| Summary: | 碩士 === 國立中興大學 === 生命科學系所 === 98 === Oncidium Gower Ramsey, an important orchid grown for cut flower production in Taiwan, is highly sensitive to ethylene leading to early senescence of flower petals and consequently reduction in the vase-life of cut flowers. To prolong the vase-life of Oncidium cut flowers by lowering the ethylene production through genetic engineering, ACC deaminase gene (NCBI database accession # DQ830987; Maruthasalam et al., 2006) was used to make two transformation vectors viz., pCAMBIA1301/ AtAP1-ACCD (controlled by a flower-specific promoter AP1 of Arabidopsis thaliana) and pCAMBIA1301/ CaMV 35S-ACCD (controlled by a constitutive promoter 35S). ACC deaminase is a bacterial enzyme which degrades ACC, an immediate precursor of ethylene biosynthesis, into equimolar amounts of α-ketobutyric acid and ammonia. The gene constructs were used for the transformation of protocorm-like bodies (PLBs) of Oncidium cultivar Gower Ramsey through particle bombardment. After bombardment, PLBs were cultured on G10 medium supplemented with hygromycin (5.0 mg/l) for obtaining transgenic plants. Through histochemical GUS, PCR and PCR-Southern analyses, two transgenic lines AtAP1-ACCD-1 and AtAP1-ACCD-2 transformed with pCAMBIA1301+ AtAP1-ACCD and one line CaMV-ACCD-1 transformed with pCAMBIA1301+ CaMV-ACCD were obtained. AtAP1-ACCD-1 and AtAP1-ACCD-2 were analyzed by RT-PCR and western blotting. The result showed a basal level accumulation of ACC deaminase transcript and protein in AtAP1-ACCD-2. The line CaMV-ACCD-1 is in the early stage of shoot elongation. After the confirmation of transgene integration, transgenic shoots were subcultured onto G10 medium without hygromycin for elongation and rooting.
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