发根农杆菌介导的铁皮石斛毛状根遗传转化体系建立及优化OA

Objective To break through the technical bottlenecks of strong dependence on traditional tissue culture and low transformation efficiency in Dendrobium officinale,this study developed a non-tissue culture ge-netic transformation system mediated by Agrobacterium rhizogenes.Methods Use stem segments of D.officinale as Explants,and A.rhizogenes strain K599(harboring plasmid pAFOE-1-eYGFPuv)as the vector,single-fac-tor and orthogonal experiments were designed to optimize infection conditions.Combined with stem segment wounding treatment and bacterial suspension injection,the exogenous reporter gene eYGFPuv was successfully introduced into plants via through molecular verification.Results The optimal combination of the transformation system was determined as follows:stem segments of two-year-old plants were selected;the bacterial suspension was adjusted to an OD600=1.0,and resuspended in MES buffer(10 mmol/L MgCl2,10 mmol/L MES,pH=6.0).The medium was supplemented with 100 μmol/L acetosyringone,0.001%Triton X-100,and 0.001%Silwet-77;ultrasonication was performed at 300 W,40 kHz and 20℃with an infection duration of 30 min.Under these conditions,the positive rate of newly regenerated roots reached 60.0%.Conclusion This method avoids the traditional tissue culture procedure,simplifies the operation process and shortens the transformation cycle.It provides an efficient and low-cost potential technical scheme for gene functional research and germ-plasm innovation of Dendrobium officinale.