Skip to main content
SpringerLink
Log in

Over Expression of Rice chitinase Gene in Transgenic Peanut (Arachis hypogaea L.) Improves Resistance Against Leaf Spot

  • Research
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

A Rice chitinase-3 under enhance version of CaMV 35S was introduced into peanut (Arachis hypogaea L.) through Agrobacterium mediation. Agrobacterium tumefaciens strain LB4404 was used harboring the binary vector (pB1333-EN4-RCG3) containing the chitinase (chit) and hygromycin resistance (hpt) gene as selectable marker. Putative transgenic shoots were regenerated and grown on MS medium supplemented with 5 mg/l BAP, 1 mg/l kinetin, and 30 mg/l hygromycin. Elongated shoots were examined for the presence of the integrated rice chitinase gene along with hygromycin gene as selectable. The integration pattern of transgene in the nuclear genome of the putative transformed plants (T0) was confirmed through Southern hybridization analysis of the genomic DNA. Survival rate of the in vitro regenerated plantlets was over 60% while healthy putatively transgenic (T0) plants with over 42% transformation frequency were produced through Agrobacterium mediated gene transfer of the rice chitinase gene and all the plants flowered and set seed normally. T1 plants were tested for resistance against Cercospora arachidicola by infection with the microspores. Transgenic strains exhibited a higher resistance than the control (non-transgenic plants). chitinase gene expression in highly resistant transgenic strains was compared to that of a susceptible control. A good correlation was observed between chitinase activity and fungal pathogen resistance.

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
Fig. 7

Similar content being viewed by others

Abbreviations

BAP:

Benzyl amino purine

SIM:

Shoot induction medium

RIM:

Root induction medium

LB:

Luria broth

References

  1. Broglie, K., Chet, I., Holliday, M., Cressman, R., Biddle, P., Knowlton, S., et al. (1991). Transgenic plants with enhanced resistance to the fungal pathogen Rizoctonia solani. Science, 254, 1194–1197.

    Article  CAS  Google Scholar 

  2. Grison, R., Grezes-Besset, B., Schneider, M., Lucante, N., Olsen, L., Leguay, J. J., et al. (1996). Field tolerance to fungal pathogens of Brassica napus constitutively expressing a chimeric chitinase gene. Nature Biotechnology, 14, 643–646.

    Article  CAS  Google Scholar 

  3. Taylor, M. G., & Vasil, I. K. (1991). Histology of and physical factors affecting transient GUS expression in pearl millet (Pennisetum glaucum (L.) R. Br.) embryos following microprojectile bombard-ment. Plant Cell Reports, 10, 120–125.

    Article  CAS  Google Scholar 

  4. Devi, P., Zhong, H., & Sticklen, M. B. (2000). In vitro morphogenesis of pearl millet [Pennisetum glaucum (L.) R. Br.]: Efficient production of multiple shoots and inflorescences from shoot-apices. Plant Cell Reports, 19, 546–550.

    Article  CAS  Google Scholar 

  5. Naeem, U. D., Mahmood, A., Shah, G. S., Saeed, I., & Hassan, M. F. (2009). High yielding groundnut (Arachis hypogaea L.) variety “GOLDEN”. Pakistan Journal of Botany, 41(5), 2217–2222.

    Google Scholar 

  6. Murashige, T., & Skoog, K. (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum, 15, 473–497.

    Article  CAS  Google Scholar 

  7. Sambrook, J., Fritsch, E. F., & Maniatis, T. (1989). Molecular cloning: A laboratory manual. New York: Cold Spring Harbor Laboratory Press.

    Google Scholar 

  8. Murray, M. G., & Thompson, W. F. (1980). Rapid isolation of high molecular weight plant DNA. Nucleic Acids Research, 19, 4321–4326.

    Article  Google Scholar 

  9. Satyavathi, V. V., Prasad, V., Khandelwal, A., Shaila, M. S., & Sita, G. L. (2003). Expression of haemagglutinin protein of Rinderpest virus in transgenic pigeonpea [Cajanus cajan (L.) Millsp.] plant. Plant Cell Reports, 21, 651–658.

    CAS  Google Scholar 

  10. Eapen, S., & George, L. (1994). Agrobacterium tumefaciens-mediated gene transfer in peanut (Arachis hypogaea L.). Plant Cell Reports, 13, 582–586.

    Article  CAS  Google Scholar 

  11. Cheng, M., Jarret, R. L., Li, Z., Xing, A., & Demski, J. W. (1996). Production of fertile transgenic peanut (Arachis hypogaea L.) plants using Agrobacterium tumefaciens. Plant Cell Reports, 15, 653–657.

    Article  CAS  Google Scholar 

  12. Rohini, V. K., & Sankara, R. K. (2001). Transformation of peanut (Arachis hypogaea L.) with tobacco chitinase gene: Variable response of transformants to leaf spot disease. Plant Science, 160, 883–892.

    Article  Google Scholar 

  13. Kone, M., Kouakou, T. H., Kone, D., Kouadio, Y. J., Zouzou, M., & Ochatt, S. J. (2009). Factors affecting regeneration of bambara peanut [Vigna subterranea (L.) Verdc.] from mature embryo axes. In Vitro Cellular and Developmental Biology—Plant, 45, 769–775.

    Article  Google Scholar 

  14. Venkatachalam, P., Geetha, N., Khandelwal, A., Shaila, M. S., & LakshmiSita, G. (2000). Agrobacterium-mediated genetic transformation and regeneration of transgenic plants from cotyledon explants of peanut (Arachis hypogaea L.) via somatic embryogenesis. Current Science, 78, 1130–1136.

    CAS  Google Scholar 

  15. Sharma, K. K., & Anjaiah, V. (2000). An efficient method for the production of transgenic plants of peanut (Arachis hypogaea L.) through Agrobacterium tumefaciens-mediated genetic transformation. Plant Science, 159(1), 7–19.

    Article  CAS  Google Scholar 

  16. Yang, H. Y., Nairn, J., & Ozias-Akins, P. (2003). Transformation of peanut using a modified bacterial mercuric ion reductase gene driven by an actin promoter from Arabidopsis thaliana. Journal of Plant Physiology, 160, 945–952.

    Article  CAS  Google Scholar 

  17. Anuradha, T. S., Jami, S. K., Datla, R. S., & Kirti, P. B. (2006). Genetic transformation of peanut (Arachis hypogaea L.) using cotyledonary node as explant and a promoterless gus:nptII fusion gene based vector. Journal of Biosciences, 31, 235–246.

    Article  CAS  Google Scholar 

  18. Mikula, A., Tykarska, T., Kuras, M., & Rybczynski, J. J. (2005). Somatic embryogenesis of Gentiana Cruciata (L.): Histological and ultrastructural changes in seedling hypocotyl explant. In Vitro Cellular and Developmental Biology—Plant, 41, 686–694.

    Article  Google Scholar 

  19. Tiwari, S., & Rakesh, T. (2008). Factors promoting efficient in vitro regeneration from de-embryonated cotyledon explants of Arachis hypogaea L. Plant Cell, Tissue and Organ Culture, 92, 15–24.

    Article  Google Scholar 

  20. Lei, S., Guiying, T., Pingli, X., Zhanji, L., & Yuping, B. (2009). High efficiency in vitro plant regeneration from epicotyls explants of Chinese peanut cultivars. In Vitro Cellular and Developmental Biology—Plant, 45, 525–531.

    Article  Google Scholar 

  21. Matand, K., & Prakash, C. S. (2007). Evaluation of peanut genotypes for in vitro plant regeneration using thidiazuron. Journal of Biotechnology, 130(2), 202–207.

    Article  CAS  Google Scholar 

  22. Grant, J. J., Gelvin, S. B., Meilan, R., & Morris, R. O. (1996). Transfer RNA is the source of extracellular isopentyladenine in a Ti-plasmidless strain of Agrobacterium tumefaciens. Plant Physiology, 110, 431–438.

    Google Scholar 

  23. Leeuwen, L. W., Ruttink, T., Borst-Vrenssen, A. W., Van, D. P., Van-Der, L. H., & Krol, A. R. (2001). Characterization of position induced spatial and temporal regulation of transgene promoter activity in plants. Journal of Experimental Botany, 52, 949–959.

    Article  Google Scholar 

Download references

Acknowledgments

The authors are highly thankful to acknowledge the cooperation of Dr. Ghulam Muhammad Ali, Chief Scientific Officer, National Institute for Genomics and Advanced Biotechnology (NIGAB), NARC, Islamabad, Pakistan and Director Barani Agricultural Research Institute (BARI) Chakwal, Pakistan for the providing the vector pB1333-EN4-RCG3 carrying rice chitinase gene expression cassette and the seeds of peanut varieties, respectively. The authors also acknowledge the support of Dr. Aish Muhammad for his guidance and support during the studies conducted at Plant Tissue Culture Lab, NARC, Islamabad, Pakistan. The authors are also very thankful to Dr. Daniel Tan, Senior Lecturer, University of Sydney, Australia for language improvement of this manuscript and Dr. Nausherwan Nobel Nawab for his valuable guidance in writing this manuscript.

Author information

Authors and Affiliations

  1. National Institute for Genomics and Advanced Biotechnology, NARC, Islamabad, Pakistan

    Muhammad Munir Iqbal, Farhat Nazir, Shaukat Ali, M. Ahsan Asif, Javaid Iqbal & Ghulam Muhammad Ali

  2. Bio-Sciences Division, Pakistan Atomic Energy Commission, Islamabad, Pakistan

    Yusuf Zafar

Authors
  1. Muhammad Munir Iqbal
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Farhat Nazir
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shaukat Ali
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. M. Ahsan Asif
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yusuf Zafar
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Javaid Iqbal
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ghulam Muhammad Ali
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Muhammad Munir Iqbal.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Iqbal, M.M., Nazir, F., Ali, S. et al. Over Expression of Rice chitinase Gene in Transgenic Peanut (Arachis hypogaea L.) Improves Resistance Against Leaf Spot. Mol Biotechnol 50, 129–136 (2012). https://doi.org/10.1007/s12033-011-9426-2

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-011-9426-2

Keywords

Search

Navigation