Somatic embryogenesis of muskmelon (Cucumis melo L.) and genetic stability assessment of regenerants using flow cytometry and ISSR markers

A new protocol for in vitro regeneration through direct somatic embryogenesis for two muskmelon cultivars (Cucumis melo L., 'Mashhadi' and 'Eivanaki') is reported. Somatic embryos were obtained culturing 4 and 8-day-old cotyledons, seeds and hypocotyls on MS medium supplemented with three different hormonal combinations never tested so far for melon (NOA+TDZ, NOA+BAP and 2,4-D+4-CPPU). Results were compared with those obtained when explants were cultivated in the presence of 2,4-D+BAP, previously used on melon. Embryogenesis occurred more successfully in 4-day-old cotyledons and seeds than hypocotyls and 8-day-old cotyledons. The best result was achieved with NOA+BAP. Genotypes significantly affected embryogenesis. The number of embryos in 'Eivanaki' was significantly higher than 'Mashhadi'. Embryo proliferation when explants were maintained in jars (9.3%) was found to be higher compared to petri dishes. For the first time, genetic stability of melon regenerated plants was evaluated using ISSR markers. Polymerase chain reaction (PCR) products demonstrated a total of 102 well-resolved bands, and regenerants were 93% similar compared to the mother plant. Somaclonal changes during embryogenesis were evaluated by flow cytometry, showing 91% of the same patterns in regenerated plants. The results suggest that the new hormone components are effective when applied for in vitro embryogenesis of muskmelon as they show a high frequency in regeneration and genetic homogeneity.