| Fungal diseases are the major factors, which limit crop production for their negative effect on yield and quality. Some toxins secreted by a few kinds of fungal pathogens often contaminate grain and affect human being's health.Agronomic practice and fungicides can only solve the problem in some extent, and some fungicides can lead to heavy environmental pollution. These make the disease resistance breeding a best selection for fighting against fungal pathogens. Conventional breeding, although contributed much to today's crop production, is time-consuming and limited for reproduction barrier among different species. Transgenic approach offers a potentially fungicide-free and environment-friendly solution for fungal pathogen control.As the most popular and widely consumed vegetable, tomato is a model species for genetic engineering research. Tomato production is affected by dozens of fungal diseases. This make the anti-fungal genetic engineering of tomato an important project. Synergistic expression of plant defense genes appears to be a preferred avenue for engineering crop protection.This research aimed at obtaining some anti-fungal transgenic tomato lines via Agrobacterium-media.ted transformation. Three pathogenesis-related genes - the bean chitinase gene (Chi), tobacco β-1,3-glucanase (Glu) gene and tobacco AP24 and two tomato cultivars - A53 and ZS5 were involved in this study. The main results are as follows:1. The effects of different concentration of foreign zeatin and IAA on tomato cotyledon regeneration were investigated. The results indicated that high level of zeatin induced the high ratio of abnormal regenerating shoots, and also inhibited the rooting of the shoots. The normal regenerating shoot ratio reached the highest (39.7%) for the cultivar A53 under 0.2mg/L of zeatin together with 1.0mg/L of IAA.2. The transformation method was improved with a two-step selective culture, in which a primary selection with high level of zeatin as 2.0mg/L was followed by a secondary selection with decreased zeatin level (0.2mg/L) supplemented with high level of IAA (1.0mg/L). The transformation efficiency was raised and the rooting process was accelerated.3. A total of 109 putative transgenic tomato plants were obtained. Among them, 67 plants including 44 transformed with AP24IChi and 23 transformed with ChilGlu wereregenerated from the cultivar A53; 42 plants including 34 transformed with AP24/Chi and 8 transformed with Chi/Glu were regenerated from the cultivar ZS5. In the 23 plants transformed with Chi/Glu, 6 were regenerated via hypocotyl explants. And 8% of the transgenic plants were predicted as tetraploidy plants from the investigation of the chlorophyll number per guard cell pair.4. The PCR analysis showed the ratio of positive transgenic plants was 93%. Southern blot analysis of the putative transgenic plants showed that the T-DNA was integrated into the tomato genome, with different copy number.5. Northern blot and disease resistance test results from some transgenic plants indicated that most of the foreign gene expressed. The resistance was improved on some plants, while the foreign genes were silent in a few transgenic plants.6. Tail-PCR was utilized to study the flanking region of the T-DNA right border integrated into the genome. This further identified the integration of the foreign gene, and the sequencing result indicated that some special phenomena such as deletion and border exceeded transfering occurred during the T-DNA integration.7. The Kanamycin spraying test method for positive transgenic tomato plants selection developed by Weide et al was improved. The modified method can be used for large-scale screening of positive plants, segregation investigation and homozygous transgenic line acquirement both in greenhouse and on open field.8. The E. coli expression vector of AP24 was constructed for further determination of the translational level of the foreign gene in the transgenic plants.9. The complete CDS of pr p23 in tomat...
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