农杆菌介导高粱成熟胚遗传转化获得耐草甘膦植株

利用萌动的成熟胚作外植体进行基因转化,可有效提高高粱转基因材料选育的效率。为了建立并完善以高粱萌动种胚作为外植体的遗传转化体系,本研究以高粱材料Is-623为转化受体,质粒pM3301UbiSpCP4为外源基因供体,采用农杆菌介导法进行遗传转化,并对转化的关键因素进行分析。结果表明,麦草畏(dicamba)诱导高粱萌动的成熟胚产生愈伤组织适宜浓度约为5 mg·L^-1;农杆菌侵染时间为2 h时遗传转化效率较高;经筛选共获得301块抗性愈伤组织,75个转化事件,经PCR、Southern blotting杂交分析、CP4-EPSPS蛋白检测试纸条检测及田间草甘膦耐性鉴定,最终获得草甘膦耐性等级1级株系6株,耐性等级2级株系3株的转基因高粱株系。本研究初步建立了以高粱萌动种胚作为外植体的遗传转化体系,为高粱遗传转化体系的研究和应用奠定了基础。

Obtaining Glyphosate-tolerant Sorghum Via Agrobacterium Tumefaciens-mediated Transformation With Mature Embryos

Acquirement of genetically modified sorghum varieties with germinated mature embryos as explants can greatly improve the efficiency of sorghum breeding. To establish and improve the genetic transformation system of sorghum embryo, in the present study, with Sorghum Is-623 as transformation receptor and plasmid pM3301UbiSpCP4 as exogenous gene donor, Agrobacterium-mediated transformation was conducted, and the key factors that affected the transformation efficiency was investigated as well. The results showed that the optimal Dicamba concentration that induce callus generation from mature embryos was about 5 mg·L^-1. The genetic transformation efficiency was the highest when embryos were inoculatd with Agrobacterium tumefaciens for 2 hours. We obtained 301 callus that showed glyphosate-resistance, and they belonged to 75 transformation events. With PCR amplification, Southern blotting hybridization and CP4-EPSPS protein test strip detection for the foreigner genes, and glyphosate-tolerance assay in the field, finally we obtained 6 transgenic sorghum lines exhibited glyphosate-tolerance level 1 and 3 lines presented glyphosate- tolerance level 2. In this study, a genetic transformation system with sorghum embryo as explant was initially established, which could benefit sorghum genetic transformation in the future.