| Seventeen varieties were used for isolated microspore culture in Chinese cabbage. The factors that influence microspore embryogenesis, microspore plant regeneration, subculture and rootage were studied in the research. At the same time, the methods of ploidy identification for microspores-derived plants and uniformity identification of horticultural characters from the DH line progenies and hybrids developed with DH lines were studied as well. The aim of research is to establish an efficient technique system of isolated microspore culture in Chinese cabbage and accelerate the speed of application of isolated microspore culture in breed practice. The results are as follows:1. The optimal sample period of microspore culture in Chinese cabbage was identified. The analysis results of the relationship between bud form indexes and microspore development degree indicates that when bud length was between 2.0-3.49 mm and the ratio of petal to anther length was between 1/2-5/4, 75% of the pollen was in the late uninucleate stage in Chinese cabbage. The stage of late uninucleate was optimal for microspore culture.2. The embryogenesis ability of different genotypes varies a lot. 15 genotypes among 17 get embryoid successfully in the research. The embryoid induction rate is 88.24%. The highest embryo yield is "caiyuan" with 244 embryos per thirty buds.3. Low salts content are benefit to Chinese cabbage embryogenesis. Compare to NLN-13 medium, reducing the content of major salts of NLN-13 medium by half can not only increase the genotypes that can be induced to get embryo but also can remarkably raise the embryoid induction rate.4. 6-BA and NAA have some effects on Chinese cabbage embryoid induction and development. The suitable content proportions are 6-BA 0.05+NAA 0.1 mg·L-1 and 6-BA 0.05+NAA 0.2 mg·L-1.5. Agarose and active carbon in suitable content can promote embryoid induction and development. The suitable content is 25 mg·L-1 agarose and 0.5 g·L-1 active carbon.6. 4℃low-temperature pretreatment can increase the frequency of microspore embryogenesis. The optimal treatment time is 24 h. Another function of low-temperature pretreatment is keeping materials fresh for isolated microspore culture temporarily.7. 33℃high-temperature pretreatment at the beginning of suspension culture is necessary to embryogenesis, and the optimal treatment time is 24h.8 The developmental stage of embryos has crucial effect on the plantlet regeneration. Cotyledon-shaped embryos are the optimal stage to transfer to plantlet regeneration medium. 9. The optimal medium for plantlet regeneration of microspore-derived embryos is "MS+3% sucrose +1.0% agar +100 mg·L-1 active carbon" and plantlet regeneration rate is 88.70%.10. "MS+3% sucrose+0.8% agar" is the optimal medium for subculture and renewal of Chinese cabbage, "1/2MS+NAA0.1 mg·L-1+3% sucrose+0.8% agar" and "MS+NAA 0.1 mg·L-1+3% sucrose+0.8% agar" are suitable mediums for rooting.11. The methods of ploidy identification of microspore-derived plant include direct identification with chromosome numbers and indirect identifications by comparing the characters of organs, tissues and cells such as stoma size, mature pollen microspore size and so on. Finally, the result can be validated by self-fruitfulness ability of microspore-derived plant.12. The distributions of haploid, diploid, polyploid and chimera microspore-derived plants from different genotype materials are different in proportion. The rate of spontaneous diploidization is in the range of 41.30～6.66%.13. Analysis of horticulture characteristic indicates that DH line obtained in the experiment and the hybrid combinations developed with it are uniform. It also indicates that the DH lines are pure lines. The requirement to uniformity of commercial hybrid can be satisfied by using it as parental lines.14. DH plants obtained in the experiment have conspicuous self-incompatibility and live up to the standard of self-incompatible line. |
