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

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