Study On Genetic Transformation Of Populus Simonii×P.nigra Against Fungal Disease

Poplar belongs to Salicaceae, Populus. There are more than one hundard kinds in the world, while more than fivty-seven in China. So, poplar is more important than other trees for not only protecting ecology but also advancing country economy development. But, poplar is damaged by fungal diaeases all the time, such as Melampsora spp., Botryosphaeria ribis, and so on, which lead to huge loss for the country economy every year. For ecology, the unremedi-ed loss is much bigger. Populus simonii×P nigra is one of the main strains popularized for its high values, such as growing fast, good quality, high adaptability etc.. So we select the Populus simonii×P. nigra as the research material. Several factors, such as pre-cultivated time, Agroba-cteria concentration, infection time, co-cultivated time and selecting methods which impact percentage of transformation were investigated in the research.Chitinase is a group of proteen which is expressed when plants are endanged by fungi. It can hydrolyze chitin which is main and important component of the cell wall of fungi except for Oomycetes in order to prohibit growing of fungi.In this study, the culture medium for leaf regeneration was MS+0.5mg/L 6-BA+0.1mg/L NAA and for rooting was WPM+0.4mg/L IBA. The target gene was foreign chitinase. Then the transformation of Populus simonii ×P. nigra was studied basing on the above preconditions. Through sensitivity experiment of leaves to Kanamycia, the Kanamycin concentration of 40 mg/L was determined when leaves differentiation, rooting by inducing bud concentration of Kanamycin was 20mg/L in rooting culture medium. Concentration of Cefazolin Sodium as antibiotic to inhibit the growth of Agrobacterium was 700mg/L and this concentration didn't have bad impacts on bud inducing and rooting for Populus simonii ×P. nigra during the experiment. Successfully established the transformation procedure Agrobacterium-mediated of Populus simonii×P nigra: cut vigorous leaves to 2-3 patches through the main vein after growing about 20 days, pre-culture time was 3⁴days;centrifuge Agrobacterium when being developped to the logarithm growth time(OD₆₀₀=0.5), then dilute Agrobacterium to the concentration of OD₆₀₀=0.3-0.4 with MS liquid culture medium or asepsis distilled water for infection;infection time was 6-15 minute;after being co-cultured 3 days, inoculate on the selecting medium to be filtrated in succession during inducing unsureness buds and rooting with Kanamycin(containing Kanamycin 40mg/L and Cefazolin Sodium 700mg/L). The target gene-foreign chitinase was transformed into Populus simonii×P nigra by Agrobacterium mediated transformation. The theory base was given for Populus simonii ×P. nigra transformation and fastness breeding.The molecule detections were done. Some transformants were identified by using PCRamplification and thirteen positive transformants were achieved. The positive transformants emergenced differential amplification strips while negative comparison didn't. Then by PCR-Southern analysis, the differential hybridizing strips appeared and the positive frequentcy was 100 percent. The results showed that the foreign gene has been integrated into genome of Populus simoniixR nigra. The highest percentage of transformation for leaves Populus simoniixR nigra was 4.12%.