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.
This is a preview of subscription content, log in via an institution to check access.
Abbreviations
- TCL:
-
Thin cell layer
- PGR:
-
Plant growth regulator
- TDZ:
-
Thidiazuron
- BAP:
-
6-Benzyl amino purine
- 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
References
Ahmad N, Faisal M (2018) Thidiazuron: From urea derivative to plant growth regulator. Springer, Singapore, pp 1–491. https://doi.org/10.1007/978-981-10-8004-3
Ali M, Mujib A, Tonk D, Zafar N (2017) Plant regeneration through somatic embryogenesis and genome size analysis of Coriandrum sativum L. Protoplasma 254:343–352. https://doi.org/10.1007/s00709-016-0954-2
Ali H, Musa IF, Abu Bakar NA et al (2019) In vitro regeneration and ISSR-based genetic fidelity analysis of Orthosiphon stamineus benth. Agronomy 9:1–12. https://doi.org/10.3390/agronomy9120778
Alizadeh M, Krishna H, Eftekhari M et al (2015) Assessment of clonal fidelity in micropropagated horticultural plants. J Chem Pharm Res 7:511–514
Al-Khayri JM, Jain SM, Johnson DV (2018) Advances in plant breeding strategies: fruits. Springer, Cham
Badgujar SB, Patel VV, Bandivdekar AH, Mahajan RT (2014) Traditional uses, phytochemistry and pharmacology of Ficus carica: a review. Pharm Biol 52:1487–1503. https://doi.org/10.3109/13880209.2014.892515
Boliani AC, Ferreira AFA, Monteiro LNH et al (2019) Advances in propagation of Ficus carica L. Rev Bras Frutic 41:1–13. https://doi.org/10.1590/0100-29452019026
Carloni E, Colomba EL, Ribotta A et al (2018) Analysis of genetic variability in vitro regenerated buffelgrass plants through ISSR molecular markers. Rev la Fac Ciencias Agrar 50:1–13
Chattopadhyaya B, Banerjee J, Basu A et al (2010) Shoot induction and regeneration using internodal transverse thin cell layer culture in Sesamum indicum L. Plant Biotechnol Rep 4:173–178. https://doi.org/10.1007/s11816-010-0133-4
Crisosto H, Ferguson L, Bremer V et al (2011) Fig (Ficus carica L.). Woodhead Publishing Limited, Cambridge
Deore AC, Johnson TS (2008) High-frequency plant regeneration from leaf-disc cultures of Jatropha curcas L.: an important biodiesel plant. Plant Biotechnol Rep 2:7–11. https://doi.org/10.1007/s11816-008-0042-y
Dessoky ES, Attia AO, Mohamed EAM (2016) An efficient protocol for in vitro propagation of Fig (Ficus carica sp) and evaluation of genetic fidelity using RAPD and ISSR markers. J Appl Biol Biotechnol 4:57–63. https://doi.org/10.7324/jabb.2016.40406
Dhage SS, Pawar BD, Chimote VP et al (2012) In vitro callus induction and plantlet regeneration in fig (Ficus carica L.). J Cell Tissue Res 12:3395–3400
Dhage SS, Chimote VP, Pawar BD et al (2015) Development of an efficient in vitro regeneration protocol for fig (Ficus carica L.). J Appl Hortic 17:160–164
Doaa AD, El-Berry IM, Mustafa NS et al (2015) Detecting drought tolerance of fig (Ficus carica, L.) cultivars depending on vegetative growth and peroxidase activity. Int J ChemTech Res 8:1520–1532
Dobránszki J, Teixeira da Silva JA (2011) Adventitious shoot regeneration from leaf thin cell layers in apple. Sci Hortic (Amsterdam) 127:460–463. https://doi.org/10.1016/j.scienta.2010.11.003
Flaishman MA, Rodov V, Stover E (2008) The fig: botany, horticulture, and breeding. Horticulutral Rev 34:113–180
George EF, Hall MA, De Klerk GJ (2008) Plant propagation by tissue culture, 3rd edn. Springer, Heidelberg
Huetteman CA, Preece JE (1993) Thidiazuron: a potent cytokinin for woody plant tissue culture. Plant Cell Tissue Organ Cult 33:105–119. https://doi.org/10.1007/BF01983223
Ibrahim H, Soliman A, Alhady MRAA (2017) Evaluation of salt tolerance ability in some fig (Ficus carica L.) cultivars using tissue culture technique. J Appl Biol Biotechnol 5:29–39. https://doi.org/10.7324/jabb.2017.50605
Japelaghi RH, Haddad R, Garoosi GA (2011) Rapid and efficient isolation of high quality nucleic acids from plant tissues rich in polyphenols and polysaccharides. Mol Biotechnol 49:129–137. https://doi.org/10.1007/s12033-011-9384-8
Jowkar A, Jafarkhani M, Mohsen K et al (2009) Cytogenetic and flow cytometry analysis of Iranian Rosa spp. Floric Ornam Biotechnol 3:71–74
Kim KM, Min YK, Pil YY et al (2007a) Production of multiple shoots and plant regeneration from leaf segments of fig tree (Ficus carica L.). J Plant Biol 50:440–446. https://doi.org/10.1007/BF03030680
Kim KM, Min YK, Pil YY et al (2007b) Production of multiple shoots and plant regeneration from leaf segments of fig tree (Ficus carica L.). J Plant Biol 50:440–446
Kislev ME, Hartmann A, Bar-Yosef O (2006) Early domesticated fig in the Jordan Valley. Science 312:1372–1374. https://doi.org/10.1126/science.1125910
Kole C, Michler CH, Abbott AG, Hall TC (2010) Transgenic crop plants, vol 1. Springer, Berlin, pp 1–315. https://doi.org/10.1007/978-3-642-04809-8
Konar S, Karmakar J, Ray A et al (2018) Regeneration of plantlets through somatic embryogenesis from root derived calli of Hibiscus sabdariffa L. (Roselle) and assessment of genetic stability by flow cytometry and ISSR analysis. PLoS ONE 13:1–17. https://doi.org/10.1371/journal.pone.0202324
Leroy XJ, Leon K (2000) A rapid method for detection of plant genomic instability using unanchored-microsatellite primers. Plant Mol Biol Report 18:9805. https://doi.org/10.1007/BF02824000
Lloyd G, McCown B (1981) Commercially-feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot-tip culture. Proc Intl Plant Prop Soc 30:421–427
Loyola-Vargas VM, Ochoa-Alejo N (2016) Somatic embryogenesis: fundamental aspects and applications. Springer, Cham
Metwali EMR, Soliman HIA, Howladar SM et al (2016) Appraisal of in vitro drought stress among three different cultivars of fig (Ficus carica L.) using RAPD and ISSR markers. Plant Omics 9:1–11
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497
Murashige T, Tucker DPH (1969) Growth factor requirements of Citrus tissue culture. Proc 1st Int Citrus Symp 3:1155–1161
Nhut DT, Da Silva JAT, Aswath CR (2003) The importance of the explant on regeneration in thin cell layer technology. Vitr Cell Dev Biol - Plant 39:266–276. https://doi.org/10.1079/IVP2002408
Nhut DT, Than HN, Trinh DN et al (2006) Latest applications of Thin Cell Layer (TCL) culture systems in plant regeneration and morphogenesis. Floriculture, ornamental and plant biotechnology: advances and topical issues. Global Science Books, Ikenobe, pp 465–471
Raji MR, Lotfi M, Tohidfar M et al (2018) Somatic embryogenesis of muskmelon (Cucumis melo L.) and genetic stability assessment of regenerants using flow cytometry and ISSR markers. Protoplasma 255:873–883. https://doi.org/10.1007/s00709-017-1194-9
Ramírez-Mosqueda MA, Iglesias-Andreu LG (2016) Direct Organogenesis of Stevia rebaudiana Bertoni Using Thin Cell Layer (TCL) Method. Sugar Tech 18:424–428. https://doi.org/10.1007/s12355-015-0391-0
Sabooni N, Shekafandeh A (2017) Somatic embryogenesis and plant regeneration of blackberry using the thin cell layer technique. Plant Cell Tissue Organ Cult 130:313–321. https://doi.org/10.1007/s11240-017-1225-4
Sharma S, Shahzad A, Mahmood S, Saeed T (2015) High-frequency clonal propagation, encapsulation of nodal segments for short-term storage and germplasm exchange of Ficus carica L. Trees Struct Funct 29:345–353. https://doi.org/10.1007/s00468-014-1114-y
Soliman HI, Gabr M, Abdallah NA (2010) Efficient transformation and regeneration of fig (Ficus carica L.) via somatic embryogenesis. GM Crops 1:40–51. https://doi.org/10.4161/gmcr.1.1.10632
Steinmacher DA, Krohn NG, Dantas ACM et al (2007) Somatic embryogenesis in peach palm using the thin cell layer technique: induction, morpho-histological aspects and AFLP analysis of somaclonal variation. Ann Bot 100:699–709. https://doi.org/10.1093/aob/mcm153
Teixeira Da Silva JA, Dobránszki J (2019) Recent advances and novelties in the thin cell layer-based plant biotechnology—a mini-review. Biotechnologia 100:89–96. https://doi.org/10.5114/bta.2019.83215
Yakushiji H, Mase N, Sato Y (2003) Adventitious bud formation and plantlet regeneration from leaves of fig (Ficus carica L.). J Hortic Sci Biotechnol 78:874–878
Yancheva SD, Golubowicz S, Yablowicz Z et al (2005) Efficient Agrobacterium-mediated transformation and recovery of transgenic fig (Ficus carica L.) plants. Plant Sci 168:1433–1441. https://doi.org/10.1016/j.plantsci.2004.12.007
Zafar N, Mujib A, Ali M et al (2019) Genome size analysis of field grown and tissue culture regenerated Rauvolfia serpentina (L) by flow cytometry: histology and scanning electron microscopic study for in vitro morphogenesis. Ind Crops Prod 128:545–555. https://doi.org/10.1016/j.indcrop.2018.11.049
Acknowledgements
The authors wish to thank Ms. Nasrin Sabooni for her kind help in ISSR molecular marker analysis and Dr. Ali Pourkhaloee for critical reading of the manuscript.
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by Ming-Tsair Chan.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Abdolinejad, R., Shekafandeh, A., Jowkar, A. et al. Indirect regeneration of Ficus carica by the TCL technique and genetic fidelity evaluation of the regenerated plants using flow cytometry and ISSR. Plant Cell Tiss Organ Cult 143, 131–144 (2020). https://doi.org/10.1007/s11240-020-01903-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11240-020-01903-5