An efficient selection and regeneration protocol for Agrobacterium-mediated transformation of oriental melon (Cucumis melo L. var. makuwa)

Abstract

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 beta-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.