利用番茄黄化曲叶病毒侵染性克隆转化烟草获得抗病突变体

Acquirement of tobacco mutant resistant to tomato yellow leaf curl virus through transformation of infectious clone of the virus

为了获得烟草抗番茄黄化曲叶病毒(TYLCV)突变体材料,以含有番茄黄化曲叶病毒侵染性克隆PTYj01的农杆菌EHA105侵染转化烟草叶片,用50 mg/L卡那霉素对愈伤组织进行筛选,愈伤组织继代培养120 d左右后进行植株再生。挑取表型正常、无TYLCV病症的再生烟草45株,经PCR鉴定均整合有TYLCV DNA序列。将表型正常的再生植株移栽,温室接种烟粉虱传毒鉴定再生烟草在整个生育期对TYLCV的抗性,共获得8株表型正常、无TYLCV病症的烟草。对这8个抗性植株T2代烟草进行农杆菌注射法和接种烟粉虱鉴定,发现T2-2、T2-7的T2代分离群体中有约75%的植株没有发病症状;对这2个株系的抗性植株进一步进行卡那霉素抗性鉴定,发现其卡那霉素的抗性也符合3︰1的分离比例,说明株系T2-2、T2-7可能发生了抗TYLCV突变。可见,通过对侵染性克隆转化的烟草进行体细胞离体筛选,获得抗TYLCV突变体的方法是可行的。

Agrobacterium tumefaciens strain EHA105 containing an infectious clone of tomato yellow leaf curl virus（PTYj01） was used to transform the leaf dishes of Nicotiana tabacum and the callus was selected with 50 mg/L kanamycin.Plantlets were regenerated after 4 mouths subculture for the purpose of variation.45 regenerated plantlets without TYLCV symptoms were selected and all the plants contained TYLCV DNA tested by PCR.Virus inoculation experiment with Bemisa babaci further confirmed that 8 individual plantlets performed normal phenotype without TYLCV symptoms when subjected to Bemisa babaci infection during the whole growth period in the greenhouse.Seventy-five percentage of the T2 generation segregation population from two individual plantlets（T2-2 and T2-7） showed resistance to TYLCV by agro-injection and Bemisa babaci inoculation,and the ratio of kanamycin-resistant plants to sensitive ones in the plants without TYLCV symptoms was 3∶1,indicating that T2-2 and T2-7 were single-point mutants resistant to TYLCV.These results suggest that it＇s possible to obtain tobacco mutant resistant to TYLCV through transformation of infectious clone.