A96 Transplasma-membrane ferricyanide reduction in sycamore cells. Characterization of the system and inhibition by some phenyl biscarbamates J.-P. BLEIN 1, M.-C. C A N I V E N C I, X. De C H E R A D E 2, M. B E R G O N 2, J.-P. CALMON 2 & R. SCALLA l ILaboratoire des Herbicides et Autres Produits Phytosanitaires, LN.R.A., B V 1540, F-21034 Dijon Cedex; :Laboratoire de Chimie Organique Biologique et de Physico-Chimie des Sols, E.N.S.A., 145 av de Muret, F-31076 Toulouse Cedex, France Plant Science, 46:77-85, 1986 Abstract. Evidence is presented for the presence of a transp|asma-membrane redox system in sycamore cells, which were found able to reduce external ferricyanide. This reduction induced a simultaneous acidification of the external medium, with a H + / e - stoichiometry of 0.925. Ferricyanide reduction was accompanied by a slight reduction of potassium uptake (15% inhibition). The transmembrane electron transport was inhibited by oligomycin and quinacrine; the effect of the latter inhibitor was only temporary, owing to its progressive absorption by the cells. At 100 #M, several derivatives of the herbicide phenmedipham were able to inhibit the growth of sycamore cells. These compounds inhibited the external acidification induced by fusicoccin, without interfering with the integrity of the plasmalemma. They had no effect on the phosphohydrolase activity of a microsomal fraction enriched in plasmalemma ATPase. They were not uncouplers of oxidative phosphorylation, and appeared as poor inhibitors of mitochondrial electron transfer (150 > 100#M). In intact cell suspensions, they inhibited both the reduction of ferricyanide and the accompanying acidification of the external medium (I50 = 32/~M). Moreover, they could induce a reversal of the functioning of the redox system, which consequently induced ferrocyanide oxidation.

Embryogenesis from microinjected single cells in a carrot cell suspension culture KOJI NOMURA & ATSUSHI KOMAMINE Institute of Biology, Faculty of Science, Tohoku University, Sendai, 980, Japan Plant Science, 44:53-58, 1986 Abstract. A microinjection method was established for intact single cells with cell walls using a carrot suspension culture system in which selected single cells differentiate to embryos at high frequency. A solution of a fluorescent dye, Lucifer Yellow CH, was microinjected into those single cells, using an inverted microscope and a hydraulic micromanipulator. In order to hold cells with cell walls and to overcome their turgor pressure, certain modification to conventional microinjection methods for protoplasts were necessary. The microinjected cells could divide and differentiate to embryos at a frequency of about 50%.

A97

Tunicamycin affects somatic embryogenesis but not cell proliferation of carrot F I O R E L L A L O S C H I A V O 1, L U I S A . Q U E S A D A - A L L U E 2 & Z. R E N E E SUNG llstituto di Mutagenesi e Differenziamento CNR, Via Svezia, 10 56100 Pisa, Italy," 2Department of Genetics and Plant Pathology, University of California, Berkeley, CA 94720, USA Plant Science, 44:65-71, 1986 Abstract. The antibiotic tunicamycin which specifically blocks the first step in the lipid-linked

oligosaccharide pathway is capable of arresting somatic embryogenesis in a reversible way. At the same drug concentration cell proliferation is not affected. The quantitative and qualitative changes induced by tunicamycin in glycolipids and glycoproteins are the same in embryogenic and nonembryogenic conditions and this might therefore indicate some proteins whose glycosylation is essential for development.

Leaf disc transformation of cultivated tomato using Agrobacterium tumefaciens

(L. esculentum)

SHEILA McCORMICK, JEANNE NIEDERMEYER, JOYCE FRY, ARLENE BARNASON, ROBERT HORSCH & ROBERT FRALEY Biological Sciences, Monsanto Company, 700 ChesterfieM Village Parkway, St. Louis, MO 63198, USA Plant Cell Reports 5:81-84, 1986 Abstract. The leaf disc transformation/regeneration system was modified for tomato (L. esculen-

tum). Both leaf explants and cotyledon/hypocotyl sections can be used to regenerate transformed plants. We have obtained over 300 transgenic plants from eight tomato cultivars. We have evidence for both single and multi-copy insertions of the T-DNA, and have demonstrated inheritance of the T-DNA insert in the expected Mendelian ratios. Several heterologous promoters function in tomato. A reduced etficiency of transformation was observed with binary T-DNA vectors as compared to co-integrate T-DNA vectors. The ease of the leaf disc method makes tomato a premier experimental organism for plant biotechnology.

A98 Plant regeneration from protoplast-derived callus of rice (Oryza sativa L.) YASUYUKI YAMADA, YANG ZHI-QI & TANG DING-TAI Research Centerfor Cell and Tissue Culture, Faculty of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606, Japan Plant Cell Reports 5:85-88, 1986 Abstract. Protoplasts isolated from cultured rice cells of an A-58 cytoplasmic male sterile line (A-58 MS)(Oryza sativa L.) were used to investigate the regeneration of rice plants. A cultured cell line (T3) of A-58 MS with a high growth rate and dense cytoplasm was selected. About 10% of the protoplasts prepared from this established cell line plated in RY-2 (a new medium) formed colonies. The calli formed shoots and roots in the regeneration medium and developed into whole plants. Protoplasts also were prepared from suspension cultures of 25 other varieties of rice using the same methods. The protoplasts isolated from two of the 25 varieties, Fujiminori and Toyotama, had high rates of cell division in RY-2 medium. Only protoplast-derived calli from Fujiminori, produced whole plants in the regeneration medium.

Plant regeneration and initiation of cell suspensions from root-tip derived callus of Oryza sativa L. (rice) JANUSZ

ZIMNY l & HORST

LORZ 2

1Botanical Garden of the Polish Academy of Sciences, Warsaw, Poland, Max-Planck-lnstitut J~r Ziichtungsforschung, D-5000 K6ln 30, FRG Plant Cell Reports 5:89-92, 1986 Abstract. Root-tip derived suspended callus of Oryza sativa cv. Thaipei showed the capacity for plant regeneration via organogenesis. Cell cultures were induced in liquid Murashige-Skoog medium containing 2 mg/1 2.4-dicholorophenoxyacetic acid. Dicamba or Picloram were effective for induction of organogenesis. Shoots and roots differentiated following subculture on medium lacking auxins but containing kinetin. At 1 and 4 mg/1 Dicamba and 1 mg/1 Picloram normal green plants were regenerated whereas with 7 mg/1 Dicamba in the medium only albino plantlets were obtained. Regenerated plantlets were grown to maturity and set seed. Cell suspension cultures, initiated from the root-tip derived calli, provided suitable material for protoplast isolation.

A99

Transformation of Solanum nigrum L. protoplasts by

Agrobacterium rhizogenes ZHI-MING WEP, HIROSHI KAMADA & HIROSHI HARADA Institute of Biological Sciences, University of Tsukuba, Sakura-mura, lbaraki-ken 305, Japan Plant Cell Reports 5:93-96, 1986 Abstract. Solanum nigrum protoplasts were co-cultivated with Agrobacterium rhizogenes harboring

agropine-type Ri plasmid (pRi15834). A large number of transformed calli were obtained on Murashige and Skoog's (MS) medium lacking plant growth regulators. Frequency of transformation was about 3.5 x 10-3. In most of the calli, hairy roots appeared on MS medium without plant growth regulator. When the hairy roots were cut into segments and subcultured on MS medium lacking plant growth regulators, calli were readily formed. Plantlets were regenerated by transferring those calli to MS medium supplemented with 1 mg/l zeatin and 0.2 mg/1 naphthaleneacetic acid. Frequency of plant regeneration was about 70%.

Transformation of Medicago by Agrobacterium mediated gene transfer MARIA DEAK, GYORGY B. KISS, CSABA KONCZ & DENES DUDITS Institute of Genetics, Biological Research Center of the Hungarian Academy of Sciences, P.O. Box 521, H-6701 Szeged, Hungary Plant Cell Reports 5:97-100, 1986 Abstract. Shoot segments of Medicago varia genotype A2 were co-cultivated with Agrobacterium

tumefaciens strain bo42 carrying pGA471, a plasmid coding for the kanamycin resistant determinant as transferable positive selection marker in plant cells (An et al., 1985). Resistant plants were regenerated at high frequency from green calli developed on inoculated stem cuttings under kanamycin selection. DNA-DNA hybridization analysis showed the presence of the structural gene of the kanamycin resistant determinant in total DNA isolated from several independent transformants. All data presented clearly demonstrate the transfer, stable maintenance and functional expression of the kanamycin resistance marker in Medicago varia cells which retain their morphogenic property.

A100

A rapid and ellicient alternative procedure for the regeneration of plants from hypocotyl protoplasts of Brassica napus T.L. B A R S B Y , S.A. Y A R R O W & J.F. S H E P A R D Allelix Inc., 6850 Goreway Drive, Mississauga, Ontario L4V 1P1, Canada Plant Cell Reports 5:101-103, 1986

Abstract. Protoplasts of several spring and winter varieties of Brassica napus were isolated from hypocotyl tissue. Protoplasts divided and formed cell colonies at high frequency, without browning when cultured in modified Shepards' medium. This high efficiency of proliferation was sustained through to plant regeneration with all varieties cultured. This has been attributed to the incorporation of a reservoir medium, the presence of 2,4-D in the proliferation medium, and the presence of kinetin in conjunction with lowering of the sucrose concentration in the regeneration medium.

An unstable anthocyanin mutation recovered from tissue culture of alfalfa (Medicago sativa) 1. High frequency of reversion upon reculture R.W. G R O O S E l & E.T. B I N G H A M Department of Agronomy, University of Wisconsin, Madison, WI 53706, USA Plant Cell Reports 5:104-107, 1986 Abstract. A white-flowered mutant ('WFM') was regenerated from tissue culture of a purple-flowered plant of tetraploid alfalfa (Medicago sativa L.). When WFM was recultured, many regenerated plants ( > 2 0 0 ) were purple-flowered. Genetic analysis established that a functional allele, C2, of a locus required for anthocyanin pigmentation was in the simplex condition (C2c2c2c2) in the donor genotype when it mutated to an unstable recessive ('mutable') allele, c2-m4, which is carried by WFM. Tissue culture experiments demonstrated that c2-m4 reverts to function at a high frequency in vitro. Results indicate that reversion occurs early in culture and may be the result of a genome shock associated with callus formation. Reversion also occurs in planta, but at a much lower frequency than in vitro. The c2-m4 allele is transmitted to progeny which revert in tissue culture. Revertant alleles, like the progenitor allele, are stable and are sexually transmitted. The action of a transposable element which is especially active in vitro is suggested.

AIOI

An unstable anthocyanin mutation recovered from tissue culture of alfalfa (Medicago sativa) 2. Stable nonrevertants derived from reculture R.W. GROOSE l & E.T. BINGHAM Department of Agronomy, University of Wisconsin, Madison, WI 53706, USA Plant Cell Reports 5:108-I10, 1986 Abstract. An unstable recessive ('mutable') allele, c2-m4, of a locus required for anthocyanin pigmentation in alfalfa (Medicago sativa L.) reverts to a stable functional state at high frequency in vitro. It was previously established that a white-flowered mutant ('WFM') and a white-flowered progeny of WFM ('WHGW3') each carry the unstable allele. More than 20% of plants regenerated from tissue cultures of WFM and WHGW3 are revertant. It is here established that most nonrevertant plants regenerated from cultures of WFM and WHGW3 are stabilized in the recessive condition. Reculture of nonrevertants of WFM and WHGW3 indicated that there are three classes of nonrevertants: (i) Nonrevertants which revert in vitro at a high frequency typical of WFM; (ii) Nonrevertants which revert upon reculture but at significantly lower frequencies than WFM; and (iii) Nonrevertants which do not revert upon reculture. These observations are discussed in terms of transposable element action in vitro.

Secondary product formation by cultures of Beta vulgaris and Nicotiana rustica transformed with Agrobacterium rhizogenes J.D. HAMILL, A.J. PARR, R.J. ROBINS & M.J.C. RHODES Plant Cell Culture Group, AFRC Food Research Institute, Norwich Colney Lane, Norwich NR4 7UA, UK Plant Cell Reports 5:111-114, 1986 Abstract. 'Hairy root' cultures of Beat vulgaris and Nicotiana rustica were established after roots were induced on plants following infection with Agrobacterium rhizogenes. The transformed cultures of B: vulgaris and N. rustica synthesised their characteristic secondary products, the betalain pigments and nicotine alkaloids respectively, at levels comparable with those of in vivo roots from the same variety. Betalains were entirely retained inside the root tissue. In contrast, a proportion of the nicotine alkaloids was secreted into the medium. The potential of this type of in vitro plant tissue culture for the production of valuable plant secondary products is identified and confirmed.

A102

Embryogenesis and plant regeneration from cotyledon protoplast culture of cucumber (Cucumis sat&us L.) SHI-RING JIA, YOU-YING FU & YUN LIN Vegetable Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China J. Plant Physiol. 124:393-398, 1986 Abstract. Protoplasts were isolated from cotyledons of six cultivars, three gynoecious lines, and two F t hybrids of cucumber. Sustained division was obtained when protoplasts were cultured in modified DPD liquid medium supplemented with 0.5mg.1-2 2,4-D and I mg.1-1 kinetin. The division frequency reached 46% after eight days in culture. After transfer of cell colonies onto modified MS agar medium supplemented with 0.01 mg.l-t 2,4-D and I mg-1-l benzylaminopurine (BA), or 0.2 mg'l-1 indoleacetic acid (IAA) and 0.5 mg. 1-1 BA, all eleven lines formed callus. The callus of the F1 hybrid 37-1G x 78 - - 50 produced a larger number of somatic embryos on medium with the hormone combinations of 0.2mg.1 -~ IAA and 5mg.1 -~ kinetin, or 2mg.1 -~ BA without auxin. When embryos were transferred onto a medium with half strength MS nutrients devoid of exogenous hormones, plantlets with shoots and roots developed.

Cytochemical studies of callus development from mierospore in cultured anther of rice S.S. TSAY l, H.S. TSAY2 & C.Y.

CHAO I

t Department of Biology, Tunghai University, Taichung, Taiwan 400, ROC; 2Department of Agronomy, Taiwan Agricultural Research Institute, Taichung, Taiwan 431, ROC Plant Cell Reports 5:119-123, 1986 Abstract. Cytochemical studies of androgenic anthers of Oryza sativa picked from the culture at 2 day intervals from 0 to 40 days have been carried out. Glutaradehyde-OsO4-fixed and plasticembedded sections were stained with TBO, SBB and PAS for acidic polymers, lipids and polysaccharides respectively. Among the population only 4% of microspores, which accumulate abundant amorphous lipid in the first few days of culture, are androgenic. Less than 30%, which have many lipid granules and some amorphous lipid, become nutritive microspores. Starch grains also accumulate in these nutritive microspores which degenerate at the stage when the androgenic multicellular microspores are in rapid development. The remaining microspores, which have no or little lipid, degenerate early. At about the 100-cell stage, each multicellular unit consists of two cell types, large and small. The large cells contain abundant amorphous lipid and starch grains which the small ones stain intensely with TBO. Our results indicate that the epidermis and endothecium of the cultured anthers are not quiescent. They can accumulate and transport lipid and polysaccharides at certain stages during the cultural period. Globular embryoid appearing structures and leaf-like protrusions can be observed at the surface of the callus in about 40-day old culture, indicating that both embryogenesis and organogenesis may take place in rice callus.

A103

Callus production from leaf protoplasts of various cultivars of bean (Phaseolus vulgaris L.) L. CREPY, L.M.G. BARROS & V.R.N. V A L E N T E CENARGEN (EMBRAPA), Cx. P. 10.2372 CEP-70770, Brasilia-DF, Brazil Plant Cell Reports 5:124-126, 1986

Abstract. High yields of viable protoplasts were obtained by enzymatic treatment from cotyledonary leaves of various greenhouse grown Phaseolus vulgaris L. cultivars. The protoplasts divided and formed cell clusters in a liquid medium. Early transfer before 10 days in the same medium was necessary for the development of cell colonies. When transferred to solid medium, the colonies gave rise to proliferating green calli. Deep green patches developed on these calli but failed to form shoots.

Isolation, culture, and cell division in cotyledon protoplasts of cotton (Gossypium hirsutum and G. barbadense) EBRAHIM FIROOZABADY & D A V I D L. D e B O E R Agrigenetics Corporation, Advanced Research Division, 5649 East Buckeye Road. Madison, WI 53716, USA Plant Cell Reports 5:127-131, 1986

Abstract. Protoplasts were isolated from cotyledons and foliage leaves of cotton (Gossypium hirsutum and G. barbadense). Cotyledon protoplasts were larger and responded to culture better than leaf protoplasts. Cotyledon derived protoplasts regenerated cell walls and formed microcolonies of 2-3 cells in G. hirsutum and 5-8 cells in G. barbadense. However, the microcolonies did not grow beyond this stage. Protoplast yield and viability, cell wall regeneration and cell division were influenced by several factors, e.g., genotype, age, tissue and growth condition of donor plant, enzyme mixture and concentration, preplasmolysis period, incubation period, and culture medium.

A104

Studies on endosperm culture of Annona squamosa Linn SREELATA NAIR, M.V. SHIRGURKAR & A.F. MASCARENHAS Division of Biochemical Sciences, National Chemical Laboratory, Pune 411 008, India Plant Cell Reports 5:132-135, 1986 Abstract. Mature endosperm tissue excised from germinated seeds (2-4 days after radicle emergence)

of Annona squamosa grew and proliferated on White's basal medium supplemented with two cytokinins, an auxin and gibberellic acid. The callus obtained could be periodically subcultured. Shoot differentiation and root induction were obtained from callus on media of different compositions. Analyses of the root and young leaf tips showed triploid number of chromosomes (3n = 21).

Anther culture in rice: IV. The effect of abscisic acid on plant regeneration L.B. TORRIZO & F.J. ZAPATA Tissue Culture Facility, The International Rice Research Institute, P.O. Box 933, Manila, Republic of the Philippines Plant Cell Reports 5:136-139, 1986 Abstract. The effect of abscisic acid (ABA) on plant regeneration in anther-derived calli of rice Oryza

sativa L. varieties Taipei 177, Taipei 309 and Fujisaka 5 was studied. ABA at concentrations up to 4 x 10-tM stimulated fresh weight increase in Taipei 309 and Fujisaka 5 while higher concentrations effected corresponding weight decreases in the three varieties tested. Relatively high ABA concentrations resulted in decreased callus size and production of more compact and whitish calli. ABA increased the frequency of calli producing green plants in Taipei 309 and the average green plant regeneration in all varieties tested.

A105 Correlation of cotyledonary node shoot proliferation and somatic embryoid development in suspension cultures of soybean (Glycine max L. Merr.) H . R . K E R N S l, U . B . B A R W A L E 2, M . M . M E Y E R , Jr. l & J . M . WlDHOLM 2 1Department of Horticulture, University of Illinois at Urbana-Champaign, IL 61801, USA;2Department of Agronomy, University of Illinois at Urbana-Champaign, IL 61801, USA Plant Cell Reports 5:140-143, 1986 Al~traet. Suspension cultures of soybean were initiated from hypocotyl or cotyledon callus tissue of several soybean genotypes. When these were grown on L2 medium with 0.4 mg/liter 2,4-D several genotypes produced numerous embryoids while others produced only a few such structures. Due to internal anatomy, no embryoid developed into a complete plant. A genotype's propensity to form normal appearing embryoids was correlated with the ability to proliferate shoots at the cotyledonary node on a medium with benzylaminopurine as determined in previous testing.

Epchrosine - a new indole alkaloid isolated from plant cell cultures of Ochrosia eUiptica LabiH KARL-HEINZ P A W E L K A l, J O A C H I M S T O C K I G T l & B R U N O DANIELI 2 ILehrstuhlJ~r Pharmazeutische Biologie, Universitiit Miinchen, Karlstr. 29, D-8000 Miinchen 2, FRG;2Dipartimento di Chimica Organica e lndustriale della Facolt?t die Scienze, Universith degli Studi di Milano, Centro CNR di Studio per le Sostance Organiche Naturali, Via G. Venezian 21, 1-20133 Milano, Italia Plant Cell Reports 5:147-149, 1986 Abstract. From plant cell suspension cultures of Ochrosia elliptica Labill. an indole alkaloid of the apparicine type has been isolated, which is not known to occur in differentiated plants. The structure of the new compound, named epchrosine, was established by UV, MS and high resolution twodimensional ~H NMR (COSY and NOESY) as (19R, 20R)-epoxyapparicine.

A106 Plant regeneration by organogenesis in Glycine max M.S. WRIGHT, S.M. KOEHLER, M.A. HINCHEE & M.G. CARNES Monsanto Agriculture Company, 700 Chesterfield Village Parkway, St. Louis, MO 63198, USA Plant Cell Reports 5:150-154, 1986 Abstract. A procedure for the regeneration of fertile plants by organogenesis from tissue cultures of soybeans, Glycine max is described. Seeds were germinated on reduced inorganic salt MS medium containing 5#M BA. Cotyledonary nodes were excised and cultured on the same medium. Presence of BA in the medium during seed germination and culture of nodal explants was required for multiple shoot and shoot-bud formation. Histological analyses established the de novo nature of shoot regeneration. Separate reduction of the concentration of inorganic salts or substitution of sucrose for fructose during culture had minimal effects on the regeneration response. Conversely, if the BA was reduced, the inhibition response could not be overcome by increased salt concentration or altered carbon source.

Control of the developmental pathway of tobacco pollen in vitro M. KYO & H. HARADA Institute of Biological Sciences, University of Tsukuba, Sakura-mura, lbaraki 305, Japan Planta 168:427-432, 1986 Abstract. We developed a new method for culture of isolated pollen. Using highly homogeneous populations of immature pollen grains of Nicotiana tabacum L. prepared by means of Percoll density gradient centrifugation, we could direct their developmental pathway by regulating certain culture conditions. When the pollen population was cultured in basal medium with glutamine, most pollen grains underwent normal maturation. On the other hand, when first cultured in basal medium without glutamine, most pollen grains did not mature but after transfer to medium with glutamine and sucrose began to divide. This method for inducing pollen cell division was possible only with midbinucleate pollen grains which are characterized by having no central vacuole and no or only a few starch grains. Evidently, some essential changes necessary for the embryogenic response can be induced by glutamine starvation only in pollen grains at a specific stage.

A107 Microeallus formation from maize protoplasts prepared from embryogenic callus C.W. IMBRIE-MILLIGAN & T.K. HODGES Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907, USA Planta 168:395-401, 1986 Abstract. Conditions have been developed that induce maize (Zea mays L.) protoplasts to resynthesize cell walls and to initiate cell divisions. Two types of embryogenic maize callus were used as a source of protoplasts: a heterogeneous callus (Type I) derived from immature embryos after three weeks in culture, and a friable, rapidly growing callus (Type II) selected from portions of the Type I callus. Many variables in the growth conditions of the donor tissue (type of medium, transfer schedule, age of callus), protoplast isolation solutions (pH, osmolarity, type and concentration of cell wall hydrolyzing enzymes, addition of polyamines) and conditions (amount of time in enzyme, amount of tissue per volume of enzyme incubation medium, agitation, preplasmolysis of source tissue, type of callus), and purification procedures (filtration and-or flotation), were found to affect both yield and viability of protoplasts (based upon fluorescein-diacetate staining). Our isolation procedure yielded high numbers of viable, uninucleated maize callus protoplasts which were densely cytoplasmic and varied in size from 20 to 50#m in diameter. Protoplasts plated in solid medium formed walls and divided several times. Of several gelling agents tested for protoplast propagation, only agarose resulted in protoplasts capable of sustained divisions leading to the formation of microcalli. Plating efficiency was established over a wide range of protoplast densities (103-107 protoplasts/ml). Highest plating efficiency (25%) was obtained at 1.106 protoplasts/ml). The resulting microcalli grew to be dense clusters of about 0.1-0.5 mm in diameter and then stopped growing. Nurse cultures of maize and carrot (Daucus carota L.), were used to establish that individual protoplasts (not contaminating cells or cell clusters) formed walls and divided. Nurse cultures also increased the efficiency of microcallus formation from protoplasts.

AI08

Regulation in tobacco callus of enzyme activities of the nicotine pathway

I. The route ornithine to methylpyrroline F. FETH, R. WAGNER & K.G. WAGNER Arbeitsgruppe Enzymologie, GesellschaftJ~r Biolechnologische Forschung, Mascheroder Weg 1, D-3300 Braunschweig, FRG Planta 168:402-407, 1986 Abstract. Nicotine synthesis was stimulated by reduction of the medium auxin concentration (induction medium) in callus tissue originating from Nicotiana tabacum cv. Samsun. The enzyme activities of the route ornithine to methylpyrroline, which are those of ornithine decarboxylase, putrescine methyltransferase and methylputrescine oxidase, were determined during callus growth in the induction medium and as a control under non-nicotine-stimulating conditions (growth medium). The enzymes were assayed by high-performance liquid chromatography. Whereas the activities of ornithine decarboxylase were very similar under nicotine-stimulating and non-stimulating conditions, those of putrescine methyltransferase and methylputrescine oxidase increased strongly in the induction medium. In addition, the pools of putrescine and methylputrescine were determined throughout the callus growth cycle. Both sets of data strongly confirm the supposition that putrescine methyltransferase is the enzyme under stringent control for nicotine biosynthesis, whereas the subsequent methylputrescine oxidase is co-regulated, although less stringently.

Regulation in tobacco callus of enzyme activities of the nicotine pathway

H. The pyridine-nucleotide cycle R. WAGNER, F. FETH & K.G. WAGNER Arbeitsgruppe Enzymologie, GesellschaftJ~r Biotechnologische Forschung, Mascheroder Weg 1. D-3300 Braunschweig, FRG Planta 168:408-413 Abstract. In tobacco callus, the induction of nicotine synthesis, which stimulates enzyme activities of the ornithine-methylpyrroline route (see the preceding paper), also leads to marked changes in the enzyme activities of the pyridine-nucleotide cycle. This cycle provides the metabolite (probably nicotinic acid) for condensation with methylpyrroline to produce nicotine. The activities of eight enzymes of the pyridine-nucleotide cycle and of quinolinic-acid phosphoribosyltransferase, the anaplerotic enzyme, were determined by high-performance liquid chromatography assays. The distinct changes of their activities upon induction of nicotine synthesis lead to the following conclusions: i) nicotinic acid is the relevant metabolite which is provided by the pyridine-nucleotide cycle and consumed for nicotine synthesis, ii) The enhancement of the nicotinic-acid pool arises in two ways, by synthesis of NAD and degradation via nicotinamide mononucleotide and by a direct route from nicotinic-acid mononucleotide (NaMN) which is degraded by a glycohydrolase with a rather high Km value. Such a Km value prevents the complete depletion of the NaMN pool.

AI09

Anthranilate synthase forms in plants and cultured cells of Nicotiana tabacum L. J.E. BROTHERTON, R.M. HAUPTMANN & J.M. WIDHOLM University of lllinois, Department of Agronomy, Turner Hall, 1102 S. Goodwin, Urbana, IL 61801, USA Planta 168:214-221, 1986 Abstract. Tobacco (N. tabacum cv. Xanthi) cell lines contained two forms of anthranilate synthase (AS; EC 4.1.3.27) which could be partially separated by gel-filtration chromatography. One form was resistant to feedback inhibition by 10#M tryptophan (trp) while the other form was almost completely inhibited by trp at the same concentration. Cell lines selected as resistant to 5-methyltrytophan (5MT) had more of the trp-resistant AS form. Only the trp-sensitive form was detected in plants regenerated from both normal and 5MT-resistant cell lines. Overexpression of the trp-resistant form in 5MT-resistant tobacco cells disappeared during plant regeneration but reappeared when callus was initiated from the leaves of these plants. The trp-sensitive form was localized in the particulate fraction and the trp-resistant form in the cytosol of tobacco cultured cell protoplasts. The trp-resistant form of AS from tobacco had an estimated MW of 200 000, determined by Sephacryl S-200 chromatography, compared to an estimated MW of 150 000 for the trp-sensitive form. The estimated molecular weights of AS from carrot and corn were 160000 and 150000, respectively. Analysis of AS activity from the diploid Nicotiana species Nicotiana otophora (chromosome number 2n = 24) by high-performance liquid chromatography showed two activity peaks identical in elution time and trp inhibition characteristics to the activity from N. tabacum (chromosome No. 48). Thus the two enzyme forms found in tobacco did not appear to have originated individually from the progenitor species genomes which combined to make up the tobacco genome.

Synchronization of protoplasts from Glycine max (L.) Merr. and Brassica napus (L.) G. WEBER, E. de GROOT & H.-G. SCHWEIGER Max-Planck-Institut J~r Zellbiologie, Rosenhof, D-6802 Ladenburg, FRG Planta 168:273 280, 1986 Abstract. Cells of Glycine max originating in a suspension culture and cells of Brassica napus prepared from hypocotyls were synchronized. Synchronization was achieved by preparing protoplasts in the usual way and subsequently letting the protoplasts regenerate into cells by removing the cell-wall-digesting enzymes. More than 70% of the cells had divided synchronously at the end of the first cycle as determined by the mitotic index. The high frequency of mitosis critically depended on the osmolality of the medium. The duration of the S-phase was estimated by measuring the activity of thymidylate kinase as well as incorporation of [3H]deoxythymidine into acid-insoluble material. The data indicate that synchronization is induced by resetting the cell cycle.

All0

Characteristic symptoms of photosynthesis inhibition by herbicides are expressed in photomixotrophic tissue cultures of Nicotiana h . C S I ~ P L t ~ & P. M E D G Y E S Y Institute of Plant Physiology, Biological Research Center, Hungarian Academy of Sciences. P.O. Box 521, H-6701 Szeged, Hungary Planta 168:24-28, 1986 Abstract. A photomixotrophic tissue culture system for Nicotiana plumbaginifolia and N. tabacum

has been developed in which a primary symptom (bleaching) of the inhibition of photosynthetic electron transport by herbicides can be observed. Photomixotrophic cultures were initiated and maintained in the light on medium containing 0.2q3.3% sucrose or glucose (low-sugar medium) as sole source of respirable carbohydrate. The usual medium for growing heterotrophic cultures contains 2-3% sucrose or glucose (high-sugar medium). Callus grown on low-sugar medium achieved a fresh weight three to four times greater in the light than in the dark and reached about half that of callus grown on high-sugar medium. Carbon-dioxide fixation rates were an order of magnitude higher in cultures grown on low-sugar medium in the light than in those grown on high-sugar medium or in any of the dark-grown cultures. The light-dependent growth and CO:fixation rates of cultures grown on low-sugar medium indicated that a major proportion of the weight increase resulted from photosynthesis. Under these photomixotrophic conditions it was found that a number of photosystem-II herbicides, at concentrations which inhibit photosynthetic electron transport, also inhibited the light-dependent component of callus growth, and caused bleaching. These effects could not be demonstrated on high-sugar medium.

Transplantation of isolated nuclei into plant protoplasts

A novel technique for introducing foreign DNA into plant cells P . K . S A X E N A l, M . M I I l, W . L . C R O S B Y 2, L . C . F O W K E l & J. K I N G l 1Department of Biology, University of Saskatchewan, Saskatoon, Sask. S7N 0 WO, Canada," 2Plant Biotechnology Institute, National Research Council Saskatoon, Sask. S7N OW9, Canada Planta 168:29-35, 1986 Abstract. The uptake of isolated nuclei from Vicia hajastana Grossh. cells into protoplasts of an

auxotrophic cell line of Datura innoxia P. Mill. was induced under the influence of polyethylene glycol and Ca 2+ at pH 6.8. The frequency of nuclear uptake varied from 0.8 to 2.3% and that of the recovery of prototrophic clones from 10 -5 to 6.10 -4. The prototrophic nuclear fusion products following nuclear uptake could be rescued by initial culture of the protoplasts in non-selective conditions and by the subsequent use of feeder cell layers to support the growth of surviving colonies on a selective medium. The presence of Vicia genomic DNA in some prototrophic clones was confirmed by dot-blot hybridization using Datura and Vicia DNA probes. In certain transformed clones, the recovery of prototrophy was accompanied by the restoration ofmorphogenetic potential. Well-developed shoots typical of wild-type Datura could be regenerated employing an appropriate regeneration medium.

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Cell-cycle arrest of plant suspension cultures by tunicamycin C. ETTLINGER, J. SCHINDLER & L. LEHLE Fakultiit J~r Biologic und Vorklinische Medizin, Universiti~t Regensburg, Universitiitstrasse 31, D-8400 Regensburg, FRG Planta 168:101-105, 1986 Abstract. The effect of tunicamycin, an inhibitor of N-glycosylation of proteins, on growth and on synthesis of D N A and protein was studied in suspension cultures from Nicotiana tabacum and Catharanthus rosea. In the presence of 0. I-I pg.M1-~ tunicamycin, cell division and D N A synthesis stopped in cells which had been proliferating logarithmically, but protein formation continued. Cytophotometric determination of the nuclear D N A content in Catharanthus cells showed that a cell-cycle arrest had occurred in G1 phase. Metabolic labelling of cells with the glycoprotein precursors glucosamine or mannose was inhibited, too. The results indicate that one or more glycoproteins are needed for the cell to pass through the G1 phase, as was recently postulated for animal and yeast cells.

TL-DNA from Agrobacterium rhizogenes plasmid pRi1855 reduces the osmotic pressure in transformed plants grown in vitro G. OOMS, J. ATKINSON, M.E. BOSSEN & R.A. LEIGH Rothamsted Experimental Station, Harpenden, Herts. A15 2JQ, UK Planta 168:106-112, 1986 Abstract. Growth, water content, osmotic pressure and solute content were examined for normal potato (Solanum tuberosum L. cv. Desiree) and a derivative (line D9X8a), which was genetically transformed with TL-DNA from Agrobacterium rhizogenes. Plants were grown (i) in vitro, (ii) in a growth chamber and (iii) in the field. In vitro, the transformed potato plants produced more biomass than the untransformed plants, partly because they had a higher water content. Potassium concentration and osmotic pressure were lower in cell sap extracted from the transformed potato shoots. In some cases the difference was as much as 50%. These differences were less clear, absent or reversed in plants from a growth chamber or from the field. In the field, however, transformed potato senesced early. It is suggested that a cellular basis for these observations may be changes induced by Ri TL-DNA expression products in plant membrane properties.

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Aberrant microtubule organization can result in genetic abnormalities in protoplast cultures of Vicia hajastana Grossh. D.H. SIMMONDS & G. SETTERFIELD Biology Department, Carleton University, Ottawa, Ont. KIA 5B6, Canada Planta 167:460-468, 1986 Abstract. Protoplast cultures of Vicia hajastana have a high division frequency. However, 20-40% of the microcolonies fail to develop beyond the 20-30-cell stage. Aneuploids and polyploids were found in early divisions and persisted in older cultures. The resulting protoplast-derived suspension culture differed karyologically from the original culture. Karyokinesis and cytokinesis were studied using simultaneous staining of microtubules (MT) by immunofluorescence, DNA by Hoechst 33258 (2-[2-(4-hydroxyphenyl)-6-benzimidazoyl]-6-[1-methyl-4-piperazyl]benzimidazole)and cell walls by Calcoftuor. Freshly prepared protoplasts showed mitoses and high frequencies of binucleate cells, which probably resulted mainly from failure of cytokinesis. In early divisions, many mitoses showed metaphase chromosomes with kinetochore MT but lacking polar MT. These aberrant mitoses probably accounted for an increase in hyperploid cells observed in protoplast cultures. Multipolar spindles, which gave rise to hypoploid cells, were also seen in the early divisions. Telophase abnormalities included dislocated phragmoplasts and incomplete formation of cross walls. Many divisions resulted in daughter nuclei of unequal size. Unequal segregation of chromosomes was detected by cytofluorimetric measurements of telophase nuclei stained with Hoechst. After 5 d of culture, 91% of the divisions with incomplete cross walls also contained different-size nuclei; conversely, 78% of the divisions with fully formed cross walls contained nuclei of equal size. The malfunctioning of spindles and phragmoplasts in the same cells indicates a functional interdependence of the different MT configurations in mitosis. During the first 24 h of culture, a high frequency of abnormalities was found in spindles, cross-wall formation and chromosome segregation; this was reduced substantially in the cells undergoing first division by 48 h. The data indicate that it may be possible to manipulate the frequency of abnormalities by controlling the onset of the first division in protoplast cultures.

Prophase bands of microtubules occur in protoplast cultures of Vicia hajastana Grossh. D.H. SIMMONDS Biology Department, Carleton University, Ottawa, Ont. KIA 5B6, Canada Planta 167:469-472 Abstract. Circumnuclear bands of microtubules (MT) have been found in the prophase of mitoses

in cultured protoplasts of Vicia hajastana. The timing of the appearance and disappearance of the prophase band of MT (PB) relative to the stage of mitosis was studied using simultaneous staining of MT by immunofluorescence and DNA by HOechst 33258. These protoplasts regenerate into unorganized tissue. Pre-prophase bands of MT have previously been found only in highly organized tissues of higher plants. The role of PB in cell division is discussed.

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Plant regeneration from callus cultures of several soybean genotypes via embryogenesis and organogenesis U . B . B A R W A L E l, H . R . K E R N S 2 & J . M . W I D H O L M 1 Department of IAgronomy and 2Horticulture, University of Illinois at Urbana-Champaign, IL 61801, USA Abstract. Using callus derived from immature embryos, regeneration of viable plants was obtained in soybean (Glycine max (L.) Merr.). Depending on the composition of the medium, regeneration occurred via embryogenesis or via organogenesis. Embryogenesis resulted when embryos were plated on Murashige and Skoog (MS) medium containing 43/~M ct-naphthaleneacetic acid. In work with the cultivar Williams 82, the addition of 5.0/~M thiamine.HC1 increased embryogenesis from 33% to 58% of the embryos plated. Addition of 30/~M nicotinic acid to the MS medium enhanced embryogenesis further to 76%. Organogenesis was obtained when medium containing 13.3#M 6-benzylaminopurine, 0.2#M and ct-naphthaleneacetic acid and four times the normal concentration of MS minor salts was used. Histological studies of these cultures confirmed the organogenic and embryogenic nature of the cultures, by demonstrating the formation of shoot buds and somatic embryos, respectively. Similar responses were obtained in all 54 genotypes tested in this manner, The cultures retained the ability to regenerate complete plants for at least 12 months and 12-15 subcultures. Seeds have been obtained from several regenerated plants and when grown in the field these produced normal-appearing fertile plants.

Intracellular compartmentation of two enzymes of berberine biosynthesis in plant cell cultures M . A M A N N I, G . W A N N E R 2 & M . H . Z E N K 1 ILehrstuhl J~r Pharmazeutische Biologie, Universitiit Miinchen, Karlstrasse 29, D-8000 Miinchen 2, FRG,'ZBotanisches Institut der Universitiit, Menzinger Strasse 67, D-8000 Miinchen 19, FRG Planta 167:310-320, 1986 AMtract. Out of the eight enzymes involved in the biosynthesis of the isoquinoline alkaloid berberine, at least, two enzymes, berberine bridge enzyme and (s)-tetrahydroprotoberberine oxidase, are exclusively located in a vesicle with a specific gravity of Q = 1.14g.cm -3 as shown by direct enzymatic assay as well as immunoelectrophoresis. Electron-microscopic examination of the enzyme-containing particulate preparation from Berberis wilsoniae var. subcaulialata cultured cells demonstrated that it is composed mainly of membranous vesicles. The protein composition of this preparation reveals the presence of only about 20 separable proteins, of which two major ones are berberine bridge enzyme and (S)-tetrahydroprotoberberine oxidase. Incubation of these vesicles with the substrate (S)-reticuline in the presence and absence of S-adenosyl-L-methionine leads to the formation of a red product which was identified as dehydroscoulerine. If the cytoplasmic enzyme S-adenosyl-L-methionine:(S)-scoulerine-9-O-methyltransferaseis added to the vesicle preparation in the presence of (S)-reticuline and S-adenosyl-L-methionine, not dehydroscoulerine but columbamine, the immediate precursor of berberine is formed. Some of the quaternary alkaloids are located inside the vesicles; fusion of these vesicles leads to vaucuoles containing the quaternary alkaloids. These vesicles are the first highly specific and unique compartment serving only alkaloid biosynthesis; they are found in members of four different plant families and in cell cultures as well as in differentiated tissue.

All4 The expression of a nopaline synthase - human growth hormone chimaeric gene in transformed tobacco and sunflower callus tissue ANDREA BARTA 1, KARIN SOMMERGRUBER t, DIANA T H O M P S O N l, K L A U S H A R T M U T W , M A R J O R I A. M A T Z K E 2 & A N T I O N I U S J.M. M A T Z K E 2 lnstitutj~r Biochemie, Universitiit Wien, Wiihringerstrafle 17, A-1090 Wien, Austria;21nstitut J~r vIolekularbiologie, Akademie der Wissenschaften, Billrothstrafle 11, A-5020 Salzburg, Austria 'lant Molecular Biology 6:347-357, 1986 Abstract. To study whether mammalian RNA processing signals function in plants, we have constructed a chimaeric gene in which the complete human growth hormone (hGH) gene is flanked by DNA fragments containing the promoter and polyadenylation site of the nopaline synthase gene. The hGH gene used contains four introns and an additional 440 bp downstream from the hGH poly(A) addition site. The transcription of this chimaeric gene was studied following its introduction into sunflower and tobacco cells using a Ti plasmid vector. Analysis ofpoly(A) ÷ RNA isolated from the transformed tumor tissue demonstrated the following: (1) a single polyadenylated transcript, 2700 bp in length, was transcribed from the chimaeric gene; (2) the transcription was initiated at the published start site of the nopaline synthase gene; (3) the hGH polyadenylation site was not used for processing of the 3' end; only the poly(A) addition site of the nopaline synthase gene was recognized, (4) no splicing of the hGH introns could be detected. We also demonstrate that the hGH pre-mRNA isolated from plant cells can be spliced in a HeLa cell nuclear extract, indicating that the hGH pre-mRNA was functional. These results show that processing signals of the hGH pre-mRNA are not recognized in these plant cells.

Restriction endonuclease studies on the chloroplast and mitochondrial DNAs of alfalfa (Medicago sativa L.) protoclones R . J . R O S E l, L O W E L L

B. J O H N S O N

& R.J. KEMBLE 2

Department of Plant Pathology, Kansas State University, Manhattan, KS 66506, USA;lPresent address: Department of Biological Sciences, The University of Newcastle, New South Wales 2308 Australia;ePresent address: Department of Plant Biology, Allelix, Inc., 6850 Goreway Drive, Mississauga, Ont., L4V 1P1 Canada Plant Molecular Biology 6:331-338, 1986 Abstract. Alfalfa protoclones were regenerated from the mesophyll protoplasts of two cloned source plants (parents), RS-K1 and RS-K2, initiated from Regen S seed. Because of the high frequency of karyotypic upset previously observed in these plants, chloroplast DNAs (cpDNA) from 23 protoclones and mitochondrial DNAs (mtDNA) from 20 protoclones were examined by restriction endonuclease analysis in order to assess recombination in their cytoplasmic genomes. Seven and four endonucleases were separately used for cpDNA and mtDNA analysis, respectively. Data were consistent with no, or a low frequency of, major sequence rearrangements in either the chloroplast or the mitochondrial genomes as a result of protocloning. However, two types of cpDNA were detected in the 23 protoclones, with only one protoclone possessing the cpDNA type of the cloned parental populations sampled. Possible explanations include a preferential selection during protocloning for one of two parental cpDNA types, an in planta sorting out of cpDNA types in the parental material or both.

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Developmental regulation of RI TL-DNA gene expression in roots, shoots and tubers of transformed potato (Solanum tuberosum cv. Desiree) GERT OOMS l, DAVID TWELL J, MARGREET E, BOSSEN 1'3, J. HARRY C. HOGE 2 & MICHAEL M. BURRELL I tDepartment of Biochemistry, Rothamsted Experimental Station, Harpenden, Herts., UK;2Department of Plant Molecular Biology, University of Leiden, Wassenaarseweg 64, 2333 AL Leiden, the Netherlands;3Present address: Department of Plant Physiol. Research, Agricultural University, Generaal Foulkesweg 72, 6703 B W Wageningen, the Netherlands Plant Molecular Biology 6:321-330, 1986 Abstract. Expression of TL-DNA from Agrobacterium rhizogenes plasmid pRi 1855 was examined in a transformed derivative of Solanum tuberosum cv. Desiree, D9X8a. Northern blot analysis identified at least nine TL-DNA coded transcripts in roots, shoots and tubers but their relative abundance differed within and between organs. This revealed a distinctive pattern of organ specified differential expression. Grafting experiments showed that the abnormal shape of tubers of transformed potato was probably determined by TL-DNA products synthesised within the tuber and not by diffusable products synthesised in other parts of the plant. The abundance of at least one transcript, tr5, was probably determined by culture conditions. Implications for functions and control of expression of Ri TL-DNA genes are discussed. It is suggested that Ri TL-DNA provides a convenient and extensive set of model genes to study variation and stability of expression of linked foreign genes introduced into plants.

Genetic transformation of Brassica campestris var. rapa protoplasts with an engineered cauliflower mosaic virus genome JERZY PASZKOWSKI, BARBARA PISAN, RAYMOND D. SHILLITO, THOMAS HOHN, BARBARA HOHN & INGO POTRYKUS Friedrich Miescher Institut, P.O. Box 2543, CH-4002 Basel, Switzerland Plant Molecular Biology 6:303-312, 1986 Abstract. A hybrid Cauliflower Mosaic Virus (CaMV) genome containing a selectable marker gene was constructed by replacing the gene VI coding region with the aminoglycoside (neomycin) phosphotransferase type II [APH(Y)II] gene from Tn5. This modified viral genome was tested for its infectivity both in planta and in a protoplast transformation system of Brassica campestris vat. rapa. Stable, genetically transformed cell lines of B. campestris var. rapa were obtained after transformation. DNA of the hybrid CaMV genome was found to be integrated into high molecular weight plant genomic DNA. Transformation was achieved only when the hybrid genome was supplied together with wild type viral DNA. A possible complementation of the modified CaMV genome with the wild type viral DNA as a helper molecule in planta and in the protoplast system is discussed.