发根农杆菌介导的茶树发根高频诱导与遗传转化

以两种野生型发根农杆菌菌株、三种共培养培养基和六种外植体为试材,建立了茶树发根高频诱导体系,最高发根诱导频率达30%以上。最佳发根诱导体系为:OD600为0.5~0.8的发根农杆菌菌液侵染已培养60~70 d苗龄无菌苗茎段10~50 min,在添加100 mmol/L乙酰丁香酮的YMB固体培养基上共培养2 d,在含500 mg/L头胞噻肟钠的MS培养基上诱导发根。发根在不含激素的LG0培养基上生长迅速,产生大量侧枝和根毛。PCR证实,发根农杆菌Ri质粒T-DNA的rolA、rolB和rolC基因已经插入到发根基因组DNA中。应用此发根诱导体系,以含携带Bt基因的双元载体质粒pCAMBIA2301的发根农杆菌15834,侵染茶树外植体诱导出发根,PCR和GUS组织化学染色证实外源基因已经插入到基因组DNA中并获得表达。

Agrobacterium Rhizogenes-Mediated High Frequency Hairy Root Induction and Genetic Transformation in Tea Plant

An efficient protocol for the establishment of transgenic high hariy root tea plant(Camellia sinensis) system induction infected by Agrobacterium rhizogenes is reported using two wild type strains of A.rhizogenes,three different co-cultivation media and six different explants.The highest induction frequency was obtained from stem segments of aseptic seedlings infected by A.rhizogenes and co-cultivated on YMB solid media with 100 mmol/L AS for 2 d.The hairy roots grew rapidly on LG0 medium without phytohormone and produced a plenty of branches and root hairs.PCR confirmed that rolA,rolB and rolC have inserted into the genomic DNA of hairy root induced by wild type A.rhizogenes.Hairy roots were also induced from explants of tea plant infected by A.rhizogenes 15834 carrying pCAMBIA2301 binary vector harboring Bt gene,transgenic root were confirmed by PCR and GUS histochemical staining that the Bt exogenous gene was inserted and expressed.