Skip to main content
SpringerLink
Log in

Optimization of regeneration and Agrobacterium-mediated transformation of immature cotyledons of chickpea (Cicer arietinum L.)

  • Original Paper
  • Published:
Plant Cell, Tissue and Organ Culture (PCTOC) Aims and scope Submit manuscript

Abstract

Immature cotyledons collected at different time intervals from four genotypes of chickpea (C 235, BG 256, P 362 and P 372) were cultured adaxially on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine, thidiazuron, kinetin, zeatin and dimethylallylaminopurine (2-iP), either alone or in combination with indole-3-acetic acid (IAA) or α-napthoxyacetic acid (α-NOA) for dedifferentiation and regeneration of adventitious shoots. Morphogenesis was achieved with explants cultured adaxially on MS medium with 13.68 μM zeatin, 24.6 μM 2-iP, 0.29 μM IAA and 0.27 μM α-NOA. Explants prepared from pods of 21 days after pollination, responded favourably to plant growth regulator treatment in shoot differentiation. Histological studies of the regenerating explants, revealed the initiation of meristematic activity in the sub-epidermal region during the onset of morphogenesis, which can be correlated with elevated activity of cytokinin oxidase-dehydrogenase, for cytokinin metabolism. The regenerated shoots were efficiently rooted in MS medium supplemented with 2.46 μM indole-3-butyric acid and acclimatized under culture room and glasshouse conditions for normal plant development leading to 76–80 % survival of the rooted plantlets. The immature cotyledon explants were used for Agrobacterium-mediated transformation with critical manipulation of cultural conditions like age of explant, O.D. of Agrobacterium suspension, concentration of acetosyringone, duration of sonication and co-cultivation for successful genetic transformation and expression of the reporter gene uidA (GUS). Integration of transgene was confirmed by molecular analysis. Transformation frequency up to 2.08 % was achieved in chickpea, suggesting the feasibility of using immature cotyledon explants for Agrobacterium-mediated transformation.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

BA:

6-Benzyladenine

CKX:

Cytokinin oxidase-dehydrogenase

DAP:

Days after pollination

IAA:

Indole-3-acetic acid

2-iP:

Dimethylallylaminopurine

MS:

Murashige and Skoog

α-NOA:

α-Naphthoxyacetic acid

PGR:

Plant growth regulator

MU:

4-Methylumbelliferone

References

  • Ainsley PJ, Hammerschlag FA, Bertozzi T, Collins GG, Sedgley M (2001) Regeneration of almond from immature seed cotyledons. Plant Cell Tissue Org Cult 67:221–226

    Article  CAS  Google Scholar 

  • Amoah BK, Wu H, Sparka C, Jones HD (2001) Factors influencing Agrobacterium-mediated transient expression of uidA in wheat inflorescence tissue. J Exp Bot 52:1135–1142

    Article  PubMed  CAS  Google Scholar 

  • Angelini RR, Allavena A (1989) Plant regeneration from immature cotyledon explant cultures of bean (P. coccineus L.). Plant Cell Tissue Org Cult 19:167–174

    Article  Google Scholar 

  • Angleton EL, Flurkey WH (1984) Activation and alteration of plant and fungal polyphenol oxidase isoenzymes in sodium dodecyl sulfate electrophoresis. Phytochemistry 23:2723–2725

    Article  CAS  Google Scholar 

  • Barna KS, Wakhlu AK (1993) Somatic embryogenesis and plant regeneration from callus cultures of chickpea (Cicer arietinum L.). Plant Cell Rep 12:521–524

    Article  CAS  Google Scholar 

  • Bilyeu KD, Cole JL, Laskey JG, Riekhot WR, Esparza TJ, Kramer MD, Morris RO (2001) Molecular and biochemical characterisation of cytokinin oxidase from maize. Plant Physiol 125:378–386

    Article  PubMed  CAS  Google Scholar 

  • Christou P (1997) Biotechnology applied to grain legumes. Field Crops Res 53:83–97

    Article  Google Scholar 

  • Davey MR, Kumar V, Hammatt N (1994) In vitro culture of legumes. In: Vasil IK, Thorpe TA (eds) Plant cell and tissue culture. Kluwer, Dordrecht, pp 313–329

    Google Scholar 

  • Dinesh Kumar V, Kirti PB, Sachan JKS, Chopra VL (1994) Picloram induced somatic embryogenesis in chickpea (Cicer arietinum L.). Plant Sci 109:207–213

    Article  Google Scholar 

  • Dita MA, Rispail N, Prats E, Rubiales D, Singh KB (2006) Biotechnology approaches to overcome biotic and abiotic stress constraints in legumes. Euphytica 147:1–24

    Article  Google Scholar 

  • Emery RJN, Leport L, Barton JE, Turner NC, Atkins CA (1998) Cis-isomers of cytokinins predominate in chickpea seeds throughout their development. Plant Physiol 117:1515–1523

    Article  PubMed  CAS  Google Scholar 

  • Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirement of suspension cultures of soybean root cells. Exp Cell Res 50:150–158

    Article  Google Scholar 

  • Ghanti SK, Sujata KG, Rao MS, Kavi Kishor PB (2010) Direct somatic embryogenesis and plant regeneration from immature explants of chickpea. Biol Plant 54:121–125

    Article  Google Scholar 

  • Hartweck LM, Lazzeri PA, Cui D, Collins GB, Williams EC (1988) Auxin-orientation effects on somatic embryogenesis from immature soybean cotyledons. In Vitro Cell Dev Biol-Plant 24:821–828

    Article  CAS  Google Scholar 

  • Hita O, Lafarga C, Guerra H (1997) Somatic embryogenesis from chickpea (Cicer arietinum L.) immature cotyledons: the effect of zeatin, gibberellic acid and indole-3-butyric acid. Acta Physiol Plant 19:333–338

    Article  CAS  Google Scholar 

  • Hoque ME, Mansfield JW (2004) Effect of genotype and explant age on callus induction and subsequent plant regeneration from root-derived callus of indica rice genotypes. Plant Cell Tissue Org Cult 78:217–223

    Article  CAS  Google Scholar 

  • Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907

    PubMed  CAS  Google Scholar 

  • Jones R, Schreiber BMN (1997) Role and function of cytokinin oxidase in plants. Plant Growth Reg 23:123–134

    Article  CAS  Google Scholar 

  • Kumar V, Davey MR (1991) Genetic improvement of legumes using somatic cell and molecular techniques. Euphytica 55:157–169

    Article  CAS  Google Scholar 

  • Kumlehn J, Serazetdinov L, Hensel G, Becker D, Loerz H (2006) Genetic transformation of barley (Hordeum vulgare L.) via infection of androgenetic pollen cultures with Agrobacterium tumefaciens. Plant Biotech J 4:251–261

    Article  CAS  Google Scholar 

  • Lazzeri PA, Hildebrandt DF, Collins GB (1985) A procedure for plant regeneration from immature cotyledon tissue of soybean. Plant Mol Biol Rep 3:160–167

    Article  Google Scholar 

  • Libreros-Minotta CA, Tipton PA (1995) A colorimetric assay for cytokinin oxidase. Anal Biochem 231:339–341

    Article  PubMed  CAS  Google Scholar 

  • Maheshwaran G, Williams EG (1984) Direct somatic embryoid formation on immature embryos of Trifolium repens, T. pratense and Medicago sativa and rapid clonal propagation of T. repens. Ann Bot 54:201–211

    Google Scholar 

  • Malik KA, Saxena PK (1992) Thidiazuron induced 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

    Article  CAS  Google Scholar 

  • Mehrotra M, Sanyal I, Amla DV (2011a) High-efficiency Agrobacterium mediated transformation of chickpea (Cicer arietinum L.) and regeneration of insect-resistant transgenic plants. Plant Cell Rep 30:1603–1616

    Article  PubMed  CAS  Google Scholar 

  • Mehrotra M, Singh AK, Sanyal I, Altosaar I, Amla DV (2011b) Pyramiding of modified cry1Ab and cry1Ac genes of Bacillus thuringiensis in chickpea (Cicer arietinum L.) for improved resistance to pod borer insect Helicoverpa armigera. Euphytica 182:87–102

    Article  CAS  Google Scholar 

  • Mohan ML, Krishnamurthy KV (2002) Somatic embryogenesis and plant regeneration in pigeonpea. Biol Plant 45:19–25

    Article  Google Scholar 

  • Mok DWS, Mok MC (2001) Cytokinin metabolism and action. Ann Rev Plant Physiol Mol Biol 52:89–118

    Article  CAS  Google Scholar 

  • Murashige T, Skoog F (1962) Revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Murthy BNS, Victor J, Singh RP, Fletcher RA, Saxena PK (1996) In vitro regeneration of chickpea (Cicer arietinum L.): stimulation of direct organogenesis and somatic embryogenesis by thidiazuron. Plant Growth Reg 19:233–240

    Article  CAS  Google Scholar 

  • O’Keefe D, Song J, Jameson PE (2011) Isopentenyl transferase and cytokinin oxidase/dehydrogenase gene family members are differentially expressed during pod and seed development in rapid-cycling Brassica. J Plant Growth Regul 30:92–99

    Article  Google Scholar 

  • Ozias-Akins P (1989) Plant regeneration from immature cotyledons of peanut. Plant Cell Rep 8:217–218

    Article  Google Scholar 

  • Parrott WA, Bailey MA, Durham RE, Mathews HV (1992) Tissue culture and regeneration in legumes. In: Moss JP (ed) Biotechnology and crop improvement in Asia. ICRISAT, India, pp 115–148

    Google Scholar 

  • Parrott WA, Durham RE, Bailey MA (1995) Somatic embryogenesis in legumes. In: Bajaj YPS (ed) Biotechnology in agriculture and forestry, somatic embryogenesis and synthetic seed II, vol 31. Springer, Berlin, pp 199–227

    Chapter  Google Scholar 

  • Pathak MR, Hamzah RY (2008) An effective method of sonication-assisted Agrobacterium-mediated transformation of chickpeas. Plant Cell Tissue Org Cult 93:65–71

    Article  Google Scholar 

  • Popelka JC, Terryn N, Higgins TJV (2004) Gene technology for grain legumes: can it contribute to the food challenge in developing countries? Plant Sci 167:195–206

    Article  CAS  Google Scholar 

  • Sagare AP, Suhasini K, Krishnamurthy KV (1995) Histology of somatic embryo initiation and development in chickpea (Cicer arietinum L.). Plant Sci 109:87–93

    Article  CAS  Google Scholar 

  • Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, New York

    Google Scholar 

  • Santarem ER, Trick HN, Essig JS, Finer JJ (1998) Sonication-assisted Agrobacterium-mediated transformation of soybean immature cotyledons: optimization of transient expression. Plant Cell Rep 17:752–759

    Article  CAS  Google Scholar 

  • Sanyal I, Singh AK, Kaushik M, Amla DV (2005) Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.) with Bacillus thuringiensis cry1Ac gene for resistance against pod borer insect Helicoverpa armigera. Plant Sci 168:1135–1146

    Article  CAS  Google Scholar 

  • Senthil G, Williamson B, Dinkins RD, Ramsay G (2004) An efficient transformation system for chickpea (Cicer arietinum L.). Plant Cell Rep 23:297–303

    Article  PubMed  CAS  Google Scholar 

  • Shri PV, Davis TM (1992) Zeatin-induced shoot regeneration from immature chickpea (Cicer arietinum L.) cotyledons. Plant Cell Tissue Org Cult 28:45–51

    Article  CAS  Google Scholar 

  • Somers DA, Samac DA, Olhoft PM (2003) Recent advances in legume transformation. Plant Physiol 131:892–899

    Article  PubMed  CAS  Google Scholar 

  • Steeves TA, Sussex IM (1989) Patterns in plant development. Cambridge University Press, New York

    Book  Google Scholar 

  • Suhasini K, Sagare AP, Krishnamurthy KV (1994) Direct somatic embryogenesis from mature embryo axes in chickpea (Cicer arietinum L.). Plant Sci 102:189–194

    Article  CAS  Google Scholar 

  • Suhasini K, Sagare AP, Krishnamurthy KV (1996) Study of aberrant morphologies and lack of conversion of somatic embryos of chickpea (Cicer arietinum L.). In Vitro Cell Dev Biol-Plant 32:6–10

    Article  Google Scholar 

  • Tetu T, Sangwan RS, Sangwan-Norreel BS (1990) Direct somatic embryogenesis and organogenesis in cultured immature zygotic embryos of Pisum sativum L. J Plant Physiol 137:102–109

    Article  Google Scholar 

  • Tewari-Singh N, Sen J, Kiesecker H, Reddy VS, Jacobsen H-J, Guha-Mukherjee S (2004) Use of a herbicide or lysine plus threonine for non-antibiotic selection of transgenic chickpea. Plant Cell Rep 22:576–583

    Article  PubMed  CAS  Google Scholar 

  • Trick HN, Finer JJ (1997) SAAT: sonication-assisted Agrobacterium-mediated transformation. Transgenic Res 6:1–8

    Article  Google Scholar 

  • Vancanneyt G, Schmidt R, O’Connor-Sanchez A, Willmitzer L, Rocha-Sosa M (1990) Construction of an intron-containing marker gene: splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium-mediated plant transformation. Mol Gen Genet 220:245–250

    Article  PubMed  CAS  Google Scholar 

  • Varshney RK, Close TJ, Singh NK, Hoisington DA, Cook DR (2009) Orphan legumes enter the genomics era. Curr Opin Plant Biol 12:202–210

    Article  PubMed  Google Scholar 

  • Veena, Jiang H, Doerge RW, Gelvin SB (2003) Transfer of T-DNA and vir-protein to plant cells by Agrobacterium tumefaciens induces expression of host genes involved in mediating plant transformation and suppression host defense gene expression. Plant J 35:219–236

  • Vernoux T, Autran D, Traas J (2000) Developmental control of cell division patterns in the shoot apex. Plant Mol Biol 43:569–580

    Article  PubMed  CAS  Google Scholar 

  • Vyroubalova S, Vaclavikova K, Tureckova V, Novak O, Smehilova M, Hluska T, Ohnoutkova L, Frébort I, Galuszka P (2009) Characterization of new maize genes putatively involved in cytokinin metabolism and their expression during osmotic stress in relation with cytokinin levels. Plant Physiol 151:433–447

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We appreciate the consistent support of Dr. C.S. Nautiyal, Director, CSIR-NBRI, Lucknow for this work. We are thankful to Dr. Dheer Singh, CRC, Gobind Ballabh Pant University of Agriculture and Technology, Pantnagar, Uttarakhand, India for generous supply of high quality seeds of chickpea. Awards of fellowship to L.T. and A.K.S. from CSIR and R.S. from UGC, New Delhi are gratefully acknowledged. This work was supported by the Council of Scientific and Industrial Research, New Delhi under the Network Project NWP-03.

Author information

Authors and Affiliations

  1. Plant Transgenic Lab, CSIR-National Botanical Research Institute, Rana Pratap Marg, P.O. Box 436, Lucknow, 226 001, India

    Aditya K. Singh, Shweta Singh, Rani Singh, Sonam Chaudhary, Indraneel Sanyal & D. V. Amla

  2. International Institute of Tropical Agriculture, P.O. Box 7878, Kampala, Uganda

    Leena Tripathi

Authors
  1. Leena Tripathi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Aditya K. Singh
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shweta Singh
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Rani Singh
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sonam Chaudhary
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Indraneel Sanyal
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. D. V. Amla
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to D. V. Amla.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Tripathi, L., Singh, A.K., Singh, S. et al. Optimization of regeneration and Agrobacterium-mediated transformation of immature cotyledons of chickpea (Cicer arietinum L.). Plant Cell Tiss Organ Cult 113, 513–527 (2013). https://doi.org/10.1007/s11240-013-0293-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11240-013-0293-3

Keywords

Search

Navigation