An efficient protocol for the regeneration of whole plants of chickpea (Cicer arietinum L.) by using axillary meristem explants derived from in vitro-germinated seedlings

  • B. Jayanand
  • G. Sudarsanam
  • Kiran K. SharmaEmail author


An efficient and reproducible protocol for the regeneration of shoots at high frequency was developed by using explants derived from the axillary meristems from the cotyledonary nodes of in vitro-germinated seedlings of chickpea (Cicer arietinum L.). Culture conditions for various stages of adventitious shoot regeneration including the induction, elongation, and rooting of the elongated shoots were optimized. The medium for synchronous induction of multiple shoot buds consisted of Murashige and Skoog basal medium (MS) with low concentrations of thidiazuron (TDZ), 2-isopentenyladenine (2-iP), and kinetin. Exclusion of TDZ and lowering the concentration of 2-iP and kinetin in the elongation medium resulted in faster and enhanced frequency of elongated shoots. Cultivation of the stunted shoots on MS with giberellic acid (GA3) increased the number of elongated shoots from the responding explants. pH of the medium played a very crucial role in the regeneration of multiple shoot buds from the explants derived from cotyledonary nodes. A novel rooting system was developed by placing the elongated shoot on a filter paper bridge immersed in liquid rooting medium that resulted in rooting frequency of up to 90%. A comprehensive protocol for successful transplantation of the in vitro-produced plants is reported. This method will be very useful for the genetic manipulation of chickpea for its agronomic improvement.

Key words

chickpea Cicer arietinum shoot regeneration tissue culture rooting 


  1. Barna, K. S.; Wakhlu, A. K. Somatic embryogenesis and plant regeneration from callus cultures of chickpea (Cicer arietinum L.). Plant Cell Rep. 12:521–524; 1993.CrossRefGoogle Scholar
  2. Barna, K. S.; Wakhlu, A. K. Whole plant regeneration from Cicer arietinum from cellus cultures via organogenesis. Plant Cell Rep. 13:510–513; 1994.CrossRefGoogle Scholar
  3. Behrend, J.; Mateles, R. I. Nitrogen metabolism in plant cell suspension cultures. I. Effect of amino acids on growth. Plant Physiol. 56:584–589; 1975.PubMedCrossRefGoogle Scholar
  4. Briggs, B. A.; McCulloch, S. M.; Edick, L. A. Micropropagation of azaleas using thidiazuron. Acta Hortic. 226:205–208; 1988.Google Scholar
  5. Chevre, A. M.; Gill, S. S.; Mouras, A.; Salesses, G. In vitro multiplication of chestnut. J. Hort. Sci. 58:23–29; 1983.Google Scholar
  6. Dineshkumar, V.; Kirti, P. B.; Sachan, J. K. S.; Chopra, V. L. Plant regeneration via somatic embryogenesis in chickpea (Cicer arietinum L.). Plant Cell Rep. 13:468–472; 1994.Google Scholar
  7. Fontana, G. S.; Santini, L.; Caretto, S.; Frugis, G.; Mariotti, D. Genetic transformation in the grain legume Cicer arietinum L. (chickpea). Plant Cell Rep. 12:194–198; 1993.CrossRefGoogle Scholar
  8. Henny, R. J.; Fooshee, W. C. Thidiazuron stimulates basal bud and shoot formation in Alocasia × Chantrieri Andre. Hort. Sci. 25:124; 1990.Google Scholar
  9. Hyndman, S. E.; Hasegawa, P. M.; Bressan, R. A. The role of sucrose and nitrogen in adventitious root formation on cultured rose shoots. Plant Cell Tiss. Organ Cult. 1:229–238; 1982.CrossRefGoogle Scholar
  10. Jodha, N. S.; Subbarao, K. V. Chickpea: world importance and distribution. In: Saxena, M. C.; Singh, K. B., eds. The chickpea. Wallingford, UK: CAB International; 1987:1–10.Google Scholar
  11. Kar, S.; Basu, D.; Ramakrishnan, N. A.; Mukherjee, P.; Johnson, T. M.; Nayak, P.; Sen, S. K. Expression of Cry IA(c) gene of Bacillus thuringiensis in transgenic chickpea plants inhibits development of pod borer (Heliothis armigera) larvae. Transgenic Res. 61:177–185; 1997.CrossRefGoogle Scholar
  12. Kar, S.; Johnson, T. M.; Nayak, P.; Sen, S. K. Efficient transgenic plant regeneration through Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.). Plant Cell Rep. 16:32–37; 1996.Google Scholar
  13. Khan, S. K.; Ghosh, P. D. Plantlet regeneration from cotyledonary nodes of chickpea. Int. Chickpea Newsl. 11:22–24; 1984.Google Scholar
  14. Krishmamurthy, K. V.; Suhasini, K.; Sagare, A. P.; Meixner, M.; deKathen, A.; Pickardt, T.; Sheider, O. Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.) embryo axes. Plant Cell Rep. 19:235–240; 2000.CrossRefGoogle Scholar
  15. Malik, K. A.; Saxena, P. K. Thidiazuron induces high frequency shoot regeneration in intact seedlings of pea (Pisum sativum), chickpea (Cicer arietinum), and lentil (Lens culinaris). Aust. J. Plant Physiol. 19:731–740; 1992.CrossRefGoogle Scholar
  16. Murashige, T.; Skoog, F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473–497; 1962.CrossRefGoogle Scholar
  17. Polisetty, R.; Patil, P.; Deveshwar, J. J.; Khetrapal, S.; Chandra, R. Rooting and establishment of in vitro grown shoot tip explants of chickpea (Cicer arietinum L.). Ind. J. Exp. Biol. 34:806–809; 1996.Google Scholar
  18. Prakash, S.; Chaudhury, J. B.; Jain, R. K.; Choudhury, V. K. Factors affecting plant regeneration in chickpea. Cicer arietinum L. Ind. J. Exp. Biol. 30:1149–1153; 1992.Google Scholar
  19. Prakash, S.; Chowdhury, J. B.; Yadav, N. R.; Jain, R. K.; Chowdhury, V. K. Somatic embryogenesis in suspension cultures of chickpea. Ann. Biol. 10:7–14; 1994.Google Scholar
  20. Preece, J. E.; Imel, M. R. Plant regeneration from leaf explants of rhododendron ‘P.J.M. hybrids’. Sci. Hortic. 48:159–170; 1991.CrossRefGoogle Scholar
  21. Rao, B. G. Regeneration from induced embryoids of gram (Cicer arietinum L.). Adv. Plant Sci. 3:299–302; 1990.Google Scholar
  22. Rao, B. G. Influence of explant and its stages of development on response for somatic embryogenesis in chickpea. Adv. Plant Sci. 4:43–47; 1991.Google Scholar
  23. Rao, B. G.; Chopra, V. L. Genotypic and explant differences in callus initiation and maintenance in chickpea. Int. Chickpea Newsl. 17:10–12; 1987.Google Scholar
  24. Rao, B. G.; Chopra, V. L. Regeneration in chickpea (Cicer arietinum L.) through somatic embryogenesis. J. Plant Physiol. 134:637–638; 1989.Google Scholar
  25. Raven, J. A. Biochemical disposal of excess H+ in growing plants? New Phytol. 104:175–206; 1986.CrossRefGoogle Scholar
  26. Riazuddin, S.; Hussain, T.; Malik, T.; Farooqi, H.; Abbar, S. T. Establishment of callus-tissue culture and the induction of organogenesis in chickpea. Pak. J. Agri. Res. 3:339–345; 1988.Google Scholar
  27. Sagare, A. P.; Suhasini, K.; Krishnamurthy, K. V. Plant regeneration via somatic embryogenesis in chickpea (Cicer arietinum L.). Plant Cell Rep. 12:652–655; 1993.CrossRefGoogle Scholar
  28. Sharma, K. K.; Bhojwani, S. S.; Thorpe, T. A. Role of the cotyledonary tissue in the in vitro differentiation of shoots and roots from cotyledon explants of Brassica juncea (L.) Czern. Plant Cell Tiss. Organ Cult. 24:55–59; 1991.CrossRefGoogle Scholar
  29. Sharma, K. K.; Ortiz, R. Program for the application of genetic transformation for crop improvement in the semi-arid tropics. In Vitro Cell. Dev. Biol. Plant 36:83–92; 2000.CrossRefGoogle Scholar
  30. Sharma, L.; Amla, D. V. Direct shoot regeneration in chickpea (Cicer arietinum L.). Ind. J. Exp. Biol. 36:605–609; 1998.Google Scholar
  31. Shri, P. V.; Davis, T. M. Zeatin induced shoot regeneration from immature chickpea (Cicer arietinum L.) cotyledons. Plant Cell Tiss. Organ Cult. 28:45–51; 1992.CrossRefGoogle Scholar
  32. Sonia; Rizvi, S. M. H.; Singh, R. P.; Sharma, K. K.; Jaiwal, P. K. In vitro regeneration and genetic transformation of chickpea (Cicer arietinum L.). In: Jaiwal, P. K.; Singh, R. P., eds. Biotechnology for the Improvement of Legumes. Dordrecht: Kluwer Academic Publishers; 2002 (in press).Google Scholar
  33. Subhadra; Vashishth, R. K.; Chowdhury, J. B.; Singh, M.; Sareen, P. K. Multiple shoots from cotyledonary node explants of non-nodulating genotype (ICC435M) of chickpea, Cicer arietinum L. Ind. J. Exp. Biol. 36:1276–1279; 1998.Google Scholar
  34. Suhasini, K.; Sagare, A. P.; Krishnamurthy, K. V. Direct somatic embryogenesis from mature embryo axis in chickpea (Cicer arietinum L.). Plant Sci. 102:189–194; 1994.CrossRefGoogle Scholar

Copyright information

© Society for In Vitro Biology 2003

Authors and Affiliations

  1. 1.Genetic Transformation LaboratoryInternational Crops Research Institute for the Semi-Arid Tropics (ICRISAT)Andhra PradeshIndia
  2. 2.Department of BotanySri Venkateswara UniversityTirupati, Andhra PradeshIndia

Personalised recommendations