College of Grassland Science and Technology, Sichuan Agricultural University, Chengdu 611130, China.
Int J Mol Sci. 2023 Jul 9;24(14):11260. doi: 10.3390/ijms241411260.
White clover is a widely grown temperate legume forage with high nutritional value. Research on the functional genomics of white clover requires a stable and efficient transformation system. In this study, we successfully induced calluses from the cotyledons and leaves of 10 different white clover varieties. The results showed that the callus formation rate in the cotyledons did not vary significantly among the varieties, but the highest callus formation rate was observed in 'Koala' leaves. Subsequently, different concentrations of antioxidants and hormones were tested on the browning rate and differentiation ability of the calluses, respectively. The results showed that the browning rate was the lowest on MS supplemented with 20 mg L AgNO and 25 mg L VC, respectively, and the differentiation rate was highest on MS supplemented with 1 mg L 6-BA, 1 mg L KT and 0.5 mg L NAA. In addition, the transformation system for -mediated transformation of 4-day-old leaves was optimized to some extent and obtained a positive callus rate of 8.9% using green fluorescent protein (GFP) as a marker gene. According to our data, by following this optimized protocol, the transformation efficiency could reach 2.38%. The results of this study will provide the foundation for regenerating multiple transgenic white clover from a single genetic background.
白车轴草是一种广泛种植的温带豆科牧草，具有很高的营养价值。白车轴草功能基因组学的研究需要一个稳定高效的转化系统。在本研究中，我们成功地从 10 个不同白车轴草品种的子叶和叶片中诱导出愈伤组织。结果表明，子叶的愈伤组织形成率在品种间没有显著差异，但在“考拉”叶片中观察到的愈伤组织形成率最高。随后，分别测试了不同浓度的抗氧化剂和激素对愈伤组织的褐变率和分化能力的影响。结果表明，在分别添加 20mg/L AgNO 和 25mg/L VC 的 MS 培养基上褐变率最低，在分别添加 1mg/L 6-BA、1mg/L KT 和 0.5mg/L NAA 的 MS 培养基上分化率最高。此外，对 4 天大的叶片介导的转化系统进行了一定程度的优化，使用绿色荧光蛋白（GFP）作为标记基因，获得了 8.9%的阳性愈伤组织率。根据我们的数据，按照这个优化的方案，转化效率可以达到 2.38%。本研究的结果将为从单一遗传背景中再生多个转基因白车轴草提供基础。
