Microspore Cultures,Application On Breeding And Epigenetics Of Brassica Campestris L.ssp.Chinesis Var.Purpurea Tsen Et Lee

Brassica campestris L.ssp.Chinesis var.purpurea Tsen et Lee is a cultivated cabbage subspecies from Brassica, which is an unique vegetable in China and very popular in the area of Chinese Changjiang River. A microspore culture technique is a method for obtaining regenerated haploid plants by the way of culturing the isolated microspores. The haploid plants obtained by microspore culture are significant and essential for genetics research of haploid and vegetable breeding. In this research, we carried out some researches on the microspore culture, epigenetics and breeding in B.campestris. The results are as follows:In this study, variants with good qualities were selected for the microspore culture. Different factors that affect the culture of regenerated embryoid were explored, including the genotypes, culture constitution, and temperature. The results revealed that different genotypes had significantly different frequencies of induced embryoids. Lower or higher temperature both influenced the induction of embryoids. A certain concentration of active carbon can obviously promote the induction of embryoids. The regeneration process of new plants was affected by several factors. The results demonstrated that low temperature treatment could increase the regeneration rate of embryoids and the concentration of agar in the solid culture medium also had significant influence on the regeneration.It was reported that the ploidy of the nucleus and chromatin arrangement were highly correlated to the cellular developmental and differential states. In this study, some cellular researches were carried out to analyze nuclei from normal tissues and embryoid in B.campestris. Fluorescent in situ hybridization experiments with several different probes were performed to investigate nucleus architecture and chromatin arrangement. The results showed that, compared to the somatic nuclei from normal tissues, nuclei from embryoid had a complicated ploidy level and had the phenomena of muti-nucleus in a single cell. The chromatin arrangement in embryoid was quite different from that in somatic nuclei, which suggested that it was correlated to nucleus ploidy and differential states. In the somatic cell, the chromatin in most of nuclei adapted an organization pattern of chromocenter, but the nuclei in embryoid did not have this feature.Some studies showed that the chromatins with specific arrangement correspondingly had unique epigenetic features. Specific epigenetic features reflected not only the structure and function of chromatins, but also the regulation of gene expression and differential states. Therefore, we investigated the epigenetics of nuclei from somatic cells and embryoid cell and explored the potential epigenetic regulation of embryoid induction and cell differentiation in microspore cultures. The chromatin immunoprecipitation and immunostaining experiments were used to detect the histone acetylation, histone methylation, and DNA methylation. The results demonstrated that as a species in Brassica, the nucleus of B.campestris had a similar chromatin arrangement and epigenetic feature with that of Arabidopis nucleus. The chromatin arrangement and nucleus ploidy in embryoid nuclei differed with that in the somatic nucleus, corresponding to the different epigenetic modification features. These cytogenetic results might provide an important instructive role to the microspore culture and breeding in B.campestris.