棉花高效体细胞胚发生及同步控制培养体系研究

棉花体细胞胚胎发生的频率低, 且体细胞胚的发育存在着不同步性, 给体细胞胚胎发生发育过程的生理学、生物化学和分子生物学的研究带来许多困难。本研究以陆地棉品种Coker201为材料, 建立了一种简单有效的棉花体细胞胚高频率发生和同步发育的培养体系。经过多次继代获得的胚性愈伤, 首先在液体培养基中振荡培养2 d使其分散, 然后过30目筛去除大的颗粒, 重新悬浮于同样的液体培养基中。悬浮培养14 d后, 过50目筛, 将筛上的胚性愈伤重悬浮于新鲜的培养基中, 并用吸管吸取悬浮液, 将其均匀地接种在表面垫有滤纸的同成分固体培养基(附加2.46 mmol L^-1 IBA和0.70 mmol L^-1 kinetin)上。培养21 d后, 用垫有滤纸的相同的固体培养基继代培养。利用这种培养体系, 获得的体细胞胚数量分别是单纯悬浮培养和固体培养(不垫滤纸)的16.5倍和4.0倍。其中, 球形胚、鱼雷形胚和子叶胚的同步发生率分别为70.2%、52.3%和73.0%。

An Efficient Culture System for Synchronization Control of Somatic Embryogenesis in Cotton(Gossypium hirsutum L.)

Low efficiency of somatic embryogenesis and asynchronous embryo development brings a lot of difficulties to the physiological, biochemical, and molecular biological studies of embryogenesis processes in cotton. In the present study, a simple and efficient method was developed to improve somatic embryogenesis frequency and synchronous development of mass somatic embryos from cultured cells of Gossypium hirsutum cv. Coker 201. The embryonic calli obtained after several rounds of subculture were scattered in liquid medium by shaking for 2 d and then resuspended in the same liquid medium after discarding the larger callus aggregates over 30 mesh-size-sieve. The suspensions cultured for 14 d were filtered through 50 mesh-size-sieve and then the aggregates over the sieve were inoculated onto the surface of Whatman filter paper placed on the solid medium containing 2.46 mmol L^-1 IBA and 0.70 mmol L^-1 kinetin to culture for 21 d followed by subculturing. The somatic embryo amount obtained by using new system was 15.5-fold higher than that by suspension culture and 3-fold higher than that by solid culture (without filter papers) at the same culture period, and in the different stages of somatic embryo development, 70.2% for globular, 52.3% for torpedo-shaped and 73.0% for cotyledonary embryos were obtained respectively during the culture. Suspension culture combined with solid culture with filter paper were considered to be beneficial for synchrony of somatic embryogenesis and mass embryo development.