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ORIGINAL PAPERAgrobacterium -mediated transformation of the winter jujube (Zizyphus jujuba Mill.)X.F.Gu ÆH.Meng ÆG.Qi ÆJ.R.ZhangReceived:16October 2007/Accepted:9April 2008/Published online:14May 2008ÓSpringer Science+Business Media B.V.2008Abstract Winter jujube,a species that originated in China,is the most prominent elite variety of jujube (Zizyphus jujuba Mill.).Due to its economic value and its recalcitrance to improvements through traditional plant breeding approaches,genetic transformation techniques may have a great potential in providing the means to transfer one or more selected desirable traits into the plant genome.We reported here an improved protocol for the Agrobacterium-mediated transforma-tion of shoot tips of winter jujube.We have identified a set of optimum transformation conditions that take into account Agrobacterium inoculum density,Agrobacterium incubation period,co-cultivation con-ditions,and vacuum (use of a vacuum pump to create a negative-pressure environment).The highest trans-formation frequency (5.2%)was obtained when the shoot-tip explants were infected for 10min and co-cultured for 4days with Agrobacterium at OD 6000.8under a negative pressure of 0.59105Pa.PCR and southern blot analyses confirmed the presence of transgenic plants and the stable integration of the target gene into the genome of regenerated plants.A histochemical staining analysis for GUS activity in the transgenic shoot tips also validated the efficiency of the transformation system.Keywords Winter jujube (Zizyphus jujuba Mill.)ÁGenetic transformation ÁShoot tip ÁNegative pressureAbbreviations BA 6-BenzylaminopurineCTAB Cetyltrimethyl ammonium bromide GA 3Gibberellic acid GUS Glucuronidase IAA Indoleacetic acid IBA Indolebutyric acidIntroductionWinter jujube,a species that originated in China,is the most prominent elite variety of jujube (Zizyphus jujuba Mill.)(Xu et al.2003).The fresh fruit of this species is highly valued among Asian consumers for its unique,delicious flavor,tenderness,juiciness,and high nutritional value.These traits have made it an economically valuable crop in Asia,especially in China (Li et al.1999).However,a key limitation to the commercial development of this crop is its relatively short-shelf-life:it is difficult maintain fresh storage quality at room temperature.(Wang et al.1999).The jujube tree and fruit are also susceptible to many disease and pests (Gao 2006).To date,winter jujube has proven relcalcitrant to improvement byX.F.Gu ÁH.Meng ÁG.Qi ÁJ.R.Zhang (&)School of Life Science,Shandong University,27Shanda Nanlu,Jinan 250100,People’s Republic of China e-mail:jrzhang@Plant Cell Tiss Organ Cult (2008)94:23–32DOI 10.1007/s11240-008-9383-zbreeding due toflower abscission,embryo abortion, and a long juvenile period(Liu et al.2004;Li et al. 2007).Genetic transformation tools,however,are not bound by the usual limitations of traditional plant breeding and,as such,provide the means to transfer one or more selected traits to winter jujube,thereby improving the commercial exploitation of this fruit. Recent studies have implicated abscissic acid(ABA) as a major player in regulating fruit ripening and senescence in plant(Cutler and Krochko1999;Sala and Lafuente2002).This suggests that the genetic modification of the endogenesis ABA biosynthetic pathway in winter jujube could result in delayed fruit ripening and increased storage and shelf-life.In addition,the introduction of genes encoding virus-resistant proteins,such as the b-1.3-glucanase gene and chitinase gene,into winter jujube would improve the plant’s resistance against fungal diseases(Li et al. 2003).Efficient and reliable in vitro regeneration and gene transformation systems are the two main requirements for the successful genetic transformation of plants.In the past,many woody species have appeared to be recalcitrant to genetic manipulation because of the difficulty in obtaining alternative explants in vitro as targets for transformation (Li et al.2007).Winter jujube has also resisted the establishment of an efficient in vitro system and, consequently,genetic modification of this plant is still a relatively inefficient procedure and has met with no large-scale success.He et al.(2003,2004)reported the transformation of in vitro internode and embryo stem explants of three jujube genotypes,but they did not provide sufficient details and the transformation efficiency was low.In an earlier publication,we reported the establishment of an efficient in vitro culture system of winter jujube for regenerating shoot tips(Gu and Zhang2005).Shoot tips were capable of regenerating cells of the shoot apical meristem that could serve as targets for genetic transformation(Dutt et al.2007).The currently preferred method for plant transformation is an Agrobacterium-mediated transformation system due to single-or low-copy integration and a high conversion efficiency–with up to85%transgenic plants being obtained in some systems(Dai et al.2001;Shou et al.2004).Here,we report on the establishment of a repro-ducible Agrobacterium-mediated transformation procedure using shoot tips of winter jujube.Various parameters of the system,including Agrobacterium inoculum density,Agrobacterium incubation period, co-cultivation conditions,and vacuum,were evalu-ated.The PCR and southern blot methods were used to confirm the development of transgenic plants. Materials and methodsPlant materials and establishment of shoot cultureMicropropagation cultures of Zhanhua winter jujube (Zizyphus jujuba Mill.)were established from dor-mant buds(Gu and Zhang2005).The dormant buds were obtained from50to55-year-old elite plants growing at the Zhanhua winter jujube institute (Zhanhua,Bingzhou,China).Regenerated shoots were cultured on shoot growth medium comprising MS medium(Murashige and Skoog1962)supple-mented with 5.77l M gibberellic acid(GA3)and 0.89l M benzylaminopurine(BA).The shoots were cultivated with30day sub-culture intervals at 25±1°C under a14/10h(light/dark)photoperiod with light supplied by cool-whitefluorescent lamps at an intensity of60l mol m-2s-1.The in vitro-grown shoot tips,approximately5–6mm in length,were then pre-cultured on shoot growth medium for7days before transformation.Sensitivity of shoot tips of winter jujubeto herbicidePrior to genetic transformation,the amount of the herbicide‘chlorsulfuron’(25%by weight in Lu¨hu-anglong;Shenyang Agricultural Chemical Company, China)required to inhibit shoot tip growth was tested. The non-transformed shoot tips were placed on shoot growth medium supplemented with0,0.125,0.25, 0.375,0.5,0.625,or0.75mg/l chlorsulfuron and subcultured three times at7day intervals.The survival rates of the explants were evaluated.The concentration of chlorsulfuron that killed most of the plants was used in subsequent transformation experiments.Agrobacterium strain and plasmidAgrobacterium strain LBA4404,which harbors plas-mid pCAMBIA1300with the betA and als genescontrolled by the cauliflower mosaic virus(CaMV) 35S promoter and terminator sequences,was used as the vector system for transformation.The als gene was cloned from Arabidopsis thaliana and confers resis-tance to the herbicide chlorsulfuron;this gene is the 197N mutation in the ALS gene encoding the enzyme acetolactate synthase,which is targeted by herbicides. The betA gene from Escherichia coli encodes choline dehydrogenase,a key enzyme in the biosynthesis of glycine betaine from choline(Fig.1).Agrobacterium cells were cultured overnight at28°C with shaking (180rpm)in YEP medium(yeast extract10g l-1, peptone10g l-1,sodium chloride5g l-1,pH7.0) supplemented with50mg l-1rifamycin and 50mg l-1kanamycin.The cells were pelleted at 1,500g for5min and resuspended in liquid MS medium with100l M acetosyringone(AS). Transformation of shoot tips with Agrobacterium Following the removal of two or three young leaves from the pre-cultured shoot tips to expose the meristem along with a few microscopic leaf primor-dia,a thin needle was used to nick the explants in the meristem tissue near the apical region.The wounded explants were then immersed in the Agrobacterium suspension for10min.The OD600was adjusted from 0.2to1.2before inoculation.To evaluate the effect of different inoculation durations,we varied the time in which the wounded explants were exposed to the Agrobacterium suspension(range:5–20min)and created negative pressure(0.59105Pa)using a vacuum pump for different infection durations(5,10, 15,and20min)at an OD6000.8Agrobacterium solution.Following inoculation,shoot tips explants were blotted dry on sterilefiler paper to remove excess Agrobacterium and then transferred to fresh shoot growth medium for2–6days co-cultivation at 25°C in the dark.After co-cultivation,the explants werefirst transferred to fresh shoot growth medium containing200mg l-1cefotaxime for7days to inhibit the growth of the Agrobacterium and then transferred onto the selection medium(shoot growth medium supplemented with0.5mg l-1chlorsulfuron)for21days at7day subculture intervals to stimulate the production of transgenic shoots.All the above processes were carried out in darkness.Surviving chlorsulfuron-resistant shoot tips were transferred onto shoot growth medium and allowed to proliferate for4weeks.The surviving shoots were rooted and transferred to soil as described by Gu and Zhang (2005).Histochemical staining for b-glucuronidaseactivityThe pCAMBIA1301plasmid contained the histo-chemical reporter gene b-glucuronidase(GUS)in which an intron sequence were inserted into the coding sequence,and the als gene driven by the CaMV35S promoter and terminator sequences were used to confirm the efficiency of the transformation system.Shoots regenerated from wild-type and infected explants were cut into segments and incu-bated in a GUS-staining solution at37°C overnight as described by Jefferson et al.(1987)and Niu et al. (2000).The plant tissues were gradually destained in 70%ethanol to remove the chlorophylls and other pigments prior to visual analysis.The results of GUS expression were documented by digital photography using an Olympus light microscope(BX51)equipped with an Olympus C500camera.PCR and southern blot analysisA CTAB protocol was used to isolate genomic DNA from young leaves removed from surviving plants that had developed roots and been grown in the greenhouse for30day(Permingeat et al.1998).The PCR amplification of the betA and als genes were carried out using als gene primer als1(50GAG GAC ACG CTG AAA TCA CC30)and als2(50GCA TCA GGG TTA GCA ACA G30),and betA gene primer bet1(CGC TAC AGG GTA AAC GCT ACA AC) and bet2(CCT CAC GGC TGC GAA TAA ATC C), respectively.The primers non-t1(50AAG CCA CTT ACT TTG CCA TCT30)and non-t2(50TTT GCT CGG AAG AGT ATG AAG30)flanking the non-T-DNA sequence of the T-DNA right border sequence of pCAMBIA1300-bet A-als were designed to determine whether there was any contamination of Agrobacterium in the transgenic plants,as verified by PCR.The PCR amplification was carried out ina Fig.1T-DNA structure of pCAMBIA1300-betA-als25l l reaction volume containing12.5ng of plasmid (used as positive control DNA)or125ng of plant DNA with12.5l M of each of the primer,5l M dNTP’s,and0.67U of Taq polymerase in19PCR buffer with25mM of MgCl2.The amplification cycling conditions consisted of one cycle at95°C for 5min,followed by35cycles(amplification at95°C for60s,56°C for60s,and72°C for60s),with a final7min extension at72°C.Southern blotting was performed to confirm the stable integration of the foreign gene in the transgenic plants.A20l g aliquot of genomic DNA was digested with EcoR I and separated by electrophoresis on a TBE-buffered0.8%(w/v)agarose gel;the excised fragments were then transferred to a Hy-bond–N+nylon membrane(Roche,Mannheim, Germany).The PCR-amplified betA gene was labeled with DIG-dUTP using a DIG-High Prime DNA Labeling and Detection kit(Roche,Mannheim, Germany).Hybridization was carried out at65°C, and immunological detection steps were performed using the DIG Nucleic Acid Detection kit according to the manufacturer’s instructions(Roche). Experimental design and data analysisEach experiment was repeated three times with at least100shoot tips for each herbicide treatment and two times for the transformation system experiment. The transformation frequency was calculated as the total number of transgenic plantlets produced relative to the total number of explants infected by Agrobac-terium.The data were analyzed using SAS version 6.12(SAS Institute,Cary,NC).Analysis of variance (ANOVA)was used to test the statistical significance, and the significance of differences among means was carried out using Duncan’s(1955)multiple range test at a significance of P=0.05.Results and discussionSensitivity of shoot-tip explants to different concentrations of the herbicide chlorsulfuronThe effects of various concentrations of the herbicide chlorsulfuron were evaluated on shoot-tip explants to determine the appropriate selection dose.Our analysis of the data revealed that fewer than20%the shoot tips were able to survive in the presence of0.5mg l-1 chlorsulfuron and that no shoots survived and no transgenic plants were obtained in the presence of higher concentrations of chlorsulfuron(data not shown).Therefore,0.5mg l-1chlorsulfuron was used in subsequent transformation experiments.Influence of Agrobacterium cell density, inoculation period and co-cultivation period,and vacuum condition on transgenic frequencyA critical factor in shoot-tip transformation systems is the density of the Agrobacterium inoculum in the inoculation medium(Li et al.2007).We obtained the best transformation frequency(3.2%)using Agro-bacterium inoculum at OD6000.8(Table1).A reduction in the mean transformation frequency was observed following inoculation with higher densities of Agrobacterium cells,possibly due to increased damage and increased production of poisons to the receptor cells.Bacterium densities of0.08–1.2have often been used in plant genetic transformation systems.Humara et al.(1999)co-cultured cotyledon explants of Pinus with Agrobacterium cells at a density of1(OD600),while Yang et al.(2005)and Miguel and Oliveira(1999)reported that the use of Table1Effect of Agrobacterium concentration(OD600value) on the transformation frequency of shoot-tip explants of winter jujube aOD600valueNumber of shoottips evaluated bNumber oftransgenicplantsTransformationfrequency(%)c0.228320.7d0.43104 1.3d0.62556 2.4ab0.82217 3.2a1.02394 1.7bc1.22643 1.1cda Shoot-tip explants were inoculated with LBA4404during an infection exposure time of10min and then co-cultured for 4daysb Each treatment consisted of two repeat experiments with at least100replicates in each experimentc Means within a column followed by the same letter are not significantly different,as indicated by Duncan’s multiple range test(P=0.05).Transformation frequency was defined as: (number of transgenic plants/total number of explants evaluated)9100%OD6000.3and0.5Agrobacterium density produced the optimal transformation rate in sugar beet and almond,respectively.As a general rule,Yu et al. (2002)found that the transformation frequency increased as the inoculum OD value(0.08–0.6) decreased in their system using hypocotyls of sweet orange and citrange as explants.It has been deter-mined that the density of Agrobacterium inoculum resulting in the highest transformation frequency is genotype-dependent.The duration of the exposure interval to Agrobac-terium cells also influences the transformation frequency of explants.Winter jujube shoot tip explants incubated for10min with Agrobacterium cells at density OD600=0.8showed a significantly increased frequency of transformation than those transformed for5min,while exposure to Agrobac-terium for more than15min resulted in a decline in transformation frequency(Table2).The exposure of the explants to Agrobacterium from1to30min as this range has been previously shown to influence transformation frequency in wood plant transforma-tion(Costa et al.2002;Yu et al.2002;Chen et al. 2006).Costa et al.(2002)selected long infection exposure times of as much as20min for the transformation of grape fruit epicotyl explants, whereas the exposure times of longer than10min decreased the transformation efficiency in Washing-ton navel orange(Bond and Roose1998).Our results are similar to those obtained in the Washington navel orange transformation system–explants of winter jujube did not show increased transformation fre-quency with an extended exposure time to Agrobacterium.Using the optimal conditions described above,the effect of varying the length of the co-cultivation period was investigated.Table3shows that the transformation frequency increased from 1.8%at 2days to3.2%at4days;however,extending the co-cultivation period to longer than4days resulted in an abundant proliferation of Agrobacterium and tissue necrosis and subsequent cell death.An increased transformation frequency has been found to be positively correlated with increases in the length of the co-cultivation up to a period of3–5days in some species(Niu et al.2000;Costa et al.2002;Kim et al. 2004).Yang et al.(2005)reported that the application of a vacuum during the transformation of sugar beet enhanced the transformation frequency.In our sys-tem,a negative pressure of0.59105Pa,created by the vacuum pump in the vacuum desiccator,resulted in a1.63-fold increase(5.2%)in the transformation frequency in comparison to that obtained at atmo-spheric pressure(Table4).It has been suggested that a vacuum pump creates a negative-pressure environ-ment that results in an increase in effective Agrobacterium volatilization,a condition conducive to the transfer of a foreign gene into plant cells.Table2Effect of the length of the exposure time to Agro-bacterium inoculum on the transformation frequency of shoot-tip explants of winter jujube aInoculation time(min)Number ofshoot tipsevaluated bNumber oftransgenicplantsTransformationfrequency(%)c53254 1.2b102217 3.2a152315 2.2ab202594 1.5ba Shoot-tip explants were inoculated with LBA4404at OD0.8 and co-cultured for4daysb Each treatment consisted of two repeat experiments with at least100replicates in each experimentc Means within a column followed by the same letter are not significantly different,as indicated by Duncan’s multiple range test(P=0.05).Transformation frequency was defined as: (number of transgenic plants/total number of explants evaluated)9100%Table3Effect of the length of the co-cultivation period on the transformation frequency of shoot-tip explants of winter jujube aDuration ofco-cultivationperiod(days)Number ofshoot tipsevaluated bNumber oftransgenicplantsTransformationfrequency(%)c22715 1.8a42217 3.2a62305 2.2aa Shoot-tip explants were inoculated with LBA4404at a density of OD0.8during an infection exposure time of10minb Each treatment consisted of two repeat experiments with at least100replicates in each experimentc Means within a column followed by the same letter are not significantly different,as indicated by Duncan’s multiple range test(P=0.05).Transformation frequency was defined as: (number of transgenic plants/total number of explants evaluated)9100%Histochemical staining of GUS activityIn order to confirm the efficiency of our transforma-tion system,we used the pCAMBIA1301plasmid containing the GUS reporter gene to transform winter jujube in our optimal Agrobacterium-mediated trans-formation system.The results of the GUS histochemical assays indicated that the GUS gene was expressed in the apical meristems of shoot tips (Fig.2a).Histochemical staining of GUS activity revealed that at least50%of the infected shoot tips were GUS positive after co-cultivation.A detailed histochemical staining for GUS activity was con-ducted to observation in vascular tissues(Fig.2b,c). Shoot tips from a transgenic plant showed strong GUS activity,while that from a wild-type winter jujube had no detectable GUS activity(Fig.2d). Regeneration of stably transformed plantsof winter jujube following Agrobacterium-mediated transformationUsing our optimal transformation procedure,we immersed the shoot tips in the Agrobacterium suspension(OD6000.8)for10min under the vacuum treatment,then co-cultured the shoot tips for4days on shoot growth medium(Fig.3a,b).The shoot tips that survived on selection medium were transferred onto Nitsch basal medium supplemented with 1.14l M indole-3-acetic acid(IAA)and 2.46l M indole-butyric acid(IBA)to induce root formation (Fig.3c,d).The rooted plants were then transplanted into plastic pots containing autoclaved vermiculite and soil(1/1,v/v)(Fig.3e)and the pots covered with plasticfilm and placed in a greenhouse maintained at 25/20°C(day/night,12/12h).The plants were irri-gated with a solution of1/10-strength MS inorganic salts at2–3day intervals and subsequently potted in soil for further growth(Fig.3f).PCR analyses were performed using als and betA gene-specific primer and genomic DNA isolated from the herbicide chlorsulfuron-resistant plants and wild-type plants.The als and betA gene fragments were amplified from chlorsulfuron-resistant plants (Figs.4,5).However,due to the possible presence of the Ti-plasmid DNA in the plant genomic DNA extracts,a technique involving only gene primers cannot be used with100%certainty to verify the presence of transgenic plants.The primers non-t1and non-t2located outside of the T-DNA region of pCAMBIA1300were therefore used to analyze the transgenic plants that had been identified by the gene-specific primer.The positive control DNA produced an amplified328-bp plasmid fragment,and some genomic DNA samples of transgenic plant did not amplify the specific target fragment(Fig.6:lanes 1–5,8).These results demonstrate that the PCR method used to analyze the transgenic winter jujube plants appears to be effective.Southern hybridization confirmed stable integration of the target gene intoTable4Effect of negative pressure on the transformation frequency of shoot-tip explants of winter jujube aInoculation time Number of shoottips evaluated b Number oftransgenic plantsTransformationfrequency(%)cNormal pressure(1.09105Pa)53254 1.2b102217 3.2a152315 2.2ab202594 1.5b Negative pressure(0.59105Pa)52395 2.1z1024913 5.2x1526011 4.2y202454 1.6za Shoot tips explants were inoculated with LBA4404at a density of OD0.8and co-cultured for4daysb Each treatment consisted of two repeat experiments with at least100replicates in each experimentc Means within a column in the normal pressure treatment or negative pressure treatment followed by the same letter are not significantly different,as indicated by Duncan’s multiple range test(P=0.05).Transformation frequency was defined as:(number of transgenic plants/total number of explants evaluated)9100%.Values in italics are significantly different in the same transformation procedure under atmospheric or negative pressure,as indicated by Duncan’s multiple range test(P=0.05)the winter jujube genome using the DIG-labeled betA gene probe.Bands of the betA gene were observed in the transgenic plants,whereas no hybridization fragments were visualized in the wild-type plants (Fig.7),indicating the stable insertion of target gene into the winter jujube genome.Woody species have often been found to be recalcitrant to the establishment of an efficient system for regenerating plantlets.Thus,the development of efficient and reliable plant transformation systems for woody plants requires the appropriate explants–for example:embryogenic calluses of Ponkan manda-rin (Li et al.2002),epicotyl segments of citrus (Ballester et al.2007),internodal stems of citrus(Gutie´rrez-E et al.1997),and shoots of cherry (Gutie`rrez-Pesce et al.1998).Winter jujubeis Fig.2Histochemicalstaining of b -glucuronidase (GUS)activity in a transgenic winter jujube plant.(a )Histochemical staining of GUS activity of the whole shoot tips of a non-transgenic (left )and transgenic plant (right )following a 4dayco-culture in MS medium containing 5.77l M gibberellic acid (GA 3)and 0.89l Mbenzylaminopurine (BA).Bar :0.4cm.(b )Histochemical staining of GUS activity of shoot tips of transgenic winter jujube plants following a 4day co-culture in MS medium containing 5.77l M GA 3and 0.89l M BA.Bar :0.3mm.(c )Histochemical staining of GUS activity of the central infected-cells of shoot tips of transgenic winter jujube plants following selection for 7days in MS medium containing 5.77l M GA 3,0.89l M BA,and 2mg l -1herbicide.Bar :0.3mm.(d )Histochemical staining of GUS activity of the shoot-tip section of a non-transgenic (left )and transgenic plant (right ).Bar :0.1cmparticularly difficult to culture in vitro.Chen et al.(2002)encountered major difficulties in regenerating plantlets from callus of winter jujube,and only a few plantlets differentiated from the callus.This lack of a system by which to produce appropriate explantscurrently limits the transformation of winter jujube.Shoot apical meristems obtained through in vitro micropropagation may be a represent alternative as the target for transformation (Dutt et al.2007).Similar beneficial results have been reported in sugar beet (Yang et al.2005),maize (Zhang et al.2005),and cotton (Lv et al.2004).The system of transfor-mation in winter jujube using shoot tips that is presented here opens the door to the genetic regula-tion of ABA biosynthesis and disease resistance in winter jujube.In conclusion,we have improved the transforma-tion protocol for shoot tips of winter jujube using an Agrobacterium -mediated method.The highest trans-formation frequency (5.2%)was obtained when the shoot tips were infected for 10min at an Agrobac-terium inoculum density of OD 6000.8underaFig.3The procedure used to obtain transformed winter jujube plants.(a )Pre-cultured shoot tips of winter jujube.Bar :1cm.(b )Shoot tips infected with Agrobacterium .Bar :1cm.(c )Selected putative transformed plantlets.Bar:1cm.(d )Rootedputative transformed plants.Bar :1.6cm.(e )Putative trans-formed plants at the soiled pot.Bar :2.4cm.(f )Transgenic plants at the soiled pot.Bar :12cmFig.4Identification of transgenic winter jujube plants based on PCR detection of the als nes 1–7:transgenic winter ne 8:wild-type winter jujube as negative ne 9:pCAMBIA1300-betA -als as positive ne M:molecular weight marks ofDL2000Fig.5Identification of transgenic winter jujube plants based on PCR detection of the betA gen nes:1–7transgenic winter jujube .Lane 8:wild-type winter jujube as negative ne 9:pCAMBIA1300-betA -als as positive ne M:molecular weight marks ofDL2000Fig.6PCR amplifications of a non-TDNA fragment to determine whether there was contamination of Agrobacterium or nes 1–7:putative transgenic winter ne 8:wild-type winter jujube as negative ne 9:pCAM-BIA1300-betA -als plasmid as positive ne M:molecular weight marks of DL2000negative pressure of 0.59105Pa,followed by co-cultivation for 4days.The PCR and southern blot analyses confirmed the insertion of the foreign gene in the winter jujube genome.Acknowledgments This research was supported by the National Natural Science Foundation of China (30300242)and the project of Department of Science and Technology of Shandong.ReferencesBallester A,Cervera M,Pena L (2007)Efficient production oftransgenic citrus plants using isopentenyl transferase positive selection and removal of the marker gene by site-specific recombination.Plant Cell Rep 26:39–45Bond JE,Roose ML (1998)Agrobacterium -mediated trans-formation of the commercially important citrus cultivar Washington navel orange.Plant Cell Rep 18:229–234Chen YQ,Lu LT,Deng W,Yang XY,McAvoy R,Zhao DG,Pei Y,Luo KM,Duan H,Smith W,Thammina C,Zheng XL,Ellis D,Li Y (2006)In vitro regeneration and Agrobacterium -mediated genetic transformation of Euonymus alatus .Plant Cell Rep 25:1043–1051Costa MGC,Otoni WC,Moore GA (2002)An evaluationof factors affecting the efficiency of Agrobacterium -mediated transformation of Citrus paradis e 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winter jujube plants.The plants genomic DNA was digested with Eco ne M:molecular weight markers of k DNA\Hin dIII ne 1:pCAMBIA1300-betA -als plasmid as positive ne 2:wild-type winter jujube as negative ne 3:transgenic plant。