ORIGINAL ARTICLE Somatic embryogenesis of muskmelon (Cucumis melo L.) and genetic stability assessment of regenerants using flow cytometry and ISSR markers Mohammad Reza Raji 1 & Mahmoud Lotfi 1 & Masoud Tohidfar 2 & Bahman Zahedi 3 & Angela Carra 4 & Loredana Abbate 4 & Francesco Carimi 4 Received: 20 May 2017 /Accepted: 2 December 2017 # Springer-Verlag GmbH Austria, part of Springer Nature 2017 Abstract A new protocol for in vitro regeneration through direct somatic embryogenesis for two muskmelon cultivars (Cucumis melo L., BMashhadi^ and BEivanaki^) 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 BEivanaki^ was significantly higher than that in BMashhadi.^ 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. Keywords Flow cytometry . Genetic stability . ISSR marker . Somaclonal changes . Somatic embryogenesis Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid 4-CPPU N-(2-Chloro-4-pyridyl)-N′-phenylurea BAP 6-Banzylaminopurine CI Culture initiation DSE Direct somatic embryogenesis EF Embryo formation ISE Indirect somatic embryogenesis ISSR Inter-simple sequence repeat MS Murashige and Skoog NOA Naphthoxyacetic acid PGR Plant growth regulator SE Somatic embryogenesis TDZ Thidiazuron Introduction Muskmelon (Cucumis melo L.) is a horticultural crop of eco- nomic value belonging to the Cucurbitaceae family, and high Handling Editor: Pavla Binarova Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00709-017-1194-9) contains supplementary material, which is available to authorized users. * Mahmoud Lotfi mlotfi@ut.ac.ir 1 Department of Horticulture, Faculty of Genetics and breeding of vegetables, University of Tehran, Pakdasht, Tehran 3391653775, Iran 2 Department of Plant Biotechnology, Faculty of Life Science and Biotechnology. G. C., Shahid Beheshti University, Tehran, Iran 3 Department of Horticulture, Faculty of Genetics and breeding of vegetables, Lorestan University, Khorramabad, Lorestan 6813717133, Iran 4 National Research Council of Italy (CNR), Institute of Biosciences and Bioresources (IBBR), 414, I-90129 Palermo, Italy Protoplasma https://doi.org/10.1007/s00709-017-1194-9