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

Abstract

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 Murashige and Skoog medium supplemented with three different hormonal combinations never tested so far for melon (naphthoxyacetic acid (NOA) + thidiazuron (TDZ), NOA + 6-banzylaminopurine (BAP), and 2,4-dichlorophenoxyacetic acid (2,4-D) + N-(2-chloro-4-pyridyl)-N′-phenylurea (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 that in “Mashhadi.” Embryo proliferation when explants were maintained in jars (9.3%) was found to be higher compared to that in petri dishes. For the first time, genetic stability of regenerated melon plants was evaluated using inter-simple sequence repeat 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.