| Tomato is a kind of staple vegetable, and it is also one of the earliest plants researched via gene engineering. Now, using biotechnology, many kinds of tomatoes have been produced, such as maturity-delaying tomato, pest and disease-resistant tomato, herbicide-resistant tomato, adversity-resistant tomato, high-quality tomato and so on. Introducing foreign genes from various species into tomato genome using molecular biology technology has been an efficient means to produce new varieties with some good characters.Defensin is a kind of Cys-rich anti-microbial peptides widely existing in animals and plants. They have broad-spectrum poisoning effects on many kinds bacteria, fungi and virus. Chitinase can degenerate chitin. It can inhibit the growth of paghogen by disrupting the new generated chitin in the tips of hypha, and so plays an important role in defending the invasion of phytopathogenic fungi. Some chitinase have the function of bacteriolysaze, and so can inhibit the growth of some bacteria species.In this study, a stable and efficient transformation procedure for tomato material Micro-Tom was established, and the alfalfa defensin gene alfAFP, the balsampear chitinase gene McChitl and their bivalent gene (AC) were introduced into Micro-Tom. The resistance of their transgenic TI generation to Ralstonia solanacearum were also investigated. The main results are as following:1. Several factors influencing transformation frequency of Micro-Tom were investigated. The results showed that, when using cotyledon as explant the highest regeneration frequency after transformation could be obtained (76.0%), compared to leaf (28.7%), and hypocotyl (19.9%); 2d were optimal for co-cultivation; Adding AS in resuspending medium MSB and co-cultivation medium was indispensable to the transformation of Micro-Tom, but its concentration was not critical:50umol/L is sufficient. This procedure was stable and efficient, with average transformation frequency of more than 20%, and transgenic plants growing normally.2.GUS histochemical staining and PCR analysis of transgenic plants with alfAFP gene, McChitl gene, and AC gene showed that, these foreign genes had been integrated into tomato genomes.3.In vitro crude protein bacteria-inhibiting experiments of three kinds of transgenic plants showed that, the alfAFP and McChitl gene have no synergistical effects on the resistance to R. solanaceanan. In the extracts of AC, alfAFP plays the main role in ihibiting bacteria; The extracts of McChitl also have effects on R. solanaceanan.4. Through the root inoculation of plantlets and the stem stabbing inoculation of full-grown transgenic plants of T| generation with AC gene, it was demonstrated that transgenic plants had enhanced resistance to R. solanaceanan than non-transgenic plants. In the experiments of the root inoculation, transgenic lines L-9, L-21, L-12 could postpone the appearing of symptoms for 1d, 2d and 4d, respectively. When infection stopped aggravating 11d after inoculation, the incidence of disease of control was 100%, and the transgenic lines L-9, L-21, L-12 were 33.3%, 44.4% and 11.1% lower than control, respectively. In the stem inoculation assay, L-21 showed increased resistance. In the first 10d after inoculation, control was infected quickly than L-21. On the tenth day, the disease index of L-21 was 38.34% lower than control. But lOd later, the disease index of control increased slowly, but L-21 was infected continuously. At the time of 30d after inoculation, the difference of the disease index of the two was not significant (5%). And the disease index of L-21 reached 100% too. After inoculation it could be seen that the area of disease spots in the stems of transgenic plants were smaller than non-transgenic plants.5. GUS staining and x2 test of the germinated seeds of TO generation selected randomly from three transgenic lines with AC gene indicated that the segregation conformed to Mendellian law. |
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