Somatic embryogenesis of muskmelon (Cucumis melo L.) and genetic stability assessment of regenerants using flow cytometry and ISSR markers
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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.
KeywordsFlow cytometry Genetic stability ISSR marker Somaclonal changes Somatic embryogenesis
Direct somatic embryogenesis
Indirect somatic embryogenesis
Inter-simple sequence repeat
Murashige and Skoog
Plant growth regulator
We would like to thank Mohsen Hamedpour-Darabi for editing the English of the manuscript.
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.
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.
- Anonymous (2014) FAOSTAT Food and Agriculture Organization of the United Nations. http://www.fao.org/faostat/en
- Baysal T, Mercati F, Ikten H, Yildiz RT, Carimi F, Aysan Y, Teixeira da Silva JA (2011) Clavibacter michiganensis subsp. michiganesis: tracking strains using their genetic differentiations by ISSR markers in Southern Turkey. Physiol Mol Plant Pathol 75(3):113–119. https://doi.org/10.1016/j.pmpp.2010.10.002 CrossRefGoogle Scholar
- Chovelon V, Restier V, Giovinazzo N, Dogimont C, Aarrouf J (2011) Histological study of organogenesis in Cucumis melo L. after genetic transformation: why is it difficult to obtain transgenic plants? Plant Cell Rep 30(11):2001–2011. https://doi.org/10.1007/s00299-011-1108-9 CrossRefPubMedGoogle Scholar
- Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure from small quantities of fresh leaf tissue. Phytochem Bull 19:11–15Google Scholar
- Farhoudi R, Saeedipour S, Mohammadreza D (2011) The effect of NaCl seed priming on salt tolerance, antioxidant enzyme activity, proline and carbohydrate accumulation of muskmelon (Cucumis melo L.) under saline condition. Afr J Agri Res 6:1363–1370Google Scholar
- Gray DJ, Mccolley DW, Compton ME (1993) High-frequency somatic embryogenesis from quiescent seed cotyledons of Cucumis melo cultivars. J Am Soc Hortic Sci 118:425–432Google Scholar
- Kulus D (2016) Application of cryogenic technologies and somatic embryogenesis in the storage and protection of valuable genetic resources of ornamental plants. In: Mujib A (ed) Somatic embryogenesis in ornamentals and its applications. Springer, India, pp 1–27Google Scholar
- Lo Schiavo F, Pitto L, Giuliano G, Torti G, Nuti Ronchi V, Marazziti D, Vergara R, Oselli S, Terzi M (1989) DNA methylation of embryogenic carrot cell cultures and its variations as caused by mutation, differentiation, hormones and hypomethylating drugs. Theor Appl Genet 77(3):325–331. https://doi.org/10.1007/BF00305823 CrossRefGoogle Scholar
- Meziane M, Frasheri D, Carra A, Boudjeniba M, D’Onghia AM, Mercati F, Djelouah K, Carimi F (2016) Attempts to eradicate graft-transmissible infections through somatic embryogenesis in Citrus ssp. and analysis of genetic stability of regenerated plants. Eur J Plant Pathol 10:1–11Google Scholar
- Moreno V, Garcia-Sogo M, Granell I, Garcia-Sogo B, Roig LA (1985) Plant regeneration from calli of melon (Cucumis melo L., cv.‘Amarillo Oro’). Plant cell, tissue and organ culture 5(2):139–146Google Scholar
- Munger HM, Washek RL (1983) Progress and procedures in breeding CMV resistant C. pepo L. Genetics Cucurbit Cooperative. http://cuke.hort.ncsu.edu/cgc/cgc06/1983toc.html report number 6. 1983
- Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15(3):473–497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x CrossRefGoogle Scholar
- Naderi D, Mousavi A, Habashi AA, Lotfi M (2011) Optimization of somatic embryogenesis induction in Iranian melon (Cucumis melon cv. Khatooni). Afr J Biotech 10:6434–6438Google Scholar
- Naing AH, Min JS, Park KI, Chung MY, Lim SH, Lim KB, Kim CK (2013) Primary and secondary somatic embryogenesis in Chrysanthemum (Chrysanthemum morifolium) cv. ‘Baeksun’ and assessment of ploidy stability of somatic embryogenesis process by flow cytometry. Acta Physiol Plant 35:2965–2974CrossRefGoogle Scholar
- Nakata E, Staub JE, López-Sesé AI, Katzir N (2005) Genetic diversity of Japanese melon cultivars (Cucumis melo L.) as assessed by random amplified polymorphic DNA and simple sequence repeat markers. Genet Resour Crop Evol 52(4):405–419. https://doi.org/10.1007/s10722-005-2258-9 CrossRefGoogle Scholar
- Nunez-Palenius HG, Cantliffe DJ, Huber DJ, Ciardi J, Klee HJ (2006) Transformation of a muskmelon ‘Galia’ hybrid parental line (Cucumis melo L. var. reticulatus Ser.) with an antisense ACC oxidase gene. Plant Cell Rep 25(3):198–205. https://doi.org/10.1007/s00299-005-0042-0 CrossRefPubMedGoogle Scholar
- Rhimi A, Fadhel NB, Boussaid M (2006) Plant regeneration via somatic embryogenesis from in vitro tissue culture in two Tunisian Cucumis melo cultivars Maazoun and Beji. Plant cell, tissue and organ culture 84(2):239–243Google Scholar
- Rocha DI, Kurczyńska E, Potocka I, Steinmacher DA, Otoni WC (2016) Histology and histochemistry of somatic embryogenesis. In: Loyola V, Ochoa-Alejo N (eds) Somatic embryogenesis: fundamental aspects and applications. Springer International Publisher, Switzerland, pp 471–494. https://doi.org/10.1007/978-3-319-33705-0_26 CrossRefGoogle Scholar
- Roustan JP, Latche A, Fallot J (1992) Enhancement of shoot regeneration from cotyledons of Cucumis melo by AgNO3, an inhibitor of ethylene action. Journal of plant physiology 140(4):485–488Google Scholar
- Sahijram L, Bahadur B (2015) Plant biology and biotechnology. In: Bahadur B, Rajam MV, Sahijram L, Krishnamurthy KV (eds). Somatic embryogenesis. Springer India, pp 315–327Google Scholar
- Sandhu JS, Kaur M, Kaur A, Kalia A (2016) Single step direct transgenic plant regeneration from adventive embryos of agro-infected sugarcane (Saccharum spp.) spindle leaf roll segments with assured genetic fidelity. Plant Cell Tissue Organ Cult 125(1):149–162. https://doi.org/10.1007/s11240-015-0936-7 CrossRefGoogle Scholar
- Siragusa M, Carra A, Salvia L, Puglia AM, De Pasquale F, Carimi F (2007) Genetic instability in calamondin (Citrus madurensis Lour.) plants derived from somatic embryogenesis induced by diphenylurea derivatives. Plant Cell Rep 26(8):1289–1296. https://doi.org/10.1007/s00299-007-0326-7 CrossRefPubMedGoogle Scholar
- Sujatha M, Visarada K (2013) Biolistic DNA delivery. In: Sudowe S, Reske-Kunz AB (eds) Transformation of nuclear DNA in meristematic and embryogenic tissues. Springer New York, pp 27–44Google Scholar
- Valladares S, Sanchez CS, Martinez MT, Ballester A, Vieitez AM (2006) Plant regeneration through somatic embryogenesis from tissues of mature oak trees: true-to-type conformity of plantlets by RAPD analysis. Plant Cell Rep 25(9):879–886. https://doi.org/10.1007/s00299-005-0108-z CrossRefPubMedGoogle Scholar
- Vicient CM, Martínez FX (1998) The potential uses of somatic embryogenesis are not limited to synthetic seed technology. Braz J Plant Physiol 10:1–12Google Scholar
- Wedzony M, Szechyńska-Hebda M, Zur I, Dubas E, Krzewska M (2014) Tissue culture and regeneration: a prerequisite for alien genetransfer. In: Pratap A, Kumar J (eds) Alien gene transfer in crop plants. Volume 1: innovations, methods and risk assessment. Springer, New York, pp 43–75CrossRefGoogle Scholar