Studies On Transformation Of Sweet Orange With PthA-NLS For Resistance To Citrus Canker And Transformation Of Ponkan With Chit42 Gene

Citrus is an important fruit crop for agricultural economy in the world, and the production is inhibited by citrus disease which severely occurres worldwide. Agronomic practice and bactericide can only solve the problem in some context, and some bactericide can cause heavy environmental pollution. These make the disease resistance breeding a best selection for fighting against the bacterial pathogens. Conventional disease resistance breeding is difficult because of polyembryony, apomixis, long juvenility, high heterozygosity and parthenogenesis; as well as limitation for reproduction barrier among different species. Resistant genes cloning and transgenic approach offers a potentially bactericide-free and environment-friendly solution for disease pathogen control. In this study, pthA-NLS gene and Chit42 gene were introduced into Citrus sinensis (Linn.) Osbeck and Ponkan (Citrus reticulata Blanco) by Agrobacterium-medialed transformation. Shoot-tip grafting was performed to complete the regeneration system.PCR and RT-PCR were used to analyze the transgenic plants.Disease resistance test indicated that resistance was improved on some transgenic strains. And the resistance mechanism of pthA-NLS in citrus was discussed. The main results are as follows:1. The plasmid of 'pthA-NLS' was constructed and transfered to Citrus sinensis (Linn.) Osbeck by Agrobacterium-mediated. Nine transgenic strains were obtained through PCR, RT-PCR and Southern blot analysis. Disease resistance test indicated that resistance to canker was obviously improved on some transgenic strains.2. An efficient regeneration system by direct organogenesis from epicotyl of Ponkan (Citrus reticulata Blanco) in vitro was set up. Seven methods were tested for regeneration, MS without any hormone resulted in the most effective one, with the rates of bud formation efficiency to 83.3% and much stronger buds were got in shortest time .100mg/l Km was used to control chimeric buds. Shoot-tip grafting was performed to complete the regeneration system. Three trangenic strains were confirmed from 18 strains through PCR analysis.3. Seven methods were tested for the sterilization of mature materials, and the combination of pre-sampling and post-sampling disinfection (innovation method) resulted in the most effective one, with the rates of the contamination and death reduced to 12.2% and 27.2%, respectively. After sterilization with the innovation method, the percentage of the alive internodal segments rates raised to 30.6%. Furthermore,the medium supplemented with 1 mg/l BA and 0.5 mg/l NAA was the best one. The regeneration frequency and bud formation efficiency reached to 12.8%. Shoot-tip grafting was performed to complete the regeneration system. It was found that the buds smaller than 5 mm in length gave higher graft success than that with longer shoots, and the best results were obtained with 1 mm buds. This probably due to more endo-fungi existed in the bigger shoots than in the shorter ones.One buds of 12 was confirmed to transfenic strain by PCR analysis.