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

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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.

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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

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Acknowledgements

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

Funding

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

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Authors and Affiliations

  1. Department of Horticulture, Faculty of Genetics and breeding of vegetables, University of Tehran, Pakdasht, Tehran, 3391653775, Iran

    Mohammad Reza Raji & Mahmoud Lotfi

  2. Department of Plant Biotechnology, Faculty of Life Science and Biotechnology. G. C., Shahid Beheshti University, Tehran, Iran

    Masoud Tohidfar

  3. Department of Horticulture, Faculty of Genetics and breeding of vegetables, Lorestan University, Khorramabad, Lorestan, 6813717133, Iran

    Bahman Zahedi

  4. National Research Council of Italy (CNR), Institute of Biosciences and Bioresources (IBBR), 414, I-90129, Palermo, Italy

    Angela Carra, Loredana Abbate & Francesco Carimi

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  1. Mohammad Reza Raji
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  2. Mahmoud Lotfi
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  4. Bahman Zahedi
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Contributions

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.

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Correspondence to Mahmoud Lotfi.

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Raji, M.R., Lotfi, M., Tohidfar, M. et al. Somatic embryogenesis of muskmelon (Cucumis melo L.) and genetic stability assessment of regenerants using flow cytometry and ISSR markers. Protoplasma 255, 873–883 (2018). https://doi.org/10.1007/s00709-017-1194-9

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