结球甘蓝小孢子胚胎发生的细胞学研究  

Cytological analysis of microspore embryogenesis in white cabbage( Brassica oleracea L. var. capitata) isolated microspore culture

作　　者：曾爱松[1,2] 高兵[1] 宋立晓[1] 张云霞[1,2] 李健绮 李英[2] 侯喜林[2] 严继勇[1] 

机构地区：[1]江苏省农业科学院蔬菜研究所,江苏南京210014 [2]南京农业大学作物遗传与种质创新国家重点实验室,江苏南京210095 [3]青岛常春藤作物科学有限公司,山东青岛266109

出　　处：《南京农业大学学报》2014年第5期47-54,共8页Journal of Nanjing Agricultural University

基　　金：江苏省农业科技支撑计划重点项目(BE2013429);江苏省农业科技自主创新资金项目[CX(13)2004]

摘　　要：游离小孢子培养即小孢子在外界胁迫诱导的条件下,从其配子体发育途径转向孢子体发育途径,诱导小孢子胚胎发生的过程。本研究通过对结球甘蓝小孢子胚胎发生的细胞学观察,为甘蓝游离小孢子培养中的可调控性及相关胚胎发育研究提供理论依据。选用3个在游离小孢子培养中胚胎发生响应有差异的甘蓝杂交种,分别采用醋酸洋红、4',6-二脒基-2-苯基吲哚(DAPI)及荧光素双醋酸酯(FDA)3种染色方法,对甘蓝小孢子体内配子体发育、游离小孢子培养中的胚胎发生过程及细胞死亡现象进行了观察。结果表明:小孢子正常发育方向是单核小孢子经历2次不对称核分裂后发育成三核花粉,而单核靠边期小孢子在体外经32.5℃热激胁迫1 d后,小孢子绝大部分死亡,培养3 d后小孢子死亡率达到90%左右。结球甘蓝游离小孢子培养胚胎发生途径以B途径为主:第1次分裂为对称分裂,其后2个核进行反复分裂形成多细胞团,依次经历球型胚、心型胚、鱼雷胚,直至子叶胚的形成。除了以上典型B途径外,培养过程中还同时存在多种发育途径:第1次不对称分裂后,生殖核继续分裂即为A-G途径;营养核和生殖核两者同时继续分裂的A-VG途径。结论:甘蓝小孢子在体外经32.5℃热激胁迫1 d后,B途径为其主要孢子体发育途径;热激胁迫迅速诱导了小孢子的死亡。Microspore embryogenesis in plants is a process that microspores can switch their developmental pathway from gametophytic to embryogenic by specific stress treatment, resulting in the formation of haploid embryos. Through cytological analysis of microspore embryogenesis in white cabbage （Brassica oleracea L. var. capitata）isolated microspore culture, this paper will be helpful for revealing significant insights into the knowledge of the particularities of embryogenesis induction in this species. Three white cabbage varieties with different response were used in this study. The microspores development and embryogenesis of three white cabbage in vivo and in vitro were studied using several staining techniques with acetocarmine, luorescein diacetate, and 4＇,6-diamidino-2-phenylindol. Flower buds containing a large number of late uninucleate stage were collected. The microspores were resuspended at a density of approximately 1.0×10^5 mL-1 in NLN-13 liquid medium（ pH= 6.0）. All cultures were treated with 32.5℃ heat shock for 1 day in darkness, and then incubated at 25 ℃. Aliquots of 100 μL were taken from the microspore suspensions for cytological examination prior to and during incubation. All cells were observed at equal intervals of 1 day during culture. For the staining test, at least four counts of 200 cells each were performed. The results showed that the microspore developmental stages from tetrad, uninucleate,bicellular to mature pollen were observed in vivo. Late uninucleate microspores processed its first asymmetrical division in vivo, giving rise to the vegetative and generative one, and finally formed mature pollen with three nuclei. There was genotypic variation in the correlation of bud length to the optimal developmental stage of microspore for embryogenesis among the three hybrids. The flesh isolated mierospores before cuhure showed low cell death levels. The proportion of dead cells increased after the 32.5 ℃ heat stress treatment for 1 day and reached high levels（ 90% ）after 3 d

关 键 词：胚胎发生 细胞程序性死亡 发育途径 结球甘蓝 

分 类 号：S635.3[农业科学—蔬菜学]