Phosphomannose-isomerase as a selectable marker to recover transgenic orchid plants (Oncidium Gower Ramsey)

Abstract

A transformation method using the phosphomannose-isomerase (pmi) gene as a selectable marker was developed for orchid Oncidium Gower Ramsey. The pmigene, which converts mannose-6-phosphate to fructose-6-phosphate allowing for selection of transgenic plants on mannose selective medium. Genetically transformed plants of Oncidium were regenerated after cocultivating protocorm-like bodies with Agrobacterium tumefaciens strain GV3101 containing the vectors pEPYON-42P and pEP-YON-42H with 35S::PMI and 35S::HPTII genes respectively. We observed that 35S::PMI (pEPYON-42P) produced high rate (27 plants) of mannose resistant transgenic plants compared to 35S::HPTII (pEPYON-42H) in which only fourteen hygromycin resistant transgenic plants were obtained. Mannose resistant transgenic plants were confirmed by PCR and Southern blot. The pmi gene expression in 35S::PMI (pEPYON-42P) transgenic plants was confirmed by RT-PCR. Furthermore, the duration of regeneration time of transgenic plants was significantly shorter in mannose selected system (4 months) than in hygromycin selected system (8 months). The pmi/mannose selection system is shown to be highly efficient for producing transgenic O. Gower Ramsey without using antibiotics or herbicides. For the first time, the pmi/ mannose-based ''positive'' selection system has been used to obtain genetically engineered O. Gower Ramsey.

Agrobacterium tumefaciens-mediated transformation of Dendrobium lasianthera J.J.Sm: An important medicinal orchid

“…Any results of transformations from previous research indicated that infection period varied from few minutes to few hours, 5 min on Artemisia carvifolia [5]; 30 min on Oncidium Gower Ramsey, Crambe abyssinica , and Dendrobium chrysotoxum Lindl [42], [43], [44]; 40 min on Cordyline fruticosa [45]; an hour on Helianthus tuberosus [46]; 4 h on Erycina pusilla [10]. The results of observation (Fig.…”

An Overview of Orchid Protocorm-Like Bodies: Mass Propagation, Biotechnology, Molecular Aspects, and Breeding

“…Using this technique, these authors reported 33-43% PLBs expressing the β-glucuronidase gene (GUS) and obtained six lineages that amplified the transgenes pepper ferredoxin-like protein (pflp) and hygromycin phosphotransferase (hpt) using the particle bombardment technique. Agrobacterium tumefasciens-mediated transformation has also been successfully used in the production of transgenic plants of Oncidium 'Sharry Baby OM8' and Oncidium Gower Ramsey using the induction of secondary PLBs from in vitro-maintained PLBs [148,149].…”

Agrobacterium-mediated transformation and shoot regeneration in elite breeding lines of western shipper cantaloupe and honeydew melons (Cucumis melo L.)

“…Timentin TM is one of the most expensive antibiotics used widely in transformation studies (Ieamkhang et al 2005;Slater et al 2011;Thiruvengadam et al 2011). Clavamox Ò is relatively inexpensive and appears to work effectively at a low dose (250 mg l -1 ).…”