Rapid Clonal Propagation And Production Of Endophytic Bacteria-free Plants Of 'Jinkui' Kiwifruit

Kiwifruit(Actinidia)is one pop ? Lar fruits in the world due to that it is rich in nutrients,especially in vitamin C.However,kiwifruit bacterial canker caused by Pseudomona syringae pv.Actinidiae(PSA)is the great threat to c P Ltivation of kiwifruit,which can destroy a kiwifruit orchard when the disease breaks out.So far,kiwifruit bacterial canker has been reported in all the main c ? Ltivation areas and main c ? Ltivars of kiwifruit in China.Establishing a protocol of in vitro plantlet regeneration of a specific kiwifruit c ?Ltivar is usef ? L not only for rapid clonal propagation and production of transgenic plants of the c ? Ltivar,but also for production virus-free and PSA-free plants of the c ? Ltivar.'Jinkui' kiwifruit is an elite c ? Ltivar of Actinidia chinensis var.deliciosa having characters of high quality,high yield,good resistance and long shelf-life,and has been c ? Ltivated as a major c u Ltivar in many provinces in China in the past two decades.In practice,'Jinkui' kiwifruit is propagated by graft,not by cutting since it is hard to root.In this study,the effects of plant growth substances and basal media on adventitious bud induction from mature leaves of 'Jinkui' kiwifruit and shoot m ? Ltiplication and rooting,and plantlet transplanting were investigated;meanwhile,the method of eliminating endophytic bacteria in 'Jinkui' kiwifruit shoots was developed.The followings are the main res ? Lts of this study.1.On 1/2 MS medium(with half-strength of macronutrients of MS medium)containing 0.5 mg/L 6-benzylaminopurine(BA)and 0.1 mg/L indolebutyric acid(IBA),about 43%of nodal sections developed new shoots,but yellow or white bacteria grew on the base of all sections.When the shoots were cut off from the original explants and cultured on fresh medium,bacteria appeared again on the base of all shoots.These data suggested that there were lots of bacteria cells inside the original nodes of 'Jinkui' kiwifruit used in this study.2.Callus and adventitious bud induction from mature leaves of 'Jinkui' kiwifruit occurred on 1/2 MS medium supplemented with 0.2 mg/L a-naphthylacetic acid(NAA)plus 1-5 mg/L BA or 1-2 mg/L thidiazuron(TDZ),but induction frequencies varied dramatically among different combinations.TDZ was much more active than BA in bud induction;on the medium with 1 mg/L TDZ,all leaf explants formed callus and 92%of them initiated adventitious buds.On the medium with 5 mg/L BA,all explants formed callus,too,but only 50%of them produced buds.3.Inclusion of 0.3-0.5 mg/L gibberellin(GA3)in the medium containing cytokinins significantly promoted elongation of shoots and therefore increased proliferation efficiency of buds by 3-4 folds.4.On the medium with 0.3 or 0.5 mg/L GA3,BA was more effective to stim,Late bud m ? Ltiplication than TDZ,kinetin and zeatin.The nodal sections cultured on the medium with 0.3-0.5 mg/L GA3 and 0.3 or 0.5 mg/L BA,each section developed a new shoot which grew and elongated normally and 1 or 2 axillary bud of the shoot grew into secondary shoots,too,res ? Lting a m ? Ltiplication coefficient of 4.5 or higher.5.When the same combination of plant growth reg ? Lators(BA and GA3),both MS medium and 1/2MS medium were better than other basal media such as B5,CF and WPM.6.All the shoots(2-3 cm long)proliferated on the m ? Ltiplication medium rooted well on the 1/2 MS medium having 0.7 or 1 mg/L IBA,and averagely a plantlet had 5.2 and 8.5 roots,respectively.However,many plantlets regenerated on the medium with 1 mg/L IBA formed big-size callus on base and most roots were initiated from callus.No obvious callus was formed on the base of the plantlets regenerated on the medium with 0.7 mg/L IBA and the roots were initiated directly from the base of the shoots.7.The plantlets regenerated on the medium with 0.7 mg/L IBA grew healthily after they were acclimated in greenhouse for one month and then placed under natural environment.The survival rate of the plantlets was 96%three month after transplantation.8.In order to obtain endophytic bacteria-free plants of'Jinkui' kiwifruit,different size shoot tips were isolated from the shoots containing endophytic bacteria and cultured on MS medium withl mg/L BA and 0.2mg/L NAA.The res ? Lts showed that the shoot tips 0.5 mm long was better in elimination of endophytic bacteria.Endophytic bacteria-free shoots were obtained by 4 successive shoot tip cultures?The elimination of endophytic bacteria from the shoots or regenerated plantlets were confirmed by bacteria cell culture and PCR.