Plant Science, 72 (1990) 245--252 245 Elsevier Scientific Publishers Ireland Ltd.

Transgenic plants of mustard Brassica juncea (L.) Czern and Coss

Helena Mathews a,b, N. Bharathan a, R.E. Litz% K.R. Narayanan a, P.S. Rao b and C.R. Bhatia b

°University of Florida, Tropical Research and Education Center, Homestead, Florida 33031 (U.S.A.) and ~Bhabha Atomic Research Centre, Bombay (India)

(Received May 9th, 1990; revision received July 16th, 1990; accepted July 19th, 1990)

The recovery of transgenic mustard plants from the R, progeny of regenerated plants using cotyledon explants treated with Agro- bacterium tumefaciens (C58C1, pGV3850:: 1103) is described. Adventitious shoots were isolated from cotyledonary callus treated with A. tumefaciens, and the shoot apices were sliced longitudinally and grown on selection medium. Regenerants were also grown on selection medium. After the second subculture, shoots with a higher ratio of green to white and/or purple were rooted. These were transplanted into soil and selfed. Seeds from R 0 generation were germinated in vitro and shoot tip and cotyledon explants were screened for kanamycin resistance. Solid green shoots were obtained from cotyledon explants on selection medium containing 20 rag/ I kanamycin. These were transplanted into soil and grown to maturity. Southern blot analysis of R 1 plants showed intact integration of T-DNA into the Brassicajuncea genome. This was further confirmed by seed germination and seedling growth on medium contain- ing 200 mg/l kanamycin.

Key words: Brassica juncea; transformation; transgenic plants

Introduction

Coty l edons are excel lent source explants for genera t ing t ransgenic p lan ts [1,2]. In Brass i ca jun - cea, adven t i t ious shoots deve lop read i ly f rom co ty ledon callus in v i t ro [ 3 1 5 ] ; bu t co-cu l t iva t ion o f co ty ledons with A . tumefac iens did no t p ro - duce direct t r a n s f o r m a n t s . Very of ten , the cells

compe ten t for t r a n s f o r m a t i o n are not necessar i ly compe ten t for r egenera t ion [6]. In r epea ted experi- ments a f te r i ncuba t ion with A . tumefaciens , coty- l edon explan ts have p r o d u c e d t r a n s f o r m e d callus bu t no t t r a n s f o r m e d shoots (our own observa- t ions) . Here we descr ibe a p r o t o c o l deve loped for the p r o d u c t i o n o f t r a n s f o r m e d m u s t a r d p lan ts

Correspondence to: Helena Mathews, University of Florida, Tropical Research and Education Center, Homestead, FL 33031, U.S.A. Abbreviations: BA, benzylaminopurine; IBA, indole-3-butyric acid; NAA, naphthalene acetic acid.

which involves j ud i c ious choice o f explants at var - ious stages, m a n i p u l a t i o n o f the mic roenv i ron- men t for sequenced p r o m o t i o n o f o rgan deve lopmen t and phased exposure to select ion pressure .

Materials and Methods

Explan t

Seeds o f Brassica j u n c e a (L.) Czern and Coss cv. Rai 5 (mus ta rd) were surface-s ter i l i_ed with 0.1070 (w/v) aqueous HgCI 2 for 15 min fo l lowed by t h o r o u g h r insing in steri le dis t i l led water . Seeds were ge rmina ted on Murash ige and Skoog m e d i u m (MS) [7] supp l e me n te d with 307o (w/v) sucrose, 550/~M inos i to l , 2 .4/~M pyf idox ine HCI , 0.3 buM th iamine HCI , 4.1 /~M nicot inic acid and 26.6 /~M glycine ( fur ther re fe r red to as basa l me d ium, BM). M e d i u m was so l id i f ied with 0.807o (w/v) Di fco Bacto agar , au toc l aved at 121 °C and 1.1. k g / c m 2 for 20 min. Cul tures were ma in t a ine d

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246

in continuous fluorescent light of 25/aE. s -1. m -2 at 25 °C.

Cotyledons from 6-day-old seedlings were cul- tured on BM with 0.54 ~ NAA and 4.4/~M BA (regeneration medium). Cotyledons showing can- logenic callus were selected for A. tumefaciens infection.

Preparation of bacterial culture A single colony of Agrobacterium tumefaciens

C58C1, pGV3850::1103 was inoculated into 5 ml of Luria Broth (LB) [8] with 50 mg/1 kanamycin (pH 7.0) overnight. This was transferred to 50 ml of LB at pH 5.6 with 30 ~M acetosyringone (AS) (Aldrich) and kept on a shaker at 200 rev./min at 28 °C for 16 h.

Co-cultivation of bacteria and explant Twenty cotyledons were cut transversely at the

region of caulogenic callus. A log phase culture of A. tumefaciens activated with AS was smeared onto the wounded surfaces of the caulogenic cal- lus attached to the cotyledon explant. These were recultured on regeneration medium for 3 days.

Tissue culture separation of transformed cell layers

Cotyledon explants were rinsed with sterile dis- tilled water after co-cultivation, blotted dry on sterile filter paper and cultured on regeneration medium supplemented with 10 mg/1 kanamycin and 500 mg/l cefotaxime. After 25 days all the shoot regenerants were excised from the cotyledon explants and were subcultured.

Tissue culture separation of transformed cell layers

Shoot tips (0.5--1.0 cm) excised from cotyle- don-derived shoot regenerants after Agrobacter- ium co-cultivation were longitudinally cut into equal haves. Each half was cultured separately on regeneration medium with 300 mg/1 cefotaxime and 20 mg/1 kanamycin. After 30 days, shoots with a high ratio of green to white and/or purple were segmented and subcultured.

Rooting and transplantation Shoot regenerants that were at least 80% green

were rooted on BM with 100 mg/l cefotaxime and

without kanamycin, and were transplanted to soil. Mature plants were self-pollinated.

Recovery and selection of putatively transformed shoots from seedling explants

Seeds from the first generation (R0) of regener- ated plants were germinated on BM as described above. Shoot tip (0.8--1.0 cm) and cotyledon explants were cultured on regeneration medium with 50 mg/1 and 20 mg/1 kanamycin, respec- tively, to suit the kanamycin tolerance of the par- ticular explant [9].

Maintenance of putatively transformed shoots Solid green shoots were cultured on regenera-

tion medium with 50 mg/l kanamycin for multipli- cation.

Rooting of putative transformants Completely green shoots derived from cotyle-

don explants on selection medium were cultured on BM with 0, 50 and 100 mg/1 kanamycin for root induction. The bases of unrooted shoots were dusted with Hormex rooting powder No. 16 (Brooker Chemical, active ingredient, IBA). The shoots were then transplanted to soil.

Southern blot hybridization DNA was extracted from young leaves (1 g) of 7

putatively transformed plants and a non-trans- formed control plant by the method of Dellaporta et al. [10]. Prior to endonuclease digestion, DNA suspensions were treated with DNase-free pan- creatic ribonuclease (RNase) to remove any con- taminating RNA [11]. Fifty microliters of an endonuclease reaction mixture contained 10/~1 of miniprep DNA, 5/A of 10 × reaction buffer, 2/al of EcoRI (20 units), 2/~1 of bovine serum albumin (BSA) and brought to volume with water. Digestion was overnight at 37 °C. Electrophoretic separation was carried out on 0.8% agarose (w/v) horizontal slab gels in TAE buffer (containing 40 mM Tris acetate and 1 mM ethylenediamine tetra- acetic acid (EDTA), pH 8.0) at 30 V for 16 h, and transferred to a nylon membrane (Amersham) by capillary blotting [12]. The DNA was immobilized on nylon membranes by baking at 80°C for 2 h in vacuum.

Hybridization probes were made from plasmid

pLGVneol l03 , maintained in E. coli, HB101. Plasmids were isolated by the alkaline extraction procedure of Birnboim and Doly [13] and digested with EcoRI and ApaI restriction endonucleases. This releases a 2 kb fragment containing the NPT- II coding region fused on its 5' end with the pro- moter region of the nopaline synthase gene and on its 3' side to a fragment of the octopine synthase gene containing the polyadenylation signal (OCS polyA) [14]. The 2-kb fragments were gel-purified and labelled by a multiprime DNA labelling sys- tem (Amersham kit, 30 /~Ci [32p]dcTP. Probe specific activities were 1 × 10~--1.5 × 109 cpm/ /~g DNA.

Prehybridization, hybridization and washes were performed at 65°C. Hybridization was in 6 × SSC, (1 × = 0.15 M NaC1/0.015 M sodium citrate, pH 7.0), 5 × Denhardt 's solution (1 x = 0.02O7e polyvinylpyrrolidone, 0.02°7o ficoll, 0.02°7o BSA), 10o70 sodium dodecylsulfate (SDS), 50o7o (v/v) formamide containing 1.1--2.2 × 107 dpm of 32p-labelled probe/ml. After overnight hybridi- zation, filters were washed twice in 2 × SSC at 65°C for 15 min and once in 1X SSC at 68°C for

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10 min followed by exposure to Kodak-x-omat AR X-ray film at - 7 0 ° C for 8 days using Cronex intensifying screens.

Screening for surviving A. tumefaciens Leaf and stem segments of the putatively trans-

formed plants (R~) were grown in liquid broth medium specific for A. tumefaciens [15]. Ten flasks, each containing five l-cm segments of stem or leaves, with 50 ml of medium, were kept on a shaker at 200 rev. /min for 7 days. Aliquots of 0.05 ml of this medium, after incubation with plant material, were plated on broth agar plate. A pure bacterial culture of C58C1, pGV3850::1103 was also plated on similar broth medium.

Progeny test Seeds from the putatively transformed plants

and a non-transformed control plant were cul- tured on BM with 200 mg/1 kanamycin and incu- bated in darkness. After 3 days the cultures were exposed to continuous fluorescent light of 25 /~E" s -~ • m -2 at 25 °C. Seedlings which were green and formed secondary and tertiary leaves in the

Cotyledonary callus of R~ generation producing solid green regenerants on regeneration m e¢lium with 20 mg/l kanamycin. Fig. 1.

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presence of kanamycin were scored as resistant and white seedlings which did not form true leaves were scored as sensitive.

Results

After 3 days of co-cultivation and reculture on regeneration medium, the wounded region of the cotyledons continued to differentiate shoots. Longitudinally sliced shoot tips on selection medium with 20 mg/l kanamycin produced shoot regenerants which were green, white, purple or a combination of the three colours. Shoots that were either completely white or white and purple were discarded, while shoots that were either completely green or green with white and /o r pur- ple were cut longitudinally and the shoot segments were subcultured. Regenerants from the second subculture were visually screened for a high ratio of green to white and /o r purple shoots. Eight

shoots that were at least 80°70 green were rooted on BM without kanamycin and were transplanted into soil. All 8 plants of the R 0 generation were selfed and seeds from 6 plants were available for further analysis. An average of 20 seeds from each of the 6 plants were germinated in vitro. Cotyle- dons and shoot tips, both from 7-day-old seed- lings, were cultured on regeneration medium with 20 mg/l and 50 mg/l kanamycin respectively. The shoot tip explants formed calli at the cut ends but did not form multiple shoots. The cotyledon explants formed caulogenic callus. From a total of 240 (20 x 2 x 6) cultured cotyledons, 15 explants produced completely green shoots (Fig. 1). These were separately subcultured on shoot multiplica- tion medium with 50 mg/l kanamycin. Although the majority of regenerants were green, occasion- ally purple or purple with white shoots were observed during multiplication of these putatively transformed shoots on selection medium with 50

Fig. 2. An autoradiogram of agarose gel showing Southern blot analysis of putative t ransformants of B. juncea probed with the ApaI and EcoRI digest (2 kb) of pLGVneo1103. Lane 1 - - homologous hybridisation to 2.0 kb probe from pLGVneo1103. Lanes 2 - - 5 and 7--9 , 5 btg each o f digested DNA from 7 t ransformants . Lane 6 is 5 #g digested DNA from a control plant. Molecular weight markers of HindIII digests are shown in (kb) to the left o f lane 1.

O

mg/l kanamycin. Green shoots were transferred to BM with 0, 50 and 100 mg/l kanamycin for root induction. Surprisingly, ~ the shoots did not form roots even on non-selection medium (0 kanamy- cin). These shoots were then dusted with Hormex rooting powder No. 16 (Brooker Chemical) at cut ends and directly transferred to soil. Of 15 such transplants of the R~ generation, 12 formed roots and produced seeds after selfing.

Southern hybridization Figure 2 shows the Southern blot results of

EcoRI cut DNA probed with a 2-kb fragment from pLGVneoll03. Hybridization bands were detected in all the lanes with transformed DNA while the control lane did not show any signal. Fragment sizes of approximately 6.5 kb and 5.2 kb were observed in all the lanes except the control lane.

Screening test for A. tumefaciens contamination Samples of liquid medium inoculated with leaf

and stem explants of putative transformants (R~), when plated on A. tumefaciens specific medium, did not show any bacterial growth. Pure bacterial cultures of C58C1, pGV3850:: 1103 readily pro- duced large numbers of colonies on the plate.

Progeny test Seeds from the 12 putative transformants of the

R~ regeneration and a non-transformed control plant were cultured on BM with 200 mg/l kanamy- cin. During 3 days of incubation in darkness, all control seeds germinated and hypocotyls were approx. 2--4 cm in length. Seeds from putatively transformed plants were of 3 types: (1) those which germinated and grew like the control; (2) seeds with barely sprouted embryos; (3) seeds which did not germinate. Kanamycin-resistant seedlings became green in the light, produced sec- ondary and tertiary leaves and grew normally, whereas the sensitive seedlings were white and did not produce true leaves (Fig. 3 and Table I). Resistant seedlings were transferred to soil for future studies.

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Fig. 3. Kanamycin resistant (left) and sensitive (right) plants 25 days after germination on BM with 200 mg/l kanamycin.

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Table I. Progeny analysis o f the R t generation.

Putatively No. o f No. o f seeds t ransformed seeds sown not germinated

plants

No. o f seeds showing arrested growth of sprouted embryo

No. o f seedlings

Resistant to kanamycin

Sensitive to kanamycin

1 49 10 0 2 86 l l 0 3 78 35 0 4 46 6 7 5 19 l 0 6 72 43 0 7 24 12 0 8 22 17 5 9 39 28 0

10 95 29 8 11 77 5 16 12 85 30 14 Control 51 0 0

(non-transformed)

3 28

5 1 4 8 1 0 3 6 6 0 0

36 47 38 32 14 21 11 0 8

52 50 41 51

Medium BM + 200 mg/I kanamycin. Observations scored after 24 days.

Discussion

Although Brassica species in general are highly susceptible to A. tumefaciens, attempts to obtain transformed plants have been reported only in B. napus [16--20]. Transformed callus lines have been reported in B. campestris [21]. In B. juncea, shoot regeneration from tumor callus and forma- tion of rooty callus by wild and mutant strains of A. tumefaciens have been reported [22,23]. In this communication we report recovery of putatively transformed plants from the R 1 generation derived from Agrobacterium-treated cotyledon explants. The intact stable integration of foreign DNA into the B. juncea genome was confirmed by Southern blot analysis.

Plant material from putatively transformed plants in A. tumefaciens specific medium was free of bacterial growth. This confirmed that the pres- ence of intact plasmid T-DNA in the plant genome was not due to residual contamination from A. tumefaciens.

Common fragments of 6.5 kb show the expected hybridization pattern of EcoRI cut gen- omic DNA transformed with pGV3850::1103 [24].

The presence of the 5.2 kb fragment could be due to the presence of additional homologous sequences in the probe outside the NPT-II sequences.

Extensive studies [9] carried out on the kana- mycin tolerance of different explants of B. juncea have clearly demonstrated that 200 mg/l kanamy- cin impairs chlorophyll formation and seedlings do not form true leaves and that 20 mg/l kanamy- cin is suffient to suppress shoot regeneration from cotyledonary explants. Accordingly, final screen- ing for kanamycin resistance was carried out at 200 mg/l kanamycin for seed germination and at 20 mg/l kanamycin for cotyledonary explants. Selection pressure was increased in a phased man- ner at the earlier stages as indicated in Materials and Methods.

Since all the cotyledons from 20 seeds each of 6 R 0 plants were pooled, independent transforma- tion events were not determined and the fre- quency of transformation could not be precisely determined. However, since at least one transfor- mation event has taken place in the 20 initial explants, the frequency of transformation is equal to or better than 5 x l0 -2. Germination at 200

mg/l kanamycin confirmed inheritance of the kanamycin resistance trait by the progeny.

Our earlier attempts to regenerate plants from B. juncea callus transformed with A. tumefaciens C58C1, PGV3850:: 1103 were unsuccessful. A sim- ilar situation has been reported in flax. Although flax hypocotyl and cotyledon segments produce shoots highly efficiently, incubation with C58C1, pGV3850::1103 results in transformed callus but no regeneration [25]. Jianping and Quiquan [26] reported transformed B. oleraceae callus only from hypocotyl segments, using C58C 1, pGV3850::1103. Feldmann and Marks [27] treated germinating seeds of Arabidopsis thaliana with A. tumefaciens and recovered transgenic plants in the second generation. A transformed plant was also recovered from the progeny of a chimaeric rege- nerated plant that resulted from DNA introduc- tion by the particle gun method into the meristems of immature soybean seeds [28].

The procedure described in this report essen- tially involved: (1) formation of a chimaera con- sisting of transformed and non-transformed cells; (2) separation of transformed and non-trans- formed cells by tissue culture; and (3) inclusion of these cells in the germinal layer of the regenerated plant enabling transmission of the transformed cells into the seeds.

The frequency of seed germination and the atypical segregation pattern of kanamycin resist- ant and sensitive seedlings in the progeny of puta- tive transformants raise a few points. Are the putative transformants of the R~ generation a mosaic of transformed and non-transformed cells? Is the higher-than-expected ratio of sensitive to resistant seedlings due to lack of expression of the Kan R gene? Considerable variability of Kan a expression has been reported in many solanaceous plants [29]. However, in the present study the high number of sensitive seedlings could be due to the presence of non-transformed cells, since the solid green shoot regenerants recovered from cotyledon explants occasionally produced purple and/or white shoots in the selection medium with 50 mg/1 kanamycin. We feel that the kanamycin-resistant plants obtained in the progeny of R 1 ought to be considered as true transformants and one should

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look at the progeny of the R 2 generation to study the inheritance pattern of the Kan R gene.

It is possible that the arrested growth of the sprouted embryos and lack of seed germination (Table I) are associated with the mutagenic role of T-DNA insertion. The insertion of foreign DNA could be functioning as a germline lethal. Embryo lethal mutants were reported in the progeny of transgenic tobacco plants [30]. The observation in B. juncea that the putatively transformed shoots were difficult to root, coupled with our earlier finding that transformed callus never underwent organogenesis indicates changes in the morphoge- netic expression of the transformed genome. Con- trol non-transformed B. juncea shoot explants root easily in BM and cotyledons produce caulo- genic calli. This suggests the possible role of T- DNA as an insertional mutagen [31]. Transgenic plants have been used to study the molecular organization of plant genes [32].

Acknowledgements

We gratefully acknowledge Dr. An Depicker, Rijkuniversiteit, Ghent for providing the Agro- bacterium tumefaciens strain used in this study. The assistance of Rose Hendrix, Sarah Wright and W.R. Graves is greatly appreciated. Florida Agri- culture Experiment Station Journal Series No. R- 0O659.

References

1 M.A.W. Hinches, D.V. Connor-Ward, C.A. Newell, R.E. McDonnell, S.J. Sato, C.S. Gasser, D.A. Fischoff, D.B. Re, R.T. Fraley and R.B. Horsch, Production of transgenic soybean plants using Agrobacterium-mediated DNA transfer. Bio/Technol., 6 (1988) 915--922.

2 R. Schmidt and L. Willmitzer, High frequency Agrobac- terium tumefaciens-mediated transformation of Arabi- dopsis thaliana leaf and cotyledon explants. Plant Cell Rep., 7 (1988) 583--586.

3 L. George and P.S. Rao, In vitro regeneration of mustard plants (Brassica juncea var. Rai-5) of cotyledon explants from non-irradiated, irradiated and mutagen treated seed. Ann. Bot., 46 (1980) 107--112.

4 R.K. Jain, J.B. Chowdhury, D.R. Sharma and W. Friedt, Genotypic and media effects on plant regeneration from cotyledon explant cultures of some Brassica species. Plant Cell, Tissue Organ Cult., 14 (1988) 197--206.

252

5 R.K. Jain, D.R. Sharma and J.B. Chowdhury, High fre- quency regeneration and heritable somaclonal variation of Brassicajuncea. Euphytica 40 (1989) 75--81.

6 I. Potrykus, Gene transfer to cereals: an assessment. Bio/ Technol., 8 (1990) 535--542.

7 T. Murashige and F. Skoog, A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiol. Plant., 15 (1962) 473--493.

8 J.H. Miller, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1972, p. 466.

9 H. Mathews, In vitro responses of Brassica juncea and Vigna radiata to the antibiotic kanamycin. Ann. Bot., 62 (1988) 671 --675.

10 S.L. Dellaporta, J. Wood and J.B. Hicks, A plant DNA minipreparation version II. Plant Mol. Biol. Rep., 1 (1983) 19--27.

11 T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1982.

12 E.M. Southern, Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol., 98 (1975) 503--517.

13 H.C. Birnboim and J. Doly, A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl. Acid Res., 7 (1979) 1513--1523.

14 R. Hain, P. Stabel, A.P. Czernilofsky, H.H. Steinbiss, L. Herrera-Estrella and J. Schell, Mol. Gen. Genet., 199 (1985) 161--168.

15 L.W. Moore, A. Anderson and C.I. Kado, Gram-nega- tive bacteria, in: N.W. Schaad (Ed.), Laboratory Guide for Identification of Plant Pathogenic Bacteria. Bacteri- ology Committee of Americal Phytopathoiogicai Society, St. Paul, Minnesota, 1980.

16 P.J. Charest, L.A. Holbrook, J. Gabard, V.N. Iyar and B.L. Miki, Agrobacterium-mediated transformation of thin cell layer explants from Brassica napus L. Theor. Appl. Genet., 75 (1988) 438--445.

17 J. Fry, A. Barnason and R.B. Horsch, Transformation of Brassica napus with Agrobacterium tumefaciens based vectors. Plant Cell Rep., 6 (1987) 321--325.

18 S. Misra and L. Gedamu, Heavy metal tolerant transgenic Brassica napus L. and Nicotiana tabacum L. plants. Theor. Appl. Genet., 78 (1989) 161--168.

19 E.C. Pua, A. Mehra-Palta, F. Nagy and N.H. Chua, Transgenic plants of Brassica napus L. Bio/Technol., 5 (1987) 815--817.

20 S.E. Radke, B.M. Andrews, M.M. Moloney, M.L. Crouch, J.C. Kridl and V.C. Knauf, Transformation of Brassica napus L. using Agrobacterium tumefaciens

developmentally regulated expression of a reintroduced napin gene. Theor. Appl. Genet., 75 (1988) 685--694.

21 J. Paszkowski, B. Pisan, R.D. ShiMto, T. Hohn, B. Hohn and I. Potrykus, Genetic transformation of Bras- sica campestris var. Rapa protoplasts with an engineered Cauliflower Mosaic Virus genome. Plant. Mol. Biol., 6 (1986) 303--312.

22 H.V. Mathews, C.R. Bhatia, R. Mitra, T.G. Krishna and P.S. Ran, Regeneration of shoots from Brassica juncea (Linn) Czern and Coss cells transformed by Agrobacter- ium tumefaciens and expression of nopaline dehydroge- nase genes. Plant Sci., 39 (1985) 49--54.

23 H.V. Mathews, P.S. Ran and C.R. Bhatia, Transforma- tion of Brassica juncea by Agrobacterium tumefaciens harbouring plasmid pTiT37 and its 'rooty' mutant pTiT37.14a/a. J. Genet., 65 (1986) 37--44.

24 P. Zambryski, H. Joos, C. Genetello, J. Leemans, M. Van Montagu and J. Schell, Ti plasmid vector for the introduction of DNA into plant cells without alteration of their normal regeneration capacity. EMBO J., 2 (1983) 2143--2150.

25 X. Zhan, D.A. Jones and A. Kerr, Regeneration of flax plants transformed by Agrobacterium rhizogenes. Plant Mol. Biol., 11 (1988)551--559.

26 Y. Jianping and S. Quiquan, Transformation of Brassica oleraceae with a Ti-plasmid derived vector. Genetic Manipulation In Crops Newsletter, 4 (1988) 76--80.

27 K.A. Feldmann and M. David Marks, Agrobacterium- mediated transformation of germinating seeds of Arabi- dopsis thaliana: A non-tissue culture approach. Mol. Gen. Genet., 208 (1987) 1--9.

28 D.E. McCabe, W.F. Swain, B.J. Martinell and P. Chris- tou, Stable transformation of soybean (Glycine max) by particle acceleration. Bio/Technol., 6 (1988) 923--926.

29 I. Potrykus, Direct gene transfer to plants, in: Applica- tions of Plant Cell and Tissue Culture. (Ciba Foundation Symposium 137), Wiley, Chichester, 1988, pp. 144-- 162.

30 E. Hebertle-Bors, B. Charvat, D. Thompson, J.P. Schernthaner, A. Barta, A.J.M. Matzke and M.A. Matzke, Genetic analysis of T-DNA insertions into the tobacco genome. Plant Cell Rep., 7 (1988) 571--574.

31 K.A. Feldmann, M. David Marks, M.L. Christianson and R.S. Quatrano, A dwarf mutant of Arabidopsis gen- erated by T-DNA insertion mutagenesis. Science, 243 (1989) 1351--1353.

32 J.S. Schell, Transgenic plants as tools to study the molec- ular organization of plant genes. Science, 237 (1987) 1176 --1183.