Development Of Pol CMS Restore Lines With Cold Resistance By Microspore Culture In Brassica Napus

Microspore culture of Brassica napus was carried out in this research, and several factors that affected microspore embryogenesis and plantlet regeneration of embryoids were explored and improved. One double haploid population derived from (zhongshuang 4R DH×P28) F1 was constructed successfully. Utilizing antifreezing identifying method identified the Fl DH population and selected stronger antifreezing lines in field.Genetic variations of the F1 DH population was analysed by AFLP in laboratory, and selected stronger antifreezing DH lines which carry with the antifreezing gene from material of P28. The main results are as follows:1. The materials of (zhongshuang 4R DH×P28) F1,P28 were performed microspore culture, and several factors that affected microspore embryogenesis were discussed in the experiment. The results are as follows: The microspore embryogenic ability is controlled by genotype, and different genotypes of Brassica napus show significant difference in ability of embryos yields, even if the same material, there is a significant difference of embryos yields among different DH lines; Different concentration of colchicine effect the embryo yields and doubling efficiency of microspore culture; Not only could Solid-liquid double-decker culturing significantly promote the embryogenic of embryoids but also are conducive to the growth and development of embryos; activated charcoal can promote the embryogenesis of embryoids remarkably, especially some materials that have poor embryogenesis ability.2. The embryoid of several Brassica napus L. was studied in the research, several factors that affected plantlet regeneration ability were discussed in the experiment. The results are as follows: The genotype affect plantlet regeneration of microspore embryoids; Different concentration of colchicine effect plantlet regeneration and doubling efficiency of microspore culture; The plant regeneration frequency was closely related to embryo developmental stage, the bigger the embryo was, the easier to become seedlings, and the seedlings mostly came from the ripe cotyledonary embryos; The low-temperature pretreation on embryoids at 5℃can promote plant regeneration of embryoids remarkably; Medium B5 with 0.1 g/16-BA can increase the frequency of plant regeneration of embryoids.3. The doubled haploid (DH) population derived from restore lines Yu-137 and Yu-127 by microspore culture were constructed successfully including 556 and 195 lines respectively. Cultured the seeds of (zhongshuang 4R DH×P28) F1 DH population in Wuhan HZAU experiment field and Gucheng mode field on the contrast in the same time. Meet the coldest temperation since 1958. Writed down the type antifreezing identifying data of (zhongshuang 4R DH×P28) F1 DH population, and analysed antifreezing index. Utilizing antifreezing identifying method identified the F1 DH population and selected stronger antifreezing 28 DH lines in in the tow fields5.The genetic variation of (zhongshuang 4R DH×P28) F1 DH population was analysed by AFLP in this research. The results are as follows: Genetic diversity of F1 DH population demonstrated that the genetic variation among DH line population is relatively big. The 20% average polymorphisms of Amplified products were detected in the experiment. the range of similarity coefficient(SC) in the DH line population varies between 0.52-0.97. The DH population was divided into 10 groups and every group was divided into many subgroups. By the AFLP technology we selected stronger antifreezing DH lines which carry with the antifreezing gene from material of P28.