Creation Of Stay Green Pure Lines By Microspore Culture In Brassica Chinensis L

Stay green refers to plants in the late of reproductive stage blade remains green without yellowing phenomenon, it has special significance for Pakchoi with green leaf organs as a product. For the purpose of rapid creation homozygous breeding materials of Pakchoi staying green, the research using Pakchoi staying green 12A446 that preliminary study to find as female material, hybridized combinations-’421’,’424’ and ’426’ are formulated with excellent inbred lines of Pakchoi. Its F1 and F2 screened excellent Pakchoi staying green as test materials, the research on the optimization of Pakchoi microspore based on the isolated microspore culture technology, by using the method of isolated microspore culture to created rapidly DH lines. The main findings are as follows:1. Different genotypes have an impact on the embryogenesis rate of Pakchoi. In the tested three pakchoi are successfully obtained the microspore embryoids, Pakchoi’426’with the highest rate of the embryo, which is 10.54 embryos per bud; Pakchoi’421’of the embryo the lowest rate, which is 2.96 embryos per bud.2. For different genotypes of pakchoi cultivars, through in NLN-13 induction medium added three kinds of histone deacetylase inhibitors-Trichostatin A (TSA). suberoylanilide hydroxamic acid (SAHA) and sodium butyrate (NaB) to improve the microspore embryogenesis. We have found that 2μM NaB in NLN-13 medium is the optimum concentration for lines ’421’ and ’426’, which resulted in the highest incidence of embryos (6.83 and 34.53 embryos per bud, respectively). And the optimum concentration of TSA for genotype’424’is found to be 0.05 μM in NLN-13 medium, which resulted in the largest embryo yields (8.54 embryos per bud). Then it also has a significant role in promoting the rate of direct plant regeneration, while reducing callus formation rate. The direct plant regeneration rate of Pakchoi’421’in the treatment of 2μM NaB reach the highest (72.90%), which is 1.60-fold compared to the control, and the formation of callus rate is also the lowest (9.96%); In 0.05μM TSA treatment, the direct plant regeneration rate of Pakchoi ’424’ is the highest (78.33%), it improved 1.17 compared with the control group, which reaches the lowest rate of callus formation (10.16%); In the treatment of 0.5μM NaB and 0.1 μM SAHA. it has the highest rate of direct plant regeneration (88.36%) and the lowest rate of callus formation (3.08%) in Pakchoi’426’.3. With Pakchoi 421,424 and 426 as tested materials, it improve its of embryogenesis microspores rate by adding two kinds of cellular antioxidants in embryogenesis medium-Vitamin C (AA) and methylene blue (MB). Among them, the appropriate concentration of AA and MB are 0.2μM and 5～10nM respectively. They also play a significant impact on somatic embryos developed into regenerated plants. The highest rate of direct plant regeneration and the lowest rate of callus formation happen in the treatment of 0.2μM AA. In lOnM MB treatment, Pakchoi ’424’ has the highest rate of direct plant regeneration(73.16%), while the ’421’ and ’426’ have the highest rate of direct plant regeneration in the treatment of 5nM MB, and rate of callus formation is as the lowest as 20.78% and 11.68% respectively.4. By adding 0.05mg·L-1 6-BA and 0.1mg·L-1 NAA NLN medium as control group in Pakchoi ’426’ , it has the highest as 20.71 embryos per bud with the treatment of adding 0.05uM TSA, and it also increases in SAHA (0.075μM) and NaB (2μM) treatment relative to the control group.5. When the plantlets root, in the conventional culture medium MS adding 0.10 mg·L-1 NAA, it is helpful for Pakchoi to rooting, and The highest are obtained with 1/2 MS,0.5 g·L-1 activated carbon and 0.5 g·L-1 IB A, the optimal combination will be beneficial to improve the survival rate of plantlets after transplanting.6. The highest natural doubling rate of Pakchoi 426 regenerated plants is 63.10%, and the lowest rate of Pakchoi 421 is 48.08%, then the average rate reaches 57.95%.7. On the basis of optimized culture system, the number of Pakchoi staying green DH lines created is 687 by using isolated microspore culture technology, which derived from ’421’ is 142, from ’424’ is 190, and the source from ’426’ is 355. It is identified the part of DH lines on horticultural traits.