Regeneration system and genetic transformation of Populus yunnanensis
[Objective]This study is aimed to establish a highly efficient system for regeneration and genetic transformation of in vitro leaves of Populus yunnanensis so as to help genetic engineering breeding research in P.yunnanensis.[Method]First,with P.yunnanensis leaves as explants,an investigation was conducted of the effects of different combinations of exogenous hormone concentrations on the induction of callus,adventitious bud differentiation and rooting so as to obtain regenerated seedlings of P.yunnanensis.Then,Agrobacterium tumefaciens mediated method was used to explore the effects of bacterial concentration,infection time and co-culture time on the genetic transformation efficiency of P.yunnanensis.[Result]The optimal medium for callus induction was 1/2 MS + 0.005 mg·L-1 thidiazuron(TDZ)+ 0.010 mg·L-1 1-naphthylacetic acid(NAA),with an induction rate of 91.7%.The optimal medium for the induction of adventitious bud was 1/2 MS + 0.002 mg·L-1 TDZ + 0.010 mg·L-1 NAA,with an induction rate of 75.0%whereas the optimal medium for rooting was 1/2 MS + 0.010 mg·L-1 NAA + 0.100 mg·L-1 3-indolebutyric acid(IBA),and the rooting rate was as high as 96.7%,with an average number of 2.57 roots.The optimal concentration of transforming bacterium D(600)was 0.2,the infection time was 5 min,and the co-cultivation time was 2 days.During differentiation,the optimal concentration of cefotaxime to inhibit the growth of A.tumefaciens was 200 mg·L-1,and the optimal concentration of kanamycin in the screening medium for resistant buds was 20 mg·L-1.The regenerated plants were identified by β-glucuronidase(GUS)staining and PCR,identified 20 positive plants,with a positive transformation rate of 45.45%.[Conclusion]The leaf regeneration system and genetic transformation system of P.yunnanensis were successfully established.[Ch,5 fig.5 tab.27 ref.]
Populus yunnanensisleafcallusregeneration systemgenetic transformation
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