玉米成熟胚胚性愈伤组织的诱导、高频再生及转化的研究

以玉米自交系CML295、CML304和18-599R的成熟胚为外植体, 结合幼胚离体培养方法, 探讨并优化了成熟胚来源的胚性愈伤组织诱导及继代培养方法。对其愈伤组织的形态和组织切片的研究结果显示, 继代过程可产生良好的Ⅱ型胚性愈伤组织。在分化培养中分别获得68.6%、75.4%和84.8%的高频再生率, 每愈伤组织块成苗数分别为2.45、2.43和2.75。利用基因枪法转化pCAMBIA1301质粒后的GUS瞬时表达效率分别为57.9%、62.5%和73.1%, 转化pCAMBIA1303质粒后检测GFP的瞬时表达效率分别为23.3%、40%和45.5%。以上3种基因型成熟胚来源的愈伤组织转化率与其对应的幼胚来源的胚性愈伤组织转化率相似。这一技术体系为玉米的遗传改良和功能基因组研究提供了重要的技术平台。

Transformation, Inducing and High-Frequency Regeneration of Embryogenic Callus Initiated from Mature Embryos of Maize(Zea mays L.)

Immature embryo-derived callus is more efficient for transgenic and plant regeneration than calli from other explant tissues. It’s reported that embryogenic callus can be initiated from mature embryos. The use of mature embryos from dry seed are easy to handle, available year round and in bulk quantities. In this study we established an efficient transgenic acceptor system and developed some new methods on maize (Zea mays L.) tissue culture, plant regeneration and genetic transformation with embryogenic callus initiated from mature embryos of three elite inbred lines CML295, CML304, and 18-599R. The calli were cultured in subculture medium of immature embryos. Tissue slice showed that the structure of callus formed from mature embryos was the same as those from immature embryos, being the typeⅡ embryogenic callus. The calli were transferred into regeneration medium of immature embryos supplemented with 2 mg L-1 6-BA. The regeneration frequencies of the calli from mature embryos of CML295, CML304, and 18-599R were 68.6%, 75.4%, and 84.8%, respectively. Vector pCAMBIA1301 was transformed into callus from mature embryos of CML295, CML304, and 18-599R by particle gun using GUS as a report gene; vector pCAMBIA1303 was transformed by particle gun using GFP as a report gene, with GUS staining frequency of 57.9%, 62.5%, and 73.1%; and GFP transient expression frequency of 23.3%, 40%, and 45.5%, respectively. The transgenic rates of embryogenic callus from mature embryos of the three inbred lines were similar to those from immature embryos. In conclusion, using embryogenic callus from mature embryos as transgenic acceptors is efficient and available.