Creation Of Excellent Germplasm By Microspore Culture In Chinese Cabbage(Brassica Campestris Spp.Pekinensis)

The aim of this study was to create DH lines which has the good traits to be applied in breeding.The effects of genotypes,plant growth regulator and organelle antioxidant on embryoid induction and plantlet regeneration were studied in 13 F1 hybrids that crossed between yellow inner leaf,clubroot resistant lines and yellow inner leaf,late bolting lines of Chinese cabbage(Brassica campestris ssp.pekinensis)with isolated microspore culture method.All obtained DH lines were analyzed for biological traits,resistance clubroot and late bolting.The results were as follows:1.Genotype plays a very vital role in microspore culture.Among thirteen cultivars,eight genotypes were successed in embryogenesis.The genotypes were ZL01,ZL 02,ZL 03,ZL 04,ZL 06,ZL 08,ZL 09 and ZL 11.The rate was 61.54%.It was suggested that there were significant differences in the induction rate of microspore embryo in different genotypes of Chinese cabbage.Genotype is one of the important factors affecting microsporeembryogenesis.2.ZL01(Easy embryogenesis),ZL01(General embryogenesis)and ZL08(Hard embryogenesis)were used to evaluate the effect of-6-benzyladenine(6-BA)and naphthyl acetic acid(NAA)on embryogenesis.Medium added with 0.1-0.2 mg L-1-6-BA and 0.05?0.1 mg L-1NAA could promote the embryogenesis and development of the embryos.The highest yield of ZL01,ZL03 and ZL08 were 5.76 embryos per bud,5.01 embryoids per bud and 4.63 embryoids per bud,respectively.3.The different concentrations of cell antioxidant(Methylene Blue)were added to the microspore culture medium of ZL01,ZL03 and ZL08.In 10nM MB treatment,the embryo induction rate and seedling rate reached the highest.The embryo induction rate of three hybrids were 6.69 embryos per bud,4.70 embryos per bud and 4.10 embryos per bud,respectively;being 1.74,1.58 and 1.62 times as much as in the control,respectively.Seedling rate of three hybrids was increased significantly.For ZL08,maximum seedling rate was 47.78%,being 1.98 times as much as in the control.By contrast,the death rate has been reduced by 1.32 times.It was suggested that the suitable concentration of MB could promote the formation of microspore embryos and plant regeneration.On the contrary,high concentration of MB had a significant inhibitory effect on embryo formation.4.Flow cytometry was used to identify the spontaneous doubling rate of regenerated plants of 8 F1 Chinese cabbage genotypes,the results showed that the doubling rate was 57.87%.For inducing roots of regenerated plants,MS added with 0.10 mg·L-1NAA was most suitable medium.5.13 DH lines were selected in this study,and the inner leaf color of them were all yellow.DBC01,DBC02,DBC07 and DBC13 were immune to root disease,other lines were highly resistance to clubroot,the incidence rate was less than 20%;from the bolting index and central stem shape showed that DBC02 and DBC03 were extremely late bolting DH lines.DBC01?DBC04?DBC05?DBC10?DBC12 were the late bolting DH lines.DBC02 with yellow heart,clubroot resistance and late bolting was created by using isolated microspore culture method.