根癌农杆菌介导的苜蓿悬浮胚性愈伤遗传转化体系的建立与优化

苜蓿基因型是限制遗传转化的关键因素之一。本实验通过对7种苜蓿胚性愈伤组织诱导，筛选出一份具有高频再生潜力的基因型公农-1号，并以该基因型为转化平台探索和建立了一套高效的苜蓿遗传转化系统。分析了影响农杆菌共培养转化苜蓿悬浮胚性愈伤的因素，优化了悬浮培养条件，建立的超声波辅助农杆菌介导苜蓿胚性愈伤的遗传转化系统为：以下胚轴诱导的愈伤组织经悬浮培养得到的胚性愈伤为转化材料，乙酰丁香酮为100 μmol·L^-1、超声波处理时间8 s、卡那霉素筛选浓度30 mg·L^-1、共培养4 d，接种于选择培养基上进行筛选和再生。最终，获得了大量的转基因植株，分子检测证实目的基因-角碱蓬液泡膜型Na⁺/H⁺逆向转运蛋白基因已经整合到苜蓿基因组中。

Establishment and Optimization of Agrobacterium-mediated Transformation of Suspension Embryogenic Callus of Alfalfa

Genotype is a key restrictive factor for genetic transformation in alfalfa. A high frequency-regeneration potential alfalfa cultivar named Gongnong No.1 was screened from 7 cultivars by embryonic callus inducing test， and was successfully used in establishing a high efficiency genetic transformation system. Additionally, we analyzed several factors affecting the genetic transformation and optimized the suspension culture conditions, and established sonication-assisted Agrobacterium-mediated transformation system in alfalfa primarily. The suspension callus from alfalfa hypocotyls were taken as explants, as concentration was determined at 100 μmol·L^-1, sonication treatment time was 8 s, kanamycin concentration was 30 mg·L^-1 and co-culture time was 4 days. Conclusively, large numbers of transgenic plants were obtained by the transformation system. Molecular analysis confirmed that exogenous vacuolar Na⁺/H⁺ antiporter gene from Suaeda corniculata was integrated into the genome of alfalfa.