| Plasmid DNA of pB1101- Chi- Bmk was mixed with fresh pollen of maize inbred line, 478, in 15% sucrose solution . The pollen suspension was treated with ultrasonication and treated pollen were collected and pollinated on maize silks. Transformants were screened by Km selection and verified by PCR amplification. The transformation rate was 1.4%.Certain factors affecting pollen-mediated transformation in maize were also studied and discussed in the thesis, which include sucrose concentration for pollen suspesion, ultrasonication intensity, and pollen germination stimulator, etc. The optimum transformation conditions in present study were 15% sucrose solution for pollen suspension and 100~200W for ultrasonication intensity.The feasibility of polled-mediated approach in maize transformation was also confirmed by histochemical and cytological observations.Comparing with other transformation methods, the pollen-mediated transformation approach could circumvent the tedious tissues culture procedures like in Agrobacterium infection and particle bombardment, etc. This approach is simple, easy to adopt, and could be widely used in breeding practice. |
