Establishment of anther and ovary cultures of rock melon (Cucumis melo L.) cv. Glamour and plantlet regeneration using various explants

The rising cost of hybrid seeds for Cucumis melo L. is increasing agricultural expenditures, highlighting the need for efficient, cost-effective production of local hybrid cultivars in Malaysia. Conventional breeding methods can take over a decade, necessitating the exploration of faster alternatives. This study investigates the use of tissue culture techniques, specifically anther and ovary cultures of C. melo L. cv. Glamour, to generate callus and evaluate the potential of various explants, including cotyledon, hypocotyl, node, petiole, and shoot, for regenerating complete clone plantlets. Additionally, the study aims to determine the ploidy level of the regenerated C. melo L. cv. Glamour plantlets or callus using flow cytometry analysis. To conduct anther and ovary cultures, male and female flowers were collected from healthy C. melo L. plants and sterilized under aseptic conditions. A Murashige and Skoog (MS) medium was prepared with hormones to induce callus formation, followed by embryogenic callus initiation. The sterilized anthers and ovaries were placed on the medium and incubated at 22-25°C. Growth was monitored, subculturing was conducted every 21 days, and the callus was analyzed for ploidy and regeneration potential. For anther culture, optimization was performed to determine the suitable hormone combination and concentration, duration of cold pretreatment, and duration of surface sterilization. No optimization was conducted for ovary culture due to a limited number of female flowers. The optimal hormone combinations identified were 5.0 mg/L 2,4-D + 0.5 mg/L BAP for anther- derived callus induction, and 4.0 mg/L BAP for embryogenic callus initiation. For ovary cultures, the combination of 1.0 mg/L BAP + 0.05 mg/L NAA proved to be most effective for embryogenic callus initiation. To evaluate the potential of various C. melo L. cv. Glamour explants (cotyledon, hypocotyl, node, petiole, and shoot) for regenerating complete clone plantlets, healthy seedlings were grown from aseptic seeds culture, and explants were excised. The explants were placed on Murashige and Skoog (MS) medium supplemented with growth hormones and incubated at 22-25°C under continuous light. Growth was monitored regularly, and the regeneration of plantlets was assessed by measuring both the height of the plantlets and the number of shoots produced. Overall, this study found that the most suitable hormone for C. melo L. plantlet regeneration is 1.0 mg/L BAP + 0.5 mg/L IAA, with cotyledon and nodal explants being the most effective. Flow cytometry revealed variations in ploidy levels, likely due to tissue culture-induced or somaclonal variations. These findings suggest that while tissue culture is a viable method for regenerating C. melo L. var. Reticulatus cv. Glamour, attention must be given to potential genetic instability. Further research is needed to understand the mechanisms behind these variations and to develop strategies to minimize their impact.