Plasmid DNA of pBI121 containing GUS gene and fresh pollen of rape Jinyou 7 were mixed in 7.5% sucrose solution and treated with ultrasonic. After that, pollens were collected, pollinated on stigmas of rape. With pollen tube growth through the style tissue and entering the ovaries, the target gene was integrated in rape genome. Transformed plants were confirmed by field character identification(Fig.2), histochemical assay of GUS expression(Fig.3), DNA dot blot hybridization(Fig.4), PCR amplification(Fig.5) and PCR-Southern blot hybridization(Fig.6).
In the study, some regeneration conditions, such as the concentration of sucrose solution, the intensity of ultrasonic and the effective of auxiliary agent for transformation system were discussed. The highly efficient transformation system on rape was established with 7.5% of sucrose solution (Fig.7) or 5% of manna sugar solution (Fig.8), 300 W of ultrasonic (Fig.9).
Compared with other approaches, the pollen-mediated transformation approach can avoid the tedious tissue culture procedures like in Agrobacterium infection and particle bombardment, etc. This approach is simple, easy to operate, and can be widely used in breeding practice.
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