An efficient selection and plant regeneration protocol for Agrobacterium-mediated transformation using cotyledon explants of oriental melon (Cucumis melo L. var. makuwa) has been developed. All six oriental melon cultivars evaluated in the study showed a >90 % shoot regeneration frequency and produced 1. 8-3. 6 shoots per cotyledon explant when cultured on Murashige and Skoog (MS) medium supplemented with 1. 0 mg L -1 benzyladenine and 0. 01 mg L -1 indoleacetic acid. Kanamycin (Km) and geneticin (Gt) in the shoot induction medium (SIM) were compared both qualitatively and quantitatively for their efficiency as a selection agent for the selection and regeneration of transgenic plants after Agrobacterium-mediated transformation. Shoot formation was completely inhibited at 50 mg L -1 Km and 10 mg L -1 Gt. Relatively high concentrations of both Gt and Km (>100 mg L -1 Km and >25 mg L -1 Gt) were necessary because large numbers of non-transgenic shoots survived during the selection process. The incorporation of a selectable marker (neomycin phosphotransferase II) into the genome of transgenic plants was confirmed using β-glucuronidase (GUS), PCR and Southern blot analysis. Shoot regeneration frequencies were 41. 2 % at 100 mg L -1 Km and 15. 2 % at 30 mg L -1 Gt 8 weeks after transformation, whereas the transformation frequencies based on the PCR were 2. 9 and 7. 1 %, respectively, 16 weeks after transformation. These results demonstrate that a large portion of the regenerated shoots on SIM supplemented with 100 mg L -1 Km consisted of non-transformed or escaped shoots, indicating that 30 mg L -1 Gt is the more suitable for the selection and regeneration of transgenic plants in oriental melon.
- Oriental melon
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