Development of an Efficient in Vitro Regeneration System for Endangered Wild Orange Citrus Chrysocarpa L.

Shamsul H. Prodhan, Md. Nazmul Hasan, Hammadul Hoque, S. S. Alam, Mohammed Raqibul Hasan, Anup Gupta, Most. Ummay Salma Khatun, Afroza Parvin, Ziaul Faruque Joy


A method for in-vitro propagation of wild type Indian orange (Citrus chrysocarpa L.) was developed by shoot organogenesis from seed. Mature seed embryos were used as explants and treated with different hormones and plant growth regulators on MS medium for callus, shoots and roots induction. For callus induction seed embryos were treated with different concentration of 2, 4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BA) and a-naphthalene acetic acid (NAA) and maximum 90.90% callus was observed on MS medium supplemented with 2, 4 -D at 16.0 M concentration.

Approximately 85% of the callus was granular, while 15% was smooth and compact. Maximum 83.33% shoot regeneration were observed on MS medium supplemented with BA at 13M concentration. Regenerated shoots were rooted on MS medium supplemented with different hormones and maximum 80% roots were observed in MS medium supplemented with 10 M Indole-3-butyric acid (IBA). The regenerated plantlets were successfully acclimatized in ex-situ condition for further study.


Citrus chrysocarpa L.; In-vitro; Callus induction; Shoots formation; Explants, Rooting; acclimatization.

Full Text:



Y. Liu, E. Heying, S. Tanumihardjo. "History, Global Distribution, and Nutritional Importance of Citrus Fruits." Comprehensive Reviews in Food Science and Food Safety, Vol. 11(6), pp. 530-545, 2012.

"Top Production of Oranges, 2013 choose "Production, Crops, World" in the left margin and picklist." United Nations, Food and Agricultural Organization, FAO Statistics, 2013. (21 June2015).

Z. Liao, M. Chen, F. Tan, X. Sun, K. Tang. Micropropagation of endangered Chinese Aloe. Plant Cell, Tissue and Organ Culture, Vol. 76, pp. 83-86, 2004.

C. Chang, C. Chen, Y. Tsai, Y. Chang. A tissue culture protocol for a rare plant Liliumspeciosum Var. gloroiosoides Baker. Botanical Bulletin- Academia Sinica Taipei, Vol. 41, pp. 139-142, 2000.

S. Kobayashi, I. Ikeda, H. Uchimiya. Conditions for high frequency embryogenesis from orange (Citrus sinensis Osb.) protoplasts. Plant Cell, Tissue and Organ Culture, Vol. 4, pp. 249259, 1985.

F. Azim, M. M. Rahman, S. H. Prodhan, S. U. Shikdar, N. Zobayer, M. Ashrafuzzaman. "Development of efficient callus initiation of malta (Citrus sinensis) through tissue culture." International Journal of Agricultural Research, Innovation & Technology, Vol. 1, pp. 64-68, 2011.

R. Mukhtar, M. M. Khan, R. Rafiq, A. Shahid, F. A. Khan. In Vitro Regeneration and Somatic Embryogenesis in (Citrus aurantifolia and Citrus sinensis). International Journal of Agriculture & Biology, Vol. 7, pp. 518520, 2005.

T. Murashige, F. Skoog. A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiologia Plantarum, Vol. 15, pp. 473-497, 1962.

A. L .P. Kiong, L.S. Wan, S. Hussein R. Ibrahim. Induction of somatic embryos from explants different of Citrus sinensis. Journal of Plant Science, Vol. 3, pp. 18-32, 2008.

N. Altaf, R. R. Khan. In vitro culture of Kinetinnow explants, Pakistan Journal of Botany, Vol. 41, pp. 597-

, 2009.

A. Das, A. K. Paul, S. Chaudhuri. Micropropagation of sweet orange (Citrus sinensis Osbeck) for the development of nucellar seedlings. Indian Journal of Experimental Biology, Vol. 38, pp. 26972, 2000.

G. S. V. Gitarani, N. A. Vinash. Callus induction and Plantlet regeneration in Sweet orange (C. sinensis L.) and Lime (C. aurantifolia). In vitro Cellular and Developmental Biology, Vol. 39, pp. 46874, 2003.

Y. J. Haoa, X. P. Wen, X. X. Deng. Genetic and epigenetic evaluations of citrus calluses recovered from slow growth culture. Plant Physiology, Vol. 161, pp. 479484, 2004.

M. N. Miah, S. Islam1, S. Hadiuzzaman. Regeneration of plantlets through somatic embryogenesis from nucellus tissue of Citrus macroptera Mont. var. anammensis ('Sat Kara'). Plant Tissue Culture, Vol. 12, pp. 167-172, 2002.

F. Begum, M. N.Amin, S. Islam, M.A.K. Azad, M.M. Rehman, In vitro plant regeneration from

cotyledon-derived callus of three varieties pummelo (Citrus grandis (L.) Osbeck). On Line Journal of

Biological Science Vol. 3, pp. 751759, 2003.

L. Pena, M. Cervera, J. Juarez, A. Navarro, J. A. Pina, N. Duran -Vila, L. Navarro L. Agrobacterium-mediated transformation of sweet orange and regeneration of transgenic plants. Plant Cell Report, Vol. 14, pp. 616619, 1995.

H. C. Chaturvedi, S. K. Singh, A. K. Sharma, S. Agnihotri. Citrus tissue culture employing vegetative explants. Indian Journal of Experimental Biology, Vol. 39, pp. 10801095, 2001.

H. Raman, S. S. Gosal, D. S. Brar. Plant regeneration from callus cultures of Citrus limon and C. jambhiri. Crop improvement, Vol. 19, pp. 100-103, 1995.

M. G. C. Costa, W. C. Otoni, G. A. Moor G.A. An elevation of factors affecting the efficiency of Agrobacterium-mediated transformation of Citrus paradise (macf.) and production of transgenic plants containing carotenoid biosynthetic genes. Plant Cell Reproduction, Vol. 21, pp. 365-373, 2002.

B. Chakravarty, B. C. Goswami. Plantlet regeneration from long term callus cultures of Citrus acida Roxb. and the uniformity of regenerated plants. Scientia Horticulturae, Vol. 82, pp. 159-169, 1999.

G. Rani, B. Singh, S. Sharma, L. Rehan, A. A. Zaidi, A. Nagpal, G. S. Virk. Micropropagation of Kinetinnow (Citrus nobilis x Citrus deliciosa) through nodal segments. Journal of Indian Botanical Society, Vol. 83, pp. 26-29, 2004.

M. Usman, S. Muhammad, B. Fatima. In vitro multiple shoot induction from nodal explants of Citrus cultivars. Journal of Central European Agriculture, Vol. 6, pp. 435-442, 2005.

V. A. Parthasarathy, V. Nagaraju. The production of novel cultivars of fruit trees using protoplast fusion. Annual Review of Plant Physiology, Vol. 12, pp. 108-111, 1999.

M. R. Hasan, A. Gupta, M. N. Hasan, H. M. Rejwan, R. Hasan, S. H. Prodhan. Efficient Regeneration System for the Improvement of Kinnow mandarin (Citrus reticulata Blanco). Journal of Biology, Agriculture and Healthcare, Vol.6, pp. 39-47, 2016.


  • There are currently no refbacks.




About IJSBAR | Privacy PolicyTerms & Conditions | Contact Us | DisclaimerFAQs 

IJSBAR is published by (GSSRR).