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作者	单位
郑楠楠，付玉洁，鲁向前，赵俊恒，孙楠楠，张国平，叶玲珍	（1.浙江大学中原研究院生物设计与育种中心, 河南郑州 450000 2.浙江大学作物科学研究所, 浙江杭州 310058）

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中文摘要:

农杆菌介导是小麦遗传转化的重要手段，但其存在转化效率低、基因型依赖性强及愈伤组织易于褐化、再生率较低等关键问题。本研究以小麦模式材料Fielder的幼胚为外植体，系统探究了胚龄、乙酰丁香酮（acetosyringone，AS）浓度、农杆菌侵染时间、草铵膦（phosphinothricin，PPT）筛选压力以及外源激素（abscisic acid，ABA；kinetin，KT）配比等对愈伤组织的诱导分化及转化效率的影响，并利用扬麦20、郑麦7698、科农199等品种对优化后的体系进行基因适用性评价。结果表明，花后12 d的幼胚是最适宜的转化受体，作为外植体诱导的愈伤组织褐化率最低（4.40%）；农杆菌侵染液OD₆₀₀=0.5、添加200 μmol·L^-1 AS、侵染愈伤组织10 min时，抗性愈伤组织绿点分化率最高（34.29%）；诱导和分化培养基中分别添加0.5 mg·L^-1 ABA和1.5 mg·L^-1 KT后，胚性愈伤组织的形成与植株再生能力显著提高； PPT最佳筛选浓度为10 mg·L^-1。在该优化体系下，Fielder的转化效率从8.16%显著提高至14.83%，扬麦20、郑麦7698和科农199的转化效率分别达到9.03%、7.32%和8.70%，接近甚至超过Fielder在优化前的水平。本研究得到的优化体系提高了小麦遗传转化效率，且对不同遗传背景的小麦品种具有较好的适用性，为小麦基因功能的解析和分子育种提供了技术支撑。

英文摘要:

Agrobacterium-mediated transformation is an important approach for genetic modification of wheat, but it still suffers from several unresolved critical technical bottlenecks, including low transformation efficiency, strong genotype dependence, severe browning of calli, and low regeneration efficiency. In this study, immature embryos of the model cultivar Fielder were used as explants to systematically investigate the effects of embryo age, acetosyringone(AS) concentration and Agrobacterium infection duration, phosphinothricin(PPT) selection pressure, and exogenous hormone(abscisic acid, ABA; kinetin, KT) ratios on callus induction, regeneration and transformation efficiency. Using the optimized protocol, applicability was evaluated in the cultivars Yangmai 20, Zhengmai 7698, and Kenong 199. The results showed that embryos harvested 12 days after anthesis exhibited the lowest browning rate(4.40%) and were most suitable as recipients for transformation. When the Agrobacterium suspension was adjusted to OD₆₀₀=0.5, supplementation with 200 μmol·L^-1 AS and a 10 min infection produced the highest induction rate of resistant green callus spots(34.29%). Addition of 0.5 mg·L^-1 ABA to the induction medium and 1.5 mg·L^-1 KT to the regeneration medium significantly enhanced embryogenic callus formation and plant regeneration. The optimal PPT selection concentration was determined to be 10 mg·L^-1. Under the optimized conditions, transformation efficiency of Fielder increased significantly from 8.16% before optimization to 14.83%; transformation efficiencies of Yangmai 20, Zhengmai 7698, and Kenong 199 reached 9.03%, 7.32% and 8.70%, respectively, approaching or exceeding the pre-optimization level of Fielder. In summary, the optimized system established in this study improves wheat genetic transformation efficiency and is broadly applicable across different genetic backgrounds, providing a technical platform for gene functional analysis and molecular breeding in wheat.

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