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Transformation of chickpea: effect of genotype, explant, Agrobacterium-strain and composition of culture medium

  • Original Papers
  • Published:
Biologia Plantarum

Abstract

Reproducible and high-frequency transgenic plant regeneration from callus and embryo axes of four different genotypes of chickpea (Cicer arietinum) was achieved after Agrobacterium-mediated transformation. Three different strains of Agrobacterium (EHA105, AGL1 and LBA4404) harboring the binary vector pCAMBIA1301 containing β-glucuronidase (GUS) and hygromycin phosphotransferase (hpt) genes under the control of a CaMV35S promoter were used. The highest number of transgenic plants was obtained from cotyledonary node-derived calli of genotype Pusa-256. A highly efficient rooting was achieved on Murashige and Skoog medium supplemented with indole-3-butyric acid. The stable integration of the gene was confirmed by molecular analyses of the transformed plants. Inheritance of GUS and hpt gene was followed through two generations and they showed the expected 3:1 inheritance.

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Abbreviations

As:

acetosyringone

BAP:

6-benzylaminopurine

Cef:

cefotaxime

CIM:

callus induction medium

CPM:

callus proliferation and regeneration medium

GUS:

β-glucuronidase

IAA:

indole-3-acetic acid

IBA:

indole-3-butyric acid

NAA:

1-napthaleneacetic acid

RIM:

root induction medium

TDZ:

thidiazuron [1-phenyl-3-(1,2,3-thiadiazol-5-YL) urea]

References

  • Chilton, M.-D., Currier, T.C., Farrand, S.K., Bendich, A.J., Gordon, M.P., Nester, E.W.: Agrobacterium tumefaciens DNA and PS8 bacteriophage DNA not detected in crown gall tumors. — Proc. nat. Acad. Sci. USA 71: 3672–3676, 1974.

    Article  CAS  PubMed  Google Scholar 

  • Cho, M.-A., Moon, C.-Y., Liu, J.-R., Choi, P.-S.: Agrobacterium-mediated transformation in Citrullus lanatus. — Biol. Plant. 52: 365–369, 2008.

    Article  CAS  Google Scholar 

  • Dayal, S., Lavanya, M., Devi, P., Sharma, K.K.: An efficient protocol for shoot regeneration and genetic transformation of pigeon pea [Cajanus cajan (L.) Millsp.] using leaf explants. — Plant Cell Rep. 21: 1072–1079, 2003.

    Article  CAS  PubMed  Google Scholar 

  • Dellaporta, S.L., Wood, J., Hicks, J.B.: A plant DNA mini preparation: version II. — Plant mol. Biol. Rep. 1: 19–21, 1983.

    Article  CAS  Google Scholar 

  • Edwards, K., Johnstone, C., Thompson, C.: A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. — Nucl. Acids Res. 19: 1349, 1991.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • Gamborg, O.L., Miller, R.A., Ojima, K.: Nutrient requirements of suspension cultures of soybean root cells. — Exp. Cell Res. 50: 151–158, 1968.

    Article  CAS  PubMed  Google Scholar 

  • Hoekema, A., Hirsch, P.R., Hooykaas, P.J.J., Schilperoort, R.A.: A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Tiplasmid. — Nature 303:179–180, 1983.

    Article  CAS  Google Scholar 

  • Hood, E.E., Fraley, R.T., Chilton, M-D.: The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBO542 outside of the T-DNA. — J. Bacteriol. 168: 1291–1301, 1986.

    CAS  PubMed  Google Scholar 

  • Huda, S., Islam, R., Bari, M.A., Asaduzzaman, M.: Shoot differentiation from cotyledon derived callus of chickpea (Cicer arietinum L.). — Plant Tissue Cult. 13: 53–59, 2003.

    Google Scholar 

  • Jayanand, B., Sudarsanam, G., Sharma, K.K.: An efficient protocol for the regeneration of whole plants of chickpea (Cicer arietinum L.) by using axilary meristem explants derived from in vitro-germinated seeds. — In Vitro cell. dev. Biol. Plant 39: 171–179, 2003.

    Article  Google Scholar 

  • Jefferson, R.A., Kavanagh, T.A., Bevan, M.W.: GUS fusion: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. — EMBO J. 6: 3901–3907, 1987.

    CAS  PubMed  Google Scholar 

  • Kant, P., Kant, S., Jain, R.K., Chaudhury, V.K.: Agrobacteriummediated high frequency transformation in dwarf recalcitrant rice cultivars. — Biol. Plant. 51: 61–68, 2007.

    Article  CAS  Google Scholar 

  • Kar, S., Basu, D., Das, S., Ramakrishnan, N.A., Mukherjee, P., Nayak, P., Sen, S.K.: Expression of Cry 1A(c) gene of Bacillus thuringiensis in transgenic chickpea plants inhibits development of pod borer (Heliothis armigera) larvae. — Transgenic Res. 6: 177–185, 1997.

    Article  CAS  Google Scholar 

  • Kar, S., Johnson, T.M., Nayak, P., Sen, S.K.: Efficient transgenic plant regeneration through Agrobacteriummediated transformation of chickpea (Cicer arietinum L). — Plant Cell Rep. 16: 32–37, 1996.

    Article  CAS  Google Scholar 

  • Krishnamurthy, K.V., Suhasini, K., Sagare, A.P., Meixner, M., Kathen, A. De Pickardt, T., Schieder, O.: Agrobacteriummediated transformation of chickpea (Cicer arietinum L.) embryo axes. — Plant Cell Rep. 19: 235–240, 2000.

    Article  CAS  Google Scholar 

  • Lazo, G.R., Stein, P.A., Ludwig, R.A.: A DNA transformationcompetent Arabidopsis genomic library in Agrobacterium. — BioTechnology 9: 963–967, 1991.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • Mok, M.C., Mok, D.W.S., Turner, J.E.: Biological and biochemical effects of cytokinin-active phenylurea derivatives in tissue culture systems. — HortScience 22: 1194–1197, 1987.

    CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Olhoft, P.M., Flagel, L.E., Donovan, C.M., Somers, D.A.: Efficient soybean transformation using hygromycin B selection in the cotyledonary-node method. — Planta 216: 723–735, 2003.

    CAS  PubMed  Google Scholar 

  • Polowick, P.L., Baliski, D.S., Mahon, J.D.: Agrobacterium tumefaciens-mediated transformation of chickpea (Cicer arietinum L.): gene integration, expression and inheritance. — Plant Cell Rep. 23: 485–491, 2004.

    Article  CAS  PubMed  Google Scholar 

  • Polowick, P.L., Quandt, J., Mahon, J.D.: The ability of pea transformation technology to transfer genes into peas adapted to Western Canadian growing conditions. — Plant Sci. 153: 161–170, 2000.

    Article  CAS  PubMed  Google Scholar 

  • Rohini, V.K., Rao, K.S.: Transformation of peanut (Arachis hypogaea L.): a non-tissue culture based approach for generating transgenic plants. — Plant Sci. 150: 41–49, 2000.

    Article  CAS  Google Scholar 

  • 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.

    Article  Google Scholar 

  • Saini R., Jaiwal, P.K.: Agrobacterium tumefaciens-mediated transformation of blackgram: an assessment of factors influencing the efficiency of uidA gene transfer. — Biol. Plant. 51: 69–74, 2007.

    Article  CAS  Google Scholar 

  • Saini, R., Jaiwal, S., Jaiwal, P.K.: Stable genetic transformation of Vigna mungo L. Hepper via Agrobacterium tumefaciens. — Plant Cell Rep. 21: 701–705, 2003.

    Google Scholar 

  • Sambrook, J., Fritsch, E.F., Maniatis, T.: Molecular cloning: A Laboratory Manual. 2nd Ed. Vol. 3. — Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989.

    Google Scholar 

  • Sarmah, B.K., Moore, A., Tate, W., Molvig, L., Morton, R.L., Rees, D.P., Chiaiese, P., Chrispeels, M.J., Tabe, L.M., Higgins, T.J.V.: Transgenic chickpea plants expressing high levels of a bean α-amylase inhibitor. — Mol. Breed. 14: 73–82, 2004.

    Article  CAS  Google Scholar 

  • Satyavati, V.V., Prasad, V., Khandelwal, A., Shailia, M.S., Lakshmi Sita, G.: Expression of hemagglutinin protein of Rinderpest virus in transgenic pigeon pea [Cajanus cajan (L.) Millsp] plants. — Plant Cell Rep. 21: 651–658, 2002.

    Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Sharma, K.K., Anjaiah, V.: An efficient method for the production of transgenic plants of peanut (Arachis hypogaea L.) through Agrobacterium tumefaciens-mediated genetic transformation. — Plant Sci. 159: 7–19, 2000.

    Article  CAS  PubMed  Google Scholar 

  • Sharma, K.K., Bhojwani, S.S., Thorpe, T.A.: Factors effecting high frequency differentiation of shoots and roots from cotyledon explants of Brassica juncea (L.) Czern. — Plant Sci. 66: 247–253, 1990.

    Article  CAS  Google Scholar 

  • Singh, R., Jat, R.S., Sahoo, P.D., Srinivasan: Thidiazuron induced multiple shoot formation in chickpea (Cicer arietinum L.). — J. Plant Biochem. Biotechnol. 11: 129–131, 2002.

    CAS  Google Scholar 

  • Somers, D.A., Samac, D.A., Olhoft, P.M.: Recent advances in legume transformation. — Plant Physiol. 131: 892–899, 2003.

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Tiwari, R.K., Trivedi, M., Guang, Z.-C., Guo, G.-Q., Zheng, G.-C.: Agrobacterium rhizogenes mediated transformation of Scutellaria baicalensis and production of flavonoids in hairy roots. — Biol. Plant. 52: 26–35, 2008.

    Article  CAS  Google Scholar 

  • Thu, T.T., Mai, T.T.X., Dewaele, E., Farsi, S., Tadesse, Y., Angenon, G., Jacobs, M.: In vitro regeneration and transformation of pigeonpea [Cajanus cajan (L.) Millsp]. — Mol. Breed. 11: 159–168, 2003.

    Article  CAS  Google Scholar 

  • Wang, M., Waterhouse, P.M.: A rapid and simple method of assaying plants transformed with hygromycin or PPT resistance gene. — Plant mol. Biol. Rep. 15: 209–215, 1997.

    Article  CAS  Google Scholar 

  • Zhang J., Xing A., Staswick P., Clemente T.E. The use of glufosinate as selective agent in Agrobacterium-mediated transformation of soybean. — Plant Cell Tissue Organ Cult. 56: 37–46, 1999.

    Article  CAS  Google Scholar 

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Acknowledgements

This work was partially supported by grants from ICGEB and Department of Science and Technology (DST), Government of India. BB acknowledges DST for the award of Scientific and Engineering Research Council (SERC) Fast Track Young Scientist Fellowship.

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  1. M. Mohan

    Present address: Department of Plant Molecular Biology, University of Delhi South Campus, Benito Juarez Marg, New Delhi, 110021, India

Authors and Affiliations

  1. International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110067, India

    B. Bhattacharjee, M. Mohan & S. Nair

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Correspondence to S. Nair.

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Bhattacharjee, B., Mohan, M. & Nair, S. Transformation of chickpea: effect of genotype, explant, Agrobacterium-strain and composition of culture medium. Biol Plant 54, 21–32 (2010). https://doi.org/10.1007/s10535-010-0004-4

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