Plant Cell Reports

Plant Cell Reports (1990) 9:26-29

c~ Springer-Verlag1990

Transformation of eggplant (Solanum melongena L.) using a binary Agrobacterium tumefaciens vector G . L . Rotino 1'2 and S. Gleddie' Plant Research Centre, Agriculture Canada, Ottawa, Ontario, K I A OC6, Canada 2 Present address." Istituto Sperimentale per l'Orticoltura, CP 48, 1-84098 Pontecagnano (SA),

Italy

Received September 18, 1989/Revised version received February 26, 1990 - Communicated by A.R. Gould

SUMMARY

Kanamycin resistant plants of Solanum melongena L. keggplant) cv. Picenfiia were obtained Following the cocultivafiion of leaf explants wifih Agrobacfierium fiumefaciens. A disarmed binary vector system confiaining the neomycin phosphotransferase ~NPTII) gene as the selectabte marker and chloramphenicol acetylfiransferase kCAT) as a reporter gene was utilized. In vitro grown plants were used as sources of explanLs to produce transgenic plants on selective medium containing 100 mg/1 kanamycin. The transformation and expression of the foreign genes was confirmed by DNA hybridizations, leaf disc assays, and by measuring NPTII and CAT enzyme activities. This technique is simple, rapid, efficient, and transgenic eggplants or this commercial cultivar have been transferred to soil where they have flowered and set seed. Key words: Solanum melongena, eggplant, A~robacterium tumefaciens, kanamycin, firansrormafiion, shoot regenerafiion. Abbreviations: CAT, chloramphenicol acetylLransferase; NS, Hurashige and Skoog; NPTII, neomycin phosphotransferase; NOS, nopaline synthase, ZEA, zeatin. INTRODUCIION

Eggplant kSolanum melongena) is an important non-tuberous Solanaceous species in many firopical, Asian, and some European countries. The recent progress in tissue and cell %Gleddie eft al. 1983), anther kDumas de Vaulx and Chambonnefi 1982; Rofiino et al. 1987), and protoplasfi culture kGleddie eft al. 1985; Sihachakr and Ducceaux 1987) suggests fiha-t in vitro techniques may contribute to the solution of several agronomic problems in this crop. Eggplant p r o d u c t i o n i s s e v e r e l y l i m i L e d by s e v e r a l soil-borne pathogens and, although sources of resistance exist in several resistant Solanaceous species, the incorporation of these firaifis into the eggplant gene pool by breeding for disease resistance has been slow. Recently, somatic hybridization between sexually-incompatible Solanum species has been employed as a potential method for inkroducing disease resistance firaifis kGleddie e_~t Offprint requests to." S. Gleddie

al. 1986; Guri and Sink 1988a; Sihachakr eft al. T989). Agrobacfierium tumefaciens-mediated gene transfer may improve the efficiency of somatic hybridization by providing dominant selectabte markers for heterokaryon selection kKomari eft al. 1989) in addition rio offering new possibilities for khe transfer of agronomically important genes. Wifih eggplanfi, Guri and Sink ~1988b) were successfui in obfiaining transgenic planfis only when using cointegrate plasmid vectors but they were unsuccessful when using binary vectors. In this paper we reporfi an efficient procedure rio transform and regenerate a commercial cultivar of eggplant, following leaf disc cocultivation with A. tumefaciens harbouring a disarmed binary vector theft confers resistance rio fihe antibiofiics kanamycin and chloramphenicol. We also demonstrate the expression and integration of the marker genes and the capacifiy of firansgenic eggplants to flower and set seed in the greenhouse. MATERIALS AND METHODS

Plant material Seeds o f ~. melongena L. cv. P i c e n t i a were o b t a i n e d from Dr. F. Restaino ( I s t i t u L o S p e r i m e n t a l e per l'Orticoltura d i Pontecagnano). Plant material for t r a n s f o r m a t i o n were grown e i t h e r i n v i t r o or i n a greenhouse. Seeds For greenhouse-grown p l a n t s were g e r m i n a t e d i n s o i l and m a i n t a i n e d at 25~ under a 16-h day c y c l e supplemented w i t h f l u o r e s c e n t lamps. Seeds f o r in vitro-grown plants were s u r f a c e sterilized by d i p p i n g i n 70% e t h a n o l f o r 30 sec f o l l o w e d by 20 min i n 7% c a l c i u m h y p o c h l o r i t e and F i n a l l y r i n s i n g t h r e e times i n s t e r i l e d e m i n e r a l i z e d water. The s t e r i l e seeds were g e r m i n a t e d i n 100 x 15 mm p e t r i dishes k25-30 per p l a t e ) on a f i l t e r paper soaked w i t h s t e r i l e d e m i n e r a l i z e d w a t e r and i n c u b a t e d i n t h e darkness at 28"C. A f t e r 7-10 days, the g e r m i n a t e d seeds were t r a n s f e r r e d i n t o s f i e r i l e GA7 boxes kMagenka C o r p . ) c o n t a i n i n g 40 ml o f haJf-sfirength B 5 medium ~Gamborg eL al. 1968) supplemenfied with I~ kw/v) sucrose and 0.8% ~w/v) bacto-agar kDifco) adjusted rio pH 5.8 ~0.2 N NaOH prior rio aufioclaving). PlanEs were maintained in a growth room at 25~ with a 16-hr daylengfih under fluorescent light ~50 uEm-2sec-1).

27 8arterial strain and growth conditions Agrobacterium tumefaciens strain GV 3101, a nopaline strain, C58 type chromosome, carrying the binary vector plasmid system pBCATI, a modified Bin 19 vector [Bevan 1984, kprovided by N. Bevan, I.P.G.R., Cambridge, U.K.)] and pGV3830 [Zambryski etal. 1983 kprovided by J. Schell, Max Planck Inst., KBTn)] was used. pGV3850 is the helper plasmid, and p8CATI contains the coding sequence of the bacterial neomycin phosphotransferase II kNPTII) gene under the control of the nopaline synthase promoter and the bacterial chloramphenicol acetyltransferase tCAT) gene under the control of the 355 promotor of cauliflower mosaic virus. Bacteria were grown at 28~ in t B medium tBacto-tryptone 10 g/L, Bactoyeast extract 5 g/L, NaC1 10 g/L) on rotary shakers to the late logarithmic stage rOD600 0.8-1.0). The bacteria were centrifuged and the pellet was resuspended to the same density in NS tHurashige and Skoog 1962) medium containing 2% tw/v) sucrose and used For the cocultivation procedure. Transformation and regeneration procedure The leaves from greenhouse grown plants t4 weeks old) were surface-sterilized for 15 min in 7% calcium hypochlorite followed by three rinses in sterile demineralized water. The younger leaves from greenhouse and in vitro grown plants were cut k10 x 15 mm) and preincubated, with the lower side in contact with the regeneration medium, in 100 x 15 mm petri dishes sealed with household plastic wrap. The regeneration medium used was MS as modified by Douglas et al. t1981) supplemented with 2 mg/l glycine, T mg-7-1 zeatin tZEA), 2% sucrose and 0.2% gelrite tKelco). The pH of the medium was adjusted to 5.6 tO.2 N KOH) prior to autoclaving at 121~ for 20 min. Filter-sterilized antibiotics were added to cooled t50-55~ autoclaved medium before pouring. All the cultures were kept in a controlled environment chamber at 25~ under SO uEm-2see-1 supplied by fluorescent lamps for a 16-hr day length. After 48 hr, the preconditioned explants were briefly dipped in the bacterial suspension, blotted dry using filter papers, and placed back in the same p l a t e s . After two days of cocultivation, the leaf pieces were transferred to selection medium tregeneration medium described above) containing 500 mg/1 cefotaxime tRousset) and 100 mg/1 kanamycin sulfate tSigma). Every three weeks the explants were transferred t o fresh selective medium. When calli were about 5 mm in diameter they were excised from the leaves and subcultured in 60 x 20 mm petri dishes containing the selection media t100 mg/1 kanamycin) with a lower concentration of cefotaxime t200 mg/l). Shoots tI-1.5 cm) were excised and transferred to GA7 boxes containing half-strength B 5 medium, I~ sucrose, 0.3% gelrite and 100 mg/t kanamycin. The shoots from a single independent callus were considered to be from a single transFormation event. The rooted shoots were transferred to peal pellets kJiffy-7), maintained in a mist chamber for 7-10 days prior to potting and maintenance in the greenhouse. The flowers were self-pollinating using a paint brush and seed was harvested by extracting the mature fruit with hot water. Expression assays Neomycin phosphotransferase tNPTII) and amphenicoi acelyltransferase tCAT) enzyme

chlor-

activities were analyzed following the procedures of McDonnell et al. t1988) and Bird et al. t1988). Leaf and callus samples for both assays were quickly ground in the extraction buffers using a plastic pestle in a microfuge tube, and then sonicated for about b sec. Kanamycin resistance was also assayed by the abiliLy of putaLiLive transformed leaf tissue to produce callus and regenerate shoots on regeneration medium containing 100 mg/l kanamycin. Southern blot anal~sis Plant DNA was exLracted from leaf tissues, digested with restriction endonucleases according to manufacturer's recommendations, electrophoretical]y separated on a 0.8% agarose gel, and transferred to NytranTM LSchleicher and Schuell) membranes according to Maniatis etal. t1982). The filter was probed with a p32-labelled kFeinberg and Vogelstein 1983) NPT II gene fragment LI.1 Kbp). RESULTS AND DISCUSSION

Leaf explants From in vitro grown plants, following cocultivations formed callus and shoots on selective media. White, friable callus was visible along the cut edges of the explants within 3-4 weeks from the cocultivation. No shoot or bud differentiation was observed at this stage. After three or more subcultures on selective medium, some regions of the calli turned green and compact green nodules were formed. Shoot primordia differentiated from most of the nodules tFig. 1). Non-cocultivated explants did not callus or regenerate shoots indicating that kanamycin at 100 mg/] was an effective level of selection. A previous report tGleddie et al. 198]) predicted that the regeneration of eggplant shoots from leaf explants of greenhouse-grown plants was very efficient and occurs within 3-4 weeks of culture. This shoot regeneration process occurs directly from the explants without callus formation. Agrobacterium cocultivation appears to delay the onset of shoot regeneration by about 4-8 weeks and an intermediate callus proliferation stage is involved. Explants taken From leaves of greenhouse-grown plants did not Form callus following cocultivation. Damage from the sterilization made the explants particularly more susceptible to A. tumefaciens infection causing an excessive colonization of the leaf tissue. Within six weeks all of the explants died and bacterial overgrowth was common.

From 195 e x p l a n t s o f i n v i t r o leaves, c o c u l t i v a t e d with A. tumefaciens, 27 resistant calli were selecL~d and planLs were obtained from 55% of these caJli. An overall transformation efficiency of 7.6% was obtained t# of Lransformed plants/# of explanLs cocultivated). The long time t8-12 weeks) necessary for the differentiation and development of transformed shoots may have caused a reduction of Lhe organogenic potential. Regenerated shoots were able to form roots in the presence of 100 mg/] kanamycin tFig. 2) and Lhe planLs when transferred Lo the greenhouse apeared phenotypically normal and set seeds tFig. 3). Leaf e x p l a n t s o f NPTII p o s i t i v e p l a n t s produced c a l l u s and shoot p r i m o r d i a on r e g e n e r a t i o n media c o n t a i n i n g 100 mg/1 kanamycin w h i l e n o n - t r a n s g e n i c leaf explants failed to callus in this media tFig. 4). One hundred and f o r t y - t w o e x p l a n t s taken

28

Fig. I. Shoot regeneration of eggplant calli following Agrobaeterium co-cultivation and selection on medium containing 100 mg/1 kanamycin and 200 mg/1 cefotaxime for three weeks. Fig. 2. A rooted transgenic eggplant on hormone-free MS medium containing 100 mg/1 kanamycin. Fig. 3. Fruit production on two mature self-pollinated transformed eggplants in the greenhouse. Fig. 4. The leaf disc assay of transformed plants is demonstrated on regeneration medium containing 100 mg/1 kanamycin. The explant on the left which has produced callus and shoot primordia is from a transgenic plant, the two explants on the right were from a non-transformed plant. Fig. 5. Progeny of a selfed non-transformed plant, left, ksensitive to 100 mg/1 kanamycin) and of a selfed transformed eggplant, right, ~which segregates resistant kR) and sensitive kS) seedlings on 100 mg/1 kanamycin). Fig. 6. NPTII dot blot assays of leaf ~L) and callus kC) extracts of 9 transformed plant lines kI,2,3,5,11,14,17,20,26). Negative control k-) extract is from the leaf of an untranaformed eggplant, positive control k+) is from the leaf of a tobacco plant transformed with the same pBCAT binary vector. Fig. 7. CAT assays of leaf kL) or callus kC) extracts of 9 transformed eggplant lines kI,2,3,5,11,14,17,20,26). Control extract is from the leaves of an untransformed eggplant. CM-C14 chloramphenicol; 1-A-CM-acetyl C 14 chloramphenicol, 3-A-CM-3 acetyl C TM chloramphenicol. Fig. B. Total DNA extracted from three transformed plants kI,2,17) and an untransformed eggplant kC) after digestion with Hind IIl and probing with a 1.1 kb fragment containing the coding region of the NPTII gene. The migration of known standards is given in kilobase kkb) pairs of DNA.

:29 from transformed plants and cultured on regeneration medium supplemented with 100 mg/l kanamycin regenerated 126 shoots, while 100 explants from non-transformed plants did not regenerate any shoots. This in vitro assay of the transformed plant's ability to regenerate on selection medium can therefore be used to confirm the presence and expression of the transferred NPTII gene. Progeny analysis of selfed-seed from five individual transformed plants revealed a total of 507 kanamycin resistant LIO0 mg/l) seedlings, 208 kanamycin sensitive seedlings with a germination rate of 60%-100% depending Upon the plant kFig. 5). CAT and NPTII activity was detected in all selected calli and shoots tested. When enzyme assays were performed on five transgenic plants, all of them expressed both CAT and NPTII activity tFigs. 6 and 7). In order to verify the transfer and integration of the NPTII gone, DNA from three plants expressing NPTII activity was restricted with several restriction enzymes and analyzed by Southern blotting. As shown in Fig. 8, the hybridization pattern confirmed the presence of the NPTII gene in the genome of the three plants tested. We were successful in the transformation of eggplant mediated by an A. tumefaciens binary vector whereas Guri and Sink --~1988b) were not This may be explained by the different eggplant genotypes and vectors used in these studies. Dramatic differences in the transformation efficiencies of various genoLypes of tomatoes kMcCormick eL al. 1986) and of Arabidopsis kSchmidt and Willmitzer 1988) have also been reported. Our results demonstrate that cocointegrate vectors are not absolutely necessary for eggplant transformation as was suggested by Guri and Sink L1988b). This allows the use of binary, disarmed Agrobacterium vectors, which may be easier for genetic manipulations, with eggplant. In our experiments the critical factor to obtain transgenic eggplant was the use of in vitro-grown donor material. It is probable that the physiological condition of the in vitro-grown plantlets provided for Agrobacterium infection, a more suitable host tissue than the greenhouse-grown plants provided.

ACKNOWLEDGEMENTS G.L.R. was the recipient of a fellowship from the Ministry of Agriculture and Forestry of Italy. REFERENCES

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C o n t r i b u t i o n No. 1247 Plant Research Centre.