Optimization For The Technical Of Isolated Microspore Culture Of Cabbage (B. Oleracea L. Var. Capitata) And Broccoli (B. Oleracea L.Var. Italica)

Cabbage and broccoli belong to biennial herb plants of Cruciferae Brassica. Because both of them are of a wide range of self-incompatibility. People usually take many years to get inbred lines by selfing during bud period. An ideal homogeneous inbred material needs6～8generations selfing by the conventional methods. We can acquire homozydous plant offspring through isolated microspore culture and plant chromosome doubling. It can not only shorten greatly the breeding process of inbred lines and new hybred varieties, but also benefit the development of the germplasm resources.Now, there are lots of reports on the plant isolated microspore culture of the plants, and the concerned technology is more and more maturing. The difference of embryo rate among genotypes and low plant regeneration rate become serious obstacles for this technique. In addition, we also lack a simple, economic, practical and reliable ploidy identification method.Therefore, the paper tries to find the factors affecting both microspore embryo rate and plant regeneration rate of17different genotypes of cabbage and10different broccoli hybrids. The method identifying the chromosome ploidy of regeneration plants through microspore culture, and the method doubling haploid plant chromosome by aritificial are developed for the selected cabbage and broccoli materials. Meanwhile the spontaneous double rates of chromosome in regeneration plant groups by microspore culture are investigated. The results are following:1. Optimizing for the factors affecting microspore culture induction in cabbage and broccoli:(1) The optimum sizes of buds among the test materials are different, the optimum bud sizes are3.5～4.5mm and3.0～3.5mm for cabbage and broccoli respectively. Most of the microspores for this size buds are in mononuclear stage, and highest yield of embryo can be obtained.(2) The microspore embryonic induction rates in different genotypes are greatly different. (3) The optimal conditions combing temperature and pH value in medium of microspore embryoid are below: For cabbage:33℃heat shock for2d+pH6.2medium; For broccoli:Treatment under4℃for1d+33℃heat shock for1d another+pH6.2medium.(4)8%mannitol in extract liquid helps the microspore embryo genesis for both cabbage and broccoli;(5)13%sucrose culture medium after2d preliminary culture in17%sugar medium can increase the embryonic induction.(6) The medium combination of NLN-13+0.05mg-L-16-BA+0.1g·L-1NAA is conductive to microspore embryonic induction in cabbage; while1/2NLN-13medium has stimulative effect for microspore embryonic induction in broccoli.(7) Pretreatment under low temperature for2d and some chemicals such as8%mannitol, appropriate concentration of CH and colchicine can promote the microspore embryonic occurence rate of cabbage.2. The factors affecting plant regeneration from microspore embryony are following:(1)7.0g-L-1aguar in B5solid medium is optimal for microspore embryony germination and plant regeneration, there are higher frequency of plant regeneration of cotyledon-shaped embryos than noncotyledon-shaped embryos.(2) Embryos with cotyledons is after25d culture in solid medium have higher plantlet rate when they are transfered to regeneration medium.(3)4.0～5.0mg-L"1of AgNO3in medium can effectively restrain the occurence of browning and vitrification of regeneration plants.3. We set up a relationship between microspore ploidy of regeneration plants and the number of chloroplast in stomatal guard cells of cabbage and broccoli. The primary results are below:the haploid plant has ten or less than ten chloroplats in its stomatal guard cell, from ten to fifteen chloroplats for diploid plant, more than fifteen chloroplats for polyploid. It’s the most effective method of microspore regeneration plant chromosome doubling to add suitable concentration of colchicine into liquid meduim during microspore culture stage.Therefore, this paper clarifies the factor affecting microspore embryonic incidence and plant regeneration rates, and developed a new accurate and fast ploidy identification method-chloroplasts ploidy boundary method. Finally it finds out a high efficient chromosome doubling method, after comparing results of several tests.