Plant Cell Reports

Plant Cell Reports (1996) 15:328--331

9 Springer-Verlag 1996

Agrobacterium tumefaciens-mediated transformation of Vigna mungo (L.) Hepper A.S. Karthikeyan, K.S. Sarma *, and K. Veluthambi Department of Plant Molecular Biology, School of Biotechnology, Madurai Kamaraj University, Madurai 625 021, India * Present address: Department of Mycology and Plant Pathology, Queen's University of Belfast, New Forgelane, Belfast BT9 5PX, Northern Ireland, U.K. Received 29 March 1995/Rcvised version received 6 July 1995 - Communicated by G. C. Phillips

Abstract : Transformed Vigna mungo (blackgrarn) calli were obtained by cocultivating segments of primary leaves with Agrobacterium tumefaciens vir helper strains harbouring the binary vector pGA472 having kanamycin resistance gene as plant transformation marker. Transformed calli were selected on Murashige and Skoog medium supplemented with 50 mg/l kanamycin and 500 mg/l carbenicillin. Transformed calli were found to be resistant to kanamycin up to 900 mg/l concentration. Expression of kanamycin resistance gene in Iransformed calli was demonstrated by neomycin phosphotransferase assay. Stable integration of transferred DNA into V. mungo genome was conru'med by Southern blot analysis. Abbreviations: BAP = 6-benzylaminopurine; 2,4-I) = 2,4dichlorophenoxyacetic acid; 2iP = 6-(~/,~-dimethylallylamino)purine; Kn = kinetin; nptlI = neomycin phosphotransferasel/; MS = Murashige and Skoog (1962) medium. Introduction Vigna mungo, commonly known as blackgrarn, is an important grain legume in tropical countries. Though it is an important pulse crop, very little work has been done towards genetic improvement of the crop. Development of reliable regeneration system and Agrobacterium-mediated transformation would provide an opportunity to improve V. mungo by genetic engineering. Only a few grain legumes such as Glycine max (Hinchee et al. 1988), Pisum sativum (Puonti-Kaerlas et al. 1989; Kathen and Jacobsen 1990; Schroeder et al. 1993), Phaseolus vulgaris (Mariotti et al. 1989; Franklin et al. 1993), Vigna unguiculata (Garcia et al. 1986, 1987) and Vigna aconitifolia (Eapen et al. 1987) have been successfully transformed using Agrobacterium. Genetic engineering of V. mungo using Agrobacterium has not been accomplished yet, We have succeeded in generating kanamycin resistant (transformed) calli from segments of primary Correspondence to." K. Veluthambi

leaves of V. mungo. Neomycin phosphotransferase activity was demonstrated in the transformed calli. Integration of transferred DNA (T-DNA) into V. mungo genome in the transformed calli was confirmed by Southern hybridization analysis. Materials and Methods Bacterial strains and culture conditions: The binary vector pGA472 (An 1986) was mobilized by Iriparental mating into A. tumefaciens vir helper strains LBA4404 (octopine type, pTiAch5) and EHA 105 (agropine type, pTiBo542). Mobilization was confirmed by Southern hybridization. A. tumefaciens slrains LBA4404 and EHA105 were received from S.B. Gelvin, Purdue University, USA. Agrobacterium strains were grown in AB medium (Chilton et al. 1974) at 28~ with 5 ~tg/ml tetracycline. Tissue culture: Certified seeds of V. mungo (L.) Hepper cv. Co5 were obtained from Tamil Nadu Agricultural University, Coimbatore, India. Mature, completely filled and unwrinkled seeds were hand-picked and washed in running tap water for a few minutes. Washed seeds were surface sterilized by treating with 70% alcohol for 5 min, sodium hypochlorite (commercial grade, 4% active chlorine) with Tween 20 (1% v/v) for 15 min, followed by 0.1% (w/v) mercuric chloride treatment for 3 rain. Treated seeds were rinsed 5 times with sterile distilled water and germinated on half-strength MS (Murashige and Skoog 1962) salts medium solidified with 0.8% agar. Callus was initiated from segments of primary leaves (0.5x0.5 cm) of 4-day-old seedlings on MS medium supplemented with 45 ttM 2,4-D and 0.015 I.tM each of BAP, 2iP and Kn. The cultures were maintained under 16/8 h light/dark regimen at 25+ 2~ Kanamycin sensitivity of leaf segments was determined in order to use kanamycin as plant selection marker. Leaf segments from 4-day-old seedlings were placed on

329 MS medium without hormones for 4 d and then transferred to callusing medium supplemented with kanamycin (Sigma Chemical Company, USA) ranging from 0 to 200 rag/1. Transformation: Leaf segments from 4-day-old seedlings were preincubated on hormone-free MS medium for 2 d. Preincubated leaf segments were collected in a Petri plate and 5 ml of Agrobacterium liquid culture grown to 1 OD at 600 nm was added and swirled gently for 5 rain. Infected leaf segments were blot-dried on sterile filter paper and cocultivated on hormone-free MS medium for 2 d. Infected leaf segments were transferred to selection medium (callusing medium supplemented with 50 mg/l kanamycin and 500 mg/1 carbenicillin) after 2 d of cocultivation. Enzyme assay: Neomycin phosphotransferase activity in transformed calli (grown on selection medium with 50 rag/1 kanumycin) was assayed by a previously reported paper chromatographic method using [7-32p]ATP and kanamycin as substrates (Roy and Sahasradbudhe 1990).

Southern analysis: Genomic DNA from transformed and untransformed calli was extracted by cetyltrimethylummonium bromide (CTAB, Sigma Chemical Company, USA) method (Rogers and Bendich 1988) and subjected to Southern analysis. Five lxg of DNA from each callus sample and 70 pg of plasmid DNA were digested with restriction enzymes (GIBCO BRL, USA) for 10 h and 3 h, respectively, and electrophoresed through 0.7% agarose gel. DNA was transferred to a nylon membrane (Bio-rad, USA) and probed with [a-32p]dCTP-labeUed probes prepared using random primer labelling kit (Amersham, UK). Hybridization was performed as described by Watson and Thompson (1988). All the radioactive chemicals were purchasedfrom B/d~C, IN-DIA ( [or-32P]dCTP-specific activity 11.1 x 1013 Bq/mmole, [T-32p] ATP-specific activity 13.69 x 1013 Bq/mmole). Results and Discussion

A concenlration of 50 mg/1 kanamycin was chosen for selection of transformants based on kanamycin sensitivity experiments. Kanamycin could be used as an effective selection marker for V. mungo, only if preincubation and cocultivation of leaf segments were performed on hormone-free MS medium (Karthikeyan et al. manuscript in preparation). Callus initiation occurred in Agrobacterium-infected leaf segments 7 to 10 d after Iransfer to selection medium. Transformation frequency with LBA4404 and EHA105 as helper slrains, was 23% and 10%, respectively, in two independent experiments.

Calli were produced as clumps trom the wounded edges of the leaf segments. Each callus dump, possibly representing a single transformation event, was separated and subcultured individually after 4 weeks. Calli, subcultured on the selection medium, proliferated rapidly and doubled in size in 2 weeks. Calli were subcultured at regular intervals of 4 weeks.

\ S- F

p _GA472

S

~

Cos

Sa

Flg.I.Restriction map ofthebinaryvectorpGA472(An 1986). Probe I represents T-DNA region and probe II represents a region outside the T-DNA. B, BamHl; Bg, BgllI; H, HindlII; P, PstI; S, Sstll; Sa, Sail; Se, ScaI; RB, Right Border, LB, Left Border.

Kanamycin resistant caUi were checked for the level of their kanamycin resistance by transferring them to media with different concentrations of kanamycin. The growth of transformed calli was visibly normal (pale green and friable) up to a kanamycin concentration of 900 mg/l. Untransformed control calli failed to grow even at 50 mg/l kanamycin concentration (Fig. 2). In an independent experiment, about 200 mg of transformed calli inoculated on callusing medium containing 0, 50, 100, 200, 400 or 800 mg kanamycin/1 increased to 1005 mg, 994 mg, 914 mg, 933 mg, 883 mg and 845 mg, respectively, registering a 3-fold increase in fresh weight even at 800 mg/l kanamycin. The transformed calli, therefore, possessed a high level of kanamycin resistance. Neomycin phosphotransferase activity was assayed in control and transformed calli using a paper chromatographic procedure (Roy and Sahasradbudhe 1990). As controls, enzyme extracts from E. coli strains JM83 (Kan) and MM294 (harbouring the plasmid pRK2013 with kanr gene) were used. Two spots with Rf values 0.66 and 0.54 were detected when the extract from E. coli MM294 with pRK2013 (Kan+) was used in the assay (Fig. 3). The fast moving spot (Rf 0.66) was also seen in extracts of E. coli JM83

330 extracts from control and transformed V. mungo calli revealed that the kanamycin-specific spot 0tf 0.54) appeared only when extracts from transformed calli were used for the assay. This spot was seen only when kanamycin was added as a substrate. The spot (Rf 0.66), not specific to kanamycin, appeared when extracts from both control and transformed tissues were used for the assay. These results provide evidence for the expression of nptII gene in the transformed calli.

Fig. 2. Tolerance of kanamycin resistant calli of V. mungo to higher levels of kanamycin. Calli in numbered plates are untransfonned and calli in plates without ntmabers are transformed calli. Kanamycin concentrations are 0(1), 50 mg/l (2), 100 mg/l (3), 200 mg/l (4), 300 mg/l (5), 500 mg/l(6), 700 meda (7) and 900 mg/l (8). Photograph was taken 30 d after transfer to kanamycin media.

Fig. 3 Paper chromatographic analysis of neomycin phosphotrans. ferasr IL A. Assay of E. coli extracts. The enzyme assay w a s performed with the extracts of E. coil JM83 (lane 1) and E. coli MM294 with pRK2013 (lane 2). B. Assay of callus extracts, Extracts from untransformed (lanes 1 and 2) and transformed (lanes 3 and 4) calli were used for assay. Lanes 1 and 3 with kanamyein a s substrate, lanes 2 and 4 without kanamycin. Each a s s a y w a s performed with 2 btCi of [y-32P]ATP.

without kanamycin resistance. The slow moving spot (Rf 0.54) was seen only when extracts from E. coli with kanamycin resistance were used for the assay. Therefore, the spot with Rf value 0.54 represents neomycin phosphotransferase activity. Analysis of

Southern hybridization analysis was carried out to verify the integration of the T-DNA with nptlI gene into V. mungo genome. A 2.3 kb BamHI fragment of the T-DNA of the binary vector pGA472 (probe I, Fig. 1) that covers the nptlI gene was used as a probe. This probe hybridized to a 2.3 kb fragment of BamHI-digested genomic DNA of transformed V. mungo calli (Fig. 4A). This probe also hybridized to the 2.3 kb BamHI fragment of purified pGA472 and total DNA from Agrobacterium harbouring the binary vector pGA472 digested with BamHI (positive controls). Genomic DNA from untransformed V. mungo caUi did not hybridize to the labelled nptlI fragment. Transfer of kanamycin resistant calli after seven subcultures to carbenicillin-free selection medium did not lead to growth of Agrobacterium, indicating the Agrobacterium contamination. absence of Agrobacterium cells that may survive the antibiotic treatment (i.e., carbeniciUin) but which are unable to produce visible colonies, may interfere with molecular and biochemical analyses and lead to the erroneous conclusion that stable integration and expression of the gene has taken place. To rule out the possible Agrobacterium contamination in transformed calli and to show that only T-DNA between borders is transferred to the genome, a 2.8 kb SstlI fragment of pGA472 (probe II, Fig. 1) which is totally outside the T-DNA borders was used as the probe. This probe hybridized only to the DNA from positive controls (purified pGA472 and DNA from Agrobacterium harbouring pGA472) and did not hybridize to the DNA from transformed V. mungo calli (Fig. 4B). These results confirm that only T-DNA sequences between borders have been transferred. Therefore, the hybridization signal observed in lanes 3, 4, 6 and 7 of Fig. 4A is due to genuine T-DNA transfer and not due to Agrobacterium contamination. To verify the stable integration of nptlI gene into the V. mungo genome and to detect junction fragments of plant DNA and the T-DNA, genomic DNA from transformed V. mungo calli was digested with PstI and probed with the 2.3 kb BamHI fragment (probe I, Fig.l). Signals corresponding to a 5.6 kb PstI

331

Fig. 4. Southernhybridizationanalysis of kanamycin resistantV. mungo calli. Approximately5 pg of genomic DNA from untransformedcalli (lane 2), LBA4404-transformedcalli (lanes 3 and 6) and EHA105-transformedcalli (lanes 4 and 7) were analysed. As controls,70 pg of purified pGA472 DNA (lane 1) and 20 ng of total DNA from Agrobacterium harbouringpGA472 (lane 5) were analysed. All the DNA samples were digested either with BamHl or Pstl, nan on 0.7% agarosegel, blotted on to nylonmembranesand hybridizedto appropriateprobes.A: Analysis of internal T-DNAfragments. The DNA samplesweredigested with BamHIand probedwith the 2.3 kb BamHIfragmentof pGA472 (ProbeI, Fig.I). B: Analysis of pGA472 sequences outside the borders. DNA samples were digested with BamHl and probed with the 2.8 kb Sstll fragment of pGA472 (Probe II, Fig.l). C: Sonthem hybridizationanalysis for junction fragmentsto confirmthe stable integrationof T-DNA into V. mungo genome.The DNA samples were digested with PstI and probedwith the 2.3 kb BamHIfragmentof pGA472(ProbeI, Fig.l).

fragment that is totally internal to the T-DNA and a 9.5 kb PstI fragment that has regions outside the T-DNA borders were seen when the probe was hybridized to purified pGA472 DNA (Fig. 4C, lane 1) and Agrobacterium total DNA (Fig. 4C, lane 5). Signal corresponding only to the 5.6 kb PstI fragment was seen when it was hybridized to the DNA from transformed calli (Fig. 4C, lanes 3, 4, 6 and 7). Sequences of pGA472 outside the borders were not found in DNA from the transformed calli. Additional bands in the lanes 6 (4.5 kb) and 7 (12.6 kb) may represent junction fragments having a defined portion of the T-DNA (close to the fight border) and portions of plant DNA. Southern analysis and nptlI assay confirm that the generation of kanamycin resistant caUi from segments of primary leaves of V. mungo represent genuine DNA transfers via A. tumefaciens. Regeneration in V. mungo has not been reported so far. Standardization of tissue culture conditions for regeneration should permit the generation of transgenic V. mungo plants using A. tumefaciens.

Acknowledgement We acknowledge the financial support from DBT, Govt. of India. We thank the Bioinformatics Centre, Madurai Kamaraj University, Madural, India for access to its facility. ASK is grateful to CSIR, Govt. of India for financial support. References An G (1986) Plant Physiol 81:86-91 Chilton M-D, Currier TC, Farrand SK, Bendich AJ, Gordon MP, Nester EW (1974) Proc Natl Acad Sci USA 71:3672-3676

Eapen S, Kob.ler F, Gerdemann M, Schieder O (1987) Theor Appl Genet 75:207-210 Franklin CI, Trieu TN, Cassidy BG, Dixon RA, Nelson RS (1993) Plant Cell Reports 12:74-79 Garcia JA, Hille J, Goldbach R (1986) Plant Science 44:37-46 Garcia JA, Hille J, Vos P, Goldbach R (1987) Plant Science 48:89-98 I-Iinchee MAW, Connor-Ward DV, Newell CA, McDonnell RE, Sato SJ, Gasser CS, Fischhoff DA, Re DB, Fraley RT, Horsch RB (1988)Bio/Technology 6:915-922 Kathen AD, Jacobsen H-J (1990) Plant Cell Reports 9:276-279 Mariotti D, Fontana GS, Santini L (1989) J Genet & Breed 43:77-82 Murashige T, Skoog F (1962) Physiol Plant 15:473-497 Puonti-Kaerlas J, Stabel P, Eriksson T (1989) Plant Cell Reports 8:321-324 Rogers SO, Bendich AJ (1988) In: Plant Molecular Biology Manual, Gelvin SB, Schilperoort RA (eds), Kluwer Academic Publishers, Dordrecht, The Netherlands, pp A 6:1-11 Roy P, Sahasradbudhe N (1990) Plant Mol Biol 14:873-876 Schroeder HE, Schotz AH, Richardson TW, Spencer D, Higgins TJV (1993) Plant Physiol 101:751-757 Watson JC, Thompson WF (1988) In: Methods for Plant Molecular Biology, Weissbach A, Weissbach H (eds), Academic Press Inc, San Diego, pp 57-75