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.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
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.
Cho, M.-A., Moon, C.-Y., Liu, J.-R., Choi, P.-S.: Agrobacterium-mediated transformation in Citrullus lanatus. — Biol. Plant. 52: 365–369, 2008.
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.
Dellaporta, S.L., Wood, J., Hicks, J.B.: A plant DNA mini preparation: version II. — Plant mol. Biol. Rep. 1: 19–21, 1983.
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.
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.
Gamborg, O.L., Miller, R.A., Ojima, K.: Nutrient requirements of suspension cultures of soybean root cells. — Exp. Cell Res. 50: 151–158, 1968.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Lazo, G.R., Stein, P.A., Ludwig, R.A.: A DNA transformationcompetent Arabidopsis genomic library in Agrobacterium. — BioTechnology 9: 963–967, 1991.
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.
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.
Murashige, T., Skoog, F.: A revised medium for rapid growth and bioassays with tobacco tissue cultures. — Physiol. Plant. 15: 473–497, 1962.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Somers, D.A., Samac, D.A., Olhoft, P.M.: Recent advances in legume transformation. — Plant Physiol. 131: 892–899, 2003.
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.
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.
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.
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.
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.
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.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10535-010-0004-4