1997 Congress on In vitro Biology Special Poster Session Abstracts

1997 Congress on In vitro Biology Special Poster Session AbstractsSource: In Vitro Cellular & Developmental Biology. Plant, Vol. 33, No. 4 (Oct. - Dec., 1997), pp.313-322Published by: Society for In Vitro BiologyStable URL: http://www.jstor.org/stable/4293148 .

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CONGRESS ON IN VITRO BIOLOGY

1997 MEETING OF THE SOCIETY FOR IN VITRO BIOLOGY

June 14-18, 1997 * Washington, DC -Special Poster Session Abstracts-

SP-1000 Production of a Diagnostic Monoclonal Antibody in Transgenic Alfalfa L.-P. VEZINA, H. Khoudi, J.-M. Ferullo, R. Bazin, R. Lemieux, S. Laberge

SP- 1001 Transformation of Short-Season Adapted Soybeans with a Wheat Oxalate-Oxidase Gene PA. DONALDSON and D.H. Simmonds

SP- 1002 Somatic Embryogenesis from Anthers and Ovaries of Six Grapevine (Vitis

sp.) Cultivars

J.R. KIKKERT, M.J. Striem, M.H. Martens, P.G. Wallace, B.I. Reisch SP-1003 A Commercially Economic Protocol ofIn Vitro Production of Chrysanthemum Cultivars

TARAKNATH CHAKRAVARTY SP- 1004 Simultaneous In Vitro Organogenesis & Somatic Embryogenesis in Coconut (Cocos nucifera L.) Immature LeafTissues

JOHN L. GRIFFIS, JR. and Richard E. Litz SP-1005 Transgene Inheritance through Individual Chimeric Plants; Toward Minimizing Tissue Culture in Maize

Transformation M. ROSS, K. Lowe, G. Sandahl, M. Miller, G. Hoerster, L. Church, L. Tagliani, D. Bond, W. Gordon-Kamm

SP- 1006 Application of Green Fluorescent Protein (GFP) as a Reporter Gene in Wheat Transformation T. HU, S.Z. Pang, J.E. Fry, M. Fromm

SP-1007 Evaluation of GFP as a Scorable Marker in Cotton Tissues

J.A. BURNS, S. Pang, T.A. Armstrong, G. Ye, M.A. Hinchee SP- 1008 AFLP Marker Analysis of Somaclonal Variation in Pecan Somatic Embryogenic Cultures

W.A. VENDRAME, G.D. Kochert, H.Y. Wetzstein SP-1009 Plant Regeneration from Protoplasts of Sweetpotato (Ipomoea batatas)

S.K. DHIR, J. Oglesby, and A.S. Bhagsari SP-1010 Transient Gene Expression in Intact Cells of Sweetpotato Following Electroporation

T.D. MICHELL, A.S. Bhagsari, P. Ozias-Akins, S.K. Dhir SP-1011 Targets for Arabidopsis Vacuum Infiltration Transformation by Agrobacterium

GUANGNING YE, Michael Fromm, Maud Hinchee SP-1012 Artificial Seed Production from Encapsulated Somatic Embryos of Carica Papaya L.

B. CASTILLO and M.A.L. Smith SP-1013 Cryopreservation: An Ex Situ Conservation Strategy for Butternut (Juglans cinerea)

TANNIS BEARDMORE, Linda DeVemo, Garry Scheer, Wendy Vong SP-1014 Studies on the New Channel for Creation of Primary Trisomics Through Anther Culture ofAutotetraploid Rice

QIN RUIZHEN and Liu Zongxian SP-1015 Studies of Somatic Embryogenesis in 20 Cultivars of Carica papaya

T.W. ZIMMERMAN, J.A. Richards, J.A. Kowalski SP-1016 Agrobacterium-mediated Genetic Transformation in Tomato (Vty Pusa ruby) (Lycopersicum Esculentum)

for Virus Resistance G. LAKSHMI SITA, M. Manoharan, C.S. Sreevidya, C.T. Ranjit Kumar, H.S. Savithri

SP-1017 Interactions of Days of Spike Storage with Days after Anthesis and Sucrose Concentrations in Promoting Wheat Somatic Embryogenesis B.-C. LI, K. Caswell, N. Leung, R.N. Chibbar

SP-1018 Evaluation of Constitutive Promoters in Brassica napus J.B. BADE, J. Van Roekel, S. Hoekstra, L.S. Melchers, M.H. Stuiver

SP-1019 Polyscias Filicifolia Bailey Cell Culture in Bioreactors A.G. KLYUSHIN, A.V. Oreshnikov, N.D. Chernyak, O.V. Reshetnyak, A.M. Nosov

SP-1020 Diterpenoid Glycosides Biosynthesis in Stevia rebaudiana Bertoni Tissue Culture N.I. BONDAREV, O.V. Reshetnyak, A.M. Nosov

SP-1021 Utilization of Biotechnological Methods in Selection of Wheat Stability to Septoria LETHY JOSE and E.A. Kalashnikova

SP-1022 Construction of Transgenic Rapeseed Plants (Brassica napus L.) Containing Crucifer Infecting Tobamovirus Movement Protein Gen. S.D. ZVEREVA, G.N. Raldugina, O.V. Borisova, Y.L. Dorokhov

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SP-1000 Production of a Diagnostic Mono- clonal Antibody in Transgenic Alfalfa. L.-P. VEZINA1, H. Khoudi3, J.-M. Ferullol, R. Bazin2, R. Lemieux2, S.

Laberge1. 1Agriculture and Agri-Food Canada, Sainte-Foy, Quebec, Canada

G1V 2J3; 2Canadian Red Cross So-

ciety, Sainte-Foy, Quebec, Canada

G1K 7B5; 3FSAA, Universite Laval, Quebec, Canada G1K 7P4.

The isolation of cDNA clones encoding for the Mabs

light and heavy chains has opened the way for the

production of antibodies in heterologous expression systems, including bacteria, insect cells and plants. However, the use of Mabs for diagnostic and thera-

peutic uses requires that the original biological source of the functional molecule (native or recombinant) be stable. For example, homologation of a Mab for

diagnostic use, generally requires that a cell bank is established from which aliquots can be retrieved and used as inoculum to initiate repeated production cycles. Repeated subculturing of bacterial strains or

hybridoma cell lines is therefore not ideal as a means of maintenance for an homologated biological source. To assess the potential of a perennial plant as a stable source of recombinant Mab, the cDNAs encoding for the light and heavy chain of a diagnostic Mab were first transferred into clones of a regenerative alfalfa

genotype. Stable expression of the fully functional multimeric protein was obtained in the progeny fol-

lowing sexual crossing of the monotransgenics. Sta-

bility and integrity of the protein was then monitored in the clonal material obtained through propagation of stem cuttings and through large scale somatic em-

bryogenesis. This study demonstrates the advantage of using the perennial characteristic of a plant in com-

parison with annual seed plants, to generate dual-

transgenics which can be fully characterized and used as a stable perennial source of recombinant material.

SP-1001 Transformation of Short-Season

Adapted Soybeans with a Wheat Ox- alate-Oxidase Gene. P.A. DONALDSON and D.H. Simmonds. Eastern Cereal and Oil- seed Research Centre, Agriculture and

Agri-Food Canada, Ottawa, Ontario Canada KIA OC6.

Short-season adapted soybeans were transformed with a wheat germin gene, which encodes an oxalate oxidase, to investigate whether the gene confers tolerance, or possibly resistance, to the oxalic acid-producing fungal pathogen Sclerotinia sclerotiorum. Cotyle- donary node explants from 4 day-old seedlings of'AC Colibri' and 'OAC Shire' were wounded, and then co-cultured in the presence of acetosyringone, with A. tumefaciens EHA105 carrying the binary vector

pRD4001 (similar to pBin 19 but with an altered nptll coding region) into which we cloned a 700 bp fragment of coding region from the wheat germin gene (gf-2.8)2 regulated by the CaMV 35S promoter, en- hancer and 3' termination sequences. Explants were co-cultured for 3 days on B5 medium with 3% sucrose + 1 mg/L 6-benzylaminopurine, and transgenic shoots were recovered 1-2 mo. later, after culture on the same medium but in the presence of 300 mg/L kanamycin. A histochemical assay for oxalate-oxidase was used for early identification of putative transformed shoots. Transgenic shoots were recovered from 0.4-2.4% of explants, but not all shoots developed to mature plants. Transgene integration and expression are confirmed thus far for three mature, fertile soy- bean plants. Integration patterns and correlation with levels of expression of the wheat 'oxalate-oxidase' gene will be described. Transmission of the gene and ex-

pression in Ti plants has been confirmed. Homozy- gous T1-derived T3 or T4 transgenic populations will be evaluated in field trials in 1998. 1Datla, D.S.S., et al. (1992) Gene 211:383-384 2Lane, B.G., etal. (1991)J. Biol. Chem. 266:10461-10469

SP- 1002 Somatic Embryogenesis from Anthers and Ovaries of Six Grapevine (Vitis sp.) Cultivars. J.R. KIKKERT, M.J. Striem, M.H. Martens, P.G. Wallace, B.I. Reisch. Cornell University, New York State Agricultural Experiment Station, Department of Horticultural Sciences, Geneva, NY 14456 USA.

Embryogenic cultures are currently the preferred tar-

get tissue for genetic transformation of grapevine because they provide a large number of regenerable cells. For the past 3 years, our laboratory has studied the initiation of somatic embryos in V vinifera 'Chardonnay', 'Merlot', and 'Pinot noir', V. Labruscana 'Concord' and 'Niagara', and the complex interspe- cific hybrid 'Chancellor'. A total of nearly 42,000

314



anthers and 9,000 unfertilized ovaries were extracted from flower buds at the uninucleate stage taken from

field-grown vines. Explants were cultured on either

Murashige and Skoog (1962) or Nitsch and Nitsch

(1969) basal medium with different concentrations of 2,4-D, NOA, BA, and/or 4-CPPU. Some of the

explants were cultured in liquid media, whereas oth- ers were cultured on media solidified with 0.25% (wt/ vol) Gelrite. Somatic embryos were obtained from all cultivars in at least one medium and one of the years. 'Chardonnay' and 'Chancellor' readily produced em-

bryos in all years and on several media, with frequencies in individual treatments as high as 24%. Embryo- genesis in the other cultivars occurred in fewer ex-

plants (highest frequency 4%) and on fewer media treatments. In general, the frequency of somatic em-

bryogenesis was higher from ovaries than from an-

thers, and on Nitsch and Nitsch basal medium with 500 mg/L glutamine, 2.5 _M 2,4-D, 2.5 _M NOA, and 5 _M4-CPPU (R. Schaefers, Ph.D. thesis, Cornell

Univ., 1995). Techniques were developed to prevent germination and to multiply embryogenic cultures of the vinifera genotypes for genetic transformation. Similar techniques have not yet been found for the Labruscana cultivars.

SP-1003 A Commercially Economic Protocol of In Vitro Production of Chrysanthe- mum Cultivars. TARAKNATH CHAKRAVARTY. Division of Plant

Physiology & Biochemistry, Indian

Agricultural Research Institute, New Delhi-110012, India.

An efficient and rapid propagation method comprising initiation of in vitro shoot tip culture from forty three field-grown Chrysanthemum cultivars Chrysan- themum Morifolium following successful in vivo root-

ing, was developed. Systematic and optimum hard-

ening conditions were studied and a cost effective protocol was worked out. A modified medium after Jaacov and Langhams 1972, comprising of MS (major and minor _) + Myoinositol 10 mg/l + Kinetin 2 mg/l + Indole Acetic Acid 0.5 mg/l + Activated Charcoal 10

mg/l + Coconut milk 5% + Sugar 3% + Agar (Quixel) 0.8%, pH 5.8, was used as the initiation as well as the

multiplication medium. The initiation time being three weeks, and the establishment phase 16 wk (pas- sage every 4 wk), shoot multiplication was 15-20 fold

in almost all cultivars. In vitro rooting, though 95%, was avoided to make the protocol cost effective, and in vivo rooting was the most efficient at 95-100%. An efficient production plan was also worked out in which a batch of 5,000 plants were produced in vitro, and

successfully transferred to the field, in the later half of

February in 25 wk. In the month of April/May, shoot

tips were excised from the micropropagated plants, and were subjected to in vivo rooting, and replanted in the field. During the planting season (June/July), rooted shoots, 1_"-2" in size, can be exploited for commercial purposes in tiny netted pots or in Hycotrays. This protocol is successful in producing more than 100,000 rooted shoots from 5,000 micropropagated plants in about 45 wk in all. In some specific cultivars, a comparative study was made between conven-

tionally grown plants and in vitro propagated plants re-

garding shoot length, flower size etc., and it was found that the vigor of the plants cultivated in vitro was supe- rior to the conventional ones in this protocol.

SP-1004 Simultaneous In Vitro Organogenesis and Somatic Embryogenesis in Coco- nut (Cocos nucifera L.) Immature Leaf Tissues. JOHN L. GRIFFIS, JR.1 and Richard E. Litz2. 1 Faculty ofAgricul- ture & Natural Resources, Africa Uni-

versity, P.O. Box 1320, Mutare, Zim- babwe; 2Tropical Research & Educa- tion Center, IFAS-University of

Florida, 18905 SW 280th St., Home-

stead, FL 33031.

This series of investigations was designed both to ex-

plore in vitro morphogenesis of selected 'Malayan Dwarf' coconut palm tissues and to study the re-

sponses of these tissues to a novel growth regulator. Immature leaf pieces were utilized as explants for these

experiments. Callus initiation and direct initiation of various organs and somatic proembryos were stimu- lated by addition of both 2,4-dichlorophenoxyacetic acid (2,4-D) and diethylstilbestrol (DES) to modified Eeuwen's Y3 medium. Effective rates of these

supplementary chemicals were 5 to 15 mg/l of DES and 25 to 50 mg/l of 2,4-D. Some immature leaf pieces formed callus on their cut edges exclusively, while oth- ers directly formed roots, miniature leaflets, apical shoot-like structures, or numerous somatic

proembryos. Some of the somatic proembryos con- tinued to grow and developed haustoria-like tissues

315



and/or roots and obvious bipolarity. Further shoot apex development did not appear to occur in the

proembryos under these specific cultural conditions. The mixture of direct organogenesis and somatic em-

bryogenesis occurring within the same explants, cultured on the same media and produced at the same time is rather unusual.

SP-1005 Transgene Inheritance through Indi- vidual Chimeric Plants; Toward Mini-

mizing Tissue Culture in Maize Trans- formation. M. ROSS, K. Lowe, G. Sandahl, M. Miller, G. Hoerster, L. Church, L. Tagliani, D. Bond, W. Gordon-Kamm. Pioneer Hi-Bred Int'l Inc., Johnston, IA 50131.

After particle-mediated DNA delivery into the maize

meristem, two approaches were used to promote transgene inheritance. The first caused extensive meristem reorganization using a selection regime. Bom- bardment of coleoptilar stage embryos with a select- able marker (UBI::NPTII::pinlI or UBI::aadA::pinII) and the gene encoding GUS was followed by hormon-

ally-induced shoot proliferation with selective pressure. This caused sufficient reorganization in the meristem to produce stable periclinal or completely transgenic plants, and resulted in transgene inheritance. The second approach used direct germination; after bombardment of coleoptilar-stage embryo mer-

istems, methods to reorganize the meristem and alter

transgenic sector fate were tested. Transgenic sectors were monitored by histochemical staining for GUS

activity (expression of UBI::uidA::pinllI). Without additional treatment, targeting the meristem produced frequent transgenic sectors in plants that typically did not persist or transmit to progeny. Treatments used to

reorganize the meristem included physical meristem

disruption and application of selective pressure (streptomycin). Both treatments resulted in larger, more

persistent transgenic sectors, and using this method

multiple events containing the aadA gene (conferring streptomycin resistance) and GUS have transmitted to progeny.

SP- 1006 Application of Green Fluorescent Pro- tein (GFP) as a Reporter Gene in Wheat Transformation. T. HU, S.Z.

Pang, J.E. Fry, M. Fromm. Ceregen, Monsanto Life Science Company, 700 Chesterfield Parkway North, St. Louis, MO 63198.

A GFP (green fluorescent protein) gene has been suc-

cessfully used as an excellent reporter gene in the wheat transformation system. GFP is an excellent tool to

optimize transformation parameters and improve ef-

ficiency. GFP is a vital, non-destructive marker.

Glyphosate selection procedure was used when CP4

gene and GFP gene were co-bombarded into immature embryos to obtain the transgenic plants. CP4 gene was confirmed to have glyphosate resistance, and used as a select marker in this paper. Several hundred green spots per bombarded callus could be seen as early as 14 hours after bombardment with a fluorescent mi-

croscope. Modified GFP construct pMON30098 (e35S/hsp70/syn S65T gfp.3aa mut gfp/nos) has bet- ter transient and stable expression than the previous version pMON30049 (e35S/hsp70/syn S65T gfp/ nos). pMON30098 with GFP produced 3-5 fold more transient spots than pMON-19468 with GUS produced with the current testing system when the two constructs were co-transformed into the same callus. Results indicate that GFP has made it possible to vi-

sually select for more transgenic plants than using glyphosate selection alone.

SP-1007 Evaluation of GFP as a Scorable Marker in Cotton Tissues. J.A. BURNS, S. Pang, T.A. Armstrong, G. Ye, M.A. Hinchee. Monsanto Co., 700 Chesterfield Pkwy. North, St. Louis, MO 63198.

Green fluorescent protein (GFP) has potential for use as a nondestructive scorable marker to replace destructive GUS assays in plant transformation systems. In addition, GFP may be useful as a new tool for track-

ing cellular growth and lineage in plant developmental studies. In this paper we test transient GFP expression in various cotton seedling tissues and zygotic embryo meristems which were microprojectile bombarded with an EnCaMV35S-gfp construct. To track

development of stable events, embryogenic suspension cultures of cotton were cobombarded with GFP and a CaMV35S-nptllI: FMV-gus construct for in vitro

kanamycin selection. High endogenous fluorescent

activity in cotton embryo axes and green tissues ob-

316



structed GFP detection under a fluorescent micro-

scope. Transient activity in etiolated tissues however was easily detected. In etiolated cotyledons using the PDS1000-He, 1800 psi allowed GFP expression in numerous cell layers, whereas 1100 psi allowed ex-

pression primarily in the outer epidermis. Epidermal cells of etiolated hypocotyls expressing GFP contin- ued rapid expansion, producing elongated fluoresc-

ing cells. The nucleus of GFP-expressing cells often fluoresced much brighter than the cytoplasm. The fate of GFP-expressing cells in kanamycin selected embryogenic cultures and ACCELL-bombarded embryo meristems will be discussed.

SP- 1008 AFLP Marker Analysis of Somaclonal Variation in Pecan Somatic Embryo- genic Cultures. W.A. VENDRAME1, G.D. Kochert2, H.Y. Wetzstein1. 1Dept. of Horticulture or 2Dept. of

Botany, Univ. of Georgia, Athens, GA 30602-7273.

Somatic embryogenic protocols have been developed for pecan [Carya illinoinensis (Wangenh.) C. Koch], in which high multiplication rates and efficient plant regeneration can be obtained. However, before somatic

embryogenesis can be exploited for clonal propagation in pecan or other species, the genetic fidelity of

regenerated plants must be evaluated and the production of true-to-type individuals verified. Current ef- forts include field testing of regenerated plants. Prob- lematic of woody species is the extensive evaluation time needed for assessments. The development of methods whereby plants could be rapidly screened for

potential tissue culture-derived genetic changes would be very valuable. A recent technique, AFLP (Ampli- fied Fragment Length Polymorphism) is relatively easy to perform, reliable, uses small amounts of tissue, and can identify significant polymorphisms at more sen- sitive levels than obtainable with RFLP or RAPD

analyses. In the present study, we evaluate the appli- cability of using AFLP to assess the genetic variability in somatic embryos of pecan. Comparisons are made

among and within embryogenic culture lines. Because

length of time in culture has been proposed to affect the degree ofsomaclonal variation, the degree ofvaria- tion in cultures initiated up to eight years ago is con- trasted.

SP-1009 Plant Regeneration from Protoplasts oFSweetpotato (Ipomoea batatas). S.K. DHIR, J. Oglesby, A.S. Bhagsari. Ag- ricultural Research Station, Fort Val-

ley State University, Fort Valley, GA 31030.

A method for regenerating plants from petiole protoplasts of in vitro raised sweetpotato (Ipomoea batatas)

cv. "Jewel" is described. Protoplast yield of 3.0-5.0 X

106 (per gm fw tissue) were obtained following 4-6 hours digestion with 1% Cellulase-R10, 2%

Macerozyme-R10, and 0.3% of Pectolyase-Y23 in

9MCPW. A plating density of 1 X 105 protoplast/ml was found optimal for culture. An initial division fre-

quency of 12-15% was obtained in liquid or agarose- gelled KP8 culture medium supplemented with 2,4- D (0.2 mg/1) and zeatin (0.5 mg/1). Colonies consist-

ing of as many as 100-200 cells were formed after 4 wk in the dark at 26_ C. The frequency of colony formation was improved by a gradual addition of fresh

liquid medium of lower osmoticum on the agarose bead or on liquid layer of cultured cells. Protocalli (1- 2 mm in diameter) were formed after 4-6 wk of culture in dark and later under continuous illumination for two weeks. Following transfer of calli onto medium containing 2,4-D (0.1-2.5 mg/l1) and BAP (0.5

mg/1), morphogenic callus started to form globular and heart-shaped structures that developed into leaf

primordia. Subsequently, shoot elongation and root-

ing were obtained on media with 2% sucrose and 1

mg/I GA3. The regenerated plantlets were successfully transplanted in the soil where plants appeared pheno- typically normal. The same petiole protoplast populations were found to efficiently express the transient

expression of introduced GUS gene using electroporation. This study indicates that petiole protoplast can be used to regenerate plants and for gene transfer study.

SP-1010 Transient Gene Expression in Intact Cells of Sweetpotato Following Electroporation. T.D. MICHELL, A.S. Bhagsari, P. Ozias-Akins1, S.K. Dhir. 1Department of Horticulture, Coastal Plain Station, Tifton, GA

31793; Agricultural Research Station, Fort Valley State University, Fort Val-

ley, GA 31030.

317



Transient expression of the B-glucuronidase (GUS) gene has been studied in leaf-derived embryogenic callus of sweetpotato [Ipomoea batatas L. (Lam.)] fol-

lowing electroporation. GUS gene expression (number of blue spots) was influenced by the several fac- tors including electroporation conditions, buffer con-

ductivity, time course of transient GUS gene expression, DNA concentration, enzyme treatment and PEG treatment. Among the different field strengths (250- 1000 V/cm) used, maximum GUS gene expression (an average of 90 blue spots) was observed after 48 h when callus pieces were preincubated with

electroporation (EPR) buffer for 1 h followed by electroporation with single electric pulses of 500 V/ cm discharged from 960 uF capacitor in the presence of 15 _g DNA/ml and 8.3 _1 NaCI (3M) Changing the electroporation buffer conductivity had only a lim- ited effect on the number of blue spots. Similarly, a time course of GUS expression study revealed that

expression of the GUS gene could be detected as early as 12 h after electroporation with a maximum activity after 72 h (112 blue spots). Increasing the amount of DNA from 5 to 50 _g/ml in the EPR buffer had a

slight effect on the GUS expression frequency (112 blue spots with 15 _g to 120 spots with 30 _g DNA/ ml). Number of blue spots was increased by enzymatic wounding of callus pieces for 10 min and by addition of 200 _1 PEG 4000 (15%) before electroporation. These results suggest that intact cell electroporation can be used for producing transgenic sweetpotato tissue.

SP-1011 Targets for Arabidopsis Vacuum Infil- tration Transformation by Agrobacterium. Guangning YE, Michael Fromm, Maud Hinchee. Monsanto Co., 700 Chesterfield

Pkwy. North, St. Louis, MO 63198.

We are interested in studying the biological mechanism(s) underlying vacuum infiltration transformation ofArabidopsis byAgrobacterium. High transformation efficiencies were achieved through optimi- zation. Our results showed that most transgenic seeds from a single infiltrated plant were from independent transformation events based on Southern analysis, progeny segregation and distribution of transgenic seeds throughout the infiltrated plants. GUS expression was monitored daily at 0 to 12 days post infiltration in mature pollens and anthers from many infil-

trated plants. In addition, all the ovules from a single plant were examined every other day. The results showed that seed transformation efficiencies correlated most with ovule transformation rather than pollen transformation in the infiltrated plants.

SP-1012 Artificial Seed Production from En-

capsulated Somatic Embryos of Carica

Papaya. L. B. CASTILLO and M.A.L. Smith. National Biotechnology Labo-

ratory, Agriculture Ministery, Santo

Domingo, Dominican Republic; De-

partment of Horticulture, University of Illinois, Urbana, IL 61801.

Single asexual embryos (0.5 cm diameter) of Carica

Papaya L. were encapsulated and tested in a liquid production system. Two types of synthetic encapsulated coating compounds were used. The frequency of regeneration from encapsulated embryos was significantly affected by: concentration of sodium alginate, the presence or absence of nutrient salts in the

capsule, and the duration of exposure to calcium chlo- ride. A 2.5% sodium alginate concentration in a half

strength MS salts base resulted in significantly higher germination frequencies than other treatments. A rela-

tively short (10 minute) exposure to CaC12 resulted in uniform encapsulation of embryos and the highest frequencies of successful germination (77.5%). Ger- minated artificial seeds produced normal plantlets.

SP-1013 Cryopreservation: An Ex Situ Conser- vation Strategy for Butternut (Juglans cinerea). TANNIS BEARDMORE, Linda DeVemo, Garry Scheer, Wendy Vong. Natural Resources Canada, Canadian Forest Service, Atlantic For-

estry Centre, P.O. Box 4000, Fredericton, New Brunswick, Canada E3B 5P7.

Butternut (Juglans cinerea) is being killed throughout its range in North American by a fungus (Strococcus

clavigignenti-Juglandaccarum). To date, control for this disease does not exist and long-term seed storage is not a viable option for this species. Cryopreservation of Butternut embryonic axes has been examined as a method of ex situ conservation for this species. Em-

bryonic axes with approximately 3 mm of cotyledonary tissue attached tolerated 24 h at 0, -5, -10, -15, -

318



40, and at -196_

C (temperature reduced at a rate of

-0.33_ C/min from 0_ C). Significant tree to tree variation was found in the embryonic axes tolerance to low

temperature. This variation correlated with the em-

bryonic axes water content; the lower the embryonic axes water content, the greater the tolerance to low

temperatures. These results suggest that cryopreservation may be a viable means for Butternut ex situ conservation.

Baartnut (. cinerea x ailantifolia) trees and their seeds have been difficult to distinguish from that of Butter- nut (. cinerea) based on morphology. Preliminary results will also be shown using Random Amplified Poly- morphic markers (RAPID) to identify species specific markers for distinguishing Butternut (V. cinerea) from Baartnut V. cinerea x ailantifolia).

SP-1014 Studies on the New Channel for Cre- ation of Primary Trisomics Through Anther Culture of Autotetraploid Rice. Qin Ruizhen and Liu Zongxian. Institute of Crops Breeding and Cul- tivation, C.A.A.S., Beijing 100081 China.

Research on the primary trisomics of rice has received

increasing attention of many researchers. Trisomics of rice were obtained mainly by using the method of trip- loid. According to the irregularity of chromosome of

autotetraploid rice PMC Meiosis and the genetic characters of gamete that can be fully expressed by pollen plant in autotetraploid rice. We explore feasibility and

cytological basis of selecting primary trisomics through Anther culture of Autotetraploid Rice.

1. 15 lines of 4390 H1 induced pollen plants from

Autotetraploid rice were selected for chromosome

study. Their PMC meiosis were observed. 272 trisomics were identified, which accounted for 6.20% of all the pollen-plants. According to the

special characters from the whole set of trisomics,

they were classified as 9 types. The 9124-7 trisomics were designated as triplo-8 by the

pachytene analysis. The frequency of trisomic appeared in self progeny of this trisomic was 34.11% and the inheritance of the characteristics was stable. Among the 9 types of trisomis, the fre-

quency of appearance of trisomic 7 was slightly higher than that of other types. Trisomics 3, 4,

and 11 were not obtained. Three kinds of trisomics were obtained from an anther culture material (14128). The frequency and type of trisomics appearance revealed the university and facts of trisomics produced from the pollen plants of tet-

raploid rice.

2. Cytological studies were done on the hybrids of

autotetraploid rice and the original tetraploid rice (indica and japanica). The results showed that (1) PMC chromosome pairing forms at diakinesis of the hybrids and original varieties were 8.46IV + 7.0111 + 0.03111 + 0.06I, 8. 1IIV + 7.7511 + 0.01III + 0.031, 7.80IV + 8.3211 + 0.041; (2) PMC meiosis of tetraploids produced irregular behavior in the first and second divisions. The ratios of irregular behavior were higher in the first division than in the second at meiosis, also higher in the varieties than in the hybrids. (3) The chromosome number of PMC at metaphase II were varied from

21--26, the type with 24 chromosomes was the most. The ratio of PMC with 25 chromosomes of the hybrids and the varieties were 6.08%, 6.00%, and 6.56%, which gave the cytological evidence of producing 2x + 1 type gamate and could be induced to trisomic plants from anther culture of

tetraploid rice.

The experimental results showed that the utilization of anther culture of autotetraploid rice for the creation of primary trisomics of rice is feasible. The procedure is simple, time- and labor-saving, and it is

worthy to be further studied.

SP-1015 Studies of Somatic Embryogenesis in 20 Cultivars of Carica papaya. T.W. ZIMMERMAN, J.A. Richards, J.A. Kowalski. University of the Virgin Is-

lands Agricultural Experiment Station, RR2 Box 10,000, Kingshill, VI 00850.

Sucrose and 2,4-D levels were evaluated for somatic

embryogenesis in 20 papaya cultivars which have production potential in the US Virgin Islands. Zygotic embryos, from 90-100 -day old immature green fruits, were cultured on 0.3% Phytagel gelled half-strength Murashige and Skoog (_ MS) salts. Sucrose and 2,4- D at 3, 6 or 9% and 20 or 40 _M respectively, were added to the medium in a factorial design. Consis-

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tently, the greatest amount of embryogenesis occurred within 90 days on _ MS containing 40 _M 2,4-D and 6% sucrose. Monthly transfers to fresh medium increased the rate of embryogenesis. After 90 days on the 2,4-D induction medium, torpedo-shaped somatic

embryos were transferred onto _

MS, 3% sucrose, 2.5% activated charcoal and gelled with 0.7 g_1-1 phytagel and 8 gl-1 agar. During a 4 week period on the charcoal containing medium, the translucent em-

bryos became white. The embryos were water stressed

by placing the opened petri plate for 2 h in a laminar flow clean bench. After the stress, the embryos were transferred to the latter medium but without charcoal for germination. The combination of the activated charcoal and water stress promoted the maturation and germination of the somatic embryos.

SP-1016 Agrobacterium-mediated Genetic Transformation in Tomato (Vty Pusa

ruby) (Lycopersicum Esculentum) for Virus Resistance. G. LAKSHIMI SITA, M. Manoharan, C.S. Sreevidya, C.T. Ranjit Kumar, H.S. Savithri. De-

partment of Microbiology and Cell

Biology and Department of Biochem-

istry, Indian Institute of Science, Ban-

galore 560012.

A method forAgrobacterium-mediated transformation and regeneration of transgenic tomato plants harbor-

ing the coat protein gene of physalis mottle virus (PhMV) was developed. The coat protein gene (CP) of PhMV was cloned into the binary vector pBI121 and transformed into the Agrobacterium having dis- armed Ti plasmid (LBA4404). The CP and NPT II are under the control of 35S promoter and NOS promoter respectively. The cotyledonary leaves were in- fected with Agrobacterial suspension (0.5 O.D.), cocultivated for 48 hours and placed on a selection medium (MS + 0.5 TDZ + 50 mg/I kanamycin and 400 mg/l cefotaxime). The callus, which formed at the cotyledonary leaves was subcultured after 2 weeks on the fresh selection medium. The kanamycin resis- tant shoots regenerated after 3 weeks. The putative transgenic shoots were transferred to the rooting medium (_ MS + 25 mg/I kanamycin and 200 mg/l cefotaxime). The transgenic nature of the regenerated plantlets was confirmed by PCR and southern hybrid- ization, using appropriate primers and labelled coat

protein probe respectively. The ability of these

transgenic plants to resist viral infection remains to be established. Results of PCR, southern hybridiza- tion etc., will be presented.

SP-1017 Interactions of Days of Spike Storage with Days after Anthesis and Sucrose Concentrations in Promoting Wheat Somatic Embryogenesis. B.-C. LI, K. Caswell, N. Leung, R.N. Chibbar. Plant Biotechnology Institute, Na- tional Research Council of Canada, 110 Gymnasium Place, Saskatoon, SK, S7N 0W9, Canada.

In an effort to increase the efficiency of somatic em-

bryogenesis of isolated scutella from spring wheat

(Triticum aestivum L.) cv Colombus, two factorial ex-

periments were conducted to determine the interac- tive effects of days of spike storage (DSS) with days after anthesis (DAA) and sucrose concentrations (SC) in culture medium on percentage of scutella producing mature embryos and total number of embryos produced per plate. In the first experiment, scutella isolated from spikes collected at 10, 11, 12, 13, 14, 15, and 16 days after anthesis and stored at 4_ C for 7, 10, 13, and 16 days were placed on MS++ medium

(MS plus 2.2 mg L-1 2,4-D and 110 mg L-1 casamino

acid) and incubated in darkness for 12-14 days and then under inflorescent lights for 2 wks. A significant DAA X DSS interaction was observed. Three DAA/ DSS combinations, 11/13 or 16 and 12/16, were most

effective, 45-50% of scutella produced embryos and a total of 21-22 embryos were produced per plate of 15 scutella. In the second experiment, scutella isolated from spikes collected at 12 DAA and stored for 15, 16, 17, 18, and 19 days were placed on MS++ medium containing 2, 3, 4, and 5 g L-1 sucrose and incubated under similar conditions as described for the first experiment. A significant DSS X SC interaction was also observed. Five DSS/SC combinations, 17/2 or 3, 18/2 or 3, and 19/2 were most effective, 50- 57% scutella producing embryos and a total of 24-31

embryos were produced per plate of 14 scutella. In conclusion, the interaction of DSS with DAA and SC in culture medium significantly affects somatic em-

bryogenesis of isolated scutella from spring wheat cv Colombus. DAA/DSS/SC combinations, 12/17/2 or 3, 12/18/2 or 3, and 12/19/2, are recommended.

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SP-1018 Evaluation of Constitutive Promoters in Brassica napus. J.B. BADE, J. Van Roakel, S. Hoekstra, L.S. Melchers, M.H. Stuiver. MOGEN International nv. Einsteinweg 97, 2333 CB Leiden, The Netherlands.

One of the research projects at MOGEN is aimed at

developing strategies for the control of fungal diseases in plants. One promising strategy consists of identify- ing antifungal proteins, and constitutive expression of the corresponding genes in transgenic plants. Based

on the success of this approach in the last few years in tomato and carrot, it was decided to broaden this tech-

nology to Brassica napus. One of the keys for fungal resistance also in this crop is high enough expression levels of the selected antifungal proteins. Various can- didate promoters to be combined with antifungal genes were selected and gus constructs were made to

get information about the functionality of these promoters in Brassica napus.

Promoter gus fusions were made with 355, Ferredoxin, MAS, and RolD sequences. Of the 35S promoter es-

pecially 4 different constructs were made to test modi- fications such as single/double enhanced and different leader sequences.

Groups of 20-30 plants per construct were evaluated for GUS expression in tissue culture and in the green- house. Data will be presented on expression levels and

patterns of the different constructs in leaf, stem, and root tissue. Each of the plant parts is scored individu-

ally on a scale of 0-5, using a quantitative histochemical analysis. Until now all chosen promoters seem to be functional in Brassica napus with some differences in tissue specificity, except for the MAS promoter. Our results also show substantial differences in expression level between the 4 different 35S constructs, which indicates the importance of designing optimal expression cassettes for genes of interest. Looking closer at the variance between plants, it seems that the ferredoxin promoter gives relatively the most equal level of ex-

pression.

Currently we are completing our set of histochemical data and just starting protein analyses using MUG as substrate.

SP-1019 Polyscias Filicifolia Bailey Cell Culture in B3ioreactors. A.G. KLYUSHIN, A.V. Oreshnikov, N.D. Chernyak, O.V. Reshetnyak, A.M. Nosov. Timiriasev Institute of Plant Physiol- ogy, Botanicheskaya Str., 35, 127276 Moscow, Russia.

There is cell culture of medicinal plant Polyscias Filicifolia Bailey in All Russian tissue culture collec- tion in Moscow Timiriasev Institute of Plant Physiol- ogy. This strain was used to produce cell suspension. Its biomass was characterized with high antistress and

antiteratogenetic activity. Therefore, working out of commercial bioreactor technology for Polyscias biomass scaling-up is of great importance. The batch cultivation was investigated in bioreactors differed in volume and construction (air-lift and stirred tank bioreactors). Maximum volume of bioreactor was 630 liters. Dry biomass productivity per 1 g sucrose was about 0.5 g. The same results were obtained when we used different variants of nutrient medium in investi-

gated bioreactors. The highest dry biomass productivity (1.0 g/l medium/day) was achieved by airlift fer- menters. The chemical analysis of the biomass found out the presence of such

aglyco, 2 triterpenoid glyco-

sides as hederagenin.

SP-1020 Diterpenoid Glycosides Biosynthesis in Stevia rebaudiana Bertoni Tissue Culture. N.I. BONDAREV, O.V. Reshetnyak, A.M. Nosov. Timiryazev Institute of Plant Physiology, Botanicheskaya Str., 35 127276 Mos- cow, Russia.

Stevia is an endemic plant from the South America

subtropics. Its leaves contain a number of diterpenoid glycosides characterized with 300 times higher sweet- ness than sucrose. The quality of Stevia glycosides is the most suitable both as an equivalent of sugar for diabetics and for general replacement of it in food in-

dustry. We investigated the biosynthesis of diterpenoid glycosides in one year old callus and suspension cultures of stevia. Subcultivations were carried out in the

Murashige and Skoog nutrient medium supplemented with 1.0 mg/l _-NAA and 0.5 mg/l 6-BAP (every 5 wk for callus and every 2 wk for suspension). Cul- tures were kept in the darkness, at 25 _ 1_ C. Callus

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and cell culture samples produced at the end of sta- tionary phase of growth (3-5 wk) were used for biotechnological analysis. Lyophilized and powdered biomass cells of Stevia were extracted twice with metha- nol. The combined filtrates of each sample was evapo- rated to dryness (in vacuo) and the residue was dis- solved in 70% (vol/vol) ethanol. Authentic samples of steviosides and extracts of cell cultures were spot- ted on glass-backed Kieselgel 60 F.254 plates (Merck, 4 X 10, 0.2 mm thick) and developed with chloro- form : isopropanol : acetic acid : water (20:20:5:5). After development, chromatograms were air-dried, then sprayed with anisaldehyde-sulfuric acid reagent and heated at 100_ C for 10 min to visualize a spots. The R1-values for steviolbioside, rebaudioside B, stevioside, rebaudioside C and A were 0.45, 0.31, 0.27, 0.23, 0.19, respectively. Thin layer chromatograms (TLC) of cell cultures extracts showed that in some samples the TLC spots appeared at the same Rf-values as steviolbioside and rebaudioside B standards. So, the biosynthesis at least of two minor diterpenoid glycosides steviolbioside and rebaudioside B is possible in stevia tissue cultures.

SP-1021 Utilization of Biotechnological Meth- ods in Selection of Wheat Stability to Septoria. Lethy JOSE, E.A. Kalashnikova. Department of Bio- technology, Timiriazev Agricultural Academy, Moscow, Russia.

Septoria nodurum is one of the widely spreaded and

dangerous pathogen of wheat Septoria, which provokes infection on all over ground organs including ear and seed. In cellular selection, as a, selective factor for disease stable plants are used cultural filtrate of pathogen, and also toxin or its mixture with ferments. Ob-

ject of our research was wheat seeds and mature em-

bryos of sort Saratovska-ya-29. S. nadurum was cultivated in solidified or liquid media which contains minerals as per as MS and 2% sucrose. Toxicity of cultural filtrate was determined on the 7th, 11th, and 14th day of cultivation of fungi in liquid media. Cul- tural filtrate concentrations were 25%, 50%, 75%, and 100%. Also determined was the toxic effect of

fungi. It was found that the selected concentrations of cultural filtrate had no toxic effect on wheat seeds. Also was shown the correlation between increasment of the toxicity of cultural filtrate and period of cultivation. Combined cultivation of isolated embryos with

3-6 pieces of agar with fungi S. nadurum carried out to the inhibition of stem and root growth in compari- son with control. Toxicity of fungi in this case was 63, 6-80%.

SP-1022 Construction ofTransgenic Rapeseed Plants (Brassica napus L.) Containing Crucifer Infecting Tobamovirus Movement Protein Gen. S.D. ZVEREVA, G.N. Raldugina, O.V. Borisoval, Y.L. Dorokhov1. Institute of Plant Physiology RAS, Botanicheskaya Str. 35, Moscow 127276, Russia; 1A.N. Belozersky Institute Physico-Chemical Biology, MSU, Moscow 119899, Russia.

We have constructed transgenic rapeseed plants containing crucifer infecting tobamovirus movement protein-gene. The crTMV 29K movement protein gene was cloned into the plant expression vector pRT101, carrying the 35S promoter and the polyA signal of CaMV strain Cabb B-D. Next step was cloning the construction flanked by 35S promoter and polyA signal into binary Agrobacterium vector for plant transformation. Resulting plasmid BL208 and binary vector alone were transferred to A. tumefaciens BPA1459 by direct transformation. Restriction analysis of A. tumefaciens-purified BL208 plasmid confirmed the positive clones. 5-Day age B.napus L. cotyledons were transformed by BPA1459 containing BL208 or binary vector alone (control plants). The regenerated, transformed seedlings (Ro) (frequency of regeneration is 10%) were identified by the NPTII activity and by western blot with 29K movement protein antibody. The transgenic plants in the phytotron conditions have normal phenotype and fertility. The next step of this work will be the determination of genetic status (copy number of insertions, the obtaining R1 plants) and their ability of systemically reproduction ofTMV UI that cannot be able to infect systemically nontransgenic rapeseed plants.

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Documents

1997 Congress on In vitro Biology Special Poster Session Abstracts