Breedubg, Application And Sterile Mechanism Of CMS451 In Cabbage (Brassica Oleracea Var. Capitata L.)

Cabbage is a leafy vegetables widely cultivated all over the world, which is a cross-pollinated crops with significant hybrid heterosis. Using male sterile line obtain hybrid cultivar is a tendency of cabbage breeding. Cytoplasmic male sterility (CMS) is an ideal method for heterosis breeding, and it has an important and practical value on cabbage breeding. CMS451 cultivated by backcrossing cabbage cytoplasmic male sterile P451.CMS451 is fully express infertility, normal nectaries physiological function, no yellow leaves at low temperature and high combinationability. In order to ascertain sterile mechanism of CMS451, we have studied it from botany, cytology, physiology and biochemistry and molecular biology. It laid the foundations for furthermore utilize the beneficial of this excellent sterility source. The main results of the research are presented as follows.1. Morphological comparison between CMS451 and its maintainer showed that CMS451 has shorter filament and whiter anther. It could not crack and spill out pollen normally, but its nectary is normal. The bud of CMS451 develops slower than its maintainer. The anther became yellow gradually when the bud length grew to 6.5 mm. The plumpness of CMS451 was lower than its maintainer.2. CMS451 and its maintainer as female parent were crossed with same male parent, the results shows that F1 generation characters of botany be similar and there was no significant difference, it has the shorter head central axis, the more heavier head and the better quality. The combining ability of CMS451 was higher than its maintainer. The nectary of CMS451 was much more active, it has fertility and attract insects normally. The sent number and stub higher than maintainer, the yield is high and the purity of hybrid was 100%, so the cost of breeding was low. We believe that CMS451 has great application and development prospects in Cabbage breeding.3. Microspore cytological observation showed that pollen abortion of CMS451 happened at uninucleate stage. The tapetal cells and middle layer cells divided gradually, the cells were vacuolization and elongation, microspore squeezed together and nutritious substance was lost. Subsequently, the tapetal cells separate from the middle layer cells completely, and then the tapetal cells to disappear in the end. Microspore stick always together with the degradation of tapetal cells, formed into deep staining material. The mature pollen can not release from the dehisced anther lead to microspore gradual disintegrated, the remains of something that form a black band.4. Enzyme-linked immunosorbent assay (ELISA) method was employed to study the changes of endogenous hormones level and the balance between hormones in different developmental stages of the bud. The results showed that the content of GA3 and ZR in buds of male sterile lines CMS451 were lower than its maintainer line, they had same changing trends in male sterile line and its maintainer. IAA content of CMS451 was higher than its maintainer, trends of IAA had a change of descend firstly then ascend while it was continuous decrease in maintainer. The content of ABA in CMS451 was higher than its maintainer at microspore mother cell stage, and the other stage just the reverse. This shows that the reason of cytoplasmic male sterility was an excessive accumulation of IAA and ABA in buds of Cabbage male sterile lines and the deficiency of ZR and GA3 in the early developmental stage.5. Analysis of the free amino acid showed that the level of free amino acid in CMS451 leaves and small buds is slightly different from its maintainer, but the difference is not obvious in total amino acid level. In CMS451 large buds, the level of free amino acid was significantly higher than its maintainer. In the leaves, small buds and the large buds, the male sterile line is higher than maintainer for the content of glycine, valine, isoleucine, leucine and phenylalanine, just the opposite for proline. The reason of male sterility supposedly was an excessive accumulation of glycine, valine, isoleucine, leucine, phenylalanine and the lack of proline in the organs of reproduction and vegetation account for male sterility in Cabbage.6. The reactive oxygen and major antioxidant enzyme activity was analysed in Cabbage male sterile line and its maintainer, results shows that rate of (?) production, the content of H2O2 and enzymes activity of MDA, POD,SOD and CAT in male sterile line buds were higher than its maintainer during different developmental stages. It was presumed that the excess generation of active oxygen in male sterile line lead to the anti-oxidant enzymes level increased to remove them. In the buds of male sterile line, SOD and CAT activity decreased at stage of mature pollen grain, while H2O2 and MDA activity increases continuously, oxidant and anti-oxidant are not to maintain in a state of equilibrium so that proteins cross-linking in the plasma lemma, this state will eventually result in metabolic disorder and damage of biomembrane lead to infertility of male.7. PCR results demonstrated that the Cabbage CMS451 mtDNA amplified the 594bp specific band. The DNA sequence of CMS451 was coincident to specific band Z12626 of Ogura CMS, Z12626 contains two open reading frame orf138 and orf158.The sequence (1-228) of CMS451 and the sequence (189-417) of orfl38 were completely the same sequences, downstream sequence of CMS451 and upstream of orfl58 were completely the same sequences. These results indicated that the CMS451 mtDNA contain orf138 and orf158 in accordance with OguCMS, the origin of CMS451 is OguCMS; in addition, genes related to cytoplasmic male sterility are highly conserved.