目的建立农杆菌介导的枳壳转化体系。方法 以枳壳实生苗上胚轴为转化外植体 ,农杆菌 Ti质粒上插入了外源目的基因——柑桔衰退病病毒外壳蛋白基因 (CTV -cp) ,转化植株用 GUS (β-葡萄苷酸酶)染色及 PCR进行鉴定。结果 枳壳转化试验中 ,2 0 d苗龄的外植体转化再生率较高 ;外植体与农杆菌共培养时间以 2～ 3d为宜 ;乙酰丁香酮能较大提高转化效率 ,转化再生频率为 2 7.5 %。转化获得的抗性植株中 ,GU S反应呈阳性所占比例为 70 .0 %。 PCR分析证实外源目的基因已整合到枳壳转化株的核基因组中。结论 成功地建立了农杆菌介导的枳壳转化系统 ,为利用基因工程的手段进行枳壳的抗病育种奠定了基础。

Object To establish an effective system for Agrobacterium mediated genetic transformation of Poncirus trifoliata Raf Methods The explants used for transformation were the epicotyls from P trifoliata; the Agrobacterium tumefaciens strain was EHA101, provided with the vector plasmid pGA482GG; the coat protein gene (CTV cp gene) was introduced into the transformation plasmid The transformation was proved by PCR and histochemical GUS assay Results In trans for mation of P tri foliata, 20 days epicotyls were suitable for transformation Shoot regeneration frequency was high when cocultivation time was 2-3 days The presence of acetosyringone during cocultivation could enhance the efficiency of transformation Histochemical GUS assay showed that 70 0% of the resistant plants were GUS positive Extra gene was proved to be transformed into P trifoliata plant by PCR analysis Conclusion An effective genetic transformation system has been established for P trifoliata This system will be useful for resistant breeding of P trifoliata.

国家自然科学基金资助课题 (3 9870 5 3 9)