Document Type : Original Article


1 Graduated of Master of Science in Plant Breeding, Faculty of Agriculture, University of Tabriz, Tabriz, Iran

2 Assistant Professor, Department of Plant Breeding and Biotechnology, Faculty of agriculture, University of Tabriz, Tabriz, Iran


Objective: n present study effect of various factors including sucrose, agar, PEG, AgNO3, activated charcoal on somatic embryo induction and its subsequent regeneration was investigated using varieties Dom siah and Nemat. Methods: 330 explant (Mature embryos) of each varieties were cultured, 33/plate (100 x 15 mm), on callus induction Murashige and Skoog (MS) medium (Murashige and Skoog 1962) supplemented with 30 g/L sucrose, 7g/L agar and 2 mg/L 2,4-D. Results: Significant difference was observed between two variety in callus induction. Somatic embryogenesis and the plant regeneration were influenced by the genotype as well as composition of the medium used. The callus induction rate and callus morphology were different depend on genotypes. High somatic embryogenesis was obtained in medium (MS medium containing 2 mg/L 2,4-D) supplemented with 3 mg/L AgNO3, 9 gr/L agar and 20 gr/L PEG, respectively. The high frequency plant regeneration was achieved from both varieties in medium supplemented with 9 and 11gr/L agar and 5 mg/L AgNO3.


Aazami, M.A., Torabi, M. and Jalili, E. (2010). In vitro response of promising tomato genotypes for tolerance to osmotic stress. African Journal of Biotechnology, 9: 4014- 4017.
Abdul-Qadir, L.H. and Al-Ka'aby, H.K. (2011). The effect of water stress on callus and somatic embryos formation of rice (Oryza sativa L.) cv. Jasmine cultured in vitro. Journal of Basrah Researches, 37: 91-99.
Akter, P. and AL-Forkan, M. (2010). Assessment somatic embryogenesisi and plant regeneration from coleoptile and root tissue of Jhum rice (Oryza sativa L.). Indian Journal of Agricultural Research, 44: 88-95.
Al-Bahrany, A.M. (2002). Callus growth and proline accumulation in response to polyethylene glycol induced osmotic stress in rice, Oryza sativa L. Pakistan Journal of Biological Sciences, 5: 1294ñ1296.
Barakat, M.N.T. and Abdel-Latif, H. (1995). Somatic embryogenesis in callus from mature and immature embryo culture of wheat. Alexandria Journal of
Agriculture Research, 40: 77-95.
Brown, G., Brooks, F.J., Pearson, D. and. Mathias, R.J. (1989). Control of embryogenesis and organogenesis in mature wheat embryo callus using increased medium osmolarity and abscisic acid. Journal of Plant Physiology,
133: 727-733.
Chen, T.H., Lam, L. and Chen. S.C. (1985). Somatic embryogenesis and plant regeneration from cultured young inflorescences of Oryza sativa L. (rice). Plant Cell, Tissue and Organ Culture, 4: 51-54. 
Cogbill, S., Faulcon, T., Jones, G., McDaniel, M., Harmon, G., Blackmon, R. and Young, M. (2010). Adventitious shoot regeneration from cotyledonary explants of rapid-cycling fast plants of Brassica rapa L. Plant Cell, Tissue and Organ
Culture, 101: 127-133.
Dennis Thomas, T. (2008). The role of activated charcoal in plant tissue culture. Biotechnology Advances, 26: 618ñ 631.
Deo, P. C., Tyagi, A. P., Taylor, M., Harding, R. and Becker, D. (2010). Factors affecting somatic embryogenesis and transformation in modern plant breeding. The South Pacific Journal of Natural and Applied Sciences, 28: 27 ñ
Ebert, A. and Taylor, H.F. (1990). Assessment of the changes of 2, 4-dichlorophenoxyacetic acidconcentrations in plant tissue culture media in the
presence of activated charcoal. Plant Cell, Tissue and Organ Culture, 20: 165ñ72.
Feng, J.C., Yu, X.M., Shang, X.L., Li, J.D. and Wu, Y.X. (2010). Factors influencing efficiency of shoot regeneration in Ziziphus jujube Mill.ëHuizaoí. Plant Cell, Tissue and Organ Culture, 101: 111-117.
Fernandes, S., Michaux-Ferriere N. and Coumans, M. (1999). The embryogenic response of immature embryo cultures of durum wheat (Triticum durum Desf.):
histology and improvement by AgNO3. Plant Growth Regulation, 28: 147ñ155.
Gairi, A. and Rashid, A. (2004). TDZ-induced somatic embryogenesis in non-responsive caryopses of rice using a shot treatment with 2,4-D. Plant Cell, Tissue and Organ Culture, 76: 29-33.
Gandonou, C.H., Errabii, T., Abrinr, J., Idaomar, M., Chibi, F. and Skali senhaji, N. (2005). Effect of genotype on callus induction and plant regeneration from leaf explants of sugarcane (Saccharum sp). African Journal of Biotechnology, 4: 1250 ñ 1255.
Ganeshan, S., Chodaparambil, S.V., Baga, M., Fowler, D.B., Hucl, P., Rossnagel, B.G. and Chibbar, R.N. (2006). In vitro regeneration of cereals based on multiple shoot induction from mature embryos in response to
thidiazuron. Plant Cell, Tissue and Organ Culture, 85: 63ñ 73.
Ge, X.J., Chu, Z.H., Lin, Y.J. and Wang, S.P. (2006). A tissue culture system for different germplasms of indica rice. Plant Cell Reports, 25: 392ñ402.
Gerdakaneh, M., Mozafari, A.A., Khalighi, A. and Sioseh-mardah, A. (2009).The effects of carbohydrate source and concentration on somatic embryogenesis of strawberry (Fragaria ananassa Duch.). AmericanEurasian Journal Agricultural and Environmental Science, 6: 76-80.
Grewal, D., Gill, R. and Gosal, S.S. (2005). Factor enhancing induction of high frequency plant regeneration from somatic embryos of indica rice (Oryza
sativa L.). Journal of Biological Sciences, 5: 697-702.
Hammatt, N. and Davey, M.R. (1987). Somatic embryogenesis and plant regeneration from cultured zygotic embryos of Soybean [Glycine max (L.) Merr]. Journal of Plant Physiology, 128: 219-226.
Hoque, Md. and Mansfield, J.W. (2004). Effect of genotype and explant age on callus induction and subsequent regeneration from root-derived callus of Indica rice genotypes. Plant Cell, Tissue and Organ Culture, 78: 217-233.
IRRI. (2006). Bringing hope, improving lives: Strategic Plan 2007ñ2015. Manila (Philippines). 61 p.
Jelodar, N. B., Davey, M. R. and Cocking, E. C. (2002). Plant regeneration from cell suspension protoplasts of two iranian japonica rice cultivars and ploidy level of regenerated plants. Journal of Agricultural Science and Technology, 4:141-149.
Jimenez, V.M., (2005). Involvement of plant hormones and plant growth regulators on in vitro somatic embryogenesis. Plant Growth Regulator, 47: 91-110.
Joshi, R.,Shukla, A. and Sairam, R.K. (2011). In vitro screening of rice genotypes for drought tolerance using polyethylene glycol. Acta Physiologiae Plantarum, 33: 2209-2217.
Kaur, A. and Gosal, S.S. (2009). Desiccation of callus enhances somatic embryogenesis and subsequence shoot regeneration in Sugarcane. Indian Journal of Biotechnology, 8: 332-334.
Kumar, V., Ramakrishna, A. and G.A. Ravishankar. (2007). Influence of different ethylene inhibitors on somatic embryogenesis and secondary embryogenesis from Coffee acanephora P ex Fr. In Vitro Cellular and Developmental Biology Plant, 43: 602ñ607.
Kuroda, S., Kato, H. and Ikeda, R. (1998). Heterosis and combining ability for callus growth rate in Rice. Crop Science, 38: 333-336.
Last, D.J. and Brettell, R.I.S. (1990). Embryo yield in wheat anther culture is influenced by the choice of sugar in the culture medium. Plant Cell Reports, 9: 14-16.
Lentini, Z., Reyes, P., Martinez, C.P. and Roca, W.M.(1995). Androgenesis of highly recalcitrant rice genotypes with maltose and silver nitrate. Plant Science, 110: 127ñ138.
Lopez-Perez, A.J., Carreno, J., Martinez-Cutillas, A. and Dabauza, M. (2005). High embryogenic ability and plant regeneration of table grapevine cultivars (Vitis vinifera L.) induced by activated charcoal. Vitis, 44: 79ñ85.
Manchanda, P. and Gosal, S. S. (2012). Effect of activated charcoal, carbon sources and gelling agents on direct somatic embryogenesis and regeneration in sugarcane via leaf roll segments. Sugar Technology, 14: 168-173.
McCoy, T. J. (1987). Characterisation of alfalfa (Medicago sativa L.) plants, regenerated from selected NaCl tolerant cell lines. Plant Cell Reports, 6: 417ñ422.
Meneses, A., Flores, MuÒoz1, D., Arrieta1, G. and Espinoza, A.M. (2005). Effect of 2,4-D, hydric stress and light on indica rice (Oryza sativa) somatic
embryogenesis. Revista de BiologÌa Tropical, 53: 361- 368.
Molina, M.D., Aponte, E.M., Cortina, H. and Moreno, G. ( 2002). The effect of genotype and explant age on somatic embryogenesis of coffee. Plant Cell, Tissue and Organ Culture, 71: 117-123.
Murashige, T. and Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum, 15: 473- 479.
Naqvi, S.M.S., Sultana, T., Yasmin, T., Mahmood, T. and Akhtar, M.H. (2006). Efficient embryogenic system from tissue culture of mature embryos for some coarse varieties of rice (Oryza sativa L.). Pakistan Journal of Botany, 38: 969-975.
Noouri-Delawar, M.Z. and Arzani, A. (2001). Study of callus induction and plant regeneration from immature embryo culture in rice cultivars. Journal of Science and Technology of Agriculture and Natural Resources, 4: 57-72.
Panjaitan, S.B., Abdullah, S.N.A., Aziz, M.A., Meon, S. and Omar, O. (2009). Somatic embryogenesis from scutellar embryo of Oryza sativa L. var. MR219. Pertanika Journal of Tropical Agricultural Science, 32: 185 -194.
Quiroz-Figueroa, F.R., Rojas-Herrera, R., Galaz-Avalos, R.M. and Loyola-Vargas, V.M. (2006). Embryo production through somatic embryogÈnesis can be used to study cell differentiation in plants. Plant Cell, Tissue and Organ
Culture, 86: 285-301.
Ramesh, M., Murugiah, V. and Gupta, A.K. (2009). Efficient in vitro plant regeneration via leaf base segments of indica rice (Oryza sativa L.). Indian Journal of Experimental Biology, 47: 68ñ74.
Sakhanokho, H.F., Rajasekaran, K. and Kelley, R.Y. (2009).Somatic embryogenesis in Hedychium bousigonianum. Hort Science, 44: 1487-1490.
Sakthivelu, G., Devi, M.K.A., Giridhar, P., Rajasekaran, T., Ravishankar, G.A., Nedev, T. and Kosturkova, G. (2008). Drought-induced alterations in growth, osmotic potential and in vitroregeneration of soybean cultivars. General
and Applied Plant Physiology Special Issue, 34: 103- 112.
Shahnewaz, SH. and Bari, M. A. (2004). Effect of concentration of sucrose on the frequency of callus induction and plant regeneration in anther culture of rice (Oryza sativa L.). Plant Tissue Culture, 14: 37-43.
Slesak, H. and Przywara,L. (2003). The effect of carbohydrate Source on the development Brassica napus L. Immature embryo In vitro. Acta Biologica Cracoviensia Series Botanica, 45: 183-190.
Tyagli, A.K., Khurana, J.P., Khurana, P., Raghuvanish, S., Gaur, A., Kapur, A., Gupte, V., Kumar, D., Ravi, V., Vij, S., Khurana, P. and Sharma, S. (2004). Structural and functional analysis of rice genome. Journal of Genetics,
83: 79-99.
Valdez, M., Garro, G. and Espinoza, A.M. (1996). Establishment of morphogenetic rice cell suspension cultures (Oryza sativa L.) in Costa Rica. Revista de Biologia Tropical, 44-45: 593-595.
Vega, R., Vasquez, N., Espinoza, A.M., Gatica, A.M. and Melara, M.V. (2009). Histology of somatic embryogenesis in rice (Oryza sativa cv. 5272). Revista de Biologia Tropical, 57: 141ñ150.
Verma, D., JOSHI, R., Shukla, A. and Kumar, P. (2011). Protocol for in vitro somatic embryogenesis an regeneration of Rice (Oryza sativa L.). Indian Journal for Experimental Biology, 49: 958-963.
Wani, S.H., Sofi, P.A., Gosal, S.S. and Singh, N.B. (2010). In vitroscreening of rice (Oryza sativa L.) callus for drought
tolerance. Communications in Biometry and Crop Science, 5: 108ñ115.
Zhang, P., Phansiri, S. and Puonti, K.J. (2001). Improvements of cassava shoot organogenesis by the use
of silver nitrate in vitro. Plant Cell, Tissue and Organ Culture, 67: 47-54.
Zuraida , A.R., Naziah, B., Zamri, Z. and Sreeramanan, S. (2011). Efficient plant regeneration of Malaysian indica
rice MR 219 and 232 via somatic embryogenesis system. Acta Physiology Plant, 33: 1913ñ1921.