AgResearch Ltd, Grasslands Research Centre, Private Bag 11008, Palmerston North 4442, New Zealand.
Plant Methods. 2013 Jul 10;9(1):25. doi: 10.1186/1746-4811-9-25.
Western clover (Trifolium occidentale) is a perennial herb with characteristics compatible for its development as an attractive model species for genomics studies relating to the forage legume, white clover (Trifolium repens). Its characteristics such as a small diploid genome, self-fertility and ancestral contribution of one of the genomes of T. repens, facilitates its use as a model for genetic analysis of plants transformed with legume or novel genes.
In this study, a reproducible transformation protocol was established following screening of T. occidentale accessions originating from England, Ireland, France, Spain and Portugal. The protocol is based upon infection of cotyledonary explants dissected from mature seed with the Agrobacterium tumefaciens strain GV3101 carrying vectors which contain the bar selection marker gene. Transformation frequencies of up to 7.5% were achieved in 9 of the 17 accessions tested. Transformed plants were verified by PCR and expression of the gusA reporter gene, while integration of the T-DNA was confirmed by Southern blot hybridisation and segregation of progeny in the T1 generation.
Development of this protocol provides a valuable contribution toward establishing T. occidentale as a model species for white clover. This presents opportunities for further improvement in white clover through the application of biotechnology.
西方三叶草（Trifolium occidentale）是一种多年生草本植物，具有作为牧草三叶草（Trifolium repens）基因组学研究有吸引力的模式物种的发展特点。其小的二倍体基因组、自育性和 T. repens 基因组之一的祖先贡献，使其成为转化豆科植物或新型基因植物的遗传分析的模型。
本研究通过筛选来自英国、爱尔兰、法国、西班牙和葡萄牙的西方三叶草材料，建立了一种可重复的转化方案。该方案基于用携带 bar 选择标记基因的载体的根癌农杆菌菌株 GV3101 感染成熟种子的子叶外植体。在测试的 17 个材料中有 9 个达到了高达 7.5%的转化频率。通过 PCR 和 gusA 报告基因的表达验证转化植物，通过 Southern blot 杂交证实 T-DNA 的整合，并在 T1 代中分离后代。
该方案的开发为将西方三叶草作为三叶草的模式物种提供了有价值的贡献。这为通过生物技术进一步改进三叶草提供了机会。
