Abstract: Leymus chinensis is an excellent forage grass species. To establish the high-frequency tissue culture regeneration system of L. chinensis "explants-somatic embryo-differentiated bud",which would provide supportive technology for the genetic transformation and establish a basic system for breeding expansion. In this study,mature seeds of L. chinensis were used as explants to carry out research on tissue culture techniques such as explant treatment,pollution rate control for tissue culture,improvement of callus growth status,and enhancement of its differentiation ability,and establish the regeneration system of L. chinensis. The results showed that the contamination rates and explants browning of tissue culture were significantly decreased when the seeds were treated with glume removal and soaked into GA₃solution (100 mg·L^-1) for 24 hours. The callus induction rate was 83.11% with the MS medium supplemented with 2.0 mg·L^-12,4-D and 0.5 mg·L^-1 KT. The induction rate of embryogenic callus was 53.26% with the MS medium supplemented with 2.0 mg·L^-12,4-D. The differentiation rate was 84.29% and the seedling rate was 52.86% after treated with the MS medium supplemented with 1.0 mg·L^-16-BA and 0.5 mg·L^-1NAA. The survival rate of the adventitious buds was 100% when transplanted into 1/2 MS medium. Using ISSR molecular marker technology,the genetic variation of the regenerated plant was identified at the DNA level,and no genetic variation was detected out,indicating that the regenerated plant can maintain genetic stability.

Key words: Leymus chinensis, Mature seeds, Regenerative system, Inter simple sequence repeat (ISSR)

摘要： 羊草(Leymuschinensis)是禾本科优良牧草，构建羊草“外植体—体细胞胚—分化芽”的高频组培再生体系，为后续遗传转化提供技术支撑并为扩繁育种建立基本体系。本研究以羊草成熟种子为外植体，开展了外植体处理、组培污染率控制、改善愈伤组织生长状态，提高其分化能力等组织培养技术的研究。结果表明，种子去稃后并在100 mg·L^-1赤霉素(GA₃)浸泡24 h，可减少组培内生菌污染和外植体褐化；诱导愈伤组织的最佳培养基为添加2.0 mg·L^-12,4-D和0.5 mg·L^-1 KT的MS培养基，诱导率为83.11%；在添加2.0 mg·L^-12,4-D的MS培养基上继代，胚性愈伤诱导率为53.26%；最佳分化培养基为添加1.0 mg·L^-16-BA和0.5 mg·L^-1 NAA的MS培养基，分化率为84.29%，成苗率达52.86%；不定芽移栽至1/2MS培养基上成活率为100%。利用简单重复序列间扩增(Inter simple sequence repeat,ISSR)分子标记技术，在DNA水平上对再生株进行遗传变异鉴定，未检测出遗传变异，表明再生植株能够保持遗传上的稳定性。

关键词: 羊草, 成熟种子, 再生体系, ISSR