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Research on Agrobacterium-mediated Transformation System of Banana (Musa Paradisiaca) Thin-cross-section

Author: HuangTongMou
Tutor: PengMing
School: Hainan University
Course: Biochemistry and Molecular Biology
Keywords: banana Agrobacterium-mediated genetic transformation thin-cross-section
CLC: S668.1
Type: Master's thesis
Year: 2010
Downloads: 40
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Abstract

Banana is the fourth big grain crop in the world and has very important status in world grain production. Many biotic and abiotic stresses such as plant disease, frost, and typhoon threatened seriously the production of banana. It is little effect for the improvement of new M. paradisiaca varieties by traditional breeding method, due to the diverse ploidy and highly sterile characteristic of M. paradisiacal. With the development of molecular biology, it is of more significant to obtain transgenetic banana idioplasm, which has resistance, disease-resistant insect pest, high production, and high fruit quality.In this study, Agrobacterium-mediated genetic transformation was performed in Musa paradisiaca.,cv.Brasil (AAA Group) and the regeneration of transgenic plants was achieved. The thin-cross-sections from low-generation test-tube seedling were co-cultured with A. tumefaciens, which contained a plasmid vector harboring the Ubi promoter, GUS gene, and the phosphinothricin as a bar gene. The bar gene was used for selection of the transformed tissues. The main results were listed as the following:1. Established the indirect organogenesis regeneration system for the thin-cross-sections of low-generation test-tube seedling and the male flower thin slices. Our results revealed that both of the thin slices could produce high regeneration rates and thus could be used as good explants to establish efficient indirect organogenesis regeneration system in the future. In this paper, different explants were used to detect the organogenesis regeneration system. Experimental results are as follows. The thin-cross-sections from low-generation test-tube seedling in the conditions of 0.5-1.0 mg/L 2,4-D+4-6 mg/L 6-BA, and the male flower thin slices in the hormone conditions of 0.5-1.0 mg/L 2,4-D+0.2-0.5 mg/L ZT were able to achieve efficient regeneration through Cauliflower body way. At the same time, cauliflower body thin slices in conditions of 1.0 mg/L 2,4-D+5 mg/L 6-BA were able to achieve efficient regeneration cycles by Cauliflower body way.2. Determined the preferable type and concentrations of Agrobacterium inhibitor and antibiotic. The thin-cross-sections from low-generation test-tube seedling were sensitive to Cefotaxime and Timentin. The budding percentage was not effected when the concentration of Cefotaxime and Timentin was 150 mg/L and 300 mg/L, respectively. The inhibitory ability of antibiotic for thin-cross-sections from low-generation test-tube seedlings follows the order:PPT>Hygromycin>Kanamycin. Our results demonstrated that kanamyin has no significant effects and so were not recommended to use as a selection agent in the direct regeneration system. The appropriate selection concentration of Hygromycin was 20-40 mg/L, and the appropriate selection concentration of PPT was 0.1-0.2 mg/L.3. Compared the banana’s sensitivity to two positive selection agents (D-Mannose and D-Xylose) by using the thin-cross-sections from low-generation test-tube seedling as explants. The results showed that banana was highly sensitive to D-Xylose. The explants showed symptoms of toxicity on the tenth day when it was cultured on the medium containing 10 g/L D-Xylose and 20 g/LSucrose. The buds of explants were gradually inhibited when 16 g/L D-Xylose and 14 g/L Sucrose were used. The explants completely died if 25 g/L D-Xylose and 5 g/L Sucrose were supplied. Therefore, D-Xylose could be used as a selection agent for banana positive selection system. D-Mannose was not significant effects on banana explants, and it can not be used as a suitable selection agent for banana positive selection system.4. Optimized the Agrobacterium infection conditions. The best transient expression of GUS was obtained under the conditions as follow:the Agrobacterium suspension was with OD600 value of 1.0 and supplemented with 100μmol/L Acetosyringone at pH 5.2, and the samples were infected for 10 min and co-cultured for 4 days at 26℃in a dark room.5. Established Agrobacterium-mediated genetic transformation system of thin-cross-section from low-generation test-tube seedling in M. paradisiaca. Using this system, the GUS gene was transformed to banana (Musa paradisiaca) thin-cross-section and transgenic seedlings were obtained. The PCR assays proved the target gene was integrated into the plant genome. The histochemical assays revealed that the GUS enzyme was steadily expressed in the transgenic plants.

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