Studies On The Technical System Of Anther And Isolated Microspore Culture In Root Mustard (Brassica Juncea Var. Megarrhiza Tsen Et Lee)

Root mustard (Brassica juncea var. megarrhiza Tsen et Lee) is a mutation of the mustard that belonged to the Brassica of family Cruciferae. Its production organelle is swelling root. Our kohlrabi breeding process lags others’, most of the cultivars are retained species. There are 5-8 years to select homogenicity inbred lines in the conventional breeding. However, the process of breeding should be accelerated using anther culture or isolated microspore culture, and obtain homozygous doubled haploid plants in 1-2 years.Two different methods were used in this study, the anther culture and the isolated microspore culture conducted with the lion head mustard, the famous root mustard varieties. The main factors that affect microspore embryogenesis, regeneration of embryoid, transplantation of regenerated plant, and ploidy identification of transplantation in the variety were studied. The main aim was to ascertain a technology for anther culture and isolated microspore culture of kohlrabi. The main results are as follows:1. The length of the bud is divided into 6 levels:under 2.0mm、2.0-2.5mm、2.5-3.0mn、3.0-3.5mm、3.5-4.0mm、beyond 4.0mm, through observations of microspores.The result showed that when the length of bud is 2.0-2.5mm, the proportion of microspores in the late uninucleus is highest, and is consideredv the best time to culture haploid cells.2. Three types of media were considered as best to study effect of different basic medium on the germ extraction rate in the progress of anther culture, MS、B5 and reformative Keller, there were no embryoids in MS-6 and B5-6 and germ extraction rate is 0.36 in reformative Keller. The results indicates that reformative Keller is more suitable for anther culture of root mustard.3. To study the effect of different pre-cooling times on germ extraction rate, the material were treated with different time including Od、1d、3d and 5d under 4 temperature. The result indicate there is significant difference in embryoid induction between 5 days treatment and 3 days treatment under 4 temperature, the induction of 5days treatment is the lowest. Though there are differences in germ extraciton quantity and germ extraction rate among 3 types treatment:pre-cooling Od、1d、3d, it can not reach the significant levels, while the germ extraction rate of 3 days treatment is the highest.4. After inoculation into the culture medium of anther, the experiment set 3 different heat shock gradient, the materials were treated with with heat shock Od、1d、2d under 32.5 temperature.The result indicates there is significant difference in germ extraction, when the materials are treated with 1d、2d under 32.5 temperature and CK. There was no significant difference between lday treatment and 2day treatments, while 2 day treatment is much better.5. By using NLN-13 liquid culture and NLN-13 solid-liquid double culture in the isolated microspore culture study, the influence of the two cultures on the isolated microspore embryogenesis was studied. The result showed no significant difference between the two cultures on embryogenesis rate.6. Pre-cooling treatment of the medium containing mannitol on isolated microspore culture of the embryo, showed that anther embryogenesis rate was highest in the anthers treatment 3 days on the B5-13 solid culture medium with 15g/L mannitol at 4℃. Compared with treatment of bud 3 days at 4℃and bud treatment 3 days on the B5-13 liquid culture medium with 15g/L mannitol a 4℃, there was significant difference; While therewas no significant difference between bud treated 3 days on the B5-13 liquid culture medium with 30g/L mannitol at 4℃and bud treaed 3 days on the B5-13 liquid culture medium with 45g/L mannitol in 4℃.7. Four hormones,6-BA、NAA、KT and 2,4-D, which are the most widely used hormones, were selected and their influence on orthogonal test and three levels of four factors on the kohlrabi embryogenesis in the isolated microspore culture was studied. The result showed that the treatment with no hormone was the best combination, and influence by the four factors from large to small is as the follows:NAA、6-BA、2,4-D and KT.8. Identifying the number of chloroplast of stomatal guard cell on ploidy of transplants, the observed number of chloroplast of normal diploid plant ranged from 12～18, and the observed number of monoploidranged from 3～13, and the observed number of diploid ranged from 12～20. The number of the chloroplast of normal diploid plant was comparatively more concentrated, and its number range from 14 to 18, in which the proportion of the five amplitude was 94.1%, and the number of guard cell chloroplast in the diploid of transplans ranged from 14～19, in which the proportion of the six amplitude was 90.1%. The number of guard cell chloroplast of monoploid ranged from 4～11, and showed a wide range of variation, in which the proportion of the eight amplitude was 92.4%, 9. Materials identitied in the normal diploid control of ploidy with flow cytometry, reveals that there were only monoploid and diploid, no polyploidy and chimera, and the natural doubling rate reached 50%.