Investigation Of Microspore Culture, Embryogenesis And Doubling Mechanism In Chinese Cabbage (Brassica Rapa L.ssp.Pekinensis)

Microspore culture technique is widely applied to plant breeding and genetic research. A remarkable progress has been made especially in Brassica crop. There are several problems unclear and needs to improve the culture technique. In these experiments pretreatments of isolated microspore, developmental pathway from gametophytic to sporophytic, the stage of natural doubling, regeneration of embryoid to plant have been investigated In order to establish good isolated microspore culture technigue of Chinese cabbage (Brassicac rapa ssp. pekinensis).During isolation of microspores, addition of mannitol to B₅ washing medium and with higher concentration of sucrose showed a signifficant effect on the diffrential development of microspores. The short-term pretreament significantly increased the frequency of Embryogenesis. In our experiments, all nine cultivars responded to the treatments.Different methods of regeneration from microspore-derived embryos were investigated. When embryos at cotyledon stage were transferred to B5 solid medium and cultured at 25°C in 16h light period, most of them deeveloped to form callus and need redifferentiation to develop the plantlets. Whereas embryos were transferred to solid B5 medium and gave an initial period of cold induction(4°C )for some day, these embryos germinated well at 25°C in the light and plants regenerated directly and quickly. In five cultivars tested, cold-pretreatments increased the regenerating rate of plants and reduced the death rate of embryoid. The optimal treatment for plant regeneration is 4°C for 10d..The optimum period of inducing embryo for microspore culture was confirmed to be the periods from mid-late to very-late uninucleate stage. The start-up sign of microspores to be induced is that the microspores noticeably swelling and the doubled nuclear chromosomes could be observed. The first symmetric division of microspores occurred after 48 hours of incubation and two cells formed. The second symmetric division occurred after 72 hours of incubation and four cells formed. There existed multi-division paths from microspores culture to embryogenesis. The main path is the division path B although division path A could be observed.