Study On Microspore Culture And Methods Of Ploidy Identification Of Plantlets In Cabbage(brassica Oleracea L. Var.capitata)

Cabbage(Brassica oleracea var. capitata) is one of the important vegetable crops in China,with a planted area of 90,0000 hectares.In the process of the conventional breeding, Cabbage belongs to a type of typical green body vernalization,it’s difficult to add generation reproduction and its breeding cycle is too long, general speaking,the purification of inbred lines takes time of at least 6 years. To obtain stable homozygote quickly and efficiently can shorten the breeding cycle greatly and improved the efficiency of breeding, The microspore culture technology can achieve the requirement in two years.regeneration plant of isolated microspore has the natural double ability. There were great differences among the ploidy of regeneration plants with the proportion of different genotypes, for the breeding,we need to identify the ploidy of regeneration plants and provide basis for artificial doubling of the plant on later.The 108 cabbage varieties were used for the isolated microspore culture. a lot of the embryoids and Regenerated plants were obtained from the isolated microspore culture.Analyzing the phenotypic of plant characteristics to study external factors of cabbage microspore embryogenesis; Flow cytometry technique, the regular of the size of cabbage leaf stoma and the chloroplast numbers of guard cell were applied to explore the efficacy methods of ploidy determination.The main results were as follows:(1) The embryonic generation ratio in early maturity, spherical type,spring cabbage are all above 50%;The number of species in middle-maturing cabbage varieties accounts for 10% of all the number of oblate spheroidal style, 6.25% of oblate spheroidal style; There were 15 types of late-maturing cabbage with no embryonic development, There were no embryonic development in 8 winter cabbage varieties. The microspores of cabbage with the characteristics of spherical type, Early-maturing variety, spring cabbage were easy to produce embryoids, cabbage with another characteristics like the oblate spheroidal style, middle-maturing and late-maturing variety, winter cabbage were difficult to produce embryoids.(2) The average naturally doubling rate of the cabbage microspore regeneration plant was 27.12%, the overall was low.And there were great differences in naturally doubling rate in different genotypes.(3) By studying the number of chloroplast surrounding the different ploidy regeneration plants,we obtained a simple method to identify the ploidy of cabbage. The rule formed by the number of chloroplasts surrounding the stoma guard cells in different microspore-derived plants is: the number of chloroplasts in haploid, diploid and tetraploid is 6-9, 9-12 and 12-23, respectively. The distribution range of the number of chloroplasts is more extensive, as the increase of ploidy, In practice, according to the number of chloroplasts observed by microscopy, the ploidy level can be determined.(4) The length range of guard cells in haploid, diploid and tetraploid is 18-23μm, 25-35μm and 35-45μm, respectively. The perimeter range of guard cells in haploid, diploid and tetraploid is 18-23μm, 25-35μm and 100-115μm, respectively. The width of guard cells largely changes as stoma size changes, but there is no obvious rule here. The method for measurement of the length of stoma guard cells was proved to be a simple way of estimating ploidy of the microspore-derived plants in the situation that the length and the perimeter of the stoma guard cells both can estimate the ploidy.