针对影响农杆菌侵染效率的主要因素进行条件优化,确立了以OD600为0.8的菌液侵染、预培养5 d后的外植体为最佳侵染条件;侵染外植体经过5 d的共培养并除菌后,转到含有250 mg/L Kan的培养基中进行再生诱导,获得了多株具有Kan抗性的再生植株;通过在共培培养基中添加20 mg/L AS,124 mg/L Na2S2O3和152.4 mg/L DTT,有效地抑制了侵染后外植体的褐化现象,提高了筛选阶段抗性芽的成活率;对具有Kan抗性的再生植株进行PCR和Southern blot验证,共获得18株包含外源基因的阳性转化株.%The main factors affecting the efficiency of Agrobacterium infection were optimized, and the optimal infection conditions were 5-day-old pre-cultured explants cultured in bacteria solution with OD600 0. 8. Infected explants after 5 d of co-culture were transferred to medium with 250 mg/L Kan for bud induction and many strains with Kan resistance ability were obtained. By adding 20 mg/L AS, 124mg/L Na2S2O3 and 152. 4 mg/L DTT in the common culture medium, browning phenomenon was effectively inhibited and survival rate of resistant shoots was enhanced. Regenerated plants with resistance to Kan were verified by PCR and Southern blot test. Total 18 positive transformants were obtained.
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