Plant Cell, Tissue and Organ Culture 26: A21-A42, 1991 10. Miscellaneous SCIENTIA (1990)

HORTICULTURAE

43:

117-128

Early testing of graft incompatibilities in apricot and lemon trees using in vitro techniques Robert Jonard, Diah Lukman, Francoise Schall and Pierre Villemur

Laboratoire de Physiologic V~g~tale Appliqu~e, Universitd des Sciences et Techniques du Languedoc, 34060 Montpellier Cddex, France Analysis of localised graft incompatibilities was carded out using several in vitro techniques, micrografts, internode association and callus fusion on two experimental models: in the Prunus species, between two apricot tree cultivars, compatible 'Luizet' and incompatible 'Canino', and a myrobolan rootstock; in the Citrus species, between two lemon tree cultivars, compatible 'Villafranca' and incompatible 'Eureka', and a cultivar 'Troyer' citrange rootstock. For these three tests, the compatible homografts and heterografts showed identical reactivity. The incompatible heterografts however, presented markedly different reactivity. In the Citrus species, the use of cell suspensions from one partner in a medium which had contained cell suspensions from another graft partner for 10 days, led to the same conclusions. Early testing of graft incompatibilities may therefore be envisaged.

J. P L A N T PHYSIOL. 1 3 6 : 1 5 - 2 3 (1990)

Using pressure-volume analysis to characterize NaCl-induced osmotic and turgor adjustment in in vitro cultures Robert M. Aug6, Ann J.W. Stodola, S. Durra Gupta, John C. Hovanesian and B.V. Conger

Department of Ornamental Horticulture, University of Tennessee, Knoxville, TN 37901, USA The applicability of pressure-volume (PV) analysis for characterizing osmotic and turgot adjustments in in vitro cultures was tested using salinized orchardgrass callus. Thermocouple psychrometry was used to derive PV curves in three experiments: (a) adjustment to varying concentrations of NaC1, (b) recovery from NaC1 stress and (c) influence of NaC1 acclimation, to an additional, increased NaCI challenge. Callus growth and water relations were unaffected by 50 mM NaC1. Harvest and zero turgor osmotic potentials were decreased about 0.7 and

A21 0.9 MPa, respectively, in callus grown for three weeks on I00 and 150 mM NaC1. Turgor was 0.2 to 0.4 MPa higher in 100 and 150 mM treatments across a range of callus water potential, compared to 0 or 50 mM NaC1. Salinization resulted in decreased estimates of callus elasticity and increased values of relative water and osmotic water content at zero turgor. The effects of NaC1 stress on elasticity and osmotic potential were still evident three weeks after transfer to fresh medium containing 0 mM NaC1. A three-week acclimation of callus to 50 mM NaC1 increased turgor and decreased osmotic potential and elasticity compared with unacclimated callus when cultures were subsequently grown on 100 mM NaC1. Apoplastic water percentages ranged from 21 to 35% and showed no response to salinization. Psychrometric PV analysis appears to offer a promising alternative for the assay of component water potentials and water contents, elasticity and apoplastic/symplastic water partitioning in callus tissue.

CROP SCI. 3 0 : 7 0 8 - 7 1 2 (1990)

Optimization of surface sterilization for legume seed G. Caetano-Anoll6s, Bauer

G.

Favelukes

and W.D.

Roots of alfalfa (Medicago sativa L.), white clover (Trifolium repens L.) and soybean [Glycine max (L.) Merr.] seedlings are frequently oontaminated with bacteria even after surface sterilization of the seeds and germination under aseptic conditions. Several seed-sterilization procedures were compared for their ability to minimize or eliminate such contamination without damaging the plant. Mercuric chloride proved the best seed disinfectant for alfalfa and white clover. Calcium hypochiorite was the best for soybean. For alfalfa seeds, treatment with 95% ethanol (v/v) for 60 rain followed by 0.2% HgCI2 (v/v) for 15 min prior to rinsing and imbibition resulted in a low frequency (<5%) of seed and root contamination. This treatment resulted in no measurable damage to alfalfa seedlings with respect to root growth, nodulation efficiency, or rate of nodule emergence following inoculation with Rhizobiura meliloti. Longer exposures to HgC12 further reduced bacterial contamination, but also caused modest reductions in seed germination. None of the surface-sterilization techniques tested completely eliminated bacterial contaminants. It appears that these sterilization-resistant contaminants are borne within the seed and proliferate on plant surfaces after germination with possible effects on plant health.

A22 HORTSCIENCE 25(7): 802-803) (1990)

A fertile tetraploid Anigozanthos hybrid produced by in vitro colchicine treatment R.J. Griesbach

U.S. Department of Agriculture, Agricultural Research Service, Florist and Nursery Crops Laboratory, Beltsville, MD 20705, USA Kangaroo paw is a new cut flower crop native to Australia. There are several interspecific hybrids with improved flower colors, heat tolerance, and growth habit. These hybrids are sterile due to divergent evolution of the parent species. Colchicine was used to double the chromosome number of one important sterile hybrid. This hybrid is everblooming, dwarf, and heat tolerant. The resdting allodiploid was fertile, and progeny are now being evaluated.

11. Morphogenesis P L A N T PHYSIOL. 9 3 : 4 4 6 - 4 5 2 (1990)

Polyamines and flower development in the male sterile stamenless-2 mutant of tomato (Lycopersicon esculentum Mill.) II. Effects of polyamines and their biosynthetic inhibitors on the development of normal and mutant floral buds cultured in vitro Rajeev Rastogi and Vipen K. Sawhney

Department of Biology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N OWO The floral organs of the male sterile stamenless-2 (sl-2/sl2) mutant of tomato (Lycopersicon esculentum Mill.) contain significantly higher level of polyamines than those of the normal (R Rastogi, VK Sawhney [1990] Plant Physiol 93: 439-445). The effects of putrescine, spermidine and spermine, and three different inhibitors of polyamine biosynthesis on the in vitro development of floral buds of the normal and sl-2/sl-2 mutant were studied. The polyamines were inhibitory to the in vitro growth and development of both the normal and mutant floral buds and they induced abnormal stamen development in normal flowers. The inhibitors of polyamine biosynthesis also inhibited the growth and development of floral organs of the two genotypes, but the normal flowers showed greater sensitivity than the mutant. The inhibitors also promoted the formation of normal-looking pollen in stamens of some mutant flowers. The effect of the inhibitors on polyamine levels was not determined.

The polyamine-induced abnormal stamen development in the normal, and the inhibitor-induced production of normal-looking pollen in mutant flowers support the suggestion that the elevated polyamine levels contribute to abnormal stamen development in the sl-2/sl-2 mutant of tomato.

PLANT SCIENCE 69: 207-214(1990)

Histological aspects of in vitro root and shoot differentiation from cotyledon explants of Brassica juncea (L.) C z e r n Kiran K. Sharma and Sant S. Bhojwani

Department of Botany, University of Delhi, Delhi 110007, India Under fairly simple culture conditions, excised cotyledons of Brassica juncea undergo high frequencies of either root formation on growth regulator-free Murashige and Skoog (MS) medium or.shoot formation on MS + 5.0 ~tMN6-benzyladenine (BA). Temporal histological events of organ differentiation processes revealed that while root formation was endogenous in origin and did not involve callusing, shoot bud differentiation occurred after the formation of meristematic nodules at the cut end. While root primordia differentiated from the petiolar cut end after 4 days in culture, the formation of a meristematic nodule at the same end occurred by 4-6 days which further lead to the differentiation of shoot meristem by 8-10 days. Although the initial accumulation of starch in organogenic cotyledons was the same during both root and shoot formation, its depletion was faster in shoot forming cultures suggesting that this process is more energy requiring than rhizogenesis.

PLANT SCIENCE 6 9 : 2 2 5 - 2 2 9 (1990)

Control of cell proliferation and differentiation by modulators of ethylene biosynthesis and action in Brassica hypocotyl explants Urmil Sethi, Atanu Basu and Sipra Guha-Mukherjee

Plant Research Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, India In Brassica cultures, proliferation could be induced by ethylene precursors, S-adenosylmethionine (SAM) or 1aminocyclopropane-l-carboxylic acid (ACC) with concomitant increase in the ethylene level, while the inhibitors of ethylene biosynthesis, aminoethoxyvinylglycine

A23 (AVG) and cobalt chloride, and the ethylene antagonist silver nitrate induced a higher percentage of shoot differentiation, with reduced ethylene level, compared to the hormone control. Putrescine (Put), spermidine (Spd) and the amino acids L-methionine or L-threonine, which increased proliferation, enhanced ethylene emanation as compared to the differentiating cultures raised on methylglyoxal-bis-(guanylhydrazone) (MGBG), L-leucine or Lisolencine.

PLANT SCIENCE 6 8 : 2 4 9 - 2 5 6 (1990)

Changes in mierotubular organization mark the transition to organized growth during organogenesis in Petunia hybrida J.A. Traas, J.P. Renaudin and B. Teyssendier de la Serve

and Skoog (MS) medium without growth regulators. The frequency of 15-17 day old embryos that.gave rise to somatic embryos increased from 8% to 29% by application of a mixture of 100 mg/l gibberellic acid, 25 mg/l a-naphthaleneacetic acid (NAA) and 5 mg/l kinetin daily to the pedicels of the developing pods. However, only callus formed on immature hybrid embryos of the reciprocal cross. These callus tissues occasionally gave rise to shoots via organogenesis when transferred to MS medium with 2 mg/l N6-benzyladenine and 0.05 mg/1 Naa, Treatment of pods with growth regulators did not influence the frequency of organogenic callus. Selfed embryos of the parents did not form somatic embryos in culture, nor did callus derived from the selfed embryos produce shoots. Thus, the ability to redifferentiate appears to be associated with interspecific hybridity.

INRA, Domaine d'Epoisses, Station de Physiopathologie Vegetale, B.V. 1540, 21034 Dijon Cedex, France

J. PLANT PHYSIOL. 1 3 6 : 5 6 - 6 0 (1990)

We have studied microtubular organization during the induction of organogenesis in callus derived from leaf protoplasts of Petunia hybrida, line PC6. The conditions for regeneration have been determined in detail and it is possible to obtain and compare calli which differ in their competence to regenerate. The main conclusions are: (1) that dividing cells in non-polarized callus have a low relative amount of preprophase bands (PPB-index), regardless of their potential to regenerate; (2) that this low PPB-index is due to the absence of PPB's in preprophase and early prophase; (3) that a 3-5-fold increase in the PPB-index marks the transition to organized growth; (4) that the interphase cytoskeleton is .unordered in the nonorganized callus and that ordered, helical arrays of interphase microtubules do not appear before polarized division has been restored.

U.S. Department of Agriculture, Agricultural Research Service, Fruit and Vegetable Chemistry Laboratory, 263 South Chester Avenue, Pasadena, California 91106, USA

In vitro flowering from Citrus limon lateral buds Brent Tisserat, Paul D. Galletta and Daniel Jones

Citrus limon (L.) Burro. f. cv. 'Eureka' vegetative lateral buds from 6-year old trees, when cultured at 14 to 20°C in vitro, flower at rates of 5 to 20% after 16 weeks and 90% within 32 weeks. Buds maintained at 25°C did not flower, and attempts to chemically induce flowering at 25°C using various growth inhibitors and promoters as well as high concentrations of sugars failed. Abscission of floral and foliar buds, as well as leaves, was common in vitro.

PLANT CELL REPORTS 9 : 8 4 - 8 7 (1990) PLANT CELL REPORTS 9 : 7 7 - 7 9 (1990)

Somatic embryogenesis and shoot organogenesis from interspe¢ific hybrid embryos of Vigna glabrescens and V. radiata H.K. Chen, M.C. Mok and D.W.S. Mok

Department of Horticulture and renter for gene research and biotechnology, Oregon State University, Corvallis, OR 97331, USA Somatic embryogenesis occurred on cotyledons of morphologically abnormal embryos derived from Vigna glabrescens × V. radiata crosses and cultured on Murashige

Regeneration of Lycium barbarum L. plants from leaf tissue, callus culture and callus protoplasts Yakov I. Ratushnyak, Vladimir A. Rudas and Nickolai M. Piven

Division of Cell Biology and Engineering, N.G. Kholodny Institute of Botany, Ukrainian SSR Academy of Sciences, Acad. Lebedev Str. 1, 252650 Kiev-GSP-22, USSR The possibility of plant regeneration from leaf tissue, callus and callus protoplasts of Lycium barbarum has been studied. Leaf segments were cultured on B5 medium (Gamborg et al. 1968) containing 1.5 mg/l 6-

A24 benzylaminopurine and 0.5 mg/l ~-naphthaleneacetic acid. Regeneration of shoots was initiated after 30 days of cultivation. Callus was obtained from leaf and internode tissues on MS medium (Murashige and Skoog 1962) containing 0.4 mg/1 of 2,4-dichlorophenoxyacetic acid. Subsequently, callus was successfully subcultured on the same medium with 1 mg/l of 2,4-dichlorophenoxyacetic acid and 0.2 mg/1 c~-naphthaleneacetic acid. Organogenesis in callus culture was obtained in the course of 40 days after transferring to TM-4 (Shahin 1984). Pmtoplasts were isolated from callus tissue grown in vitro using an enzymatic method. Cell colonies, minicallus formation and organogenesis were obtained. Shoots were rooted on Murashige and Skoog medium containing 0.1 mg/l ~x-naphthaleneacetic acid. Regenerated plants were transferred to soil and were grown to maturity. Regenerated plants carried normal morphological traits.

Solanum carolinense can be induced when stem segments are cultured on medium containing benzyladenine. When cultured on medium containing 2,4-dichlorophenoxyacetic acid, stem explants produced only callus. Using two-dimensional gel electrophoresis, total soluble protein and protein labeled in vivo with [35S]-methionine were extracted from regenerating and nonregenerating cultures and the gels or autoradiographs computer analyzed to identify different sets of proteins associated with development in vitro. Overall, the level of expression of 51 stained and 40 labeled proteins was altered, and it was shown that in vitro morphogenesis of stem segments of S. carolinense is associated with major quantitative changes in protein expression. Qualitatively, stage-specific polypeptides related to callus formation and in vitro organogenesis were also identified.

EUPHYTICA 4 8 : 1 0 3 - 1 1 0 (1990)

JOURNAL OF EXPERIMENTAL BOTANY 41 (227): 745-748 (1990)

In vitro culture of tissues, cells and protoplasts of Trifolium alexandrinum L. (Egyptian clover) M.N. Barakat

In vitro production of stigma-like structures from stigma explants of Crocus sativus L. K.S. Sarma, K. Maesato, T. Hara and Y. Sonoda

Tissue Culture Laboratory, Department of Crop Science, Faculty of Agriculture, University of Alexandria, Egypt

Biotechnology Laboratory, Department of Agricultural Chemistry, Faculty of Agriculture, Gifu University, Gifu 501-11, Japan

Callus induction derived from root, hypocotyl and cotyledon explants were investigated for four cultivars of Trifolium alexandrinum L. The Murashige & Skoog (1962) medium containing 2.0 mg/L NAA and 0.5 mg/L BAP was able to induce callus from different explants. A wide range of culture media were tested to determine the morphogenetic potential of different callus types derived from different explant types. Shoot morphogenetic development was observed with the cultivars Sakha 4and Giza 10. Cell suspension cultures were established from hypocotyl derived callus. Methods for isolation and culture of protoplasts from cotyledons are described.

Stigma-like structures (TC stigmas) were produced in tissue cultures from stigma explants of Crocus sativus under defined conditions. MS medium supplemented with NAA (10 mg din-3) + BA (1.0 mg dm-3) induced the optimum response. NAA was found to be an important addendum to achieve a good response. The TC stigma regeneration response as a function of explant age showed significant differences (except between stage 1 and stage 4). A culture temperature of 20°C seems to be better than 25°C with reference to all parameters. Crocin and picrocrocin pigments, responsible for colour and bitter taste, respectively, were extracted, identified and quantified from the TC stigmas. Safranal was not detected in fresh samples.

J. PLANT PHYSIOL. 1 3 6 : 2 1 3 - 2 1 8 (1990)

A two-dimensional electrophoretic analysis of protein synthesis and accumulation during adventitious shoot formation in somatic tissue cultures of Solanum carolinense L. Thomas L. Reynolds

Department of Biology, the University of North Carolina, Charlotte, North Carolina 28223, USA Adventitious shoot formation from tissue cultures of

PHYSIOLOGIA (1990)

PLANTARUM

79:

267-274

Changes in the starch content during organogenesis in in vitro cultured Begonia rex stem explants B.S. Mangat, M.K. Pelekis and A.C. Cassells Stem explants, excised from greenhouse-grown Begonia

A25 rex plants, were cultured on basal medium (T. Murashige and F. Skoog, Physiol. Plant. 15: 473-497, 1962) contained in sterile Petri dishes. The medium was supplemented with benzyladenine (0.1 mg 1-1), naphthaleneacetic acid (0.01 mg 1-1) and, according to experimental requirements, with either sucrose (3%) or mannitol (3%). Histochemical and biochemical examination of the starch content of the explant was carded out over several days. There was no starch deposition or organogenesis in tissue cultured on mannitol and carbohydrate-free growth medium. The most dramatic finding was the heavy accumulation of starch in tissue cultured on sucrose medium. This copious accumulation preceded any organ formation and was mainly in regions which ultimately gave rise to shoot primordia. The heavy build-up of starch preceding organogenesis was also observed when explants previously cultured on mannitol medium were transferred to medium containing sucrose. During shoot primordia development there was a decrease in the starch content of the cultured tissue indicating the utilization of the polyglucan in the organogenic process.

H O R T S C I E N C E 25(6): 684-687 (1990)

Regeneration of Laehenalia species from leaf explants Josephina G. Niederwieser and Bela M. Vcelar

Vegetable and Ornamental Plant Research Institute, Private Bag X293, Pretoria, 0001 Republic of South Africa The physiological stage of donor plants determined to a great extent the morphogenic potential of Lachenalia (Jacq.) hybrid leaves, but the optimal stage for various cultivars was different. Contact of the bud-forming adaxial epidermal ceils with the medium did not significantly stimulate in vitro bud formation on Lachenalia leaf explants, but resulted in the formation of callus from the buds of certain hybrids. Wounding on either the adaxial or the abaxial side of leaves had a stimulating effect on certain hybrids, but others did not respond significantly. A reduction in the length of explants from 10 to 3.3 mm resulted in an increase in the total number of buds formed by a specific amount of explant tissue (width of explant = 15 ram).

E U P H Y T I C A 4 8 : 2 6 9 - 2 7 4 (1990)

High frequency plant-regeneration through direct shoot development and somatic embryogenesis from immature inflorescence cultures of finger millet (Eleusine coracana Gaertn.) Leela George and Susan Eapen

Plant Biotechnology Section, Bhabha Atomic Research Centre, Trombay, Bombay-400085, India Plant regeneration from cultured immature inflorescence segments of Eleusine coracana was obtained by direct shoot development and somatic embryogenesis. Direct development of shoots from cultured inflorescence segments occurred on MS medium supplemented with 2,4-D in combination with zeatin. Inflorescences with well developed spikelets differentiated at a low frequency (<5%) from callus cultures initiated on media supplemented with 2,4-D in combination with zeatin or coconut water or picloram + kinetin. Somatic embryogenesis was also induced in callus cultures growing on MS + picloram + kinetin at the end of four passages. Supplementation of the media with different concentrations of sucrose showed 3% sucrose as the best concentration for plant differentiation from somatic embryos. The majority of the regenerated plants showed the diploid chromosome constitution in their root tips. The regenerants were in general shorter with an increased number of tillers compared to the control.

12. Nutrition P L A N T PHYSIOL. 9 3 : 1 0 7 1 - 1 0 7 7 (1990) Boron deficiency in cultured pine cells: Quanti-

tative studies of the interaction with Ca and Mg Robert Dixon Teasdale and Dianne Katherine Richards

Centre for Plant Biotechnology, School of Science and Technology, Bond University, Gold Coast, Qld 4229, Australia A pronounced interaction between calcium, magnesium, and boron was found in growth studies with Pinus radiata cell cultures. Quantitative isoactivity data for the interaction was analyzed in terms of selected simple and plausible theoretical models. The data was found to be consistent with a model in which a critical acceptor molecule is activated only by binding both Ca and B at separate sites; Mg competitively displaces Ca to inactivate the acceptor. It was found that B is, surprisingly, not bound strongly (Kdiss = 450 __ 80 micromolar) and that the affinity for Ca is two orders of magnitude stronger than for Mg. Therefore only a small proportion of the acceptor will be boronated under natural conditions. Moderate levels of mannitol were found to aggravate B deficiency due to its effective removal by direct chemical

A26 complexation. At higher concentrations of mannitol (or other sugars), where osmotic contribution is significant, little B was needed to overcome growth inhibition - a result consistent with B having a primary role in cell wall biosynthesis.

13. Protoplasts PLANT SCIENCE 6 8 : 2 3 1 - 2 3 8 (1990)

Somatic embryogenesis and plant regeneration from hypocotyl protoplasts of Brassica juncea (L.) Czern & Coss Eng-Chong Pua

Institute of Molecular and Cell Biology, National University of Singapore, 10 Kent Ridge Crescent, Singapore 0511, Republic of Singapore

embryos developed into green embryos, and 33% initiated calluses. Other pro-embryos dcdifferentiated into calluses which later redifferentiated embryos. Sixteen percent of the embryos developed directly into plants, whereas 81% produced plants indirectly via secondary embryos. The remaining 3% of the primary embryos failed to develop into plants. The lowest plating efficiency for direct embryogenesis was 0.3%. The high percentage of direct embryogenesis observed was related to the genetic nature of the clone, low density of liquid medium, low protoplast culture density, and the composition of culture media.

PLANT CELL REPORTS 9 : 5 1 - 5 3 (1990)

Regeneration of fertile plants from embryogenic suspension culture protoplasts of Sorghum vulgare Zhi-ming Wei and Zhi-hong Xu

A novel system for plant regeneration from hypocotyl protoplasts of Brassicajuncea cv Leaf Heading and India Mustard was developed. Protoplasts divided rapidly and gave rise to pro-embryos at frequencies of 3-6% for Leaf Heading and 15-20% for India Mustard after 17 days on Murashige & Skoog's medium supplemented with 6% glucose and benzyladenine (BA) in combination with 2,4-dichlorophenoxyacetic acid each at 1 or 2.5 [tM. Abundant plantlets were regenerated from embryogenic cultures of India Mustard grown on hormone-free medium, whereas regeneration of Leaf Heading was low. The presence of AgNO3 at 30 lxM in conjunction with 10 ~tM BA and 2.7 I.tM naphthaleneacetic acid considerably enhanced plant regeneration of Leaf Heading. After acclimatization all protoplast-derived plants of India Mustard and 91% of Leaf Heading were phenotypically normal.

Plant Molecular Genetics Laboratory, Shanghai Institute of Plant Physiology, Academia Sinica, 300 Fenglin Road, Shanghai 200032, People's Republic of China

P L A N T CELL REPORTS 9 : 2 1 - 2 5 (1990)

PLANT CELL REPORTS 9 : 4 2 - 4 6 (1990)

Direct embryogenesis from single mesophyli protoplasts in alfalfa (Medicago sativa L.)

Stress responses in alfalfa (Medicago sativa L.) IV. Expression of defense gene constructs in electroporated suspension cell protoplasts

Jiasheng Song, Edgar L. Sorensen and George H. Liang

Department of Agronomy and USDA-ARS, Kansas State University, Manhattan, KS 66506-5501, USA We used a tetraploid clone derived from an anther culture operation of 'Ladak' alfalfa to study the pathway of direct embryogenesis from leaf-mesophyll protoplasts. About 72% of the protoplasts divided, and 7% of those produced pro-embryos. Approximately 38% of the pro-

Protoplasts were isolated from immature inflorescencederived embryogenic suspension cultures of two cultivars of Sorghumvulgare. The protoplasts were cultured in a modified KSP liquid medium. They started to divide after 4-5 days of culture, and achieved 16.8% division frequency by 10 days. Protocalli proliferated further upon transfer to C1 solid medium. After that, they were moved to C1 differentiation medium to induce shoot formation, followed by whole plant regeneration. So far, 60 plants have been obtained, with only two albinos. Some of these have been transplanted to soil in pots and grown to flowering and have set seeds.

Arvind D. Choudhary, Helmut Kessmann, Christopher J. Lamb and Richard A. Dixon

Plant Biology Division, The Samuel Roberts Noble Foundation, P.O. Box 2180, Ardmore, OK 73402, USA We have investigated conditions for the uptake and expression of chimeric genes in protoplasts of alfalfa (MedicagosativaL.). Constructs containing the bacterial

A27 reporter gene chloramphenicol acetyltransferase (CAT) under the control of either the cauliflower mosaic virus 35S promoter or a bean chalcone synthase (CHS) promoter were introduced into protoplasts by electmporation in the presence of polyethyleneglycol. The extent of expression in the absence of added inducers depended on the conditions for isolation, electroporation and subsequent culture of the protoplasts. Expression of the CHS promoter construct was increased on exposure of the protoplasts to a fungal elicitor or reduced glutathione. The relative levels of induced expression in relation to either basal expression or the type of elicitor used depended on the age of the suspension cultures from which the protoplasts were isolated. Electroporation of protoplasts with a construct from which bean CHS antisense transcripts were synthesized under the control of the 35S promoter resulted in the inhibition of appearance of elicitor-induced endogenous alfalfa CHS activity. The suitability o f the alfalfa protoplast system for analysis and potential identification of defense response genes is discussed.

PLANT CELL REPORTS 9 : 1 7 - 2 0 (1990)

Genotype- and promoter-induced variability in transient B-glucuronidase expression in pea protoplasts Shaun L.A. Hobbs, Jennifer A. Jackson, David S. Baliski, Catherine M.O. DeLong and John D. Mahon

Plant Biotechnology Institute, National Research Council of Canada, Saskatoon, Saskatchewan, S7N OW9, Canada Leaf mesophyll protoplasts isolated from pea (Pisun sativum L.) genotypes Century and PI244253 showed transient expression of l~-glucuronidase (GUS) when electroporated with plasmid DNA containing various promoter-leader sequence constructs driving the GUS gene. The optimum conditions for transient expression were: using protoplasts isolated from leaf material that had been kept in the dark for 90 h; electroporating at 250 V and 960 p.F; and using 125 ~tg of calf thymus carrier DNA and 75 ~tg of plasmid DNA. PI244253 had 5 to 20 times the GUS activity levels of Century. Similar levels of transient expression were obtained using either the nopaline synthase or cauliflower mosaic virus 35S (35S) promoters. These levels were lower than that obtained using a duplicated 35S promoter derivative. The presence of an untranslated coat protein mRNA leader sequence from alfalfa mosaic virus between each promoter and the

GUS gene resulted in increased GUS activity. Leaf mesophyll protoplasts and root protoplasts of PI244253 did not differ in levels of transient expressioh.

PHYSIOLOGIA (1990)

PLANTARUM

79:

347-353

Cell wall synthesis in carrot cells: Comparison of suspension-cultured cells and regenerating protoplasts Hans-Peter Mock, Michael Emmerling and Hanns Ulrich Seitz The synthesis of cell walls and extracellular material during the regeneration of carrot (Daucus carota L. ssp. sativus) protoplasts was examined. Cell walls and extracellular material were analysed for their carbohydrate content. In cell walls, the amount of carbohydrate increased 4- to 5-fold with only minor changes in neutral sugar composition. Glucose was abundant, during cultivation, making up to 70% (w/w) followed by mannose, which accounted for 17%. This indicates the formation of a glucomannan. The neutral sugar composition of extracellular polysaccharides showed greater variety with a significant increase of arabinose and galactose during cultivation. This feature is probably connected to the occurrence of arabinogalactan proteins in the culture medium. Hydroxyproline, an indicator for extensin and arabinogalactan proteins, showed an increase parallel to the formation of cell walls and extracellular polysaccharides. Results are compared with corresponding data from suspension-cultured cells used for protoplast isolation.

PLANT CELL REPORTS 9 : 6 1 - 6 4 (1990)

Isolation, culture and plantlet regeneration from cotyledon and mesophyil protoplasts of two pickling cucumber (Cucumis sativus L.) genotypes Z.K. Punja, F.A. Tang and G.G. Sarmento

Biotechnology and Genetic Engineering Section, Campbell Institute for Research and Technology, Campbell Soup Company, Route 1, Box 1314, Davis, CA 95616, USA Optimal protoplast yields from Cotyledons (2.0 x 106 protoplasts/0.5 g tissue) and from true leaves (5.0 x 106 protoplasts/g tissue) of two Cucumis sativus genotypes were obtained following a 16 h digestion with, respectively, 1.25% pectinase + 0.5% Cellulysin and 0.5% pectinase + 1.0% Cellulysin. Enzyme solutions were

A28 prepared in modified MS medium containing half-strength major salts, full complement of minor salts and vitamins, 2% sucrose and 0.25 M mannitol. A plating density of 3.5--4.0 x 104 protoplasts/ml or higher was required for sustained division, with first division occurring in 6-7 days, second-third division in 8-9 days, and minicalli formation by day 13. Embedding in 0.4% agarose provided the highest plating efficiency (proportion that formed minicalli) of mesophyll protoplasts, which was 28.3% for genotype 3672 and 15% for genotype 3676. By comparison, liquid culture and droplet culture gave lower plating efficiencies (10-19%). Cotyledon and mesophyll protoplasts of one genotype formed minicalli on MS medium containing 2,4-D/BA at 1.0/2.5 IxM and 5.0/5.0 laM, respectively, within 21 days, while mesophyll protoplasts of the second genotype formed minicalli on MS medium containing NAA/BA at 5.0/5.0 I~M within 12 days. Shoot buds or somatic embryos were obtained upon subculture of calli to MS medium containing lower concentrations (0.05-0.01 lxM) of 2,4-D/BA or NAA/BA and a few plantlets, ca.. 18, were recovered on hormone-free medium, Abbreviations: BA, benzyladenine; 2,4-D, 2,4-dichlorophenoxyaceticacid; MS, Murashige and Skoog medium; NAA, naphthaleneacetic acid.

BIO/TECHNOLOGY 8 : 7 3 6 (1990)

Genetically engineered fertile Indica-rice recovered from protoplasts Swapan K. Datta, Alex Peterhans, Karabi Datta and Ingo Potrykus

Swiss Federal Institute for Technology, Plant Sciences, ETH-Zentrum, CH-8092, Ziirich, Switzerland We have established an efficient protocol for plant regeneration from haploid Indica-type rice protoplasts. Incubation of these protoplasts with the selectable hygromycin phosphotransferase (hph) gene expressed under control of the 35S promoter of cauliflower mosaic virus (CaMV) and polyethyleneglycol (PEG), and subsequent culture in the presence of hygromycin B, led to the recovery of numerous resistant clones from which 77 plants were regenerated. Data from Southern analysis and enzyme assays proved that the transgene was stably integrated into the host genome and expressed, and that it was inherited in offspring.

PLANT CELL REPORTS 9 : 1 0 9 - 1 1 2 (1990)

Plant regeneration from protoplasts of Felicia and Brachycome R.S. Malaure, M.R. Davey and J.B. Power

PLANT CELL REPORTS 9 : 1 0 5 - 1 0 8 (1990)

Temporary inhibition of cell wall synthesis improves the transient expression of the GUS gene in Brassica napus mesophyll protoplasts Marc Chapel and Kristina Glimelius

Department of Plant Breeding, Swedish University of Agricultural Sciences, Box 7003, S-750 07 Uppsala, Sweden Transformation of Brassica napus mesophyll protoplasts was performed with the l~-glucuronidase gene fusion system. After electroporation, transient expression in protoplasts transformed directly after isolation was about 1 to 2 per million. By the use of 2,6-dichloro-benzonitrile, a non-toxic inhibitor of cell wall synthesis, and in the presence of 5% polyethyleneglycol, transformation of the cell material was performed three days after isolation. At that time, about 25-30% of the protoplasts had reached the first S-phase of the mitotic cycle. A 1000-fold increase of protoplasts expressing the l~-glucoronisidase gene transiently was obtained, in the partly synchronized protoplasts, compared to those transformed directly after isolation.

Plant Genetic Manipulation Group, Department of Botany, University of Nottingham, Nottingham NG7 2RD, UK Protoplasts were isolated from leaves of axenic shoot cultures of Felicia bergeriana (Kingfisher Daisy) and Brachycome iberidifolia (Swan River Daisy) and from callus cultures of Felicia. Plants were regenerated from all three sources and since both species are of ornamental value (blue flowered) the establishment of plant regeneration provides a basis for their incorporation in somatic hybridisation programmes involving important omamentals such as Chrysanthemum. PLANT CELL REPORTS 9 : 1 2 9 - 1 3 2 (1990)

Direct analysis of RNA transcripts in electroporated carrot protoplasts Elizabeth E. Murray, Wallace G. Buchholz and Benjamin Bowen

Agrigenetics Advanced Sciences Corporation, 5649 Buckeye Road, Madison, WI 53711, USA We describe a method for direct analysis of RNA transcribed from DNA induced into carrot cells by electro-

A29 poration. Octopine synthase RNA transcribed from the plasmid p35SOcs was detected in total and poly A+ RNA on Northern blots and in RNA protection assays. The highest level of octopine synthase transcript was detected at approximately 8 hrs post-electroporation, although RNA could still be detected after 48 hrs. This method allows detection of foreign gene expression in a plant system and bypasses the need for reporter genes.

The efficiency of co-transformation was generally 20-30%, i.e. the frequency of kanamycin-resistant calli having both the neo and gusA active genes. Southern blot analysis using a probe for gusA indicated integration of several copies of the gene, often as head to tail tandem repeats. JOURNAL OF EXPERIMENTAL BOTANY 41 (228): 769-774 (1990)

P L A N T CELL REPORTS 9 : 1 3 9 - 1 4 2 (1990) Callus formation from root protoplasts of Quercus rubra L. (red oak) M. Brison and A. Lamant

Laboratoire de Physiologic Cellulaire V#g#tale, Universit# de Bordeaux I, Avenue des Facult#s, F-33405 Talence Cedex, France Root protoplasts of Quercus rubra L. were isolated from 12 day old seedlings with an enzyme mixture containing Cellulose R10 + Rhozyme HP150 + Macerozyme R10, supplemented with cysteine and bovine serum albumin. Protoplasts were purified by a Ficoll density gradient centrifugation and cultured at low density in a liquid medium. The modified woody plant medium, containing 2.2 lxM benzyladenine + 1.8 IJ2VIzeatin + 5.3 ~tM c~naphthaleneacetic acid + 2.2 ~ dichlorophenoxyacetic acid, allowed sustained divisions and formation of microcalluses. Protoplast-derived microcallus developed into green and compact callus when transferred to an agarose solidified medium, supplemented with casein hydrolysate and indole 3-acetic acid (devoid of 2,4-dichlorophenoxyacetic acid) and placed under low illumination.

Variation in net photosynthesis, Rubiseo activity and chloroplast ultrastructure among somatic hybrids of Solanum tuberosum and S. brevidens E. Pehu, A. Nurmi and M.A.J. Parry

Biochemistry and Physiology Department, AFRC Institute of Arable Crops Research, Rothamsted Experimental Station, Harpenden, Herts. AL5 2JQ, UK The effect of ploidy, parental chloroplast type and parental nuclear genome dosage on net photosynthesis, Rubisco activity and chloroplast ultrastructure was studied among somatic hybrids of diploid S. brevidens and dihaploid S. tuberosum. An increase in nuclear ploidy resulted in an increase in net photosynthesis and specific leaf weight. There were no significant differences in net photosynthesis or Rubisco activity between the hybrids having different parental chloroplast type. Examination of the hexaploid hybrids indicated that Rubisco activity was affected by nuclear-organelle genome incompatibility, the most affected combination being tuberosum chloroplast type with brevidens nuclear genome. Examination of chloroplast ultrastructure revealed wide variation in the size of chloroplasts, starch granules, plastoglobuli and in grana stacking among the hybrids and between fusion parents.

PLANT CELL REPORTS 9 : 1 6 8 - 1 7 2 (1990)

Co-transformation of indica rice protoplasts with gusA and neo genes Jianying Peng, Leszek A. Lyznik, Lisa Lee and Thomas K. Hodges

Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907, USA Protoplasts of the indica rice (Oryza sativa L.) variety, 117,54, were transiently transformed with the gusA gene and stably transformed with both the neo and gusA genes. We show that PEG-mediated co-transformation of protoplasts with two genes on separate plasmids coupled with selection on kanamycin is an effective way of transferring foreign gene(s) into the indica rice genome.

BULLETIN OF THE NATIONAL RESEARCH INSTITUTE OF VEGETABLES ORNAMENTAL PLANTS AND TEA 3 : 6 7 - 6 9 (1989) Protoplast culture in vegetable crops - Development and improvement of culture procedure Takeshi Nishio, Yoshiteru Sakata, Hiroshi Yamagishi, Yutaka Tabei, Takanori Sato and Kenji Takayanagi Methods for the isolation and culture of protoplasts were examined using various vegetables in 17 plant species. Efficient culture procedures for plant regeneration from protoplasts were developed in Brassica oleracea, Sola-

A30

num melongena, Lactuca sativa and Cichorium endivia. Improvement of the protoplast culture method led to constant plant regeneration but with a relatively low efficiency in Brassica campestris and Lycopersicon esculentum. Plants were regenerated from protoplasts of artificially synthesized Brassica napus 'Hakuran', Petasites japonicus, Cucumis melo and Daucus carota. Calli were obtained from protoplasts in Raphanus sativus, Capsicum annuum, Arcticum Lappa and Cucumis sativus, but they did not regenerate plants. Protoplasts did not divide normally in Colocasia esculenta, Dioscorea opposita and Allium sativum Protoplast culture procedures of these vegetables were compared and analysed to identify simple and efficient methods of culture.

PLANT CELL REPORTS 9 : 1 1 7 - 1 2 0 (1990) Controlled cell wall regeneration for efficient

microinjections of Nicotiana tabaeum var. Carlson protoplasts S. Joshi and A.M. Vincentini

Department of Microbiology, University of Toronto, 100 College Street, #214, Toronto, Ontario MSG 1L5, Canada Nicotiana tabacum var. Carlson protoplast culture conditions were modified to contain a cell wall inhibitor, 2,6-dichlorobenzonitrile, in order to delay cell wall regeneration and to allow efficient nuclear and cytoplasmic microinjections. Under modified conditions, the protoplast preparations appeared healthier as compared to the control protoplasts and showed no resistance at all during microinjection: Furthermore, the duration of protoplast microinjection was extended for up to 3-4 days. In order to set up nuclear microinjections, the nuclei of these protoplasts were stained either before or.after immobilization without any adverse effect on their mitotic activity. Successful cytoplasmic microinjections were demonstrated by injecting Alfalfa mosaic virus (AMV) RNA, which resulted in viral infection of 14% of the injected protoplasts.

14. Somaclonal variation PLANT SCIENCE 6 9 : 2 3 1 - 2 3 7 (1990) The selection of chlorsulfuron-resistant cell lines of independent origin f r o m an embryogenic cell suspension culture of Brassica napus L. Praveen K. Saxena, David Williams and John King

Department of Horticultural Science, University of Guelph, Guelph, Ontario N1G 2W], Canada A simple method has been developed to isolate chlorsulfuron-resistant cell lines of independent origin from an embryogenic cell suspension culture of Brassica napus cv Jet Neuf. Clonal cell cultures were used as the source of cells for the isolation of resistant variants. The selection procedure involved plating of cells, mutagenized with ethyl methane sulfonate, on a medium supplemented with 10-s or 5 x 10-s M chlorsulfuron and rescuing the cell colonies which survived after 7 weeks of incubation. The selected cell lines were 10-1000-fold more resistant than the wild-type to chlorsulfuron and another sulfonylurea herbicide sulfometuron methyl. The activity of the enzyme acetolactate synthase, the site of action of sulfonylureas, isolated from resistant cells was not inhibited by those concentrations of both the herbicides which inhibited the wild-type enzyme. Subculture of variant calli on agarose-solidified medium was necessary for the induction of embryogenesis, the further development of embryos, and the maintenance of resulting shoot cultures. Stem explants from regenerated shoots of the resistant variant formed callus on chlorsulfuron-supplemented medium indicating that the resistance was not lost during organogenesis.

PLANT PHYSIOL. 9 3 : 1 1 9 0 - 1 1 9 5 (1990) In vitro selection of calli resistant to a triazole cytochrome-P-450-obtusifoliol-14-demethylase

inhibitor from protoplasts of Nicotiana tabacum L cv Xanthi Pascale Maillot-Vemier, Hubert Schaller, Pierre Benveniste and Genevieve Belliard

Laboratoire de Biologic et Biochimie du Ddveloppement des Plantes, URA CNRS 1182, Universitd Louis Pasteur, 28 rue Goethe, 67083 Strasbourg Cedex, France The selection of biochemical mutants has been undertaken in order to elucidate regulatory and functional aspects of sterol biosynthesis in plants. 2-(4-Chloro-

A31 phenyl)- 3-phenyl- 1-(1H- 1,2,4-triazol- 1-yl)- 2,3-oxidopropane (LAB170250F), an experimental fungicide of the triazole family, was used as a selective agent. Indeed, this compound is a strong inhibitor of the cytochrome-P450-obtusifoliol-14-demethylase in sterol biosynthesis. The selection strategy consisted of screening large populations of microcalli derived from ultraviolet-mutagenized protoplasts of Nicotiana tabacum L. cv Xanthi for resistance to a lethal concentration of LAB170250F. The best selective conditions were first determined, i.e. strength of the selection pressure as well as the time and duration of its application in the developmental process from protoplast to whole plant. Selection experiments resulted in the recovery of 40 resistant calli. These calli were divided into three classes according to the modification of their sterol content in response to LAB170250F. Some of these calli might be impaired in sterol biosynthesis, but most have a sterol profile identical to that of the control calli. This suggests that the toxic properties of LAB170250F are due to the parallel inhibition of sterol biosynthesis and of at least one additional unidentified target in the plant cell. P L A N T PHYSIOL. 9 3 : 3 8 4 - 3 8 8 (1990)

Carbon use efficiency and cell expansion of NaCIadapted tobacco cells Sherry Rae Schnapp, Ray A. Bressan and Paul M. Hasegawa

Center for Plant Environmental Stress Physiology, Department of Horticulture, Purdue University, West Lafayette, Indiana 47907, USA Carbon use efficiencies (gram cell organic dry weight accumulated per gram sugar assimilated from the medium) of unadapted and NaCl-adapted (428 millimolar) cells of tobacco (Nicotiana tabacum L. var Wisconsin 38) were determined to evaluate metabolic costs associated with growth and survival in a saline environment. No net increase in carbon costs was associated with salt adaptation. At low substrate levels, carbon use efficiencies of unadapted and NaCl-adapted cells were not appreciably different (0.495 and 0.422, respectively) and at higher substrate levels carbon use efficiency of NaC1adapted cells was clearly higher than that of unadapted cells. These results indicate that a homeostasis of metabolic efficiency is established after cells have adapted to NaC1. Altered carbon availability does not cause the reduced cell volume that results from adaptation to NaC1. This does not preclude, however, the possibility that altered intracellular partitioning of carbon affects cell expansion.

P L A N T CELL REPORTS 9 : 8 0 - 8 3 (1990)

Growth, ammonia accumulation and glutamine synthetase activity in alfalfa (Medieago sativa L.) shoots and cell cultures treated with phosphinothricin Lori C. Krieg, Mark A. Walker, Tissa Senaratna and Bryan D. McKersie

Department of Crop Science, University of Guelph, Guelph, Ontario, NIG 2W1, Canada Phosphinothricin is a non-selective herbicide which inhibits glutamine synthetase (EC 6.3.1.2) activity causing an overaccumulation of ammonia in higher plants. Alfalfa (Medicago sativa) shoot tissue and petiole-derived callus exposed to phosphinothricin show 50 and 70% reductions, respectively, in glutamine synthetase activity with a concomitant rise of 10- and 20-fold, respectively, in endogenous ammonia. The diffusibility of ammonia may limit the use of a detoxifying gene, phosphinothricin acetyltransferase, as a selectable marker for alfalfa transformation. However, the addition of up to 40 times the standard levels of ammonium nitrate to the culture media used in this study had no effect on callus growth, although glutamine synthetase activity was inhibited by 50% and endogenous ammonia increased 27-fold. Therefore, ammonia accumulation may not be the primary cause of cell death in alfalfa after exposure to phosphinothricin. It follows that diffusion of ammonia from cell to cell would not restrict the selection for phosphinothricin acetyltransferase transformed cells, thereby indicating that this enzyme could be used as a selectable marker in transformation experiments.

P L A N T CELL REPORTS 9 : 1 3 3 - 1 3 6 (1990)

Specific differences in tolerance to exogenous berberine among plant cell cultures Hideharu Sato, Yoshinori Kobayashi, Hiroshi Fukui and Mamoru Tabata

Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto 606, Japan The tolerance of plant cells to exogenously administered berberine, an antimicrobial isoquinoline alkaloid, was studied using berberine-producing and non-producing cell suspension cultures. Both Coptisjaponica and Thalictrum flavum cells, which have an intrinsic ability to synthesize berberine, took up exogenous berberine from the culture medium by an energy-requiring active transport to accumulate it exclusively in vacuoles. By contrast T. minus cells, which excrete indigenous berberine most-

A32 ly into the medium, did not take up exogenously supplied berberine, indicating that the alkaloid transport in this species is unidirectional, No inhibition of cell growth by exogenous berberine was observed in the three berberineproducing cell cultures. On the other hand, a small amount of exogenous berberine strongly inhibited cell growth in the berberine-free cultures of Datura innoxia, Catharanthus roseus, and Paeonia albiflora. The berberine taken up actively by Datura cells could not be transported into vacuoles but was dispersed in the cytoplasm, causing a severe inhibition of cell growth.

of both cultivars maintained higher levels of K + than non-selected lines on all external NaC1 levels. Selected lines of Blue Crop had higher levels of Na ÷ and C1- than that of Denise Blue. The results suggest Na + and C1accumulation could be a mechanism allowing better growth in selected lines at moderate salinity levels (5075 mM NaC1).

CAN. J. P L A N T SCI. 70 547-550 (1990)

R.K. Jain, Sunita Jain, H.S. Nainawatee and J.B. Chowdhury

Heritability of in vitro regeneration in wheat (Triticum aestivum L.) This study measured the heritability of in vitro regeneration in wheat (Triticum aestivum L.). Immature embryos of 28 F2-derived families of the spring wheat cross HYg12 x HY320 were cultured in the F 3, F4 and F 5 generations. Highly significant (P<0.01) genotypic differences were observed for embryo germination, callus induction, embryogenic callus formation, and planflet regeneration. Comparisons of generation means and midparent values suggest that additive variance is an important factor in the inheritance of in vitro regeneration.

P L A N T CELL REPORTS 9 : 1 5 1 - 1 5 5 (1990)

Growth characteristics and ion contents of nonselected and salt-selected callus lines of highbush blueberry (Vaccinium corymbosum) cultivars Blue Crop and Denise Blue Morley S. Muralitharan, Reinhard F.M. Van Steveninck and Stephen F. Chandler

School of Agriculture, La Trobe University, Bundoora, 3083, Australia Non-selected and sodium chloride selected callus lines of Vaccinium corymbosum L.cv Blue Crop and cv. Denise Blue were grown on media supplemented with 0-100 mM NaC1. For both cultivars, fresh weight and dry weight yields were greater in selected lines on all levels of NaC1. Selected lines of Blue Crop displayed better growth than selected lines of Denise Blue at most concentrations of NaC1. Internal Na+ and C1- concentrations in selected and non-selected lines of both cultivars increased as external concentration was raised. However, selected lines of Blue Crop and Denise Blue accumulated more Na + and C1- than non-selected lines. Selected lines

E U P H Y T I C A 4 8 : 1 4 1 - 1 5 2 (1990)

Salt-tolerance in Brassica juncea L. I. In vitro selection, agronomic evaluation and genetic stability

Department of Genetics, Department of Chemistry and Biochemistry, Haryana Agricultural University, Hisar-125 004, India In vitro selection of salt tolerant plants of Brassicajuncea L. (Indian mustard) cv. Prakash has been accomplished by screening highly morphogenic cotyledon explant culrares on high NaCI media. Out of a total of 2,620 cotyledons cultured on high salt medium, 3 survived, showed sustained growth and regenerated shoots. They were multiplied by axillary bud culture on NaC1 free medium. The salt-selected shoots retained salt tolerance following 3 month of growth and multiplication on control medium. While two of these somaclones flowered and set seeds, the third one grew slowly, had abnormal leaf morphology and was sterile. The seed of the two fertile plants were sown in the field to raise R1 segregating generation. Data were recorded for field, other agronomic components and oil content. The somaclonal lines, both selected salt-tolerant and non-selected, showed tremendous amount of variation for all the characters studied. One of the two tolerant somaclones invariably showed reduced height, longer reproductive phase and higher 1000 seed weight. Based on the agronomic performance of R1 plants of these somaclones, some plants were selected and their progeny were evaluated for agronomic performance under standard field conditions and salt-tolerance in the greenhouse using sand pot culture method. Most of the lines bred true for their specific characteristics. In the greenhouse, selected salt-tolerant somaclones (SR-2 and SR-3) performed better for plant growth, yield and other agronomic traits at higher salt treatments, indicating thereby that salt-tolerance trait selected in vitro was expressed in the whole plants and is genetically stable and transmitted onto the progeny. The two tolerant lines, however, differed in their salt-tole-

A33 rance during vegetative and reproductive phases as indicated by their salt-tolerance and stress susceptibility indices. The mechanism of salt-tolerance is not clear and needs to be further investigated.

E U P H Y T I C A 4 8 : 1 8 9 - 1 9 6 (1990)

In vitro separation of chimerai pears into their component genotypes H. Abu-Qaoud, R.M. Skirvin and E. Chevreau

University of Illinois, Department of Horticulture, 1707 So. Orchard, Urbana, IL 61801, USA An adventitious shoot regeneration system for Pyrus communis was used to separate two chimeral pears into their component genotypes. The two cultivars were a variegated type, 'Louise Bonne Panach6e' and a red fruited mutant, 'Red Hardy'. Leaves of these cultivars were placed onto a regeneration medium consisting of Nitsch & Nitsch (1969) salts supplied with various levels of Thidiazuron (TDZ) and NAA. After two months the regenerants were moved onto a proliferation medium of Lepoivre salts. Later they were evaluated for their chimeral status. Among the regenerants of the variegated type, 100% segregation occurred, most shoots were green, a few were albino. Regeneration was more efficient for dissociating the variegated chimera than rapid shoot multiplication and physical injury. In 'Red Hardy', after two months on the regeneration medium, 20 to 33% of the regenerants were green, the rest were red. The stability of the red and the green regenerants were assessed on media supplemented with various levels of sucrose and by total anthocyanins measurement. Both types were stable.

CROP SCI. 3 0 : 7 8 1 - 7 8 5 (1990)

Salinity tolerance in Sorghum. II. Cell culture response to sodium chloride in S. bicolor and S.

halepense Y.W. Yang, R.J. Newton and F.R. Miller

Department of Forest Science and Department of Soil and Crop Sciences, Texas Agric. Exp. Stn., The Texas A&M Univ. System, College Station, TX 77843-2135, USA Grain sorghum [Sorghum bicolor (L.) Moench] is reported to be less tolerant to salt than the noxious weed and potential biomass energy crop, johnsongrass [S. halepense (L.) Pers.]. The objective was to compare callus growth and osmotic responses to salinity of these

two sorghum species. Callus was grown for 2 wk in a liquid Murashige and Skoog medium to which NaC1 was added to f'mal concentrations of 0.05, 0.1, and 0.15 M. Relative fresh weight growth of callus in response to increased salinity in the culture medium was greater in S. halepense than in S. bicolor. After 2 wk of NaC1 treatment, brown coloration and apparent necrosis were observed on callus of S. bicolor but not on that of S. halepense. The Na/K ratios were lower in callus of S. halepense than that of S. bicolor at all NaC1 concentrations. Total amino acids in salinized callus did not change, but proline increased; this increase was greater in S. halepense callus than in that of S. bicolor. Levels of Na and CI were larger in S. halepense callus treated with 0.15 mM NaC1 and these were associated with a q~p of 0.40 MPa, whereas a Up of 0.26 MPa was observed in callus of S. bicolor. Accumulation of NaC1 accounted for three-fourths of the solutes involved in osmotic adjustment in the callus of both species. Growth and Na/K ratios in salinized callus in this study were correlated with whole plant responses determined in another study. Callus growth and Na/K ratios could therefore be used as indicators of whole plant salt tolerance in Sorghum.

P L A N T CELL PHYSIOL. 31(3): 325-331 (1990)

Immunochemical analysis of multiple subunit polypeptides of glutamine synthetase in methionine sulfoximine-sensitive and tolerant tobacco cell cultures Tornoyuki Yamaya, Hirohiko Ishida, Kazunari Kamachi and Kunihiko Ojima

Laboratory of Plant Cell Engineering, Department of Agricultural Chemistry, Faculty of Agriculture, Tohoku University, 1-1 Amamiyamachi-Tsutsumidori, Sendai, Miyagi, 981 Japan A methionine sulfoximine (MSX) tolerant cell line of tobacco (Nicotiana tabacum L. cv. Xanthi) cells was selected by culturing the wild-type cells in suspension media in the presence of MSX at a step-wise increase in its concentration (0.3 to 5 ~tM). Fifty per cent inhibition of growth occurred at 0.18 [tM MSX for the wild-type cells whereas 4.65 ~tM was required for the tolerant cells. The tolerant cells possessed about 1.5-fold increase in glutamine synthetase (GS) activity. Kinetic experiments showed that an inhibitor constant for MSX was identical between GS isolated from these two cell types. Subunit polypeptides of GS in both cell types were analyzed with an immunoblotting method by using polyclonal antibody raised against a chloroplastic GS in spinach. A single

A34 polypeptide (41 kDa) was recognized by the antibody in wild-type cells, whereas two predominant polypeptides of 41 and 40 kDa were seen in the MSX tolerant cells. When the GS subunit polypeptides in the wild-type cells were examined with two-dimensional gel electrophoresis, two major and four minor polypeptides associating distinct charge at 41 kDa were detected. The extract from the MSX-tolerant cells had the same set of polypeptides at 41 kDa and in addition two major and some minor spots at 40 kDa. These results indicate that 1) tobacco GS is consisted of heterogeneous subunit polypeptides in surface charge and 2) MSX causes formation of additional multiple 40 kDa polypeptides which may be related to the tolerant nature of the selected cell line.

SCIENTIA HORTICULTURAE 4 2 : 2 1 - 2 8 (1990)

Cultivar differences in shoot-forming capacity of hypocotyl tissues of chilli pepper (Capsicum annuum L.) cultured in vitro Neftali Ochoa-Alejo and Leticia Ireta-Moreno

Centro de lnvestigaci6n y de Estudios Avanzados del I.P.N. Unidad Irapuato, Apartado Postal 629, 36500 Irapuato, Gto, Mexico Cultured hypocotyl segments of 16 cultivars of chilli pepper (Capsicum annuum L.) were tested for their ability to form adventitious shoots. Explants were grown on six culture media prepared with the basal medium formulated by Murashige and Skoog, supplemented with four combinations of indoleacetic acid (IAA)/benzyladenine (BA) or with three combinations of IAA/2-isopentenyladenine (2iP). Cultivar differences were observed with regard to their capacity for in vitro differentiation of shoots and approximately one-third (5/16) of the cultivars tested exhibited relatively high differentiation capabilities (determined by the number of shoots per explant and by the frequency of shoot formation). Optimal shoot regeneration medium varied with cultivar. Cultivar 'Anaheim TMR-23' displayed the highest shootregeneration response.

15. Somatic embryogenesis J. PLANT PHYSIOL. 1 3 5 : 3 0 6 - 3 1 2 (1989)

Somatic embryogenesis in Trifolium: Protein profiles associated with high- and low-frequency regeneration J.D. McGee, E.G. Williams, G.B. Collins and D.F. Hildebrand

Department of Agronomy, University of Kentucky, Lexington, KY 40546, USA A one-step regeneration system was developed for Trifolium rubens and Trifolium pratense using petiole segments of shoot cultured in vitro. Isogenic cell lines of T. rubens showing high- or low-frequency somatic embryogenesis (18 and 0.2 embryos per explant respectively) were isolated from a single seedling genotype by successive cycles of selection and subculture. Variation in the frequency of embryogenesis apparently arose by somaclonal variation in callus cultures; and in basal callus regions of shoot cultures. Using two-dimensional gel electrophoresis, the polypeptide translational profiles of cultured petioles from T. rubens isolines were examined over a 20-day interval covering the induction period for somatic embryogenesis. Two different genotypes of T. pratense showing high frequency or zero somatic embryogenesis in the same culture system, were also included in the protein analysis. Two polypeptides exhibiting appropriate behavior for association with somatic embryogenesis were detected in the three regenerating lines. These same two components were also detected in the non-regenerating T. pratense genotype. In this latter genotype, however, they were found to exhibit a translational pattern different from that expected of proteins involved in somatic embryogenesis.

PLANT CELL REPORTS 8 : 3 7 5 - 3 7 8 (1989)

Morphogenic and genetic stability in long-term embryogenic cultures and somatic embryos of Norway spruce (Picea abies [L.] Karst) L.H. Mo, S von Arnold and U. Lagercrantz

Department of Forest Genetics, Swedish University of Agricultural Sciences, Box 7027, S-75007 Uppsala, Sweden; Department of Plant Breeding, Swedish University of Agricultural Sciences, Box 7003, S-75007 Uppsala, Sweden Embryogenic cultures were initiated from mature zygotic embryos of Picea abies. The somatic embryos in the

A35 embryogenic cultures were first stimulated to mature and then either to develop further into plantlets or to differentiate new embryogenic cultures. The procedure was repeated three times during two years. The ability to give rise to new embryogenic cultures or to develop into plantlets was similar for all somatic embryos irrespective of how long they had been cultured in vitro. The nuclear DNA content, measured in a flow cytometer, was estimated at 32 pg/G1 nuclei in seedlings developed from zygotic embryos. Nuclei isolated from embryogenic cultures and from plantlets regenerated from somatic embryos had the same DNA content as those isolated from seedlings.

PLANT SCIENCE 6 9 : 2 3 9 - 2 4 7 (1990)

Comparative analysis of translatable mRNA populations in zygotic and pollen-derived embryos of barley (Hordeum vulgare L.)

differentiated embryoids were then exposed to high concentrations of both ABA and indole-3-acetic acid, after which samples were desicated to approx. 10% tissue moisture. Incubating cultures in 3.2 mmol/1 O2 (approx. 9%, low-O2) increased embryoid formation sixfold in one wheat line and nearly threefold in another. In the former line low-O2 caused the formation of mostly embryogenic callus. Low-O2 also decreased precocious germination of immature embryos, decreased callus growth, and improved development and viability of the resultant embryoids. Including 1.9 ~tmol/l ABA in the callus-induction medium reduced germination of immature embryos and reduced the incidence of embryoids with visible abnormalities. Despite the improved morphology, significantly fewer of the embryoids produced on ABA-containing medium germinated. Desiccation significantly enhanced germination of these embryoids as well as those produced an ABA free medium.

Patricia Higgins and Dianna J. Bowles

Department of Biochemistry, University of Leeds, Leeds, LS2 9JT, UK

PLANT SCIENCE 6 9 : 2 1 5 - 2 2 4 (1990)

Zygotic embryos of barley have been compared to those formed in anther culture. Polysomal RNA has been isolated from these two classes of embryo at different stages of development and translated in vitro. 35S-Translation products have been analysed by 2-D gel electrophoresis. Comparison of the profiles indicates that substantial differences exist in the pattern of gene expression in zygotic and pollen-derived embryos. Many translation products characteristic of the pollen-derived embryos are also present in callus.

E. Lain6 and A. David

PLANTA 1 7 5 : 4 1 7 - 4 2 4 (1988)

Improved somatic embryogenesis in wheat by partial simulation of the in-ovulo oxygen, growth-regulator and desiccation environments J.G. Carman

Plant Science Department, Utah State University, Logan, UT 84322-4820, USA The effects of 02, growth-regulators and desiccation on callus growth and somatic embryo (embryoid) development were investigated in cultures of immature embryos of two lines of Triticum aestivum L. Callus and embryoid formation were induced on media that contained N 6furfurylaminopurine (kinetin) and either 2,4-dichlorophenoxyacetic acid or 3,6-dichloro-o-anisic acid, either with or without abscisic acid (ABA). Cultures containing

Somatic embryogenesis in immature embryos and protoplasts of Pinus caribaea Labo Biotechnologies Vdgdtales, Facultd des Sciences, Universitd de Picardie, 33, rue Saint-Leu, 80039 Amiens Cedex, France Somatic embryogenesis was obtained from cultured immature embryos of Pinus caribaea Morelet var. hondurensis on media containing 2,4-dichlorophenoxyacetic acid (2,4-D), N6-benzyladenine (BA) and/or kinetin. The stage of zygotic embryo development for optimal somatic embryogenesis was found to be the early stage of polyembryony. Embryogenic calli have been subcultured for more than ten months and have retained their embryogenic ability. Different callus lines (different gene types) expressed distinct nutritional requirements for growth. Embryos bearing well differentiated cotyledons were recovered after transfer of calli on a modified Hakman and yon Arnold (J. Plant Physiol., 121 (1985) 149-155) medium supplemented with abscisic acid and devoid of organic nitrogen. Rooted plantlets were obtained after transfer to an hormone-free medium. Protoplasts were isolated from embryogenic cell suspensions. Sustained divisions of protoplast-derived cells and regeneration of somatic proembryos were achieved using first the medium designed for the culture of cotyledon protoplasts of the same species (E. Lain6 et al. Physiol. Plant., 72 (1988) 374-379) and then dilution with the cell suspension medium. The appearance and nutritional require-

A36 ments of the established protoplast-derived cell suspension were similar to those of the original cell suspension. Cell aggregates were transferred to solid medium for callus development. Fully differentiated embryos with cotyledons and hypocotyl were obtained 5 months after protoplast isolation.

ANNALS OF B O T A N Y 6 6 : 1 7 - 2 1 (1990)

Factors affecting formation of somatic embryos and embryogenic callus f r o m u n e m e r g e d inflorescences of a graminaceous crop Pennisetum Manjubala Talwar and A. Rashid

Department of Botany, University of Delhi, Delhi 110007, India Differentiation of somatic embryos was dependent on the concentration of auxin and the mineral medium. Low levels of auxin 2,4-D in N 6 medium, a low ammonium nutrient, favoured the formation of somatic embryos, while on MS medium containing high ammonium compact tissues appeared. At higher levels of auxin, irrespective of nutrient medium, compact tissues were formed. The origin of compact tissue on N6 medium could be traced to somatic embryo-like structures. This tissue regenerated into somatic embryos on hormone-free N6 medium whereas on MS medium thalloid structures appeared.

P L A N T CELL REPORTS 9 : 3 4 - 3 7 (1990)

Somatic proembryo production from excised, wounded zygotic carrot embryos on hormonefree medium: evaluation of the effects of pH, ethylene and activated charcoal David L. Smith and Abraham D. Krikorian

Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, NY 117945215, USA Wounded zygotic embryos of cultivated carrot produce somatic proembryos on hormone-flee nutrient medium containing 1 mM NH4+ as the sole nitrogen source. Continued maintenance of proembryos on this medium leads to a 'pure' culture of preglobular stage proembryos (PGSPs). Ethylene had no effect on this process. Also, somatic embryo production was not affected by growing cultures on activated charcoal-impregnated filter papers. However, somatic proembryos initiated on activated charcoal papers were not maintainable as PGSPs and devel-

oped into later embryo stages. Normally, medium pH dropped from 5.7 to 4 during each subculture period, but when using activated charcoal papers the pH endpoint was around 6-7 due to a leachable substance(s) within the filter papers. When powdered, actiVated charcoal was used in the medium as an adsorbent of products potentially released after wounding, pH dropped at the normal rate and to the expected levels; proembryos did not mature into later embryo stages and were maintainable exclusively as PGSPs. Low pH (=4) is detrimental to proembryo production, but is essential to maintaining PGSPs on hormone-free nutrient medium, whereas a sustained pH > 5.7 allows continued development of PGSPs into later embryo stages.

P L A N T CELL REPORTS 9 : 4 7 - 5 0 (1990).

Production of isolated somatic embryos from sunflower thin cell layers Bernard P61issier, Ouafa Bouchefra, R6gis P6pin and Georges Freyssinet

Rh~ne-Poulenc Agrochimie, Biologic Mol~culaire et Cellulaire Vdg~tale, BP 9163, F-69263 Lyon Cedex 09, France We describe here a two step procedure which allows the easy isolation of somatic embryos from Sunflower (Helianthus annuus L.) hypocotyl tissues. Thin cell layers composed of the epidermis plus 3 to 6 parenchyma cell layers were incubated for 5 days in a basal Murashige and Skoog medium using an auxin to cytokinin weight ratio of 1/1. The epidermis layers were then transferred to a Gamborg medium containing a high level of sucrose. After one week of incubation in this medium, many somatic embryos started to be released from the parental epidermal tissue. Even though the germination of these embryos is difficult, we have been able to induce secondary embryos and regenerate fertile plants.

CAN. J. BOT. 6 8 : 1 0 8 6 - 1 0 9 0 (1990)

Synchronous and high frequency germination of interior spruce somatic embryos following p a r tial d r y i n g at high relative humidity D.R. Roberts, B.C.S. Sutton and B.S. Flinn Forest Biotechnology Centre, British Columbia Research Corporation, 3650 Wesbrook Mall, Vancouver, B.C., Canada V6S 2L2 The germination of mature somatic embryos of interior spruce was limited by the low frequency of root emer-

A37 gence. In addition, development was abnormal, since elongation and greening of the hypocotyl and cotyledons preceded root emergence by 1-2 weeks. Pretreatment of the embryos on water-saturated Kim-paks increased the frequency of root emergence but did not alter the abnormal pattern of germination. Somatic embryos do not survive desiccation at room humidity, but partial drying at high humidity promoted germination up to 90%. Furthermore, this treatment decreased the time required for root emergence such that elongation of the root and hypocotyl-cotyledon was synchronized over a period of 5-6 days. This germination closely resembled that of excised zygotic embryos. Drying over a range of humidities indicated that humidities of 81% and lower were lethal to the embryos, whereas germination was enhanced following treatment at humidities greater than 95% relative to untreated controls. The best germination and root elongation occurred on one-half strength basal media containing 2-3.4% sucrose. Of the plantlets derived from treated embryos, 50% survived transfer to soil compared with only 5% of the untreated controls.

J. AMER. SOC. HORT. SCI. 115(4): 691-696 (1990) Somatic embryos derived from cotyledons of

P L A N T CELL REPORTS 9 : 9 3 - 9 6 (1990)

High frequency embryoid and phmtlet formation from tissue cultures of the Finger milletEleusine coracana (L.) Gaertn. Prasanna Sivadas, S.L. Kothari and N. Chandra

Department of Botany, University of Rajasthan, Jaipur, India Compact nodulated embryogenic callus differentiated from cultured seeds of Eleusine coracana(Finger Millet) on Murashige and Skoog (1962) basal medium with 2,4-dichlorophenoxyaeetic acid (1.0, 3.0 mg 1-1). This embryogenic callus was maintained on a medium with a lower level of 2,4-diehlorophenoxyacetic acid. At every subculture the embryogenic callus had some preexisting embryoids in it. With this method of subculture the callus has retained its morphogenic potential for four years. Following transfer to media with different levels of auxins and cytokinins, the callus showed varied patterns of growth and morphogenesis. Embryoids could be germinated in profusion to form plantlets which could be transferred to the field. Shoot buds also differentiated from the whole surface of the embryoid or from the flattened medstemoids.

cucumber

H O R T S C I E N C E 25(7): 795-797 (1990)

Rebecca M. Cade, Todd C. Wehner and Frank A. Blazich

Plant recovery from sweetpotato somatic embryos

Department of Horticultural Science, North Carolina State University, Raleigh, NC 27695-7609, USA

Raymond P. Ch6e, Jonathan R. Schultheis and Daniel J. Cantliffe

Two studies were conducted to test the effects of various tissue culture media on somatic embryogenesis from cotyledon tissue of cucumber (Cucumis sativus L.). The two best media for embryo initiation were Murashige and Skoog (MS) salts and vitamins containing either ! or 2 mg 2,4-D/liter and 0.5 mg kinetin/liter. In the second study, embryos developed more normally. More plantlets developed when tissue was removed from the initiation medium after 3 weeks and transferred to MS containing 1 mg NAA/liter and 0.5 mg kinetin/liter for 3 weeks, rather than leaving the embryos on a medium containing 2,4-D. Histological evidence indicated that the embryos were multicellular in origin. Charcoal in the maturation medium inhibited embryo development. Chemical names used: (2,4-dichlorophenoxy)-acetic acid (2,4-D); N-(2furanylmethyl)- 1H-purine-6-amine (kinetin); 1-naphthaleneacetic acid (NAA).

Vegetable Crops Department, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL 32611, USA Plant formation from somatic embryos in response to BAP, NAA and sucrose was studied in sweetpotato [Ipomoea batatas (L.) Lam.]. A maximum of 15% embryos at the torpedo stage of development formed plants on agar-solidified basal medium containing 3% sucrose and no growth regulators. The percentage of embryos forming shoots was increased to 53% by 4 p.M BAP, but BAP reduced whole plant formation and promoted callusing at the root axis end of embryos. The frequency of plant development was increased to 38% by adding 0.1 ~tM NAA to the basal medium. Reducing sucrose concentration to 1.6% in basal medium increased the frequency of plant development to 32%. Chemical names used: 6-benzylaminopurine (BAP); ct-naphthaleneacetic acid (NAA).

A38 HORTSCIENCE 25(7): 792-793 (1990)

High frequency of somatic embryogenesis and recovery of fertile cucumber plants Paula P. Chee

Molecular Biology Research, The Upjohn Company, 301 Henrietta Street, Kalamazoo, MI 49007, USA A simple procedure for regeneration of cucumber plants (Cucumis sativus L. cv. Poinsett 76) from cotyledon and hypocotyl explants has been developed. Somatic embryogenesis was induced on Murashige and Skoog (MS) salts and vitamins medium supplemented with 2,4-D at 2.0 mg.liter-1 and kinetin at 0.5 mg.liter-L Development of embryos was accomplished on MS medium with NAA at 1.0 mg.liter-~ and kinetin at 0.5 mg.liter-1. Eighty-five percent of the mature somatic embryos formed showed a typical bipolar structure. All developed into morphologically normal plantlets when transferred to MS medium containing no growth regulators. Chemical name used: 2,4-dichlorophenoxyacetic acid (2,4-D).

16. Genetic engineering PLANT PHYSIOL. 93:857-863 (1990) A polyethylene glycol-mediated protoplast t.ransformation system for production of fertile trans-

genic rice plants Akio Hayashimoto, Zhijian Li and Norimoto Murai

Department of Plant Pathology and Crop Physiology, College of Agriculture, Louisiana State University, Baton Rouge, Louisiana 70803-1720, USA We have established an efficient procedure for protoplast transformation and regeneration of fertile transgenic plants of rice (Oryza sativa L.) cultivars Nipponbare and Taipei 309. Protoplasts were mixed with a plant-expressible hygromycin resistance gene and treated with 25% (w/v) polyethylene glycol. Stringent selection of transformed colonies was applied to 14-day-old regenerated protoplasts in the presence of 95 micromolar of hygromycin B for 12 days. After selection, 450 and 200 resistant colonies were recovered per million treated Taipei 309 and Nipponbare protoplasts, respectively. Southern hybridization analysis of hygromycin-resistant cell lines and regenerated plants indicated that 1 to 10 copies of transferred DNA were integrated at 1 to 4 loci of the rice genome. Southern DNA analysis suggests that the introduced plasmid DNA may form concatemers by

intermolecular recombination prior to integration. Four Taipei 309 and 39 Nipponbare transgenic rice plants were regenerated and grown to maturity in the greenhouse. Two Taipei 309 and 35 Nipponbare plants set viable seeds. Agronomic traits of Taipei 309 transgenic plants and inheritance of the hygromycin resistance trait by progeny of the selfed transgenic plants were analyzed.

PLANT CELL REPORTS 9 : 1 0 - 1 3 (1990)

Agrobacterium-mediated transformation

of

strawberry calli and recovery of transgenic plants Narender S. Nehra, Ravindra N. Chibbar, Kutty K. Kartha, Raju S.S. Datla, William L. Crosby and Cecil Stushnoff

Department of Horticulture Science, University of Saskatchewan, Saskatoon, S7N OWO, Saskatchewan, Canada Transformed calli and shoots of strawberry (Fragaria × ananassa Duch.) cv. Redcoat were obtained using Agrobacterium tumefaciens carrying plasmid pBI121. Inoculated leaf explants produced transgenic calli at a frequency of 3% on selection medium containing 50 ~tg/ml kanamycin. Twenty per cent of selected calli regenerated, giving rise to transgenic shoots. All transgenic calli and shoots expressed substantial amounts of GUS and NPT-II activity. The Southern blot analysis confirmed the insertion of both marker genes into the strawberry genome as single and multiple copy inserts. The transgenic shoots elongated on rooting medium in the presence of 25 ~tg/ml kanamycin, but exhibited reduced rooting ability. PLANT PHYSIOL. 93:1110-1116 (1990) Factors influencing the tissue culture and the

Agrobacterium tumefaciens-mediated transformation of hybrid aspen and poplar clones Marc De Block

Plant Genetic Systems N.V., Jozef Plateaustraat 22, 9000 Gent, Belgium Tissue culture conditions and transformation have been established for both aspen and poplar. The use of previously described culture conditions resulted in shoot tip necrosis in the shoot cultures and necrosis of stem and leaf explants. Shoot tip necrosis could be overcome by buffering the medium with 2-(N-morpholino)ethanesulfonic acid and Ca-gluconate and by growing the shoots below 25°C. Necrosis of the explants was probably due

A39 to an accumulation of ammonium in the explants and could be overcome by adapting the NO3-/NH4+ ratio of the media. Stem explants of established shoot cultures of tbe aspen hybrid Populus alba x P. tremula and of the poplar hybrid Populus trichocarpa x P. deltoides were cocultivated with Agrobacterium strains having chimeric bar and neo genes on their disarmed tDNAs. Transformed aspen shoots were obtained from 30 to 40% of the explants, while transformed poplar shoots were obtained from 10% of the explants. Extracts from the transformed trees contained high phosphinotricin acetyltransferase and neomycin phosphotransferase activities, and the trees contained one to three copies of the chimeric genes. The transformed trees were completely resistant to the commercial preparations of the herbicide phosphinotricin (glufosinate), while control trees were not.

B I O / T E C H N O L O G Y 7 : 2 7 3 (1989)

The genetic engineering of two commercial p o t a to cultivars for resistance to potato virus X Andr6 Hoekema, Marianne J. Huisman, Lucy Molendijk, Peter J.M. van den Elzen and Ben J.C. Cornelissen

Mogen International N.V., Einsteinweg 97, 2333 CB Leiden, The Netherlands We efficiently transformed the commercial cultivars of potato (Solanum tuberosurn) Bintje, Desiree and Escort after optimizing the conditions for regeneration from potato tuber discs. For transformation, tuber discs were cocultivated with Agrobacteriura turnefaciens using a disarmed binary vector system. This system allowed the introduction of a chimaeric gene encoding the coat prorein (CF) of potato virus X (PVX) into two cultivars most susceptible to this virus, Bintje and Escort. Five transgenie plant lines with expression levels of CF higher than 0.1% of soluble leaf protein were analyzed for resistance to a challenging inoculation with PVX (1 ~tg/ml). We observed a delay in symptom development as well as a drastic reduction in the accumulation of virus. Furthermore, we found a correlation between the expression level of the CP-gene and the reduction in virus accumulation. Cytogenetic analysis of 62 independently obtained transgenic lines showed the normal tetraploid number of chromosomes (2n = 4x = 48) in 97 percent of the examined plants. Phenotypically all these plants appeared normal. One plant line. exhibited an abnormal phenotype and contained about the octaploid number of chromosomes (4n = 8x = 96). These results and preliminary data on morphological characteristics, as these are determined

in the official variety registration procedure, indicate that potato cultivars can be genetically engineered to contain new desirable traits with preservation of their intrinsic properties.

P L A N T CELL REPORTS 9 : 1 6 0 - 1 6 4 (1990)

Agrobacterium tumefaciens mediated transformarion and regeneration of muskmelon ~plants Guowei Fang and Rebecca Grumet

Department of Horticulture, Michigan State University, East Lansing, MI 48824, USA Transgenic muskmelon (Cucurais melo L.) plants were produced efficiently by inoculating cotyledon explants with Agrobacterlum tumefaciens strain LBA4404 bearing a Ti plasmid with the NPT II gene for kanamycin resistance. After co-cultivation for three days, explants were transferred to melon regeneration medium with kanamycin to select for transformed tissue. Shoot regeneration occurred within 3-5 weeks; excised shoots were rooted on medium containing kanamycin before transferring to soil. Morphologically normal plants were produced in three months. Southern blot analysis confu'med that ca. 85% of the regenerated plants contained the NPT gene. Dot blot analysis and leaf callus assay of progeny of transgenic plants verified transmission of the introduced gene(s) to the next generation. Factors affecting transformation efficiency are discussed.

P L A N T CELL REPORTS 9 : 8 8 - 9 2 (1990) Recovery of morphologically n o r m a l transgenic

tobacco from hairy roots co-transformed with Agrobacterium rhizogenes a n d a b i n a r y vector plasmid H. Hatamoto, M.E. Boulter, A.H. Shirsat, E.J. Grey and J.R. Ellis

Department of Biological Sciences, University of Durham, South Road, Durham DH1 3LE, UK Co-transformation of tobacco (Nicotiana tabacum) leaf explants with Agrobacterium rhizogenes harbouring pri1855 and the binary vector pBinl9 was achieved at a frequency of 67%. The kanamycin resistant hairy roots were cultured via a callusing phase to regenerate plants which were partially fertile when out-crossed with wildtype pollen. Phenotypic and molecular analysis of the F 1 progeny demonstrated the efficient segregation of the hairy root marker from the kanamycin resistance marker, enabling morphologically normal plants to be recovered

A40 which retained the binary vector marker gene. This co-transformation strategy provides a means of introducing non-selectable genes into plants in cases where antibiotic resistance markers are undesirable.

CROP SCI. 3 0 : 8 6 6 - 8 7 1 (1990)

Cell biology and molecular genetics: Engineering of herbicide-resistant alfalfa and evaluation under field conditions Kathleen D'Halluin, Johan Botterman and Willy De Greef

table marker and chloramphenicol acetyltransferase (CAT) as a reporter gene was utilized. In vitro grown plants were used as sources of explants to produce transgenic plants on selective medium containing 100 mg/l kanamycin. The transformation and expression of the foreign genes was conf'mned by DNA hybridizations, leaf disc assays, and by measuring NPTII and CAT enzyme activities. This technique is simple, rapid, efficient, and transgenic eggplants of this commercial cultivar have been transferred to soil where they have flowered and set seed.

Plant Genetic Systems N.V., J. Plateaustaaat 22, B-9000 Gent, Belgium

JOURNAL OF EXPERIMENTAL BOTANY 41 (226): 529-536 (1990)

The recent development of gene transfer systems for higher plants and progress in identifying genes encoding important agronomic traits have opened new possibilities for improvement of crop species. Our objectives were to establish a transformation procedure for alfalfa (Medicago sativa L.) line RA-3 using Agrobacterium-mediated T-DNA transfer on stem and petiole discs, compare the applicability of three selectable marker genes, and introduce a trait conferring resistance to a broad-spectrum herbicide. Fifty-nine transgenic lines carrying the bialapbos resistance gene bar encoding resistance to the herbicide glufosinate-ammonium [ammonium-DL-homoalanin-4-yl (methyl) phosphinate] were analysed under greenhouse and field conditions. The plants expressing bar under control of the cauliflower mosaic virus CaMV35S promoter showed the highest levels of resistance, whereas plants carrying bar under control of the T-DNA TR2' promoter generally exhibited only a tolerance under field conditions. Our data clearly demonstrate the necessity of combining molecular analysis and field evaluation of individual transgenic lines.

Transformation of sugarbeet (Beta vulgaris) by Agrobacterium tumefaciens K. Lindsey and P. Gallois

Leicester Biocentre, University of Leicester, Leicester LE1 7RH, UK A method has been developed for the regeneration of transformed plants of the commercially important crop sugarbeet (Beta vulgaris L.), using Agrobacterium tumefaciens. Binary vectors were used, carrying both screenable and selectable genes. Plant regeneration from shootbase tissues was found to be relatively rapid and frequent compared with petioles or leaf tissue. Inoculation of cultured shoot-base tissues resulted in the production of transformed plants, as determined by (1) introduced resistance to kanamycin, (2) introduced CAT or GUS activity, and (3) Southern blot analysis to show the integration of foreign DNA. The transformation frequency was found to be dependent upon explant source, plant genotype and selection conditions used.

BOT. MAG. TOKYO 1 0 3 : 1 1 - 2 3 (1990) P L A N T CELL REPORTS 9 : 2 6 - 2 9 (1990)

Transformation of eggplant (Solanum melongena L.) using a binary Agrobacterium tumefaciens vector G.L. Rotino and S. Gleddie

Plant Research Centre, Agriculture Canada, Ottawa, Ontario, KIA 0C6, Canada Kanamycin resistant plants of Solanum melongena L. (eggplant) cv. Picentia were obtained following the cocultivation of leaf explants with Agrobacterium tumefaciens. A disarmed binary vector system containing the neomycin phosphotransferase (NFFII) gene as the selec-

Observation by SEM of the attachment of Agrobacterium tumefaciens to the surface of vinca, asparagus and rice cells Nobuyuki Terouchi, Seiichiro Hasezawa, Hisashi Matsushima, Yasuko Kaneko and Kunihiko Sy6no

Department of Pure and Applied Sciences, College of Arts and Sciences, University of Tokyo, Meguroku, Tokyo 153, Japan Transformation of vinca cells was performed by the co-cultivation of cell-wall regenerated vinca protoplasts with Agrobacterium tumefaciens. Using this in vitro and single cell system, attachment of the bacteria to the

A41 surface of vinca cells was observed by scanning electron microscopy (SEM). Figures of the bacteria polarly binding to the plant cell wall were often observed. As Escherichia coli does not attach to the plant cells at all, the observed attachment of A. tumefaciens is suggested as a characteristic feature in crown gall induction. Even though no evidence of transformation was obtained by the co-cultivation methods, a similar attachment was observed in the cell-wall regenerated protoplasts of rice. The bacteria also attached to the surface of isolated mesophyll cells of asparagus and root hairs of rice. From these observations, we concluded that the attachment is not the limiting step of crown gall induction by A. tumefaciens in monocotyledonous plants. Extracellular fibrils like pili were observed with a few strains of A. tumefaciens for the first time. These fibrils were observed regardless of their ability of attachment and infectivity.

seems to differ with potato cultivar. The addition of a macroelement solution to the culture medium of disks clearly stimulated the growth of tumor tissues but did not increase the number of tumors. GA3 acted in the same way when it was added to the medium or to the inoculum. The effects of gibberellic acid and mineral elements are additive. However, tumors quickly become difficult to number when they grow too intensively. The other growth regulators studied (IAA, 2,4-D, kinetin) did not show any positive effect on tumorigenesis. These results are discussed in the context of the present knowledge on the effect of the studied factors on tumongenesis.

P L A N T CELL REPORTS 8 : 3 8 7 - 3 9 0 (1989)

Successful cocultivation of Brassica napus microspores and proembryos with Agrobacterium Paul M. Pechan

ANNALES DES SCIENCES NATURELLES, BOT A N I Q U E 13(10): 134-148 (1989)

The potato disk bioassay. Effect of some parameters related to the inoculum and to the growth medium on the development of tumors Messaoud Boudjeniba and Gerard Hunault

Universit~ P. et M. Curie, UER 59, Laboratoire de Vitroculture Exp~rimentale et Appliqu~e, Station de Biologie V~g#tale 'A. de Richelieu', Le Haut-Buisson, F-72400 Cherre, France We described previously (Boudjeniba and Hunault, 1988) the effect of various factors related to the explants and to the tubers on the development of crown gall tumors on potato disks grown in vitro. In this paper are given some results dealing with the efect of variations in the composition of the inoculum and of the growth medium of these disks. All the five Agrobacterium tumefaciens strains studied proved able to induce the appearance of tumors on potato disks. The number of tumors, however, varied widely with the strain. B6 and B6S3 strains gave the best results. The wild strains from Vine crown gall were less effective. With B6S3 strain, the highest number of tumors was obtained when the bacterial concentration of the inoculum reached 106 CFU/ml. Higher bacterial densities proved unnecessary. The more suitable pH values of the inoculum were between 5.0 and 6.0. pH higher than 6.0 reduced the tumorigenesis. Addition of DMSO to the inoculum, as advocated for the use of this system in the detection of antitumor activity of plant extracts, induced a strong decrease in tumor number (>20% for 12.5% DMSO in the inoculum). Sensitivity to DMSO

University of Guelph, Guelph, Ontario NIG 2W1, Canada Brassica napus microspores and microspore-derived proembryos were cocultivated with Agrobacterium turnefaciens harbouring a binary vector. The vector contained selectable genes for kanamycin and hygromycin antibiotic resistance. Microspores and proembryos survived the cocultivation procedure and subsequent antibiotic selection. Thousands of plantlets can be regenerated from a single experiment. Biochemical analysis indicated up to 7.3% of plants exhibited neomycin phosphotransferase II enzyme activity. Success of the cocultivation procedure depended largely on choosing the proper coculture conditions while allowing microspore embryogenesis to proceed.

B I O T E C H N O L O G Y 8 : 7 5 0 (1990)

Field resistance of transgenic Russet Burbank potato plants to effects of infection by potato virus X and potato virus Y Wojciech Kaniewski, Cliff Lawson, Bernard Sammons, Lisa Haley, Jesse Hart, Xavier Delannay and Nilgun E. Tumer

Plant Sciences Department, Monsanto Company, 700 Chesterfield Village Parkway, St. Louis, MO 63198, USA Transgenic Russet Burbank potato plants expressing the coat protein genes of potato virus X (PVX) and potato virus Y (PVY) were transplanted into the field after propagation in tissue culture. Virus resistance, plant

A42 growth and tuber yield were determined. Our results show that expression of PVX and PVY CP genes confer a very high level of resistance to PVX and PVY infection in clone 303, one of the four different transgenic potato clones tested. After inoculation with PVX and PVY, tuber yield of control Russet Burbank plants decreased, while the yield of clone 303 was unaffected. Four different uninoeulated transgenic clones had tuber yields as high as control Russet Burbank. These results confirm that resistance to PVX and PVY is effective in the field and can prevent yield losses due to dual infection by these viruses.

P L A N T CELL REPORTS 8 : 4 2 9 - 4 3 2 (1989)

Transient expression of chloramphenicoi acetyltransferase (CAT) gene in barley cell cultures and immature embryos through microprojectile bombardment K.K. Kartha, R.N. Chibbar, F. Georges, N. Leung, K. Caswell, E. Kendall and J. Qureshi

Plant Biotechnology Institute, National Research Council of Canada, 110 Gymnasium Road, Saskatoon, S7N OW9, Saskatchewan, Canada Transient expression of chloramphenicol acetyl transferase gene has been detected in cultured barley (Hordeum vulgare L. cv. Heartland) ceils and freshly isolated immature zygotic embryos (cv. Ellice) following the introduction of the gene by microprojectile bombardment. The DNA expression vector used to introduce the CAT gene, pCaMVI1CN, is a pUC8 derivative and consisted of a CaMV35S promoter, a fragment of alcohol dehydrogenase intronl, a CAT coding region and NOS polyadenylation region. The inclusion of the Adhl intronl was essential for the expression of CAT activity in cultured cells as well as immature zygotic embryos. Expression of CAT activity, which was dependent upon the DNA concentration used, could be detected as early as 20 h after bombardment. The results also suggested that the recipient cells have to be in an active state of cell division in order for the introduced gene to be expressed since mature zygotic as well as somatic embryos failed to reveal any gene expression. The effect of other param, eters which influence the expression of the introduced gene as well as the potential of this novel technology for cereal transformation are also discussed.

E U P H Y T I C A 4 8 : 1 5 3 - 1 5 7 (1990)

Expression in ritro of resistance to Heterodera schachtii in hairy roots of an alien monotelosomic addition plant of Beta vulgaris, transformed by Agrobacterium rhizogenes H. Paul, J.E.M. van Deelen, B. Henken, Th.S.M. de Bock, W. Lange and F.A. Krens

Foundation for Agricultural Plant Breeding, SVP, P.O. Box 117, 6700 AC Wageningen, The Netherlands Hairy roots, induced by Agrobacterium rhizogenes, were obtained of nematode susceptible beet plants (Beta vulgaris) and of the nematode resistant alien monotelosomic addition AN5, carrying a telosome from B. Patellaris. The additional telosome was found to be stably present in vitro in the roots of AN5. The hairy root cultures were inoculated with larvae of the beet cyst nematode Heterodera schachtii. On the root culture of AN5 significantly less cysts developed than on the other root cultures. These results indicate that the resistance to the beet cyst nematode is expressed in the roots after transformation and can be monitored under in vitro conditions.

BIOCHEM. PHYSIOL. P F L A N Z E N 1 8 5 : 1 3 5 - 1 4 0 (1989) Transformation of field bean (Viola faba L.)

cells: Expression of a chimaeric gene in cultured hairy roots and root-derived callus Joachim Schiemann and Gisela Eisenreich

Zentralinstitut fiir Genetik und Kulturpflanzenforschung der Akademie der Wissenschaften der DDR, Gatersleben, GDR A procedure for transformation of Vicia faha cells has been developed. Inoculation of bean seedlings with Agrobacterium rhizogenes resulted in the formation of roots at the inoculation sites. Transformation was monitored by detection of GUS activity resulting from the stable expression of a chimaeric l~-glucuronidase gene cotransferred with the T-DNA of Agrobacterium rhizogenes. The GUS gene was provided by a disarmed binary vector derived from Agrobacterium tumefaciens. GUS-positive roots were propagated in vitro on hormone-free solidified medium. Callus established on transformed roots maintained GUS activity during the culture period.