Abstract: L. chinensis is a high-quality forage grass with significant ecological and feed values， serving as a vital genetic resource for stress-resistant breeding in cereal crops. However， functional genomic studies have been severely hindered by the lack of an efficient and stable genetic transformation system. In this study， using immature spikes and mature seeds of the Zhongke series as explants， we systematically optimized sterilization protocols， phytohormone combinations， Agrobacterium-mediated infection parameters， and selection pressure to establish a rapid and reliable transformation platform. The results showed that sulfuric acid pretreatment combined with calcium hypochlorite disinfection effectively reduced contamination and improved explant survival. Supplementation of the regeneration medium with phytosulfokine and CuSO₄ significantly enhanced embryogenic maintenance and differentiation potential. Through large-scale genotype screening， 52 elite callus lines were obtained， with average differentiation rates of 62.19% and 40.69% for immature spikes and mature embryos， respectively. Among these， the line “Zhongke No.1-95” maintained a 100% differentiation rate even after more than 10 subculture passages. Furthermore， a vacuum-ultrasonic-assisted infiltration strategy coupled with gradient glufosinate selection increased the average transformation efficiencies of calli derived from immature spikes and mature embryos to 19.12% and 11.03%， respectively， and the entire period from callus induction to transgenic plantlet recovery was shortened to 70 days. Collectively， this study overcomes the technical bottleneck in L. chinensis genetic transformation and provides a highly efficient and reliable platform for molecular breeding， functional validation of stress-related genes， and gene-editing-based germplasm innovation in this important forage species.

Key words: Leymus chinensis, Embryogenic callus, Agrobacterium tumefaciens, Genetic transformation

摘要： 羊草（Leymus chinensis （Trin.） Tzvel.）是兼具生态与饲用价值的优质牧草，也是禾本科作物抗逆育种的重要基因资源，但其功能基因组的研究因缺乏高效、稳定的遗传转化体系而受到严重制约。本研究以中科系列羊草幼穗和成熟种子为外植体，系统优化消毒方案、培养基激素配比、农杆菌侵染条件及筛选压，旨在建立快速稳定的遗传转化平台。结果表明，采用硫酸去稃结合次氯酸钙消毒可有效降低污染率并提高外植体存活率；在再生培养基中添加植物磺肽素与硫酸铜显著提升了愈伤组织的胚性维持能力与分化潜能。通过大规模基因型筛选，获得52个优异愈伤系，幼穗和成熟胚的平均分化率分别达到62.19%和40.69%，并从中鉴定出继代10次以上仍保持100%分化率的优良胚性愈伤系“中科1号-95”。进一步采用真空-超声辅助侵染结合草铵膦梯度筛选策略，使幼穗和成熟胚来源愈伤组织的平均转化效率分别达到19.12%和11.03%，并将从愈伤诱导至获得转基因苗的周期缩短至70天。该体系的建立有效突破了羊草遗传转化的技术瓶颈。综上，本研究为羊草分子育种和功能基因研究提供了关键技术支撑。

关键词: 羊草, 胚性愈伤组织, 根癌农杆菌, 遗传转化