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Research Article

volume 40 issue 5 (october 2017) : 785-794,   Doi: 10.18805/LR-3615

In vitro callus induction and regeneration of multiple shoots through callus derived from leaf and epicotyl explants in pigeon pea (Cajanus cajan L.)

P
Padmavathi
A
A.V.Thangella
B
B. Fakrudin
1Department of Biotechnology, University of Agricultural Sciences, Dharwad-580 005, Karnataka, India.

  • Submitted18-09-2015|

  • Accepted05-11-2016|

  • First Online 18-09-2017|

  • doi 10.18805/LR-3615

Cite article:- Padmavathi, A.V.Thangella, Fakrudin B. (2017). In vitro callus induction and regeneration of multiple shoots through callus derived from leaf and epicotyl explants in pigeon pea (Cajanus cajan L.). Legume Research. 40(5): 785-794. doi: 10.18805/LR-3615.

ABSTRACT

An efficient in vitro protocol was developed for callus induction, high frequency plant regeneration through callus cultures derived from cotyledonary leaf and epicotyl explants, rooting of shoots derived from callus and establishment onto the natural conditions in two cultivars of pigeon pea; ICPL 87119 and ICPL 8863. Cotyledonary leaf and epicotyl explants were tested for callus induction across 48 different combinations and concentrations of auxins and cytokinins in MS medium, wherein, higher doses of auxins (15 mg/1 NAA) in combination with lower doses of cytokinins (0.5 mg/l kinetin) induced regenerable callus from leaf explants while lower doses of auxins (0.2 mg/1 NAA) in combination with higher doses of cytokinins (8 mg/1 kinetin) induced regenerable callus from epicotyl explants in both the genotypes. Plantlet regeneration from leaf and epicotyl derived callus was optimized at 0.05 mg/l TDZ in both genotypes. Rooting was optimized on ½ MS + 0.5 mg/1 IBA media in both genotypes. Well-rooted plants were acclimatized and established successfully into natural conditions in potting mixture-containing soil: FYM in 1:1 ratio resulting in 48.01 per cent survivability. Regenerated plants were uniform morphologically with normal leaf shape and growth. This protocol finds its significance in rapid multiplication of transgenic plants.

REFERENCES

  1. IIPR (Indian Institute of Pulses Research, 2016) http://www.iipr.res.in/e-pulse-data-book.html
  2. Dayal, S., Lavanya M., Devi P. and Sharma K.K.. (2003). An efficient protocol for shoot regeneration and genetic transformation of pigeon pea [Cajanus cajan (L.) Millsp.] by using leaf explants. Plant Cell Reports., 21: 1072-1079.
  3. Delporte, F., Mostade, O., Jacquemin. J.M. (2001). Plant regeneration through callus initiation from thin mature embryo fragments of wheat. Plant Cell Tissue Organ Cult., 67: 73-80
  4. Eapen, S. and George. L. (1993). Plant regeneration from leaf discs of peanut and pigeon pea: Influence of benzyladenine, indoleacetic acid and indoleacetic acid-amino acid conjugates. Plant Cell Tissue Organ Cult., 35: 223-227.
  5. Eapen, S., George, Tivarekar L. (1998). “Thidiazuron-induced shoot regeneration in pigeon pea (Cajanus cajan L.)”. Plant Cell Tissue Organ Cult., 53: 217-220.
  6. Franklin, G., Jeyachandran, R. and Ignacimuthu, S. (2000). Factors affecting regeneration of pigeon pea (Cajanus cajan L. Millsp) from mature embryonal axes. Plant Growth Regul., 30: 31–36.
  7. Geetha, N., Venkatachalam, P., Prakash, V. and Sita., L.G. (1998). High frequency induction of multiple shoots and plant regeneration from seedling explants of pigeon pea (Cajanus cajan L.). Curr Sci., 75: 1036-1041.
  8. George, L., and Eapen., S. (1994). Organogenesis and embryogenesis from diverse explants in pigeon pea (C. cajan L.). Plant Cell Reports. 13: 417-420.
  9. Gomez, K. A. and Gomez., A. A. (1984). Statistical Procedures in Agricultural Research. 2nd edition New York, Chichester. Wiley pp. 680. 
  10. Husain, M.K., Anis., M. and Shahzad., A. (2007). In vitro propagation of Indian Kino (Pterocarpus marsupium Roxb.) using thidiazuron. In Vitro Cell Dev Biol Plant. 43: 59-64. 
  11. Khaleda. L., Al-Forkan., M. (2006). Genotypic variability in callus induction and plant regeneration through somatic embryogenesis of five deepwater rice (Oryza sativa L.) cultivars of Bangladesh. African Journal of Biotechnology. 5(16): 1435-1440. 
  12. Krishna, G., Reddy, P.S., Ramteke, P.W., Bhattacharya, P.S. (2010). Progress in tissue culture and genetic transformation research in pigeon pea (Cajanus cajan (L.) Millsp.). Plant Cell Reports. 29: 1079-1095.
  13. Kumar, S., Reddy, T.P. and Reddy, G.M., (1983). Plantlet regeneration from different callus cultures of pigeon pea (Cajanus cajan L.). Plant Sci Lett., 32: 271-278. 
  14. Lawrence, P.K. and Koundal., K.R. (2001). Agrobacterium tumefaciens mediated transformation of pigeon pea [Cajanus cajan (L.) Millsp.] and molecular analysis of regenerated plants. Curr Sci., 80: 1428-1432. 
  15. Mehta, U., Mohan Ram., H.Y. (1980). Regeneration of plantlets from the cotyledons of Cajanus cajan L. Indian J Exp Biol., 18: 800–802.
  16. Mohan, M.L., and Krishnamurthy., K.V. (1998). Plant regeneration in pigeon pea [Cajanus cajan (L.) Millsp.] by organogenesis. Plant Cell Rep. 17: 705-710.
  17. Murashige, T and Skoog, F. (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant., 15: 473-497. 
  18. Naidu, R.B., Kulkarni, D.D. and Krishnamurthy, K.V. (1995). Genotype dependent morphogenic potentiality of various explants of a food legume, the pigeon pea (Cajanus cajan L.). In Vitro Cell Dev Biol Plant. 31: 26-30. 
  19. Nene, Y.L., Sheila, V.K. (1990). Pigeon pea: Geography and Importance. In: The Pigeon pea, Nene YL, Hall SH, Sheila VK (Eds.). CAB International, Wellingford, UK pp: 1-14.
  20. Rao, B.G. and V.L. Chopra. (1989). Regeneration in chickpea (Cicer arietinum L.) through somatic embryogenesis. J Plant Physiol. 134: 637-638.
  21. Sanyal, I., Singh, A.K., Kaushik, M. and Amla, D.V. (2005). Agrobacteriu m- mediated transformation of chickpea (Cicer arietinum L.) with Bacillus thuringiensis cryIAc gene for resistance against pod borer insect Helicoverpa armigera. Plant Sci., 168: 1135-1146
  22. Saxena, K.B. (2008). Genetic improvement of pigeonpea—a review. Trop Plant Biol., 1: 159–178.
  23. Saharan, V., Yadav, R.C., Yadav, R.N., Chapagain. B.P. (2004). High frequency plant regeneration from desiccated calli of indica rice (Oryza Sativa L.). Afr J Biotechnol., 3(5): 256-259.
  24. Shiva Prakash, N., Pental, D. and Bhalla-Sarin, N. (1994). Regeneration of pigeon pea (Cajanus cajan) from cotyledonary node via multiple shoot formation. Plant Cell Rep. 13: 623–627. 
  25. Singh, N.D., Sahoo, L., Neera, B.S. and Jaiwal, P.K.(2003). The effect of TDZ on organogenesis and somatic embryogenesis in pigeon pea (Cajanus cajan L. Millsp). Plant Sci. 164: 341–347.
  26. Tyagi, P., Comai, L. and Byers, B. (2001). Comparison of plant regeneration from root shoot and leaf explants in pigeon pea (Cajanus cajan) cultivars. SABRAO J. 33: 59–71, 
  27. Villiers, S.D., Emongor, Q., Njeri, R., Gwata, E., Hoisington, D., Njagi, I., Silim, S. and Sharma, K. (2008). “Evaluation of the shoot regeneration response in tissue culture of pigeon pea [Cajanus cajan (L.) Millsp.] varieties adapted to eastern and southern Africa”. Afr J Biotechnol., 7: 587–590.
  28. VishuKumar, V., and Patil., M.S. (1998). Tissue culture studies in pigeonpea. Karnataka J Agric Sci., 13(1): 20-28. 
  29. Yadav, P.B.S. and Padmaja, V. (2002). Shoot organogenesis and plantlet regeneration from leaf segments of pigeon pea. Plant Cell Tiss Org Cult. 73: 197–200.
  30. Varshney, R.K., Hoisington, D.A., Upadhyaya, H.D., Gaur, P.M., Nigam, S.N., Saxena, K., Vadez V., Sethy, N.K,. Bhatia, S, Aruna, R., Gowda, M.V.C., Singh, N.K. (2007). Molecular genetics and breeding of grain legume crops for the semi-arid tropics. In: Varshney RK, Tuberosa R, editors. Genomics assisted crop improvement, vol II: genomics applications in crops. Dordrecht: Springer pp. 207–242. 
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    Research Article

    volume 40 issue 5 (october 2017) : 785-794,   Doi: 10.18805/LR-3615

    In vitro callus induction and regeneration of multiple shoots through callus derived from leaf and epicotyl explants in pigeon pea (Cajanus cajan L.)

    P
    Padmavathi
    A
    A.V.Thangella
    B
    B. Fakrudin
    1Department of Biotechnology, University of Agricultural Sciences, Dharwad-580 005, Karnataka, India.

    • Submitted18-09-2015|

    • Accepted05-11-2016|

    • First Online 18-09-2017|

    • doi 10.18805/LR-3615

    Cite article:- Padmavathi, A.V.Thangella, Fakrudin B. (2017). In vitro callus induction and regeneration of multiple shoots through callus derived from leaf and epicotyl explants in pigeon pea (Cajanus cajan L.). Legume Research. 40(5): 785-794. doi: 10.18805/LR-3615.

    ABSTRACT

    An efficient in vitro protocol was developed for callus induction, high frequency plant regeneration through callus cultures derived from cotyledonary leaf and epicotyl explants, rooting of shoots derived from callus and establishment onto the natural conditions in two cultivars of pigeon pea; ICPL 87119 and ICPL 8863. Cotyledonary leaf and epicotyl explants were tested for callus induction across 48 different combinations and concentrations of auxins and cytokinins in MS medium, wherein, higher doses of auxins (15 mg/1 NAA) in combination with lower doses of cytokinins (0.5 mg/l kinetin) induced regenerable callus from leaf explants while lower doses of auxins (0.2 mg/1 NAA) in combination with higher doses of cytokinins (8 mg/1 kinetin) induced regenerable callus from epicotyl explants in both the genotypes. Plantlet regeneration from leaf and epicotyl derived callus was optimized at 0.05 mg/l TDZ in both genotypes. Rooting was optimized on ½ MS + 0.5 mg/1 IBA media in both genotypes. Well-rooted plants were acclimatized and established successfully into natural conditions in potting mixture-containing soil: FYM in 1:1 ratio resulting in 48.01 per cent survivability. Regenerated plants were uniform morphologically with normal leaf shape and growth. This protocol finds its significance in rapid multiplication of transgenic plants.

    REFERENCES

    1. IIPR (Indian Institute of Pulses Research, 2016) http://www.iipr.res.in/e-pulse-data-book.html
    2. Dayal, S., Lavanya M., Devi P. and Sharma K.K.. (2003). An efficient protocol for shoot regeneration and genetic transformation of pigeon pea [Cajanus cajan (L.) Millsp.] by using leaf explants. Plant Cell Reports., 21: 1072-1079.
    3. Delporte, F., Mostade, O., Jacquemin. J.M. (2001). Plant regeneration through callus initiation from thin mature embryo fragments of wheat. Plant Cell Tissue Organ Cult., 67: 73-80
    4. Eapen, S. and George. L. (1993). Plant regeneration from leaf discs of peanut and pigeon pea: Influence of benzyladenine, indoleacetic acid and indoleacetic acid-amino acid conjugates. Plant Cell Tissue Organ Cult., 35: 223-227.
    5. Eapen, S., George, Tivarekar L. (1998). “Thidiazuron-induced shoot regeneration in pigeon pea (Cajanus cajan L.)”. Plant Cell Tissue Organ Cult., 53: 217-220.
    6. Franklin, G., Jeyachandran, R. and Ignacimuthu, S. (2000). Factors affecting regeneration of pigeon pea (Cajanus cajan L. Millsp) from mature embryonal axes. Plant Growth Regul., 30: 31–36.
    7. Geetha, N., Venkatachalam, P., Prakash, V. and Sita., L.G. (1998). High frequency induction of multiple shoots and plant regeneration from seedling explants of pigeon pea (Cajanus cajan L.). Curr Sci., 75: 1036-1041.
    8. George, L., and Eapen., S. (1994). Organogenesis and embryogenesis from diverse explants in pigeon pea (C. cajan L.). Plant Cell Reports. 13: 417-420.
    9. Gomez, K. A. and Gomez., A. A. (1984). Statistical Procedures in Agricultural Research. 2nd edition New York, Chichester. Wiley pp. 680. 
    10. Husain, M.K., Anis., M. and Shahzad., A. (2007). In vitro propagation of Indian Kino (Pterocarpus marsupium Roxb.) using thidiazuron. In Vitro Cell Dev Biol Plant. 43: 59-64. 
    11. Khaleda. L., Al-Forkan., M. (2006). Genotypic variability in callus induction and plant regeneration through somatic embryogenesis of five deepwater rice (Oryza sativa L.) cultivars of Bangladesh. African Journal of Biotechnology. 5(16): 1435-1440. 
    12. Krishna, G., Reddy, P.S., Ramteke, P.W., Bhattacharya, P.S. (2010). Progress in tissue culture and genetic transformation research in pigeon pea (Cajanus cajan (L.) Millsp.). Plant Cell Reports. 29: 1079-1095.
    13. Kumar, S., Reddy, T.P. and Reddy, G.M., (1983). Plantlet regeneration from different callus cultures of pigeon pea (Cajanus cajan L.). Plant Sci Lett., 32: 271-278. 
    14. Lawrence, P.K. and Koundal., K.R. (2001). Agrobacterium tumefaciens mediated transformation of pigeon pea [Cajanus cajan (L.) Millsp.] and molecular analysis of regenerated plants. Curr Sci., 80: 1428-1432. 
    15. Mehta, U., Mohan Ram., H.Y. (1980). Regeneration of plantlets from the cotyledons of Cajanus cajan L. Indian J Exp Biol., 18: 800–802.
    16. Mohan, M.L., and Krishnamurthy., K.V. (1998). Plant regeneration in pigeon pea [Cajanus cajan (L.) Millsp.] by organogenesis. Plant Cell Rep. 17: 705-710.
    17. Murashige, T and Skoog, F. (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant., 15: 473-497. 
    18. Naidu, R.B., Kulkarni, D.D. and Krishnamurthy, K.V. (1995). Genotype dependent morphogenic potentiality of various explants of a food legume, the pigeon pea (Cajanus cajan L.). In Vitro Cell Dev Biol Plant. 31: 26-30. 
    19. Nene, Y.L., Sheila, V.K. (1990). Pigeon pea: Geography and Importance. In: The Pigeon pea, Nene YL, Hall SH, Sheila VK (Eds.). CAB International, Wellingford, UK pp: 1-14.
    20. Rao, B.G. and V.L. Chopra. (1989). Regeneration in chickpea (Cicer arietinum L.) through somatic embryogenesis. J Plant Physiol. 134: 637-638.
    21. Sanyal, I., Singh, A.K., Kaushik, M. and Amla, D.V. (2005). Agrobacteriu m- mediated transformation of chickpea (Cicer arietinum L.) with Bacillus thuringiensis cryIAc gene for resistance against pod borer insect Helicoverpa armigera. Plant Sci., 168: 1135-1146
    22. Saxena, K.B. (2008). Genetic improvement of pigeonpea—a review. Trop Plant Biol., 1: 159–178.
    23. Saharan, V., Yadav, R.C., Yadav, R.N., Chapagain. B.P. (2004). High frequency plant regeneration from desiccated calli of indica rice (Oryza Sativa L.). Afr J Biotechnol., 3(5): 256-259.
    24. Shiva Prakash, N., Pental, D. and Bhalla-Sarin, N. (1994). Regeneration of pigeon pea (Cajanus cajan) from cotyledonary node via multiple shoot formation. Plant Cell Rep. 13: 623–627. 
    25. Singh, N.D., Sahoo, L., Neera, B.S. and Jaiwal, P.K.(2003). The effect of TDZ on organogenesis and somatic embryogenesis in pigeon pea (Cajanus cajan L. Millsp). Plant Sci. 164: 341–347.
    26. Tyagi, P., Comai, L. and Byers, B. (2001). Comparison of plant regeneration from root shoot and leaf explants in pigeon pea (Cajanus cajan) cultivars. SABRAO J. 33: 59–71, 
    27. Villiers, S.D., Emongor, Q., Njeri, R., Gwata, E., Hoisington, D., Njagi, I., Silim, S. and Sharma, K. (2008). “Evaluation of the shoot regeneration response in tissue culture of pigeon pea [Cajanus cajan (L.) Millsp.] varieties adapted to eastern and southern Africa”. Afr J Biotechnol., 7: 587–590.
    28. VishuKumar, V., and Patil., M.S. (1998). Tissue culture studies in pigeonpea. Karnataka J Agric Sci., 13(1): 20-28. 
    29. Yadav, P.B.S. and Padmaja, V. (2002). Shoot organogenesis and plantlet regeneration from leaf segments of pigeon pea. Plant Cell Tiss Org Cult. 73: 197–200.
    30. Varshney, R.K., Hoisington, D.A., Upadhyaya, H.D., Gaur, P.M., Nigam, S.N., Saxena, K., Vadez V., Sethy, N.K,. Bhatia, S, Aruna, R., Gowda, M.V.C., Singh, N.K. (2007). Molecular genetics and breeding of grain legume crops for the semi-arid tropics. In: Varshney RK, Tuberosa R, editors. Genomics assisted crop improvement, vol II: genomics applications in crops. Dordrecht: Springer pp. 207–242. 
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