCM) by ~10% and levels of MMP-9 in E-10 SFCM by 45%. In the presence of 20 IJM CMT-3, levels of MMP-2 in COLO 205 SFCM were reduced by 65% and levels of MMP-9 in E-10 SFCM were reduced by 60%. In contrast, CMT-3 did not affect TIMP-1 levels in E-10 SFCM. As detected by gelatin zymography, enzyme activities of both MMPs were inhibited by CMT-3 as welI.COLO 205-mediated ECM degradation was inhibited ~40% in the presence of 20 pM CMT-3. From cytotoxicity assays using MTS (a tetrazolium salt), neither 10 nor 20 pM CMT-3 was toxic to E-10 cells. However, 20 pM CMT-3 promoted ~50% cell death in COLO 205 cells. Thus, we propose that this reduction in ECMdegradative activity reflects reduced levels of expression as well as enzymatic activity of MMPs released by cells in the presence of CMT-3. This reduction of MMP expression correlates with the cytotoxic effect of CMT-3 on COLO 205 cells, but not on E-10 cells. By its selective effect on MMP expression, CMT-3 diminished the MMP/TIMP ratio in E-10 cells from -2.2 to -1. Therefore, we conclude that CMT-3 is not cytotoxic to E-10 cells, but it caused reduction of both free MMP-9 levels and enzymatic activity. [NIH DE-10985]

Pravastatin and simvastatin, drugs which lower cholesterol by inhibiting HMG CoA reductase (HMGR/), have been associated with a low incidence of skeletal myopath), in humans and in rats. A new potent HMGRI, atorvastatm, induced myotoxicity in dogs. Previous studies have indicated that myotoxicity is directly related to the potency o f the HMGRI and the depletion o f mevalonate. Results from a recent in vivo study in the rat documented that atorvastatin is three-fold more potent an inhibitor o f HMG CoA reductase in muscle cens man pravastatin, and that at least two-fold higher concentrations o f atorvastatin can be measured in muscle cells. Atorvastatin is extensively metabolized in vivo to two long-lived principal metabolites, BMS-243887 and BMS-241423, which have some HMGRI activity. The objective o f this study was to compare the myotoxicity o f atorvastatin and its principle metabolites with pravastatin in cultures of primary rat skeletal myotubes. Endpoints measured following 48 hours exposure to the test article included indicators o f membrane damage (CPK, LDH, and AST), total cell protein, protein synthesis (3H-leucine incorporation), and energy status (ATP). Simvastatin was included as a reference standard because o f its known myotoxicity. Four o f the 5 compounds tested induced concentration-related reductions in ATP and protein synthesis (PS). Pravastatin (PS, IC50=452 [aM) was 20-75 fold less myotoxic than atorvastatin (PS, ICs0=l 1.7 laM), BMS-243887 (PS, IC5o=22.3/aM), or simvastatin (PS, ICs0 = 6.1 p.M). BMS241423 was not myotoxic. Total protein or enzyme leakage were not affected by any HMGRI tested. The results o f thxs study indicate that atorvastatin and one o f its principal metabolites, like simvastatin, are significantly more myotoxic than pravastatin.

T-1007

T-1008

Alteration of Intracellular pH in the Syrian Hamster Embryo Cells in culture with Phenobarbital. Y. Oshiro~, P. G. Gunasekar2, P. S. Balwierz~, D. L. Morris~and C. L. Alden~. 1 Product Safety Assessment, Seade, Skokie, IL 60077 and 2 Department of Pharmacology and Toxicology, Purdue University, West Lafayette, IN 47907. Phenobarbital, a non-genotoxic carcinogen, induces morphological transformation in the pH6.7 Syrian hamster embryo (SHE) cell transformation assay. The mechanism of morphological transformation in SHE cells upon exposure to non-genotoxic carcinogens has been associated with the promotional effects of the compound. However, the mechanism of transformation by non-genotoxic carcinogens has not been clearly understood. Maintenance of intracellular pH is critical for cellular processes. Basal metabolism, DNA and RNA synthesis, protein synthesis, cellular transport and fertilization are all highly pH dependent processes. We have expored the possibilityof intracellular pH perturbation in SHE cells by phenobarbital using a pH-sensitive fluorescent compound. The pH 6.7 SHE cell transformation assay was carried out following a published method. The intracellular pH of individual SHE cells was measured by utilizinga fluorescence ratio method with BCECF. We found that the intracellular pH was not altered by phenobarbital at 0.25 mM where cell transformation was not observed. On the contrary, the intrecellular pH was lowered by as much as 0.33 pH unit at phenobarbital concentration of 1.00 mM where cell transformation was observed. The intracellular pH change in SHE cells may alter the activities of some pH sensitive enzymes which may be involved or required in the process of morphological transformation.

Congress On In Vitro Biology

Effect of Small Halogenated Organics on Gap Junction Communication in Normal Rat Liver Cells (Clone 9). S.G. BENANE, C.F. Blackman, A.M. Richard, D.E. House, NHEERL, USEPA, Research Triangle Park, N.C. 27711-2055 Gap junction communication (GJC) plays an important role in the normal functioning of cells. Some reports suggest that GJC plays a role in suppression of tumor promotion, with GJC inhibition implicated in the etiology of carcinogenesis. In previous studies we have reported that some halogenated organic compounds inhibit GJC in Clone 9 cells, and we derived a quantitative structure-activity relationship (QSAR) to account for potency variation within the haloacid subclass. Our finding that a number of small halogenated organic molecules from diverse chemical classes inhibited GJC motivated us to test additional structurally related halogenated organics to further explore the structure-activity space associated with GJC inhibition. As in the previous studies, GJC was quantified using the scrape-load lucifer-yellow dye-transfer assay in a normal rat liver epithelial cell line (Clone 9) obtained from ATCC, subsequent to 24-hour chemical treatment. The following chemicals were tested in this study: 2,2,2-tribromoethanol (TBE), trichloroethylene (TCE), dibromoacetic acid (DBA), monochloromonobromoacetic acid (MCMB), 3-bromopropionic acid (BPA), 3chloropropionic acid (CPA), 1,3 dibromo-l-propene (DBP), 1,1dichloropropene (DCP), 1,1,3-trichloropropene (TCP), 2,2bis(chloromethyl)l,l-propene (CMP), methyl dichloroacetate (MDC), methyl trichloroacetate (MTC), and ethylene glycol monobutyl ether (EG). MCMB, BPA, and CPA did not influence GJC at concentrations below toxic levels. Experiments were done with other chemicals to determine GJC-50 values, the dose (mM) that would cause a 50% reduction in GJC. The relative GJC-50 values were: EG (10) > DCP (9) = CMP (9) = MDC (9) > TCE (6.5) > DBA (5) > MTC (3) > TBE (0.75) > TCP (0.03) > DBP (0.015). These results will be used to further probe relationships between chemical structure and GJC inhibition both within and across classes of small halogenated organics. This abstract does not reflect EPA policy.

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Abstracts

T-1009 Persistence of HIV Proviral DNA Sequences during Establishment of Immortalized B-Cell Cultures from Peripheral Blood. C.M. BEISWANGER, L.H. Toji, P.K. Bender, L Leonard, M. Manduka, J. Coccia, C. Libert, R. Overton, J. Baxter, A. Palmer, and I.C. Beck. Coriell Institute for Medical Research, and Cooper Hospital/University Medical Center, Camden, NJ08103. E-mail: Coriell Cell Repositories has tested over 5200 EBV immortalized B-cell cultures for HIV proviral DNA sequences using a PCR method. Although several studies have reported HIV production in immortalized B-cell lines, our assays did not identify any HIV-positive cultures among the CCR cell lines tested. It was not known whether our negative screening results were related to CCR transformation protocols or to the sampling of a select population in which the incidence of HIV infection is low. To assess the question of HIV in immortalized B-cell cultures directly, cultures were established from peripheral blood samples collected from HIV infected patients. Plasma HIV RNA load ranged from 103 to 2x103 copies/ml and CD4 counts from 13 to 844 cells/cmm. Duplicate cultures were established from each sample using Epstein-Barr virus and either phytohemagghitinin (a T-cell mitogen) or cyclosporin A (a T-ceil toxin) for transformation. Cellular composition of the cultures was assayed by flow cytometry at each passage using antibodies to CD3, CD4, and CD19. Population doubling levels were also tracked for each culture. Each sample was assayed for HIV proviral sequences using a PCR method. The results of this study indicate that HIV proviral sequences persist for some time in culture, irrespective of the mitogen employed for transformation. There appeared to be a trend toward decreasing HIV viral sequences with succeeding passages and continued screening is warranted. (Supported by NIH contract NO1GM4-2102)

T-1011 Effects of Smokeless Tobacco on Extracellular Matrix Production of R22 Rat Heart Smooth Muscle Cells In V#ro. DA. DABYDEEN, S.R. Simon, and E.J. Roemer. Dept. Of Pathology, SUNY at Stony Brook, Stony Brook, NY 11794-8691 Smokeless tobacco (ST) adversely affects oral mucosa, leading to gingival recession and mucosal lesions at sites of placement. This study examined the effects of ST extracts on the growth and biosynthetic capacity of R22 rat heart smooth muscle cells. Viability of confluent R22 cells cultured for eight days in the presence of ST extract was assessed by two markers: MTS, a tetrazoUum salt, and Alamar Blue, a redox indicator. Smokeless tobacco extracts were not markedly cytotoxic until concentrations in excess of 2% were reached. For selective radiolabeling of extracellular matrix (ECM) components, cells were grown in medium contain Lng one of two pairs of radiolabeled precursors: [3H]-fucose and [~ S]-methionine-cysteine (-rranco S-label), or [3 H]proline and [ S]-sulfate. Incorporation of precursors into ECM components was quantified by sequential enzymatic digestion. At subcytotoxic levels of chewing tobacco (1S1), dry snuff tobacco (1S2) and moist snuff tobacco (1S3) extracts, incorporation of radiolabels into ECM components was significantly inhibited, with an especially marked loss of collagenase sensitive matedal in the ECM of extract-exposed cells. Moreover, all three ST extracts inhibited incorporation of sulfate into glycosaminoglycans. In order to determine whether diminution of ECM collagen is due to failure to incorporate soluble collagen into insoluble ECM or failure to secrete soluble tropocollagen units, we examined supematant medium from R22 cells cultured in the absence or presence of ST extract. It appears that the decrease in ECM collagen is paralleled by a diminution in soluble proline-containing polypeptides. From these resutts, we conclude that loss of ECM collagen content after culture of confluent R22 stromal cells in the presence of smokeless tobacco products is not due solely to a failure to incorporate soluble collagen into the matrix. [NIH DE-10985; STRC #0610]

Congress On In Vitro Biology

T-1010 A Novel Human Hepatocyte Cell Line for Application in In Vitro Toxicological Assays to Prevent Animal Tests. H. Schulz, K. Tbxom, H. HI~IBNER and R. Buchholz, Depa.nment of Bioprocess Engineering, TU Berlin, 13353 Berlin, Seestr.13, E-mail: In Germany every year more than 1,2 million animals are killed for various scientific reasons. A big amount is taken by toxic test procedures of the drug industry to evaluate the toxicity of new products. Questions about the correctness of transferring results from animal to human and the problem of animal experiments in general are always discussed. Meanwhile there are a lot of In Vitro assays with different species of hepatocytes. Unfortunately these cells lose their biological function very fast or they are not completely differentiated. Behind this backround a new hepatocyte cell line was generated. The hepatocytes* derived from human were immortalized by manipulation of apoptoses genes. We want to introduce this new cell line as a high potential tool for In Vitro toxicological assays with a number of advantages in contrast to known cell lines. The cell line growes adherent in fibroblastic-like manner and shows all biological functions of detoxification. The so called phase I of detoxification is stable at least over a six-month-period of cell culturing and is easy to induce. Now there is the possibility of long time cell culturing to investigate slow-active substances, too. The phase II we have to investigate further. These hepatocytes are easy to handle and all experiments can be performed with identical cell material. *obligingly given by Prof. Straug, Max-Delbrack-Center Berlin. We would like to thank the Fond der Chemischen Industrie.

T-1012 Cell recovery / tissue model couple for in vitro toxicological assay of biomaterials. K. IMAI and M. NAKAMURA. Department of Biomaterials, Osaka Dental University. 81, Kuzuha hanazono-cho, Hirakata-City, Osaka 573-1121 .E-mail: We performed cytotoxicity tests used a tissue model (MATREX TM) which was an in vitro reconstructed cell culture system. This tissue model was exposed to seven kinds of dental monomers for 3, 10 or 30 minutes, then cells were rinsed and recovered for 48 hours with fresh medium. Quantitative assays for adenosine triphosphate (ATP) lactate dehydrogenase (LDH) and nitric oxide (NO) were performed to studying the possibility of application of this system to in vitro cytotoxicity testing. Bioluminescent assay of ATP and total NO concentration were used as biomarkers of cell recovery. ATP assay was useful to examine changes with high sensitivity at three minutes after treatment. Amounts of ATP were greatest with D-2.6E, followed by UDMA > Bis-GMA > TEGDMA > EDMA > HEMA > Bis-MPEPP in descending order. In the contrast, it took 30 minutes to obtain a comparable response in the LDH assay or by examining NO. This method might enable to examine early signs of celt injury, and thus may be a candidate for efficient endpoints.

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Abstracts

T-1013 Organotypic (Raft) Epithelial Culture System for Contact Dermatitis Testing. C. MEYERS. Dept. of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, PA 17033.. Email: . In response to ~environmental insult, keratinocytes can play a pivotal role in allergic contact dermatitis (ACD), as well as irritant contact dermatitis (ICD). We have developed a working hypothesis that an in vitro keratinocyte tissue culture system can be efficacious for assessing the capacity of industrial, cosmetic, environmental, and therapeutic products to induce ACD and ICD. A crucial component for testing our working hypothesis is the use of a proper in vitro keratinocytes system. An assortment of techniques have been tried by various investigators to culture epithelial cells. The organotypic (raft) culture system has been shown to most accurately mimic the in vivo physiology of the epidermis. Growing epithelial cells in the raft system has allowed for a complete differentiation program in contrast to what is achieved in monolayer cultures. We have evaluated compounds well characterized in their capacity to induce irritant contact dermatitis and allergic dermatitis for their ability to effect the expression of cytokines, chemokines, and adhesion molecules in full thickness human epidermis. Additionally, changes in the morphological and biochemical differentiation of the epithelium following treatment will be correlated with the expression of these immunoregulatory proteins. We are attempting to develop this human epithelial organotypic (raft) culture system as a mechanistically-based replacement for animal testing.

T-1014 Increased Acetaminophen Cytotoxicity to Human and Rat Hepatocytes by In Vitro Induction with Ethanol or Phenobarbital. +D.E. MENKING* *C.J. Cao, +R.J. Mioduszewski, A.T. Eldefrawi, +J.J. Valdes. *Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, MD 21201, +Research and Technology Directorate, U.S. Army ERDEC, Aberdeen Proving Ground MD 21010 E-mail: aeldefra @umarvlar}d.edu There have been a few incidents of severe liver damage and death from acetaminophen taken in conjunction with alcohol. The Cytosensor microphysiometer was used to examine the potential of synergistic effects between the two drugs in human and rat liver cell lines. This potentiometric biosensor monitors changes in cellular metabolism of glucose by measuring changes in the rates at which cells excrete acidic metabolites in response to activation or inhibition of celt surface receptors, enzymes or other cellular components. Excellent correlations were found between the IC50 values obtained for 10 drugs and those from in vitro fluorescence assay and [3H]thymidine uptake (Cao et al., 1997, Tox. in Vitro 11:285). Exposure of human hepatocytes to acetaminophen (2-40 mM) for 2-30 min produced reversible dose-dependent stimulation of metabolism (20 to 80%) but reduced it in rat hepatocytes. Higher concentrations and longer exposure inhibited it in both and caused cell death in a dose-dependent manner. Pre-incubation of hepatocytes for 3 days with ethanol or phenobarbital, enhanced the acetaminophen-induced metabolic changes, while N-acetylcysteine and glutathione blocked them. The data suggest induction of enzymes that bioactivate and increase acetaminophen toxicity.

T-1015 In Vitro Toxicology of Carmoisin and Amaranto on CHO Cells. J.M. POZUELO ~, D. Mufioz-Mingarro*, F. Llinares ~, T. Dominguez*, R. Arriazu *, A. Lopezz and A. SantaMaria 2. tUnivers.San Pablo CEU. P.Box 67 Boadilla del Monte 28660 Madrid. Spain. 2Inst.Salud Carlos III. Madrid. Spain. E-mail: An important aspect of cosmetic products is the determination of their capacity to produce adverse effects, such as irritation and toxicity. The Carrnoisin and Amaranto are two colorants widely used and currently marketed as component in cosmetics. This work was designed to investigate the toxic effects of these compounds on a cell line, chinese hamster ovary (CHO). The cells were treated with relevant concentrations of Carmoisin (0.05, 0.1, 0.15 and 0.2 mg/mL) and Amaranto (0.5, 1.0, 1.5 and 2 mg/mL) and incubated for 24 h at 37 °C. Two end pints were measured as markers of toxicity, MTT reduction and neutral red uptake (NR). Results of citotoxicity obtained of both test were compared and there was an excellent correlation between data. The most sensitive index of cytotoxicity was found in the MTI" test. CHO cells were more sensitive to Carmoisin than Amaranto, although morphological variations not were observed. The inhibitory effect of Carmoisin on the cell growth was more pronounced with increasing concentrations and the highest concentration used (0.2 mg/mL) indicated the lowest cell growth. We also evaluated the G-6PDH activity and the results show an increment of enzymatic activity with the increase of Carmoisin concentration, not so with the Amaranto. These results suggest that Carmoisin affect inhibiting cell growth and modifies the physiological functions of the pentose phosphate pathway.

Congress On In Vitro Biology

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Abstracts

1-1001

1-1002

Production of Selected Baculoviruses in Newly Established Cell Lines. C. L. GOODMAN, A. H. Mclntosh, G. N. El Sayed and B. Stiles. USDA, ARS, Biological Control of Insects Research Lab, 1503 S. Providence Rd., Columbia, MO 65203; American Cyanamid Co., Agricultural Research Div., P.O. Box 400, Princeton, NJ 08540. E-mail: , One key to the mass production of baculoviruses is the development of new insect cell lines capable of high production levels. Selected newly generated cell lines were evaluated for baculovirus production, with some of these data being reported here. The species of baculoviruses examined included: alfalfa looper virus (AcMNPV), velvetbean caterpillar virus (AgMNPV), bollworm virus (HzSNPV), and diamondback moth virus (PxMNPV). Extracellular virus titers (ECV) were determined using TCIDs0 assays and percent cells generating occlusion bodies (OB) was assessed. For AcMNPV production, of the 22 new cell lines tested, 3 lines produced moderate ECV titers (> 10~TCID~/ml) and 7 produced occlusion bodies. For AgMNPV, of the 5 new celt lines tested, 4 lines produced high ECV titers (> 107 TCID~o/ml) and all produced OBs. For HzSNPV of the 5 new cell lines tested, only one line produced moderate ECV levels and none generated OBs. For PxMNPV, of the 6 new cell lines tested, 4 lines generated high ECV levels and 5 produced OBs. Thus, numerous new celt lines were found to produce a variety ofbaculoviruses.

Production of Baculovirus by Microencapsulation. H. HOBNER and R. Buchholz. Technical University of Berlin, Bioprocess Engineering, Seestr. 13 Sekr. GG2, 13353 Berlin, Germany. Commercial baculovirus pesticide production had been performed in larval production systems mainly. Unfortunately viral pesticides do have a slow speed of action and it takes from 5 to 15 days postinfection to kill an insect pest. An obvious solution to this problem was genetic engineering of baculoviruses. The idea was to add a new gene to the virus that would allow the virus to kill insects quickly and to prevent them from feeding after infection. The addition and expression of pesticidal genes, especially neurotoxins, to baculovirus through is rather simple. Unfortunately virus production of these genetically engineered viruses can no longer be performed in larval production systems. These viruses have to be produced in vitro, because the cell cultures are not affected by the additional foreign gene product in contrast to the larvae. Although cell culture bioreactors have been improved it seems to be quite difficult to achieve and maintain high cell densities in vitro and there are many reports suggesting that high cell densities are incompatible with high polyhedra production rates. In contrast to that we can show that microencapsulation can be used to reach cell densities above 5x107 cells/mI. Further more the ceils maintain their full ability to produce polyhedra, yields over 10~ potyhedra/mi are possible. We thank the BMBF (BEO 22-0317717) for funding in part.

1-1004

1-1003

How sticky are developing insect midgut cells? Immunocytological studies with fibronectin and integrin ~i antibodies on developing Heliothis virescens midgut stem cells cultured in vitro. MJ LOEB and RS HAKIM. Insect Biocontrol Laboratory, USDA, Beltsville MD 20705; Dept of Anatomy, Howard University School of Medicine, Washington DC 20059. E-mail:

Role of p35 in Cellular Response to AcMNPV Inoculation. G.F. CAPUTO, S.R. Palli and S.S. Sohi, Great Lakes Forestry Centre, Canadian Forest Service, P.O. Box 490, 1219 Queen Street East, Sault Ste. Marie, ON P6A 5M7 Canada. E-Mail: [email protected] Recently, we reported that wild-type Autographa californica nucleopolyhedrovirus (wtAcMNPV) induces apoptosis-like death in CF-203 cells that is not blocked by p35, a suppressor of apoptosis (Palli et al., 1996, Virology 222:201-213). Subsequently, we found that modifiedAcMNPV (AcMNPVp35"), that lacks p35 gene, caused significantly less death in these cells. To study the role of p35 in cellular response to AcMNPV, we tested 10 cell lines for wtAcMNPV and AcMNPVp35- replication. Based on their response, the cell lines could be divided into three groups: (1) wtAcMNPV replicated well in SF-21, MD-108, CO-21 and CF-70 cells and produced occlusion bodies (OB) in 94, 88, 39 and 30% of the cells, respectively. On the other hand, AcMNPVp35°did not produce any OB in these four cell lines but induced apoptosis in 58, 83, 13 and 2 1 % of the cells, respectively. (2) wtAcMNPV induced apoptosis-like death in 100, 95 and 25 % of CF-203, CF-60 and CF-50 cells, respectively; but AcMNPVp35° induced death in only 13, 12 and 3 % of these cells, respectively. (3) Neither wtAcMNPV nor AcMNPVp35" replicated or induced apoptosis-like death in CF-124T, CF-2C1 and CF-27 ceils. Thus, AcMNPV produces hardly any effect (less than 1% infection or death) in some cell lines (e.g. CF-27), replicates well in other lines (e.g. SF-21), and induces apoptosis-like death in yet some other cell lines (e.g. CF-203). This data shows that lack of p35 results in the induction of apoptosis in permissive cell lines such as SF-21, but p35 is necessary for the induction of apoptosis-like death in nonpermissive lines such as CF-203. Supported by the National Biotechnology Strategy Fund and the Canadian Forest Service.

Congress On In Vitro Biology

Lepidopteran midgut stem cells, located at the bases of the existing epithelium in vivo, multiply just prior to the molt, and move into the existing monolayer of midgut epithelium, and differentiate there to mature columnar and goblet cells. The existing pattern in which goblet cells are surrounded by columnar cells is maintained and the midgut is enlarged to suit the growing larva. Fibronectin and its receptor, ~ integrin, indicate cell surface adhesive sites. Here we show most stem cells immunologically positive to fibronectin and integrin ~i subunit antibodies, but negative as they elongate. Mature columnar cells exhibit a network of positively staining material overall, with an apically positive concentration to which weakly active young cells appear to attach in vitro. Mature goblet cells stain more weakly than columnar cells; unstained goblet cells are seen attached to more intensely stained columnar cells. Thus a hierarchy of stickiness seems to exist which may influence placement of developing columnar and goblet cells in the gut integument.

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Abstracts

1-1005 DNA content in Manduca midgut cells in vitro and in vivo. RS HAKIM, MJ LOEB, and F.T. Hakim. Dept of Anatomy, Howard University School of Medicine, Washington DC 20059; Insect Biocontrol Laboratory, USDA, Beltsville MD 20705; Medicine Branch, National Cancer Institute, National Institutes of Health, Bethesda MD 20892.E-mail: When larval midgut stem cells grow and then differentiate in culture, both the size of the cell nuclei and their DNA content changes. The columnar cells have the largest DNA content, stem cells the smallest, as measured by fluorescent activated cell sorter analysis. In the current study, we provide further cell sorter analysis of the DNA content of the stem, goblet and columnar cells, as measured against the DNA content of Manduca sperm. We also present data from whole mounts of midgut prepared for Feulgen analysis, which shows some of the variation, within the columnar cells, in nuclear size, and shows qualitatively, size and distribution differences between columnar, goblet and stem cells.

Congress On In Vitro Biology

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Abstracts

P-1001

P-1002

A study of 4-Hydroxy-2-Nonenal And Malondialdehyde In Daucus Carota Callus Cultures of Different Ages and Morphogenetic Competencies. ~EE BENSON, IDH Bremner, IWJ Magill, IHJ Staines, 2/',1.Deighton, 2S Millam, ZUniversity of Aberta~: Dundee, Bell St. Dundee, Scotland, UK, DDI 1 H G ; Scottish Crop Research Institute, Invergowrie, Dundee, Scotland, UK, DD2 5DA. E-mail: [email protected]. Free radical-generated, aldehydic lipid peroxidation products have been associated with animal cell ageing *. This study, has profiled the formation2'3 of the lipid peroxidation products 4-Hydroxy-2-nonenal (HNE) and malondialdehyde (MDA) in D. carota cv Autumn King callus cultures at approximately 12 and 36 months after culture initiation. HNE levels were up to 10 times higher in the embryogenic, younger cultures compared to the aged cultures which had completely lost morphogenetic competence. A comparison was made between two different callus clones of Autumn King which had been initiated at the same time. One of these was embryogenic, whereas the other, had never shown morphogenetic capability. The embryogenic line had higher levels of HNE compared to the non-embryogenic line of the same age. Differences in MDA profiles were less obvious, however, ratios of HNE:MDA were, in general, higher for the embryogenic compared to the non-embryogenic clones. Ongoing studies are constructing HNE and MDA profiles for a wider genotype range and the antioxidant status of the cultures is being assessed. The objective of future research is to determine the role of lipid peroxidation in plant ageing and development. 'Zollner, H, Schauer, RJ, Esterbauer, H (1991) pp 337-369 in Oxidative Stress: Oxidants and antioxidants (ed. H Sies). Acad.Press Lon. 2Deighton,N, Magill, WJ, Bremner, DH, Benson, EE (1997) Free Rad.Res. 27 255-265, ~Bremner, DH, Magill, WJ, Deighton, N, Benson, EE (1997) Phyton, 37, 39-44.

P-1003

Effects Of 4-Hydroxv-2-Nonenal And Malondialdehyde On Dedifferentiated Plant Tissue Cultures. ~LK ADAMS, XDH Bremner, ~EE Benson, *HJ Staines, ZN. Deighton, "~S Millam, *Universityof Abertay Dundee, Bell St. Dundee, Scotland, UK, DDI 1HG; ~ Scottish Crop Research Institute, lnvergowrie, Dundee, Scotland, UK, DD2 5DA. E-mail: [email protected]. 4-Hydroxy-2-nonenal (HNE) and malondialdehyde (MDA) are lipid peroxidation products known to exhibit toxic activities in animal cells ~. Recently2'3 the authors reported, for the first time, the definitive identification and detection of MDA and HNE in plant cells. These studies show that embryogenic competence in Daucus carota may be associated with MDA and HNE profilesz'3. This investigation evaluated the effects of exogenously applied acetal derivatives of HNE and MDA on the growth of embryogenic and non-embryogenic callus of D. carota. Callus exposed to exogenously applied MDA and lINE (range 0.42-420 nM) showed a reduction (6 times less than controls) in cell proliferation. However, growth recovered after the aldehydes were removed from the media, demonstrating that the effects of HNE and MDA are reversible. Embryogenic and nonembryogenic cultures responded differently following aldehyde removal and recovery growth was 50% greater in nonembryogenic, compared to embryogenic cultures. Endogenous levels of MDA (uptu 501aM) and HNE (upto 0.2/aM) have been detected in D. carota cultures2'3. The finding that exogenously applied aldehydes influence plant growth may have important consequences for in vitro manipulations. ~Zollner, H, Schauer, RJ, Esterbauer, H (1991) pp 337-369 in Oxidative Stress: Oxidants and antioxidants (ed. H Sies). Acad.Press Lon. 2Deighton,N, Magill, WJ, Bremner, DH, Benson, EE (1997) Free Rad.Res. 27 255-265, 3Bremner, DH, Magill, WJ, Deighton, N, Benson, EE (1997) Phyton, 37, 39-44.

P-1004 Sweetening the Xylem to Produce More Cellulose. DAVE ELLIS, Bao Xue, Bob Gawley, Margarita Gilbert, Craig Newton, and Ben Sutlon. BC Research Inc. 3650 Wesbrook Mall, Vancouver, BC, Canada V6S 2L2. E-mail:

Over Expression of Pectin Methylesterase Does not Alter Cell Expansion in Transgenic Tobacco Cell Lines. M. TIZNADO, J. Gaffe and A.K. Handa.. Department of Horticulture, Purdue University, West Lafayette, IN. 47907-1165. E-mail: Pectin Methylesterase (PME), a ubiquitous plant enzyme, modifies the pectin chemistry of cell wall. We had characterized a PME gene (pmeul) that expresses throughout plant development (Gaffe et. ai., Plant Physiol. 114:1547, 1997) to determine the role of PME in plant cell growth. Transgenic tobacco cell lines expressing 13-glucuronidase gene (GUS) under the control of pmeul promoter (pmeul::GUS) show that pmeul promoter is more active in cells during expansion than early growth phases, suggesting a role for pmeul in cell expansion. However, maximum endogenous PME activity in control and pmeul::GUS cells are observed in the early log phase cells and declined thereafter. Differential behavior of the endogenous PME activity and GUS under the pmeul promoter could be due to expression of other PME isozymes. We over expressed pmeul under the control of CaMV 35S promoter in tobacco cells to determine if higher PME activity regulates cell expansion. Transgenic tobacco cell lines containing 5- and 10- fold higher PME activities compared to control cell lines gained fresh weight at a rate similar to control, but showed 5 to 10 % increase in dry weight during the early growth phases. Experiments are in progress to determine if this gain in dry weight is due to altered pectin chemistry of cell over expressing pmeul. Taken together, these results indicate that over expressing pmeul alone is not enough to alter ceil expansion. This research was supported by USDA/NRI 9437304-1110 grant.

Congress On In Vitro Biolog7

Cellulose, an unbranched chain of glucose residues derived from UDP-glucose, is the major economic component in trees. Interest in increasing or modifying cellulose microfibrils has been a target of numerous studies. Research with starch, also a chain of glucose residues, has demonstrated that levels of these po!ysaccharides can be manipulated in specific tissues. This was done by modifying the availability of ADP-glucose, a precursor to starch biosynthesis. Using a similar approach, we have introduced a bacterial UDPG-PPase gene into tobacco to increase the level of UDP-glucose, the precursor to cellulose. With ectopic expression of a bacterial UDPG-PPase gene using a CaMV 35S promoter, significant increases in height, biomass and cellulose were observed. Since our interest was in modifying cellulose content in the stem, a xylem-specific parsley 4CL promoter was used to direct the expression of the UDPG-PPase gene to the xylem. Like ectopic expression, tobacco transformed with this xylemspecific construct also had significant increases in height, biomass and cellulose. However, in contrast to ectopic expression where increases in biomass were distributed throughout the leaves, stem, and roots, significant increases in biomass with the 4CL promoter were confined to the stem. These data support the suggestion that carbon reallocation within the plant can be used to increase cellulose content. Current emphasis is the testing of this construct in trees.

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Abstracts

P-1005

P-1006 Bacterium-Stimulated High Frequency Somatic Embryogenesis in Geranium (Pelargonium x hortorum Bailey) Hypocotyl Cultures. B.N.S. Murthy and P.K. SAXENA. Department of Horticultural Science, University of Guelph, Guelph, Ontario, NIG 2WI, Canada.

Differential Display of Novel mRNAs in Shoot Organogenic Cultures of Loblolly Pine. YINGHUA HUANG, Yah Zhang, and C.G. Tauer. Department of Forestry, Oklahoma State University, Stillwater, OK 74078. E-mail: Plant organogenesis is a complex developmental process that involves specific changes in gent expression and cellular metabolism. During the organanogenesis process the developmental and biochemical events are coordinated by both the genetic program and extemal stimuli such as plant growth regulators and the culture environment. A shoot-forming system of loblolly pine has been developed for studying molecular mechanisms of this developmental process. In an effort to discover genes responsible for regulation of morphogenesis, mRNAs from organogenic cultures at various development stages were amplified by reverse transcription polymerase chain reaction (RT-PCR) and compared by differential display. Undifferentiated cells were included as a control. A large number of differential display fragments showed up, indicating many changes in transcriptional activities at different stages of organogenie development. We have identified, out of a total of around 25,000 eDNA bands, about 51 fragments where the expression of the corresponding genes seems to be associated with various developmental time points. Most of these cDNA fragments have been cloned and sequenced. The results indicate that the differential display technology is a powerful tool to study shoot organogenesis and other developmental processes. We will also report successful isolation and characterization of these differentially expressed genes.

P-1007

Co-cultivation of hypocotyl cultures of hybrid seed geranium (Pelargoniura X hortorum Bailey) with the bacterium (GSEPB; Bacillus circulans) remarkably promoted somatic embryo formation and improved both the frequency and quality of somatic embryos. This bacterium was isolated as an in vitro contaminant from cultures of geranium seedling explains. In the ev Ringo Rose, the least responsive among a large number of cuhivars screened on different regeneration media, the embryogenic response of hypocotyl explants was more than four times that of axenic cultures (control, without bacterial cocultivation). The highest number of somatic embryos was induced with bacterial concentration of 0.47 x 10~ cfu/ml. Bacterial stimuli was equally effective in inducing the somatic embryos on explants cultured on both solid and liquid media, under light and dark incubation. Induction of high frequency somatic embryogenesiswas not attainable using the spent medium. However, the addition of some phenolic compounds like apigenin, quecertin and kaemperon to culture medium supplementedwith thidiazuron (TDZ) and co-cultivation synergistically promoted embryogenesis. Among the various treatments tried (use of culture filtrate, autoclaving, sonication, sonication + filtration, HPLC fractions of bacterial inoculum) to evaluate the stimulatory effect using cell free extracts (to exclude live bacterial cells from the culture) only the use of a few selected HPLC fractions promoted embryogenesisto a certain extent. It was also observed that co-cultivation of the explants with bacterium during the first week was crucial for obtaining high frequency embryogenesis, indicating the role of bacterial stimuli in the somatic embryo induction process. The bacteria modulated endogenous growth regulator level of explants, including auxin and cytokinins probably to an optimum level resulting in improved somatic embryogenesis.

P-1008 A Novel Approach to Microspore Embryogenesis in Brassica napus L.: Too Much or Too Little Sugar? KATICA ILI~-GRUBOR ~, Stephen M. Attree 2, Larry C. Fowke 1. ~Department of Biology, University of Saskatchewan, Saskatoon, Saskatchewan, S7N 5E2 Canada. 2Pacific Biotechnologies Ine, 455 Gorge Road East, Victoria, BC V8T 2 W l Canada

Haploid embryos were induced from isolated microspores of Brassica napus L. cv. Topas cultured on high concentrations (22-

25% wt/vol) of polyethylene glycol (PEG 4000). PEG replaced sucrose as osmoticum in the attempt to uncouple osmotic from nutritional requirements in this embryogenic system. Sucrose in low concentration (0.1% wt/vol) was provided as a carbohydrate source in the medium. Within two weeks, PEG embryos reached torpedo stage and the embryo yield appeared comparable to the sucrose control (13% wt/vol). When placed under light, two-week-old PEG embryos quickly changed color from yellow to dark green and were strikingly similar to dissected immature zygotic embryos developed in ovulo (14-15 DPA) in their size, shape and internal structure. In contrast, large sucrose embryos slowly attained a pale green color, had small and sometimes aberrant cotyledons, elongated hypocotyls, and cells filled with abundant starch grains. During germination, the majority of sucrose embryos developed normal roots; however, hypocotyls became thick and swollen, bearing numerous secondary embryoids. These structures were not observed on elongated hypocotyls of PEG plantlets. Assessment of the subsequent growth of haploid plantlets is in process. With further improvement this new method might be suitable for other Brassica species and furthermore for microspore and anther culture of other Angiosperms, with a great potential application in breeding programs.

Congress On In Vitro Biolog 7

Plant Regeneration from Cell Suspension Cultures of Ins germanica cv. 'Skating Party'. Y. WANG and T. H. H. Chen. Department of Horticulture, Oregon State University, Corvallis, OR 97331, USA. Email: efficient plant regeneration protocol of Iris germanica cv. 'Skating Party' was explored through

An

using its cell suspension cultures. The suspension cultures were established from leaf bases and were maintained for four years by subculturing monthly. The effect of plant growth regulators (2,4dichlorophenoxyacetic acid and Kinetin), age and size of suspension culture aggregates on plant regeneration were examined. Our results showed that the regeneration potential of the cells was optimized when 6 week old cell aggregates of less than 520 p.m were treated with 5 p.M of 2,4-dichlorophenoxyacetic acid and 0.5 p.M of kinetin. This treatment allowed the reliable regeneration of 1000 to 8000 shoots from 1 gram screened cultures.

44-A

Abstracts

P-1009

P-1010

A Revised Protocol for Shoot Organogenesis from Watermelon Cotyledons. M.E. COMPTON, School of Agriculture, University of Wisconsin-Platteville, Platteville, WI 53818. E-mail: .

Applications of tissue and cell culture for seedless watermelon improvement. D.J. GRAY 1, M E Compton 2, N.J. Barnett 1 and RB. Carle 1. ~CFREC/IFAS, University of Florida, Leesburg, FL 34748; 2School of Agriculture, University of Wisconsin, Platteville, WI 53818. E-mail:

A revised protocol for shoot organogenesis from watermelon cotyledon explants was devised by comparing the major differences that exist among several published reports. The main factors that were examined included the light environment in which seedlings were grown before explant preparation and the region of the cotyledon used for explants. Seeds from four watermelon cultivars ('Crimson Sweet', 'Minilee', 'Sweet Gem' and 'Yellow Doll') were incubated for seven days in vitro either in darkness or 16 hr photoperiod. Four types of cotyledon explants were prepared from seedlings grown in both environments. Apical and basal halves were obtained by making a cut across the cotyledon width. Apical and basal quarters were made by cutting apical and basal halves longitudinally. All explants were incubated on shoot regeneration medium for six weeks followed by three weeks on shoot elongation medium. The percentage of explants with shoots was 1.7-fold greater for cotyledons derived from seedlings incubated in darkness than those germinated in light. Shoot formation was about 10-fold greater for explants from cotyledon basal halves and quarters than apical halves and quarters. Based on these results, optimum shoot regeneration frequencies could be expected when using explants derived from the cotyledon base of seedlings incubated in the dark for seven days before explant preparation.

Seedless watermelon plants are triploid F 1 hybrids, produced by fertilizing a tetraploid plant with diploid pollen. Triploid seeds for seedless watermelon are among the most costly of any crop due to problems with germplasm improvement and seed production. Tetraploid parents are usually produced from diploid seedlings by chemically-treating the plumule to induce chromosome doubling; however, chimeric plants are a common result and the few true breeding tetraploids obtained usually exhibit low self-fertility. Triploid seed production is costly, since tetraploid plants are hand-pollinated to prohibit selfing. These problems have been reduced by integration of two distinct in vitro regeneration systems (shoot organogenesis and axillary shoot proliferation) into seedless watermelon breeding. Organogenesis is used to generate nonchimeric tetraploid shoots at a higher frequency than the conventional method. Axillary shoot proliferation is utilized to maintain and increase selected tetraploids in culture and to rescue elite lines from the field. The in vitro systems also permit development and maintenance of self-sterile/cross fertile tetraploid lines, which can be used for open pollinated production of hybrid triploid seed.

P-1011

P-1012

Genetic Control of Regeneration in Immature Cotyledons of Melon Varies with Physical Microenvironment. Jeffrey Adelberg and Jin Feng Chen, Dept. of Horticulture, Clemson University, Clemson SC 29634. E-mail: Immature cotyledons of three inbred genotypes of melon, Cucumis melo L., and all hybrid cross-combinations, were placed on both agar and liquid/membrane system (MS with 10 gM BA) for 6 week regeneration treatment. Genotype and physical microenvironment had interactive effect on regeneration indicating genetic control was expressed differently on agar than the liquid/membrane system. In general, regeneration was better on agar medium with the exceptional response from the inbred 'Ogen', and its progeny. In the cross involving 'Ogen' and 'Rocky Ford Green', regeneration rate(%) was similar for both parental inbreds on agar, but greater for cotyledons from the hybrid. With the membrane, the hybrid had the same regeneration rate as 'Ogen', the superior inbred in that microenvironment. This effect was mirrored when counting numbers of buds per cotyledon. Regeneration is therefore heterotic for this cross on agar, but heterosis is not expressed in the membrane system. Cotyledons from hybrids of the inbred 'Ogen' with the inbred 'Green Ice~, in both reciprocal combinations were intermediate with respect to regeneration on agar medium (multigenic or co-dominant), but on the membrane system hybrids with 'Ogen' as maternal parent were similar to 'Ogen' but hybrids with 'Green Ice' as maternal parent had fewer buds then either parent (heterotic with cytoplasmic factors). In hybrids of 'Green Ice' and 'Rocky Ford Green', the more buds per cotyledon on agar medium was observed when 'Green Ice' was maternal parent than from either parent (heterotic with cytoplasmic factors). On the membrane system heterosis was negative when 'Green Ice' was maternal parent. These results imply a complex environmental component with cytoplasmic factors, affects the genetic control of regeneration. Furthermore, statements about superior or recalcitrant genotypes need to be made relative to physical microenvironment.

Congress On In Vitro Biology

Transformation of 'Bose' pear (Pyrus communis L.) with the rolC gene from Agrobacterium rhizogenes. R.SCORZA, R.L. Bell, and C. Srinivasan. USDAARS Appalachian Fruit Research Station, 45 Wiltshire Rd.,Keameysville, WV 25430. E-mail: [email protected] Tree size control is an objective in the genetic improvement of pear. Traditionally, manipulating tree size and vigor in established cultivars is achieved through the use of size controlling rootstocks. There are no completely satisfactory size controlling rootstocks for pear. Genetic transformation provides an approach to developing new size controlling rootstocks and also to directly alter the growth of the scion variety. We have developed a gene transfer system for pear. While the major objective is to transfer genes for fire blight (Erwinia amylovora) resistance into major pear cultivars, there also exists the possibility of manipulating tree form and vigor through the use of genes that affect plant growth, including rolC. Leaves of in vitro-grown 'Bose' pear were transformed with Agrobacterium tumefaciens EHA101 carrying a pGA482based plasmid containing NPTII, GUS, and rotC genes. Plants were regenerated under kanamycin selection. Four clones were isolated that were kanamycin resistant and GUS positive. PCR assays and DNA blots indicated the presence of the rolC gene in these clones. Each transgenie clone has been multiplied in vitro and planted in the greenhouse where growth is being evaluated in relation to untransformed controls.

45-./t

Abstracts

P-lO13

P-1014 The Use of Glufosinate as a Selective Agent in Transformation of Soybean. Z. ZHANG ~, A. Xing2, P. Staswick1 and T.E. Clemente2. ~Department of Agronomy and 2Center for Biotechnolgy, University of NebraskaLincoln, Lincoln, NE68588. EmaiI:.

Agrobacterium-Mediated Genetic Transformation of Western

Agrobacterium-Mediated

A routine soybean transformationprocedure has been developed using an Agrobacterium-cotyledonary node transformation system and the bar gene as selectable marker coupled with Glufosinateas a selectiveagent. Soybean cotyledonary explants were derived from 5 day old seedlings and co-cultivated with Agrobacterium tumefaciens strains for 3 days. Explants were cultured on Bs/B5medium supplementedwith 1.7 mg/l BAP and with Glufosinatelevels ranging from 3.3 rag/1 to 6.7 mg/l for 4 weeks. A~er 4 weeks explants were subcultured to MS/B5 medium supplementedwith 1 mg/1zeatin-riboside,0.5 mg/1GA3 and 0.1 mg/1 IAA amended with 1.6 mg/l to 2.0 rag/1 Glufosinate. Elongated shoots were rooted on a MS/B5 rooting medium supplemented with 0.5 mg/1 NAA without further selection. Plantlets were transplantedin soil and set seeds in the greenhouse. Primary transformants and their progeny were characterized by Southernblot analysis and leaf paint assay.

P-1015

Canadian Pea Genotypes. P.L. POLO'WICK, K. Mei, D.S. Baliski and J.D. Mahon. National Research Council Canada, Plant Biotechnology Institute, 110 Gymnasium Pl.,Saskatoon, SK, S7N 0W0, Canada. E-mail: Since 1995, researchers at the Plant Biotechnology Institute have been producing transgenic pea plants by inserting genes into the processing pea cultivar Greenfeast, using modifications of a method developed at CSIRO Australia (Schroeder eta/., 1993 Plant Physiology 101:751). In these transgenic plants, the new genes are functional and inherited. However, Greenfeast is not a useful pea for western Canadian farmers. Thus, an important question is: can transform cultivars that are more suitable, or is Greenfeast unique? Three sets of genetic constructs were used in attempts to transform 7 advanced breeding lines from 3 pea breeding programs. Each Agrobacterium strain had a reporter gene (uidA; GUS) as well as different chemical selection genes. Strain LBG71 contained a pat gene, which confers resistance to Lphosphinothricin (L-PPT). Strain LBG66 had a gus::npt//fusion gene that produced a single protein with both GUS reporter and kanarnycin resistance activities. Strain LBG75 contained a mutant acetolactate synthase (a/s3r) gene that provided resistance to chlorsulfuron. Transgenic plants were recovered from all genotypes tested and from each Agrobacterium construct used. A total of 39 independent transformation events was recorded, with the largest number from LBG75. Between 1 and 45 clones of each independent type were rooted, for a total of 323 plants. When the number of independent transgenic plants is calculated as a percentage of the pea seeds used to produce them, the average for the cultivar material is 0.6%, equal to that for Greenfeast. The average time, from co-cultivation to planting in soil of the first plant from each event, was 186 days. Segregation analysis of the T1 progeny from a number of the transgenic peas showed that the GUS reporter gene segregated according to normal Mendelian inheritance patterns. Also, the levels of GUS activity in the T1 generation were similar to those determined in the original transgenic plants. On the basis of these results, we are now confident that the PBI group can introduce foreign genes into a variety of pea types.

P-1016 Plant Regeneneratlon, and GUS-Gene Expression in Leaf Callus of Pigeonpea. K.SREENIVASU* P. Ananda Kumar and RP Shanna. NRC on Plant Biotechnology, IARI, Pusa,New Delhi, INDIA -I 10 012. *Present address NRC for Sorghum, Rajendranagar, Hyderabad (AP) INDIA- 500 030. E-mail: NRCSOR(][email protected] nic.in

Genetic modification of phenylpropanoid pathway of soybean and Astragalus plants. V. LOZOVAYA, HeeSook Song, Hyeon-Je Cho, V. Krasnyanskaya, Hong-Jae Park and J. Widholm. University of Illinois at UrbanaChampaign, ERML, 1201 W.Gregory Dr. Urbana IL 61801. E-mail:[email protected] Soybean and Astragalus plant genetic modification was performed in order to alter the cell wall phenolic composition aimed at understanding the biological importance of wall phenolics and enhancing plant disease resistance. We have chosen for transformation a gene that operates at the level of general phenylpropanoid metabolism (phenylalanine ammonia liase - PAL5), a gene that operate specifically at the lignin branch pathway (hydroxycinnamyl alcohol dehydrogenase - CAD1) and also a gene of the final polymerization step (laccase). For the transformation process we have used soybean embryogenic cultures, soybean and Astragalus sp. hairy roots. The genes have been placed in constructs with PAL2 promoter that should direct root and stem expression mainly and the CaMV35S promoter that should cause expression in all tissues of the plant. Agrobactedum rhizogenesmediated Astragalus sinicus and soybean transformation system was used for foreign genes expression with kanamycin resistance gene being used as a selectable marker and GUS as a reporter gene. In addition the particle bombardment of immature somatic embryos was performed to place phenolic metabolism genes into soybean and bombarded tissues were selected on hygromycincontaining medium. Kanamycin and hygromycin- resistant plant matedal was screened by GUS and PCR for the presence of foreign genes. PAL and CAD activities were estimated in transgenic and untransformed plants. It was found that PAL activity level varied in a considerable range in transgenic material, being at the level of control plants in many samples tested. PAL activity of other samples exceeded that of control significantly (3-9 times). Data on enzyme activity will be discussed in connection with cell wall phenolics alteration in transgenic plants. This work is supported by Illinois Soybean Program Operating Board, the North Central Soybean Research Program and Illinois Agdcultural Experiment Station.

CongressOn In Vitro Biology

Efficient plant regeneration via somatic embryogenesis has been developed fiom leaf and cotyledon explants of six genotypes of pigeonpea, which is a recalcitrant grain legume having high protein content. Leaf and cotyledon explants from 8-10 day old seedlings produced embryo genic callus and somatic embryos, when cuhured on Murashige & Skoog (MS) medium supplemented with growth regulator thidiazuron (TDZ). Subsequent withdrawal of hormone resulted in tile maturation and growth of embryos into plantlets on MS basal meditun. Histological analysis showed somatic embryos as well as shoot-buds originating from callus surface. The rooted plantlets were transferred and acclimatised in field, where. they showed normal morphological characters. The leaf discs of pusa-606 were co-cultivated with Agrobacter/um tumefaciens containing pTOK 233. Callus obtained after 8 weeks on the selection medium containing hygromycin showed GUS gene expression. Present protocol of plant regeneration and genetic transformation may be usefi, l in producing transgenic pigeonpea, resistant to major insect pest (Heliothis armigera) leading to crop improvement.

46-A

Abstracts

P-1017

P-1018

Transgenic papayas to the rid& new cultivars and new propagation methods. M. FITCH~, P. Moore 2, T. Leong2, and D. Gonsalves3, 1USDA,ARS, Aiea, Hawaii USA 96701, 2HARC, /de.a, Hawaii USA 96701, 3Comell University, Geneva, New York USA 14456. Email: [email protected] Transgenic Hawaiian papayas resistant to papaya ringspot virus (PRSV) will be available in the marketplace shortly. The cultivar SunUp, a homozygous line containing the coat protein gene of a mild Hawaiian strain of PRSV, and Rainbow, an F1 hybrid between SunUp and Kapoho, will be the first transgenic papayas released in the USA. Additional cultivars are currently being transformed with several virus resistance genes. Transgenic Kamiya containing either the translatable or untranslatable PRSV coat protein (CP) gene or the PRSV replicase (NIB) gene have been regenerated. Three lines were apparently immune or highly resistant to PRSV. Although more transgenic lines await inoculation tests, because of tetraploidy in the first three Kamiya resistant lines, we are also hybridizing and backcrossing the deregulated resistance gene from SunUp into Kamiya and other cultivars. Superior individuals will be backcrossed for a total of three rounds, resulting in virus resistant transgenic plants with 94% of the backcross cultivars' traits. In conjunction with field evaluations of the introgressed hybrids, we are comparing field plantings of micropropagated Rainbow plants with seedlings in order to determine if advantages other than preferred sex expression (hermaphrodite) will be gained by growing clonally propagated materials. If micropropagated plants can be produced economically we will be able to develop elite complex papaya hybrids containing the virus resistance gene(s).

Transformation and plant regeneration have been reported in papaya, Caricapapaya L.; both Agrobacterium and the particle gun have been used to produce transgenic plants. However, the efficiency of transformation reported is too low to allow the routine production of transgenic plants expressing desirable phenotypes, i.e., sense suppression of useful genes in papaya. Therefore, we have improved the efficiency and speed of transformation and regeneration significantly to allow production of large numbers of transgenic plants in a reliable manner. Embryogenic tissues were cocultivated for 2-3 days with A. tumefaciens disarmed strain C58 or LBA4404 carrying a binary vector containing either an ALS gene conferring resistance to chlorsulfuron or the NPTII gene conferring resistance to geneticin (G418) or kanamycin. After cocultivation and a recovery period, tissues were selected on chlorsulfuron, G418, or kanamycin and transferred to somatic embryo conversion media to produce completely transformed plants. Transformation has been confirmed by GUS assay, polymemse chain reaction (PCR), and by Southern hybridization. About 150 independent transformed lines can be obtained from a gram of embryogenic tissue inoculated with Agrobacterium. About 70 independently transformed plants have been transferred to the greenhouse to evaluate genetic stability. We are characterizing this population for possible culture-induced variation.

P-1019

P-1020

Improvement of transformation and regeneration in papaya. E. FIROOZABADY, Y. Moy, P. Oeller and N. Gutterson. DNA Plant Technology, 6701 San Pablo Ave., Oakland, CA 94608.

Evaluation of Plasmid Size and a Supervirulent Plasmid on Rice Shoot Tip Transformation. S.H. Park, B.-M Lee, C. Zapata, V. Choi, and R.H. SMITH. Department of Soil and Crop Sciences, Texas A&M University, College Station, TX 77843. E-mail: Oryza sativa cv. Jefferson is a commercially grown rice cultivar in Texas. A method to rapidly and efficiently transform rice cultivars without modification of the genotype has been established in this laboratory. However, it is desirable to increase transformation efficiency. This report examines the length of the foreign gene construct and a supervirulent plasmid, pAD1289, using the rice seedling shoot apex and Agrobacterium mediated gene transfer to increase transformation efficiency. Initial experiments using a 6.7 or 8.4 kb gene construct with the pat and Bt genes did not indicate a significant difference in transient gene expression (5.2 vs 8.3% shoot apex survival on selection medium). Reducing the gene construct to 4.3 kb with only the pat gene resulted in significantly higher levels of survival (25.2%). The addition of a supervirulent plasmid resulting in constituitive expression of the vir gene to the pat, Bt, 8.4 kb construct increased survival from 8.3 to 11.5%. Phenotype analysis of R1 progeny indicates that the primary transformed plants were chimeric. Progeny from 5 primary plants were examined. Three plants had a total of 10 tillers out of 45 containing seed which germinated on 3 mg/l glufosinateammonium. Data will be presented on both pat and Bt phenotype expression as well as molecular analysis.

CongressOn In VitroBiology

Chloroplast Transformation of Rice. V. RUDRASWAMY ~, S. Varmaz, H. Daniellz and N.A. Reichert ~. ~Department of Plant and Soil Sciences, Box 9555, Mississippi State, MS 39762; 2Department of Botany and Microbiology, 101 Life Sciences Building, Auburn University, Auburn, AL 368495407. E-mail: Availability of chloroplast (plastid) transformation protocols in an economically important crop like rice would be preferable over nuclear transformation due to the following: containment of transgene transfer via pollen (eg., the weed red rice is sexually compatible with commercial rice) and enhanced expression of the transgene due to thousands of copies of foreign genes being present per transgenic plant cell. A biolistic plastid transformation protocol has been developed for rice by utilizing the universal plastid transformation vector pSBL-ctV2 constructed at Auburn University (contains the aadA gene conferring resistance to spectinomycirgstreptomycin) in combination with selection/regeneration protocols using mature rice seeds. DNA delivery via the PDS-1000/He was optimized using nuclear expression vectors pAHC25 and pActFz. Targets included excised mature embryos and those present in intact rice seeds. The plastid transformation protocol was combined with transfer onto a cytokinin-rich Murashige and Skoog-based multiple shoot proliferation medium containing spectinomycin and streptomycin sulfate two days post-bombardment. Green shoots were selected successively under a series of selection cycles. Integration of foreign genes into chloroplast genomes was verified via PCR analyses using gene- and chloroplast genome-specific primers.

47-A

Abstracts

P-1021

P-1022 Production of Transgenic Oil Palm (Elaeis guineensis Jacq.) Via Microprojectile Bombardment. G.K.A. PARVEEZ1;, F. Tahir1, K. Harikrishnaz, S. Napis2, S.C. Cheah 1, P. Christou3. 1Plant Science & Biotechnology Unit, Palm Oil Research Institute of Malaysia, P.O. Box 10620, 50720 Kuala Lumpur, Malaysia; 2Dept. Of Biotechnology, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia; 3John lnnes Centre, Norwich NR4 7UH, United Kingdom. E-mail: < [email protected] >

Oil palm is an important economic crop of Malaysia and the highest oil yielding plant in the world. Modification of oil quality via genetic engineering is an important target for the country to remain competitive in the global oils and fats market. Gene transfer into oil palm remains the bottleneck for achieving this target. In this report we describe the successful regeneration of transgenic oil palm after bombardment of embryogenic callus cultures. Optimization of DNA delivery conditions (physical and biological parameters), selection of efficient promoters and determination of minimal inhibitory concentration of selection agents have been determined. Transformed calli were selected using the herbicide Basta and confirmed by molecular analysis (PCR and Southern blot hybridization). The transformed calli were successfully regenerated into whole plants. Molecular and protein analyses confirmed the transgenic status of the regenerated plants. Problems faced during this study and their subsequent solutions will be presented.

P-1023

Transformation of Recalcitrant Barley Cultivars: Improvement in Regenerability and Decreased Albinism. M.-J. CHO, W. Jiang and P.G. Lemaux. Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA. E-mail: . Albinism and loss of regenerability are two problems that occur during in vitro culture of most barley cultivars, especially during the prolonged periods necessary for transformation efforts. To minimize these problems, a reproducible system to maintain highly regenerative cultures over extended times was developed. Callus derived from immature scutellar tissues of three spring barley cultivars, Golden Promise (GP), Galena, and Harrington, was grown for 3 to 4 weeks on callus-induction medium containing 2.5 mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D) in combination with 6-benzylaminopurine (BAP; 0.01 or 0.1 mg/L) and 5.0 laM cupric sulfate. The addition of BAP and copper resulted in the induction of shiny, compact and slightly brown-colored callus with highly regenerative structures. When these structures were maintained on medium containing 0.1 mg/L BAP in combination with 2,4-D and copper and exposed to dim light, the resulting green regenerative tissues could be maintained for more than a year and still be triggered to form multiple green shoots without marked loss in regenerability or evidence of albinism. Using media containing 2,4-D, BAP and copper as an intermediate step between initiation/maintenanceand regeneration, multiple green shoots could be induced from transgenic and nontransgenic GP calli initiated on either 2,4-D or dicamba alone. Using the intermediate step, the frequency of shoot regeneration increased from 2.4 to 11.4 times for nontransgenic and transgenic calli of GP. The use of these improved culture and regeneration protocols resulted in successful transformation of previously recalcitrant, commercially important barley cultivars.

P-1024 Subcellular Targeting of Barley Hordein PromoteruidA Fusions in Transgenic Barley Seed. M.-J. Cho 1, C.D. HA2, B.B. Buchanan1 and P.G. Lemaux 1. 1Dept. of Plant and Microbial Biology and 2Dept. of Molecular and Cell Biology, Univ. of California, Berkeley, CA 94720, USA. E-mail: .

Barley (Hordeum vulgare L.) hordeins are major seed storage proteins that accumulate in protein bodies of the developing starchy endosperm. B-hordeins account for about 70 to 80% of the total hordein fraction and they are encoded by a multigene family comprising 10 to 20 members. In order to test the functionality in barley of the B l-hordein regulatory regions and the signals that direct subcellular localization of proteins in the endosperm, barley was transformed with two chimeric hordein-uidA (g-us) gene constructs with or without a 19 amino acid signal peptide sequence. Developmental and tissue-specific GUS expression was observed in T1 barley seeds transformed with both constructs as determined by histochemical and fluorometric assays. GUS expression from the two hordein-uidA constructs was very evident in developing endosperm, especially in peripheral cells of endosperm tissue and endosperm tissue near the scutellar side. Stronger GUS activity was detected in transformed endosperm tissue with the hordein-uidA gene construct containing the signal sequence than that without the signal sequence. GUS activity was not observed in nontransformed endosperm tissues. The uidA gene driven by the endosperm-specific B Ihordein promoters was stably inherited and expressed in T2 progeny of the transgenic lines. Analysis of the role of the hordein 19 amino acid NH2-terminal sequence on the subceUular localization of GUS expression using immunogold labeling is in progress.

Congress On In Vitro Biology

Fertile, Transgenic Oats Recovered Following Visual Selection of Cells Expressing the Green Fluorescent Protein Gene. H.F. KAEPPLER1, G.K. Menon1,A.M Nuutila2, and RW. Skadser~.l.Dept. of Agronomy, Univ. of Wisc., 2.USDA-ARS Cereal Crop Quality Res. Unit, Madison, Wl, 53706. E-mail: Alternative selectable marker genes are needed for cereal monocot transformation. The green fluorescent protein (gfp) gene has many advantages over currently available marker genes and shows promise as a marker for visual selection of transformed cultures. The objective of this research was to determine if fertile, transformed oat plants could be recovered following visual selection for gfp expressing cell lines. A modified gfp gene was delivered into friable, embryogenic oat tissue cultures via microprojectile bombardment. Gfp expression in bombarded tissues was monitored using a dissecting microscope equipped for fluorescence microscopy. Cells expressing gfp were manually selected at each subculture. To date, seven oat cell lines highly expressing gfp have been recovered following visual selection for gfp expression. Plants were regenerated from each of those lines. Fertile plants have been produced in all lines that have reached the flowering stage. Mature seeds were harvested for seedling analysis. PCR and Southern analyses confirm transgene integration in primary regenerants. Research is being conducted to determine if, and how many, chimeric plants are present among those regenerated. Similar selection experiments are underway in barley and maize.

48-A

Abstracts

P-1025

P-1026 Green Fluorescent Protein as a Reporter for Orchardgrass Leaf Transformation. H.A. RICHARDS and B.V. Conger, Dept. of Plant and Soil Sciences, University of Tennessee, Knoxville, TN 37901-1071. E-mail: [email protected]

The green fluorescent protein (GFP) gene is preferable to other reporters because the assay is nondestructive. This project examined the potential application of GFP as a reporter for orehardgrass transformation. Orehardgrass leaves were split in half along the midvein, the basal 22 mm cut transversely into 4 m m x 4 mm segments, and plated on SH medium without growth regulators. The leaf segments were transferred to SH medium containing 150 gl~ sucrose 4 h before bombardment. Tungsten particles, coated with plasmid DNA, were delivered via a particle inflow gun. The segments were assayed for GFP expression after 48 h. The number of cells expressing GFP were counted to determine the level of gene expression. Several gene and promoter combinations were compared including GFP and mGFP driven by the 35S promoter, GFP driven by the ubiquitin promoter, and sGFP driven by the actin promoter. The mGFP gene is a modified version for expression in Arabidopsis, and sGFP is a synthetic sequence for enhanced expression in maize. The GFP and mGFP genes did not produce detectable fluorescence while sGFP did. The sGFP gene was inserted into a vector containing the phosphinothriein resistance gene, bar. The psGFP-BAR construct was used in transformation experiments and transient sGFP expression was detected. The combination of sGFP and bar gives a nondestructive reporter and a seleetable marker for use in our transformation experiments.

Cold Storage of Mint Germplasm In Htro. BARBARA M. REED. U S D A / A R S National Clonal Germplasm Repository, 33447 Peoria Rd. Corvallis, O R 97333-2521 E-mail: [email protected] Storage time for in vitro cultures of three mint species and one cultivar was determined using three Murashige and Skoog (MS) medium (1962) nitrogen concentrations and four storage conditions. Cultures were stored in tissue-culture bags on MS with 25%, 50% and 100% of the standard nitrogen concentration (MS-N). Storage conditions were: 4°C and -1°C in darkness; 4°C with a 12 hr photoperiod Q0 u M m 2 s l ) ; and 25°C with a 16 hr photoperiod (25 ~Mm'%" 1). Culture condition was rated on a 0-5 scale with 0 indicating dead and 5 indicating dark green. All four genotypes stored at 25°C were in excellent condition at 6 months but declined to repropagation levels (2 or below) by 18 months. The condition of 25°C stored plantlets varied with genotype and MS-N. Ratings for all four genotypes on 50% MS-N at 4°C with light remained higher than 2 at 30 months but declined to below 2 by 36 months. Live cultures were retrieved from all four genotypes even after 54 months storage, but plants in most treatments were dead and the remaining plantlets were in very poor condition (rating means 0.03 to 0.7). Cultures stored at 4°C with a 12 hr photoperiod on 50% MS-N survived longer than those held under other conditions. We recommend storing mint cultures on MS medium with 50% MS-N at 4°C with a 12 hr photoperiod. This regime should provide a minimum storage of 24 to 36 months.

P-1028

P-1027 Developing Cryopreservation Strategies For Genebanks of VegetativelyPropagated Crop Plants ~DJ, DUMET, ~EE Benson, 2B Reed, 3K Harding, 3S Millam, 3RM Brennan, ~Universityof Abertay Dundee, Bell St. Dundee, Scotland, UK, DDI 1HG; USDA-ARS, National Clonal Germplasm Repository, 33447 Peoria Road, Corvallis, Oregon, OR 97333, USA; 3Scottish Crop Research Institute, lnvergowrie,Dundee, Scotland, UK, DD2 5DA. E-mail: [email protected]. Cryopreservation has an important application for the conservationof vegetatively propagated crops and advances in cryopreservation techniques are now enabling the transfer of cryo-conservation technologiesto genebanks. This study reports the current progress in developing cryopreservation methods for in vitro germplasm of the crop species Solanum tuberosum~ (potato) and Ribes nigrum2 (black currant). Cryopreservation, using encapsulation/dehydration, vitrification and controlled freezing methods has been applied to a range of potato and black currant cultivars and validation of conservation strategiesbetween genebanks is now in progress. Studies using DifferentialScanningCalorimetry(DSC) have an importantrole in the developmentof eryopreservationmethods based on vitrification protocols. Using thermal analysis it is possible to determinethe stability of glasses in cryopreserved shoot-tips of R. nigrum and Solanum spp. This is particularly important in the development of cryoprotectivedesiccationstrategies. Studiesof post-storage molecular stability have been performed on plants regenerated from cryopreserved shoot-tips of potato and, so far indicate that cryopreserved plant germplasm is genetically stable. IBenson, EE, Wilkinson, M, Todd, A, Ekuere, Lyon, J (1996) Cryo-Letts. 17, 119128.2Benson, EE Reed, BM, Brennan, RM Clacher, KA, Ross, DA (1996) Cryo-Letts. 17 347-362.

Congress On In Vitro Biology

In vitro Collection and Culture of Tropical Species of Piperaceae, Rubiaceae, and Melastomataceae. V.C. PENCE, CREW, Cincinnati Zoo and Botanical Garden, 3400 Vine Street, Cincinnati, Ohio, 45220, Together, the families Piperaceae, Rubiaceae and Melastomataceae include approximately 14,000 tropical species, including species used for medicines, beverages, condiments, dyes, lumber and horticulture. In vitro collection (IVC) techniques can be used to collect plant germplasm w h e n seeds are not available. In order to examine the applicability of IVC to these families, leaf tissue was collected from six Piperaceae, four Rubiaceae, and four Melastomataceae species from sites in Trinidad and Peru using IVC techniques. In some cases, flower and immature fruit tissues were collected as well. Tissues were collected onto MS m e d i u m with 0.5 m g / L BAP, 0.5 m g / L N A A and were subcultured to several media upon return to the lab. Shoot regeneration from leaf tissue occurred from all species of Piperaceae collected and from three of the four Rubiaceae species. Leaf tissue of Melastomataceae produced only callus under these conditions, but plants were obtained from immature seeds germinating in vitro. These results suggest that at least some Piperaceae and Rubiaceae species may regenerate readily from leaf tissue, but that species of Melastomataceae may be less responsive. Alternate tissues, such as immature seeds can be useful, if available at the time of collection.

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Abstracts

P-1029

P-1030

Walnut as Model to Study Developmental Processes: From the Available In Vitro Systems to Gene Cloning and Genetic Transformation. C. BRETON, P. Capelli, D. Cornu, I. Cou4e,J.P., Charpentier, E. Germain and C. Jay-Allemand.Institut National de la Recherche Agronomique, Station d'Amdioration des Arbres Forestiers,45160 Ardon, France.

Studies on Hyperhydration ofDianthus caryophyllus in an Acoustic Window Mist Reactor. M.J. CORRELL and P.J. Weathers. Biology/Biotechnology Dept., Worcester Polytechnic Institute, Worcester MA 01609.

Walnut trees (,Juglanssp.) produce high quality timber, valuable fruits and secondary products (oil, alcohol, dyes). However, vegetative and sexual propagation of these trees is difficult so that demand remains superior to supply. Different in vitro systemswere developedto produce plants or study developmental processes: (i) somatic embryogenesis, (ii) adventitious root formation from cotyledon fragmentsand (iii) micropropagation.The genetic origin of the material used is controlled (J. regia; J. nigra x J. regia). It was obtained from immature zygoticembryos,mature embryoaxis or axillary buds from selected adult trees. Improvementof medium and cultural conditions used for micropropagationresultedin multiplicationrates of 1.5-2.5 / 3 weeks and 25 to 90% rooting efflciencies.Subsequentradial growth of the stem can also be obtained in vitro under specific culture conditions. Cotyledon fragments were used to determine the origin of adventitious roots and study their formation through enzyme markers (Duroux et al., BiochemJ, in press) and cDNA cloning. Several embryonic lines have also been stabilized. Oue hybrid line was used for transformation. Six transgenie lines expressing antisense chs gene showed contrasted flavonoidcontents and rooting abilities (El Euch et al., Plant Mol Biol, in press). Interestingly,three J. regia lineswere obtained from "earlymature tree" phenotypes.Microshootspresentingputative terminal flower buds were observed after 7 subcultures. Walnut in vitro systemsare complementaryand easilyconnectedone to another. They provide good opportunities to study key processesin one recalcitrantwoody species: root development,cambiumactivityand wood formation,flowerinduction.

A major problem associated with in vitro tissue culture is the low survival rates o f micropropagated plants in vivo. For example, carnation (Dianthus caryophyllus L.) grown with high humidity and low light often becomes hyperhydrated. These hyperhydrated plants have an undeveloped cuticle, low chlorophyll content, fewer stomata, and appear glassy. The acoustic window mist reactor (AWMR) is an excellent system to study the causes o f hyperhydricity because all culture conditions, including gases and humidity, can be controlled. Dianthus is a good model to study how relative humidity, CO2, and other factors control different aspects o f hyperhydration. The duration and frequency o f mist cycles on Dianthus growth were investigated using the AWMR. Increasing the mist cycle from 2 to 10 min per hour increased hyperhydration from 5 to 68%. Concurrent decreases in chlorophyll occurred. Research is underway to study the effects o f hyperhydration on cuticle formation, stomata development, and chlorophyll content in

P-1031 In Vitro Clonal Propagation o f "AL-Belehi" Pomegranate (Punica granatum L.). Abdelrahman S. A. AL-Wasel. Dept o f Hort. and Forestry, College o f Agric. and Vet. Medicine, King Saud Univ., AL-Qassim, Saudi Arabia. E-mail: Q26F001 @SAKSU00 In vitro clonal propagation o f pomegranate (Punica granatum L.) cv AI-Belehi was achieved by culturing nodal segments from one-year-old plants grown in the greenhouse on three different Murashige and Skoog (MS) media (solid, liquid and double-phase). Solid MS medium alone or with 0.5 or 1.0 mg naphthaleneacetic acid (NAA) produced the highest number o f bursting buds and the longest shoots. Shoot multiplication was also achieved on MS medium supplemented with combinations o f BA (0.0, 0.5, 2, 4 mg/L) and NAA (0.0, 0.1, 0.5 m g / L ) o r T D Z ( 0 . 5 , 1.5,2.5,3.5, 4.5 mg/L) and NAA (0.0, 0.1 mg/L). Thehighest shoot proliferation rate occurred on auxin-free media containing 2 or 4 mg 6-benzyladenine (BA) (3.33 and 3.4 shoots/explant, respectively), while thidiazuron (TDZ), especially at high levels, resulted in a notable reduction in shoot number and shoot length. The optimal combination o f growth regulators was 0.5 mg BA and 0.1 mg NAA. Considerable adventitious rooting occurred on half strength MS salts. The highest rooting percentage (92%) was obtained with 0.5 mg NAA. Above 80% of the rooted shoots were transferred successfully to the soil.

Congress On In Vitro Biology

Dianthus.

P-1032 DED-Elicitor Induced Gene Expression in Cells from Disease-Tolerant and Disease-Susceptible American Elms. J.C. KAMALAY, S.M. Eshlta and D.W. Carey. USDA Forest Service, Delaware, OH 43015. Selected American elms (e.g., 'Valley Forge" and 'New Harmony') can tolerate artificial inoculations with the Dutch elm disease fungus Ophiostoma novo-ulmi which kill other American elms. We postulate that American elm DED tolerance is mediated at the cellular level. We have established cell suspension cultures from DED-tolerant and DED-susceptible elms to model the progression of DEn pathogenesis and to determine the cellular mechanisms which allow DED-tolerant selections to escape the disease. A complex elicitor was prepared from O. novo-ulmi according to the procedure described by Albersheim, et al., (1975) for a heat-released wall fraction. Since disease tolerance mechanisms are unknown, we initially conducted a broad-based search for tolerance markers including cellular bioassays, examination of putative defensive and signaling chemicals, and differential gene expression. Changes in cell growth were monitored dlrectly by light absorbance and indirectly by media conductivity, and cell viability was determined using fluorescein diacetate staining. Altered accumulation of putative defense response metabolites (t-cinnamic acid and p-coumaric acid) and defense signal metabolites (salicylic acid and benzoic acid) were monitored using HPLC. Changes in gene expression were monitored by examination of intracellular proteins and secreted proteins after SDS-PAGE and 2-D gel electrophoresis and by demonstrating alteration in messenger RNA populations by differential display of RT-PCR products. The investigation revealed differences in cellular and biochemical responses in the different American elm genotypes due to time in culture and some dramatic responses when the elicitor preparation was administered. A testable model for the disease tolerance response mechanism will be formulated based on the timing of elicitor-induced changes in both known defense reactions and those novel to DED-tolerant American elms.

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Abstracts

P-1033

P-1034

Incorporation of an Antifungal Protein Gene in Oat Varieties by Genetic Transformation. G.K.MENON, H.F.Kaeppler, R.W.Skadsen and A.M.Nuutila, Univ. of Wisconsin and USDAARS, Madison, WI-53706. Email:<¢[email protected]> Crown rust is a widespread and damaging disease of oat causing significant yield reduction. Improved resistance to crown rust in oats by traditional breeding has been racespecific and usually breaks down in time. Incorporation of genes encoding antifungal compounds, such as members of the thaumatin-like, PR-5 proteins, known as 'permatins,' may produce durable crown rust resistance. Our objective is to transform oat with a permatin gene and to evaluate the transformants for resistance to crown rust. Oat embryogenic cultures initiated from varieties GP-1, Dane and Belle were co-transformed with the permatin gene and a selectable marker gene, NPTII (Neomycin phosphotransferase) or GFP (Green Fluorescent Protein) using microprojectile bombardment. Forty four putative permatin transformants have been regenerated so far. Preliminary PCR data has identified two independent transformed lines, further evaluation of the transformants are being conducted using Southern and western blots. Evaluation of resistance to crown rust will be conducted by inoculating the transgenic plants with rust spores.

In vitro selection of grape for resistance to the anthracnose fungus, Elsinoe ampelina. S. JAYASANKAR ~, D. J. Graf, R. E. Litz2, ~CFREC/IFAS, University of Florida, Leesburg, FL 34748; 2TRECfIFAS, University of Florida, Homestead, FL 3303 t .Email:

The fungus, Elsinoe ampelina (de Bary) Shear. causes anthracnose or bird's eye spot disease on almost all aerial parts of grape including the ripe berries. This disease is a major factor that precludes the cultivation of Vitis vinifera varieties in the humid tropics. Because conventional breeding of grape is difficult and cumbersome, we attempted in vitro selection. Highly embryogenic grape cell suspension cultures of V.vinifera, 'Chardonnay', were selected repeatedly against fungal culture filtrate. Most of the culture material did not survive the exposure to culture filtrate; however, a few cells multiplied, which were established as separate culture lines and plants were regenerated through embryogenesis. Somatic embryos from selected cultures inhibited the causal fungus in a dual culture assay, whereas nonselected embryos did not. Similarly, spent medium from resistant embryos also inhibited the fungus suggesting the involvement of extracellular compounds. Analysis of spent medium revealed the appearance of new peptides and enhanced secretion of chitinase compared to medium from unselected cultures grown under similar conditions. Chitinases continued to be detected at least 7-8 months after withdrawal of selection pressure suggesting that the changes are not epigenetic. Resistance was further suggested by the unimpeded growth of selected mature somatic embryos in a medium containing 40% (v/v) culture filtrate, under which conditions, nonselected embryos succumbed within 3 days. We have regenerated more than 100 plants from each of three independent lines, which are being evaluated for resistance using in vitro assays as well as greenhouse studies. Our results indicate the potential of in vitro selection as a valuable tool for inducing resistance in perennial species.

P-1036

P-1035 Engineered resistance to tomato spotted wilt virus in peanut. Z.V. MAGBANUA l, J.K Roberts l, H.D. Wilde L, K. Moore 2, M.K.U. Chowdhury 3, H.Y. Wetzstein3, and W.A. Parrott t. ~Department of Crop and Soil Sciences, University of Georgia, Athens, GA 30602, 2Agratech Seeds Inc., PO Box 664, Ashburn, GA, 3Department of Horticulture, University of Geogia, Athens, GA 30602 E-mail: Repetitive somatic embryo cultures of peanut (Arachis hypogaea L.) cultivar AT120 were cotransformed via microprojectile bombardment. The first plasmid contained the antisense coding sequence of the nucleocapsid (N) gene from a peanut isolate of the tomato spotted wilt virus (TSWV), driven by a double CaMV 35S promoter. The second plasmid contained the GUS gene and the hph gene for hygromycin resistance, both driven by the CaMV 35S promoter. Embryos resistant to hygromycin were matured and converted into plants. Leaves of young plants were screened for the presence of the N gene using PCR. Forty percent of the hygromycin resistant lines also had the N gene, of which those from four lines were fertile. Southem blot analysis of these four lines revealed N gene insert numbers ranging from two to eleven. Northern blot hybridization analysis confirmed transcription in at least one line, which has 11 inserts, while no transcripts were detected in the other lines. Nuclear run-offs are being performed on these lines to help determine a possible mechanism of resistance. Although these plants have remained virus-free for a year, additional resistance tests are being rur them and their progeny.

Congress On In Vitro Biology

Engineering Crops for Durable Disease Resistance with an R Gene. X.-Y. Tang, G.B. Martin, and J.-M. ZHOU. Department of Plant Pathology, Kansas State University, Manhattan, KS 66506. E-mail: . The tomato disease resistance gene Pto naturally confers resistance to races of the Pseudomonas syringae pv tomato bacterium carrying the avrPto gene. However, several transgenic tomato lines that express Pto at higher levels showed racenonspecific resistance to P.s.tomato in the absence ofavrPto. When inoculated with a P.s.tomato strain without avrPto, these plants exhibited inhibited bacterium growth (up to two order of magnitude lower than in nontransgenic plants), and developed few disease lesions on the leaf. The Pto gene encodes a serine/threonine protein kinase and interacts with several proteins postulated to mediate various defense responses. These proteins include a second serine/threonine kinase, which play a role in the HR development, and three transcription factors that may function in coordinated regulation of PR gene expression. The elevated expression of the Pro kinase may have constitutively activated the downstream components. In fact, leaves of these transgenic plants developed mild spontaneous necrotic lesions at the microscopic level, and constitutive activation of SAR may be involved in the observed non-specific resistance. We are currently determining the efficacy of this resistance to other pathogens on tomato. We are also developing a genetic screening for Pto variants that activate defense gene expression without provoking cell death. The potentials of using the modified resistance gene in durable disease resistance will be discussed.

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Abstracts

P-1038

P-1037

The Influence of Fluridone on the Antioxidant Enzyme Signal Transduction Pathway. D.R. GOSSETT, B. Bellaire, S.W. Banks, M.C. Lucas and A. Manchandia Louisiana State University-Shreveport, Shreveport, LA 7A 115, E-mail:
Genetically Engineered Plants Show Improved Cd Tolerance and Accumulation. YONG LIANG ZHU, Elizabeth A.H. Pilon-Smits, Norman Terry. Dept. of Plant and Microbial Biology, University of California at Berkeley, CA 94720. E-mail: . Phytoremediation is the use of plants to cleanup polluted waters and soils. Our goal is to increase the efficiency of plants to phytoremediate heavy metals through phytoextraction, i.e. accumulation in plant biomass. The objective of this study was to develop transgenic plants with increased production of heavy metal binding peptides ( glutathione and phytochelatins ). The gene gshH encoding glutathione synthetase (GS) from E. coli was transformed to Brassicajuncea (Indian mustard), using Agrobacterium tumerfaciens. Transgenic line GS7 showed higher gshlI transcription level than line GS10. When grown on toxic levels of cadmium (Cd), GS7 grew better than wildtype; growth of GS10 was intermediate between GS7 and wildtype. Cd concentrations in GS7 shoots were significantly higher than in wildtype shoots; GS10 showed intermediate Cd concentrations. Biochemical analysis showed that the GS plants had higher concentrations of glutathione, phytochelatins and total thiols than wildtpe plants. It is concluded that overexpression ofgshlI increases the capacity of B. juncea to tolerate and accumulate Cd. These results show promise for heavy metal pbytoremediation.

Callus tissue from a NaCl-tolerant cell line was transferred to culture tubes containing 150 mM NaC1 and media containing 150 mM NaC1 + 0.2 gM fluridone. Following preincubation, the tubes were amended with NaCI to a final concentration of 250 mM NaCI, 0.1 mg/L ABA, 0.1 laM paraquat, or 0.01% H202. The callus tissue was harvested at 30 rain, 1 hr, 2 hr, 4 hr, and 8 hr intervals and analyzed for ascorbate reductase (AP) and glutathione reductase (GR) activity. All treatments except hydrogen peroxide and hydrogen peroxide plus fluridone induced significant increases in AP activity above the control activity levels within 1 hour after treatment. Pretreatment with fluridone failed to suppress the increase in AP activity in all treatments except when used with hydrogen peroxide. Except for fluridone and hydrogen peroxide plus fluridone, all treatments resulted in significant increases in GR activity above the control levels. In contrast to AP, pretreatment with fluridone suppressed or significantly delayed the increase in GR activity in all but the paraquat treated tissues. These data suggests that AP activity is rapidly induced by almost any stress metabolite completely independent of ABA, while stress-induced increases in GR activity are generally slower and partially, at least in the case of NaCl stress, associated with ABA concentrations.

P-1039 Assessments of Hydroxyl Free Radical Activity and Antioxidant Status in Freeze-recalcitrant and Freeze-sensitive Microalgae: Implications For The Crvopreservation Of Al~;al Culture Collections. ~EE BENSON, I'2RAFleck, *DH Bremner, 2jG Day *Universityof Abertay Dundee, Bell St. Dundee, Scotland, UK, DD1 1HG; 2Culture Collection of Algae and Protozoa, Institute of Freshwater Ecology, Far Sawrey, Ambleside,Cumbria, LA22 OLP, UK. E-mail: [email protected].

P-1040

The abilityof in vitro culturesof mieroalgaeto survivecryopreservation has important consequences for the ex situ conservation of this important group of organisms. Many microalgae are freeze-recalcitrant and to improve cryopreservation methodology it is essential to determinewhich factors in the cryogenicprocess predisposethe cells to stress. Superoxidedismutaseactivityand sulthydrylgroup content were found to increase in Haematococcuspluvialis and Euglena gracilis cells exposed to cryogenic treatments, indicating that both organisms are able to enhance their antioxidantstatus in response to cryoinjury. However, as E. gracilis was freeze-recalcitrant compared to H. pluvialis, the productionof hydroxylradicals(OH) by frozen/thawedE. gracilis cells, was also evaluated using a non-destructivefree radical detection methodk Highestlevels of OH activity were detected in cells exposed to -60°C, (the subzero temperature employed in the two-step cryopreservation protocol) and -196°C; 'OH production was greatest within the first 24h-48h recovery period. Application of the chelator desferrioxamine2 (which reduces OH production by inhibiting Fenton Chemistry)was found to enhancesurvivalin cryopreserved K gracilis. tBenson, EE and WithersLA (1987)Cryo-Letts. 8, 35-36; ZBenson,EE, Lynch, PT, Jones, J (1995) Plant Sci. 110,249-258.

Congress On In Vitro Biolog7

Biolistic-mediated Transformation and Regeneration of Soybean. L.L. MCDOUGALD ~, Y.H. Dan t'2 and N.A. Reichert I. 1Department of Plant and Soil Sciences, Box 9555, Mississippi State, MS 39762; 2Current address: Monsanto Corporation, 700 Chesterfield Parkway North, St. Louis, MO 63198. Email:

We previously developed a unique, genotype-independent organogenic regeneration protocol utilizing hypocotyl sections excised fxom seven day old soybean seedlings. The regeneration protocol was combined with an optimized biolistic-mediated transformation protocol using the PDS-1000/He apparatus. Bombardments were conducted on genotypes representing three maturity groups (IV - VI). Hypocotyl sections were bombarded with plasmid pAHC25 [contained chimeric [3-glucuronidase (GUS) and bar (phosphinothricin resistance) genes], delivering two shots at a helium pressure of 1350 psi. Transient and stable expression of GUS was determined histochemically. Tissues were placed on selection media containing 3.0 - 5.0 mg/l Ignite (a phosphinothricin-based herbicide). Putative transformants have been generated and are being analyzed for the presence of the bar gene via PCR analyses.

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Abstracts

P-1041

P-1042

In Vitro Culture Conditions for the Regeneration and Transformation of Lupins and Other Legume Species. J.E. BARTON, N.F. Fletcher, P.C. Smith*, C.A. Atkins* and J. Hamblin. Centre for Legumes in Mediterranean Agriculture (CLIMA) and Department of Botany (*), University of Western Australia. Nedlands, WA 6907 AUSTRALIA. E-mail: . Legumes are grown in rotation with cerealsin Western Australia. The narrow leafed lupin (Lupinus agustifolius)is the most important grain legume in the regionwith a millionhectaresunder cultivation.An Agrobacteriumbasedgene transfer system for these lupins has been successfully developed and introduced. Outcomesfrom this initiativeincludethe field testingof a number of transformedlupin lines and the developmentof gene constructs for use in the gene transfer program. The introduction of other legume speciesinto the cereal:lupin rotation such as chickpeas,peas, faba beans, white lupins, yellow lupins and lentilshas led to the modificationof the lupin gene transfersystem for the transformationof a range of legumespecies.In vitro culture procedures have been developed for each species. Shoots can be regenerated from the wounded excisedembryonicaxesof germinatedchickpea,pea, faba bean, white lupin, yellow lupin and lentil seeds, the morphologyof each embryonic axis determiningthe target tissuesused for transformationexperiments.Media used for the propagation of white lupin and lentil shoots have been modified in order to promote shoot growth and recoveryunder herbicideselection.Seed is recovered from rooted shoots in solution culture. Embryo rescue procedures have also been adopted to recover putative T1 transformants. The opportunities created by these protocols for the gene transfer program in Australia are discussed.

Using GFP as a Reporter Gene in Soybean. A.E. BRZOZOWSKI, T. Ponappa, and J.J. Finer, Department of Horticulture & Crop Science, Ohio State University/OARDC, Wooster, OH 44691 The jellyfish (Aequorea victoria) green fluorescent protein (GFP) provides a new tool to monitor gene expression in plants. Unlike the GUS histochemical assay, which results in tissue death, GFP expression can be observed over an extended period of time in living tissue following UV or blue light excitation. DNA constructions used in this study were modified to enhance solubility of the protein and/or modify its fluorescence properties. The objectives of this study were to identify GFP constructions suitable for soybean cultures, to evaluate transient GFP expression in soybean over time, and to examine the effect of prolonged blue light exposure on GFP activity. Embryogenic suspension cultures of soybean (Glycine max [L.] Merrill.) were bombarded (~ 10 clumps/bombardment) using several GFP gene constructions and expression was monitored under blue light. Transient expression was observed with all DNA constructions tested, but was greatest in quantity and intensity with a soluble modified red-shifted GFP (smRS-GFP). Expression of smRS-GFP was visible as early as 1.5 h (-135 spots/bombardment) following bombardment. Peak expression was seen at approximately 6.5 h (~600 spots/bombardment) before decreasing to less than 1.5 spots/clump 7 days after bombardment. Prolonged exposures of high intensity blue light did not alter the number of transient events. We are currently evaluating smRS-GFP as a reporter gene for stable transformation in soybean.

P-1044

P-1043 Developmentof an In Vitro Transformation System for Lupinus luteus H. LI, K. Hoffmann, S. Wylie, K. Ryan, L. Liu, M.G.K. Jones. State Agricultural Biotechnology Centre (SABC), Murdoch University, Perth 6150, Australia, and Centre for Legumes in Mediteranean Agriculture (CLIMA), University of Western Australia, Perth 6907, Australia. E-mail, Li Hua @ central.murdoch.edu.au Lupins are a major crop in Western Australia, grown on 2.4 million acres of sandy soils in r~edium to nigh rainfall areas in the wheatbelt region. The major species grown is narrow-leafed lupin (Lupinus angustifolius). A further 1 million acres of poorly drained silty soils with low pH and a high aluminium content (Wodjil soils) exist on the eastern side of the wheatbelt where narrow-leafed lupins grow poorly. However, yellow lupins (L. luteus) tolerate such soils and one cultivar 'Wodjil', was released to growers in the 1997 season. 'Wodjil' is resistant to cucumber mosaic cucumovirus but susceptible to bean yellow mosaic potyvirus (BYMV), an important virus in the region. As genes for resistance to BYMV have not been detected in the yellow lupin germplasm, genetic engineering was seen as a method for introducing such genes, as well as other domestication traits. An in vitro transformation and regeneration system was developed for this species. Genes for herbicide resistance (bar) and BYMV resistance (Nla gene from BYMV) were introduced via Agrobacterium to hypocotyl segments which were grown in vitro until the shoots were big enought to graft to nontransgenic lupin rootstocks. Approximately 10% of explants produced roots /n vitro upon treatment with IBA. Grafted or rooted explants were transferred to hydroponic medium in the glasshouse. Seeds were harvested and grown on in soil. Transgenic plants were analysed by a number of methods including PCR, Southern analysis, bromocresol purple assay and PAT assay. T4 generation plants were subjected to BYMV challenge through manual and aphid inoculation and ELISA and visual observation indicated that resistance or delayed virus accumulation occurred in two transgenic lines.

Congress On In Vitro Biology

Is GUS Really a Reliable Reporter Gene for Higher Plants-A Case Study in Pigeonpea CHARU SUDAN, Shivaprakash N. and Neera BhallaSatin. Plant Dev. Biol Lab. School of Life Sciences, Jawaharlal Nehru University,N.Delhi-110067, INDIA. E-Mail:Neera@j nuniv.emet .in.,[email protected] Pigeonpea (Cajanus cajan), is an important grain legume of the tropics and subtropics, which needs to be genetically engineered against biotic and abiotic stresses. GUS is the most commonly used reporter gene to monitor transformation events in plants. A putative GUS like activity was first observed in control untransformed explants of pigeonpea during transformation studies and was further confirmed in almost all tissues of pigeonpea using fluorescertse assays. The observed activity was truly enzymatic and non-microbial in origin. The rate of MUG cleavage was substrate dependent and a function of protein concentration in the assay. Linear, time dependent cleavage of MUG in presence of chloramphenicol, ruled out the presence of inducible GUS activity from microbial contaminants. Bacterial GUS was biochemically distinguishable from the endogenous enzyme. The endogenous GUS activity was resistant to 10raM SL (Saccharie acid 1-4 lactone, a specific inhibitor of bacteria[ GUS) and selectively suppressed by 10% ethanol. These differences offer a means to design a differential assay for transgenic GUS activity while suppressing the endogenous enzyme. In anthers the endogenous activity is temporally and spatially regulated, pH dependent and tolerant to 10mM SL. A cautious analysis of transformation events using GUS as a reporter gene in pigeonpea, is therefore required. The role of endogenous GUS and its substrate in pigeonpea needs to be investigated.

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Abstracts

P-1045

P-1046

Biolistic Transformation, Expression and Inheritance of Bovine B-casein in Soybean. P.J. MAUGHAN, R. Philip, M.J. Cho, LM. Widholm, and L.O. Vodkin. University of Illinois, Urbana, IL 61801. E-mail: 1-vodkin @uiuc.edu A 630 bp fragment encoding a 24 kD bovine milk protein 13casein was cloned into a seed specific lectin promoter expression cassette and introduced into soybean somatic embryos via particle bombardment. Four hygromycin resistant embryogenic cell lines were selected. A1] four lines were PCR and Southern blot positive for the 13-casein gene, however, only one line did not show extensive rearrangement of the 13-casein gene and/or lectin promoter. The presence of the 13-casein gene in the genome of the four plants regenerated from this culture was confirmed via Southern blot hybridization analysis. Gene copy experiments and progeny inheritance analysis indicate that the plants contain four to eight tandem copies of the 13-casein gene and that the insertion occurred at a single genetic locus. Bovine 13-casein mRNA was highly expressed in developing cotyledons. A very low level of 13-casein mRNA was found in leaf tissues although endogenous lectin transcripts are not found in the same samples. Bovine 13-casein protein was highly expressed in cotyledons of transformed plants, but was not detectable in leaf tissues of transformed plants by Western blot analysis. Bovine 13-casein produced in transgenic plants migrated as multiple bands very similar to pure bovine 13casein, with a main band at approximately 24 kD. This experiment represents the first report of the expression of a milk protein in soybean and opens the way for the general improvement of protein quality in soybean directed by seed specific promoters.

P-1047 Transforming Spring Barley Using the Enhanced Regeneration System and Microprojectile Bombardment. B.J. WEIR ~, S. Ganeshan 2, K.J. Lai ~, K. Caswell ~, B.G. Rossnagel ~, and R.N. ChibbarL ~National Research Council of Canada, Plant Biotechnology Institute, Saskatoon, SK, Canada, S7N 0W9, and 2Crop Development Centre, University of Saskatchewan, Saskatoon, SK, Canada, S7N 5A8 The enhanced regeneration system (ERS) is based on culturing isolated scutella derived from immature zygotic embryos of cereals. This system works with a wide variety of wheat and barley genotypes but at different efficiencies. To increase the efficiency of the ERS for barley, we examined the effect of preisolation storage and media composition on the induction of embryogenic callus and subsequent somatic embryo development for two barley genotypes. The two genotypes differed in their response to the above parameters, but both induced more embryogenic callus and shoots per calli on media containing maltose. The coupling of the ERS with micro-projectile bombardment resulted in several barley transformants. Isolated barley scutella were bombarded with gold particles separately coated with two gene constructs (pBARGUS and pRC62). Both gene constructs contain the scorable marker gus. In addition, pBARGUS contains the bar gene which confers resistance to glufosinate ammonium and in pRC62 the npt11 gene confers resistance to aminoglycosidic antibiotics (e.g. Geneticin). Molecular and histochemical assays were used to confirm the expression and integration of the inserted DNA into transformants and in advanced generations of two field-tested, transformed barley lines.

Congress On In Vitro Biolog7

High-Frequency Transformation of Oat Via Microprojectile Bombardment of Embryogenic Callus-derived Highly Regenerative Cultures. M.J. Cho, W. JIANG and P.G. Lemaux. Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA. E-mail: . A highly efficient and reproducible transformation system for oat (Avena sativa L. cv. GAF/Park) has been developed using microprojectile bombardment of highly regenerative cultures derived from seed-derived embryogenic callus. Callus was induced from mature seeds after completely removing shoots and roots; induction was on callus-induction medium containing 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzyl aminopurine (BAP) and high cupric sulfate under dim light conditions. Highly regenerative cultures were induced from embryogenic callus and used as a transformation target. From 327 individual explants bombarded with a 13-glucuronidase (uidA) gene and a hygromycin phosphotransferase (hpt) gene, 84 independent transgenic events were obtained using a 8- to 12-week selection period for hygromycin resistance; all the events were regenerable, giving a 26% transformation frequency. Co-expression of GUS activity was detected in 70% of the transgenic clones. Stable integration of the foreign genes in To and TI plants was confirmed by polymerase chain reaction (PCR) amplification and DNA blot hybridization. The fertility of the transgenic lines is being analyzed.

P-1048 Regeneration ofBiolistic-mediated Transgenic Maize from Scutellar Nodal Sections. V. RUDRASWAMY and N.A. Reichert. Department of Plant and Soil Sciences, Box 9555, Mississippi State, MS 39762. Email:

We previously developed a unique, genotype-independent organogenic regeneration protocol utilizing scutellar nodal sections excised from three day old maize seedlings. In transient assays, these explants were also determined competent to DNA delivery via the PDS-1000/He apparatus. For determination of stable transformation events, nodal sections were excised from two genotypes (Pioneer 3085 and 3156). Plasmid pAHC25 [contained chimeric [3-glucuronidase (GUS) and bar (phosphinothricin resistance) genes] was used in bombardments and delivered, after precipitation onto 1.6 ~tm tungsten microprojectiles, at a helium pressure of 650 psi. Postbombardment, nodal sections were placed on Murashige and Skoog-based selection medium 'A' supplemented with pchlorophenoxyacetic acid (4-CPA) plus 6-benzyladenine (BA) and 2.0 mg/l Ignite (a phosphinothricin-based herbicide). Putative transformants were selected and are being analyzed for the presence of the bar gene via PCR analyses.

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Abstracts

P-1049

P-1050

Transgenic Tropical Maize Plants with cryIAb, eryIAe and eryIB Genes for Insect Resistance

Wheat Genetic Engineering at CIMMYT hi. Bohorova. S. Fennell, S. McLean, L. Diaz, M.E. Ramos, M. Pacbeco, J.J. Olivares, D. Hoisington. CIMMY'r, Lisboa 27, Apdo Post-641, 06600 Mexico D.F., Mexico E-mail: [email protected]

N. Bohorova I, W. Zhang 1, S. McLean 1, R. Frutos2, M. Royer 2, M. Salgado l, B. Luna 1, R.M. Brito 1, L. Diaz 1, M.E, Ramos I , P. Estanol I , R.V. Ordonez 1, M. Pacheco I , C.R. Castillo 1, G. Vergara 1, I. Fonseca 1, M. A. Moreno 1, D. Hoisington I. 1 CIMMYT, Lisboa 27, Ap.Post. 6-641, 06600 Mexico D.F., Mexico., 2 BIOTROP/IGEPAM, CIRAD, 2477 Av. du Val de Montferrand, BP 5035, 34032 Monpellier Cedex l, France. E-mail: [email protected] Under the UNDP-sponsored project for developing insect resistance in tropical maize germplasm via genetic engineering we have developed the techniques to transform elite CIMMYT tropical inbreds. The group has identified CIMMYT maize inbred lines with the ability to regenerate whole plants through tissue culture and to stably integrate foreign genes. In order to study the genes which are most appropriate in the generation of transgenic plants we have screened the toxic activity of native isolates of Bacillus thuringiensis (Bt) as well as specific cryl proteins against four major maize pests: corn earworm (CEW), fall armyworm (FEW), southwestern corn borer (SWCB) and sugarcane borer (SCB). Based on the results, we collaborated with Dr. R. Frutos to synthesize a truncated crylB gene and have developed several crylB gene constructs. To generate transgenic plants we have established methods for biolistic bombardment, selection and regeneration of immature embryos and calli with plasmids containing crylAb; crylAc and crylB genes which are carried by different promoters. Transgenic plants resistant to the herbicide BasraTM contained the expected bands for the cry, bar and gus genes from Southern blot analyses. A simple leaf bioassay presented varying levels of resistance to Southwestern corn borer to transgenic plants with crylAc and crylB gene. Analyses of the progenies confirm the sexual transmission of the introduced genes and their stable expression. For Agrobacterium - mediated transformation the bacterial strains LBA4404 and EHAI05 containing plasmids with crylAb and crylB are being used for inoculation of immature embryos and calli from tropical maize inbreds and hybrids. Different parameters, such as co-cultivation conditions, sensitivity during the selection and regeneration are being investigated. The first transformants confirmed with PCR and Southern analysis provide evidence that Agrobacterium- mediated transformation of tropical hybrids is Oossible.

The techniques of plant genetic engineering applied to agriculture provide breeders with new opportunities to improve the efficiency of production and to increase the utility of agricultural crops. The main activities in the attempt to obtain transgenic wheat at CIMMYT are: l) to define the in vitro culture conditions necessary for regeneration of a range of bread and durum wheat germplasm; 2) to develop a more effective biolistic gene transfer system with selectable genes and to determine the optimal conditions for Agrobacterium transtbrmation; 3) to utilize the synergistic effect of Chitinase (Chi), l~-l,3Glucanase (Glu), and Ribosome inactivating proteins (RIP) using biolistic transtbrmation and to generate germplasm that posses effective resistance against a range of fungal pathogens; 4) to increase the level of expression of seed storage proteins in wheat endosperm (the unique bread-making characteristic of wheat flour) using a native high -molecular-weight glutenin subunit (HMW-GS) genes. Highly regenerable bread wheat genotypes - Attila, Kauz, Baviacorn, Luan, Opata, Bobwhite and durum wheat genotypes - Minimus, Ariza, Altar, Bajio were selected for transformation studies. Using the optimal microprojectile bombardment system transgenic bread wheat have been produced using marker/reporter gene constructs confirmed with PCR and Southern blot analyses. It has been observed that the transformed plants with a bar/gus gene construct do not express the integrated genes. However the analyses of marker gene expression in the progeny show non-Mendelian segregation. PCR confirm the presence of the Chi and the Glu genes in transgenic plants from bread wheat Attila, Baviacora and Bobwhite. Further Southern blot analyses are being carried out to the progenies of the plants obtained from the embryos cotransformed with anti-fungal gene constructs. Transient gus expression has been observed in the plants regenerated from the embryos of Baviacora, cotransformed with 1Axl and bar/gus gene constructs. Further SDS-PA gel electrophorase analyses are being carried out for the seeds. Agrobacterium transtbrmation experiments are being carried out with bread wheat genotypes using different bacterial strains and culture conditions, and transient gus expression is detected in most of the immature embryos and calli used.

P-1051

P-1052

Tissue Culture and Gene Transformation in Bahiagrass. M.F. GRANDO 1, R.. Shatters 1,2, Jr. and C.I. Franklin 3. I Dept. of Agronomy, University of Florida, P.O.Box 110300 Gainesvillle, FL 32611-0300.2USDA, ARS, P.O.Box 14565 Gainesville, FL 32604, 3 Dept. of Biology, Savannah State University, P.O.Box 20060, Savannah, GA 31404. Bahiagrass (Paspalum notatum Flugge) is an important warm season forage grass in ~ e southern U.S. and South America. We are developing a Biolistic TM gene introduction method for this grass to allow the use of biotechnology in the production of improved cultivars. Conditions for embryogenic callus (e-callus) induction and plant regeneration were optimized for use in these experiments. Comparison of explant tissues and media types showed that immature inflorescences were the most prolific in production of e-calli, with best results (50% of the inflorescenees producing e-callus) from the use of MS medium containing 10 p.M 2,4-D. However, initiation of e-ealli from germinating seeds was less labor intensive and MS medium containing Dieamba or low levels of 2,4-D, both with 5~aM 6-benzilaminopurine (BAP), produced the highest frequency of seeds that formed embryogenic tissues producing e-callus. From 9734 seeds 65.7% germinated and 21.4% of these produced e-calli. Callus induced from seeds in MS + 6.6 mg L -1 Dicamba + BAP possessed the best regeneration potential with 67% of calli producing shoots. Shoot formation was best when mature calli was transferred to MS + llaM BAP + ll.tM GA 3 with 1,640 shoots formed per gram fresh weight of callus tissue. Optimal root formation was obtained when shoots were transferred to SH medium. In order to produce transgenic bahiagrass with increased nutritional value, 5,900 e-callus pieces were bombarded with 1 gold particles coated with pAHC25 carrying bar and gus genes and another plasmid containing the vegetative storage protein (vsp gene) from soybean. Transient expression o f g u s reporter gene was observed in all sampled calli using a histological assay. Putative transformants from bombarded calli resistant to bialaphos are currently being tested.

Congress On In Vitro Biology

Genetic transformation of pineapple (Ananas comosus [L.] Merr.) via particle bombardment. G.-L. N a n and C. Nagai, Hawaii Agriculture Research Center, Aiea, HI 96701, E-maih A n efficient transformation system based on particle b o m b a r d m e n t has been developed using a phenotypically stable, yet transformable pineapple tissue culture, namely the protocorm-like body (plb). These plb cultures were initiated and maintained in liquid medium. Following bombardments with D N A constructs containing GUS/NPTII genes, transformed plbs were selected in the presence o f geneticin first in liquid medium followed by plant regeneration on agar-solidified medium. An average o f one transformation event per one to two bombarded plates o f plb was obtained. The transgenic plant lines contain G U S / N P T I I gene insertions at one to three loci, as determined by Southern hybridization. These transgenic pineapple lines have been multiplied and planted in a greenhouse for further evaluation. The established system is being used to transform pineapple with agronomically important genes, e.g., disease resistance.

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Abstracts

P-1053

P-1054 Transforming Embryogenic Cell lines of Gladiolus with either the bargusA Fusion Gene or Cotransformation. K. KAMO, *A. Blowers and 2D. McElroy. Floral & Nursery Plants Research Unit, U.S. National Arboretum, Beltsville, MD 20705. *Sanford Scientific, Inc., 2DeKalb Genetics, Corp. E-mail:.

Genetic transformation and regeneration of transgenic 'Stella de Oro' daylily. Jing-Tian Ling, Jing Huang and Roger Sauve. Cooperative Agricultural Research Program, Tennessee State Univ. Nashville, TN 37209 Suspension culture cells of Hemerocallis x 'Stella de Oro' were transformed with phosphinothricin acetyltransferase (PAT) gene under control of the CaMV 35S promoter via microprojectile bombardment. Selection of transformed calli were initiated 2-3 weeks after bombardment on a medium that contained 4 mg/1 phosphinothricin (PPT). Transgenic shoots were regenerated from PPT-resistant calli on a medium that contained 2 rag/1 thidiazuron (TDZ) and 0.01 mg/1 naphthaleneacetic acid (NAA), and rooted on a half-strength MS medium. Stable transformation was confirmed by detection of the PAT gene using Southern analysis. The development of an efficient transformation protocol for Hemerocallis will now allow the genetic improvement of Hemerocaltis flower longevity by genetit, eligincering teclmiques.

P-1055 Molecular Analysis of TO Plants Transformed by Agrobacterium and Comparison of Agrobacteriummediated Transformation with Bombardment Transformation in Maize. Z.-Y. ZHAO, W. Gu, T. Cai, L.A. Tagliani, D. Hondred, D. Bond, S. Krell, M. Rudert, W.B. Bruce and D.A. Pierce, Pioneer Hi-Bred Intl., Inc., Johnston, IA 50131. E-mail: [email protected]. Stably-transformed, genetically engineered plants and their progeny containing the bar and Gus genes were obtained through Agrobacterium-mediated T-DNA delivery in maize Hi-lI. Agrobacterium strain LBA4404 that carried the "super-binary vector" created by Japan Tobacco, Inc. was used in this study. Optimization of culture and transformation conditions resulted in a significant increase the transformation frequency to a range of 3367% in Hi-lI. TO plants generated from Agrobacterium delivery were analyzed by Southern and Gus expression assays where 58% of analyzed embryo-derived events showed low copy number and simple insertion pattern for both bar and Gus genes. In addition, more than one independent event can be recovered from a single embryo. The data further indicate that fewer than 10% of events contained backbone DNA sequences including antibiotic resistance genes of the super-binary vector. All of these events with backbone DNA sequences were in the multiple copy/complex insertion category. The data indicate no obvious relationship between Agrobacterium concentration (10s - 101° cfu/ml tested) and copy number/insertion pattern. Gus expression was also analyzed in TO plants. Analysis of transgenic events using Agrobacterium and particle gun DNA delivery indicate several important advantages of Agrobaeterium transformation over bombardment.

CongressOn In VitroBiolog7

Embryogenic cell lines of Gladiolus were bombarded with plasmid DNA containing either the bargusA fusion gene under the CaMV 35S promoter (pDM327) or cobombarded with gusA under the CaMV 35S promoter (pBCG) and bar under the CaMV 35S promoter (pDM307). Over 500 cell lines were isolated for either the fusion gene or co-bombarded cells following selection on Murashige and Skoog's medium supplemented with 2 mg/1 2,4-D and 6 mg/l phosphinothricin. The optimum DNA concentration for isolating stable transformants was one tenth that for optimum isolation of lines with gusA expression. Six times as many cell lines were isolated following co-bombardment as compared to bombardment with the bargusA fusion gene. In contrast, three times as many cell lines (72% of the cell lines) containing the bargusA fusion gene expressed gusA as compared to co-transformed cell lines (23%) following histological staining. GusA expression ceased after 6 months in culture for 4% of the cell lines containing the fusion gene and 5% of the co-transformed cell lines. Enhanced gusA expression was demonstrated using the bargusA fusion gene.

P-1056 Persistence of Agrobacterium following genetic transformation of Zea mays and Arabidopsis. D.T. TOMES, C. Mertensotto, J.P. Ranch, and G. Huffman. Grain and Agronomic Traits Departments, Pioneer Hi-bred Intl. Inc., Johnston, IA 50131 We have examined the persistence of Agrobacterium in two contrasting transformation systems, one which includes an intentional extensive period of callus culture counter-selection against Agrobacterium (corn), and a second in which transformation occurs without selection in immature flower buds exposed to Agrobacterium (Arabidospsis). Agrobacterium grows poorly on the plant tissue culture medium used for corn embryogenic callus production, and over 100 independent events growing as callus, and 150 transgenic plants before transfer to the greenhouse were PCR negative for Agrobacterium, and by inability to recover live cells. A high throughput 96 well format screening system was developed to test greenhouse grown transgenic plants of corn and Arabidopsis. This system could detect single Agrobacterium in macerated plant tissue of corn, and Arabidopsis. Agrobacterium was detected at a low frequency in treated Arabidopsis plant tissues including leaves and seed from primary treated plants. Trays underneath primary treated Arabidopsis plants also had detectable Agrobacterium at low frequency. However, 96 samples of transgenic T2 Arabidopsis seed did not show contamination by Agrobacterium. One plant of 94 tested corn plants in the greenhouse was Agrobacterium positive. Subsequent testing of this plant by PCR and extensive growth tests for Agrobacterium were all negative. We conclude that the frequency of Agrobacterium in transgenic corn and T2 seed in Arabidopsis is below the detection limit using standard microbiological testing methods.

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Abstracts

P-1057

P-1058 Abstract Withdrawn

Biolistic and Agrobacterium-mediated Transformation of Wheat and Sorghum. QINGLI-MI, 3eoung-Mee, S. Muthukrishnan and G. H. Liang. Kansas State University, Manhattan, KS 66506. E-mail: A rice chitinase gent (chill) was introduced into wheat (Bobwhite and Pavon 76) and sorghum (Tx 430 and C401) using a biolistic procedure. The gene encoding a thaumatin-like protein (TLP, an anti-fungal protein) was also incorporated into wheat using Agrobacterium tumefaciens (LBA 4404). Immature zygotic embryos were bombarded with tungsten particles coated with plasmid DNA containing the rice chitinase gene and the selectable marker bar. Transformed wheat and sorghum plants expressed the rice chitinase constitutively and most of their progeny segregated in a Mendelian fashion. Co-expression of the rice chitinase gene and the bar gene was observed. Expression of the rice chitinase gene was detected by Western blotting which detected a 35 kDa band expected for the rice chitinase. The presence of the chitinase transgene was confirmed by Southern blot analysis. However, the level of expression of the gene varied from plant to plant and silencing of the gene was occasionally encountered. To lower the copy number of the transgene and to stabilize its expression, an improved transformation protocol using Agrobacterium tumefaciens was developed and tested on sorghum and wheat. Calli derived from immature zygotic embryos were cocultivated in the presence of acetosyringone and the Agrobacterium carrying the vector pMON410 containing hpt or bar gene driven by the ubiquitin promoter and the chill or tip gene under the control of CaMV 35S promoter. Expression oftlp gene in transgenic wheat plants was confirmed by Western and Southern blot analysis.

P-1059 Effects of Sonication, Plasmid Size and a Supervirulent Plasmid on Transient Gent Expression in Cotton Shoot Tips Transformation. B.-M. Lee, S.H. Park, C. Zapata, V. Choi, and R.H. SMITH. Department of Soil and Crop Sciences, Texas A&M University, College Station, TX 77843. E-mail:

Gossypium hirsutum cv. Sphinx is a commercially grown Texas cotton cultivar. A method to rapidly and efficiently transform cotton cultivars without modification of the genotype has been established in this laboratory. However, it is desirable to increase transformation efficiency. This report examines sonication, the length of the foreign gent construct and a supervirulent plasmid, pAD1289, on transient expression of the cotton shoot tip on selection medium. Initial experiments using a 6.7 or 8.4 kb gene construct with the pat and Bt genes did show a difference in transient gene expression (1.7 vs 0.8%, shoot apex survival on selection medium, respectively). The addition of a supervirulent plasmid, pAD 1289, resulting in constituitive expression of the vir gent to the/vat, Bt, 8.4 kb construct increased survival from 0.8 to 9.4%. The combination of the supervirulent plasmid, pAD1289, and 0, 5, 10, 15, 30, and 60 see sonication resulted in 8, 21, 18, 15, 12, and 16% survival of shoot apices on selection medium with 2.5 mgO glufosinateammonium, respectively. Data will be presented on both p a t and Bt phenotype expression as well as molecular analysis.

CongressOn In VitroBiology

P-1060 Efficient Transformation of Commercial Strawberry Cnltlvars. C. BAKER, M. Lea, D. Engler and A. Morgan. DNA Plant Technology Corporation, 6701 San Pablo Ave., Oakland, CA 94608, E-mail: . The introduction of each gene of interest into strawberry requires a large number of primary transformants in order to obtain optimum copy number and expression (suppression or over-expression) of the gene; and for the transformant to be free of somaclonal variation. We have optimized our transformation system such that the transformation efficiency(total number of events produced/total number of explants) is at least 20%, and is 150% for some cultivars. A range of explant sources has been used (in vitro leaves and greenhouse-grown leaves, petioles, and runners) as well as a range of counterselection agents and selectiblemarker genes. Shoots can be regenerated with either BA or TDZ, but TDZ is more efficient for regeneration and normalization. Cotransformation ofexplants with two Agrobacteriacontaining different genes of interest will be discussed. Both single and double selection have been successfulfor the introduction of multiple genes, but frequenciesof cotransformation are low.

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Abstracts

P-1061

P-1062 Expression of Soluble Invertase in Transformed Carrot. YUAN-YEU YAU and Philipp W. Simon. Department of Horticulture, University of Wisconsin-Madison, 1575 Linden Drive, Madison, WI 53706. E-mail: .

Eleetroporation of Embryogenic Protoplasts of Sweet Orange and Regeneration of Transgenic Plants. R. P. NIEDZ and W. L. McKendree

U.S. Horticultural Research Laboratory, 2120 Camden Road, Orlando, FL 32803. E-mail: .

Electroporation conditions using a single discharge exponential decay waveform from a 960 pF capacitor were optimized for protoplasts isolated from an embryogenic cell line of sweet orange (Citrus sinensis (L.) Osbeck cv. Hamlin). Electric field strength (375-450 V/cm), vector DNA concentration (100 gg/ml ), carrier DNA concentration (100 og/ml),

electroporation buffer (pH 8), and protoplast heat shock (5 rain @ 45 ° C) were optimized using a plasmid pBI221 containing the GUS coding sequence under the control of the CaMV 35S promoter and measuring GUS activity 24 h after electroporation. All variables significantly improved electroporation efficiency and when combined resulted in 7,714 pmol 4-MU reinring protein4 compared to 8.7 pmol 4-MU minamg protein"t or an 886 fold increase in GUS activity over the control. Transgenic plants were regenerated when protoplasts were electroporated with plasmid pBI-505 that contained the GUS gene under the control ofa 35S-35S promoter containing 33 bp of the untranslated leader sequence from alfalfa mosaic virus. GUS ÷ protoplast-derived embryos were selected by staining with X-glue in a relatively nontoxic buffer. Stable integration of the transgene in these plants was confirmed by Southern blot analysis and GUS enzymatic assay.

The beta-fructofuranosidases (invertases) cleave sucrose and related sugars into fructose and glucose. This enzyme is present in most plant tissues in multiple forms. Carrots contain an insoluble invertase and soluble invertases. The Rs locus in carrot conditions the accumulation of sucrose, fructose, and glucose. Two different sizes of invertase isozyme II bands have been identified from Rs/and rs/rs plants based on genomic DNA PCR amplification, 4 kb and 6 kb respectively. The inbreds B493, B4367 and B7262Yc are homozygous for the recessive allele (rs/rs) and accumulate high levels of sucrose, while most carrots accumulate glucose and fructose (Rs/-). Increased consumer interest in sweeter carrots requires greater understanding of sucrose metabolism and its biochemistry. We established a gene transformation system for carrots using an Agrobacterium-mediated method. Intact soluble invertase isozyme I and isozyme II cDNAs were generated from B493 and B4367 via RT-PCR. The cDNA fragments were clot~ed into pBI121. B493 calli and tobacco plants were transformed by Agrobacterium tumefaciens LBA4404 containing the pB1121 vector driven by the 35S promoter. Transformed calli have been obtained and regenerated into plants. Invertase expression was compared in transformed and non-transformed rs/rs carrots. The expression of carrot invertase in other species (tobacco) will be studied. These results improve our understanding of the role of soluble invertase in sucrose metabolism of carrot.

P-1064

P-1063 Expression of the roIC Gene of Agrobacterium rhizogenes in a Regal P e l a r g o n i u m Reduces Plant Height, Leaf Area and Petal Area. M.R.BOASE, C.S. Winefield and N.K Borst. N e w Z e a l a n d Institute for Crop & Food Research Limited, Private Bag 4005, Levin, NewZealand. Email:. .

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The phenotypic effects of the roIC gene u n d e r the CAMV35S promoter w e r e a s s e s s e d for f o u r i n d e p e n d e n t transformants of a regal p e l a r g o n i u m , Pelargonium Xdomesticum cultivar 'Dubonnet'. After one y e a r of growth, r e p r e s e n t a t i v e control plants averaged 51 cm high, with leaves of 41 cm 2, and petals of 8.3 cm 2. In comparison, the smallest rolC plant was only 7cm high, with leaves 3.4 cm 2. The other three i n d e p e n d e n t rolC transformants were of intermediate height (34cm), w i t h leaves 13.5 cm 2 and petals of 5.0 c m 2. Results of Southern analyses to confirm the presence of the rolC transgene and northern analyses to confirm expression of rolC will be presented.

CongressOn In VitroBiology

Optimized Parameters in Agrobacterium-mediated and Biolistic Transformation of Kenaf. M.M. YOUNG and N.A. Reichert. Department of Plant and Soil Sciences, Box 9555, Mississippi State, MS 39762. Email: Kenaf (Hibiscus cannabinus L.) leaf section explants were utilized in plant transformations. In Agrobacterium tumefaciens-mediated transformation, explants of two cultivars were precultured, then inoculated with a disarmed strain of Agrobacterium tumefaciens containing chimeric [3-glucumnidase (GUS) and neomycin phosphotransferase II (NPT II) genes. Explants were placed on selection medium two days after coculture with GUS activity assayed histochemically seven days later. Explant preculture and inoculation times, along with the promoter controlling GUS influenced transformation efficiencies. GUS constructs containing the double CaMV 35S promoter yielded the greatest overall area of GUS-positive sectors on the leaf explants. The presence of acetosyringone could enhance transformation efficiencies, but was dependent on chimeric gene construct and cultivar. Enhanced wounding using the PDS-1000/He apparatus prior to inoculation decreased GUS activity. Leaf explants were also utilized in biolistic transformations using plasmids containing chimeric GUS and NPT II genes. Transient expression of GUS activity was assayed three days post-bombardment. A target distance of 7.5 cm and pressure of 900 psi yielded the greatest numbers of GUSpositive sectors. Other factors influencing GUS activity included gap distance, DNA amount and preculture time. Optimized parameters are currently being employed to obtain genetically transformed kenaf.

5&A

Abstracts

P-1065

P-1066

Transgenic Sweetpotato Plants Expressinga Synthetic Protein Gene Shows High Levelsof Native Proteins and Normal Yield in a Field Test. M. EGNIN, J. Jackson, A. McKenzie,M. Walker, J. Lewisand C.S. Prakash. Center for Plant Biotechnology Research, Tuskegee University,Tuskegee,AL 36088. E-mail . The goal of this study was to increase the nutritional quality of sweetpotato (Ipomoea batatas L.) protein by enhancing the profile of essentialamino acids. Five transgenic sweetpotato lines were developed using Agrobacterium tumefacienscontaining a synthetic asp-1 gene coding for a storage protein rich in essentialamino acids. The transgenicplants were tested by PCR, Southern, Northern, and Protein analyses. The transgenic plants demonstrated high levels of the essentialamino acids: ttyptophan, lysine, methionine, threonine, and isoleucine compared to the untransformed control. Interestingly, the transgenic lines also had a 2.5 to 5 fold increasein the total protein content in storage roots and leaves. In a SDS-Pagegel study, the increasedprotein content was due to the enhanced levels of native proteins especially sporamin, the dominant protein in sweetpotatostorage roots. The reasonsfor such increasein protein content remains to be investigated.The transgenic and control plants were field-tested in the Summer of 1997 to evaluate their growth and yield traits. Although three of the five transgenic lines grew slower than the nontransgenic control and produced fewer storage roots, two transgenic lines with the highest protein levels did not show any such yield reduction and had normal growth. Application of nitrogenous fertilizer did not make any significantdifferencein the yield of these two lines. Animal feedingstudies are now underway to test the in vivo nutritive value of the high-protein sweetpotato lines. Our studies show that it is possible to develop sweetpotato lines with substantial increasein the protein content without any yield penalty through biotechnology.

P- 1067

Abstract Withdrawn

Comparison of EGFP Expression in Tissue Culture and Transgenic Plants Using the Standard CaMV35S Promoter and a Duplicated-Enhancer CaMV35S Promoter Containing an AMV RNA4 Leader Sequence. R.G. SHATTERS, JR., Jan A. Sasser, and S.H. West. USDMARS, P.O.Box 14565 Gainesville, FL 32604 The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is an excellent, reporter molecule that can be detected in living cells without the need to kill the cells that are expressing it. Cells containing the molecule can be readily differentiated from non-GFP expressing cells by their fluorescence when excited with UV/bfue light. Mutant variations of the wild-type GFP (wtGFP) protein are available with altered fluorescent characteristics. One such mutant EGFP contains a mutation increasing fluorescence intensity approx. 35 times greater than wtGFP. It also contains multiple silent mutations to adjust codon usage preference for optimized human cell expression. Fortuitously, mutations in EGFP have also removed the criptic intron recognition/cleavage site that has been shown to reduce efficient wtGFP expression in plants. We are using the EGFP as a marker for selecting transgene expressing cells during the development of transgenic plants. Binary vectors derived from pBl 121 were constructed that contain EGFP under the promotion of either a standard CaMV35S promoter or a duplicateenhancer CaMV35S promoter containing an AMV RNA4 leader sequence. These vectors were used to compare expression levels of EGFP in transgenic, callus, embryos and regenerated alfalfa plants after standard Agrobacterium-mediated DNA transfer, and in bahiagrass callus culture bombarded with the constructs using the Biolistiestm procedures. High levels of EGFP fluorescence were observed in callus of both Agrobacterium infected alfalfa and bombarded bahiagrass. Results with transgenic alfalfa and comparison of EGFP expression using the different promoters will be presented.

P-1068 Systemic Acquired Resistance o f Carica Papaya against Phytophthora Palmivora. Y. JUDY ZHU ~, Susan Schenck i and Paul Moore2. 1Hawaii Agriculture Research Canter, Aiea, HI 98701, 2USDA, ARS, Aiea, HI 96701. E-mail:. Acquired resistance is an inducible defense mechanism exhibited by many plants that provides protection against a broad range o f pathogens. Systemic acquired resistance (SAR) has been induced by treatment with chemical substances such as salicylic acid (SA) and benzo(1,2,3)thiodizaole-7-carbothioic acid Smethyl ester (BTH) in both dicotyledenous and moncotyledonous plants. We are exploring the possibility o f using SA-or BTH-induced SAR as an alternative approach for control o f the root rot and fruit rot diseases caused by Phytophthora palmivora. Here we report that in tropical fruit papaya, Carica papaya, chemical treatments with SA and B T H can induce SAR against Phytophthora palmivora. Young papaya plants pretreated with 15 m M SA showed 35-50% fewer lesions and smaller infected areas two weeks aider inoculation with P. palmivora than similar plants without SA treatment. B T H increased papaya plant resistance to P. palmivora at concentration as low as 1.0 raM. B T H at this concentration exhibited slight toxicity to the papaya seedlings but gave complete protection against P. palmivora whereas control plants treated with water showed over 70% mortality 5 days after inoculation. Studies on the effectiveness o f a range o f concentrations o f B T H will be reported. Enzyme activities o f the pathogenesis-related proteins chitinase and 13-1,3-glucan~e increased more than six-fold following B T H treatment indicating that B T H is acting as a chemical inducer o f SAR in papaya.

CongressOn In VitroBiology

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Abstracts

P-1069 DNA Delivery into Sweet Potato Cells: Electroporation vs. Particle Bombardment. NEELY. SHAH*, A. S. Bhagsari, and S. K. Dhir, *Warner Robins High School, Warner Robins, GA 31088, Fort Valley State University, Fort Valley, GA 31030. E-mail: . For the sweet potato (lpomoea batatas L.) to reach its maximum agricultural potential, the crop must overcome barriers such as diseases and pests. New advances in recombinant DNA technology have made it possible to introduce genes containing valuable traits into the sweet potato genome through processes such as electroporation and particle bombardment. The objectives of the present study were to: (1) evaluate the effect of partial cell wall digestion on DNA delivery using electroporation, (2) determine the optimal explant for transient gene expression using particle bombardment and (3) compare the techniques of particle bombardment and electroporation for DNA transfer, pBI121 plasmid DNA (containing GUS and NPTII genes) was transferred by electroporation into young leaf-derived embryogenic callus. An enzyme solution containing 1.0% Cellulase, 0.5% Macerozyme, and 0.1% Pectolyase in 9MCPW was used for partial cell wall digestion from incubations of 1 to 30 minutes. The maximum number, on average 350 blue spots were observed (consisting of approx. 1-50 cells/spot) when tissue pieces (50 mg fw/sample) were incubated with the enzymes solution for 30 min followed by electroporation of a single electric pulse (500 V/cm, 150 ~tF) with EPR-buffered DNA. Next, the plasmid DNA was precipitated onto 1 I~m gold particles at an equimolar amount and bombarded onto leaves (whole and segmented) and petioles. After 48 hours, in all three explants tested, on average 300-400 blue spots were observed/bombardment. Based on these studies, electroporation and particle bombardment can both be successfully used to transfer DNA into sweet potato cells.

P-1071 Suppression of Microbial Contamination of cultures of Valencia Orange Buds Derived from field grown trees. M.G.BAUSHER and R.P. Niedz. U.S. Horticultural Research Laboratory, Orlando, F L 32803. Emall A serious impediment to the culture of plant material from field and greenhouse sources is the presence of residual microorganisms after disinfestation treatment. Antibiotics and fungicides have been used to suppress microbial growth in tissue cultures, but problems with specificity, phytotoxicity, selection for resistance, and high cost discourage their widespread use. An alternative is the use of bioeides which offer broad spectrum control o f bacteria and fungi. We describe a system combining the use of standard disinfestation techniques in combination with the use of industrial biocides to allow the aseptic culture of field grown plant materials. Standard disinfestation techniques were compared with and without the isothiazolone biocide PPM. With a standard disinfestation treatment, (sulfuric acid dip, followed by a sodium bicarbonate dip, 95 % ETOH dip, dilute Na hypochlorite soak containing Silwet ® and a sterile water rinse) 84% of the buds were contaminated within 30 days. Substituting hydrogen peroxide for Na hypochlorite increased the contamination rate to 100%. Dipping the buds in 0.2 % agar slurry containing 10-20 ml/L PPM and 5 ml/L in the culture media resulted in no apparent contamination after 30 days. and 5 mg/L in the culture media. Some phytotoxicity occurred at high concentrations of 20 ml/L PPM in the culture media.

Congress On In Vitro Biology

P-1070 The effect ot tissue culture conditions on auxin stability in culture media. Jhy-Jhu Lin, Jiu-Lin Xia, Jianqing Lan, & Nacyra Assad-Garcia. Life Technologies, Inc.(GIBCO BRL), R&D. Medical Center Dr. Rockville, MD 20854. Auxin stability in culture media is critical for understanding the mechanism of plant regeneration. Degradation of auxins in MS medium is related to pH, salt concentration, light exposure, medium sterilization and involvement of plant tissues or calli. Inconsistent results have been reported on auxin stability. In this study, we use capillary electrophoresis (CE) to analyze IAA in the MS medium which is reconstituted from 50X concentrated liquid MS medium. Both qualitative and quantitative analyses of various auxins such as CPA, IAA, NAA, 2,4-D, MeolAA, 5-CUAA were carried out. In a Magenta container, 50% IAA degradation was observed after 7 days at 25°C for solutions containing 2 mg/l of IAA in the MS medium under continuous light. In contrast, only 30% IAA loss was found after 3 weeks for the same solutions under a cycle of 16 h light and 8 h dark. The complete IAA degradation was not observed until 8 weeks. The auxin degradation is much more rapid when tobacco leaf discs are involved. About 50% IAA or NAA was lost after 1.5 weeks under the same cycle of 16 h light and 8 h dark. In addition, after examining various plastic or glass containers for light absorbency, a wide range of light absorbency (from 277 nm to 355 nm) was observed. Therefore, our results demonstrate that (1) the rapid degradation of auxins in tissue culture is primarily derived from the presence of plant tissues; (2) the slower IAA degradation than literature reports may be due to the difference of medium formulation, medium preparation, tissue culture containers, and incubation condition.

P-1072 Effect of Different Cytokinin Types and Concentrations on Micropropagution of Eastern Hibiscus. M.M. Jenderek, A. J.OLNEY 1 and Slawomir Kolosowski 2. ~Califomia State University Fresno, CA.93740, z Research Institute of Vegetable Crops, Skiemiewiee,PL. E-mail:[email protected] The influence of 3 various eytokinins (BA, 2iP and TDZ) on tissue culture propagation of H.syriacus vat. Aphrodite was investigated. Aseptically germinated seedling fragmems (root and hypocotyl) were cultured on McCown's woody plant basal salt medium supplemented with potassium nitrate (800mg/L), adenine sulfate (80mg/L) and MS vitamins containing one of the 3 eytokinins at 5 different levels (0.5, 1.0, 2.2, 4.4 and 10 ~t M). The explants were ineculated horizontally in petri dishes and kept in a growth chamber with 16 hr. photoperiod, at 26 *C. Callus was induced abundantly by all 3 eytokinins at any concentration investigated however the callus color varied. Explants grown on TDZ exmtaining media produced deep green callus whereas callus induced on BA and 2iP supplemented media was cream-beige with some white fragma~. The biggest and most numerous number of adventitious buds was formed on medium containing 0.5 and lp M TDZ. About 50 to 100 % of leaves initiated on BA media were yellow (with the highest % for media with > 2.2 ~ M ) a s compared to 0 to 50 % observed on explants eultored on 2iP media and 0 to 10 % recorded for media with 0.5 and lla M of TDZ. 2iP at exmomtmtions of 2.2 and 4.4 p. M promoted formation of small leaves originating from callus where 5 to 20 % of explants on media with 2.2, 4.4 and 101aM of BA developed short shoots without callus formation. However, at low rates, explants on BA containing media produced the highest shoot number for all cytokinins investigated, TDZ induced the most healthy and vigorous appearing leaves, shoots and adventitious buds and hence is a promising cytokinin in further improvement of hibiscus micropropagation.

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Abstracts

P-1073

P-1074

Long-Term Maintenance o f Date Palm in Vitro Cultures. J.M. AL-KI-IAYRI, and C.M. Bautista. Date Palm Research Center, King Faisal University, AI-Hassa 31982, Saudi Arabia.

A micropropagation System For Primula scotica: A rare And At Risk Sconish Species Endemic To Orkne..x EE BENSON, JE Danaher, I Pimbley, J Wake, S Daiey. University of Abertay Dundee, Bell St. Dundee, Scotland, UK, DDI 1HG; E-mail: [email protected].

A decline in the quality and quantity o f plant regeneration is often associated with a prolonged maintenance o f in vitro cultures. On regeneration medium, date palm (Phoenix dactylifera U ) cultures initially produce multiple shoots, but eventually they become brown and cease multiplication in spite o f subculturing onto a fresh medium. The objective o f this study was to prolong the regeneration capacity o f date palm in vitro cultures in order to maximize the number o f regenerants. The system used consists o f four types o f media differing in the concentration o f growth regulators and activated charcoal. In addition to 30 g L "l sucrose and 6 g L "l agar, MS medium was supplemented with (in mg L'l): A) 3 2-iP and I00 2,4-D, B) 30 2-iP and 10 NAA, C) 6 2-iP and 10 NAA, and D) 0.2 kinetin, 0.1 BAP, 0.1 2-iP, 0.1 NAA, 0.1 IAA, and 0.1 NOA. Activated charcoal was added, at 3 g L "l, to medium A and 1.5 g L t to media B and C. Medium C induced both embryogenesis and organogenesis. Organogenesis was enhanced and predominated by transferring the cultures to medium D. Maintaining the cultures on either medium C or D, however, eventually caused browning and regeneration was terminated. The cultures were rejuvenated and multiplication resumed by alternating the cultures between media D and C. This protocol successfully maintained regeneration o f date palm cultures for 3 years. Plantlets appeared phenotypically normal but genotypic variation, often associated with prolonged in vitro culturing, cannot be excluded.

Primula scotiea Hook. Is a rare Scottish plant endemic to the

P-1075

P-1076 Effects of Photoperiod and Alternating Temperature on

Regeneration of Epimedium grandiflorum "White Queen' from Rachis Explanu. S.M. AL-MATAR, M.M. Abo El-Nil, J.M. AlKhyri and F.H. Huang. Kuwait Institute for Scientific Research, BiotechnologyDept., P.O. Box 24885-Safat 13109, Kuwait. E-mail: . Epimedium grandiflorum was micropropagated from rachis explants. The explants were cultured on Murashige and Skoog (MS) basal salts medium supplementedwith 2 mgtL pyridoxine-HCL2 mglL nicotinicacid, 100 mglL myo-inositol, 0.40 mglL thiamine.HCL, and (wt/vol) 3% sucroseand 0.2% phytageI. The medium also contained 2,4-dichlorophenoxyaceticacid (2,4-D) at 0.01, 0.05, 0.10, 0.20, or 0.25 mg/L combined with either benzylaminopurine (BAP) or 2, isopentenyladenine (2ip) at 2.5, 5.0, or 10.0 mg/L. Cultureswere maintainedat a 16-h photoperiod (40 mmol/m2/sl) and 23 ± 2° C. Callogenesisincreasedsignificantlyas the 2,4-D concentration increased (0.01 to 0.25 rag/L). The highest number of shoots was obtained in treatmentscontaining0.25 mg/L 2,4-D combinedwith 2.5, 5.0, or 10.0 mg/L BAP, but the shoot number waS not significantly different among all the treatments tested.When 2ip was replacedby BAP,longershoots resulted(0.39 cm). Shootswere separatedand movedto elongationmediumcomposedof 0.1 mg/L 2,4-D and 5.0 mg/L BAP. To produce whole plants, shoots were separated and rooted on 1.0 g/l charcoal. Rachis providedan excellentsource of explant for Epimedium micropropagation and can be used for callus induction and shoot formation.

Congress On In Vitro Biology

Orkney Islands, The species is restricted to a very specific niche environment and changes in habitat usage have placed the species at risk. The mature plant is perennial, however, even in niches where P, scotica is endemic, severe winters enhance mortality in young plants and, as a result, the incidence of flowering plants can be low. All these factors have contributed to a decline in the natural populations of this species. This study reports the development of an in vitro micropropagation system for P. scotica which may have potential applications in assisting the conservation of this rare Scottish plant. Seedling cultures of P.scotica were initiated from seed surface-sterilised with sodium hypochlorite. Rapid shoot propagation was achieved by transfer of the germinated seedlings to Murashige and Skoog medium supplemented with BAP and 1AA. Proliferative capacity was genotype dependent and plantlet multiplication rates of 5-8 were achieved within a 4-6 week culture cycle. In exceptional cases, plant multiplication rates of >10 were possible for some genotypes. In vitro shoot cultures produced roots and plantlets which were able to survive transfer to a range of ex vitro growth substrates. Future studies will optimise ex vitro transfer strategies for this species.

Cryopreservation and Cold Hardiness o f In Vitro-Grown Pyn~s Mefistems YONGJIAN CHANG l and Barbara M. Reed2. ~Department of Horticulture, Oregon State University, Corvallis, OR 97331, E-mall: [email protected] 2USDA/ARS National Clonal Germplasm Repository, 33447 Peoria Rd. Corvallis, OR 97333-2521. Alternating (22°C 8 hr/-l°C 16 hr) or constant low temperature (4°C) pretreatment combined with an 8 hr photopefiod or darkness were tested for their effects on cryopreservation and cold hardiness o f two Pyrus species. Plantlets were pretreated for 0, 1, 3, 5, 8, 12, or 16 weeks then cryopreserved by slow freezing or tested for cold hardiness. Both alternating and constant low temperature pretreatments improved "the regrowth of P. cordata and P. pashia meristems following cryopreservation compared with growth-room cultured controls. The alternating temperature pretreatment with either an 8 hr photoperiod or darkness was significantly better than a constant low temperature treatment for both genotypes. Regrowth following cryopreservation increased with the duration of the pretreatment for the first 5 weeks. Regrowth reached 50-100% at the fi~h week with alternating temperature treatments, and 2042,5% with constant temperature pretreaments. Meristem regrowth was not influenced by photoperiod. Alternating temperature pretreatments also affected plant cold hardiness (CH). Plants pretreated with 10 wks alternating temperatures reached LT50 at -25°C; those with constant low temperature at -12.5°C; and controls at -10°C An alternating temperature pretreatment o f 5 to 16 wk duration was the most effective for inducing CH in tissue cultured pears. The optimum length ofpretreament varied with genotype.

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Abstracts

P-1077

P-1078

Hydrogel Encapsulation of African Violet Shoot Primofidia. M.H. RENFROE and D.T. Bolick. Dept. of Biology, James Madison University, Harrisonburg, VA 22807. E-mail: [email protected] Mass propagation and distribution of commercially-important plants which do not typically set seeds may be accomplished by encapsulation of shoot tips. Leaf explants of African violet were cultured on a shoot regeneration medium. Shoot primordia were excised from the axenic cultures and encapsulated in a hydrogel matrix consisting of 2 % sodium alginate eomplexed with t00 mM CaNe3. Encapsulated shoots were planted into test tubes containing sterile vermiculite wetted with a shoot growth medium. Emergence and growth of shoots was 100%. The plants rooted spontaneously and were transferred to potting soil and acclimatized. Subsequently, planting of capsules was attempted in non-sterile conditions. The treatments compared with the control group were (1) inclusion of carbendazim, a fungicide, into the alginate and (2) a treatment of alginate dissolved into one-half strength MS macronutrients instead of water. As a control, shoots were encapsulated in a matrix consisting of 2% sodium alginate complexed with 100raM CaNe3. Encapsulated shoots were planted into non-sterilized greenhouse flats filled with medium grade vermiculite. Shoots emerged and developed into propagules in all three groups. Encapsulation of shoot meristems into the alginate gel provided protection during handling and still allowed emerging leaves to break through. Propagation by encapsulation of shoot primordia could help reduce over-collection of plants from the wild and help preserve germplasm of rare and endangered plant species.

In v i t r o Multiplication of Lttchi chtnensJs Sonn. ( L i t c h i ) . DILIP KUMAR DAS, N.Shiva P r a k a s h and Neera B h a l l a - S a r i n , School of L if e Sciences, Jawaharlal Nehru U n i v e r s i t y , N e ~ Delhi-f10067, India. Litchi c h i n e n s i s Sonn. ( L i t c h i ) is a valuable h o r t i c u l t u r a l cash crop. It is a tropical nut fruit tree with limited seed v i a b i l i t y (ca.3-7 d a y s ) m d low 8 e r m i n a b i l i t y (24). As .Litchi is h i g h l y heterozygous, it is d i f f i c u l t to maintain the purity of the elite varieties. The conventional method of v e g e t a t i v e multiplication by a i r l a y e r i n g , c u r r e n t l y in vogue, is extremely slow and inefficient. Clonal propagation vi__.aa tissue culture might help overcome these problems. In the present investigation, the germination percentage of s e e d s was improved by giving a cold treatment (0 ° C) for four weeks. The s e e d l i n g s germinated u n d e r a s e p t i c conditions were used for the induction of multiple shoots from a x i l l a r y buds by i n planta treatment ~ i t h 6, benzylamino purine (BAP) o v e r a period of 30 d a y s . An a v e r a g e of ca. e i g h t shoot buds could be formed from each node. These shoots were f u r t h e r elongated and rooted on h a l f s t r e n g t h B5 meOlum supplemented with 0.I m8/l ot naphthalene a c e t i c a c i d (NAA). These p l a n t l e t s ace being hardened f o r t r a n s f e r to the s o i l . At t h i s stage no somaclonal v a r i a t i o n s have been detected and all plantlets are uniform in a r>pearance.

P-1080

P-1079

Mieropropagation of Salvia officinalis from different type of aseptic seedling explants. P. C.

Micropropagation of Centaurium erytraea from different type of aseptic seedling explants'. L. R.

Santos-Gomes and M FERNANDES-FERREIRA. Department of Biology, University of Minho, Largo do Pace, 4709 Braga; Codex, PORTUGAL

Amorim and M. FERNANDES-FERRE1RA Department of Biology, University of Minho, Largo do Pa~o, 4709 Braga, Codex, PORTUGAL

Salvia officmafis is used as an antiseptic and antiphiogistic agent in inflammations of oral cavity and gingivits. This species is included in a project* of agricultural production and transformation of medicinal plants for industrial purposes which involves clonal propagation and chemical characterization of active extracts. In vitro cultures of Salvia officinalis were established on MS medium containing 2% (W/V) sucrose, solidified with agar (0.8%) and supplemented with 2,4-D and one of the citokinins: KIN, BA or ZEA. Four types of primary explants from aseptic seedlings were tested: stem segments, leaves, root segments, and nodal shoot segments. Some of the cultures were maintained in dark conditions while others were submitted to a 16h day/lighi 8h dark photoperiod In both cases temperature was 24-25°C. Calli was the only type of culture induced from root segments independently of dark or light conditions used. Calli and shoots were induced from stem segments at percentages depending on light regime and phytohormone supplementation. Nodal shoot segments were the most effective for direct shoot regeneration through organogenesis. Rhizogenesis was induced when the regenerated shoots were transferred to MS medium devoid of phytohormones.

Centaurium erytraea is a medicinal plant used by their healing, tonic and febrifuge properties. The establishment of m v i v o cultures of this species by sowing in Nature has proved difficult with low seed germination rates*. We started in vitro cultures in aseptic environment from sterilised seeds and we obtained germination rates of 100%. The seedlings were used as source of primary explants for establishment of in vitro cultures. Four different type of primary explants were used: entire leaves, proximal segment of leaves, middle segment of leaves, and root segments. The explants were laid on LS or MS medium containing 2% sucrose, solidified with 0.8% agar and supplemented with IAA or IBA and K1N or BA. About 50% of the cultures were maintained in dark and 50% with photoperiod of 16h day/light and 8h dark Shoot organogenesis occurred from all type of primary explants tested, at different rates depending on the basal medium, hormonal supplementation and light regime used. Within the range of essays performed, the highest rates of shoot induction were obtained from entire leaves cultured on LS medium supplemented with IAA and KIN. Induction rates higher than 50 shoots per primary explant were obtained in these conditions. * Project PLANTAMEDI, P-1304, supported by PRAXIS XXI/Adl

* PLANTAMEDI, P-1304, supported by PKAXIS XX/Adl

Congress On In Vitro Biology

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Abstracts

P-1081

P-1082 Evaluation of promoters for biolistic transformation of embryogenic P#rus strobus. A_M. DINER1, A. Zipf~ and R.Ward z, ~U.S.D.A. Forest Service, ~Dept. of Plant and Soil Science, Alabama A&M University, Normal, AL 35762. E-mail:.

Analysis of tobacco anthranilate synthase gene promoter using Agrobacterium rhizogenes-mediated Astragalus sinicus transformation system. H.-J. CHO*, H.-S. Song, J.E. Brotherton and J.M. Wclholm. Department of Crop Sciences, University of Illinois, Urbana, IL 61801. Email:

Anthranilate synthase [AS] is a key enzyme in the tryptophan biosynthetic pathway that catalyzes the first reaction in the pathway which synthesizes tryptophan from chorismate. AS is feedback inhibited by the end product, tryptophan. Tryptophan analogs, such as 5-methyltryptophan, have been used to select resistant plant cell cultures that usually have increased free tryptophan levels due to the presence of a feedback-insensitive AS. In previous unpublished work, we found a wild type (unseiected) tobacco (Nicotiana tabacum) cell line (AB-15-12-1) which was resistant to 5-methyltryptophan and contained feedback-insensitive AS that was expressed in the cultured cells but not in regenerated plants. We have reported on the cloning and characterization of the ASA2 gene that encodes a feedback-insensitive AS o~-subunitand on studies of the promoter region of this gene. An appro~mately 2.3kb ASA2 promoter has been cloned from the 5M-rresistant AB15-12-1 genomic DNA. In this expedment, we further characterized stable expression of the gus marker gene controlled by the various ASA2 promoter regions using Agrobacterium rhizogenes-mediated transformation of a leguminous plant, Astragalus sinicus, a system which shows high transformation efficiency. The A.rhizogenes DC-AR2 strain harboring a binary vector was used to transform plant cells in wound sites and transformation was monitored by histochemical GUS assay. Histochemical GUS assays showed that the activity of the 1.356kb ASA2 promoter fused to a gus gene was strong and showed constitutive expression patterns in hairy roots, callus, and wounded stem and leaf tissues. GUS activity of the 2.252 kb ASA2 promoter fused to a gus gene was Very weak compared to that of 1.356 ASA2 and CaMV 35S promoters but was strong especially in the vascular cylinder and root tip. Whole plants transformed with 1.356kb ASA2-gus showed very similar constitutive expression patterns compared to the CaMV 35S promoter. Whole plants are being regenerated for further ASA2 promoter expression pattern studies. This work was supported by funds from the Illinois Agricultural Experiment Station and the Illinois Council on Food and AgdcuRural Research.

Three gene promoters individually linked to the GUS marker gene were evaluated in somatic embryogenic cultures of eastern white pine (Pinus strobus L). Cultures were bombarded using the Biolistic T M PDS-1000 particle delivery system PDS-1000 (I)uPont, Wilmington, DE). Promoters were monocot-enhanced ubiquitin promoter (Ubil), Chlorella viral adenine methyltransferase promoter (amt), and monocot-enhanced (maize) alcohol dehydrogenase promoter (adhI). Cultures were bombarded once at 6.3 cm with 1 ug gold particles coated with 7 ug DNA, 5 days following subculture to freshly prepared maintenance medium. Assays were two (adh), one (amt) and seven (Ubil). Replicates per assay ranged from 4 to 15. Each replicate consisted of a 2.5 cm diameter culture centrally located on plated medium. Foci of GUS-expressing transformed sites were assayed after 1 to 20 days. Sample size assayed per bombarded culture was approximately one-quarter of the total tissue per target culture sample. All bombarded tissues expressed GUS at 24 hours. Numbers of sites expressing GUS were determined for each bombarded culture. GUS activity was expressed in single cells and in small clusters of 36ceils, the latter suggesting mitotis following transformation of single cells. Irrespective of the promoter employed, a diffuse blue color frequently extended through cells neighboring a strongly GUSpositive cell in a tissue. Promoters differed significantly in frequency of GUS expression. The Ubil promoter showed greater frequency of GUS-expression than did the amt promoter (0.7 x Ubil) which, in turn,was more effective than the dhl promoter (0.05 x Ubil). Expression decreased through 22 days to an undetectable level.

P-1083

P-1084 Cloning and Expression of a soybean eDNA Encoding Cystathionine- 7-Synthase. C.A. HUGHES 1' J.S. Gebhardt 2 , A. Reuss 2 , M. MacDonald 2, A. Samuels ~ and B.F. Matthews 2, ~Morgan State University, Dept. o f Biology, Baltimore, Maryland 21251 and 2USDA/ARS, SARL, Beltsville, Maryland 20705 E-mail: [email protected]

Transient expression of a beta-glucuronidase (GUS) gene under the control of a peanut metallothionein-like gene promoter in green beans. D. V. BELIAEV and R. L. Smith, Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 32611. E-mail: To isolate a peanut promoter specifically active in pods we carried out a differential display experiment using RNA isolated from pods and seeds of peanut cv. Altica. Cloned cDNAs differentially amplified in pods but not in seeds were hybridized to RNA from various peanut tissues. From them POD3 cDNA was chosen for further analysis because it hybridized only to RNA isolated from pods. A genomic clone and the U-end of the cDNA corresponding to it were isolated and sequenced. A 65aa long P O D 3 0 R F is homologous to the metallothioneinlike gene family. Primer extension experiments pointed out two transcription start sites 5bp apart from each other. A translational fusion of the POD3 to the GUS gene was made in such a w a y that 32 N-terminal amino acids of P O D 3 0 R F plus 8 more amino acids were fused to GUS protein. The fusion took place in the third exon of the POD3 genomic sequence, therefore, for the chimeric mRNA to be correctly translated the two introns of the POD3 have to be spliced out. The construct contains 1.2kb of the promoter region. Its efficient expression in different tissues of the green bean and peanut fruits has been demonstrated. Since the transient expression is difficult to quantify, w e are currently carrying out the experiments on stable transformation of alfalfa and peanut with this construct.

Congress On In Vitro Biology

The essential amino acid content of dietary proteins is very important because they are required to ensure proper growth in humans and livestock that are unable to synthesize them. We are interested in determining the regulatory mechanism(s) that control the biosynthesis of an essential amino acid, methionine, which is not well understood in plants. Since methionine content is deficient in soybean, it is important to understand the role o f CS, a branch point enzyme that leads to the synthesis o f methionine. A soybean leaf cDNA library was screened using a radiolabeled Arabidopsis CS cDNA probe. A soybean cDNA clone was identified that encodes a protein o f 536 amino acids with a predicted molecular weight o f 58 kDa. The deduced amino acid sequence of the encoded protein corresponds well to the Arabidopsis (80% identity), but not to E. coti and other microorganisms. The first 152 amino acids are rich in serine and threonine representing about 20% o f the amino acids and probably depicts a transit peptide for targeting the protein to the chloroplast. Northern blot analysis reveled that CS mRNA was detected in extracts o f 8day old leaves and cotyledons, light grown material contained more CS mRNA, especially light grown cotyledons. [Supported by NSF Grant # IBN-9602190].

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P-1085

P-1086 Production of Saffron in Tissue Cultures of Crocus sativus L. A.V. LOSKUTOV, C.W. Beninger, G.L. Hosfield, and K.C. Sink. Department of Horticulture and USDA Agricultural Research Service, Michigan State University, E. Lansing, MI 48824. E-mail: .

Investigations into the Molecular Events Occurring During Germination in Lima Beans (Phaseolus lunatus, L.). Emily Kathleen Adldson, Central High School, Macon GA. Germination is a complex series of events beginning with imbibition and ending with the emergence of the root and stem. These events are the result in changes which occur in the seed when different proteins are made after genes are turned on. The purpose of these studies has been to gain a better understanding of what occurs during germination by comparing a dormant seed, the control, with seeds at different time periods during imbibition. The lima bean, Phaseolus lunatus, L., has been used for these studies. The specific approaches of these studies has been to (1) investigate protein expression, (2) investigate mRNA expression, (3) investigate differences in specific mRNA expression, and (4) characterize a specific eDNA. Results from these studies have confirmed the variation in protein and mRNA expression during imbibition. They have also demonstrated the presence of mRNAs in the dormant seed. Sequence analysis of a clone isolated from a eDNA library has regions of homology with Brassica napus and a chicken pie-alpha globin gene. These regions suggest that this eDNA may form a protein that binds to DNA.

P-1087 Continuous Production of Paclitaxel by Encapsulated Cell Culture. JORG WORLITSCHEK, Holger Hiibner and R. Buchholz, Department of Bioprocess Engineering, TU-Berlin, I3353 Berlin, Seestr.13; E-mail: Wide spread experiments to realize the production of the anticancer drug Paclitaxel by plant cell culture have shown the enormous possibilities of this production method. Here a high yield of taxanes and a minimization of by-products can be achieved. Nevertheless the main problems are remaining: Paclitaxel is extracted out of the cell biomass and the media by a proceeding where the ceils are killed by physical damage and toxic effects of the solvents. This batch proceeding combined with a self limitation of the taxane production at higher concentrations leeds to the main restriction of a large scale taxane production by cell culture technology. The aim of our research lies in the development of a process that enables a continous extraction of taxenes out of the cell culture system. This can be achieved by an encapsulation of the cell cultures in microspheres combined with an in-situ extraction of the product. Our institute is working with such an immohilisation system for several years and has developed a patent for capsules with variable pore sizes. Experiments of our group with a reference system of Cruciata glabra that produces the dye antrachinon show an increase of the factor 6 to 10 between the amount of secundary metabolite extracted in the batch process and the amount extracted in this kind of continuously running system. We would like to thank the Fond der Chemischen Industrie.

Congress On In Vitro Biology

Studies are underway to produce structures in tissue culture that will yield the important biochemical constituents naturally found in the stigmas of autumn crocus. Explants of floral tissues were evaluated for the proliferation of stigma-like-structures (SLS) by culture on 5 basal media supplemented with different combinations and concentrations of hormones and vitamins. Optimum proliferation of SLS was observed on CR3-B5 using half-ovary explants. Initiation of SLS occurred 50-60 days after inoculation and explants on CR3-B5 had less browning, higher response per explant, and faster subsequent growth of SLS. The periodic removal of brown tissue, all non-SLS and sub-dividing the ovaries into sectors allowed the production of SLS up to 1.5 year with 3 harvests. Harvested SLS were dried for 30 min at 80C. The presence of crocin, crocetin, picroerocin and safranal in SLS was estimated by comparison with natural stigmas using TLC and HPLC profiles. Commercial inaccessible standards crocin and picrocrocin - were obtained by TLC of the natural saffron extract. To quantify the important secondary metabolites, HPLC was used. A new method of safranal quantification was developed. The concentration of each compound in SLS samples was the same or higher than in natural saffron. Work continues to optimize the in vitro culture conditions for SLS production and explore the possible use of callus and suspension culture.

P-1088 In-Vitro Stimulation of Lycopene Biosynthesis in Tomato Fruit. BE'TIT .K. ISHIDA, Western Regional Research Center, USDA, ARS, 800 Buchanan Street, Albany, CA 94170 Tomato calyces (Lycopersicon esculentum, cv. VFNT Cherry), cultured in vitro, develop into fruit tissue at temperatures between 16 and 22°C. Calyces become swollen, produce ethylene, and become red and succulent. Production of flavor volatiles and changes in cell wall ultrastmcture, color, and sugar characteristic of ripening fruit also occur (Ishida BK, Plant Cell 3: 219- 223, 1991; Ishida BK et al, Physiol. Plant. 89: 861-867, 1993). Recently (Ishida BK et al., Plant Mol. Biol. 36: 733739, 1998) we showed that ripening VFNT Cherry tomato calyces have high tomato AGAMOUS (TAG1) gene expression• TAG1 RNA levels also increase in ripening tomato fruit. In both tissues, TAG1 expression was correlated with degree of ripening. We also found (submitted to Physiol. Plant.) that lycopene concentrations in fruit cultured in vitro at 16°C are high ( 580 + 70/.tg g4 fresh weight), approximately ten times higher than in standard field and greenhouse tomatoes. Addition of 2-(4-chlorophenylthio)-triethylamine (CPTA) further increased lycopene concentration by 14% to 660 + 49 /.tg g4 fresh weight. Some of the carotenoid-derived volatile flavor compounds, as well as some not related, increased with CPTA addition. In our studies of tomato, lycopene concentration correlates well with TAG1 expression, suggesting a causal relationship. Tomato calyces and fruit, cultured at 16 and 26°C + CPTA and in the greenhouse, were analyzed for lycopene and [~-carotene. The largest increase in lycopene resulted from a cool temperature-induced mechanism in which TAG1 is activated. This activation seems to play a key role in ripening and induction of the carotenoid biosynthetic pathway. The smaller increase in lycopene induced by CPTA might be result from inhibiton of cyclases, which blocks ~-carotene synthesis.

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Abstracts

P-1089 Accumulation and Release of Tropane Alkaloids in Hairy Root Cultures of B ~ Candida: Use of Chitosan as Elicitor. T.C. SPOLLANSKY; S.I Pitta-Alvarez and AM. Giulietti, A.M. C~edra de Microbiologia .Industrial y Bioteenologia, Facultad de Farrmcia y Bioquimica, Universidad de Buenos A ~ J u r ~ 956 6to. piso, Buenos Aires (1113), ARGE2q'HNA Hah-y roots of Brugmans/a amd/da (Solanaceae) produce the a~pane alkaloids s c o p o l a n ~ and hyos,~r~fine. The effects of chitosan on growth, alkaloid a~umulation and release into the medkun were tested. Crab shell chitosan was added to hairy root cultures in three final concentrations (all at pH 5.5) : 10, 100 and 1000 mg/L Chitosan in concentrations of 100 and 1000 mg/l were also tested under two lower pH conditions: 4.5 and 3.5. The samples were taken alter 24, 48 and 72 hs. Chitosan 10 mg/l (pH 5.5) significantly increased scopolamine (100%) and h)oscyamine (200°6) accumulation in roots alter 24 and 48 ks, with no effect on a~kaloid release. Chitosan 1000 mg/1 (pH 5.5) ~x:reased significantly the accumulation (100-200%) and release (500%) of both alkaloids. Chitosan 1000 mg/1 (pH 4.5) significantly decreased scopolamine and hyoscyam~ production,compared to the control at pH 5.5, but there was a dm.,mtic h~uease in the release of scopolan~ne to the medium (100-fold). Chitosan 1000 mg/l (pH 3.5) also deca'eased alkaloid accumulation in roots and increased the release into the medium. These eff~ts were sh-/lar to those seen h the controls at pH 4.5 and 3.5, suggesting that the lower pH could be ha part respons~le for these observatiom. However, at both pH levels, the release of akkaloids ioto the medium at 48 ks was enhanced by chitosan. Results observed with chitosan 100 mg/l were similar to those cleon'bed for chitosan 1000 m ~ .

P-1091

P-1090 Effects of Epi-Brassinolide on Growth and Purple-Red Pigment Formed in Onosma paniculatum Cell Cultures. Y.H. YANG, J. Yao and R.Q. Cao. Department of Biological Science and Technology, Nanjing University, Nanjing 210093, P.R. China. E-mail: . Onosma paniculatum is a species peculiar to China, containing purple-red pigment in the roots, which has traditional medical applications in China. Epibrassinolide (epi-BR) with other plant growth regulatorswas used to study the effects on the growth and formation of the pigment in Onosma panicuhtum cell cultures. The resultsshowed that epi-BR had a significanteffect on the cell growth and the pigment formation. Epi-BR at 0.0l ppb promoted significantly Relative Fresh or Dry Weight Increase (~'W] or RDW'I) of the cultures in the light. But epi-BR had no manifest effect on RFW'I and RDWI in the culture without light. Epi-BR with a higher concentration at 1.0 ppb was better to increase the pigment formation, when cultured in modified M9 medium in the dark. The pigment formed is naphthoquinone compound consisting of shikonin and its derivatives, which are very close to those of shikonin derivativesin the intact roots based on Rfvalues, column chromatography and structure analysis. Therefore. a new system (one-stagedculture) for production of the pigment was suggestedby adding epi-BR in the culture. In addition, the reasons for differentialeffectsof epi-BR between light and dark cultures for the cell growth were also discussedin this paper.

P-1092 Production of Transgenic Barley Plant (T~) Resistant to BaYMV (Barley Yellow Mosaic Virus). T. HAGIO 1, S. Kashiwazaki2 and Y. Ohkawak ~ National Institute of Agrobiological Resources, Tsukuba 305 Japan. National Agriculture Research Center, Tsukuba 305 Japan.

Transgenic barley plants (T1 lines) resistant to barley yellow mosaic virus (BaYMV) were obtained by particle bombardment (PDS1000/He, Bio Rad) using the cDNA of BaYMV coat protein (CP) gene. Immature embryos (cv. New Golden) were bombarded with DNA coated gold particles. After the selection culture, transgenic #lants regenerated and were examined. To plants. Ninety-five plants regenerated after selection culture on the media containing hygromycin. Southern analysis and PCR of genomic DNA confirmed the presence of the CP gene in 12 plants out of 95 tested. All the plants possessing the CP gene reached maturity and self-fertilized seeds were obtained. T1 plants. To examine the transmission of the BaYMV CP gene, five independent To plants were chosen. Five to fifteen seeds were randomly selected from one ear of each To plant and were seeded in pots. Thirty-nine seedlings were obtained and genomic DNA was isolated. Southern hybridization of the PCR products showed the presence of the CP gene in all the 39 plants. In Western blot analysis, these 39 plants showed the bands which reacted with the BaYMV-CP antibody. After mechanical inoculation of the virus onto the seedlings, 37 plants showed no symptoms while two plants showed reduced symptoms. Developed severe symptoms were observed in non-transformed control plants. All the 39 T1 plants grew normally and self-fertilized mature seeds were obtained.

Congress On In VitroBiology

Transformation of Barley with a Permatin (Thaumatin-like) Gene in the Sense and Antisense Orientations. A M . NUUTILA, R.W. Skedsen, M. O'Connor and H.F. Kaeppler. USDA-ARS Cereal Crops Research Unit, Madison, WI 53703 and Department of Agronomy, University of Wisconsin, Madison, WI 53706. E-mail: In recent years, barley (Hordeum vulgare L.) has been challenged by Fusarium outbreaks in the upper Midwest. Fusarium infection reduces seed yield and makes seeds unfit for use in food, feed and beverages. One defense against infections is the synthesis of pathogenesis-related proteins (PRPs). PRPs are synthesized in response to infections and/or are deposited in fruits and storage organs during normal development. PRPs are classified into five families. PR-5 proteins include thaumatin-like proteins (TLPs). A Stoup ofTLPs, permatins, has been found in barley and other cereal seeds. Further knowledge of their properties could lead to more effective antifungal strategies. Genes for permatin proteins from the starchy endosperm of barley and oats were cloned and used to transform barley in the sense and antisense orientations. Transformation was done using microprojectile bombardment. Excised immature embryos of barley cv. Golden Promise were used a s target material. After the bombardment, the embryos were cultured on callus induction medium with a selective agent, bialaphos. After four subcultures, resistant callus cultures were transferred on regeneration medium with bialaphos. Green plantlets were transferred into Magenta boxes containing rooting medium with bialapbos. When the plantlets reached the top of the box they were transferred in soil. The effects on growth and permatin expression will be discussed.

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Abstracts

P-1093

P-1094 Cristulariella moricola Resistant Kenaf Generated via In Vitro Selection Protocols. N.A. REICHERT and T.-K. Lira. Department of Plant and Soil Sciences, Box 9555, Mississippi State, MS 39762. E-mail: < [email protected] >

Overexpression of Plant Defense Genes in Tran.sgenic Soybean Lines. T.S. KO, S.S. Korban, and J.M. Widholm. Dept. of Natural Resources & Environmental Sciences and "Dept. of Crop Sciences, University of Illinois, Urbana, IL 61801. E-maih . We are interested in improving the general defense system of soybean against various fungal and bacterial pathogens. Transformation vectors including p35SPGLU, p35SMRIP, p35SMGLU and p35SMCHI were constructed by adding a peach [5-1,3-glucanase genomic DNA, maize ribosomereactivating protein (RIP) cDNA , maize [~-l,3-glucanase and maize chitinase cDNAs, respectively into the cloning vector pBI221. A hygromycin gene cassette, used as a selcctable marker for soybean transformation, was added into each of the constructs. Somatic embryos of soybean cvs. Jack, Yale, Iroquois, and Century were bombarded using the Particle Inflow Gun (PIG) with a single or a combination of these plant defense gene constructs. Bombarded somatic embryos were incubated in an FG medium without shaking for 3 days. After 6 to 7 weekly subcultures onto a selective medium, individual green clumps were selected, and individually proliferated by subculturing them onto an FG medium. A total of 66 putative transgenic lines, with either maize glucanase, peach glucanase, maize chitinase, maize RIP or a combination of these genes, were generated. The frequency of transformation, based on number of PCRanalyzed embryogenic lines, was 70%. To date, 83 putative transgenic plants from 22 transgenic lines have been recovered, and these are growing in the greenhouse. Most of th'e transgenic plants (To) appear to be fertile. We are currently conducting P C R a n d genomic Southern analyses to confirm stable integration of the transgenes. Enzyme activity assay and disease resistance response of T1 plants will be presented.

Kenaf (Hibiscus cannabinus L.) is an annual fiber crop grown in the southern U.S. Cristulariella moricola Hino, the causal agent of the fungal disease zonate leaf spot (ZLS), has become a serious problem in some kenaf production areas by causing massive defoliation which can dramatically reduce fiber yields. Fungicide treamaents are ineffective once ZLS symptoms appear. Due to no known resistance to ZLS in kenaf germplasm, in vitro selection strategies were combined with previously developed adventitious regeneration protocols (using leaf explants) to induce and select for kenaf mutants which display resistance to C. moricola infection. Oxalic acid is believed to be one elicitor of infection, so cell-free fungal extracts (fungal culture filtrates) were utilized in long-term selection protocols by incorporation into shoot initiation media. A short-term selection protocol utilized dual cultures of C. moricola and kenaf explants on twosided petri plates with compartments separated by cellimpermeable membranes. Shoots selected from both systems were rooted and transplanted to soil. The selected regenerants were screened in greenhouse inoculations and detached leaf assays to determine level(s) of resistance. Initial screenings indicate some regenerants display resistance to infection by C. moricola.

P-1095

P-1096

Transgenic Tobacco Plants Express an Oat cDNA Coding for Polyanfine Oxidase NIU DONG, J. Y. Gardener, G. D. Kuehn, and Gregory C. Phillips, Dept. of Agronomy & Horticu[mr and Molecular Biology Program, New Mexico State University, Las Cruces. NM 88003. E-mail: ndong@nmsuedu Thermophilic and halophilic bacteria, as well as certain plants tolerant to abiotic stresses, accumulate uncommon polyamines. These uncommon polyamines may protect nucleic acid and protein molecules under extreme environmental conditions. Two key enzymes are involved in converting common polyamines to uncommon polyamines, polyamine oxidase and schiff base reductase. In plants, polyamine oxidase is widely distributed among cereals and other monocots, but is limited among dicots. A cDNA clone derived from oat which encoded the polyamine oxidase was used to replace the GUS gene in a binary, vector pDGiDkr, thereby constructing pD(PAO)DKr. "Xanthi'" tobacco leaf discs were transfonned with Agrobactcrimn tttmc~tciett.s EHAI05 harboring pD(PAO)DKr. Thirtv-eiuht ,_,reen plantlets grew out of MS plus 2 m~L BA medium containing 250 m~L clafaron and 100 m ~ L kanamicin Only one transformant line exhibited high polyamine oxidase activity, two lines showed intermediate activity, and the other 35 lines expressed v e ~ low activity. Nontransformed controls and transtbrmed controls lacking oat polyamine oxidase cDNA showed no detectable polyamine oxidase activity. We are analyzing the transformants with Southern blots and PCR-junction sequence searching to examine a possible position effect on gene expression Some transformants were very slow growing, and it is possible that those lines suffered from depletion of essential spermidine and spermine due to transgenic expression of the polyamine oxidase cDNA

Congress On In Vitro Biology

Improved Water Use Efficiency in Transgenic Wheat Expressing the Barley HVA1 Gene. ELUMALAISIVAMANP, Ahmed Baheildin2, Jon M. Wraith ~, Thamir AI-Niemi I , William E. Dyer a, Tuan-Hua David Ho 3 and Rongda Qu4. L- Department of Plant Soil and Environmental Sciences, Montana State University, Bozeman, MT 59717; 2 _ Agricultural Research Center, AGERI, Egypt; 3 _ Department of Biology, Washington University, St. Louis, MO 63130; 4 _ Department of Crop Science, North Carolina State University, Raleigh, NC 27695- 7620. Email: [email protected] The group 3 late embryogenesis abundant (LEA) protein gene HVA1 from barley (Hordeum vulgare L.) was introduced into wheat (Triticum aestivum L.) cv. Hi-Line using the biolistic bombardment method. A total of ten primary transgenic lines were obtained. Constitutive expression of the HVA1 gene as regulated by the maize ubiquitin promoter led to high protein expression levels in leaves and roots. T3 progeny were tested under greenhouse conditions for drought tolerance. Plants were grown at 20 and 500 kPa soil matric potentials to simulate moderate drought and normal moisture conditions, respectively. Transgenic plants expressing the HVA1 gene had significantly higher water use efficiency values ( 1.73 X 103) as compared to 1.41 X 103 and 1.39 X 103, respectively for the controls (p = 0.01) under moderate drought conditions. Transgenic plants expressing the HVA1 gene also produced significantly higher numbers of tillers and root fresh weights than controls.

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Abstracts

P-1097 EFFECTS OF PACLOBUTRAZOL ON SOYBEAN (Glycine max) Leslie A. Jennings, (Dr. Sibdas Ghosh), Department o f Biological Sciences, University o f Wisconsin-Whitewater, 800 West Main Street, Whitewater, WI 53190. Email: [email protected]. This research is supported by a grant from UW-Whitewater to LJ & SG. Paclobutrazol, a triazole compound, is used as a growth regulator (retardent) in a variety o f agronomic crops. Oneweek-old soybean (Glycine max) seedlings were treated with paclobutrazol o f varying concentrations ranging from 0.01 m M to 0.50 m M per pot for 14 days. In treated seedlings, leaf areas, fresh and dry weights o f both primary and trifoliate leaves were significantly lower than in the controls. Paclobutrazol treated seedlings were found to be more resistant to water stress. The increased stress resistance is evidenced by reduced height and increased levels o f chlorophyll, carotenoid and protein contents o f the leaves o f treated seedlings.

P-1099

P-1098 Effect of Hysterine on the Growth and Peroxidase (ECI.I 1.1.7) Levels in Phaseolus vulgaris L. Callus. J. Taboada ~, M. Torres, H. GonzalezCerezo2, 1. Brunner3 and A. RUBLUO3. qnstituto de Quimica, UNAM 2UAM-lztapalapa, Jlnstituto de Biologia, UNAM, Mdxico, D.F. e-mail: [email protected]. Hysterine a sesquiterpene lactone isolated from Parthenium bipinatifidum show cytotoxicity to animal cells lines Hep2 and L929 in culture at low concentrations (5-10 mgL-t). Peroxidases are ubiquitous in the plant kingdom and their activity on in vitro systems has been mentioned as a useful tool for investigating differential metabolism Due to the interest into dilucidate the possible role of hysterine into cytotoxic and metabolic changes in plant systems, the aim of this work was to evaluate the effect of this lactone on the growth of Phaseolus vulgaris callus as well as on the peroxidase activity as an early response of the metabolic action of these substances on a plant tissue system. Callus of P. vulgaris were obtained from the culture of sterile young leaves on MS basal media enriched with 2,4-D (2mgL-*).Other explants were cultured similarly but in presence of hysterine (25-225mgL% Callus production was evaluated weekly and analyzed by an Annova and a Dunnet multiple rank test. Hysterine was extracted from MS media and determined by TLC. Peroxidaes activity was measured by the oxygen evolved by the reaction of H:O, with an homogenated containing the enzyme. Young bean leaves and callus grown without and in presence of hysterine were homogenized and electrophoresed for peroxidase presence. Results show an inhibition effect expressed as callus percentage induced by hysterine after 125mgL-~ dose of Iactone. Considering fresh weight, a drastic fall was observed after 75 mgLL and this drop increased with the dose. These results make evident a toxic effect on plant tissue cultures that was dose-dependent. Similarly the peroxidase levels decreased, and the isozyme pattern changes, implying that hysterine is an inhibitor of the viability and reproduction of plant cells as well as causing drastic changes in their enzymatic activity that would give in turn, an expression in the plant development.

P-1100 In Vitro Production and Long Term Storage o f Maize

Cytosolic Calcium in Zea Mays Sperm Cells. P. J. HALL, G. Zhang and D. D. Cass Department o f Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9. Email: We are investigating the role o f calcium in the double fertilization in Zea mays. Isolated Zea mays sperm cells are known to take up calcium and fuse when exposed to high calcium. Cytosolic calcium concentration and localization within living sperm cells can be observed in real time using cell permeant fura-2 am. Cells in calcium solution initially have an increase in cytosolic calcium, followed by a decrease likely due to sequestering o f calcium into organelles. Ionophores and EGTA have been used to investigate their effects on cytosolic calcium. Freshly loaded sperm cells with fura-2 have a defined region o f high calcium between the nucleus and the cell membrane. During fusion a region o f high calcium forms at the point o f fusion between two sperm cells. We expect to see the same event during fusion between a sperm cell and a fura-2 loaded egg cell.

Congress On In Vitro Biology

Zygotic Embryos Derived from Isolated Embryo Sacs. J.D. LAURIE *, G. Zhang j, L.E. McGann 2 and D.D. Cass *. *Dept. of Biological Sciences, 2Dept. o f Lab. Medicine and Pathology, University o f Alberta, Edmonton, Alberta, Canada T6G 2E9. E-mail: . A novel embryo sac isolation method was developed employing mechanical sectioning o f maize ovaries using a Vibratome. Isolated sections containing intact embryo sacs were viable and developed in vitro on MS media producing endosperm (90%) and embryos (75%). Embryos (10-14 DAP) were frozen to 196°C in liquid nitrogen (LN). Root and shoot apices exhibited differential sensitivity to cooling rate. To overcome this problem embryos were vitrified in VSY2 (10% propylene glycol, 15.5% ethylene glycol, 20.5% dimethyl sulfoxide, 6M polyethylene glycol). Aider a 48 hour incubation on MS containing 15% sucrose and 50p.M abscisic acid (ABA) to increase desiccation tolerance 46% o f the embryos survived vitrification, storage in LN and warming. This method was then applied to in vitro produced embryos derived from 1 DAP embryo sacs. Survival (82%) was higher for these embryos indicating a greater degree o f desiccation tolerance. Immediate applications are the use o f isolated embryo sacs as targets for genetic manipulation, and vitrification as a means to preserve transgenic embryos.

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Abstracts

P-1101

P-1102

Microinjection of Sperm Cells into Isolated Maize Embryo Sacs Results in In Vitro Fertilization. G. Zhang, J.D. LAURIE, P.J. Leedell, and D.D. Cass. Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9. E-mail: . We have developed an in vitro fertilization protocol for maize by microinjecting isolated sperm cells into unfertilized embryo sacs thus bypassing pollen and stigma interactions. Embryo sacs were exposed by sectioning of ovules using a Vibratome. Isolated sperm cells were microinjected into embryo sacs in an attempt to fertilize eggs and central cells. Endosperm development was observed 5 days after microinjection and grew to a diameter of 1.5 mm by 14 days. Microscopic observation showed fully cellular endosperm together with a developing embryo. Our in vitro fertilization protocol provides an experimental tool for studying fertilization events in flowering plants.

Transient GFP Expression in Sperm Cells and Zygotes of Zea mays. P.J. LEEDELL, G.Zhang, D.D.Cass. Department of Biological Sciences, University of Alberta, Canada, T6G 2E9. E-mail: . Most methods of monocot transformation rely on a callus culture step to ensure regeneration of non-chimeric plants. However, this method has the disadvantage of producing uncontrolled genetic variation in the resultant plants including possible silencing of introduced genes. An alternative method involves the use of isolated sperm cells expressing a recombinant plasmid in in vitro fertilization, followed by regeneration of normal, non-chimeric, transgenic plants. Sperm cells of Zea mays isolated by density gradient centifugation were transfected with a constitutive GFP expressing plasmid by electroporation, and incubated at 26 °C overnight. The optimum conditions for electroporation of sperm ceils were determined by monitorin~ uptake of high molecular weight rhodamine labelled dextran. Following incubation, cells were observed by both fluorescence microscopy, and by confocal laser scanning microscopy to visualize the presence of cytoplasmic GFP protein which was confirmed by Western blot analysis. Further, sperm cells shown to be expressing GFP were fused in vitro by calcium mediated fusion to isolated egg cells of Zea mays.

P-1103

P-1104

Towards Production of Transgenic Maize by Microinjecting Exogenous DNA into Zygotes and Two-Celled Proembryos. G. Zhang, J.D. LAURIE and D.D. Cass. Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9. E-mail: .

Production of Altered Endosperm by Microinjection of Exogenous DNA into the Central Cell of Maize Embryo Sacs. G. Zhang, J.D. LAURIE and D.D. Cass. Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9. E-mail: .

We have developed a protocol to produce transgenic Zea mays (maize) by microinjecting zygotes or two-celled proembryos with exogenous DNA followed by direct zygotic embryogenesis. Embryo sacs were isolated by sectioning ovules using a Vibratome. Zygotes within intact embryo sacs were microinjected with plasmid DNA containing the 13glucuronidase (GUS) reporter gene. Histochemical assay 6 days after microinjection showed GUS expression, with a 10% transformation rate. Sectioning and microscopic observation of developing embryo sacs revealed GUS expression in both endosperm and embryos. When the upper cell of two-celled proembryos was microinjected, 35% transient expression was observed. Regenerants were obtained through direct zygotic embryogenesis in tissue culture and grown into fertile plants. To date both histochemical and PCR analysis of regenerants have been negative. We are currently working on obtaining transgenic plants and are optimistic since we have observed such a high rate of transient expression after microinjection.

An in vitro system enabling endosperm transformation and subsequent observation of gene expression during endosperm development has been developed. The present study describes the way in which microinjection is used to deliver exogenous DNA into central cells of maize (Zea mays L.) embryo sacs. Embryo sacs were isolated by sectioning ovules using a Vibratome. Central cells of intact embryo sacs were subsequently microinjected with plasmid DNA containing the 13-glucuronidase (GUS) reporter gene. Endosperm growth was observed after microinjection, and histochemical examination of developing endosperm revealed transient GUS expression in 17.5% of injected embryo sacs. Microscopic observation of endosperm showed uniform GUS expression. Stable transformation was observed after initiating long-term cell cultures from transiently expressing endosperm. The potential biological significance of endosperm transformation and culture is discussed.

Congress On In Vitro Biology

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Abstracts

P-1 105

P-1106

Adventitious Shoot Regeneration In Vitro from Leaf Explants o f Eight Important Blackberry Cultivars. CHIH-WEI V. TSAO. Department o f Horticulture, Oregon State University, Corvallis, OR 97331. E-mail: and Barbara M. Reed. USDA/ARS National Clonal G-ermplasm Repository, 33447 Peoria Rd. Corvallis, OR 97333-2521. E-mail: Eight commercially important blackberry cultivars, 'Marion', 'Thornless Evergreen', 'Hull Thornless', 'Cherokee', 'Kotata', 'Waldo', 'Chester' and 'Shawnee' were tested for regeneration using the top 1 to 6 leaves from in vitro plantlets. Adventitious shoots were regenerated from halfleaf explants following pretreatment for one week in the dark on Murashige and Skoog (1962) medium with 1 lxM Nphenyl-N'-(1,2,3-thidiazol-5-yl)urea (TDZ), and further growth on MS medium with indole-3-butyric acid (IBA), Nebenzyladenine (BA) and TDZ with a 16-hour photoperiod. Adventitious shoots were successfully produced on all eight cultivars after the fourth week on regeneration media with 2.9 I.dVl gibberellic acid (GA3)plus 0, 0.5 or 5 g M IBA, and 0.5 to 10 IxM BA, or TDZ. Regeneration media with TDZ induced more adventitious shoots on 'Waldo', but BA was better for the other seven cultivars. We recommend 5 or 10 BM BA and 0.5 I.dVlIBA for in vitro shoot regeneration from blackberry leaves.

P-1107

Development of an AdventitiousShoot Regeneration Procedure for Transformation of Eastern White Pine. C.H. Michler and T.M. HUBACHER. USDA Forest Service, North Central Forest Experiment Station, 5985 Highway K, Rhinelander,WI 54501. Email: . The ability to regenerateadventitiousshoots from transformedconifer cells or tissues is limited more by lack of adequate regenerationprocedures than our ability to stablytransform tissues. In addition, another important limitation tc~ conifer transgenictechnologyis the protracted time requirementfoliowingthe transformationevent to produce a rooted transformedshoot that can be tested ex vitro. We attempted to overcome both limitations by combining microparticle bombardment transformationof competent Eastern white pint (Pinus strobus L.) organogeniccell initials with a tissue culture procedure th:tr utilized germinating zygotic embryos as nurse tissue for transformc~ adventitious shoots thereby greatly reducing the time in vitro. With ouc regeneration procedure, the in vitro phase required for adventitious shot,~ regeneration and elongation was reduced by 69% over similar publisbed procedures.With ex vitro rooting, 72% of all cuttings that ranged from 2 tmn to 5.9 cm rooted after 6 weeks. This compared to other published reports where rooting after 6 weeks.This compared to other published reports where rooting percentagewas only 40% after 6 months in vitro and 0% with ex vitro rooting attempts. We will present resultsof in vitro treatments for stimulation of nurse tissuegrowth, adventitiousshoot production, and ex vitro rooting. In addition,we will discussresultsof transformationexperimentsusing an Eastern white pine chltinasepromoter-GUSfusionconstruct.

P-1108 Overcoming Hybrid Incompatibility between Nicotiana Trigonophilla Dunal and N. Tabacum L. Using In Vitro Technigues. D.N. NIKOVA, Physical Department , Technical University Munich, D - 85747 Garching , GERMANY. E mail: < [email protected] >.

The wild species Nicotiana trigonophilla Dunal, possesses genes for resistance to some diseases. N. trigonophilla was successfully included in hybridisazation with N. tabacum. The obtained capsules were with small amount of badly developed seeds. They were grown in vitro, using embryo culture method. Murashige and Skood (MS) nutrient medium was used with supplements of sucrose (30 g/l) yeast extract (500 mg/1), casein-hydrolisate (500 rag/l), inositol (100 rag/l), gibberellic acid (0,1 mg/l), kinetin (KIN) - 0,05 mg/1, naphthylacetic acid (NAA) - 0,05 rag/1 and agar (7 g/l). Viable Ft plants were obtained, that reached florescence. Ft hybrid plants were completely (male and female) sterile. To overcome the sterility of the hybrid tissue culture methods are used. Explants of stem parenchyma were cultivated to MS nutrient medium, supplemented with 0,5 mg/l KIN and 2,0 rag/1 NAA for callus induction. Organogenesis was stimulated on the same medium with 2,0 mg/l KIN and 0,5 rag/1 NAA and rooting - on MS medium enriched with 2,0 rag/1 ferulic acid. The regenerants obtained from passages I - V were sterile. In five plants, 45% viable pollen was found and seed capsules were formed as a result of self - pollination. These plants are in the proccess of investigation.

Congress On In Vitro Biology

Organogenic Regeneration o f Soybean From Hypocotyl Explants and Progeny Analyses. Y.H. Dan L'-', L.L. MCDOUGALD ~, J. M.Tyler ~, and N.A. Reichert 1. ~Dept. o f Plant and Soil Sciences, Box 9555, Mississippi State, MS 39762; 2Current address: Monsanto Corp., 700 Chesterfield Parkway North, St. Louis, MO 63198; ~USDA-ARS, Soybean Research Unit, P.O. Box 196, Stoneville, MS 38776. E-mail:

A unique, genotype-independent organogenic regeneration protocol was developed for soybean using hypocotyl explants. Plants from 13 genotypes, representing maturity groups IV - VI, were successfully regenerated. All responded by producing adventitious shoots on the acropetal end o f hypocotyl exptants excised from seven day old seedlings a~er placement on a medium containing 5.0 - 10 ~zM 6-benzyladenine (BA). Histological analyses confirmed the adventitious nature o f arising shoots, but incompletely excised cotyledonary buds also contributed to shoot initiation. Explants were maintained on shoot initiation media in the dark, then transferred to shoot elongation media containing 0.36 ~ M BA for an additional four weeks. Excised shoots were then rooted on media containing 12.5-29.2 ~zM indole-3-butyric acid. Numerous regenerants were successfully transplanted to soil, acclimatized and grown to maturity in the greenhouse. No aberrant phenotypes or fertility problems were noted. R~ progeny harvested from regenerants o f four cultivars are currently being analyzed in greenhouse screenings.

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P-1110

P-1109 Microshoot Proliferation of Oryza sativa L. for Successful Clonal Propagation. V. RUDtLASWAMY and N.A. Reichert. Department of Plant and Soil Sciences, Box 9555, Mississippi State, MS 39762. Email:

Embryos excised from mature rice seeds were analyzed for multiple shoot responses in vitro. Six rice genotypes adapted for growth in Mississippi (two cultivars and four breeding lines) were utilized. Responses were compared in Murashige and Skoogbased (MS) liquid media containing four different concentrations of6-benzyladenine(BA). Microshoot production began within 12 days after culture initiation. Sizable clusters of microshoots were noted within 8 weeks after initiation. Different genotypes displayed distinct culture morphologies. However, the numbers ofmicroshoots produced per explant appeared more correlated to the concentration of BA in the medium. Separated microshoots rooted in liquid MS-based medium containing IBA within 10 days after transfer. Regenerants from all six genotypes were transplanted to soil, acclimatized and grown to maturity in the greenhouse. No phenotypic variations were detected. Progeny screenings are currently underway. This microshoot proliferation protocol provides an efficient way to produce true-to-type rice plants and therefore, can be incorporated into genetic transformation systems.

P-1111 IN Vitro Regeneration Studies On Phlox drummondii Hook. and P. Paniculata Linn. (Polemoniaceae). VARSHA RAJA and S.N. Raina, Laboratory of Cellular and Molecular Cytogenetics, Depatment of Botany, University of Delhi, Delhi -7, India. E-mail : varsha@ biochem.iisc.emet.in Phlox drummondii (an annual) and p. paniculata (a perennial) are universal ornamental species cultivated for their colourful flowers and long period of blooming. Callus cultures were induced from internodal segments of both species on Murasshige and Skoog (MS) medium supplemented with an auxin and/or a cytokinin in certain combinations only. Neither 2, 4-D nor kinetin could induce callus formation in either species. Massive callus growth occurred in 6 to 7-week-old explant cultures of p. drummondii on MS+NAA+BAP and of p. paniculata on MS+NAA only. Normal leafy shoots regenerated copiously from callus cultures of p. paniculata transferred to MS alone, and of p. drummondii transferred to MS+BAP. Quantitative data indicate that rapid multiplication of both the ornamental species of phlox through aseptic cultures is a feasible proposition.

Congress On In Vitro Biology

In Vitro Development and Ontogeny of Multiple Shoots from Mature Caryopses of Switehgrass. J.K. McDANIEL, S. Dutta Gupta, and B. V. Conger. Department of Plant and Soil Science, The University of Tennessee, Knoxville, TN 37901 - 1071. E-mall:. An efficient and reproducible in vitro culture system has been developed for regeneration of multiple shoot clumps from mature caryopses of both lowland and upland cultivars of switchgrass (Panicum virgatum L.). The multiple shoots were induced on MS medium supplemented with various combinations of 2,4-D and thidiazuron (TDZ). Maximum response was obtained with 4.5 ~tM 2,4-D and 18.2 ~tM TDZ. Regenerated shoots proliferated and rooted efficiently on MS medium without growth regulators. The developmental pattern of the multiple shoot formation was studied by light and scanning and transmission electron microscopy. These studies indicated that the multiple shoot meristems were formed directly from shoot apices of the germinating earyopses. The initial process involved inhibition ofintemode elongation and delayed development of the apical meristem leading to the formation of tissue strata with multiple shoot meristems. Subsequent reprogramming of these meristems into supernumerary shoot buds resulted in the formation of multiple shoots. The simplicityof the protocol and direct production of multiple shoots make this a potentially attractive system for microprojectile mediated gene transfer. Research supported in part by Lockheed Martin Energy Systems, Inc. under Contract No. IlX-SL 129(2.

P-1112 Induction of Somatic Embryogenesis in Three Genotypes of Soybean. NICOLLE E. H O F M A N N and S.S. Korban. Department of Natural Resources and Environmental Sciences, University of Illinois, Urbana, IL 61801. E-maih The i n f l u e n c e of media c o m p o n e n t s on the induction of somatic embryogenesis in three genotypes of soybean was investigated. Genotypes under investigation included Iroquois, Macon, and Savoy. Media modifications included sucrose concentration, type and concentration of auxin, and pH level. Immature cotyledons were used as the source of explant. Cotyledons were placed on a medium containing MS salts, B5 vitamins, sucrose, and auxin. Gelrite (0.2%) was used as the solidifying agent. Sucrose concentrations of 1, 2, 3, 4.5, or 6% were used. The auxins, 2,4-D and NAA, were used at five concentrations. The pH of the medium was adjusted with 1N NaOH to either 5.7 or 7.0. Induction efficiency of somatic embryos varied in response to the different treatments tested. Frequency of embryogenesis also varied among the different genotypes used in this study.

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P-1113 Investigation o f Continuous Somatic Embryogenesis in Alfalfa. L.-N. TIAN i, D.C.W. Brown 1, E. Watson ~and J. Webb 2. ISouthem Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, Ontario, Canada N5V 4T3, 2Department o f Biology, Carleton University, Ottawa, Ontario, Canada K1S 5B6. Alfalfa is one o f the first major crop plants in which plants were regenerated from tissue culture. In spite o f a well developed system, cultures lose embryogenic capacity quickly over subsequent subcultures and the new cultures for embryogenesis must be started with new explants each time; this requires extensive labour and time. A system for continuous embryogenesis was investigated in alfalfa. Petiole explants were cultured in basal B5h medium and the basal medium contained a higher than normal level of potassium (50 mM) plus proline (25 mM) and thioproline (0.4 mM) (SH4K medium). The callus induced on the basal medium was yellow-brown in colour but callus formed on SH4K was cream in colour. Embryos did not form and develop on the callus induced on SH4K medium unless the callus was transferred to growth regulator-free medium. Embryos induced on the basal medium could grow to maturity. The number o f embryos induced in SH4K was about ten times higher that that induced on the basal medium (i.e., 50 vs 5). Callus induced on SH4K medium could be subcultured onto fresh induction medium and embryogenic capacity was retained for at least six subculture cycles. The majority o f the embryos (50-60%) induced on SH4K medium were morphologically normal. Fertile plants developed from embryos taken from the subcultured tissues.

P-1115 Somatic Embryogenesis of Papaya (Carica papaya) from Root, Petiole and Leaf Tissues. X. Y. LI, A. K. Yadav, and J. L. Robbins, Agricultural Experiment Station, Fort Valley State University, Fort Valley, Georgia 31030. E-mail: . Somatic embryogenesis from vegetative tissues instead of zygotic embryos has been investigated in an attempt to regenerate true-totype papaya (Caricapapaya) clones. Petiole, root and leaf tissues were used as explants to initiate embryogenic cultures. Various culture media were used to determine the requirement of salts and plant growth regulators for initiation of embryogenic cultures and embryo development. Petiole tissues have shown much more response to the initiation media, containing MS or V2 MS basal salts, 2,4-D or NAA, and Kinetin or BA, than leaf tissues. Embryogenic tissues have been initiated from petioles but not from leaf tissues. Root tissues were highly responsive to the medium containing BA. Embryogenic tissues were induced from roots on the medium with MS salts and 0.5 mg/liter BA. After the embryogenic tissues were transferred to the medium containing Gamborg's B-5 macro-nutrients but depleting (NH4)2SO 4, MS micro-nutrients, MS vitamins, 1 g/liter L-glutamine, 0.5 rag/liter BA, 30 g/liter sucrose and 8 g/liter agar, a large number of cotyledonary embryos were produced. In contrast, no embryos were induced on the medium with MS salts and vitamins, 0.5 rag/liter BA, 30 g/liter sucrose and 8 g/liter agar. In conclusion, root tissues may be a better source of explants than petiole and leaf tissues for initiating embryogenic cultures. For embryo development, macro-nutrients and/or nitrogen source may have impact on embryo maturation in papaya.

Congress On In Vitro Biology

P-1114 Somatic embryogenesys in cowpea [Vigna unguiculata (k) Walp] A. PELLEGRINESCHI. Biotechnology Research Unit ~nternationa~ Institute of Tropical Agriculture, c_Jo CIAT PO Box 025443 Miami, Florida 33102 A method for inducing somatic embryogenesis in cowpea [Vigna unguiculata (L.) Walp.] has been developed, starting from mature cotyledon explants. Induction of somatic embryogenesis was influenced by basal media composition, pH, hormone concentration, gelling agents and incubation environment. Cotyledons and embryos from two cultivars, Ife brown and 83D-442, were excised from green immature pods. The proximal part of the cotyledon was removed and transferred to several induction media with a range of hormone concentrations, pH and basal media for four weeks. The induced cotyledons were transferred to hormone-free liquid medium for 1 week and then transferred to the same medium containing 0.2% phytagel. Several somatic embryos developed within 6 weeks from the wounded region of the cotyledons. The embryos were isolated and dehydrated for 4 days and transferred again to the same solid medium for germination. After germination the plantlets were transferred to soil and developed into normal fertile plants. The optimised protocol was effective in both varieties.

P-1116 Regeneration of Daylily via Somatic Embryogenesis.JOHNNY CARTER and Seema Dhir. Agricultural Research Station, Fort ValleyState University,Fort Valley,GA 31030-3298. Daylily (Hemerocallisspp.) a popular perennial,has been used more and more in commerciallandscapingbecauseof its plant form, ability to provideseasonal color and ease of culture. Nearly 38,000 hybrids of daylily are registeredand many are availablecommercially.Although, easilypropagated vegetatively,the process is very slow, thus delayingthe releaseof new cultivars by severalyears. Therefore, mass-propagationof superior daylilycultivars via tissue culture and its future application in rDNA technology for genetic improvement is desirable. We have developed a simple procedure for regenerationof daylily plants via somaticembryogenesis.Youngflowerbuds were sterilizedwith 20% clorox for 30 minutes. Filament pieces were used to initiate cultures on Murashige & Skoog's (MS) basal alone or in combination with benzylaminopurine (BAP; 1, 2 or 3 rag/l) and indole-3-aceticacid (IAA;0.5 mg/l). In 4-8 weeks, globular, heart or torpedo shaped embryoswere formed on respondingfilament explants.The percentageof explants forming embryos ranged from none on MS basal, 20% on 1.0 mg/l BAP, 52% on 2.0 mg/l BAP and 68% on 3.0 mg/l BAP. The number of embryos per embryogenicdump also increasedwith increasingBAP levels.On average4-5 embryoswere visible on BAP 1.0 mg/l, 17 on BAP 2.0 mg/l and 44 on BAP 3.0 mg/l. The cultures were routinelysubculturedon the same medium. Once embryosturned green, the clumpswere subdividedto allow their further developmentinto plants. In 4-6 weeks, plants developed a healthy root system and were transferred into pots. So far, 27 plants have been transferredto soil, all are growingwell under greenhouseconditions.

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P-1117

P-1118 Development of Regeneration Protocols for Mississippi Rice Lines. L. CHEN ~, D. S. Luthe2 and N. A. Reicherd. ~Department of Plant and Soil Sciences, Box 9555, ZDepartment of Biochemistry and Molecular Biology, Box 9650, Mississippi State, MS 39762. Email:

As part of a genetic improvement project, somatic embryogenic regeneration protocols were developed for rice cultivars and breeding lines adapted for growth in Mississippi. Those tested included three Mississippi cultivars [Jackson, newly released Litton (1996) and Priscilla (1997)] and six breeding lines. The three cultivars plus breeding line RU9604077 were compared for embryogenic callus initiation and plant regeneration responses. Mature seed explants were placed on three callus initiation media and subsequently transferred onto each of two regeneration media. The callus initiation medium (CIM) which contained auxins 2,4dichlorophenoxyacetic acid (2,4-D) and 1-naphthaleneacetic acid (NAA) produced the greatest amount of embryogenic callus. When used in combination with maturation medium N6R [contained NAA and 6-benzyladenine (BA)], plantlets could be regenerated in four to six weeks post-initiation. All nine Mississippi genotypes (those listed above plus five breeding lines) screened on the CIM/N6R combination produced plantlets. Regenerants were transplanted to soil and all displayed normal morphologies as well as R~ progeny of Litton and Priscilla analyzed in greenhouse screenings. Development of a regeneration protocol specifically for Mississippi genotypes should allow direct introduction of genes into those lines which, in turn, should shorten the time required to bring enhanced cultivars to the rice growers. P-1119

The Influence of Osmotic Pretreatment on the Ebryogenie Capacity of Switchgrass (Panicum virgatum L.) Suspension Cultures. M.K. ODJAKOVA and B.V. Conger. Dept. of Plant and Soil Science, The University of Tennessee, Knoxville, TN 37901-1071. Email: We examined the influence of osmotic pretreatment on the initiation of suspension cultures and induction of somatic embryogenesis in 'Alamo' switchgrass. Somatic embryos produced from in vitro cultured inflorescences at different developmental stages were used as explants to initiate suspension cultures. They were precultured for 30 h on osmotic medium containing 0.1, 0.2 or 0.3 M each of sorbitol and manitol. They were then transferred to MS liquid medium with 9/aM 2,4-19 and 5/aM BAP and cultured for 27 d. Results showed that cultures initiated from younger embryos produced a higher embryogenic response than those from older embryos. A higher concentration of the osmoticum resulted in more embryos produced than lower concentrations. Cultures initiated with embryos at later developmental stages produced nonembryogenic clusters and the number of embryogenic cells was low. In contrast, less developed somatic embryos showed a very high embryogenic potemialespecially when precultured on the highest osmoticum and produced almost 100% organized structures, including somatic embryos. Research supported in part by Lockheed Martin Energy Systems. Inc. under Contract No I IX-SYl61C.

P-1120

Somatic Embryogenesis in Sweet Potato (Ipomoea batatas (L.) Lain.). S. ALMAZROOEI ; N. Taylor; G. Henshaw and D. Blakesely. Department of Biological Sciences, Kuwait University, P.O.Box 5969 Safat 13060, Kuwait E-mail < Mazrooei @ kuc01, kuniv, edu. kw > Somatic embryogenesis in sweet potato (Ipomoea batatas) was successfully induced in all of the ten genotypes that were investigated and they responded at frequencies in the range from 4% to 90%. To improve the efficiency of induction of embryogertic tissues different factors were investigated, including the type and concentration of auxin, type and size of the explant, state of the mother plant and the incubation conditions. The most responsive explant was the 0.5-1 mm sized axillary buds excised from in vitro-grown plantlets and the induction rates were highly genotype dependent; the most effective induction medium was Murashige & Skoog basal medium supplemented with 5/aM 2,4-D or 2,4,5-T, depending on the genotype. "The same medium was also found to be the most effective for proliferation and maintenance of the embryogenic tissue. The subsequent maturation of somatic embryos and planttct regeneration were optimised by subculture of the embryogenic tissue onto a series of media with increasing sucrose concentrations in the range 0.1M to 0.7M, followed by transfer to medium with no auxin, or with a lowered auxin concentration (1 ~tM). Media supplemented with BAP, NAA, GA3 neither improved embryo maturation nor plantlet formation; ABA on the other hand improved the formation of mature embryos, which however failed to develop into plantlets upon transfere to the regeneration medium.

Congress On In Vitro Biology

Fast rotation clinostat for studying gravitational effects of somatic embrygenesis from orchardgrass leaf cultures. AVASILENKO, J.K. McDaniel, and B.V. Conger. Dep. of Plant & Soil Science, University of Tennessee, Knoxville, TN 379011071. E-mail: . To understand the role of gravity in the regulation of plant somatic embryogenesis, it is necessary to study effects under microgravity (spaceflight) conditions. Microgravity significantly reduced somatic embryo development from orchardgrass (Dactylis glomerata L.) leaf cells during a space shuttle experiment (STS-64). Since omnilaterally gravistimulated plants do not respond gravitropically, it may be assumed that a clinostat can produce nearly the same biological results as microgravity. Also, the possibilities to perform experiments in space are very limited. In a previous experiment with a slow rotation clinostat (1 rpm), no differences were observed in somatic embryogenesis between rotated and nonrotated segments. A fast rotation clinostat (50 rpm) may be superior for simulating the effects of microgravity. Therefore, we designed such a clinostat to conduct experiments with leaf segments oriented vertically utilizing a horizontal axis of rotation. This apparatus maintained acceleration forces in the range of approximately 10-3g. Results indicate about 48% reduction in embryogenesis from clinorotated segments compared to nonrotated controls. The research was supported in part by NASA grant NAG10-0221.

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P-1121

P-1122

Applications (Non. Transgenics) for Introgression of Late Blight Resistance {Phvthoohthora i.nfestans)in Potato. G.CIPRIANI, R. Espejo. and A. Golmirzaie. International Potato Center, Apartado 1558-Uma 12, Peru, Late Blight is an important disease especially in potato production regions. Present control methods are largely based on fungicide applications which increase the yield costs of production and have a deleterious effect on the environment An alternative for potato, complementary to conventional plant breeding, is to create lines with natural resistance introgressed from wild species, through protoplasts fusion. Conditions were optimized for the isolation and culture of potato protoplasts. Leaf tissue was obtained from in vitro plants of DEsir6e and Spunta cultivars, and the wild specie So/anum pap#a was used as explant A new isolation method was developed,employingan enzymatic digestion lrealment of 16-18 hours with Celullase R-10, Macerozyme R10, Calcium Chlodde and MES under gentle agitation. Large quantitiesof protoplasts were obtained (10s -106cel/mL) which were then embedded in alginate beads to facilitate their manipulation. More than 75% of the protoplasts divided during the first two weeks. Cell divisions led to the formation of colonies in approximately 70% of the samples. For each milliliter of protoplast suspension 18-20 calli were obtained. These were transferred to a regeneration medium, where multiples shoots formed in 90-95% of the samples. Regenerated plants developed with normal appearance. They will be transferred to greenhouse conditions to evaluate their morphology and citogenetic stability. Having necessary conditionsfor isolation,culture and regenerationfrom protoplasts,we will next carry out somatic fusions induced by polietilenglycol and eleclrofusion. Hybdd lines showing resistance could be incorporatedinto breeding programs and disfributedto nationalprograms. Potential

Biotechnological

P-1123 Growth and Viability of Buffelgrass (Cenchrus ciliaris L.) Cell Suspension Cultures on Salt Stress. E. RUIZ, E. C&rdenas, C.G.S. Vald6s; F. Zavala, U. Lbpez and R Vfizquez. Fac. de Agronomia U.A.N.L. Apdo. Postal # 358 San Nicolfis de los Garza, N.L. MEXICO. Buffelgrass ( C e n c h r u s ciliaris L.) is an apomictic perennial grass that has good forage characteristics and drought tolerance. Many cultivars have been selected for large semiadd extensions in Mexico, nevertheless most cultivars not show salt tolerance. Tissue culture techniques have been applied to study cellular level stress response. The object of this study was to identify buffelgress cellular lines with salt tolerance. Caryopses of PI240170 accesion were used for callus induction. Culture medium was MS (1962) supplemented with 1.0 mg.1-1 2,4-D; 0.3 mg.1-1 IAA; 30 g.1-1 sucrose and 5.0 g.1-1 agar-gel. Cell suspension cultures were initiated by placing 0.5 g of friable callus into a 125 ml Erlenmeyer flask containing 30 ml of same medium without agar-gel. Cellular suspensions were grown on media amended with either O, 60, 120 and 180 mM NaCI. Samples were taken every six days to evaluate fresh weight and cell viability. Treatment with 60 and 120 mM NaCI showed the best growth pattern.

CongressOn In VitroBiology

Effect Of Carbohydrate Stress And Infection By The Plant Pathogenic Bacteria Pseudomonas ~yringae pv. syringae R-32 On Cowpea Calli (Vigna unguiculata). L. ALCAK.~-MELENDEZ, S. REAL COSIO and Y. BASHAN. Exp. Biol., CIBNOR, La Paz, B.C.S., Mdxico, CP.23000. E-mail:. Sucrose is normally supplied as a carbohydrate source to cultured plant cells. If not, the plant tissues undergo stress. Pseudomonas syringae pv. syringae R-32, a pathogenic bacteria that forms brown spots on leaves of inoculated cowpea plants also causes plant stress. Stress can be detected by measuring the production of primary metabolites like proteins and specific activity of some enzymes that help protect plant tissues. we wished to quantify the production of proteins and three enzymes as plant defense variables, on submitting cowpea calli to two stresses, carbohydrate and pathogen stress. The effects were measured ten days after inoculation with the pathogen on supressed carbohydrate calli. Changes in total proteins, phenolic acids, and the enzymes peroxidase, polyphenol oxidase, and glucose-6-phosphate dehydrogenase occurred in the calli tissues. The results suggest that carbon source stress and pathogen attack in cowpea tissue culture enhanced all enzymes associated with plant defense mechanisms, but did not induce increased production of an additional quantity of proteins. The latter were significantly increased in nonstressed calli to high levels after the pathogen attack as was the accumulation of phenolic acids. We conclude that carbon-nonstressed calli are better models to study pathogenesis of Pseudomonas ~yringae pv. syringae R-32.

P-1124 ~sozyme patterns seen in two species of lichens (i.e. Parmefia sulcata Tayl. and P, caperata (L.) Ach.). Shirley-Jean Hassett and Peter M. Bradley, Department of Biology, Worcester State College, Worcester MA O1602-2597. We wish to culture lichen phycobionts and mycobionts and study the lichenizing process as these components are brought together in culture. As part of this work, we wanted to know if isozymes can be detected and used as markers in lichen-forming experiments. For this preliminary study, two species of Parmelia were collected from a location in Barre, Massachusetts that was pasture prior to about 1900 and has since reverted to forest. Lichens were obtained from a rocky substrate (i.e. schist) and also from the bark of oak trees. Specimenswere air dried in the dark for 2-4 days and then rehydrated in water for 2h. Proteins were extracted from pieces of thallus by grinding in cold buffer (Le. O.1M Trizma, 0.02% ~-mercaptoethanol, 0.1% bovine serum albumin, 1% polvinyl pyrrolidone, pH=7.4). Proteins were separated by horizontal electrophoresis using a gel buffer of 5mM L-histidine pH=7.0 (i.e. 7% polyacrylamide + 2% starch gel; and electrophoresis for 20 mins. at lS0V + 16 -18h at 80V at 4°C). Gels were stained to locate the following isozymes: glucose 6phosphate dehydrogenase,phosphoglucosa isomerase, diaphorase, alkaline phosphatase and urease. The results showed that the enzymes were present in both lichens. In most cases the isozyme forms were different between specimens of the same species collected from different substrates. This may be due to colonization of the substrate at different times by different variants of the lichens (i.e. the rocks were available before the trees had grown), tsozyme analysis could be useful to detect components in experimentally produced lichens.

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P-1125

P-1126

In Yitro Propagation of Some Woody Species as a Part of the US-Hungarian Horticultural Research Program for Stress Tolerant Ornamental Plants. EKIKA SZENDP,AK 1'2, Paul E. Read I and Elizabeth Jfimbor-Benczair2, 1Universityof NebraskaLincoln, Department of Horticulture, NE 68583-0724, USA; 2Universityof Horticulture and Food, Budapest, Hungary. Because of the similarity of the climate and. geophysical characteristics of Nebraska and Hungary, a joint project was initiated in 1994 to examine the potential of Hungarian-developed stress-tolerant ornamental plants for adaptation to conditions endemic to Nebraska and the northern Great Plains region of the United States. A reciprocal component of this project includes studies of the ability of ornamental plants developed in the U.S. to tolerate the challenging conditions found in urban and other stressful locations in Hungary. Specific research has focused on propagation technologies for production of sufficient plant numbers to facilitate greenhouse, nursery and field testing of candidate genotypes. Several Hungarian genotypes have been successfully propagated at the University of Nebraska-Lincoln, Department of Horticulture and Statewide Arboretum (NSA) facilities. Micrnpropagation was accomplished for Salix matsudana 'Golden Spiral' and Populus alba x grandidentata 'Favorit'. Our objective was to establish contamination flee in vitro cultures of these cultivars, proliferate and finally root and acclimatize them. Cultures were initiated via forcing from dormant twigs of these cultivars at the University of Horticulture and Food at Hungary. Dormant branches were forced in a 2% sucrose solution supplemented with 200 mg 8hydroxyquinoline-cilrate/l and then the sotb,vood outgrowths were used as explants. The explants were disinfested with a series of bleach and/or ethanol solutions and rinsed in sterile distilled water before placing them onto the medium. The disinfested one-bud long explants were cultured directly on a shoot proliferation medium for one month, tested for disease and then the clean in vitro cultures were sent to the Department of Horticulture at the Universityof Nebraska-Lincoln. ARer several experiments we found that the best proliferation medium for these cultivars is full strength woody plant medium (WPM, Lloyd and McCown, 1981) supplemented with 0.01 mg NAA/1,0.2 mg BA/I, 5g agar/l and 1.6g gelrite/l with the pH adjusted to 5.6. Using this medium we were able to prevent the problem of vitrification, and achieved a relatively longer culture period with high multiplication rate and healthy plantlets. Our current work focuses on rooting and acclimatization.

High Frequency Plant Regeneration From Callus Cultures of Tvpha anzustifolia. K. S. Sarma, Kazumi Hodono and Suzanne Dethier Rogers, Department of Bioscience, Salem-Teikyo Univ., Salem1 WV 264261 Rogers~Salem.WVNet.edu Typha angustifolia (Narrow Leaf Cattail) is a freshwater wetland monocot of high biomass production. Typha species are widely used for the remediation of polluted waters. Genetic engineering of Typha~ with genes coding for decontamination activities, has the potential to increase the remediation abilities of these ecologically important plants. Reliable tissue culture and transformation protocols are necessary for such genetic manipulations. No tissue culture systems are available for T. angustifolia. Here we present a system with high frequency plant regeneration from callus cultures of T. angustifolia. Seeds were surface sterilized with bleach followed by mercuric chloride, and placed under continuous light for 3 days to induce germination. Callus was induced, in the dark, from seedlings planted on gelled MS medium supplemented with picloram. Callus initiation was observed, after 10 days, from the root and shoot juncture. Four weeks old calli, upon transfer to BA containing medium, regenerated shoots at a high frequency. Roots were induced on shoots cultured on medium containing NAA, and plants were established in the greenhouse. Factors affecting callus growth, shoot regeneration, rooting and greenhouse establishment of plants are described.

P-1127 Embryogenesis of Wheat (TJ'iticum aestivum L.) Microspores is Enhanced b y t h e Stress Treatment o f Anthers. M. Y. ZHENG. Department o f Biology, Houghton College, Houghton, N Y 14744. Stress has been found to elicit in vivo responses in many plant species. The present study was designed to exploit the effects of starvation and temperature on in vitro embryogenesis o f isolated wheat microspores. Tillers o f the spring wheat cultivar Pavon 76 were sampled when most microspores within anthers in the middle of the spike were at the uninucleate stage. They were then starved under 5, 28, 33 C or a combination o f two temperatures for 2 through 7 days prior to the isolation o f microspores by a micro-blender. When microspores with a star-like cytoplasm, an early indicator o f embryogenesis, were counted immediately following the isolation, starvation at 33 C for 48 hr increased the count by 13%; while 28 C for 48 hr!ed to an 18% increase; the combination o f 5 C for 5 days and 33 C for 48 hr had the most increase at 28%. Subsequent culture with CHB-2 medium plus low melting temperature agarose ;howed that over 90% o f microspores with star-fike cytoplasm had undergone cell divisions leading to the formation o f embryoids. The final embryoid yields were directly correlated with the number ofmicrospores showing star-like cytoplasm. A m o n g all embryoids, 85% regenerated into plants. With stress treated microspores, a much lower density of 3.5 x 104/ml rather than a 3 x 106/ml as previously reported, was required to achieve embryogenesis. The results indicate that the starvation is critical for embryogenesis induction and that high temperature may accelerate its effects. L o w temperature, on the other hand, may shift the embryogenesis to a later stage, explaining the combined effect of low plus high temperature starvation.

Congress On In Vitro Biology

P-1128 High Plant Regeneration from In Vitro Explants by Somatic Embryogenesis in Sweetpotato (Ipomoea batatas (L.) Lain.). G CIPRIANI, S. Diaz and A. Golmirzaie. International Potato Center, Apartado 1558, Lima 12, Peru. Factors influencing plant regeneration from somaGc embryos in sweetpotato were studied in several experiments using in vitro plants of cultivars Maria Angola, Jonathan and Morada Inta as explant sources. Two types of explants were tested: leaf segments (3x5 ram) and complete leaves with an X-type cut in the cen~'al part of the lamina. From both type of explants, embryogenic calli and somatic embryos were obtained, although, fenolizatJon was observed in the borders of complete leaves, causing some loss of those explants. On induction media, concentrations of 2,4 D were tested, including 0.01, 0.05, 0.1 and 0.5 rag/1.. The best embryogenic plant regeneration results were obtained with a 0.05 mg/L concenb'ation. Once the most suitable 2,4 D concenl~ation was determined, the effect of supplementing induction and regeneration media with gibberelic acid was studied, testing concentrations of 0.05, 0.1 and 0.5 mg/L. With 0.1 mg/L more regenerants were obtained and they had the best conformation. Another factor evaluated was duration of induction period, testing exposures of 35 and 55 days. More plants per explant were formed when induction period was shorter. Cul~var Maria Angola showed the best response with 16 to 22 plants per explant. Plant regeneration was observed at 3.5 months in cultivars Maria Angola and Morada Inta, and at 5 months in Jonathan. The maximum regeneration expressed as a percentage {number of regenerating explants/number of total explants) for Maria Angola was 100 and for Jonathan was 96%. With cultJvar Morada Inta, only organogenic regenerationwas obtained with a 35-day exposure, and a maximum embryogenic regeneration was 20% at 55 days.

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P-1130

P-1129 S t a n d a r d i z e d In V i t r o E x p e r i m e n t a l Protocol f o r High Frequency Mass M l c r o p r o p e g a t l o n of Jo~olo~. (Simmondsln ch Lmensts (LInk) S c h n e i d e r ) . Anees Khanam, Y B Nersing Rao end S A F a r o o k , Cytogenetics end Tissue C u l t u r e L a b o r a t o r y , Department of Botany, Osmnnls k'3ntverslty, Hydernbad 500 007. In the present study an attempt was made on micro p r o p a g a t i o n o f female jo~oba plant through tissue culture techniques using female nxillary bud e x p l e n t s as e x p e r i m e n t a l m a t e r i a l . Standard e x p e r i mental p r o t o c o l s and t e c h n i q u e s were f o l l o w e d under a s e p t i c c o n d i t i o n s . The s i n g l e stem fragment h a v i n g o p p o s i t e a x I l t o r y buds was inoculated in MS BM media with 3% sucrose and 0.8 ngnr. 5ubculturinc was c a r r i e d on in MS med|n f o r t i f i e d w i t h BAP concentration ranging from 1 mg/l to 10 mg/l. The percentngn f r e q u e n c y of shoot p r o l l f o r s t Ion was optimum when media was suppIemented with 4 mg/I of BAr. Successful rooting wns o b s e r v e d from shoot bunches when inocuIated in r o o t i n g media, supplemented w i t h B A r , IBA end a c t i v a t e d c h a r c o a l . Results i n d i c a t e t h a t c u l t u r e medium supplemented w i t h 4 mg/I of BAr was found to be ldeaI f o r micro propagation of JoJobe and t h i s may w e l l s e r v e as a s t a n d a r d e x p e r i m e n t a l p r o t o c o l f o r high f r e q u e n c y mass propagation of female Jo.loba p l a n t l e t s .

P-1131

GUS Expression in. Typha latifolia (Cattail) Cells Transformed with Agrobacterium. SUZANNE DETHIER ROGERS Dept. of Bioscience, SaleJn-Teikyo Univ., Salem, WV 26426-0500 Ro~ersf~Salem.WVNet.edu Typha latifolla is a freshwater wetland monocut common throughout open marshes of temperate and tropical regions. It is widely planted in constructed wetlands used for the phytoremediatlon of polluted waters. Research has lead to isolation of genes coding for specific pollutant decontamination activities. Typha is a potential target for genetic transformation because is highly productive and able to tolerate a wide range of environmental conditions. While numerous monocot species have been transformed by Agrobacterium to date no studies have been published on genetically transforming Typha. The objectives of this study were to determine if Typha cells could be genetically transformed by Agrobacterium and if early GUS expression could be used to facilitate identifying conditions that yielded expression. Typha suspension cultures were grown in MS medium with picloram. Cells were wounded by cutting with a scalpel blade or by mashing with a spatula. After 48 his the cells were inoculated with Agrobacterium carrying an intronGUS reporter gene. The cells were cocultivated for 2 days, rinsed with MS medium containing 250 mg/I c e f o ~ e , resuspended in MS medium and grown for 2 and 5 days. The cells were then stained for GUS activity to detect early transformation events. Blue cells or dusters of cells occurred at low frequencies in cultures that had been wounded by cutting or mashing, and grown 2 or 5 days after cocultivation. No GUS positive cells were detected in unwounded cells. In a relatively short period of time the GUS gene could be used to determine if transient Agrobacterium transformation occurred in this plant.

P-1132

Exogenous Application of Auxin Enhances Agrobacteriummediated Transformation Efficiency of Etiolated Internodes from 'Royal Gala' Apple, Q. Liu and FoA. Hammerschlag. USDA/ARS, Fruit Laboratory, BeltswiHe, MD 20705. Email: [3.glucurouldase ((;US) expression and the production of transgenie 'Royal Gala' apple (Ma/us x domest/ca Borkh,) were evaluated following cocaltivafion of etiolated internodes with A g r a ~ u m tumefac/ens in the presence or absence of amfin. Early stages of the transformation process were monitored by counting the number of GUS expressing leaf zones immediately after cocultivalion with A. tumefac/ens strain EHA105 (p35SGUSint), as well as, by counting the number of GUS expressing cam developing after 2 weeks of postcultivation on kanamycin selection medium. Cocultivatiun of explants on medium with auxin yielded more than two-fold as many GUS expressing zones and caUi compared to those on medium without am6n. Anatomical and histochemical observation showed that cells expressing GUS zones were located on the cut surface, inner cortex and vascular bundles, and less frequently in the epidermal and subepidennal layers where adventitious shoot initiation occurred. Eight transgeni¢ apple plants with the GUS gene were obtained from 356 interuodal explants after cocultivation with A. tumefac/ens strain EHA101 (pEHA101/pGT100) and regeneration on medium with 2.7 pM NAA compared with none from 363 explants on regeneration mediumwithout auxin. The integration of the GUS gene into the apple genome was confirmed by PCR and Southern blot analyses.

-CongressOn In Vitro Biolog7

Genotype-Dependent embryogenesis, Organogenesis and Agrobaeterium mediated transformation in Pigeonpea (Cajanus cajan L.) R.V. RAMANA, CH Venu, T. Jayasree, A. Sadanandana, Plant cell and tissue culture lab, Department of Botany, Kakatiya University, Warangal - 506 009, A.E hldia. The morphogenic response o f seven different genotypes o f Pigeonpea to various growth regulators was evaluated. The explants like immature cotyledons were used for the induction o f direct somatic e m b r y o g e n e s i s on Blades m e d i u m with 2,4,5-T or 2-4D in combination with BA or KN. Within 2 to 3 weeks embryos were induced directly from the explants without an interveining callus phase. The greatest number of embryos/explant were obtained with LRG-332 on 2,4,5T + BA medium. Immature embryonal axes were used for the organogenesis on Blades medium with 1.125 mg/L BA. Within one week callus is formed from the explants followed by initiation o f shoot buds. Among the tested cultivars MRG-66 shown the best response with 9.3 + 0.04 shoots/explant, The shoots elongated within 15 days on basal medium and rooted on 0.2 mg/L NAA. The protocol described for organogenesis was suitable for Agrobacterium mediated genetic transformation by the co-cultivation technique. The immature embryonal axes of MRG-66 & ICPL 332 were removed from the field grown pods and cocultivated with Agrobacterium tumefaciens strain LBA 4404. These explants were transferred to regeneration medium with 200 mg/L Cefotoxime. After one week the explants placed on final selection medium wi'th 1.125 mg/L BA + 200 mg/L Cefotoxime + 100 mg/L Kanamycin. the putative transformants were rooted on rooting medium with 50 mg/L Kanamycin, The embryo axes with developing shoots were stained histochemically for the detection of Gus expression. High frequency of transformation (25%) was obtained with MRG 66.

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P-1133

P-1134 The Cryopreservationof In Vitro Cultured Apple Shoot Tips by Drop-let YANHUA ZHAO1, Yongjie Wu1, Fli~rent Engelmann2, Mingde Zhou3, Shuangying Chen1, Lyndsey A. Withers2 and Deming ZhangI IChangh Institute of Pomology, Heibei A~ademy of Agricultural and Forestry Sciences, Changli, Hebci 066600, P. R. China; 2IPGRLVia delle Sette Chiese 142, 00145 Rome, Italy; 31PGRIOffice for East Asia, CAAS, 30 Baiskiqian Road, Beijing 100081, P. 1L China.

The Cryopreservation of In Vitro Cultured Apple Shoot Tips: The Effect of Water Content of Material Shoot Tips YONGJIE WU ~, Yanhua Zhao~, Florent Engelmann2, 1~fmgde Zhou3, Shuangying Chen1, Lyndsey A. Withers2 and Deming Zheng~ ~ChangliInstitute of Pomology, Hebei Academy of Agricultural and Forestry Sciences, Changli, Heibei 066600, P. R. China; 2IPGRI, Via delie Sette Chiese 142, 00145 Rome, Italy; 3IPGRI Office for East Asia, CAAS, 30 Baishiqiao Road, Beijing 100081, P. R. China.

The in vitro cultured apple shoot tips were successfully

cryopreserved by using drop-let method. One optimal program: DMSO (1.0%) preculture, the containment of shoot tips in 5~ droplet with 15% (v/v) DMSO on aluminum foil, two-step freezing method, fast thawing and directly subculture on standard condition, was proposed. The effects of material subculture days, DMSO preculture, different cryoprotaetant concentrations and prefreezing temperatures on the survival rate of shoot tips alter LN were studied. With DMSO 0.0%) preculture, the higher survival rate (90%) of shoot tips was got. But the more clearly effect of DMSO concentration on the survival rate was found in the droplet. Following the increasing of DMSO concentration (0.25, 0.5, 1.0, 3.0, 5.0 and 15%),the survival rate increased from 4.7% (0.25%) to 86% (15%). By plungingthe samples directly into LN, no shoot tips survived from cryopreservation,while by prefieezing the samples to -40°C before fast freezing, the survival rate of shoot tips arrived to 81%. Followedthe longing of the subculture days of materials, the survival rate of shoot tips increased from 44% (subculture 40 days) to 80% (subculture 100 days). By using the established program, shoot tips of 5 apple coltivars were successfully cryopreserved, the highest survival rate after LN arrived to 92%.

The in vitro cultured apple shoot tips were cryopreserved by encapsulation dehydration method. The water content of shoot tips was tested and the effects on the cryopreservation of apple in vitro shoot tips were studied. Following the subculture of materials, the water content of material shoot tips decreased. After 3 weeks subculture, the water content of material shoot tips is about 85%, while after 6 months subculture, the water content decreased to 64%. The lower water content of material shoot tips improved the cold resistance and dehydration resistance of shoot tips during cryopreservation. When the water content is about 85%, the survival rate of shoot tips after LN is only about 59°,6; while the survival rate increased to 80°,6 when the water content decreased to 69°,6, at the same time, the survival rate of shoot tips after LN without sucrose preculture arrived to 28%. By selecting the materials with about 69°,6 shoot tip water content, shoot tips of 11 freely selected apple cultivars were successfully cryopreserved, the highest survival rate arrived to 80%.

P-1136

P-1135 The Cryopreservation of In Vitro Cultured Apple Shoot Tips by Simple Encapsulation-dehydration YANHUA ZHAO ~, Yongjie Wu ~, Florent Engelmann2, Mingde Zhou3, Shuangying Chen1, Lyndsey A. Withersz and Deming Zhang~ ~ChangliInstitute ofPomology, Hebei Academy of Agricultural and Forestry Sciences, Changli, Heibei 066600, P. R. China; :IPGRI, Via delle Sette Chiese 142, 00145 Rome, Italy; 3IPGRI Office for East Asia, CAAS, 30 Baishiqiao Road, Beijing 100081, P. IL China

The Effect of Cold Hardening on the Cryopreservation of In Vitro Cultured Apple Shoot Tips YONGJIE WU I, Yanhua Zhao 1, Florent Engelmann2, Mingde Zhou3, Shuangying Chenl, Lyndsey A. Withers2 and Denting Zhang~ ~Changli Institute of Pomology, Hebei Academy of Agricultural and Forestry Sciences, Changli, Heibei 066600, P. R. China; 2IPGRL Via delle Sette Chiese 142, 00145 Rome, Italy; 3IPGRI Office for East Asia, CAAS, 30 Baishiqiao Road, Beijing 10008 I, P. R~ China.

The in vitro cultured apple shoot tips were successfully cryopreserved by using the simple encapsulation-dehydration method. The effect of culture duration were studied. The result demonstrated that for cryopreservation, the shoot tips of subcultured 3 weeks materials must be precultured with 0.1M, 0.3M, 0.7M and 1.0M sucrose daily and dehydrated in air-flow for 4 hr before fast freezing. The survival rate of shoot tips (Malus. rubusta and M. domestica cv.Tsugaru) after LN is 88.3% and 70°,6. While with subcultured 25 weeks materials, the survival rate of shoot tips after 1.0M sucrose preculture for 1 day and 1 hr dehydration is about 80°,6 and 71% , even without sucrose preculture, only by 1 hour dehydration, the survival rate of shoot tips also arrived to 81.8% and 71.4%. By controlling the subculture duration of materials, one simple encapsulation-dehydration method was established: the encapsulation of shoot tips, 1.0M sucrose preculture or without sucrose preculture, dehydration, fast freezing and thawing, and directly subculture on standard conditions.

The in vitro cultured apple shoot tips were cryopreserved by using simple encapsulation-dehydration method including encapsulation, 1.0M sucrose preculture, dehydration, fast freezing and thawing, and directly subculture on standard condition. The effect of cold hardening on the cryopreservation of in vitro cultured apple shoot tips has been studied. Though the cold hardening (5°C, 8h light/16h dark) did not change the water content of material shoot tips and the survival rate of shoot tips before LN, the survival rate of shoot tips after cryopreservation were improved. Without cold hardening, the survival rate of shoot tips after LN was only about 7%, while after two to three weeks cold hardening, the survival rate increased to 85%. But with longer cold hardening, the survival rate decreased. Also when the materials were cold hardened for two weeks, even without sucrose preculture, the survival rate of shoot tips a~er LN could arrive to 21%. The experiment demonstrated that as one of the important factbrs in the cryopreservation of apple in vitro shoot tips, cold hardening could improve the dehydration and cold resistance of shoot tips.

Congress On In Vitro Biology

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P-1137 In Vitro Clonal Propagation of an Elite Cultivar of P o m e g r a n a t e (Punica granatum L. cv. Ganesh) using Nodal Explants from M a t u r e Tree. S. K. NAIK and P. K. Chand, Plant Tissue and Cell Culture Facility, P. G. Department of Botany, Utkal University, Bhubaneswar 751004, Orissa India.

P-1138 T r o p h i c a n d H o r m o n a l F a c t , s In0.uence o n Stevia r e b a u d i a n a Bertoni s n o o t s c / r o w t h in t h e Roller Bioreactor. N.I. B O N D A R E V a n d A . M . N o s o v . Timiryazew Institute of Plant Physiology, B o t a n i c h e s k a y a Str., 35, 127276 M o s c o w , R u s s i a . E-mail: v l a d i m i r @ a d . p l a n t p h y s . m s k . r u

Pomegranate (Punica ~ranatum L.), of the family Punicaceae, i s an i m p o r t a n t fruit tree of tropics. Moreover, the tree is also valued for its pharmaceutical properties. Conventional methods of propagation of these plants through stem cuttings is time consuming and tedious while seed propagation does not ensure true-totypeness. We report here an efficient procedure for in vitro clonal propagation of an elite cultivar of pomegranate (cv. Ganesh) using nodal stem segments from a mature tree. Axillary shoot proliferation was induced in the nodal segments in Murashige and Skoog's (1962) m e d i u m (MS) supplemented with 1.0 - 2.0 mg/l zeatin (Z) or 0.5 - 1.0 mg/l benzyladenine (BA). High frequency bud break and maximum number of shoot bud formation were recorded in MS containing 2.0 mg/l Z. These shoots were excised and rooted in half-strength MS medium (I MS) containing 1.0 mg/l indole 3 - butyric acid (IBA). The plantlets were hardened off and successfully established in soil.

Large-scale bioreactor cultivation o f disease-free stevia p l a n t s is very perspective for its m a s s cloning especially b e c a u s e o f s o m e difficulties in its seed p r o p a g a t i o n . S o m e t i m e s it c o u l d be used as a n alternative for p r o d u c i n g p l a n t r a w materials in t h e p l a n t a t i o n s . B u t c o n d i t i o n s o f stevia s h o o t s cultivation in a such system h a v e n o t b e e n s t u d i e d yet. T h a t is w h y we investigated the influence o f s o m e t r o p h i c ( c a r b o h y d r a t e s a n d mineral s u b s t a n c e s c o n t e n t s ) a n d h o r m o n a l ('NAA, BAP, G A 3 ) f a c t o r s o n t h e growth o f stevia s h o o t s in the bioreactor. Two-leaves s t e m m i c r o c u t t i n g s were used as initial explants. M i c r o s h o o t s cultivation was carried o u t in t h e roller bioreactor a p p a r a t u s . Special c~,lindrical vessels were used. Stevia m i c r o s h o o t s grew d u r i n g 5 weeks at the light in liquid M S m e d i u m u n d e r 25+1 C. T h e m o s t i m p o r t a n t biotechnological o..rowth character~tics s u c h as fresh a n d dry matter, a n d s-hoots lengh were 1,5-2 times m o r e in t h e rolIer bioreactor, t h a n in t h e control tubers. T h e increase o f m i n e r a l M S salts f r o m I to 2 s t a n d a r d s i m p r o v e d 1,5 times m o r e s h o o t s lengh, r o o t s lengh a n d q u a n t i t y , the n u m b e r o f leaves pairs a n d their fresh a n d dr)' m a t t e r . 15-20% increase o f dry m a t t e r was achieved in n u t r i e n t m e d i u m s u p p l e m e n t e d by 3% s u c r o s e c o m p a r i n g to t h e s a m e c o n c e n t r a t i o n s o f g l u c o s e a n d fructose. Rising sucrose c o n t e n t s f r o m 2 to 3% resulted in 60% increase o f dry m a t t e r , a n d its g r o w t h f r o m 3 to 5% developed it by 25-30%. S u p p l e m e n t i n g this M S m e d i u m with 0,1 mg/I B A P or 0,1 mg/l B A P + 0,1 mg/l N A A s t i m u l a t e d the g r o w t h o f s h o o t s n u m b e r . Last h o r m o n a l v a r i a n t p r o d u c e d 1,5 times m o r e s h o o t s . O n t h e c o n t r a r y , their roots were poor. W h e n cultivating in MS m e d i u m with GA3 (I,0 mg/l), fresh m a t t e r grew by 66% a n d dry m a t t e r by 15% at the expense o f s t e m s e l o n g a a t i o n .

P-1139

P-1140

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The Effect of PPM on Carnivorous Plant Tissue Culture Media. ERIC CUMBEE 7th grade, Ben Hill County Middle School, Fitzgerald, GA 31750. E-mail: . In the tissue culture process,contamination of media by bacteria and fungi is a major problem. The objective of this project was to field test a plant preservative material (PPM) to observe the effectivenessof it as an antibiotic and fungicide on tissue culture media. The usefulnessof this study is of value to the company, and to the entire tissue culture community. By gathering data gained from these studies, the company can determine the effectivenessof the product. To the tissueculture community, including commercialand research, the development of a low cost plant preservativematerial will virtually change the procedures of tissue culture. It will prevent monetary and time loss by contaminated cultures. The hypothesisformulatedwas that PPM would reduce or prevent mold, fungus, and bacteria growth in tissue culture media. The procedures used were to placecarnivorousplant seeds and plant tissue in media with PPM and without PPM. In addition, six flasks of prepared media with and without PPM were exposed to air for different lengths of time. The outcome of the experimentationconcluded that the flaskscontaining PPM did prevent contamination of cultures and that the flaskswithout PPM contained varying degrees of contamination. With further study, several questions are remaining to be answered. Questions include: What is the effect of PPM on plant growth? How long will the PPM protect growing media from contamination? What are the correct amounts of PPM to be added to media for different types of plants?

Congress On In Vitro Biology

Engineering of Chloroplast Genome for the expression of Biopolymer, Herbicide and Insect resistance genes. H. DANIELL Department of Botany and Microbiology, Aubum University, Auburn AL 36849-5407. E-mail: daniehe~,,mail.aubum.edu Major advantages of chloroplast transformation include high levels of expression and containment of foreign genes because plastid transgenes are not transmitted by pollen. Protein based polymers can be prepared of varied design and composition through genetic engineering and can be made biodegradable with chemical clocks to set their half-lives such that they can be environmentally friendly over their complete life cycles of production and disposal. Compositions tested to date have been shown to be extraordinarily biocompatible, allowing for numerous non-medical and medical applications, including the prevention of post-surgical adhesions, tissue reconstruction and programmed drug delivery. Expression of the PBP(GVGVP)~21 in bacteria, fungi, cultured plant cells and transgenic plants (via chloroplast and nuclear genomes) will be presented. Genetic engineedng of herbicide resistance by stable integration of the EPSPS gene into either the large single copy region (tobacco vector) or the inverted repeat regions (universal vector) of the tobacco chloroplast genome and engineering insect resistance using the cryllA gene will be presented. Southem blot analysis shows stable integration of the foreign genes into all of the chloroplast genomes (5000-10,000 copies per cell) of transgenic plants. While control plants were extremely sensitive to glyphosate, transgenic plants survived sprays of high concentrations. Insects 40,000 fold resistant to crylAc, and a thousand fold resistant to cryllA showed 100% mortality when fed with transgenic leaves due to high levels of CRYIIA expression.

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