Genetic fidelity of regenerated plants via shoot regeneration of muskmelon by inter simple sequence repeat and flow cytometry

Abstract

A new and efficient method of direct shoot proliferation was developed here in two muskmelon genotypes (Cucumis melo L.), including ‘Khatooni’ and ‘Sooski Sabz’ (100%) using 4-day-old cotyledonary explants in vitro. The regeneration of muskmelon was evaluated on Murashige and Skoog (MS) medium supplemented separately with 17 treatments as combinations of two auxins (1-Naphthaleneacetic acid [NAA] and Naphthoxyacetic acid [NOA]) and four cytokinins (Benzylaminopurine [BAP], Thidiazuron [TDZ], Kinetin [KIN], and N-(2-Chloro-4-pyridyl)-N′-phenylurea [CPPU]). The treatments were experimented on 4- and 8-day-old cotyledon explants. A significant degree of organogenesis occurred in 4-day-old cotyledons (P < 0.01) than in 8-day-old cotyledons and shoots grew significantly (P < 0.01) by increasing the concentration of cytokinins in the media. The highest regeneration frequency was obtained in media supplemented with 0.6 mg/l BAP and 0.025 mg/l NOA. Derivatives were transferred to the greenhouse after complete adaptation. For the first time in muskmelon, genetic fidelity was evaluated in mother plants and in regenerated plants by inter simple sequence repeat (ISSR) markers. Ten primers amplified 112 well-resolved bands with an average of 11 bands per primer and ranged from 7 to 13 bands. According to the results of molecular analysis, no genetic variation was observed among the ex vitro and mother plants. The stability of the ploidy level through plant regeneration was verified by a flow cytometry analysis which indicated monomorphic patterns in the regenerated and mother plants. More than other treatments, the combination of BAP and NOA appeared more suitable for the tissue culture of muskmelon.

Keywords

• Muskmelons (Cucumis melo L.)
• Shoot regeneration
• Genetic fidelity
• ISSR marker
• Flow cytometric