Abstract:

By comparative analysis on the different explant tissues, concentrations of bialaphos for selection, pre-culture and inoculation time, etc, an efficient Agrobacterium-mediated transformation system for an elite alfalfa (Medicago sativa L.) cultivar Debao was established. The results showed that cotyledon was the best explant for callus induction and transformation. The optimal bialaphos concentration for selection of transgenic calli and plants was 2.5 mg/L in the selection medium. The pre-culture of cotyledon before inoculation with Agrobacteria cells was a key factor affecting transformation efficiency, ranging from less than 5% without pre-culture to about 30% with 3 days of pre-culture. Three days of inoculation was optimal for transformation. More than one hundred independent transgenic plants were obtained, 30 were randomly selected for PCR amplification of bar gene and all the 30 plants were confirmed to be transgenic. The PCR positive plants survived and grew well after spray of 5‰ Basta solution. The Agrobacterium-mediated transformation system described in this study was proved to be reliable and efficient.

Key words: Agrobacterium-mediated transformation, Basta resistant, genetic improvement, Medicago sativa L.

摘要：

以农艺性状优良的紫花苜蓿品种“德宝”为转化受体,通过对外植体选择、bialaphos筛选浓度、预培养与共培养时间等因素进行优化,成功建立了高效苜蓿组织培养再生体系和农杆菌介导的遗传转化体系。试验结果表明,子叶作为外植体愈伤组织诱导频率最高,可达97%;bialaphos作为筛选标记,获得转化愈伤组织与转基因植株的最佳筛选浓度为2.5 mg/L;子叶在农杆菌浸染前的预培养对于对于抗性愈伤组织产生频率甚为关键,可由低于5%(未经过预培养)提高至30%以上(预培养3 d);子叶与农杆菌的共培养时间以3 d为最佳。按最佳条件转化共计获得100余株T0代抗性株系。PCR检测其中30个抗性株系,全部表现为阳性。对抗性植株和野生型植株叶片喷施5‰除草剂Basta溶液,阳性植株全部表现为除草剂抗性,进一步证实了建立的紫花苜蓿转化体系是高效、可靠的。

关键词: 农杆菌介导的遗传转化;Basta抗性;遗传改良;苜蓿