Observation Of Microspore Development And Research On The Regenerated Embryo Cultivation System For High Oil Rapeseed(Brassica Napus L.)

Rape isolated microspore culture technique has important application value in rapeheredity and breeding. Through this technology, we can not only quickly gain homozygotes,shorten the breeding years to improve the breeding efficiency; but also can use embryoids andregenerated plants to construct the genetic map, gene direction, and mutation breeding.Sixteen F1hybrids of high oil content were used as meterials, and the forms ofmicrospores in each developmental stage were observed. in isolated microspore cultivation,the pH variation in sterilized process of B5medium were explored,and studied the factors onmicrospore induction cultivation,such as microspore developmental stages, genotype, budlength, bud low temperature pretreatment, hormone, colchicine and so on. Trying to prove upthe best condition and essential technology of the high embryo rate and embryoid survivalrate, in order to establish the regeneration system of the high oil content in Brassica napusL..The main results of the studies are:1. After sterilization15²0min, the pH of B5medium decreased from0.40to0.55.The medium B₅-13used for isolated microspore is appropriate in the pH of6.20⁶.40beforesterilization.2. The morphological characteristics of small spores from the tetrad stage to maturitywere observed, and the microspore shape is relatively different at different developmentalstages. The late period mononuclear microspore nucleus was near the cell wall and nuclearstaining was apparent, the shape of a nearly circular crack-like cell wall outer layer with awavy plastic pattern. Most strains of small spores late mononuclear corresponding bud lengthwere in the2.5³.0mm.3. The affection of low-temperature pretreatment to materials embryo rate was notobvious, and moisture and the whole inflorescence preservation were benefit for extendingthe flower bud culture time.4. The cell enlargement rate significantly increased after treating by the50mg/Lcolchicine for48h, and it can promote the formation of embryoid. Small spores in12-28h hada large number of cell enlargement, but in the end, only a few swollen cells growed into embryoids.5. Embryoid induction rate of different genotypes were significantly different. Eight ofthe16tested materials embryoid were obtained, in which y138×y145had the highestembryonic rate, reaching in19/bud;1479×HZ had the lowest embryonic rate, reaching in0.6/bud.6. Different spores' materials respond differently to the hormone type and concentration.By comparing the two combination, four hormones concentration ratio on microspore culture,we obtained the results as follows: add0.1mg/L2,4-D and0.05mg/L (or0.1mg/L),6-BAconducive y138×y145and other4material in embryonicin NLN to add0.05mg/L NAA,0.05mg/L6-BA is conducive to1438×HZ and other4materials the embryo;7. In B5solid medium, added0.1mg/L2,4-D and0.1mg/L6-BA compared with0.1mg/L GA3is more beneficial for embryoids to rapid growth. The closer the embryo shaped ascotyledon, the faster growth after inoculation, and higher seeding rate can be obtained.