Regeneration Capacity Of Different Explants From ’Fengmiguan’(Zizyphus Jujuba Mill.) And Transformation Of Bt Gene

Jujube (Zizyphus jujuba Mill.) is an important economic fruit treeof China. The small flowers, difficult to pollinate, lower rate of fruit-bearing andhigher rate of embryo abortion were existed in jujube. Meanwhile, the development ofjujube industry was seriously affected by insect pests. The traditional method ofbreeding is very difficult to get generations, so genetic engineering has become aneffective means for modem plant breeding improvement. In this study, we use freshjujube varieties--Z.jujuba’fengmiguan’ as materials. Our objective was to obtain anefficient and stable regeneration system using different explats from ‘fengmiguan’.It’s a foundation for jujubes genetic transformation. Moreover, the pBar13-Cry2A*and pBar13-Cry1C*vector were transformed into pBI121in order to resolve theproblem of the Bastar sensitivity with jujube. The jujube insect-resistant transgeniclines would be obtained by using the regeneration system of different explants from‘Fengmiguan’ and the new vector included Bt gene. The main results are as follows:1. Establishment of different explant regeneration system in ’fengmiguan’ jujube(1) The mature embryo culture. The optimal medium for the epicotyl from the matureembryo culture is WPM+GA30.3mg/L. Completed plants were obtained by culturingin30d.(2) Establishment of regeneration system with stem. MS as the basic culture meiun,the effects of6-BA and IAA on stem regeneration adventitious buds were studied. Theresults showed that the frequency of callus formation and regeneration rate were up to80%and100%in MS medium supplemented with1.5mg/L6-BA and0.1mg/LIAA,respectively. MS+6-BA1.5mg/L+IAA0.1mg/L+AgNO30.5mg/L was theoptimal medium for the stem regeneration.In the study of the effects of IBA and NAAon rooting culture,1/2MS+0.5g/LAC as the basic culture meium,the results showedthat,1/2MS+0.5g/LAC+IBA0.5mg/L was the best medium for rooting,thefrequence of rooting achieving to60%. (3) The regeneration of adventitious bud from leaf and cotyledon. The leaves whichwere cut2~3knives along vertical to the main veins not to hurt leaf margin wereinoculated in the WPM which contained NAA0.2mg/L+TDZ0.5mg/L+AgNO30.5mg/Land the cotyledon the abaxial surface of which touching the medium and were cut2~3knives were inoculated in the WPM which contained NAA0.2mg/L+TDZ0.5mg/L+AgNO30.5mg/L.After10d cultured in darkness,there were many calluses.Then30d later cultured in normal condition, the adventitious bud were induced.(4) The regeneration of adventitious shoots from epicotypl segments.The effects of6-BA and NAA on epicotypl segments regeneration adventitious buds were studiedusing MS+AgNO30.5mg/L as the basic culture medium. The result revealed that thefrequency of callus formation and regeneration rate were up to94%and63%,respectively, in MS medium supplemented with1.0mg/L6-BA and0.1mg/LNAA.The perfect medium for the epicotyl regeneration was found to be MS+6-BA1.0mg/L+NAA0.1mg/L+AgNO30.5mg/L.(5)The regeneration of adventitious shoots from hypocotyl segments. Hypocotylsegments was inoculated in three different culture meium,The result indicated that thefrequency of callus formation were up to94%in MS+2,4-D1.5mg/L+6-BA0.5mg/L, appearing adventitious bud point after culture15d,formate of adventitious budafter culture30d, the regeneration rate were up to29%.(6) The regeneration of adventitious bud from root. The completed root was culturedin MS+6-BA0.5mg/L+NAA0.5mg/L+PVP1.0g/L medium, callus formationculture in10d, and adventitious bud formation culture in30d.2. Study of transformation vector and transformation of leaves on jujube with Bt(1) Transformation of leaves on jujuba with Bt: The leaves of Z.jujuba’fengmiguan’asthe transformation explants. The results displaied that5regenerated adventitious budswere obtained under the condition with a concentration of OD600=0.3and infectiontime10min in50d.(2) Transformation vector: Identification of pBI121-Cry2A clones by PCRamplification and enzymatic digestion. The results indicated that Cry2A and Cry1Chave been insterted into the vector pBI121by enzymatic digestion, respectively.