Transformation of Montmorency sour cherry Gisela 6 cherry rootstock mediated by Agrobacterium

Plant Cell Rep(2006)25:117–123

DOI10.1007/s00299-005-0038-9

GENETIC TRANSFORMATION AND HYBRIDIZATION

Guo-Qing Song·K.C.Sink

Transformation of Montmorency sour cherry(Prunus cerasus L.) and Gisela6(P.cerasus×P.canescens)cherry rootstock mediated by Agrobacterium tumefaciens

Received:23March2005/Revised:25May2005/Accepted:9July2005/Published online:21December2005

C Springer-Verlag2005

Abstract Sour cherry(Prunus cerasus L.)scion cv.Mont-morency and rootstock cv.Gisela6(P.cerasus×P. canescens)were transformed using Agrobacterium tumefa-ciens strain EHA105:pBISN1carrying the neomycin phos-photransferase gene(npt II)and an intron interrupted?-glucuronidase(GUS)reporter gene(gus A).Whole leaf ex-plants were co-cultivated with A.tumefaciens,and selection and regeneration of transformed cells and shoots of both cultivars was carried out for12weeks on selection medium containing50mg l?1kanamycin(Km)and250mg l?1 timentin.These media were[Quoirin and Lepoivre(Acta Hortic78:437–442,1977)]supplemented with0.5mg l?1 benzylaminopurine(BA)+0.05mg l?1indole-3-butyric acid(IBA),and woody plant medium[Lloyd and McCown (Proc Int Plant Prop Soc30:421–427,1980)]containing 2.0mg l?1BA+1.0mg l?1IBA for cv.Montmorency and cv.Gisela6,respectively.Seven out of226(3.1%)explants of cv.Montmorency and?ve out of152(3.9%)explants of cv.Gisela6produced30/39GUS-and PCR-positive shoots from the cut midribs via an intermediate callus.Southern analysis of the GUS-and PCR-positive transformants con-?rmed stable integration of the transgenes with1–3copy numbers in the genomes of seven lines of cv.Montmorency and?ve of cv.Gisela6.The selected transformants have a normal phenotype in vitro.

Keywords Agrobacterium.Sour cherry(Prunus cerasus).Sweet cherry(Prunus avium).Transformation. Woody plant

Abbreviations AS:Acetosyringone.BA:Benzylamino purine.GUS:β-Glucuronidase.IBA:Indole-3-butyric acid.Km:Kanamycin.MS:Murashige and Skoog medium.NPTII:Neomycin phosphotransferase. Communicated by W.A.Parrott

G.-Q.Song·K.C.Sink( )

Department of Horticulture,Michigan State University,

Plant Transformation Center,East Lansing,MI48824,USA

e-mail:sink@https://www.sodocs.net/doc/2c2196666.html,

Tel.:+1-517-355-5191

Fax:+1-517-432-8825PCR:Polymerase chain reaction.TDZ:Thidiazuron. WPM:Lloyd and McCown woody plant medium Introduction

Sweet cherry(Prunus avium L.)and sour cherry(Prunus cerasus L.)are two important woody fruit crops belonging to the Cerasus subgenus within Prunus.Conventional ap-proaches for cherry breeding,such as hybridization,clone selection,and mutagenesis,are generally dif?cult and long-term processes due to heterozygosity,polyploidy,length of ?eld trials,and the interval between generations.Thus, transformation of cherries offers an attractive approach to complement these breeding methods by ef?ciently intro-ducing single or multiple desired traits such as improved fruit quality and resistance to insects,diseases,and/or her-bicides.

Several reports are devoted to the development of regen-eration systems for sour cherry(Mante et al.1989;Dolgov and Firsov1999;Tang et al.2000,2002),sweet cherry (Yang and Schmidt1992;Bhagwat and Lane2004),and black and wild cherries(Hammatt and Grant1998).How-ever,these regeneration systems do not appear suitable for stable transformation of these cherry cultivars.To date, no transgenic cherry plant with a normal phenotype has been obtained;however,three reports attempted to regener-ate transgenic cherry plants using different transformation methods(Druart et al.1998;Guti`e rrez-Pesce et al.1998; Dolgov and Firsov1999).Particle bombardment of meris-tems,Agrobacterium rhizogenes-mediated transformation of shoots,and Agrobacterium tumefaciens-mediated trans-formation of embryogenic calluses were among the strate-gies for genetic transformation of cherry plants despite the lack of molecular evidences(Druart et al.1998).Using A.rhizogenes,transgenic plants of the cherry rootstock cv.Colt(P.avium×P.pseudocerasus)containing the non-disarmed pRi1855T-DNA were obtained but showed abnormal phenotypes such as enhanced rooting capacity, shortened internodes,and wrinkled leaves(Guti`e rrez-Pesce et al.1998).When four A.tumefaciens strains were tested

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for transformation of a sour–sweet hybrid,3/58regenerants selected with5mg l?1hygromycin were GUS positive, but molecular studies of the regenerates were not reported (Dolgov and Firsov1999).Therefore,additional efforts appear warranted to achieve a successful stable transfor-mation protocol for cherry cultivars.

We previously devised an ef?cient shoot regeneration system using leaf explants from in vitro stocks of sour cherry cv.Montmorency(Song and Sink2005).The opti-mum tissue culture protocol was linked to transient gus A expression studies to determine the optimum A.tume-faciens strain,among EHA105,LBA4404,and GV3101 each with pBISN1,co-cultivation media and time,and use of acetosyringone(Song and Sink2005).This progress formed the basis of studies reported herein to obtain sta-ble,transgenic cherry plants.In this report,we describe the successful stable transformation of sour cherry scion cv. Montmorency and a dwar?ng cherry rootstock cv.Gisela6 (P.cerasus×P.canescens)using A.tumefaciens. Materials and methods

Plant materials and stock cultures

Ten1-year-old,shoot tips of sour cherry(P.cerasus L.)cv. Montmorency or cv.Gisela6(P.cerasus×P.canescens), each1–2cm in length,were cultured per85mm×100mm glass jar each containing100ml medium,capped with the bottom of a glass Petri dish and sealed with plastic wrap(Song and Sink2005).The stock culture medium for both cvs.was that of Quoirin and Lepoivre(1977)con-taining0.5mg l?1benzylaminopurine(BA),0.05mg l?1 indole-3-butyric acid(IBA),and2%(w/v)sucrose(herein QLBI).All solidi?ed media used in this study contained 0.6%(w/v)Bacto-agar(Becton,Dickinson and Co.,Sparks, MD).Media pH was adjusted to5.2before autoclaving (121?C,20min at105kPa)except as otherwise noted.Ace-tosyringone(AS)and all antibiotics were?lter-sterilized (0.22μm)and added to media cooled to50–60?C after autoclaving.

Stock cultures were maintained by subculture at6 week intervals on QLBI under a16h photoperiod of 40μmol m?2s?1from cool white?uorescent tubes at25?C. Uniform size leaves with midribs,1.5–2.0cm in length, were harvested from6-week-old stock cultures,each cut transversely and equidistant four times through the midrib, and used as explants.

Shoot regeneration

We previously devised an ef?cient,adventitious shoot re-generation system for cv.Montmorency via direct organo-genesis(Song and Sink2005),which was employed in this study.Brie?y,liquid Murashige and Skoog(1962)medium (MS)+0.1mg l?1thidiazuron(TDZ)+3%(w/v)su-crose was used as a24h pretreatment(herein MST). Shoot regeneration was carried out on Quoirin and Lep-oivre(1977)medium supplemented with3.0mg l?1BA, 0.5mg l?1naphthaleneacetic acid(NAA),and4%(w/v) sucrose(herein RM-M).

For shoot regeneration from leaf explants of the cv. Gisela6,the method established for cv.Montmorency was tested and found ineffective.Subsequently,TDZ(1.0or 2.0mg l?1)and BA(1.0or2.0mg l?1)were evaluated separately in woody plant medium(WPM:Lloyd and Mc-Cown,1980)supplemented with1.0mg l?1IBA and3% (w/v)sucrose for improvement of shoot induction.WPM supplemented with2.0mg l?1BA,1.0mg l?1IBA and 3%(w/v)sucrose(herein RM-G)was found effective and subsequently was used for transformation of cv.Gisla6. Using the above optimal regeneration media RM-M and RM-G,kanamycin(Km)(mg l?1:0,10,25,50,75and 100)+250mg l?1timentin were evaluated on shoot re-generation of cvs.Montmorency and Gisela6,respectively, to determine the concentrations required for selecting Km resistant shoots.

In all regeneration experiments,freshly prepared leaf ex-plants were incubated,abaxial side up,on these test media for1week in the dark,followed by culture at25?C under a16h photoperiod of40μmol m?2s?1from cool white ?uorescent tubes.Six Petri dishes,each with?ve explants, were used as replications for each treatment.Regeneration responses are described by percentage of leaf explants that regenerated shoots and the mean number of shoots per re-generating explant,documented8weeks after the start of each experiment.The experiments were repeated two times and arranged in completely randomized designs.Data were analyzed for signi?cance by AOV with mean separation by Duncan’s;PROCGENMOD(SAS8.2,SAS Institute,Cary, NC)was used.

Agrobacterium strain and binary vector

A.tumefaciens strain EHA105(Hood et al.1993),with the pBISN1plasmid(Ni et al.1995),was used.The binary vector pBISN1,a derivative of pBI101,con-tains the neomycin phosphotransferase gene(npt II)di-rected by the nos promoter and an intron interrupted ?-glucuronidase gene(gus A)driven by the chimeric super promoter(Aocs)3AmasPmas.The portable intron,cloned from potato ST-LS1,allows expression of GUS only in transformed plant cells and not bacterial cells(Narasimhulu et al.1996).EHA105:pBISN1was streaked and incubated on solid YEB medium(Vervliet et al.1975)supplemented with50mg l?1Km at28?C to obtain single colonies. Transformation

Single colonies of EHA105were cultured in10ml liquid YEB(Vervliet et al.1975)+50mg l?1Km at28?C in the dark for48h with rotary shaking(300rpm).The cells were collected by centrifugation,2min,2,500×g,and re-suspended to a?nal OD600of0.5in liquid co-cultivation media with100μM AS.Fresh leaf explants of cv.Gisla

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6and MST pretreated leaf explants of cv.Montmorency

were immersed in10ml bacterial suspension for30min

at28?C,blotted dry on sterile?lter paper,and transferred

onto a?lter paper disk laid abaxial side down on solidi?ed co-cultivation medium+100μM AS,and incubated in the

dark at25?C.Two co-cultivation media,MST and WPM +2.0mg l?1IBA+3%(w/v)sucrose(herein WPMI), were both tested for cv.Montmorency.Likewise,two co-

cultivation media,WPMI and RM-G,were tested for cv.

Gisla6.

After4days co-cultivation,leaf explants were washed two times(5min each)in liquid co-cultivation medium +500mg l?1timentin,rinsed two times(3min each) in the same medium without timentin to eliminate excess bacterial cells,and then blotted dry on sterile?lter paper. Subsequently,20such inoculated leaf explants were sub-jected to histochemical GUS assay.The other10per Petri dish were placed abaxial side up on Km selection RM-M or RM-G for cv.Montmorency and cv.Gisela6,respectively. Selection was carried out in the dark at25?C for2weeks. During this period,using forceps,the leaf explants were pressed gently to the agar medium surface every3days. Subsequently,the leaf explants were transferred onto fresh selection medium and cultured under a16h photoperiod of 40μmol m?2s?1.Subculture on fresh selection medium was performed at3week intervals.Sixty to one hundred explants were used for each treatment,and the experiments were repeated three times.The number of explants pro-ducing Km-resistant calluses and/or shoots was recorded after12weeks selection.Shoots from an individual explant of each cv.were excised and cultured separately on QLBI containing50mg l?1Km+250mg l?1timentin as50ml in each Magenta(Magenta Corp.,Chicago Ilinois)GA7. After6weeks the resistant shoots,2–3cm in length,were excised and inserted into50ml of WPM+1.0mg l?1 NAA+50mg l?1Km+250mg l?1timentin for root-ing in GA7’s.All regenerated plants were cultured under the same environmental conditions as used for the stock cultures.

Km-resistant regenerants that also had GUS-and PCR-positive leaves were considered putative transfor-mants.Transformation frequency was calculated as the percentage of inoculated leaf explants producing at least1 GUS-and PCR-positive transformant.

Histochemical GUS assay

Histochemical GUS assay of plant tissues was performed as described in Jefferson et al.(1987).Expression of GUS was monitored at selected steps:leaf explants inoculated with A.tumefaciens following co-cultivation,Km-resistant calluses,leaves excised from Km-resistant shoots,and rooted Km-resistant plantlets.All tissues were stained in 2mM X-Gluc(100mM phosphate buffer,100μM EDTA, and100mM Triton X-100)overnight at37?C.Chloro-phyll was removed from the tissues using70%ethanol rinses.Polymerase chain reaction(PCR)

and Southern analyses

Total genomic DNA was isolated using DNeasy Plant Maxi Kits(Qiagen,Valencia)from the leaves of GUS-and PCR-positive lines growing on QLBI+50mg l?1 Km+250mg l?1timentin.The primers corresponding to a377bp fragment of the code region of gus A were 5 -gatcctcgcattacccttacgc-3 and5 -gtgagcgtcgcagaacattac-3 ;and the reaction conditions included94?C for2min, 35cycles of94?C for10s,55?C for 1.5min and 72?C for2min,with a?nal10min extension at72?C. The primers for a600-bp fragment of the npt II coding region were5 -GAGGCTATTCGGCTATGACTG-3 and 5 -ATCGGGAGCGGCGATACCGTA-3 ,and the reaction conditions included94?C for2min,35cycles of94?C for30s,60?C for1.5min and72?C for3.5min,with a ?nal10min extension at72?C(Donaldson and Simmonds 2000).Ampli?ed products were separated on0.8% agarose gel containing ethidium bromide,visualized and photographed under UV light.

For Southern hybridization,DNA(20μg)was digested with Hin dIII,electrophoresed in0.8%agarose gel,and subsequently transferred to a N+-nylon membrane(Amer-sham,Arlington Heights,IL).A377-bp fragment contain-ing the gus A coding region was ampli?ed by PCR from plasmid pBISN1using the primers indicated above.The ampli?ed fragment was puri?ed using QIAquick gel extrac-tion kit(Qiagen,Valencia,CA).The377-bp fragment was then used as a probe after labeling with[32P]-dCTP using Random Primers DNA Labeling System(Invitrogen,Carls-bad,CA).Hybridization was carried out at56?C overnight. The membranes were washed in prewarmed buffers,twice with2×SSC+1%SDS and twice with0.1×SSC+0.5% SDS(30min per wash)at60?C.Membranes were exposed to X-ray?lm for3d at?80?C.

Results and discussion

Shoot regeneration of cv.Gisela6

Following the optimized regeneration protocols devised for cv.Montmorency(Song and Sink2005),only20–30%of leaf explants of cv.Gisela6produced shoots via direct shoot organogenesis from the cut edges of midribs.This regenera-tion frequency is much lower than that of cv.Montmorency. This supports previous reports that cherry plant regenera-tion is generally genotype-dependent(Mante et al.1989; Yang and Schmidt1992;Hammatt and Grant1998;Dolgov and Firsov1999;Tang et al.2000,2002;Bhagwat and Lane 2004;Song and Sink2005).To improve shoot regeneration for cv.Gisela6,WPM+2.0mg l?1BA+1.0mg l?1IBA (RM-G)was found to be more effective than TDZ in re-generating shoots(Table1).Under the optimal regeneration conditions,a few calluses developed from the cut edges in2 weeks,and then all shoot regeneration occurred via primary callus from the wounded midribs in8weeks(Fig.1).Our result is consistent with previous reports that the competent

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Table 1Regeneration of Gisela 6on WPM with

1.0mg l ?1IBA and different concentrations of BA or TDZ after 8weeks culture Plant growth regulator (mg l ?1)%Regenerated explants Mean number of shoots per regenerated explant BA TDZ 1.0036.7a ? 1.6a

2.0068.3b

3.5b 0 1.00.0c 0.0c 0

2.0

30.0a

2.1a

?

Letters in the same column denote signi?cant differences at p ≤0.05by Duncan’s

test

Fig.1Adventitious shoot regeneration from leaf explants of cv.Gisela 6after 8weeks culture.Bar:1cm

cells for shoot regeneration in leaf explants of cherries are located at the wounded midribs (Tang et al.2002;Bhagwat and Lane 2004;Song and Sink 2005).Thus,RM-G was subsequently selected for transformation of cv.Gisela 6.Transformation and recovery of regenerants

Km at 25mg l ?1had been previously tested to select the transformed cells of a sour–sweet cherry hybrid,although no transgenic plants were obtained (Dolgov and Firsov 1999).To determine the minimum Km concentration re-quired for selection of transformants under the regenera-tion conditions that we used,Km at six levels (mg l ?1:0,10,25,50,75and 100),each combined with 250mg l ?1timentin,was evaluated on shoot regeneration of uninoc-ulated leaf explants of cv.Montmorency and cv.Gisela 6.When Km was at or over 50mg l ?1,leaf explants of both cvs.died without shoot regeneration in 8weeks (Table 2).Thus,Km at 50mg l ?1was used to select transformed cells and tissues.

Using the transformation protocol described for cv.Montmorency,co-cultivation media were found to have an in?uence on production of Km-resistant regenerants (Table 3).WPMI yielded a higher frequency of regener-ation (3.1%)than MST (0.8%).During the ?rst 2weeks

of selection in the dark following co-cultivation on WPMI,most of the leaf explants placed on RM-M became twisted at the edges toward the agar,and tiny callus clusters were observed from the cut edges.After 12weeks selection,50.2%of the inoculated leaf explants yielded Km-resistant calluses from the cut edges.Intensive GUS-staining was de-tectable in presumed Km-resistant calluses (Fig.2a ).Seven out of 226(3.1%)inoculated leaf explants produced 16shoots,of which 12(75%)had GUS-positive tissues,via a primary callus stage from the wounded midribs.When MST was used for co-cultivation,15.1%of the inoculated leaf explants had Km-resistant calluses;2out of 251(0.8%)leaf explants produced GUS-positive regenerants from the wounded midribs via direct shoot regeneration on selection RM-M after 12weeks (Fig.2b ).Allowing cells to divide vigorously from the wounded midribs via a callusing stage on WPMI during co-cultivation is probably the main rea-son for the improvement in transformation frequency of cv.Montmorency.On the other hand,much higher production frequency (50.2%)of Km-resistant calluses versus lower production frequency (3.1%)of Km-resistant shoots indi-cates that lower shoot regeneration is the key factor for ef-?cient production of transgenic plant of cv.Montmorency.Leaf explants of the rootstock cv.Gisela 6were highly susceptible to A.tumefaciens EHA105under the conditions https://www.sodocs.net/doc/2c2196666.html,rge blue foci indicative of GUS expression were observed in the wounded midribs of >90%of leaf explants after 4days co-cultivation.Shoots were regener-ated from the wounded midribs via a primary callus stage on selection RM-G after 12weeks (Fig.2c ).Co-cultivation media showed no signi?cant in?uence on transformant fre-quencies.A total of 16shoots were produced,12of which had GUS-positive tissues (Table 3).

Regenerants of cv.Montmorency and cv.Gisela 6were excised and cultured on QLBI containing 50mg l ?1Km +250mg l ?1timentin for proliferation.All 30regenerants

Table 2Effect of Km on shoot regeneration from leaf explants of Montmorency and Gisela 6after 8weeks culture %Regenerated explants Km (mg l ?1

)053.3a ?40.0a 1030.0b 21.6b 2511.7c 11.7c 500.0d 0.0d 750.0d 0.0d 100

0.0d

0.0d

?

Letters in the same column denote signi?cant differences at p ≤0.05by Duncan’s test

121

Table 3

Summary of transformation experiments on Montmorency and Gisela 6

Co-cultivation medium Selection medium Infected explants Km-resistant shoots Shoots with

GUS-positive leaves Explants with GUS-and

PCR-positive regenerants no.(%)Montmorency MST a RM-M 251762(0.8)WPMI b RM-M 22616127(3.1)Gisela 6WPMI RM-G 152754(2.6)RM-G c

RM-G

152

9

7

5(3.3)

a

MST:MS +0.1mg l ?1TDZ b

WPMI:WPM +2.0mg l ?1IBA c

RM-G:WPM +2.0mg l ?1BA +1.0mg l ?1IBA

that had GUS-positive tissue survived and proliferated,whereas the other nine regenerants that had no GUS-positive tissues stopped growing and turned white after 6weeks (Table 3,Fig.2d ).Each of the regenerants that had GUS-positive tissues was considered a transgenic line.The escapes (9/39)were most likely caused by the poor contact of the inoculated leaf explants with the selection medium.On rooting medium containing 50mg l ?1Km +250mg l ?1timentin,transformants with GUS-positive leaves rooted in 4weeks.They were not morphologi-cally distinguishable from the non-transformed stock cul-tures (Fig.2e and f ).Expression of (Aocs)3AmasPmas –GUS was detectable in all tissues;being strongest in the roots,whereas no blue staining was observed in non-transformed plantlets (Fig.2e and f ).The expression pat-tern of (Aocs)3AmasPmas –GUS in the leaves of cherry plants is different from that observed in blueberry plants where a gradual decrease of blue staining was observed in leaves from the base toward the shoot tip (Song and Sink 2004

).

Fig.2a–f Histochemical GUS assays in transformed plant tis-sues of cv.Montmorency and cv.Gisela 6.a GUS expression in uninoculated (upper)and inoculated (lower)leaf explant tissues of cv.Montmorency after 8weeks culture on selection medium.b Re-covery of Km-resistant shoots of cv.Montmorency after 12weeks selection.c Recovery of Km-resistant shoots of cv.Gisela 6after 12weeks selection,arrows:adventitious shoot(s).d Regrowth of shoots regenerated from an inoculated leaf explant of cv.Gisela 6on QLBI

containing 50mg l ?1Km and 250mg l ?1timentin.(1)A shoot with GUS-negative leaves and (2)a shoot with GUS-positive leaves.e Rooted transgenic plants (1and 2)and non-transformed plant (N)of cv.Montmorency before (upper)and after (lower)GUS staining.f Rooted transgenic plants (1,2,and 3)and non-transformed plant (N)of cv.Gisela 6before (upper)and after (lower)GUS staining.Bars:1cm

122

Fig.3Southern analysis for the GUS reporter gene.a Linear map of the T-DNA region of plasmid pBISN1indicating the ap-proximate positions of the Hin dIII restriction sites (not to scale).b Blots of transgenic clones of cv.Montmorency (lanes 1–7)and cv.

Gisela 6(lanes 8–12).DNA samples were digested with Hin dIII.N1Non-transformed control of cv.Montmorency.N2Non-transformed control of cv.Gisela 6

Molecular analyses of putative transformants

All putative Km-resistant and GUS-positive transformants,18for cv.Montmorency and 12for cv.Gisela 6,were screened by PCR for npt II and gus A.The predicted frag-ments,600bp of npt II and 377bp of gus A,were detected in all transformants and were absent in non-transformed plants (data not shown).

Fourteen Hin dIII-digested genomic DNA samples were hybridized to the 377bp probe derived from an internal coding sequence of gus A.One or more hybridization bands was detected in each of the transgenic lines but was absent in non-transformed controls (Fig.3),indicating stable in-tegration of gus A.Among the ?ve transgenic lines of cv.Gisela 6,one line had three bands and four lines each had a single band with three different patterns.Among the seven transgenic lines of cv.Montmorency,two lines each had two bands with different patterns and ?ve lines each had single bands with four patterns.Hin dIII cuts outside of the gus A and an uncertain site in the genomic DNA.Therefore,the differences in the patterns of the hybridization bands most likely represent independent transformation events as well as random integration of gus A.

In conclusion,our studies demonstrate the potential for obtaining stable transformed cherry plants with a normal phenotype using the A.tumefaciens -mediated transfor-mation method.Our transformation protocols can be summarized:(1)Leaf explants excised from in vitro stock cultures are cut transversely and equidistant four times through the midrib,and inoculated with EHA105directly (cv.Gisela 6)or after a TDZ pretreatment (cv.Mont-morency);(2)Co-cultivation is carried out for 4days in the dark.For cvs.like Gisela 6where regeneration usually occurs via a primary callus stage,the optimal co-cultivation medium can be just the same as their regeneration medium;

for cvs.like Montmorency that have a direct shoot regener-ation system,a callus induction medium may be preferable to regeneration medium as the co-cultivation medium;(3)Selection and regeneration of transformed cells is con-ducted on regeneration media containing 50mg l ?1Km and 250mg l ?1timentin;and (4)Regrowth and proliferation of regenerants is carried out on QLBI +50mg l ?1Km +250mg l ?1timentin.Following this protocol,stably trans-formed plants of sour cherry cv.Montmorency and cherry rootstock cv.Gisela 6are obtained after 12weeks.These transformation protocols,as mediated by A.tumefaciens ,may be feasible to transform other cherry cultivars.

Acknowledgments We thank Dr.S.Gelvin,Purdue University,for providing plasmid pBISN1.This research was supported by MSU Project GREEEN (Generating Research and Extension to Meet Eco-nomic and Environmental Needs).

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