Indian Journal of BiotechnologyVol 2. January 2003, pp 138-146

Agrobacterium-mediated Transformation Efficiency in Blackgram and RiceEnhanced by Multiple Copies of pTiBo542 virB and virG

V Balaji, P Rajamuni, G Sridevi and K Veluthambi*Department of Plant Biotechnology, School of Biotechnology, Madurai Kamaraj University, Madurai 625 021, India

Broad host-range Inc W plasm ids pBAL3 with 3' end of virB and complete virG of pTiBoS42 and pToK47 withcomplete virB and virG were introduced into Agrobacterium tumefaciens vir helper strains harbouring a binaryvector pBAL2 (Inc P). Transformation efficiencies of these strains were evaluated by transforming the primary leafsegments of black gram [Vigna mungo (Linn.) Hepper cv. CoS] and the scutellum-derived calli of rice [Oryza sativaLinn. cv. Pusa Basmati 1]. Transformation was evaluated on the basis of GUS staining in stably transformed calli. Inthe octo pine vir helper strain LBA4404 harbouring pBAL2, the plasmid pBAL3 with the 3' end of virB and completevirG of pTiBoS42 increased transformation efficiency by 141 % to 192%. The plasmid pToK47 that carried pTiBoS42virB in addition to virG did not promote transformation of blackgram any further. In the L, L-succinamopine virhelper strain EHAI0S, pBAL3 did not improve blackgram transformation. In rice, transformation ofLBA4404(pBAL2) occurred only when multiple copies of pTiBoS42 vir genes were present. Transformationefficiencies with pBAL3 and pToK47 were 10 and 18%, respectively. While, multiple copies of 3'end of virB and virGof pTiBoS42 harboured on a compatible replicon were effective in blackgram, complete virB was additionallyrequired for efficient transformation of rice.

Keywords: Agrobacterium tumefaciens, virG, GUS expression, blackgram, rice, transformation

IntroductionAgrobacterium tumefaciens Ti plasmid vectors are

highly suitable for transforming a wide range ofplants (Gelvin, 2000). However, many importantcrops such as grain legumes and monocots arerelatively recalcitrant to A.-mediated transformation(Schroeder et al, 1993; Chan et al, 1993). A numberof strategies have been deployed to overcome thelimitations in transforming these plants (Hiei et al,1994; Jaiwal et al, 2001). The type of virG and itscopy number in Agrobacterium were found to be

*Author for correspondence:Tel: 91-2452-2458683; Fax: 91-2452-2459105E-mail: veluthambi@mrna.tn.nic.in

Abbreviations:Ap: Ampicillin; BAP: 6-Benzylaminopurine; BCIM: Blackgramcallus-induction medium; BRIT: Board of radiation and isotopetechnology; Cx: Cefotaxirne; 2,4-D: 2,4-Dichlorophenoxyaceticacid; gusA: j3-glucuronidase; Hm: Hygromycin; hph: hygromycinphosphotransferase; Inc: Incompatibility group; 2ip: 6-(y, y-dimethylallylamino) purine; Km: Kanamycin; KN: Kinetin; LB:Left border: NAA: o-Naphthaleneacetic acid; nptl/: neomycinphosphotransferase; RB: Right border; RCIM: Rice callus-induction medium; Rm: Rifampicin; RSIM: Rice shoot-inductionmedium: Sm: Streptomycin; Sp; Spectinomycin; Tc: Tetracycline.

important contributing factors. Rogowsky et al (1987)showed that an increase in the copy number of virGresulted in a proportional, acetosyringone-indepen-dent increase in vir gene expression. Jin et al (1987)found that a 2.S kb DNA fragment harbouring virGand 3' end of the virB operon from pTiBoS42 wasresponsible for the supervirulence phenotype of theL,L-succinamopine type A. tumefaciens strain A281.They also reported that virC, virD and S' end of thevirB operon are not involved in the supervirulence.Komari (1990) showed that A. tumefaciens strainsharbouring complete virB, virG and virC of pTiBoS42were able to transform cell suspension cultures ofChenopodium quinoa, which were not transformed bycommon binary vectors, as pGA437 and pGA472.Multiple copies of virG in A. tumefaciens greatlyenhanced the transient transformation efficiency ofcelery, carrot and rice tissues (Liu et al, 1992).

Multiple copies of pTiBoS42 virB, virG and virChave been reported to increase the efficiency ofAgrobacterium-mediated transformation of manycrops (Hiei et al, 1994; Ishida et al, 1996). Jaiwal etal (2001) generated transgenic Vigna radiata by usingthe superbinary vector pTOK233 with multiple copiesof pTiBoS42 virB, virG and virC. introduction of a

BALAJI et at: RICE AND BLACKGRAM TRANSFORMATION WITH pTiBoS42 vir GENES

ternary plasmid carrying a constitutive virG mutantinto A. tutnefaciens strain in combination with astandard binary vector resulted in highertransformation efficiencies in different plant species(van der Fits et al, 2000).

Multiple fold increase in the efficiency of transienttransformation of rice and soybean has beendemonstrated with constitutive virG mutant carried onthe binary vector (Ke et al, 2001).

To date, the generation of transgenic Black gram(Vigna 111Ul1g0(Linn.) Hepper cv. C05 plants by A. -mediated transformation has not been reported.Karthikeyan et al (1996) reported the generation oftransformed blackgram calli through Agrobacterium-mediated transformation at a low frequency.Generation of transgenic rice plants usingAgrobacterium has been previously reported (Hiei etaI, 1994; Rashid et al, 1996; Mohanty et al, 1999;Khanna & Raina, 1999; Kumria et al, 2001). In amajority of studies on rice transformation, thesuperbinary vector pTOK233 that carries pTiB0542virS, virG and virC has been used to improve theefficiency of Agrobacterium-mediated transformation.In the present study, a ternary system, in which abroad host-range plasmid that is compatible with thebinary vector and harbours the pTiB0542 vir genes inmultiple copies is evaluated, and usefulness ofcomplete virS and virG of pTiB0542 for ricetransformation, and 3' end of virS and virG fortransformation of blackgram is also reported.

Materials and MethodsSeed Material

Certified seeds of blackgramcv. C05 wereobtained from Tamil Nadu Agricultural University,Coimbatore, India. Seeds were surface-sterilized asdescribed by Karthikeyan et al (1996). Seeds ofindica rice cv. Pusa Basmati 1 (PB 1) were obtainedfrom Dr George Thomas, Southern PetrochemicalsIndustries Corporation (SPIC) Science Foundation,Chennai. Mature rice seeds were dehusked andsurface-sterilized as described previously(Vijayachandra et al, 1995).

Bacterial Strains and Growth ConditionsA. tumefaciens and Escherichia coli strains and

plasmids used in this study are listed in Table 1.A. tumefaciens was grown at 28°C either in YEP or inAB minimal medium (Chilton et al, 1974), and E. coliwas grown in Luria-Bertani medium (Miller, 1972) at37°C. The concentration of antibiotics (mg rl) used

139

for culturing A. tumefaciens strains on solid mediawere as follows: rifampicin (10), tetracyclinehydrochloride (5), streptomycin sulphate (300),spectinomycin dihydrochloride (100) andcarbenicillin (100 mg rl for EHA 105 & 5 mg r' forLBA4404). Liquid cultures of A. tumefaciens wereraised in medium supplemented with half theconcentration of the antibiotics specified above.Kanamycin monosulphate (50), ampicillin sodium salt(50) and tetracycline hydrochloride (15) were used forE. coli. These antibiotics were bought from SigmaChemical Co, St Louis, USA.

Plasmid ConstructionsDNA cloning and transformation of E. coli DH5a

with plasmid DNA was performed as described byManiatis et al (1982). The plasmid pBAL2 is a binaryvector derived from pGA472 (An et al, 1985) thatcarries nptII as a plant selection marker.

The binary plasmid pBAL2 was constructed asfollows: a 1.7 kb hph cassette from pTRA151 (Zhenget al, 1991) was cloned as an EcoR I fragment intopBIG (Table 1). Following transformation, thecolonies were initially selected on 50 mg r' ampicillinand subsequently patched on 50 mg l' hygromycinwith the appropriate controls. The colony whosegrowth was comparatively equivalent to the positivecontrol (pTRA1S1) was chosen, plasmid DNA wasextracted and subjected to detailed restrictionanalysis. The recombinant plasmid containing hphand gusA gene cassettes was named as pBALI.

The plasmid pBALl was digested with Xho I andcloned into pMHlO02, a pGA472 derivative (An et al,1985). Colonies were selected on LB mediumcontaining 50 mg r' ampicillin and 15 mg r'tetracycline. Detailed restriction analysis of theplasmid DNA from the selected colonies confirmedthe clone. The appropriate orientation of the insert inthe clone was confirmed by Kpn I digestion. ThepBSIIKS+ backbone was removed from plasmidDNA by Kpn I digestion and self-ligation. Followingtransformation, the colonies were initially selected on15 mg r' tetracycline and patched on 50 mg r'ampicillin. Clones with ampicillin sensitivity andtetracycline resistance were chosen. The resultantbinary plasmid with nptII and hpti as plant selectionmarkers and gusA with catalase intron as a reporterwas named as pBAL2 (Fig. 1).

The wide host-range replicon pBAL3 (Fig. 2) with3' end of virB and complete virG was constructed asfollows: a 4.7 kb Sal I fragment 10 representing a

140 INDIAN J BIOTECHNOL, JANUARY 2003

Strain or plasmid

A. tumefaciensLBA4404

EHAI05

Plasm ids

pMHlO02

pUCD2

pSBGA281-G

pBAL2

pBAL3

pToK47

pBIG

pTOK233

Table I-Bacterial strains and plasmids used

Characteristics

A vir helper, harbours disarmed Ti-plasrnidpAL4404, a T-DNA deletion derivative ofpTiAch5, octopine type (Rm')A vir helper, L, L-succinamopine type, harboursT-DNA deletion derivative ofpTiBo542, asupervirulent-type Ti-plasrnid (Rm')

A derivative of the binary vector pGA472harbouring a multiple cloning site (Tc'), Inc PA wide host-range replicon of the incompatibilitygroup, Inc W (Tc', Km', Ap' & Sp'/Sm')Harbours the Sal I 10 fragment of pTiBo542containing the 3' end of virll, complete virG,virC and the 5' end of virD in pUC119 (Ap')A pGA472-based binary vector harbouring nos-nptll intowhich CaMV 35S-hph from pTRA 151and CaMV 35S-gusA with catalase intronwere introduced (Tc'), Inc PThe Sal I fragment 10 of pTiBo542 having the3' end of virB, complete virG, virC and the5' end of virD in pUCD2 (Ap', Krn' & Sp'/Sm'), Inc WA pUCD2 derivative, harbouring the 15.8 kb Kpn Ifragment of pTiBo542 containing the entirevirB, virG and virC (Ap', Tc' & Sp'/Sm'), Inc WHarbours the CaMV 35S-gusA gene with catalaseintron as Hind IIVEcoR I fragment in pBSIIKS+ (Ap')A superbinary plasmid harbouring nos-nptll,CaMV 35S-hph and CaMV 35S-gusA withcatalase intron within the T-DNA and pTiBo542 virB,virG and virC genes in the region outside the T-DNA (Tc')

References

Hoekema et ai, 1983

Hood et ai, 1993

Lab collectionAn et al, 1985Close et ai, 1984

Gelvin, 2000

This workZheng et ai, 1991Ohta et al, 1990

This work

Jin et al. 1987

S B Gelvin, 2000Ohta et ai, 1990Hiei et al, 1994

portion of the pTiB0542 vir region (containing the 3'end of virb, virG and virC) was purified frompSBGA281-G (Table 1) and introduced into pUCD2at the Sal I site resulting in the insertional inactivationof the tetracycline resistant gene. Clones werescreened on the basis of kanamycin resistance andtetracycline sensitivity. The confirmed plasmid wasnamed as pBAL3 (Fig. 2).

Introduction of Plasmids into A. tumefaciensPlasmids from E. coli were introduced into

A. tuniefaciens by triparental mating using pRK2013a"?:_...mobilization helper (Ditta et al, 1980). Thepresence of plasmids in the transconjugants wasconfirmed by Southern analysis (Southern, 1975).

Plant TransformationTransformation of primary leaf segments of black

gram was performed as described previously(Karthikeyan et al, 1996). Trar.sformed calli wereselected on BCIM supplemented with 50 mg ]"1

kanamycin and 250 mg r' cefotaxime (Table 2). Sinceleakiness to kanamycin selection may rarelycontribute to kanamycin resistance, transformationefficiencies on the basis of GUS+ staining areexpressed.

Surface-sterilized PB 1 rice seeds were cultured onRCIM in the dark for three weeks (Table 2). Non-embryogenic sectors were removed and the callus wassub-cultured on the same medium and preincubated inthe dark for four days. A. tumefaciens strains were

BALAJI et al: RICE AND BLACKGRAM TRANSFORMATION WITH pTiBo542 vir GENES

p

estS

pBALZ18.&Kb

S St

Fig. I-Restriction map of the binary vector pBAL2. RB: rightborder; LB: left border; tet: tetracycline resistance gene; oriV:RK2 origin of replication; cos: cos site of lambda phage; B:BamH I; Bg: Bgl II; C: Cia I; E: EcoR I; Ec: EcoR V; H: Hind III;K: Kpn I; N: Nco I; P: Pst I; S: Sal I; Sc: Sac I; Sn: Sl1aB I; St: SstII; X· Xba I; Xh: Xho I.

grown to 1 OD at 600 nm in liquid AB minimalmedium (Chilton et al, 1974) with appropriateantibiotics (Table 1). The bacterial cells were pelletedat 1100xg at 25°C and resuspended in AA-ASmedium (Rashid et al, 1996). Infection of thepreincubated calli and cocultivation were performedas described by Rashid et al (1996). Aftercocultivation, the infected calli were transferred toselection medium-RCIM (Table 2) supplemented with50 mg r' hygromycin and 250 mg }"I cefotaxime.Selection of hygromycin-resistant calli was carriedout for eleven weeks. First sub-culture of the calli to afresh selection medium was performed after twoweeks. Thereafter, sub-culturing was done at three-week intervals. The proliferated hygrornycin-resistantcalli obtained were subjected to GUS histochemicalstaining (Hiei et al, 1994).

141

pBAL3(17·7kb)

Fig. 2- Restriction map of pBAL3. pSa: pSa ongm ofreplication; pMBI: pMBl origin of replication; Ap': ampicillinresistance gene; Km': kanamycin resistance gene; Sp':spectinomycin resistance gene; B: BamH I; Bg: Bgl II; C: Cia I;E: EcoR I; Ec: EcoR V; Bs: BstE II; H: Hind III; K: Kpti I; P: PSI

I; Pv: Pvu II; S: Sal I; ScI: Sac I; ScII: Sac II.

Hygromycin-resistant and GUS-positive calliobtained upon transformation with A. tumefaciensstrain LBA4404(pTOK233) were transferred to RSIMsupplemented with 40 mg l' hygromycin and250 mg r' cefotaxime (Table 2). Regenerated rootedplants were acclimatized to soil and maintained in atransgenic greenhouse.

DNA Isolation and Southern Blot AnalysisTotal DNA was extracted from the leaves of

LBA4404 (pTOK233)-transformed and non-transformed control rice plants as described by Rogersand Bendich (1988). DNA concentration wasdetermined by fluorometry (TKO 100, HoeferScientific Instruments, San Francisco, USA) using theHoechst dye 33258. Control and To plant DNA (20ug) was digested with Hind III for 12 hrs and

Medium

Table 2-Media used for tissue culture and transformation of black gram and rice

Composition

MS salts and vitamins (Murashige & Skoog, 1962),30 g r' sucrose, 45 f.!M

2,4-D, 0.015 f.!M each of BAP, 2ip and KN, 8 g r' agar, pH 5.7

MS salts, B5 vitamins, 30 g r' sucrose, 300 mg r' casarninoacids, 500 mg r'proline, 11.3 f.!M 2,4-0, 2.25 g r' Phytagel, pH 5.8MS salts and organic supplements, 30 g r' sucrose, 300 mg r' casaminoacids,13.9 f.!M KN, 8.1 f.!M NAA, 4 g r' Phytagel, pH 5.8

BCIM

RCIM

RSIM

142 INDIAN J BIOTECHNOL, JANUARY 2003

electrophoresed through 1% agarose gels. The DNAwas blotted onto the Zeta-Probe membrane (Bio-Rad,Hercules, California) and hybridized with the 0.9 kbnptlJ coding sequence (RB probe) and 1.1 kb hphcoding sequence (LB probe). The probes werelabelled with [a-32PJdCTP (BRIT, Mumbai, India)using a random primer labelling kit (AmershamPharmacia Biotech, Little Chalfont, England).

Results and DiscussionMultiple Copies of 3' end of virB and virG ofpTiB0542 Enhance the Transformation Efficiencyin Blackgram

The effect of multiple copies of 3' end of virB andvirG of pTiBoS42 by introducing the ternary Inc Wplasmid pBAL3 (Table 1; Fig. 2)) into theA. tumefaciens strain LBA4404 (Hoekema et al,1983) carrying the Inc P binary vector pBAL2 (TableI, Fig. 1) was evaluated. The plasmids pBAL2 andpBAL3 are compatible with each other. Stabletransformation efficiencies (Km' calli) and percentageof GUS+ calli are presented in Table 3. In a previousreport, which included Southern analysis to study T-DNA integration (Karthikeyan et al, 1996), kana-mycin-resistant proliferation of calli was establishedas a reliable index of stable transformation.A. tumefaciens strain LBA4404(pBAL2) without

extra copies of vir genes transformed 12% to 17% ofthe leaf segments in two independent experiments. Inthe presence of pBAL3 with the 3' end of virB andcomplete virG of pTiB0542, the transformationefficiency increased from 12% to 35% and 17% to41% (Table 3). The increase in transformationefficiency contributed by pBAL3 was 192% in thefirst experiment and 141% in the second experiment.When pToK47 with multiple copies of pTiB0542 virBand virG was used, transformation efficiency did not

increase over the levels achieved with pBAL3.Therefore, multiple copies of 3' end of virB and virGof pTiBo542 seem to be adequate to elevatetransformation efficiency in blackgram.

Supplementation of a plasmid containing the virregion of pTiB0542 in the octopine strains increasedtumourigenesis (supervirulence) on tobacco andtomato plants (Hood et al, 1986b). Increasedtumourigenesis was achieved in Nicotiana glauca leafdiscs by providing 3' end of virB and virG ofpTiB0542 in an octopine strain A348 (Jin et al, 1987).Jaiwal et al (2001) reported the transformation ofV. radiata using pTOK233 with the pTiB0542 virB,virG and virC. However, they did not compare thetransformation efficiencies with and without thepTiB0542 vir genes. The efficiency ofAgrobacterium-mediated transformation of blackgramin the presence or absence of pBAL3 (3' end of virB,virG and virC of pTiB0542, Table 1) was evaluated inthis study.

van der Fits et al (2000) reported that expression ofa constitutive virG mutant on a ternary plasmidgreatly enhanced Agrobacterium-mediated transienttransformation efficiencies in a diverse range of plantspecies. However, in Dolichos lablab (a grainlegume), no transient transformation was observedwith the A. tumefaciens strain LBA4404 with andwithout constitutive virG on a ternary plasmid. Thesupplementation of pTiB0542 virG on a ternaryplasmid pBAL3 in LBA4404(pBAL2) increased thestable transformation of blackgram.

Multiple Copies of virG and the Type of Ti-plasmidInfluence the Transformation Efficiency in Black-gram

Whether the increase in transformation efficiencyof black gram observed in Table 3 is linked to the

Table 3-Effect of multiple copies of pTiB0542 virB, virG and virC on the transformation efficiencyin primary leaf segments of blackgram

A. tumefaciens strain Number of leaf % leaf segments % leaf segmentssegments infected with Km' calli with GUS+ calli

Exptl Expt2 Exptl Expt2 Exptl Expt2

LBA4404(pBAL2) 129 106 33 50 12 17LBA4404(pBAL2, pBAL3) 96 112 50 67 35 41LBA4404(pBAL2, pToK47) 110 117 43 62 22 30

Scoring of kanamycin resistant (Km') calli and GUS histochemical analysis was performed after 25 days in the selection medium-BCrM (Table 2) supplemented with 50 mg r' kanamycin and 250 mg r' cefotaxime

BALAJI et al: RICE AND BLACKGRAM TRANSFORMATION WITH pTiB0542 vir GENES

pTiBoS42 vir background or to multiple copies ofpBAL3 with the 3' end of virB and virG genes ofpTiBoS42 was evaluated. PBAL3 was introduced intoA. tumefaciens vir helper strains, LBA4404 (octopine-type) and ERAlOS (L,L-succinamopine-type) bothharbouring the binary vector pBAL2. Transformationof primary leaf segments of black gram wasperformed. LBA4404 (pBAL2) without pBAL3showed 11% transformation, which increased to 31%in the presence of pBAL3 (Table 4). A 182% increasewas contributed by pBAL3 in LBA4404.Surprisingly, only a marginal increase of 9% wasobserved for ERA 105 (pBAL2, pBAL3) overEHAlOS (pBAL2) (Table 4). Thus, pBAL3 with the3' end of virB and complete virG of pTiBoS42improved transformation in LBA4404 but did notimprove transformation in EHAlOS.

Multiple copies of virG and the type of Ti-plasmidharboured by A. tumefaciens strains can influence thetransformation efficiency in different plant species.Liu et al (1992) observed that supplementation ofmultiple copies of octopine virG or pTiBoS42 virG innopaline Ti-plasmid backgrounds did not bring abouta significant increase in transient transformation ofcelery and rice. However, a five-fold increase in thetransient transformation efficiency was obtained byintroducing additional copies of octopine virG orpTiBoS42 virG in L,L-succinamopine-type Ti-plasmid background. The effect of additional copiesof pTiBoS42 virG in octopine-type Ti-plasmid wasnot evaluated by Liu et al (1992). It was observed thatsupplementation of multiple copies of 3' end of virBand virG of pTiBoS42 as a ternary plasmid (pBAL3)in LBA4404 (octopine strain) increased the stabletransformation efficiency of blackgram by 182%, buta similar increase was not observed for EHAI0S

143

(Table 4). This is understandable, since EHA 105 byitself is a vir helper derived from the supervirulentstrain A281 containing pTiBoS42.

These results agree with the previous observationmade by Jin et al (1987) and Krishnamohan et al(2001). Multiple copies of pTiBoS42 virG and 3' endof virB did not increase the virulence of A281 (L,L-succinamopine strain) but increased virulence ofA348 (octopine strain) on N. glauca leaf discs (Jin etal, 1987). Krishnamohan et al (2001) reported thatadditional copies of 3' end of virB and virG ofpTiB0542 in LBA4404 increased tobaccotransformation efficiency by 65%, but a similarincrease was not observed for EHAlOS.

Multiple Copies of virB and virG Enhance theStable Transformation Efficiency in Rice

Evaluation of the relative importance of virB, virGand virC (pToK47) and 3' end of virB and completevirG (pBAL3) in a ternary system on stabletransformation of rice was done (Table 5). LBA4404(pBAL2) without the pTiBoS42 vir genes did nottransform rice. Supplementation of multiple copies of3' end of virB and complete virG of pTiBoS42 in theform of pBAL3 in LBA4404 (pBAL2) resulted instable transformation efficiencies of 8% and 10%.Transformation efficiency increased further from 8%to 18% when pToK47 with virB, virG. and virC ofpTiBoS42 was supplemented in LBA4404 (pBAL2).The superbinary vector LBA4404 (pTOK233)transformed rice at a slightly higher efficiency of22%. Thus, complete virB in addition to virG ofpTiBoS42 is required for achieving hightransformation efficiency in rice.

Liu et al (1992) reported that additional copies ofoctopine and L,L-succinamopine virG genes in

Table 4-Effec:t of multiple copies of pTiB0542 virG and the type of Ti-plasrnid on the transformation efficiency in primary leafsegments of blackgram

A. tumefaciens strain vir helper Presence or Number of % leaf % leaftype absence of leaf segments segments segments

extra copies of infected with Km' with GUS+virG from calli calli

pTiB0542 (pBAL3)

LBA4404(pBAL2) Octopine 209 23 11LBA4404(pBAL2. pBAL3) Octopine + 207 45 31EHA105(pBAL2) L. L-Succinamopine 144 32 22EHA105(pBAL2. pBAL3) L. L-Succinamopine + 137 31 24

Scoring of kanamycin resistant (Kmr) calli and GUS histochemical analysis was performed after 25 days in the selection medium-BCIM (Table 2) supplemented with 50 mg r' kanamycin and 250 mg r' cefotaxime

144 INDIAN J BIOTECHNOL, JANUARY 2003

Table 5-Effect of multiple copies of pTiB0542 virB, virG and virC on stable transformation of scutellum-derived calli of indica rice cv. Pusa Basmati 1 using A. tumefaciens

A. tumefaciens strain % Hmrcalli % GUS+calli

Expt 1 Expt2 Expt 1 Expt2

LBA4404(pBAL2) 0 0 0 0LBA4404(pBAL2, pBAL3) 14 14 8 10LBA4404(pBAL2, pToK47) 20 NO 18 NOLBA4404(pTOK233) 24 26 22 22

For transformation with each Agrobacterium strain, 50 scutellum-derived calli were taken for cocultivationNO-Not done

A. tumefaciens strains contairung the L,L-succin-amopine-type Ti-plasmid enhanced the efficiency oftransient transformation of rice. They did not evaluatethe role of complete virB and the effect of additionalcopies of pTiBo542 virG in A. tumefaciens straincontaining an octopine-type Ti-plasmid. From ourresults, it is clear that in addition to virG, supple-mentation of complete virB of pTiBo542 is essentialfor obtaining a high transformation efficiency in rice.Hiei et at (1994) demonstrated that the pTiBo542virB, virG and virC contributed to a hightransformation efficiency in rice, but did not comparethe transformation efficiency in the absence of thecomplete virB of pTiBo542. The observationspresented in Table 5 show that complete virB, inaddition to virG of pTiBo542, is required forachieving a high transformation efficiency in rice.~

Southern Analysis of To Rice PlantsSouthern analysis (Southern, 1975) was done in To

rice plants generated from GUS+ calli to ensure thatGUS-staining in rice reflects genuine transformation.To plants obtained from LBA4404 (pTOK233)-infected calli were chosen for Southern analysis toconfirm the integration of T-DNA into the ricegenome. Genomic DNA (20 ug) from the controlplant and eight To plants was digested with Hind IIIand hybridized with nptll and hph coding sequencesas probes to detect right and left border junctionsequences, respectively (Fig. 3). The nptll probe isexpected to hybridize to the junction sequences longerthan 5.65 kb. Hybridization with the nptII proberevealed the presence of two T-DNA copies in plants8, 10, 12, 17,21 and 28. Three junction fragments ofvarying sizes were detected in plants 26 and 35.Surprisingly the To plants 12,21 and 28 which carriedtypical junction fragments longer than 5.65 kb alsoshowed shorter junction fragments. These may either

be due to truncated T-DNA transfer or due toscrambling of T-DNA prior to integration.

Left border junction sequence analysis with the hphprobe revealed that the T-DNA copy numbers weresimilar to those derived from nptII probe analysis. Ina larger proportion of the plants analyzed, junction

AC U 8 10 12 17 21 26 2835 L Pkb

lI I I I I I I I I I I I kb

23.1-_12·09.4-1

6.6- - -6·04.4_ - -50

-4·0_30

2·3_2·0- _2.0

_1.6

B

23.1- _12·09·4- - -6·06·6- -504·4- -4.0

_3·02.3- .2.02·~ _1.6

Fig. 3-Southern analysis of To rice plants transformed withLBA4404(pTOK233). (A): Analysis of right border junctionsequences with nptll probe. (B): Analysis of left border junctionsequences with hplt probe. Numbers on the top refer to To lines.Twenty microgram aliquots of plant DNA were digested ~ ithHind III and analyzed. Lanes-C: DNA from untransformedcontrol plant digested with Hind III; U: Undigested DNA from Toplant 35; L: kb ladder; P: Total DNA from LBA4404(pTOK233)digested with Hind III (100 ng was loaded in panel A and 500 ngwas loaded in panel B). Positions and sizes of A-Hind 1lIfragments (left) and kb ladder (right) are marked.

BALAJI et al: RICE AND BLACKGRAM TRANSFORMATION WITH pTiB0542 vir GENES

fragments greater than 5.0 kb were observed.However, some plants revealed bands shorter than 5.0kb. Hybridization patterns were similar in plants 8 and10 with both probes suggesting that they are siblingsthat might have arisen from a single transformationevent. All the seven independent transgenic plantsthat regenerated from GUS+ calli carried integrated T-DNA. Thus, GUS+ calli give a reliable score fortransformation.

Transformation of different plant species wasperformed with LBA4404 (pTOK233) which containsvirB, virG and virC of pTiB0542 and the T-DNA onthe binary vector (Hiei et al, 1994; Mohanty et al,1999; Jaiwal et al, 2001). In this study, additionalcopies of vir genes (virB, virG and virC) of pTiB0542were introduced on a wide host-range ternaryplasmids (pBAL3 & pToK47; Table 1). Ternaryplasmids can be used in combination with existing A.tumefaciens and standard binary vectors. In thisrespect, it offers a clear advantage over otherimproved A. tumefaciens vector systems that providemultiple vir genes on the binary vector itself. Thebinary vector pT0K233, for example, contains thehypervirulence region of pTiB0542, therebyincreasing the size to over 50 kb (Hiei et al, 1994). Tointroduce genes of interest on the T-DNA of suchlarge binary vectors is often not possible withstandard molecular cloning techniques. The Inc Pplasmid pSBl (virB, virG and virC of pTiB0542),cannot be used in A. tumefaciens strains carryingstandard Inc P binary vectors for improving thetransformation efficiency. Ternary Inc W plasmids(pBAL3 & pToK47) reported in our study enhancedthe transformation efficiency of blackgram and ricewhen contained in A. tumefaciens carrying Inc Pbinary vectors.

Thus, ternary plasmids expressing multiple copiesof virB and virG of pTiB0542 greatly enhanceAgrobacterium-mediated stable transformationefficiencies in blackgram and rice. Multiple copies of3' end of virB and complete virG of pTiB0542 on aternary plasmid (pBAL3) were found to be adequateto increase transformation efficiency of blackgram.A. tumefaciens strains harbouring multiple copies ofcomplete virB and virG genes of pTiB0542transformed rice at the highest efficiency, indicatingthat complete virB of pTiB0542 in addition to virG isnecessary for achieving a higher transformationefficiency in rice. In contrast to improvedAgrobacterium strains having hypervirulence regions

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added on the binary vector backbone, the ternaryplasmid pBAL3 reported in this study is moreconvenient for use in standard A. tumefaciens strainsin combination with existing binary vectors.

AcknowledgementThe authors gratefully acknowledge Dr S B Gelvin,

Purdue University, USA for providing the p\asmidspSBGA281-G and pBIG and A. tumefaciens strainsLBA4404 and EHA105. They also thank Dr C [Kado, University of California, Davis, USA forpUCD2, Dr T Komari, Japan Tobacco Inc, Japan forLBA4404 (pTOK233), Dr K Nakamura, NogoyaUniversity, Japan for the plasmid pIG221 with intron-gus and Dr E W Nester, University of Washington,USA for the plasmid pToK47. Thanks are also due toDr K Dharmalingam, for his kind permission to usehis instrumentation facilities, and Dr S Krishnaswamyfor permitting to use the Stereo Microscope.

VB and PR thank CSIR,New Delhi for researchfellowships.This work was supported by theDepartment of Biotechnology (DBT), New Delhi,Govt of India and Indo-Swiss Collaboration inBiotechnology jointly funded by DBT, Govt. of Indiaand Swiss Agency for Development and Cooperation(SDC).

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