| Peanut is one of the important oil and economic crop. A flavus infection, an worldwide problem, affects the production, processing and trading of peanut, and is serious in South China because of the special climate. As no germplasm highly resistant or immune has been found out so far, the problem can not be solved by traditional breeding method, but it could possibly be solved by transforming antifungal gene to peanut cultivars. Based on this method, the study has transformed the binary antifungle genes, Chitinase and P -1,3-Glucnase gene, mediated with A. tumefaciens into peanut cultivars, Quanhua No. 10 and Jinhua 1012, to improve their anti-invasion potential. The results were showed as below.The regeneration system of four kinds of peanut explants were partially modified and complemented in this research, including advantitious shoots(As), somatic embryogenesis(Se), embryo leaflet(El) and cotyledon adventitious shoots(Cs). MS medium containing 5mg/L BA was demonstrated again as the relative optimum medium to induce shoots differentiation and growth. It has some merit to delay shoots elongation when minifing the remnant cotyledon in A.s explants preparation, for it made the explants earlier to assimilate the nutrition in MS medium without ill effect on shoots induction number. And, many adventitious shoots were seen differentiating from the leaflets sampleing from the above shoots in MS with high BA. There was close relationship between Se induction efficiency and peanut post-harvest time. The result showed that the indued buds number and velocity from Se explants of peanut seed stored for about 4 to 6 months were more than that of newly mature seed harvested. Much callus and many somatic embryos could be induced and differentiated in MS medium containing 40mg/L 2,4-d and 0.5mg/L KT, and the regeneration period could be shrunk 10d earlier for somatic embryos when being transferred to MS+5mg/L medium to induce buds elongation directly. MS medium containing both 5mg/L BA+2mg/L NAA or 5mg/L BA+3mg/L NAA could induce much callus and adventitious buds from El explants, and the latter medium could induce much more callus and buds than the former, but with more degree of browning.Km-resistance trial showed that buds elongation could be inhibited to the maximal extend in 200mg/L of Km in medium, and roots could not grow out in50mg/L of Km. The transgenic research studies were made with four kinds of explants as mentioned above, results showed that 21 positive transformants were got assayed by PCR method for As transgenic method, 11 plants in Quanhua No. 10 and 10 in Jinhua No. 1012. It was verified that the target genes had been integrated into the Genomic DNA of transformants with PCR-Southern blot method. Now, several descendant seeds from the TO transformants are being propagated in culture medium to be assayed inheritance and diverse of the target genes, and the resistance to Aflavus. It was not good to invade the explants more than 15 minutes with A. tumefaciens liquid. In addition, no or only Id pre-cultivation could improve transformation efficiency, but 2d pre-cultivation didn't show such result. Transformants have not yet been got through Se and El transformation ways but several scores of Km-resistance buds have been obtained and needed following induction and assay, and the better transformation efficiency was seen with 10~20min invasion in A. tumefaciens liquid and 72hr co-cultivation after it. Especially, it was found that more transformants would be got when Km screening more carried in buds elongation stage in Se transformation way. Although labouring transformtion trials were conducted in Cs method, only 1 Km-resistance explant was got, and the most limited factor was browning problem except for seed vigor and cultivars difference. In addition, there were some difference between the two used peanut cultivars during transformation. The A. tumefaciens strains was exchanged as the former strain was insensitive to biotics.In order to observe the similarity and compare the different transformat... |
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