农杆菌介导的葡萄风信子愈伤组织遗传转化体系的优化

Optimization of Agrobacterium Tumefaciens -Mediated Genetic Transformation System based on Callus of Grape Hyacinth

葡萄风信子(Muscari spp.)为百合科葡萄风信子属植物的总称,该属植物多具有稀少的正蓝色花色,是研究单子叶植物花青苷合成机理的模式材料。为开发一种高效的葡萄风信子遗传转化体系,本试验以葡萄风信子常见种亚美尼亚(M.armeniacum)、栽培品种‘黑眼睛’(M.aucheri‘Dark Eyes’)为材料,用叶片和花蕾分别诱导获得愈伤组织,对愈伤组织的潮霉素敏感性进行测试,对根癌农杆菌介导的愈伤组织遗传转化体系进行优化。试验结果表明:葡萄风信子亚美尼亚和‘黑眼睛’愈伤组织的潮霉素临界浓度分别为150 mg·L^(-1)和120 mg·L^(-1)。GUS染色结果显示不同基因型、不同农杆菌菌株及不同功率超声处理的愈伤组织瞬时转化效率差异不显著。而液体培养的花源愈伤组织能获得更高的瞬时转化效率,为葡萄风信子遗传转化的最优受体。其最高瞬时转化效率为98.73%。试验共获得了209块抗性愈伤组织,经PCR和RT-PCR检测,阳性率可达90%。本研究为在葡萄风信子中快速有效的开展基因功能研究奠定了技术基础。

Grape hyacinth(Muscari spp.)included all species in the Muscari genus of Liliaceae,and is a model plant for the mechanism study on anthocyanin synthesis in monocots due to its rare blue color.In this study,the hygromycin sensitivity of callus induced from a common species M.armeniacum and the cultivar of M.aucheri‘Dark Eyes’were tested,and an optimized genetic transformation system based on callus and Agrobacterium tumefaciens was developed.The results showed that the critical concentrations of hygromycin were 150 mg·L^(-1)and 120 mg·L^(-1)for leaf-and-flower-derived calli of M.armeniacum and M.aucheri‘Dark Eyes’,respectively.There were no significant differences of transformation efficiencies among different genotypes,among different Agrobacterium strains and among different ultrasonic power,based on GUS staining.The highest transient genetic transformation efficiency of 98.73%was received for flower-derived calli cultured in liquid culture method.A total of 209 pieces of resistant calli were obtained,and the transformation success rate was 90%based on examinaitons of PCR and RT-PCR.The present study provides an efficient technical support for the functional researches of genes in grape hyacinth.