玉米单倍体胚性愈伤的诱导与鉴定

为建立一套适合玉米单倍体胚性愈伤培养和快速筛选体系,以单倍体诱导系MT-1为父本,18-599红为母本进行单倍体诱导,设置暗培养、全光照培养和光暗周期培养3种光照培养方式,均分别培养0、1、5、10、20、40 h后,观察幼胚形态和颜色。结果表明,自交系18-599红的单倍体和二倍体幼胚均能正常诱导形成胚性愈伤组织;不同光照培养方式对紫色(二倍体)愈伤率的检出效果依次为光培养>光暗周期培养>暗培养。通过光照筛选的方法可在早期鉴定愈伤组织,其二倍体愈伤的筛选率在培养20 h时可达68%,40 h时为71%,剔除了大部分非单倍体愈伤,综合分析确定光照强度为2 000 lx、20℃处理20 h为最适光照筛选处理。染色体压片技术获得的拟单倍体愈伤中有二倍体愈伤的检出,但经流式细胞仪检出获得的拟单倍体愈伤再经染色体压片检测,无二倍体愈伤,表明流式细胞仪检测单倍体愈伤的准确性高于染色体压片技术。通过光照初步筛选结合流式细胞仪的精确鉴定,迅速从3 000个单倍体愈伤中获得110个单倍体愈伤,单倍体愈伤率3.67%。本研究结果为以玉米单倍体愈伤为转基因受体,快速获得转基因植株提供了一定的技术支撑和理论参考。

Induction and Identification of Haploid Embryogenic Callus in Maize(Zea mays L.)

In order to establish maize haploid embryogenic callus culture and rapid screening system, MT-1 was used as inducer to induce haploid on maternal material 18-599(red). And 0, 1, 5, 10, 20 and 40 h were cultured under dark light and light/dark photoperiod for haploid callus induction. The results showed that calluses could be induced from all young embryos of inbred 18-599(red). The effects of light culture on the callus rate of purple (diploid) were: light culture > light/dark alternate culture > dark culture. Haploid callus could be identified early by light screening, and the detectable rates were 68% and 71% for exposed 20 h and 40 h, respectively, which could delete eliminate most of none haploid callus. It was concluded that illumination intensity 2 000 Lx, and treatment at 20℃for 20 h was the best screening process. Furthermore, the remaining putative haploid calluses were observed through flow cytometer and chromosome tableting technology, and some diploid calluses were detected from candidate haploid calluses after tableting technology with flow cytometer, but no diploid callus were detected after flow cytometer, which suggest that flow cytometer is more accurate than tableting technology in detecting haploid calluses. By illumination screening and flow cytometer detection, 100 haploid callus were obtained rapidly from 3 000 haploid callus, and the haploid callus rate was 3.67%. The result of this study provided a reference for utilizing maize haploid embryogenic callus as a transgenic receptor and also lays the foundation for obtaining transgenic plant rapidly.