| The Brassica oleracea L.var.botrytis is a variation of Brassica oleracea . It is accepted by customers because of its taste, anti-cancer and cancer prevention. At present, China owns the biggest planting area and is the highest output country for Brassica oleracea L.var.botrytis, but its output and quality is seriously influenced by many kinds of plant disease. TheB.nigra is one of the three Brassica and always grows in wild. The research showed that BB genome ofB.nigra has many kinds of plant disease resistant genes. At the same time, studies on somatic hybridization of Brassica oleracea L.var.botrytis and B.nigra have definited genetic background and obtained addition lines, and analysis were conducted with more resistant black mustard broccoli somatic hybrid and asymmetric into and back into the offspring groups in morphology, chromosome structure and the molecular level. It has an important significance of molecule identification and modifing the genetic diversity of Brassica oleracea .L through Somatic Hybridization.The study results were described as fellows:1. Study on the regeneration of protoplast, the result shows that there are many factors affecting the regeneration frequency of B.nigra and B.oleracea protoplasts, and illumination, osmotic of medium and culture conditions had a significant impact on cell division and shoot regeneration frequency. But the illumination influence is the most in these factors, in light culture, the activity and division of protoplast are bad, and there are many breakage. But in dark culture, the division of protoplast was very strong. Therefore, it was the important condition of obtaining the high production of protoplast in dark culture. In addition, there was no different shapes in using different radiation, but the higher dose of radiation, the easier for the protoplast fusion to breakdown and the ratio of hybrid cell was lower.2. The analysis of the somatic cell hybrid was carried on through the morphology, cytology, and in situ hybridization.The result showed that 35% DNA content of hybrids was lower than the DNA content of parents, and 34% the number of chromosome of hybrids was lower than that of the parents. Because that the statistical results of two methods were quite close, therefore it was discovered that the number of chromosome and the DNA content were positive correlation through the SPASS. Finally this research determined the gene composition of addition lines through GISH, and discovered that the different hybrid or the identical hybrid contained the different number of chromosome, in general, it had 2-8 chromosome of B. nigra. It explained that the chromosome had the equational division. 3.In this research, the DNA-methylation variations of three selfing offspring (S2, S3 and S4) and three backcrossingoffspring(BC1, BC2andBC3) derived from Cauliflower- black mustard somatic hybrids were analyzed by using MSAP. The results showed that it had no significant difference among three selfing offspring (S2, S3andS4)and backcrossing offspring(BC1, BC2andBC3). But the cytosine methylation level of backcrossing offspring was higher than that of selfing offspring. Further analysis of DNA methylation variation among parents and different offspring showed that the methylation variations were mostly classified as B type (e.g.offspring had the same type of sites with one of the parents). This methylation partiality indicated that DNA-methylation variation was not irregular, but tended to be a variation of the parental types, this may be involved parental dominance in crossing process, and it was also the possible result of exclusion of those negative variation in the formation of new species. |
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