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

Author:
Choi, Jun Young, Shin, Jeong Sheop, Chung, Young Soo, Hyung, Nam-In
Source:
Plant cell, tissue, and organ culture 2012 v.110 no.1 pp. 133-140
ISSN:
0167-6857
Subject:
Cucumis melo, Southern blotting, benzyladenine, cotyledons, cultivars, geneticin, genome, indole acetic acid, kanamycin, kanamycin kinase, melons, polymerase chain reaction, shoots, transgenic plants
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 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.