Plant genetic transformation has arguably been the core of plant improvement in recent decades. Efforts have been made to develop in planta transformation systems due to the limitations present in the tissue-culture-based methods. Herein, we report an improved in planta transformation system, and provide the evidence of reporter gene expression in pollen tube, embryos and stable transgenicity of the plants following pollen-mediated plant transformation with optimized sonication treatment of pollen. The results showed that the aeration at 4°C treatment of pollen grains in sucrose prior to sonication significantly improved the pollen viability leading to improved kernel set and transformation efficiency. Scanning electron microscopy observation revealed that the removal of operculum covering pollen pore by ultrasonication might be one of the reasons for the pollen grains to become competent for transformation. Evidences have shown that the eGfp gene was expressed in the pollen tube and embryos, and the Cry1Ac gene was detected in the subsequent T1 and T2 progenies, suggesting the successful transfer of the foreign genes to the recipient plants. The Southern blot analysis of Cry1Ac gene in T2 progenies and PCR-identified Apr gene segregation in T2 seedlings confirmed the stable inheritance of the transgene. The outcome illustrated that the pollen-mediated genetic transformation system can be widely applied in the plant improvement programs with apparent advantages over tissue-culture-based transformation methods.

The aim of this work was to explore the potential toxicological effects on rabbits of transgenic poplar (Populus cathayana Rehd.) leaves with binary insect-resistance genes used as feed. Fifty-four 40-d-old weaned New Zealand White Rabbits (Oryctolagus cuniculus) (live weight 0.98±0.1 kg) were fed for 70 consecutive days with a common pelleted diet and fresh poplar leaves containing a chitinase-BmkIT gene combination or untransformed counterparts (60 g/d). Rabbit body weight and hematological and biochemical data in blood samples were recorded. Organ histological structures were observed and the organ weights in the 2 groups were also measured. The results of the growth study revealed no significant differences (P>0.05) for final mean BW of rabbits, intake of the combined feed and poplar leaves or feed conversion ratio between the 2 groups. No obvious pathological changes were observed in the small intestine, stomach, spleen, kidney, lung, heart, bladder, pancreas, prostate and ovary. Electron microscopic observation of liver cells and renal cells showed they were both normal in the 2 groups. All hematological and biochemical data tested fell within the normal range in the 2 groups after 70 d of feeding. We conclude that the poplar leaves with the chitinase-BmkIT gene combination had no obvious harmful effects on rabbits.

We conclude that the processed poplar leaves with foreign chitinase-BmkIT genes had no obviously harmful effects on rabbits.

Two germinating seed transformation methods i.e., scarifying and puncturing methods, were used to transfer the bar gene into an inbred maize line. We generated transgenic glufosinate-tolerant maize plants, and conducted a comparison of the two transformation methods. We used the plasmid pCAMBAR.CHI.11 as the donor and germinating maize seeds as the recipients. The germinating seeds were subjected to puncture or scarifying treatment after being soaked in water for 12 h. The injured seeds were co-cultivated with Agrobacterium for 24 h and then sown in the experimental plots. Putative transgenic maize plants were selected by basta tolerance screening and PCR detection, and were further confirmed by Southern blotting. Following glufosinate tolerance assay and molecular detection of transgenic lines of the transgenic seedling (T0), first generation of transgenic seedling (T1) and second generation of transgenic seedling (T2), we have confirmed that the bar gene was stably inherited and expressed. The PCR amplification result for T2 transgenic plants showed that the genetic segregation ratio of the bar gene followed the 3:1 ratio of a single dominant Mendelian factor. The study proved that both the germinating seed treatment approaches are rapid and simple plant transformation methods. In particular, the application of the puncture method can expand the tissue culture free transformation to dicotyledonous plants.