棉花体细胞增殖和胚胎发生中的细胞程序性死亡

棉花组织培养中愈伤组织褐化可能与细胞程序性死亡(PCD)有关.对棉花组培中不同时期的愈伤组织DNA进行琼脂糖电泳,观察到:仅在第一次愈伤组织继代后10 d左右和愈伤组织第一次继代到分化培养基中培养10 d左右,愈伤组织的DNA产生了180 bp左右的片断或其整数倍片断大小的DNA带,呈DNA梯(DNA ladder)状电泳,说明在这两个时期PCD达到了高峰.在这两次PCD高峰后1周均出现愈伤组织大规模的褐化死亡.显微镜观察到第一次PCD发生高峰的愈伤组织存在许多管状、内含物少的细胞,这些细胞分布在愈伤组织的边缘和内部;第二次PCD发生高峰观察到体细胞胚胎分化、PCD细胞的木栓化和水渍化.对体细胞胚胎分化时期的原生质体进行荧光染色(DAPI),荧光显微镜下观察到发生细胞程序性死亡的细胞核浓缩、染色质凝聚、结块、边缘化和形成PCD小体.

Programmed Cell Death During Somatic Proliferation and Embryogenesis of Cotton (Gossypium hirsutum L.)

The mechanism of callus browning during cell proliferation and somatic embryogenesis of cotton was studied and was found to involve programmed cell death (PCD). By DNA ladder electrophoresis technique it was found that PCD happened only during the first subculture and the fifth subculture (Fig.1), leading to cell proliferation and embryo differentiation, respectively. Clear DNA ladder was observed 10 days after subculture when large-scale cell death happened. The brown calluses were sampled for observation under microscope and the first PCD peak was found to be followed by the formation of many small tube-shaped cells (Fig.2C) near the surface of the callus, and the second was by corkification (Fig.2D,E) and hydrophanous outlook (Fig.2F) during embryo formation. DAPI staining of isolated PCD cells showed that the chromatin in the nucleus moved to the peripheral zone of the cell and formed many vesicles or apoptosis bodies (Fig.3A-F). To our knowledge, this is the first report about PCD leading to callus browning during cell culture and somatic embryogenesis of cotton.