超声波辅助农杆菌介导SeNHX1基因转化紫花苜蓿的研究

为建立高效快速的紫花苜蓿遗传转化体系，以紫花苜蓿6个栽培品种为材料，采用超声波辅助农杆菌介导法，对影响紫花苜蓿遗传转化体系的若干因素进行了研究。结果显示，6个栽培品种对卡那霉素敏感性不同，维多利亚为40 mg/L，而其他5个品种为50 mg/L；通过梯度实验表明：根癌农杆菌侵染浓度OD600为0.4~0.5，侵染时间为10~15 min，超声波处理时间为10 min是实现紫花苜蓿子叶转化及抗性愈伤组织形成较为有利的组合。卡那霉素抗性愈伤组织DNA的PCR扩增显示，转化体和质粒DNA分别扩增出长度大约为546 bp的清晰条带，初步表明耐盐SeNHX1基因已整合至紫花苜蓿愈伤组织中。

Transformation of SeNHX1 Gene in Alfalfa (Medicago. sativa) by Sonication Assisted Agrobacterium-mediated Gene Transfer

In order to establish a high frequency and rapid regeneration system for the genetic transformation of alfalfa. Six cultivars of alfalfa were applied to investigate some factors which affect alfalfa genetic transformation system, such as ultrasonication, the concentration and time of agrobacterium tumefaciens infection and so on, by sonication assisted agrobacterium-mediated. Experimental results showed that, the best combination for alfalfa cotyledon transformation and resistant callus information were as follows: the concentration of agrobacterium tumefaciens infection OD600 was 0.4-0.5, and the appropriate time of infection was 10-15 min, with 10 minutes’ of ultrasonication in the meantime. PCR amplification of the DNA of kanamycin resistance callus revealed that, transformant and plasmid DNA amplified about 546 bp distinct stripe with the length respectively, which means salt tolerance gene SeNHX1 have integrated into alfalfa callus.