农杆菌介导圆叶葡萄胚发生组织的转化和植株再生

GENETIC TRANSFORMATION AND HYBRIDIZATIONAgrobacterium -mediated transformation of embryogenic cultures and plant regeneration in Vitis rotundifolia Michx.(muscadine grape)S.A.Dhekney ÆZ.T.Li ÆM.Dutt ÆD.J.GrayReceived:6November 2007/Revised:10January 2008/Accepted:18January 2008/Published online:7February 2008ÓSpringer-Verlag 2008Abstract A method to produce transgenic plants of Vitis rotundifolia was developed.Embryogenic cultures were initiated from leaves of in vitro grown shoot cultures and used as target tissues for Agrobacterium -mediated genetic transformation.A green fluorescent protein/neomycin phosphotransferase II (gfp/nptII )fusion gene that allowed for simultaneous selection of transgenic cells based on GFP fluorescence and kanamycin resistance was used to optimize parameters influencing genetic transformation.It was determined that both proembryonal masses (PEM)and mid-cotyledonary stage somatic embryos (SE)were suitable target tissues for co-cultivation with Agrobacterium as evi-denced by transient GFP expression.Kanamycin at 100mg l -1in the culture medium was effective in sup-pression of non-transformed tissue and permitting the growth and development of transgenic cells,compared to 50or 75mg l -1,which permitted the proliferation of more non-transformed cells.Transgenic plants of ‘‘Alachua’’and ‘‘Carlos’’were recovered after secondary somatic embryo-genesis from primary SE explants co-cultivated with Agrobacterium .The presence and stable integration of transgenes in transgenic plants was confirmed by PCR andSouthern blot hybridization.Transgenic plants exhibited uniform GFP expression in cells of all plant tissues and organs including leaves,stems,roots,inflorescences and the embryo and endosperm of developing berries.Keywords Genetic engineering ÁGrapevine ÁSomatic embryos ÁTransgenic Abbreviations BA BenzyladenineGFP Green fluorescent protein NAA Napthyleneacetic acidIntroductionVitis rotundifolia Michx.the muscadine grape,is native to the southeastern United States where it is cultivated for fresh fruit,jelly,juice and wine production.It is an excellent source of health beneficial compounds (Percival and Sims 2003).Wine produced from V.rotundifolia contains 10–12times more resveratrol than that produced from V.vinifera L.(Ector et al.1996).Morphologically,V.rotundifolia differs significantly from V.vinifera (Olien 2001).The stem of V.rotundifolia has a smooth and thin bark that exfoliates in scales from older branches.Tendrils are unbranched.Berries are borne in small clusters of two to ten and possess a distinct fruity flavor.In contrast,the stem of V.vinifera has a rough bark that exfoliates in strips from older branches.Tendrils are forked and berries are borne in large clusters.V.rotundifolia possesses a greater degree of tolerance to pest and diseases and performs better in the hot and humid conditions of the southeastern US coastal plains (Galet and Norton 1990).Communicated by K.Kamo.S.A.Dhekney ÁZ.T.Li ÁM.Dutt ÁD.J.Gray (&)Mid-Florida Research and Education Center,University of Florida/IFAS,2725Binion Road,Apopka,FL 32703-8504,USA e-mail:djg@ufl.eduPresent Address:M.DuttCitrus Research and Education Center,University of Florida/IFAS,700Experiment Station Road,Lake Alfred,FL 33850,USAPlant Cell Rep (2008)27:865–872DOI 10.1007/s00299-008-0512-2Although conventional breeding has resulted in improved varieties of V.rotundifolia(Fry1967),they still possess undesirable berry characteristics such as thick skin, large number of seeds and poor storage life(Himelrick 2003).Transfer of certain traits,such as seedlessness,from V.vinifera to V.rotundifolia could lead to varieties with improved fruit qualities.However,the development of hybrids between V.vinifera(2n=38)and V.rotundifolia (2n=40)is hampered due to marginal sexual compati-bility between the two species,which is attributed primarily to the difference in chromosome number(Patel and Olmo1955).Low yield in F1hybrids of interspecific crosses greatly restricts the transfer of desirable traits between the two species.Crop improvement in V.rotundifolia might be achieved using genetic transformation to facilitate transfer of desirable traits,such as seedlessness,from V.vinifera (Colova-Tsolova et al.2003,2005).In addition,genetic transformation will allow identification and isolation of novel disease resistance genes from V.rotundifolia utiliz-ing reverse genetics as previously reported for Arabidopsis (Sessions et al.2002).Agrobacterium-mediated transformation is routinely used to produce transgenic plants from V.vinifera and a number of Vitis species and hybrids(reviewed by Gray et al.2005); however,to date,there have been no reports describing the procedure for V.rotundifolia.Somatic embryogenesis and plant regeneration was previously reported in V.rotundifolia (Colova-Tsolova et al.2007;Gray1992;Gray and Hanger 1993;Robacker1993),suggesting that embryogenic cultures might be used in genetic transformation as in earlier studies with V.vinifera(Li et al.2001,2006).We present here thefirst report of transgenic plant recovery from V.rotundifolia,which was achieved by Agrobacterium-mediated transformation of embryogenic cultures.We studied factors,including the developmental stage of embryogenic cultures amenable to transformation and kanamycin sensitivity of embryogenic cultures to recover transgenic plants from V.rotundifolia‘‘Alachua’’. Materials and methodsEmbryogenic culture initiation and maintenanceEmbryogenic culture induction medium consisted of Nitsch and Nitsch(1969)salts and vitamins,0.1g l-1 myo-inositol,20g l-1sucrose,9l M2,4-D and4.4l M BA.Medium pH was adjusted to5.5prior to the addition of 7.0g l-1TC agar(Phytotechnology laboratories,LLC, Shawnee Mission,KS,USA).The medium was autoclaved at120°C and 1.1kg cm2for20min,and then25ml medium was dispensed into each100915mm Petri dish and allowed to cool.In vitro shoot cultures of V.rotundi-folia‘‘Alachua’’were prepared as described by Gray and Benton(1991)for use as explants.Unopened in vitro grown leaves1.5–5.0mm long were placed abaxial surface down,on induction medium and incubated in dark at26°C for5–7weeks.Resulting callus cultures were transferred to basal MS medium lacking glycine and containing10.7l M NAA and0.9l M BA and maintained at26°C in light (65l m s-1m-1)for5weeks.Developing embryogenic cultures were transferred to growth regulator free X6 medium consisting of a modified MS medium lacking glycine and supplemented with 3.033g l-1KNO3and 0.364g l-1NH4Cl(as the sole nitrogen source),60.0g l-1 sucrose,1.0g l-1myo-inositol,7.0g l-1TC agar(Phyto-technology laboratories,LLC,Shawnee Mission,KS, USA),0.5g l-1activated charcoal and a pH value adjusted to5.8prior to autoclaving.Cultures were maintained by careful transfer of proliferating proembryonal masses (PEM)to fresh X6medium at4–6weeks interval.Proembryonal masses growing on X6medium were used to initiate suspension cultures as previously described for other Vitis species(Jayasankar et al.1999).The liquid medium was composed of B5major salts(Gamborg et al. 1968),MS minor salts and organic compounds,400mg l-1 glutamine,60g l-1sucrose and4.5l M2,4-D.The pH was adjusted to5.8prior to dispensing35ml into each 125ml Erlenmeyerflask,which was sealed with aluminum foil and autoclaved as before.Flasks were inoculated with 0.5g of PEM and several layers of Parafilm were used to seal the aluminum foil cover to theflask.Cultures were maintained in the light at26°C on a rotary shaker at 110rpm and subcultured every2weeks.Cultures were passed through a900l m sieve to separate the developing somatic embryos(SE)from thefine fraction of PEM. Sieving was carried out whenever necessary to remove developing SE and synchronize culture development.PEM were used for co-cultivation with Agrobacterium or trans-ferred to X6medium for SE development;these SE were also used in transformation experiments. Transformation vector and bacterial strainA bifunctional marker gene consisting of an in-frame translational fusion between an egfp gene and a neomycin phosphotransferase(nptII)gene(Li et al.2001)was placed under the control of a double Cassava Vein Mosaic Virus (CsVMV)promoter and cloned into the T-DNA region of a pBIN-19derived binary vector(Datla et al.1991).The binary vector(designated as pLC214)was introduced into A.tumefaciens strain EHA105by the freeze–thaw method (Burrow et al.1990).The Agrobacterium culture was grown overnight in MG/L medium(Garfinkle and Nester1980)at26°C on a rotary shaker (185rpm)containing 20mg l -1rifampicin and 100mg l -1kanamycin.Prior to transfor-mation,the bacterial cells were pelleted by centrifugation at 6,000rpm for 8min,resuspended in 25ml liquid X2medium (same as X6medium but with 20g l -1sucrose)without any antibiotics and cultured continuously under the same conditions for 4h.The bacterial culture (OD 600=0.8–1.0)then was used to inoculate embryogenic cultures.Transformation of embryogenic culturesCultures composed of either PEM (Fig.1a)or mid-cotyle-donary stage SE (Fig.1b)were co-cultivated with Agrobacterium to determine the earliest stage of develop-ment amenable to transformation.A typical mid-cotyledonary stage SE is shown in Fig.1b (inset).Wounding of cultures during co-cultivation was avoided to reduce subsequent post-co-cultivation necrosis.Cultures were submerged in bacterial solution for 8min and then blotted dry on filter paper to remove excess bacterial solution.They were subsequently placed in Petri dishes on two layers of filter paper soaked in liquid DM medium (Li et al.2001).Cultures were co-cultivated in the dark for 3days at 26°C.GFP expression in PEM and SE was recorded using a Leica MZFLIII stereomicroscope equipped for epi-fluorescence (Leica Microscopy System Ltd.,Heerbrugg,Switzerland).Percent transient GFP expression was recorded as the number of PEM or SE exhibiting GFP versus the total number of explants co-cultivated with Agrobacterium .A PEM or SE with ten or more GFP expressing cells was scored as positive for transient expression.Effect of kanamycin on growth and proliferation of embryogenic culturesIn order to optimize the selection of stably transformed callus and transgenic SE,kanamycin at three concentrations(50,75and 100mg l -1)was tested in DM medium (Li et al.2001).SE at the mid-cotyledonary stage were co-cultivated with Agrobacterium for 3days as described above.SE then were washed for 3days by transfer to 125ml Erlenmeyer flasks containing 30ml liquid DMcck 15medium (DM medium containing 200mg l -1each of carbenicillin and cefotaxime,and 15mg l -1kanamycin)on a rotary shaker at 110rpm.Washed SE were transferred to Petri dishes containing 25ml solidified DMcck 50,DMcck 75or DMcck 100medium (DM medium containing 200mg l -1each of carbenicillin and cefotaxime and either 50,75or 100mg l -1kanamycin).Thirty SE were placed in each Petri dish,which was replicated three times for each kanamycin concentration.Developing cultures were grown in the dark for 90days.Cultures then were trans-ferred to X6cck 50,X6cck 75or X6cck 100medium (X6medium containing 200mg l -1each of carbenicillin and cefotaxime and either 50,75or 100g l -1of kana-mycin)and subcultured at 30days intervals until secondary SE were observed.Experiments were repeated three times.Analysis of stable transgene expression at different kanamycin concentrationsStable transformation frequency was estimated based on the number of SE explants producing a GFP positive callus (transgenic callus line)versus the total number of co-cul-tivated primary SE explants.GFP positive SE that developed from a GFP positive callus were isolated and designated as an independent transgenic SE line.The number of transgenic callus lines were recorded after 60days on DMcck 50,DMcck 75and DMcck 100medium while the number of transgenic SE lines and non-trans-formed SE (SE which did not express GFP)were recorded after transfer to X6cck 50,X6cck 75and X6cck 100medium,respectively.Fig.1Embryogenic cultures of V.rotundifolia ‘‘Alachua’’used for transformation.aProembryonal masses (PEM)and b Somatic embryos (SE)at the mid-cotyledonary stage of development.Note A single mid-cotyledonary stage SE (inset ).Scale bars Fig.a ,b =10mm,Fig.b inset =0.5mm.Transgenic plant recoveryFive embryos from each independent transgenic SE line were transferred to germination medium,which consisted of MS salts lacking CaCl2and supplemented with709mg l-1 Ca(NO3)2Á4H2O,0.845mg l-1MnSO4ÁH2O, 1.0g l-1 myo-inositol,1.0l M BA and7g l-1T.C.agar.Cultures were placed for6weeks in light(65l mol s-1m-1)with a 16h photoperiod.Plants with a shoot and root system were acclimatized to ex-vitro conditions in a growth chamber and then transferred to a greenhouse.Tissues and organs from transgenic plants were observed for GFP expression and compared with non-transformed plants,which emitted chlorophyll induced red autofluorescence.Molecular analysis of transgenic plant linesTotal genomic DNA was isolated from young leaves of three transgenic plant lines and a non-transformed plant according to the method of Lodhi et al.(1994),but modified with increased EDTA(100mM)in extraction buffer.PCR was performed to detect specific DNA sequences in transgenic plants that corresponded to the egfp/nptII fusion gene.A forward primer EG-51(50-ATG GTGAGCAAGGGCGAGGAGCTGT-30)and a reverse primer EG-32(50-CTTGTACAGCTCGTCCATGCCG AGA-30)were used to amplify a717bp DNA fragment from the egfp/nptII fusion gene.Conditions for PCR reactions were:1cycle at95°C for4min,39cycles at 94°C for1min,58°C for1min,72°C for1min and a final cycle at72°C for4min.Plasmid DNA used in transformation served as a positive control while DNA from non-transformed plants was used as a negative control.Southern blot hybridization was performed as previ-ously described(Li et al.1997).Three microgram of DNA was digested with Hin dIII,separated by electro-phoresis in a0.7%(w/v)agarose gel,and transferred onto a nylon membrane(Schleicher and Schuell,Inc., Keene,NH,USA).A717bp DNA fragment corre-sponding to the egfp gene was labeled with digoxigenin and used as a probe for hybridization according to the manufacturer’s instructions(Roche Molecular Biochemi-cals,Mannheim,Germany).The hybridized membrane was washed,subjected to chemilluminescent development using CDP-Star substrate,and then exposed tofilm (Kodak Biomax Light1).A molecular marker was con-structed using egfp containing plasmid fragments of known sizes and used as a positive control while geno-mic DNA from a non-transformed plant served as a negative control.Statistical analysisExperimental results were analyzed using one way analysis of variance(ANOVA)and Student’s t-test to determine mean separation between treatments.ResultsProliferation of embryogenic culturesProembryonal masses proliferation occurred in liquid medium.Sieving led to synchronous development of cul-tures.However,maintenance in liquid medium beyond 12weeks led to browning of cultures,even with biweekly transfers and when cell density was lowered.Cultures could be maintained by transferring PEM to solid X6 medium,which also was used for development of SE.To date,embryogenic cultures grown on solid medium have been maintained for17months by transfer of small PEM to fresh medium every6weeks.Transformation of embryogenic cultures Proembryonal masses and SE were amenable to transfor-mation by Agrobacterium.Transient GFP expression occurred in PEM and SE3–5days after co-cultivation (Fig.2a),then gradually decreased in intensity.Fifty-eight percent of SE exhibited transient GFP expression(data not shown).Severe tissue necrosis occurred after co-cultivation as evidenced by culture browning.GFP-positive callus began to appear from co-cultivated explants after30days of culture on DMcck50,DMcck75and DMcck100 medium.Callus proliferation occurred for approximately 60days before transgenic PEM began to appear(Fig.2b). PEM gave rise to transgenic SE30days after transfer to X6cck50,X6cck75and X6cck100medium(Fig.2c).Effect of kanamycin concentration on growthand development of embryogenic culturesPreliminary experiments compared the effect of kanamycin levels on the number of stable transgenic SE lines recov-ered from PEM and SE explants.No differences in response were observed between PEM and SE;therefore only data for the later are presented(Table1).SE explants on DMcck50medium exhibited rapid growth of both transgenic and non-transgenic callus as judged by GFP expression;this resulted in a20%recovery of transgenic callus(Table1).Similarly62%of the secondary SEultimately recovered from this treatment were non-trans-genic.In contrast,kanamycin at 75and 100mg l -1suppressed growth of non-transgenic callus and SE while permitting the proliferation of transgenic cells.There were no significant differences between the number of trans-genic SE lines due to 75mg l -1(43.3%)or 100mg l -1kanamycin (39%).However,100mg l -1kanamycin resulted in significantly fewer non-transgenic SE (14.4%),compared to 75mg l -1(31.1%).Transgenic somatic embryo germination and plant developmentEighty-eight percent of transgenic SE germinated as evi-denced by development of pigmentation,enlargement of the hypocotyl,expansion of cotyledons and emergence of the radicle.Resulting plants appeared to be true to type (Fig.2d).A total of 21transgenic plant lines were obtained from 72embryo lines.Green fluorescence was observed from cells of all mature plant tissues and organs includingleaves (Fig.2e),stems,roots (data not shown),inflores-cences (Fig.2f),and anthers (Fig.2g).GFP expression was not observed in the exocarp or mesocarp tissues of devel-oping berries (Fig.2h).Developing seeds exhibited GFP expression in the endosperm and embryo (Fig.2h),but not the hardening seed coat (data not shown).Molecular analysis of transformantsGenomic DNA was extracted from three transgenic plant lines and used for amplification of the egfp gene.Ampli-fication of a 717bp fragment corresponding to the egfp gene following PCR was observed in all three transgenic lines (Fig.3,lanes 1–3)and the positive plasmid control (Fig.3,lane P),whereas no amplification was observed from the non-transformed plant line (Fig.3,lane 4).Genomic DNA from three randomly selected transgenic plant lines and a non-transformed plant was digested with restriction endonuclease Hin dIII and analyzed for trans-gene integration and copy number using SouthernblotFig.2Production of transgenic plants of V.rotundifolia ‘‘Alachua’’.a Transient GFP expression in SE explants4days after co-cultivation with Agrobacterium .b GFPexpressing transgenic callus and PEM 60days after co-cultivation.c Transgenicsecondary SE expressing GFP 90days after co-cultivation.d A true to type transgenic ‘‘Alachua’’plant in the greenhouse.Uniform GFP expression in mature leaf (e ),inflorescence (f )and anthers (g ).h GFP expression in developing berry.Note that the berryexocarp and mesocarp,which is devoid of GFP,emits red chlorophyll-inducedautofluorescence whereas the embryo and endosperm show uniform GFP expression.Scale bars :Fig.a ,f ,g ,h =0.5mm,Fig.b ,c ,e =5mmhybridization.The presence of a single Hin dIII restriction site upstream of the probe sequence within the T-DNA region of pLC214ensured that any hybridization fragments produced were due to a downstream Hin dIII restriction site in the plant genome and subsequently corresponded to the number of integrated T-DNA sequences.Digested genomic DNA samples along with the molecular marker were subjected to hybridization using a 717bp DNA fragment corresponding to the egfp gene.No hybridization was observed in the non-transformed control plant (Fig.4,lane 1).Transgenic plant AL214-1(Fig.4,lane 2)showed a single hybridization fragment (6.0kb)whereas AL214-3(Fig.4,lane 4)showed two hybridization fragments (6.0and 11.42kb),thereby indicating the insertion of one and two transgenes,respectively.The transgenic plant AL214-2(Fig.4,lane 3)produced three hybridization fragments (2.72,3.97and 6.0kb)indicating the insertion of multiple transgenes.Among these DNA fragments,ahybridization signal of greater intensity (corresponding to approx.five copies of the plasmid fragment)was observed in the 6.0kb fragment.DiscussionThis is the first report of genetic transformation and plant regeneration from V.rotundifolia .The egfp/nptII reporter-marker fusion gene facilitated study and optimization of the transformation process.Both PEM and SE were determined to be suitable targets for Agrobacterium infection as indi-cated by transient GFP expression.Proliferation of embryogenic cultures in V.rotundifolia occurs by division and fragmentation of PEM (Gray 1992),which give rise to SE (Gray and Mortensen 1987;Jayasankar et al.2003).Thus,transformation of PEM cells can result in formation of transgenic SE lines and subsequently plants.The use of suspension cultures facilitated rapid proliferation of embryogenic tissue,thereby generating a large amount of material for transformation experiments in a short time.PEM were used previously for genetic transformation of V.vinifera (Mauro et al.1995;Vidal et al.2003).Similarly,we have demonstrated that SE are suitable targets for transformation (Li et al.2001).Proliferation of grapevine SETable 1Effect of kanamycin concentration on growth and recovery of stably transformed V.rotundifolia ‘‘Alachua’’callus,SE lines and non-transformed SE after co-cultivation with Agrobacterium Kanamycin conc.(mg l -1)Transgenic callus lines (%)Transgenic SE lines (%)Non-transformed SE (%)5020.0c 16.6b 62.0a 7551.0a 43.3a 31.1b 10044.0b39.0a14.4cEach treatment consisted of 30SE explants in a Petri dish containingDMcck medium,which was replicated three times.Values represent mean percentage of observations from three replications for each treatment.Data were recorded 60days (transgenic callus)and 90days (transgenic and non-transformed SE)after co-cultivation.Statistical analysis was carried out using one-way ANOVA.Mean within columns followed by the same letter were not significantly different according to Student’s t test (P =0.05)Fig.4Southern hybridization analysis of genomic DNA isolated from leaves of a non-transformed plant and three randomly selected independent transgenic plant lines of V.rotundifolia ‘‘Alachua’’.Three micro gram genomic DNA from plants was digested with restriction enzyme Hin dIII and separated by electrophoresis on a 0.7%(w/v)agarose gel along with plasmid fragments as a molecular marker (M ).A film image showing varying size and copy numbers of integrated egfp transgenes from digested genomic DNA of a non-transformed plant (lane 1),transgenic plants AL214-1,AL214-2,AL214-3(lanes 2–4)and the control molecular marker (M).Fig.3PCR analysis of genomic DNA isolated from leaves of non-transformed and transgenic V.rotundifolia ‘‘Alachua’’plants.A 717bp DNA fragment corresponding to the egfp gene sequence was amplified from three randomly selected transgenic plants (lanes 1–3)and a control plasmid (P )but not from a non-transformed plant (lane 4)occurs via secondary embryogenesis with secondary embryos arising from single epidermal or sub-epidermal cells(Gray1995).Such cells represent suitable targets for transformation and plant recovery via the process of sec-ondary somatic embryogenesis in V.rotundifolia as well.The egfp gene permitted visual screening of cultures for identifying transformants while the nptII gene allowed for optimum concentrations of kanamycin to be determined. Kanamycin at50mg l-1was not effective since it allowed non-transformed callus and SE proliferation and resulted in recovery of relatively fewer transformants.In comparison, 100mg l-1kanamycin suppressed non-transformed tissue and allowed greater percentage of transformed SE and transgenic plants to be recovered.This kanamycin level is higher than that used by Vidal et al.(2003)(15mg l-1) and Li et al.(2006)(75mg l-1)to transform Vitis.Even though a few non-transformed SE developed at100mg l-1 kanamycin,they were easily identified by their lack of GFP emission.Thus,a combination of egfp along with nptII allowed for effective screening of embryogenic cultures and confidence in recovery of stable transformants.Only 21independently transformed plant lines were recovered from72transgenic SE lines.Low plant recovery from SE of V.rotundifolia was previously attributed to the frequent development of large fused cotyledons,which inhibited shoot development(Gray1992).The presence and stable integration of transgenes in the plant genome was confirmed by PCR and Southern blot hybridization.Among the transgenic plants analyzed, insertion of single and multiple copies of transgenes was observed.No visual difference in GFP expression was observed among transgenic plants with varying transgene copy number.Among the three hybridization fragments observed in transgenic plant line AL214-2,greater signal intensity was found to be associated with a6.0kb frag-ment.Since an equal amount of genomic DNA was loaded for all samples during electrophoresis(data not shown),the greater signal intensity observed may have resulted from the integration of transgenes at a single genomic locus as concatamers.The presence of such concatamers has been previously reported in studies using both microprojectile bombardment(Finer and McMullen1990;Hadi et al.1996) and Agrobacteriu m-mediated transformation(Wenck et al. 1997).A high level of GFP expression continued to be observed in this transgenic plant and its clones produced by vegetative propagation,2years after regeneration,thereby indicating no gene silencing due to multiple copy insertions as has been previously discussed(Muskens et al.2000).In addition to the quantitative results reported here for ‘‘Alachua’’,transgenic plants of V.rotundifolia‘‘Carlos’’were recovered as well.Studies are ongoing to extend transformation technology to additional cultivars and begin the testing of functional genes.Acknowledgments This research was supported by the Florida Agricultural Experiment Station and the Florida Department of Agri-culture and Consumer Services’Viticulture Trust Fund.D.J.Gray and Z.T.Li own stock in Florida Genetics LLC,which may license resulting patents and,as such,may benefitfinancially as a result of the outcome of research reported in this publication.ReferencesBurrow MD,Chlan CA,Sen P,Murai N(1990)High frequency generation of transgenic tobacco plants after modified leaf disk cocultivation with Agrobacterium tumefaciens.Plant Mol Biol Rep8:124–139Colova-Tsolova VM,Lu J,Perl A(2003)Cyto-embryological aspects of seedlessness in Vitis vinifera L.and exploiting recombinant DNA technology as an advanced approach for introducing seedlessness into Vinifera and Muscadine grapes.Acta Hortic 603:195–199Colova-Tsolova VM,Lu J,Perl A(2005)Genetically customized seedless grapes for fresh market industry.Acta Hortic689:481–484Colova-Tsolova VM,Bordallo PN,Phills BR,Bausher M(2007) Synchronized somatic embryo development in embryogenic suspensions of grapevine Muscadinia rotundifolia(Michx.) 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