Optimization of explants, media, plant growth regulators and carbohydrates on callus induction and plant regeneration in Citrus jambhiri Lush.
Keywords:Callus, carbohydrates, Citrus jambhiri, explants, redifferentiation
Callus induction was attempted from the four explants viz. root, cotyledon, epicotyl, and leaf segments excised from in vitro raised seedlings of C. jambhiri. Among various MS media supplementations with growth regulators and carbohydrates, the maximum (95.50%) and the earliest (8.30 days) callogenesis was obtained in epicotyl segments, when cultured on MS medium supplemented with NAA (10.0 mgl-1) + BAP (1.0 mgl-1) + sucrose (8%). The modified MS (macro and micro-nutrients reduced to half) fortified with BAP (5.0 mgl-1) + GA3 (3.0 mgl-1) recorded maximum shoot regeneration (43.10%) from callus, with an average of 5.30 shoots per callus after 35.50 days of culturing. However, prolonged exposure to GA3 resulted in thin elongated shoots and leaves. The age of the callus substantially influenced the plant regeneration frequency. The potency of the callus to regenerate decreased significantly with an increase in the age of the callus. Shoot regeneration was recorded maximum (43.43%) in 60 days old calli, followed by 90 days old (30.48%) calli, whereas it was minimum (10.46%) in 150 days old calli. The maximum (79.50%) shoot proliferation was recorded in MS medium supplemented with BAP (1.0 mgl-1) + Kin (0.5 mgl-1) with an average of 5.06 shoots per culture. The MS medium fortified with NAA (1.0 mgl-1) + IBA (1.0 mgl-1) induced maximum (77.33%) rooting, with an average of 3.19 roots per shoot after 13.4 days of culturing. Rooted plants were hardened and survived the best (83.6%) on the potting mixture consisting of cocopeat + vermiculite + perlite (2:1:1).
Ali, S. and Mirza, B. 2006. Micropropagation of rough lemon (Citrus jambhiri Lush.). I: Effect of explant type and hormone concentration. Acta Bot. Croatica, 65: 137-46.
Bitters, W.P., Murashige, T., Rangan, T.S. and Nauer, E.1970. Investigations on established virus-free plants through tissue culture. Calif. Citrus Nurs. Soc., 9: 27-30
Button, J. and Bornman, C.H. 1971. Development of nucellar plants from unpollinated and unfertilized ovules of the Washington navel orange in vitro. J. S. Afr. Bot., 37: 127-134
Bhansali, R.R. and Arya, H.C.1978. Differentiation in explants of Citrus pardadisi Macf. grown in culture. Indian J. Expt. Biol., 16: 409-416.
Button, J.1978. The effects of some carbohydrates on the growth and organization of citrus ovular callus. Z. Pflanzenphysiologie 88: 61-68. DOI: https://doi.org/10.1016/S0044-328X(78)80118-4
Carimi, F., Pasquale, F. and Crescimanno, F.G.1995. Somatic embryogenesis in citrus from styles culture. Pl. Sci. Limerick., 105: 81-86. DOI: https://doi.org/10.1016/0168-9452(94)04032-C
Carimi, F., Tortorici, M.C, Pasquala, F.D. and Gulio, F.1998. Somatic embryogenesis and plant regeneration from undeveloped ovules and stigma/style explant of sweet orange navel group [Citrus sinensis (L.) Qsb.]. Plant Cell Tissue Organ Cult., 54: 183-189. DOI: https://doi.org/10.1023/A:1006113731428
Chaturvedi, H.C., Chowdhury, A.R. and Mitra, G.C. 1974. Morphogenesis in stem callus tissue of Citrus grandis in long term culture- A biochemical analysis. Curr. Sci. 43: 139-42.
Chaturvedi, H.C., Singh, S.K., Sharma, A.K. and Agnihotri, S. 2001. Citrus tissue culture employing vegetative explants. Indian J Exp Biol., 39(11): 1080-1095.
Das, A., Paul, A.K. and Chaudhuri, S. 2000. Microporpagation of sweet orange (Citrus sinensis Osbeck.) for the development of nucellar seedlings. Indian J. Exp. Biol., 38: 269-272.
Dhatt, A.S. and Grewal, H.S.1997. Somatic cell culture and shoot regeneration from callus in citrus. Proc III Agricultural Science Congress, March 12-15, 1997, Punjab Agricultural University, Ludhiana. pp. 213-14.
Gill, M.I.S.1992. Studies on somatic cell and protoplast culture in Mandarins. Ph.D Dissertation, Punjab Agricultural University, Ludhiana.
Gill, M.I.S. and Gosal, S.S. 2002. Micropropagation of Pectinifera (Citrus depressa Hayata) a potential citrus rootstock for sweet orange. Indian J. Citricul., 1: 32-37.
Gill, M.I.S, Singh, Z., Dhillon, B.S. and Gosal, S.S. 1995. Somatic embryogenesis and plantlet regeneration in mandarin (Citrus reticulate Blanco). Sci. Hortic., 63:167-174. DOI: https://doi.org/10.1016/0304-4238(95)00812-8
Grinblat, U. 1972. Differentiation of citrus stem in vitro. J. Am. Soc. Hort. Sci., 97: 559-603. DOI: https://doi.org/10.21273/JASHS.97.5.599
Hagagy, N.A.A., Khafagy, S.A.A. and Alli, H.M.M. 2001 Trials to improve propagation of some citrus rootstocks by using embryo culture techniques. Annals Agric. Sci., 39: 2281-96.
Handaji, N., Larsalane, N., Lamarti, A., Dambier, D., Benyahia, H., Maiguizo, H., Cheikh, O.Y. and Ollitrault, P. 2005. Induction of somatic embryogenesis and plantlet regeneration in mandarin (Citrus reticulata L.). Al. Awamia., 114: 107-121.
Hao, Y.J., Wen, X.P. and Deng, X.X. 2004. Genetic and epigenetic evaluations of citrus calluses recovered from slow growth culture. J. Pl. Physiol., 161: 479-484. DOI: https://doi.org/10.1078/0176-1617-01102
Hill, K. and Schaller, G.E. 2013. Enhancing plant regeneration in tissue culture A molecular approach through manipulation of cytokinin sensitivity. Pl. Signal. Behav. 8: 10, e25709. DOI: https://doi.org/10.4161/psb.25709
Hiregouda, L., Kumar, A.H.G. and Murthy, M.N. 2005. In vitro culture of Feronia lemonia (L.) Swingle from hypocotyl and internodal explants. Biol. Plantarum., 49(1): 41-45. DOI: https://doi.org/10.1007/s10535-005-1045-y
Kayim, M. and Koc, N.K. 2006. The effects of some carbohydrates on growth and somatic embryogenesis in citrus callus culture. Sci. Hort., 109: 29-34. DOI: https://doi.org/10.1016/j.scienta.2006.01.040
Kaur, S. 2018. In vitro somatic embryogenesis and regeneration from epicotyl segments of Rough lemon (Citrus jambhiri Lush.) Inter. J. Chem. Stud., 6(1): 2082-2091.
Khan, E. U. and Nafees, A. 2005. Somatic embryogenesis and regeneration of plants from nucellar tissue of Kinnow mandarin (Citrus reticulata Blanco.) Indus J. Pl. Sci., 4: 362-368.
Khosroushahi, A.Y., Hossein, N.M. and Simons, H.T. 2011. Effect of antioxidants and carbohydrates in callus cultures of Taxus brevifolia: evaluation of browning, callus growth, total phenolics and paclitaxel production. BioImpacts, 1(1): 37-45.
Kochba, J., Spiegel-Roy, P., Neumann, H. and Saad S. 1978. Stimulation of embryogenesis in citrus tissue culture by galactose. Naturwissenschaften, 65: 261-262. DOI: https://doi.org/10.1007/BF00368574
Kumar, K., Kaur, H., Gill, M.I.S., Rattanpal, H.S., Kanika, Gosal, S.S. 2011. An efficient regeneration protocol from callus culture in rough lemon (Citrus jambhiri Lush.). Indian J. Agric. Sci., 81(4): 324-329.
Litz, R.E., Moore, G.A. and Srinivasan, C. 1985. In vitro system for propagation and improvement of tropical fruits and palm. Hortic. Rev., 7: 157-200. DOI: https://doi.org/10.1002/9781118060735.ch4
Maheshwari, P. and Rangaswamy N.S. 1958. Polyembryony and in vitro culture of embryos of Citrus and Mangifera. Indian J. Hortic. 15: 275-282.
Mukhopadhyay, A. and Bhojwani, S.S. 1978. Shoot bud differentiation in tissue cultures of leguminous plants. Z. Pflanzenphysiol., 88 : 263-68. DOI: https://doi.org/10.1016/S0044-328X(78)80248-7
Murashige, T. and Tuker D.P.H. 1969. Growth factor requirement of citrus tissue culture. In: International citrus symposium, 1.Riverside. Proceedings. Riverside: University of California, pp.1155-1169.
Moniruzzaman, M., Zhong, Y., Huang, Z., Yan, H., Yuanda, L., Jiang, B. and Zhong, G. 2021. Citrus cell suspension culture establishment, maintenance, efficient transformation and regeneration to complete transgenic plant. Plants, 10:664. DOI: https://doi.org/10.3390/plants10040664
Parkash, O. 2003. Studies on in vitro plantlet production and acclimatization of rough lemon (Citrus jambhiri Lush). M.Sc Thesis, Punjab Agricultural University, Ludhiana.
Raman, H. 1990. Somaclonal variation and distant hybridization for canker resistance in Citrus species. Ph.D. Dissertation, Punjab Agricultural University, Ludhiana.
Raman, H., Gosal S.S. and Brar D.S. 1992. Plant regeneration from callus culture of Citrus limon and Citrus jambhiri. Crop imp., 19: 100-103.
Ramsundar, V., Sathiamoorthy, S. and Thamburaj, S. 1998. Rapid plant regeneration from callus culture of acidlime (Citrus aurantifolia). South Indian Hort., 46: 196-197.
Rashad, M, Khan, M.M., Ramzan, R., Adnan, S. and Khan, F.A. 2005. In vitro regeneration and somatic embryogenesis in Citrus aurantifolia and Citrus sinensis. Int. J. Agric. Biol., 7: 518-520.
Reinert, J. and Bock, D. 1968. Control of totipotency in plant cells growing in vitro. Nature., 220: 1340-1341. DOI: https://doi.org/10.1038/2201340a0
Rattanpal, H.S., Kaur, G. and Gupta, M. 2011. In vitro plant regeneration in rough lemon (Citrus jambhiri Lush) by direct organogenesis. Afr. J. biotech., 10(63): 13724-13728. DOI: https://doi.org/10.5897/AJB09.1264
Russo, R., Sicilia, A., Caruso, M., Arlotta, C., Di Silvestro, S., Gmitter, F.G., Nicolosi, E., Lo Piero, A.R. 2021. De Novo transcriptome sequencing of rough lemon leaves (Citrus jambhiri Lush.) in response to plenodomus tracheiphilus infection. Int. J. Mol. Sci., 22:882. https://doi.org/10.3390/ijms22020882 DOI: https://doi.org/10.3390/ijms22020882
Salonia F, Ciacciulli, A., Poles, L., Pappalardo, H.D., La Malfa, S. and Licciardello, C. 2020. New plant breeding techniques in Citrus for the improvement of important agronomic traits. A review. Front. Plant Sci., 11:1234. DOI: https://doi.org/10.3389/fpls.2020.01234
Savita, Virk, G.S. and Nagpal, A. 2010. Effect of explant type and different plant growth regulators on callus induction and plantlet regeneration in Citrus jambhiri Lush. Inter. J. Sci. Tech., 5: 97-106.
Pati, P.K., Virk, G.S. and Nagpal, A., 2015. An efficient somatic embryogenesis protocol for Citrus jambhiri and assessment of clonal fidelity of plantlets using RAPD markers. J. Plant Growth Regul., 34: 309-319. DOI: https://doi.org/10.1007/s00344-014-9465-6
Singh, S. and Rajam, M.V. 2009. Citrus biotechnology: Achievements, limitations and future directions. Physio. Mol. Biol. Plants. 15: 3-22. DOI: https://doi.org/10.1007/s12298-009-0001-2
Singh, B., Sharma, S., Rani, G., Zaidi, A.A., Hallan, V., Nagpal, A. and Virk, G.S. 2006. In vitro production of Indian citrus ringspot virus-free plants of kinnow mandarin (Citrus nobilis Lour × C. deliciosa Tenora) by nucellus culture. DOI: https://doi.org/10.3923/ppj.2006.274.282
Plant Pathol. J., 5: 274–282.
Snijman, D.A., Noel, A.R.A., Bornman, C.H. and Abbott, J.G. 1977. Nicotiana tabacum callus studies. II: Variability in cultures. Z. fur pflanzenphysiologie., 82: 367-370. DOI: https://doi.org/10.1016/S0044-328X(77)80213-4
Tomaz, M.L., Mendes, B.M.J., Mourao, F.F.A., Demetrio, C.G.B., Jansakul, N., Rodriguez, A.P.M. and Mourao, F.F. 2001. Somatic embryogenesis in Citrus spp. I : Carbohydrate stimulation and histodifferentiation. In vitro Cellular Develop Bio Pl., 37: 446-452. DOI: https://doi.org/10.1007/s11627-001-0078-y
Vasil, V. and Hildebrandt, A.C. 1965. Differentiation of tobacco plants from single isolated cells in micro culture. Sci., 150: 889-92. DOI: https://doi.org/10.1126/science.150.3698.889
Wu, G.A., Terol, J., Ibanez, V., López-García, A., Pérez-Román, E., Borredá, C., Domingo. C., Tadeo, F.R., Carbonell-Caballero, J., Alonso, R., et al. 2018. Genomics of the origin and evolution of Citrus. Nature., 554: 311–316. DOI: https://doi.org/10.1038/nature25447
Yeoman, M.M. and Forche, E. 1980. Cell proliferation and growth in callus cultures. Int. Rev. Cytol. Suppl., 11: 1-24.
Yun, J., Kim, B., Jin, S., Ahn, S., Kim, Y.O., Moon, D. and Song, K. 2006. Factors affecting embryogenic culture initiation from undeveloped seeds of satsuma mandarin (Citrus unshiu). Hort. Environ. Biotech., 47: 276-79
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