Studies On The Technical System Of Anther And Isolated Microspore Culture Of Brassica Campestris Ssp.pekinensi

The Chinese cabbage ( Brassica campestris ssp. pekinensi ) breeding took on a new look along with the development of biotechnology-. Homozygous lines established by anther and isolated microspore culture could provide new solutions for improvement of Chinese cabbage breeding. Thirteen cultivars of spring and summer Chinese cabbage were used in the study of anther and isolated microspore culture. Meanwhile, ploidy identification, propagation, chromosome doubling of regenerated plants by colchicine and regeneration of leaf were also studied.The main results are as follows:1. The cytological observations of pollen in different developmental stages of Chinese cabbage were undertook. The results showed that most of the pollen were in the late uninucleate stage when the ratio of petal to anther length was between 1/2-4/5 in Chinese cabbage. The stage of late uninucleate was optimal for anther and isolated microspore culture.2.Anther culture: The effects of genotype and cultural medium on embryoid induction were investigated in spring and summer Chinese cabbage. The results showed it was optimal to induce the embryoid in Keller medium with double organic ingredients and 10% sucrose. The capacity of embryoid production varied significantly in different genotypes, and genotype 126 was an ideal material for embryoid induction.3.Isolated microspore culture: Embryoids were obtained from four cultivars, and the embryoid induction frequency varied significantly among different cultivars. Low-temperature pretreation on donor plant (3-5 "C for 48 hours) in NLN liquid medium did not yield more embryoids.The embryoid induction frequency reached the highest when isolated microspores were cultured in NLN-13 supplemented with 6-BA 0.2mg/L and NAA0.05mg/L.4. Regeneration from embryoids. Propagation and transplantation: The frequency of plants regenerated from embryoids was 72.73% for anther culture, while 54.61% for isolated microspore culture. Propagation of regenerated plants: a large number of plantscould be obtained by induction of shoot in the 85 medium supplemented with 6-BA1.0mg/L and NAA0.25mg/L. More than 90% of the plantlets survived during transplantation after acclimation in plastic pot for more than a week.5 .Ploidy identification: The ploidy character of the plantlets were primarily identified by observing the phenotypic appearance as well as the stoma density on the leaf surface. The ploidy feature were further determined by chromosome microscopical observation and Flow Cytometer technology. Chromosome identification showed that the number of haploid plantlets chromosome is n=10,while 2n=20 for diploid. The DNA count of haploid plantlets was equal to half of diploid .6.Chromosome doubling: The ratios of natural chromosome doubling of varied from 40% to 70% for different genotypes. It was optimal for chromosome doubling inducement by culturing the plantlets in 85 medium with colchicine 100mg/L for 10 days .7.Regeneration of leaf: The experiment showed it was optimal for leaf regeneration when the treatmeat was in the 85 medium supplemented with 6-BA 2.0mg/L, NAA 0.5mg/L, GA1.0mg/L, ABA 0.25mg/L and AgNO3 5.0mg/L.