Acta Biologica Hungarica

, Volume 62, Issue 1, pp 34–44 | Cite as

Effect of In Vitro Culture Conditions on Somaclonal Variation in Cowpea (Vigna Unguiculata Walp.) Using Rapd Markers

  • P. SivakumarEmail author
  • S. Rajesh
  • R. Gnanam
  • A. Manickam


We report a high frequency regeneration protocol in cowpea (Vigna unguiculata Walp. var. C 152) via somatic embryogenesis from 10-d-old primary leaf explants. A study was conducted to examine the effect of somaclonal variations in in vitro derived cowpea plants under field conditions. The regenerated plantlets were successfully transferred to field after hardening in vitro and grown for collecting R0, R1 and R2 seeds. The seeds of R1 and R2 generations were subsequently, grown under field conditions and their various biometrical traits were compared and evaluated with non-tissue cultured cowpea plants as check. There was no detectable somaclonal variation induced in R0–R2 in any of the biometrical traits. The results indicate that the inclusion of different plant growth promoters at specified concentrations and duration in our earlier tissue culture work did not induce any detectable mutation. The RAPD analysis also shows that there is no genetic variation among R2 cowpea plants. The somatic embryogenesis protocol we report could thus be safely applied for high frequency true-to-type regeneration and transformations protocols without any somaclonal variation.


Cowpea plant growth regulators somaclonal variation 



2,4-dichlorophenoxyacetic acid


6-benzylamino purine


casein hydrolysate


L-Glutamic acid-5-amide


isopentenyl adenine




Murashige & Skoog’s medium


MS salts with B5 vitamins






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  1. 1.
    Al-Zahim, M., Ford-Liod, B., Newbury, H. (1999) Detection of somaclonal variation in garlic (Allium Sativum L.) using RAPD and cytological analysis. Plant Cell Rep. 18, 473–477.Google Scholar
  2. 2.
    Amitha, K., Reddy, T. P. (1996) Regeneration of plant lets from different explants and callus cultures of cowpea (Vigna unguiculata L.). Phytomorph. 46, 207–211.Google Scholar
  3. 3.
    Barwale, U. B., Widholm, J. M. (1987) Somaclonal variation in plants regenerated from cultures of soybean. Plant Cell Rep. 6, 365–368.PubMedGoogle Scholar
  4. 4.
    Bingham, E. T., Mccoy, T. G. (1986) Somaclonal variation in alfalfa. Plant Breeding Review 4, 123–152.Google Scholar
  5. 5.
    Bingham, E. T., Mccoy, T. J., Walker, K. A. (1988) Alfalfa tissue culture. In: Hanson, A. A., Barnes, D. K, Hil, R. R. (eds) Alfalfa and Alfalfa Improvement. ASA-CSSA-SSA, Madison, W., pp. 903–929.Google Scholar
  6. 6.
    Chen, W., Chen, T., Fu, Y., Hsieh, R., Chen, W. (1998) Studies on somaclonal variation in Phalaenopsis. Plant Cell Rep. 18, 7–13.Google Scholar
  7. 7.
    Chintapalli, P. L., Moss, J. P., Sharma, K. K., Bhalla, J. K. (1979) In vitro culture provides additional variation for pigeonpea. In Vitro Cell. Dev. Biol.–Plant. 33, 30–37.Google Scholar
  8. 8.
    Evans, D. A., Sharp, W. R. (1988) Somaclonal and gametoclonal variation. In: Evans, D. A., Sharp, W. R., Ammirato, P. V. (eds) Handbook of Plant Cell Culture. Macmillan Publishing Company, New York, pp. 97–132.Google Scholar
  9. 9.
    Evans, D. A. (1989) Somaclonal variation-genetic basis and breeding applications. Trends in Genetics 5, 46–50.PubMedGoogle Scholar
  10. 10.
    Freytag, A. H., Rao Arelli, A. P., Anand, S. C, Wrather, J. A., Owens, L. D. (1989) Somaclonal variation in soybean plants regenerated from tissue culture. Plant Cell Rep. 8, 199–202.PubMedGoogle Scholar
  11. 11.
    Gale, J., Boll, W. G. (1979) Growth of bean cells in suspension cultures in the presence of NaCl and protein-stabilizing factors. Can. Bot. 57, 777–782.Google Scholar
  12. 12.
    Gamborg, O. L., Miller, R. A., Ojima, K. (1968) Nutrient requirements of suspension cultures of soybean root cells. Experimental Cell Res. 50, 151–158.Google Scholar
  13. 13.
    Gautheret, R. J. (1955) The nutrition of plant tissue cultures. Ann. Rev. Plant Physiol. 6, 433–484.Google Scholar
  14. 14.
    Gosal, S. S., Bajaj, Y. P. S. (1979) Establishment of callus tissue cultures and induction of organogenesis in some grain legumes. Crop Improvement 6, 154–160.Google Scholar
  15. 15.
    Graybosch, R. A., Edge, M. E., Deianny, X. (1987) Somaclonal variation in soybean plants regenerated from the cotyledonary node tissue culture system. Crop Sci. 27, 803–806.Google Scholar
  16. 16.
    Gulati, A., Jaiwal, P. K. (1990) Culture conditions effecting plant regeneration from cotyledons of Vigna radiata (L.) Wilczek. Plant Cell Tiss. Organ Cult. 23, 1–7.Google Scholar
  17. 17.
    Gulati, A., Jaiwal, P. K. (1992) In vitro induction of multiple shoots and plant regeneration from shoot tips of mungbean (Vigna radiata L. Wilczek). Plant Cell Tiss. Organ Cult. 29, 199–205.Google Scholar
  18. 18.
    Gulati, A., Jaiwal, P. K. (1993) Comparative salt responses of callus cultures of Vigna radiata (L.) Wilczek to various osmotic and ionic stresses. J. Plant Physiol. 141, 120–124.Google Scholar
  19. 19.
    Gyulai, G., Kiss, E., Csillag, A., Heszky L. E. (1993) Developmental analysis of primary and secondary somatic embryogenesis in soybean tissue culture. Acta Biol. Hung. 44, 189–196.Google Scholar
  20. 20.
    Gyulai, G., Mester, Z., Kiss, J., Szeman, L., Heszky, L., Idnurm, A. (2003) Somaclone breeding of reed canaygrass (Phalaris arundinacea L.). Grass Forage Sci. 58, 210–215.Google Scholar
  21. 21.
    Hughes, K. W. (1986) Tissue culture derived crossing barriers. Am. J. Bot. 73, 323–329.Google Scholar
  22. 22.
    Kulothungan, S., Ganapathi, A., Shajahan, A., Kathiravan, K. (1995) Somatic embryogenesis in cell suspension culture of cowpea Vigna unguiculata (L.) Walp. Israel J. Plant Sci. 43, 385–390.Google Scholar
  23. 23.
    Lacroix, B., Assoumou, Y., Sangwan, R. S. (2003) Efficient in vitro direct shoot organogenesis and regeneration of fertile plants from embryo explants of Bambara groundnut (Vigna subterranea L. Verdc.). Plant Cell Rep. 21, 1153–1158.PubMedGoogle Scholar
  24. 24.
    Larkin, P. J., Scowcroft, W. R. (1981) Somaclonal variation–a novel source of variability from cell cultures for plant improvement. Theor. Appl. Genet. 60, 197–214.PubMedGoogle Scholar
  25. 25.
    Libbenga, K. R., Torrey, J. G. (1973) Hormone induced end reduplication prior to mitosis in cultured Pea root cortex cells. Am. J. Bot. 60, 293–299.Google Scholar
  26. 26.
    Mathews, A. H., Rao, P. S., Bhatia, C. R. (1986) Somaclonal variation in cotyledonary plants of mungbean. Z. Pflanzenphysiol. 96, 169–173.Google Scholar
  27. 27.
    Mendoza, A. B., Futsuhara, Y. (1990) Varietal differences on plant regeneration by tissue culture in mungbean Vigna radiata (L.) Wilczek. Jap. J. Breeding 40, 457–467.Google Scholar
  28. 28.
    Mitra, J., Steward, F. C. (1960) Growth and organized development of cultured cells. IV. The behavior of the nucleus. Am. J. Bot. 47, 357–368.Google Scholar
  29. 29.
    Muthukumar, B., Mariamma, M., Gnanam, A. (1995) Regeneration of plants from primary leaves of cowpea. Plant Cell Tiss. Organ Cult. 42, 153–155.Google Scholar
  30. 30.
    Murashige, T., Skoog, F. A. (1962) Revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant. 15, 473–497.Google Scholar
  31. 31.
    Murashige, T., Nakano, R. (1965) Morphogenetic behavior of tobacco tissue cultures and implication of plant senescence. Am. J. Bot. 52, 819–827.Google Scholar
  32. 32.
    Nagarajan, P., Walton, P. D. (1987) A comparison of somatic chromosomal instability in tissue culture regenerates from Medicago media Pers. Plant Cell Rep. 6, 109–113.PubMedGoogle Scholar
  33. 33.
    Nagarajan, P., Walton, P. D. (1989) Evaluation of R1 alfalfa somaclones for herbage yield and morphological characteristics. Plant Breeding 102, 333–337.Google Scholar
  34. 34.
    Pandey, R., Ganapathy, P. S. (1984) Isolation of sodium chloride tolerant callus of Cicer arietinum L. cv. BG203. Plant Cell Rep. 3, 45–47.PubMedGoogle Scholar
  35. 35.
    Prem Anand, R., Ganapathi, A., Anbazhagan, V. R., Vengadesan, G., Selvaraj, N. (2000) High frequency plant regeneration via somatic embryogenesis in cell suspension cultures of cowpea, Vigna unguiculata (L.) Walp. In Vitro Cell. Dev. Biol.-Plant 36, 475–480.Google Scholar
  36. 36.
    Ramakrishnan, K., Gnanam, R., Sivakumar, P., Manickam, A. (2005) In vitro somatic embryogenesis in cell suspension cultures of cowpea Var. C152. Plant Cell Rep. 24, 449–461.PubMedGoogle Scholar
  37. 37.
    Rubluo, A., Kartha, K. K., Mroginski, L. A., Dyck, J. (1984) Plant regeneration from pea leaflets cultured in vitro and genetic stability of regenerants. J. Plant Physiol. 117, 119–130.PubMedGoogle Scholar
  38. 38.
    Sanchez, P. D., Ruiz, M., Maldonado, S. H. G., Pineda, E. V., Chavira, V. G., Velazquez, J. A. A., Gallegos Aviles, A. M. (2006) An organogenic plant regeneration system for common bean (Phaseolus vulgaris L.). Plant Sci. 170, 822–827.Google Scholar
  39. 39.
    Singh, B. D., Kao, K. N., Miller, R. A. (1975) Karyotypic changes and selection pressure in Haplopappus gracilis suspension cultures. Can. J. Genet. Cytol. 17, 109–116.Google Scholar
  40. 40.
    Scowcroft, W. R. (1985) Somaclonal variation: the myth of clonal uniformity. In: Hohn, B., Dennis, E. S. (eds) Genetic Flux. Springer-Verlag, Vienna, pp. 217–245.Google Scholar
  41. 41.
    Shoemaker, R. C., Amberger, L. A., Palmer, R. G., Oglesby, L., Ranch, J. P. (1991) Effect of 2,4-dichlorophenoxyacetic acid concentration on somatic embryogenesis and heritable variation in soybean (Glycine max [L.] Merr.). In Vitro Cell. Dev. Biol.–Plant 27, 84–88.Google Scholar
  42. 42.
    Stephens, P. A., Nickell, C. D., Widholm, J. M. (1991) Agronomic evaluation of tissue culture-derived soybean plants. Theor. Appl. Genet. 82, 633–635.PubMedGoogle Scholar
  43. 43.
    Torrey, J. G. (1967) Morphogenesis in relation to chromosomal constitution in long-term plant tissue cultures. Physiol. Plantarum 20, 265–275.Google Scholar
  44. 44.
    Torrey, J. G. (1977) Cytodifferentiation in cultured cells and tissues. Horticult. Sci. 12, 138–139.Google Scholar
  45. 45.
    Wang, H., Holl, F. B. (1988) In vitro culture and incidence of somaclonal variation in regenerated plants of Trifolium pratense L. Plant Sci. 55, 159–167.Google Scholar
  46. 46.
    Zhao, Y., Grout, B., Crisp, P. (2005) Variations in morphology and disease susceptibility of micropropagated rhubarb (Rheum rhaponticum) PC49, compared to conventional plants. Plant Cell Tiss. Organ Cult. 82, 357–361.Google Scholar

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© Akadémiai Kiadó, Budapest 2011

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Authors and Affiliations

  • P. Sivakumar
    • 1
    Email author
  • S. Rajesh
    • 2
  • R. Gnanam
    • 3
  • A. Manickam
    • 3
  1. 1.Department of Crop ImprovementADAC & RITiruchirappalli-India
  2. 2.Plant Molecular Biology LaboratoryNational Research Centre for SoybeanIndoreIndia
  3. 3.Centre for Plant Molecular BiologyTamil Nadu Agricultural UniversityCoimbatoreIndia

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