Study On Tissue Culture And Agrobacterium-mediated Genetic Transformation Of Acacia Crassicarpa

This paper concentrated the studies on the optimization of Acacia crassicarpa tissue culture system, establishment of genetic transformation receptor systems and Agrobacterium-mediated genetic transformation. The main results indicated as follows:1. The tissue culture system of Acacia crassicarpa was optimized by the orthogonal design and a series experiments. The test results showed that: After using 98% concentrated sulphuric acid treatmented 30 min and boiling water bath treatmented 10 min and using 75% alcohol treatmented 30s and 0.1 % HgCl₂ treatmented 10 min to Acacia crassicarpa seeds for sterilization, and then using the way of embryo down for inoculation and 1/2 MS medium for the seeds culture , the germination rate of the seeds achieved to 69%.In the first culture, the medium formula was MS+6-BA 2.0mg / L+KT 1.0mg / L+NAA 0.1 mg / L. In the first subculture, the medium formula was MS+6-BA 1.0mg / L+KT 1.0mg / L+NAA 0.1mg/L. In the second subculture, the medium formula was MS+6-BA 1.5mg / L+KT 1.5mg / L+NAA 0.5mg / L. In the rooting culture, the medium formula was MS+NAA 1.0mg / L+IAA 1.0mg / L. In the sound plantlet culture, the medium formula was MS+KT 0.6mg / L+ NAA 0.1 mg / L. When the plantlets were transplanting, smithfield and nursery medium (1:1) was used as the matrix.2. The receptor system on genetic transformation of Acacia crassicarpa was established by the experiments of hormone combinations and the experiments of antibiotics selection. The test results showed that: Using 6-BA combined with IBA had the better effect of adventitious buds induction than that of using 6-BA alone and 6-BA combined with NAA or IAA, the best using concentration combination was 6-BA 1.5mg / L+IBA 0.5mg / L and the differentiation rate of leaf-plate reached to 62%. The recovery selection pressure of kanamycin to Acacia crassicarpa leaves showed 50 mg / L, the rooting selection pressure was 150 mg / L. Using cephalosporins adriamycin on the leaf-plate differentiation had few effects, but using streptomycin sulfate and ampicillin affected the differentiation greatly. Therefore, cephalosporins adriamycin was identified as the main antibacterial antibiotics and the using concentration was showed 100～200mg / L.3. The genetic transformation system of Acacia crassicarpa by Agrobacterium-mediated GUS gene was established by the comparing experiments of different bacterial strain, bacterial concentration, infestation time, preculture(cocultivation) time and selection mediums. The test results showed that: The preculture or dark culture was used for 5d. The bacterial strain that named At06 was used for the infestation, which the strain concentration showed that OD₆₀₀ value was 0.5 and the infestation time was 10 min. The cocultivation was taken for 3d . The medium was added AS 50 mg / L and sugar 30 g / L, which the pH value showed 5.8. After cocultivation for 3d, the genetic transformation of Acacia crassicarpa was taken by the way of slection medium . After the culture on the selection medium for 5d, the explants was taken to detect the transient expression rate of GUS gene, which the transient expression rate was fond higher and it reached to 92%. After the first selection culture, bud elongation culture (the second selection), rooting culture (the three selection), 4 kanamycin resistance plantlets were got from 200 explants.