Preliminary Study On The Establishment Of Genetic Transformation System In Maize Parthenogenesis Haploid Embryos

According to the diploid embryo regeneration and genetic transformation conditions, this study used maize parthenogenesis haploid embryos as explant, and optimized some of the conditions. Ultimately, this study successfully explored the methods of ploidy identification of haploid immature embryos and obtained haploid regenerative and positive transgenic plants, in addition, preliminary established the parthenogenesis haploid immature embryo genetic regeneration and transformation system. Besides, using haploid mature embryos as receptors for genetic transformation without the phase of callus culture, this study directly differentiated and regenerated haploid plants. Therefore, this study explored a set of new genetically modified method, provided a new possibility and direction for the application of haploid breeding. At the same time, the differences of subcellular structure between haploid and diploid and hybrid in the process of genetically modified were analyzed in this study, which provided a certain theoretical support for the haploid genetic transformation system. Specifically, this study got the following results:(1) This experiment induced haploid by the cross between EDI as male parent and 18-599R as female parent. Using the characteristics of inducer’s marker genes, this experiment screened the callus that cultured from immature embryos by the means of light. Finally, the callus of heterozygous individuals showed purple, the callus of haploid individuals were still yellow. The analytical result showed that light method for screening the haploid individuals was not significant, but different light time reached extremely significant level. The haploid callus could be successfully screened under the light time of 20 and 40 h, But because callus appeared green under the level of 40 h, it was not conducive to the cultivation of the later experiment, we finally confirmed the light condition for 20 h,2000 lx.(2) The light screen could not completely get rid of hybrid individuals and the expression of marker genes associated with the gene type and maturity, we needed to further determine accurate haploid individuals, so this experiment utilized a set of method including light, chromosome observation and flow cytometry detection. Eventually this experiment screened out 110 haploids from 3000 immature embryos, haploid rate was 3.67% that was similar to the EDI induction rate (3.5%), so as to guarantee the consistency of materials.(3) The experiment optimized the hormones ratio through orthogonal design in the process of differentiation and found that the haploid rate of treatment 13 is significantly higher than other disposes. Analyzed the different factors of different treatment concentration, the results were not significant. Therefore, at present the experiment only got the optimal dispose of the haploid callus differentiation:KT (0.5 mg/L)+6-BA (0.5 mg/L)+ABA (0.5 mg/L)+2,4-D (1 mg/L)+NAA (0.1 mg/L). So the further study should extend the range of non-significant factors’level, so as to screen out the best level of main factors.(4) This experiment successfully got 13 haploid regeneration plants,15 regeneration double haploid plants and 4 haploid positive individuals which regenerated and transformed from the 18-599R haploid immature embryos. At the same time, the experiment used Hill haploid mature embryos as the receptor of genetic transformation that pollinated by EDI after 12-14 d. Then regenerated plants directly, skipping the mature embryo difficult process of callus induction and culture stage. Finally, the haploid mature embryos genetic transformation got 14 haploid positive individuals.(5) Through the observation analysis of haploid and diploid and hybrid callus subcellular structure, the experiment got the following results:the average cell wall thickness:haploid> diploid>hybrid. The nucleus size of callus is very different. The haploid had more mitochondria in the observation area. One hand, the thicker cell wall of haploid callus could protect the fragile haploid cells. On the other hand, it also could provide a certain theoretical basis to optimize the conditions of haploid genetic transformation, accordingly agrobacterium infection of concentration and time would be changed. More mitochondria might because the haploid weaker vitality need to make sure its strong respiration so that the haploid could adapt and resist the influence of the external environment for its survival.