, Volume 255, Issue 3, pp 873–883 | Cite as

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

  • Mohammad Reza Raji
  • Mahmoud Lotfi
  • Masoud Tohidfar
  • Bahman Zahedi
  • Angela Carra
  • Loredana Abbate
  • Francesco Carimi
Original Article


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.


Flow cytometry Genetic stability ISSR marker Somaclonal changes Somatic embryogenesis 



2,4-Dichlorophenoxyacetic acid






Culture initiation


Direct somatic embryogenesis


Embryo formation


Indirect somatic embryogenesis


Inter-simple sequence repeat


Murashige and Skoog


Naphthoxyacetic acid


Plant growth regulator


Somatic embryogenesis





We would like to thank Mohsen Hamedpour-Darabi for editing the English of the manuscript.

Author contribution

ML, FC, and MT conceived and designed research. MRR conducted molecular experiment. MRR and LA conducted flow cytometry experiment. MRR and AC conducted tissue culture experiment. MRR, BZ, and LA analyzed data. MRR, AC, and FC wrote the manuscript.

Funding information

This study was financially supported by grant no. 951002 of the Biotechnology Development Council of the Islamic Republic of Iran.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

709_2017_1194_MOESM1_ESM.docx (16 kb)
ESM 1 (DOCX 16 kb)
709_2017_1194_MOESM2_ESM.docx (18 kb)
ESM 2 (DOCX 18 kb)


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Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2017

Authors and Affiliations

  • Mohammad Reza Raji
    • 1
  • Mahmoud Lotfi
    • 1
  • Masoud Tohidfar
    • 2
  • Bahman Zahedi
    • 3
  • Angela Carra
    • 4
  • Loredana Abbate
    • 4
  • Francesco Carimi
    • 4
  1. 1.Department of Horticulture, Faculty of Genetics and breeding of vegetablesUniversity of TehranPakdashtIran
  2. 2.Department of Plant Biotechnology, Faculty of Life Science and Biotechnology. G. C.Shahid Beheshti UniversityTehranIran
  3. 3.Department of Horticulture, Faculty of Genetics and breeding of vegetablesLorestan UniversityKhorramabadIran
  4. 4.National Research Council of Italy (CNR)Institute of Biosciences and Bioresources (IBBR)PalermoItaly

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