Agrobacterium–Mediated Transformation Of Dendrobium Broga Giant Orchid With Chitinase Gene

• Main Author: Mohammad, Jasim Uddain
• Format: Thesis
• Language: English
• Published: 2016
• Subjects: QH1 Natural history (General - Including nature conservation, geographical distribution)
• Online Access: http://eprints.usm.my/31882/1/JASIM_UDDAIN_24%28NN%29.pdf; http://eprints.usm.my/31882/
• Institution: Universiti Sains Malaysia

Dendrobium Broga Giant (Dendrobium Bobby Messina × Dendrobium superbiens) ialah orkid hibrid terbaru di Malaysia. Kajian ini telah menghasilkan protokol transformasi perantaraan-Agrobakterium menggunakan jasad seperti protokom (JSP) untuk menghasilkan orkid transgenik Dendrobium Broga Giant (DBG) dengan gen anti-kulat. Satu teknik mikropropagasi yang mudah dan kompeten telah dihasilkan untuk percambahan JSP dari DBG. Media Murashige dan Skoog (MS) dengan setengah kekuatana separa pepejal ditambah dengan 1.0 mg/L BAP dan 0.5 mg/ L NAA menghasilkan percambahan JSP tertinggi. Interaksi antara Agrobakterium dan JSP dinilai berdasarkan pergerakan bakteria dan keupayaan pelekatan. JSP yang separa luka memaparkan pergerakan Agrobacterium serta kapasiti pelekatan yang lebih tinggi berbanding dengan rawatan lain. JSP yang berjaya ditranformasi dinilai dengan menggunakan ejen pemilihan seperti geneticin (G-418), kanamycin dan neomycin untuk menentukan kepekatan perencatan minimum. Geneticin menunjukkan keberkesanan tertinggi dengan 50 mg/L membunuh sepenuhnya JSP yang tidak ditranformasi dan membenarkan kemandirian JSP yang berjaya ditransformasikan. 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.