Biologia plantarum 51:69-74, 2007 | DOI: 10.1007/s10535-007-0014-z

Agrobacterium tumefaciens-mediated transformation of blackgram: An assessment of factors influencing the efficiency of uidA gene transfer

R. Saini1,*, P. K. Jaiwal1
1 Department of Biosciences, M.D. University, Rohtak, India

Agrobacterium tumefaciens strain EHA105 carrying a binary vector pCAMBIA2301, which contains a neomycin phosphotransferase gene (nptII) and a β-glucuronidase (GUS) gene (uidA) interrupted with an intron, was used for transformation of Vigna mungo cotyledonary node explants. Various factors such as preculture and wounding of explants, manipulations in inoculation and co-cultivation conditions were found to play a significant role in influencing tissue competence, Agrobacterium virulence and compatibility of both, for achieving the maximum transformation frequencies. The stable transformation with 4.31 % efficiency was achieved using the optimized conditions. The transformed green shoots that were selected and rooted on medium containing kanamycin and tested positive for nptII gene by polymerase chain reaction were established in soil to collect seeds. GUS activity was detected in leaves, roots, pollen grains and T1 seedlings. Southern analysis of T0 plants showed the integration of nptII into the plant genome.

Keywords: cotyledonary node; GUS activity; transgenic plants; Vigna mungo
Subjects: Agrobacterium tumefaciens; blackgram; cotyledon, cotyledonary node; glucuronidase; in vitro culture, regeneration, proliferation, differentiation; nutrient medium, Murashige and Skoog (MS); polymerase chain reaction (PCR); Southern blot analysis; transgenic plants; Vigna mungo

Received: February 10, 2005; Accepted: October 15, 2005; Published: March 1, 2007Show citation

ACS AIP APA ASA Harvard Chicago IEEE ISO690 MLA NLM Turabian Vancouver
Saini, R., & Jaiwal, P.K. (2007). Agrobacterium tumefaciens-mediated transformation of blackgram: An assessment of factors influencing the efficiency of uidA gene transfer. Biologia plantarum, 51(1), 69-74. doi: 10.1007/s10535-007-0014-z.
Download citation
  • BibTeX (.bib)
  • Bookends (.ris)
  • EasyBib (.ris)
  • EndNote (.enw)
  • EndNote 8 (.xml)
  • ISI WoS (.isi)
  • Medlars (.medlars)
  • Mendeley (.ris)
  • MODS (.xml)
  • Papers (.ris)
  • RefWorks (.txt)
  • RefManager (.ris)
  • RIS (.ris)
  • MS Word (.xml)
  • Zotero (.ris)
    • Open full article

    References

    1. Atkinson, R.G., Gardner, R.: Agrobacterium-mediated transformation of pepino and regenration of transgenic plants.-Plant Cell Rep. 10: 208-212, 1991. Go to original source...
    2. Bean, S.J., Gooding, P.S., Mullineaux, P.M., Davies, D.R.: A simple system for pea transformation.-Plant Cell Rep. 16: 513-519, 1997. Go to original source...
    3. Bidney, D., Scelonge, C., Martich, J., Burrs, M., Sims, L., Huffman, G.: Microprojectile bombardment of plant tissues increases transformation frequency by Agrobacterium tumefaciens.-Plant mol. Biol. 18: 301-313, 1992. Go to original source...
    4. Binns, A.N., Thomashow, M.F.: Cell biology of Agrobacterium infection and transformation of plants.-Annu. Rev. Microbiol. 42: 575-606, 1988. Go to original source...
    5. De Bondt, A., Eggermont, K., Druart, P., De Vil, M., Goderis, I., Van der Leyden, J., Broekaert, W.: Agrobacterium-mediated transformation of apple (Malus × domestica Borkh): an assessment of factors affecting gene transfer during early transformation steps.-Plant Cell Rep. 13: 587-593, 1994. Go to original source...
    6. De la Riva, G.A., Cabrera, J.G., Padron, R.V., Pardo, C.A.: Agrobacterium tumefaciens: a natural tool for plant transformation.-Eur. J. Biotechnol. 1: 1-18, 1998. Go to original source...
    7. Geetha, N., Venkatachalam, P., Laxmi Sita, G.: Agrobacterium-mediated genetic transformation of pigeonpea (Cajanus cajan L.) and development of transgenic plants via direct organogenesis.-Plant Biotechnol. 16: 213-218, 1999. Go to original source...
    8. Holford, P., Hernandez, N., Newburg, H.T.: Factors influencing the efficiency of T-DNA transfer during co-cultivation of Antirrhinum majus with Agrobacterium tumefaciens.-Plant Cell Rep. 11: 196-199, 1992. Go to original source...
    9. Jaiwal, P.K., Kumari, R., Ignacimuthu, S., Potrykus, I., Sautter, C.: Agrobacterium tumefaciens-mediated genetic transformation of mungbean (Vigna radiata L. Wilczek) - a recalcitrant grain legume.-Plant Sci. 161: 239-247, 2001. Go to original source...
    10. Jaiwal, P.K., Singh, R.P. (ed.): Focus on Biotechnology. Vol. 10A: Improvement Strategies for Leguminosae Biotechnology.-Kluwer Academic Publisher, Dordrecht 2003. Go to original source...
    11. Janssen, B.J., Gardner, R.C.: The use of transient GUS expression to develop on Agrobacterium-mediated gene transfer system for kiwifruit.-Plant Cell Rep. 13: 28-31, 1993. Go to original source...
    12. Jefferson, R.A., Kavanagh, T.A., Bevan, M.W.: GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.-EMBO J. 6: 3901-3907, 1987. Go to original source...
    13. Kaneyoshi, J., Kobayashi, S., Nakamura, Y., Shigemoto, N., Doi, Y.: A simple and efficient gene transfer system of trifoliate orange.-Plant Cell Rep. 13: 541-545, 1994.
    14. Li, H.Y., Zhu, Y.M., Chen, Q., Conner, R.L., Ding, X.D., Li, J., Zhang, B.B.: Production of transgenic soybean plants with two anti-fungal protein genes via Agrobacterium and particle bombardment.-Biol. Plant. 48: 367-374, 2004. Go to original source...
    15. Lin, J.J., Garcia-Assad, N., Kuo, J.: Effect of Agrobacterium cell concentration on the transformation efficiency of tobacco and Arabidopsis thaliana.-Focus 16: 72-77, 1994.
    16. Mohan, K.L., Krishnamurthy, K.V.: Plant regeneration from decapitated mature embryo axis and Agrobacterium mediated genetic transformation of pigeon pea.-Biol. Plant. 46: 519-527, 2003. Go to original source...
    17. Muthukumar, B., Mariamma, M., Veluthambi, K., Gnanam, A.: Genetic transformation of cotyledon explants of cowpea (Vigna unguiculata L. Walp.) using Agrobacterium tumefaciens.-Plant Cell Rep. 15: 980-985, 1996.
    18. Popelka, J.C., Terryn, N., Higgins, T.J.V.: Gene technology for grain legumes: can it contribute to the food challenge in developing countries.-Plant Sci. 167: 195-206, 2004. Go to original source...
    19. Rogers, S.O., Bendich, A.J.: Extraction of DNA from plant tissues.-In: Gelvin, S.B., Schilperoot, R.A. (ed.): Plant Molecular Biology Manual. Pp. 1-11. Kluwer Academic Publisher, Dordrecht 1988. Go to original source...
    20. Saini, R., Jaiwal, S., Jaiwal, P.K.: Stable genetic transformation of Vigna mungo L. Hepper via Agrobacterium tumefaciens.-Plant Cell Rep. 21:851-859, 2003. Go to original source...
    21. 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. Go to original source...
    22. Somers, D.A., Samac, D.A., Olhoft, P.M.: Recent advances in legume transformation.-Plant Physiol. 131: 892-899, 2003. Go to original source...
    23. Stachel, S.E., Messens, E., Van Montagu, M., Zambryski, P.: Identification of signal molecules produced by wounded plant cells that activate T-DNA transfer in Agrobacterium tumefaciens.-Nature 318: 624-629, 1985. Go to original source...
    24. Stachel, S.E., Nester, E.W., Zambryski, P.: A plant cell factor induces Agrobacterium tumefaciens vir gene expression.-Proc. nat. Acad. Sci. USA 83: 379-383, 1986. Go to original source...
    25. Uranbey, S., Sevimay, C.S., Kaya, M.D., Ipek, A., Sancak, C., Basalma, D., Er, C., Ozcan, S.: Influence of different co-cultivation temperatures, periods and media on Agrobacterium tumefaciens-mediated gene transfer.-Biol. Plant. 49: 53-57, 2005. Go to original source...
    26. Zambryski, P.C.: Chronicles from the Agrobacterium-plant cell DNA transfer story.-Annu. Rev. Plant Physiol. Plant mol. Biol. 43: 465-490, 1992. Go to original source...

    Return to the content