The effects of different explants, basal media and growth regulators on regeneration of carob (Ceratonia siliqua L.)

Abstract

Ceratonia siliqua L. is a slow growing evergreen tree of the family Fabaceae used for the rehabilitation of marginal and submarginal dry areas of the Mediterranean basin due to it’s resistant to drought and salt tolerance. In this study, the effects of different basal media (Woody Plant Medium and Murashige and Skoog medium), explant types (cotyledon and hypocotyl) and growth regulators (BA, Kinetin and NAA) on in vitro callus formation, differentiation of callus to shoot and root formation were investigated. High frequencies of caullogenesis were obtained and the best medium for callus induction was WPM supplemented with 1.0 mg L-1 BA + 0.5 mg L-1 Kinetin + 0.5 mg L-1 NAA and 0.5 mg L-1 BA + 1.0 mg L-1 Kinetin + 0.5 mg L-1 NAA for hypocotyl explants and 0.5 mg L-1 BA + 1.0 mg L-1 Kinetin + 0.5 mg L-1 NAA; 0.5 mg L-1 BA + 0.5 mg L-1 Kinetin + 0.5 mg L-1 NAA and 1 mg L-1 Kinetin + 0.5 mg L-1 NAA for cotyledon explants. Callus induction was more readily obtained from hypocotyl explants than cotyledon explants. It was determined that explant types as significant on shoot formation, statistically. The shoot ratio was obtained from cotyledon explants in WPM as 10%. The best regeneration was obtained from cotyledon explants placed on WPM (30%) instead of MS medium.

Keywords

• Callus,in vitro,plant hormone,tissue culture,woody plants

References

1. [1] Ansar A, Touqeer A, Nadeem AA, Ishfaq AH (2009). Effect of different media and growth regulators on in vitro shoot proliferation of olive cultivar ‘Moraiolo’. Pak. J. Bot. 41(2): 783-795.
2. [2] Barracosa P, Osorio J, Cravador A (2007) Evaluation of fruit and seed diversity and characterization of carob (Ceratonia siliqua L.) cultivars in Algarve region. Sci Hortic 114:250–257.
3. [3] Batista MT, Estudo Fitoquı´Mico E (2009). Ultraestrutural da semente em Ceratonia siliqua L. (In Portuguese). Ph.D. Thesis Coimbra: Faculty of Pharmacy, University of Coimbra.
4. [4] Batlle I, Tous J (1997). Carob tree. Ceratonia siliqua L. promoting the conservation and use of underutilized and neglected crops. International Plant Genetic Resources Institute, Rome. 92 pp.
5. [5] Brugaletta M, La Malfa S, Gentile A, Almeida R, Romano A (2009). “In vitro culture establishment of Ceratonia siliqua (L.) mature trees from cultivars of different Mediterranean countries”, III. International Symposium On Acclimatization and Establishment Of Micropropagated Plants, Acta Horticulturae, 812:113-120.
6. [6] Canhoto JM, Rama SC, Cruz GS (2006). Somatic embryogenesis and plant regeneration in carob (Ceratonia siliqua L.). In Vitro Cell. Dev. Biol. Plant. 42:514-519.
7. [7] Carimi F, Di Lorenzo R, Crescimanno FG (1997). Callus induction and somatic embryogenesis in carob (Ceratonia siliqua L.) from ovule culture. Sci. Hort. 70:73-79.
8. [8] Cruz GS, Canhoto JM, Abreu MA (1990). Somatic embryogenesis and plant regeneration from zygotic embryos of Feijoa sellowiana berg. Plant Sci. 66:263-270.

9. [9] Custodio L, Martins-Loucao MA, Romano A (2004). Influence of sugars on in vitro rooting and acclimatization of carob tree. Biologia Plantarum, 4:3,469-472.
10. [10] Custodio L, Romano A (2006). In vitro morphogenesis in zygotic embryo cultures of carob tree (Ceratonia siliqua L.). Proceedings of the Vth International Symposium on In Vitro Culture and Horticulture Breeding, Vols 1 and 2, Acta Horticulturae:725:477-481
11. [11] Custodio L, Escapa AL, Fajardo A, Aligue R, Albericio F, Neng N, Nogueira JMF, Romano A (2011a). Antioxidant and cytotoxic activities of carob tree fruit pulps are strongly influenced by gender and cultivar. J Agric Food Chem 59:7005–7012.
12. [12] Custodio L, Escapa AL, Fernandes E, Fajardo A, Aligue R, Alberıcio F, Neng N, Nogueira JMF, Romano A (2011b). Phytochemical profile, antioxidant and cytotoxic activities of carob tree (Ceratonia siliqua L.) germ flour extracts. Plant Foods Hum Nutr 66:78–84.
13. [13] Dunstan DI, Tautorus TE, Thorpe TA (1995). Somatic embryogenesis in woody plants. In: Thorpe TA. Ed. In vitro embryogenesis in plants. Dordrecht, The Netherlands: Kluwer Academic Publishers. 471-538.
14. [14] El Ferchichi HS, Naghmouchi DJ, Walker E, Correal MB, Khouja ML (2008). Variability in the pod and seed parameters and nuclear DNA content of Tunisian populations of Ceratonia siliqua L. Agrofor. Syst. 74:73-81.
15. [15] Endres L, Souza BM, Mercier H (2002). In vitro nitrogen nutrition and hormonal pattern in bromeliads. In vitro Cell Dev. Biol. 38:481-486.
16. [16] Goncalves S, Correia PJ, Martins-Loucao MA, Romano A (2005). A new medium formulation for in vitro rooting of carob tree based on leaf macronutrients concentrations. Biologia Plantarum. 49(2):277-280.
17. [17] Gubbuk H, Tozlu I, Dogan A, Balkıc R (2016). Environment, industrial use and human health aspects of carob. Ziraat Fakültesi Dergisi, Mustafa Kemal Üniversitesi, 21(2), 207-215. (In Turkish)
18. [18] Hakim L, Islam MR, Mamun ANK, Ahmed G, Khan R (2010). Clonal propagation of carob (Ceratonia Siliqua L., Fabaceae), Bangladesh Journal Of Botany, 39(1):15-19.
19. [19] Hammatt N (1996). Fraximus excelsior L. (Common Ash). In: Bajaj, Y.P.S. (Ed.), Biotechnology in Agriculture and Forestry, Vol. 35, Trees, IV. Springer, Berlin, 172-193.
20. [20] Hammatt N, Grant N.J (1998). Thidiazuron: A potent cytokinin for woody plant tissue culture. P. avium L. (Wild Cherry). Plant Cell Rep. 17:526-530.
21. [21] Haq N (2006). Ceratonia siliqua, Carob. p. 387–391. In: J. Janick and R.E. Paull (eds.), Encyclopedia of Fruit & Nuts. CAB Intl. Oxford, UK.
22. [22] Hillcoat D, Lewis G, Verdcourt B (1980). A new species of ceratonia (leguminosae-caesalpinoideae) from Arabia and The Somali Republic. Kew Bull. 35:261-271.
23. [23] Hsina T, El Mitili N (2009). In vitro micrografting of mature carob tree (Ceratonia siliqua L.). Open Hortic. J. 2:44-48.
24. [24] Ibanez A, Valero M, Morte A (2003). Influence of cytokinins and sub culturing on proliferation capacity of single axillary bud micro cuttings of Vitis vinifera L. cv. ‘Napoleon’. Anales De Biologia. 25: 81-90.
25. [25] Kadota M, Niimi Y (2003). Effects of cytokinin types and their concentrations on shoot proliferation and hyperhydricity on in vitro pear cultivar shoots. Pl. Cell Tissue Organ Cult. 72: 261-265.
26. [26] Ksia E, Harzallah-Skhiri F, Verdeil JL, Gouta H, Alemanno L, Bouzid S (2008). Somatic embryo production from immature seeds of carob (Ceratonia siliqua L.): histological evidence, Journal Of Horticultural Science & Biotechnology, 83:4-401-406.
27. [27] Lakshmi Sita G (1999). Somatic embryogenesis in rosewood and other Indian tree legumes. In: Jain, S. M.; Gupta, P. K.; Newton, R. J., Eds. Somatic Embryogenesis In Woody Plants, Vol. 5. Dordrecht, The Netherlands: Kluwer Academic Publishers. 95-112.
28. [28] Lee CL, Paul JL, Hackett WP (1977). Promoting of rooting in stem cuttings of several ornamental plants by pretreatment with acid or base. Hort Science. 12:41-42.
29. [29] Lo Gullo M, Salleo S (1988). Different strategies of drought resistance in three mediterranean sclerophyllous trees growing in the same environmental conditions. New Phytol. 108:267-276.
30. [30] Lloyd G, McCown (1980). Commercially-feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot-tip culture. B., Int. Plant Prop. Soc. Proc. 30, 421.
31. [31] Maalej M, Chaar AR, Drira N (2006). Contribution to the improvement of olive tree somatic embryogenesis by mineral and organic analysis of zygotic embryos. Euphytica, 151: 31-37.
32. [32] Makris D and Kefalas P (2004). Carob pods (Ceratonia siliqua L.) as a source of polyphenolic antioxidants. Food Technol Biotechnol. 42:105–108.
33. [33] Manso T, Nunes C, Raposo S, Lima-Costa M (2010). Production of the biocontrol agent pantoea agglomerans PBC-1 in a stirred tank reactor by batch and fed-batch cultures. World J Microbiol Biotechnol. 26:725-735.
34. [34] Martins-Loução MA, Duarte P (1987). Effect of ammonium and nitrate nutrition on the growth of carob (Ceratonia siliqua L.) plants. - In: Ullrich WR, Aparicio PJ, Syrett, PJ and Castillo F. (Ed.): Inorganic nitrogen metabolism. Springer-Verlag Berlin 251-253.
35. [35] Martins-Loução MA, Cruz C (1999). The role of N source on carbon balance. In: Srivastava HS and Rana P. (Ed.): Modes of nitrogen nutrition in higher plants. 231-282.
36. [36] Murashige T, Skoog FA (1962). Revised medium for rapid growth and bioassays with tobacco cultures. Physiol Plant. 15:473-497.
37. [37] Nas MN, Read PE (2004). A hypothesis for the development of a defined tissue culture medium of higher plants and micropropagation of hazelnuts. Sci. Hort. 101:189-200.
38. [38] Naghmouchi S, Khouja ML, Rejeb MN, Boussaid M (2008). Effect of growth regulators and explant origin on in vitro propagation of Ceratonia siliqua L. via cuttings. Biotechnologie Agronomie Societe Et Environnement, 12:3,251-258.
39. [39] Naghmouchi S, Khouja ML, Romero A, Boussaid M (2012). Micropropagation of carob, Ceratonia siliqua L., by apex culture. Acta Botanica Gallica, 159:3, 357-361.
40. [40] Osório ML, Osório J, Gonçalves S, David MM, Correia MJ and Romano A (2012). Carob trees (Ceratonia siliqua L.) regenerated in vitro can acclimatize successfully to match the field performance of seed-derived plants. Trees, Springer Berlin Heidelberg, 26(6), 1837-1846.
41. [41] Parrott WA, Durham RE, Bailey MA (1995). Somatic embryogenesis in legumes. In: Bajaj, Y. P. S., Ed. Biotechnology in Agriculture and Forestry, Vol. 31. Berlin: Springer-Verlag: 199-227.
42. [42] Pekmezci M, Gübbük H, Eti S, Erkan M, Onus N, Biner B, Adak N, Karassahin I (2005). Dogu Akdeniz Bölgesinde kültür ve yabani formda yetisen keciboynuzu tiplerinin seleksiyonu. Bahce 34, 73–82.
43. [43] Pekmezci M, Gübbük H, Eti S, Erkan M, Onus N, Karaşahin I, Biner B, Adak N, 2008. Batı Akdeniz ve Ege bölgesinde yabani ve kültür formunda yetişen keçiboynuzu tiplerinin seleksiyonu. Akdeniz Üniversitesi Ziraat Fakültesi Dergisi, 21(2): 145-153. (In Turkish)
44. [44] Preece JE (1995). Can nutrient salts partially substitute for plant growth regulators. Plant Tissue Culture Biotechnol. 1:26- 37.
45. [45] Radi A, Echchgadda G, Ibijbijen J, Rochd M (2013). “ In vitro propagation of Moroccan Carob (Ceratonia siliqua L.)”, Journal of Food Agriculture & Environment, 11:1, 1103-1107.
46. [46] Ramsay JL, Galitz DS (2003). Basal medium and sucrose concentration influence regeneration of easter lily in ovary culture. Hort. Sci. 38(3): 404-406.
47. [47] Romano A, Barros S, Martins-Loucoa MA (2002). Micropropagation of the mediterranean tree Ceratonia siliqua. Plant Cell Tiss. Organ Cult. 68:35-41.
48. [48] Russo G, Polignano GB (1996). Variation of seed and fruit characters in Ceratonia siliqua L. cultivars. Genet. Resour. Crop Evol. 43:525-531.
49. [49] Ružić D, Sarić M, Cerović R, Ćulafić LJ (2004). Contents of macronutrients and growth of sweet cherry rootstock. In vitro-Biol. Plant. 47:463-465.
50. [50] Sakcali MS, Ozturk M (2004) Eco-physiological behavior of some mediterranean plants as suitable candidates for reclamation of degraded areas. J Arid Environ 57:1–13
51. [51] Sanchez S, Lozano LJ, Godı´nez C, Juan D, Perez A, Hernandez FJ (2010) Carob pod as a feedstock for the production of bioethanol in mediterranean areas. Appl Energy 87:3417–3424.
52. [52] Saïdi R, El Bouzdoudi B, Kbiach MB, Lamarti A, Maouni A (2015). Effects of macroelements and auxins on the micropropagation of Carob tree (Ceratonia siliqua L., Leguminosae) by shoot tip cultures. Journal of Materials and Environmental Science, Volume 6:8, 2330-2337.
53. [53] Saïdi R, El Bouzdoudi B, El Kbiach MB, Maouni A, Badoc A, Lamarti A (2016). Micropropagation of Carob tree (Ceratonia siliqua L.) by cotyledonary buds. Journal of Materials and Environmental Science, 7:12, 4850-4859.
54. [54] Sebastian KT, Mccomb JAA (1986). Micropropagation system for carob (Ceratonia siliqua L.). Sci. Hort. 28:127-131.
55. [55] Seçmen Ö (1974). Ceratonia siliqua L’nın Ekolojisi. Bitki,1 (4):533-543.
56. [56] Soumendra KN, Pattnaik S, Chand PK (2000). High frequency axillary shoot proliferation and plant regeneration from cotyledonary nodes of pomegranate (Punica granatum L.). Sci. Hort. 85:261-270.
57. [57] Tang H, Ren Z, Reustle G, Krczal G (2002). Plant regeneration from leaves of sweet and sour cherry cultivars. Scient. Hort. 93:235-244.
58. [58] Taiz L, Zeiger E (2002). Mineral nutrition. In: Plant Physiology. 2nd Ed. Sinauer Associates Inc. Pub. P. 67-86.
59. [59] Tefera W, Wannakrairoj S (2006). Synergistic effects of some plant growth regulators on in vitro shoot proliferation of korarima (Afram omum corrorima (Braun) Jansen). Afr. J. Biotech. 10:1894-1901.
60. [60] Te-chato S, Susanon T, Sontikun Y (2006). Cultivar, explant type and culture medium influencing embryogenesis and organogenegenesis in Anthurium spp. Songklanakarin J. Sci. Technol. 28(4): 717-722.
61. [61] Tetik N, Turhan I, Oziyci HR, Gubbuk H, Karhan M, Ercisli S (2011). Physical and chemical characterization of Ceratonia siliqua L. germplasm in Turkey. Scientia Horticulturae 129: 583-589.
62. [62] Tomas WV, Motyka MS, Schmulling T (2001). Regulation of plant growth by cytokinins. Sci. Hort. 6: 36-39.
63. [63] Tous J, Romero A, Batlle I (2013). The Carob Tree: Botany, Horticulture, and Genetic Resources, in Horticultural Reviews Volume 41 (ed J. Janick), John Wiley & Sons, Inc., Hoboken, New Jersey. doi: 10.1002/9781118707418.ch08
64. [64] Trigiano RN, Buckley LG, Merkle SA (1999). Somatic embryogenesis in woody legumes. In: Jain SM, Gupta PK and Newton RJ. Eds. Somatic embryogenesis in woody plants. Dordrecht, The Netherlands: Kluwer Academic Publishers. 4:198-208.
65. [65] Turhan I, Bialka KL, Demirci A, Karhan M (2010) Ethanol production from carob extract by using Saccharomyces cerevisiae. Bioresource Technol 101:5290–5296
66. [66] Vardar Y, Secmen O, Ozturk M (1980). Some distributional problems and biological characteristics of ceratonia in Turkey. Portug. Acta Biol. (A) 1.4 (XXI), 75–86.
67. [67] Yang HY, Schmidt H (1992). Untersuchungen zur adventivxpro bregeneration in vitro bei kirschen. II. Adventivsprobbildung an in vitro-Blattern Verschieder Prunus avium Idiotypen. Gartenbauwissenchaft 57:7-1.