Wong, Jovita Jun (2018) Genetic transformation of Sabah traditional rice for fungal resistance using Agrobacterium-mediated technique. Masters thesis, Universiti Malaysia Sabah.
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Abstract
Tadong is one of Sabah traditional paddy. It bears purplish-black grain and cultivated at highland area. It contains high level of anthocyanin which is an antioxidant, anti-hyperglycemic agent. However, the production of Tadong is low due to the fungal invasion, such as rice blast fungus - Magnaporthe oryzae. Genetic transformation technique is a biotechnological approach for the production of rice variety with enhanced ability on fungal resistance, hence improving yield. Thus, the aim of this study was to develop a transformation protocol of Tadong rice with fungal resistant gene through Agrobacterium-mediated technique. First, callus was induced from sterilised seed cultured on N6 medium (1975) containing different concentrations of 2,4-D (0, 0.5, 1.0 and 1.5 mg/L) and BAP (0, 1.5, 2.0 and 2.5 mg/L). Second, three days pre-cultured calli were selected and inoculated with liquid culture of Agrobacterium tumefaciens strain LBA4404 harbouring plasmid pMDC140-GUS-wwin2-hptII. Factors influencing the transformation efficiency, such as co-cultivation periods (0, 1, 3 and 5 days), infection periods (10, 20 and 30 minutes), pH of co-cultivation medium (5.3, 5.7 and 6.0) and incubation temperature during co-cultivation process (23, 25 and 30°C), were evaluated. The expression of GUS gene in infected callus was tested using histochemical GUS assay after 3 days of co-cultivation period. Next, calli infected with the optimised transformation were selected on selection medium for 10 weeks. Total RNA of hygromycin-selected T0 transgenic calli were extracted and the RNA concentrations were quantified. The integration of wwin2 and hptII genes were confirmed using reverse-transcriptase PCR (RT-PCR) while the expression of the integrated wwin2 and hptII genes were checked using real-time PCR (qPCR). Results showed the maximum callus induction (74.0% ± 0.07) was obtained from the seeds cultured on N6 medium containing 1.0 mg/L 2,4-D and 1.5 mg/L BAP, while combination of 4.0 mg/L BAP and 0.1 mg/L NAA produced the highest shoot regeneration frequency (63.3% ± 0.14). The best transformation efficiency of Tadong callus (93.3% ± 0.01) was achieved with 3 days co-cultivation period, 30 minutes of immersion period, medium pH of 5.7 and 25°C of co-cultivation periods. After 10 weeks of selection periods, 2 calli out of 1057 transformed calli (0.18%) survived in medium containing 10 mg/L hygromycin and 25 mg/L cefotaxime. RT-PCR and qPCR analysis of these calli had proven the presence and expression of wwin2 and hptII genes in transgenic Tadong callus. Results from gel electrophoresis for cDNA of wwin2 and hptII after PCR process produced 65 bp and 77 bp of amplicon sizes, respectively. Real-time PCR analysis on both genes in tested transgenic callus showed expression [Ct hptII = 27.64(TC1) and 22.70 (TC2), Ct wwin2 = 29.80 (TC1) and 24.28 (TC2)]. In conclusion, this transformation protocol can be used to transform the genome of Tadong. Several modification, especially during pre- and post transformation, need to be carry out to increase the transformed callus recovery of transformed cells, and regeneration frequency Tadong species in the future study.
| Item Type: | Thesis (Masters) |
|---|---|
| Keyword: | Genetic transformation, Tadong, Agrobacterium, Regeneration, Transformation efficiency |
| Subjects: | S Agriculture > SB Plant culture > SB1-1110 Plant culture > SB183-317 Field crops Including cereals, forage crops, grasses, legumes, root crops, sugar plants, textile plants, alkaloidal plants, medicinal plants |
| Department: | FACULTY > Faculty of Science and Natural Resources |
| Depositing User: | DG MASNIAH AHMAD - |
| Date Deposited: | 24 Mar 2025 16:26 |
| Last Modified: | 24 Mar 2025 16:26 |
| URI: | https://eprints.ums.edu.my/id/eprint/43307 |
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