Loquat Anther-derived Embryos Histological Study, Transformation Mediated With Agrobacterium Tumefaciens And EMS Chemical Mutation

In this experiment, the origin of loquat anther-derived embryos was investigated by histology. After the establishment of a protocol for loquat anther-derived embryo transformation, CycD2 gene was transformed to loquat anther-derived embryos with Agrobacterium tumefaciens mediation. Loquat anther-derived embryos were used for EMS chemical mutation and molecular changed after mutation treatment in some materials. The main results were as follows:1. Histological studies revealed that the embryos developed through the typical globular, heart, torpedo and cotyledon stages. The meristematic region came into being after the torpedo embryo was formed, and it was much more visible when embryo developed into the cotyledonary stages. When the secondary embryogenesis underwent the direct pathway, embryos originated from the epidermal cell between mesocotyl and hypocotyls. When the secondary embryogenesis underwent the indirect pathway, embryos arose indirectly from subepidermal cells of calluses derived from primary embryos. These calluses were composed of small cells with densely stained nucleus and cytoplasm. The cell walls of the embryogenic cells thickened and their cytoplasm and nuclei gradually became condensed and visilble under light microscope. 2-cell proembryos, 4-cell proembryos and muti-cell proembryos were formed with the division of embryogenic cells.2. After co-culture with Agrobacterium tumefaciens EHA52, loquat anther-derived embryos were transferred to selection medium. Four antibiotics were evaluated for their effects on eliminating the Agrobacterium tumefaciens EHA52 from loquat anther-derived embryos and the production of secondary embryos. Exposure of the embryos to carbenicilin did not eliminate the Agrobacterium tumefaciens, and ampicilin, cefotaxime, gentamicin sulfate were effective for eliminating the Agrobacterium tumefaciens. Secondary embryo production was significantly reduced by all the antibiotics. 500-1000mg/L ampicilin was the optimal antibiotic concentration to select the loquat anther-derived embryos after transformation.3. The selectable marker CycD2 gene was used for multiplex PCR, and the results showed that the transformed embyos expressed the CycD2 gene. So we could draw a conclusion that the CycD2 gene was successfully transferred into the anther-derived embryos.4. The death rate of loquat anther-derived embryos was very high when the EMS concentration ranged 0.1%-0.9% and the time of treatment was 0.5h, 1h and 2h, respectively. Most of the loquat anther-derived embryos were black-surfaced and dead. The percentages of the survival embryos after EMS treatment were all under 50%. Maximal survival rate was obtained in the treatment of 0.3% EMS for 0.5h and minimal survival rate in by 0.7% EMS for 2h. In comparison with the CK, the secondary embryo induction rate decreased after the EMS treatment.5. There were 11 treatment differences from the contrast when using s55 as the primer, 11 treatment differences when using s232 as the primer, 13 treatment differences when using s377 as the primer, and 3 treatment differences when using s362 as the primer. 0.3% EMS for 2h was the most effective in all treatments.