Agrobacterium–Mediated Transformation Of Dendrobium Broga Giant Orchid With Chitinase Gene
| dc.contributor.author | Mohammad, Jasim Uddain | |
| dc.date.accessioned | 2017-02-02T06:23:16Z | |
| dc.date.available | 2017-02-02T06:23:16Z | |
| dc.date.issued | 2016-02 | |
| dc.description.abstract | Dendrobium Broga Giant (Dendrobium Bobby Messina × Dendrobium superbiens) is a new hybrid orchid in Malaysia. This study has established Agrobacterium-mediated transformation protocol using protocorm-like bodies (PLBs) to produce transgenic Dendrobium Broga Giant (DBG) orchid with an antifungal gene. A simple and competent micropropagation technique was established for the successful PLBs proliferation of DBG. Half-strength of Murashige and Skoog (MS) semi-solid media supplemented with 1.0 mg/L BAP and 0.5 mg/L NAA produced the highest PLBs proliferation. The interaction between Agrobacterium and PLBs was evaluated based on bacterium motility and attachment capability. Mildly wounded PLBs displayed higher Agobacterium motility as well as attachment capacity compared to other treatments. Successfully transformed PLBs were evaluated using selection agents such as geneticin (G-418), kanamycin and neomycin to determine the minimal inhibitory concentration required. Geneticin displayed highest effectiveness with 50 mg/L completely killed the non-transformed PLBs and allowed the survival of the transformed PLBs. Several factors (PLB size, wounding, immersion time, co-cultivation period, Agrobacterium density, acetosyringone, L-cysteine and silver nitrate concentrations) were evaluated via transient gusA gene expression for the optimisation of transformation protocol for DBG orchid. Based on the transient gusA gene expression results, 3-4mm size mildly wounded PLBs immersed for 15 minutes in the Agrobacterium suspension (OD600 nm 0.8) and co-cultivated for 2 days in dark condition on MS medium containing 200μM acetosyringone, 60μM silver nitrate and 200 mg/L L-cysteine produced highest gusA gene expression in DBG orchid. Finally, chitinase genes wwin1 and wwin2 were introduced to DBG’s PLBs via Agrobacterium-mediated transformation system to produce fungal resistance transgenic DBG orchid. Number of putative transformants with wwin1 and wwin2 genes was obtained at 18.13% and 17.60% after one month under geneticin selection. Final confirmation of transgenic lines was confirmed by polymerase chain reaction (PCR) and polymorphic analyses were determined by DAMD molecular marker. The maximum polymorphic level (13.51%) was obtained in wwin1 carrying transgenic line 4 and wwin2 carrying transgenic line 8. However, the minimum polymorphic level (5.41%) was recorded from the wwin1 gene carrying transgenic line 1. Biochemical analyses such as total chlorophyll, carotenoid, porphyrin, carbohydrate and proline were determined in each transgenic line and shown higher contents than the non-transformed control plantlets. Hence, the successful transformation of DBG’s PLBs revealed that all transgenic lines are considered potentially fungal resistant and it is also possible to introduce other genes for improvement of agronomic traits in this orchid hybrid. | en_US |
| dc.identifier.uri | http://hdl.handle.net/123456789/3648 | |
| dc.language.iso | en | en_US |
| dc.publisher | Universiti Sains Malaysia | en_US |
| dc.subject | Orchid | en_US |
| dc.title | Agrobacterium–Mediated Transformation Of Dendrobium Broga Giant Orchid With Chitinase Gene | en_US |
| dc.type | Thesis | en_US |
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