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• W89321464 abstract "The aim of the project was to lengthen the shelf life of orchid flowers to getsuperior quality flowers. The strategy used was by retarding the internalethylene biosynthesis pathway through transferring the ACC oxidase genein the reverse orientation (antisense) into the orchid cells of DendrobiumSavin White and Oncidium Gower Ramsey. This is complimented byisolation of ACC oxidase gene fragments from Oncidium for future geneticmanipulation.A tissue culture system was established to provide plant materials fortransformation work. Protocorm-like bodies (plbs) of Dendrobium andOncidium were used to induce callus on half strength MS (Murashige andSkoog, 1962) medium. In Dendrobium, unwounded plbs or wounded plbswere tested to induce callus with Picloram (0, 0.6, 0.7, 0.8, 0.9 mg/L) incombination with Kinetin (0, 0.6, 0.7, 0.8, 0.9 mg/L). Oncidium callus wasinduced with Picloram (0, 12, 20, 30, 40, 50 mg/L) or 2,4 Diphenoxyaceticacid (2,4-D) at concentrations of 0, 5, 10, 15, 20, 25 mg/L separately. Thehighest rate of Dendrobiurn callus (42%) was obtained using unwounded plbswith 0.9 mg/L Picloram combined with 0.8 mg/L Kinetin. UnwoundedDendrobium plbs produced the highest rate of callus (17%) withcombinations of 0.8 mg/L Picloram and 0.7 mg/L Kinetin or 0.9 mg/LPicloram and 0.9 mg/L Kinetin. The most effective callus induction (43.3%)for Oncidium was obtained with 5mg/L of 2,4-D. Picloram at 50 mg/L hadthe highest rate of callus induction (36.7%). Histological observationsrevealed that callus cells were undifferentiated whereas plbs had distinctivemeristematic areas. Regeneration of Dendrobium and Oncidium callus wassuccessfully obtained.Before transformation, a protocol was established for the selection ofputative transgenic cells using hygromycin. Optimization of particlebombardment parameters (helium gas pressure and target/macrocarrierdistance) was done with GUS assay. Helium pressure of 1100 psi (7580 kPa)with platform levels 1,3 or 1,4 was found suitable. ACC oxidase antisenseconstruct (pPhACOAS1) was used for transformation and after hygromycinselection; one transgenic line of Dendrobium was obtained and regenerated.Confirmation of the transformed lines was done by Polymerase ChainReaction (PCR) and Southern Blot.wwwks.Jm-W Y 8 AACC oxidase gene was isolated from pollinated Oncidium flowers. Physicalchanges during senescence of pollinated flowers were observed andribonucleic acid (RNA) was isolated from various stages after pollination (0hr, 18 hrs, 24 hrs, 36 hrs, 48 hrs, 72 hrs) and unpollinated flowers. ACCoxidase expression from the RNA samples was analyzed through NorthernBlot and showed increased levels of expression over time. The Reverse-Transcription Polymerase Chain Reaction (RT-PCR) technique was used toisolate ACC oxidase gene fragments from the RNA samples and wassuccessfully amplified from three stages (unpollinated, 18 hours and 48hours after pollination). The gene fragments were then cloned into vectors,sequenced and characterized. The nucleic sequence and deduced amino acidsequence obtained from the three different stages had high homology withother ACC oxidase sequences in the Genebank. The analysis of the positiveclones obtained showed two versions of ACC oxidase sequences (OncACO1and OncAC02) which were successfully isolated." @default.
• W89321464 created "2016-06-24" @default.
• W89321464 creator A5078500096 @default.
• W89321464 date "2005-09-01" @default.
• W89321464 modified "2023-09-27" @default.
• W89321464 title "Biolistic Transformation of Selected Orchid Hybrids for Improved Shelf Life and Cloning of Partical ACC Oxidase Gene from Oncidium Gower Ramsey" @default.
• W89321464 hasPublicationYear "2005" @default.
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