| Poplar is an important sylvicultural and commercial tree, which is very popular throughout china and other places of the world.G1 (Populus deltoids) is a kind of hybrid popular.It is the main strain of been popularized for its high values such as growing fast, good quality, high adaptability etc..So we select the Populus deltoids as the research material.Several factors such as hormone concentration were investigated to optimize the leaf regeneration system in vitro.A high frequency regeneration system in culture has been established.Chitin and glucans are the most important components of the cell wall of many fungi.Chitin, which is a biopolymer of N-acetyl-D-glucosamine in a β-1,4 linkage, can be degraded to GlcNAC by chitinase.β-1,3-glucanase is a protein that catalyzes the hydrolysis of glucans which is a polymer of glucose.Many experiments show that these two hydrolyses present strong inhibition to fungal growth both in vivo or in vitro.In our study, two expression vectors contain chitinase gene and β-1,3-glucanase gene respectively were constructed.Based on the high frequency regeneration system of Populus deltoids, we obtained lots of transgenic plants through the developed Agrobacterium tumefaciens mediated transformation method.Conclusions are drawn and shown as following:First, a high frequency regeneration system of Populus deltoids has been established.The results shown that different culture medium can greatly effect on Populus deltoids's growth, differentiation, regeneration, root growth etc.The best shoot elongation medium is MS supplemented with 0.15mg/L 6-BA, 0.1mg/L NAA, 0.1mg/L KT, 20mg/L Ad; the best leaf regeneration medium is MS supplemented with 0.5mg/L 6-BA, 0.1mg/L NAA; the best root differentiation medium is 1/2 MS supplemented with 0.1mg/L NAA.The effect caused by other physical factors, such as pH value, concentration of ager, temperature, humidity, also have been analyzed.Second, we developed the traditional Agrobacrium tumefaciens mediated transfomation method and made it more efficient.Chitinase gene and β-1,3-glucanase gene had been transformed into plants by continuous transformation method.Lots of transgenic plants have been obtained.Third, the transformants were identified by the PCR analysis and PCR-Southern analysis.The results showed that these two genes had been integrated into Populus deltoids genome.Forth, poplar disease fungi can be inhibited by the transgenic plants's cell extract in vitro.The effect of inhibition which had been caused by the transgenic plants with chitinase gene and β-1,3-glucanase gene is better than that only with one gene of the obove two.It is proved that the transgenic plants have had resistance to fungal disease. |
