| Galanthus nivalis agglutimn (GNA) is an ideal antigen for the prevention and the cure to the harm made by pests with piercing-sucking mouthparts. The plant expression vector pLG66m was constructed by inserting the gna gene into the upper part of the gus gene which is located in the vector pBI121, and it was then transformed into Agrobacterium tumefaciens LBA4404 by freeze-thaw method to construct a binary vector system LBA4404/pLG66m for transforming Medicago. In this system, gna and gus gene were linked together and formed a fusion gene which be transcribed by the same promoter CaMV35S. Consequently, this strategy allowed to monitor the transfer and integration of T-DNA in plant chromosome by histochemical detection of GUS activity. The propose of this study is looking for a simple, economic method to transform foreign gene into medic.The transformation method involves soaking of young seedlings with Agrobacterium, is a new, simple and rapid transformation method. It can avoid tissue culture-based regeneration systems. Medicago truncatula has a relatively small genome and is diploid and autogamous, as a model legume for molecular genetic analyses, it was selected as experimental plant to find optimal conditions of this transformation method to Medicago. Regarding quantity of the bactera absorbed and infiltrated in plant cell as an index, the vernalization time, the concentrations of Silwet L-77 and the time of soaking seedlings with bacteria were considered synthetically during experiments, the optimal condition for enhancing the ability of infection of A. tumefaciens was concluded. The optimal treatment of bacteria as following: the seeds dealt for 24 hours for germination at 25℃, and then soaked in solutions of Agrobacterium suspended in 0.05% Silwet L-77 for 5-10mins.According to the studies on distribution of A. tumefaciens on plant cell walls, the expression of vir gene, and the transient expression of gus gene, the feasibility of this method for transforming to barrel medic was tested. Observation in Confocal Laser Scanning Microscopy, the bacteria could enter into the cellular gap and gather to mass on the cell wall. The revulsant such as sucrose and acetosyringone could obviously stimulate vir gene to express during soaking seeds with A. tumefaciens. The transient expressing efficiency of gus gene in young treated seedlings was more than 90%. The expression of the gene was going to decrease obviously when the third real leaves outgrew. Expression of gus gene in medic was detected by histochemical method. This proved that foreign genes could enter plant cellular nuclei and express efficiently through seedlings soaked directly in A. tumefaciensThe feasibility of soaking treatment method for other alfalfa transformation was experimated. During the experiments, 15 common cultivated varieties of alfalfa were selected as experimental materials dipped directly in A. tumefaciens. By histochemical detection of GUS expression, there were only 7 varieties appearing with blue spot or streak among the 15 varieties, M. saliva cv.'Zhaodong' was the most sensitive, which the ratio of GUS expression was 10.5% , but its expression was much lower than that on the model legume. From above, I concluded that the factors and conditions applicable fortransforming M. truncatula were not suitable for all of the Medicago genotype. Therefore, the simple transformation method with seedlings soaking in A. tumefaciens need further improve in practical application.
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