Indirect regeneration of Ficus carica by the TCL technique and genetic fidelity evaluation of the regenerated plants usi
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ORIGINAL ARTICLE
Indirect regeneration of Ficus carica by the TCL technique and genetic fidelity evaluation of the regenerated plants using flow cytometry and ISSR Ruhollah Abdolinejad1 · Akhtar Shekafandeh1 · Abolfazl Jowkar1 · Ali Gharaghani1 · Abbas Alemzadeh2 Received: 1 June 2020 / Accepted: 31 July 2020 © Springer Nature B.V. 2020
Abstract The fig (Ficus carica L.), is known as a precious fruit tree for its nutrition and medicinal values, economic importance and for sustainable production in the semi-arid and arid areas. Expanding the cultivation of fig in new vulnerable areas and the breeding programs in fig need a reliable high-efficient system for in vitro morphogenesis to meet future demands. This study was carried to develop an efficient protocol for indirect regeneration of F. carica L. cultivars ‘Sabz’ and ‘Torsh’ using thin cell layer (TCL) technique. The genetic fidelity of the regenerated plants was also evaluated using flow cytometry technique and ISSR markers. Stem segments of 10 mm in diameter were taken from mature plants, then explants were transversally cut into layers of 0.5–0.8 mm thickness. Callus induction was successful using Murashige and Tucker (MT) medium supplemented with 9.08 μM TDZ plus 9.8 μM IBA (IM3 medium) which resulted in 50 ± 6.11% calli in ‘Sabz’ cultivar. Morphogenic calli were cut into small pieces and cultured on Murashige and Skoog (MS) medium for shoot development. Maximum shoot regeneration (45%) was observed in 17.68 μM BAP in combination with 4.54 μM TDZ and 1.07 μM NAA (RM2 medium), with an average of 6.9 shoots per explant. Flow cytometry and ISSR molecular marker analyses confirmed the stability of ploidy level and genetic identity of indirectly regenerated plants in both cultivars. The results of this study demonstrate that indirect regeneration of F. carica L. by the use of TCL system is a reliable and promising approach for future mass propagation programs as well as possible in vitro breeding objectives. Key message A rapid and high-efficient in vitro method for mass propagation via callus culture in two F. carica cultivars was established by using TCL technique for the first time. Flow cytometry and ISSR molecular markers confirmed the clonal identity of regenerants in both cultivars. Keywords Fig (Ficus carica L.) · Flow cytometry · Molecular markers · Morphogenic callus · Plant growth regulators Abbreviations TCL Thin cell layer PGR Plant growth regulator TDZ Thidiazuron BAP 6-Benzyl amino purine Communicated by Ming-Tsair Chan.
KIN Kinetin 2iP 6-(γ,γ-Dimethylallylamino) purine IBA Indole-3-butyric acid NAA 1-Naphthalene acetic acid 2,4-D 2,4-Dinitrophenylhydrazine GA3 Gibberellic acid ISSR Inter simple sequence repeats CTAB Cetyl trimethyl ammonium bromide
* Akhtar Shekafandeh [email protected] 1
Department of Horticultural Science, College of Agriculture, Shiraz University, Shiraz, Iran
Department of Plant Genetics and Production, College of Agriculture, Shiraz University, Shiraz, Iran
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