Abstract
Blackgram (Vigna mungo (L.) Hepper), an important grain legume crop, is sensitive to many fungal pathogens including Corynespora cassiicola, the causal agent of corynespora leaf spot disease. In the present study, plasmid pGJ42 harboring neomycin phosphotransferase (nptII) a selectable marker gene, the barley antifungal genes chitinase (AAA56786) and ribosome-inactivating protein (RIP; AAA32951) were used for the transformation, to develop fungal resistance for the first time in blackgram. The presence and integration of transgene into the blackgram genome was confirmed by PCR and Southern analysis with an overall transformation frequency of 10.2 %. Kanamycin selection and PCR analysis of T0 progeny revealed the inheritance of transgene in Mendelian fashion (3:1). Transgenic plants (T1), evaluated for fungal resistance by in vitro antifungal assay, arrested the growth of C. cassiicola up to 25–40 % over the wild-type plants. In fungal bio-assay screening, the transgenic plants (T1) sprayed with C. cassiicola spores showed a delay in onset of disease along with their lesser extent in terms of average number of diseased leaves and reduced number and size of lesions. The percent disease protection among different transformed lines varies in the range of 27–47 % compare to control (untransformed) plants. These results demonstrate potentiality of chitinase and RIP from a heterologous source in developing fungal disease protection in blackgram and can be helpful in increasing the production of blackgram.
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Abbreviations
- BAP:
-
6-Benzylaminopurine
- IBA:
-
Indole-3-butyric acid
- nptII:
-
Neomycin phosphotransferase
- RIP:
-
Ribosome-inactivating protein
- RM:
-
Rooting media
- SR:
-
Shoot regeneration medium
References
Jeswani, L. M., & Baldev, B. (1990). Publication & information division. New Delhi: Indian Council of Agricultural Research.
Singh, R. A., Gurha, S. N., & Ghosh, A. (2005). In T. D. Thind (Ed.), Diseases of field crop and their management (2nd ed., pp. 179–204). Delhi: Daya Publishing House.
Singh, R. S. (1990). Plant diseases (6th ed.). New Delhi: Oxford and IBH.
Fillippone, E. (1993). Grain Legumes, 2, 20–21.
Punja, Z. K. (2001). Canadian Journal of Plant Pathology, 23, 216–235.
Phuntumart, V. (2003). In C. N. Stewart Jr. (Ed.), Transgenic plants: current innovations and future trends (pp. 180–215). Wymondmam, England: Horizon Scientific Press.
Grover, A., & Gowthaman, R. (2003). Current Science, 84, 330–340.
Stefani, F. O. P., & Hamelin, R. C. (2010). Environmental Reviews, 18, 441–475.
Ceasar, S. A., & Ignacimuthu, S. (2012). Biotechnology Letters, 34, 995–1002.
Rohini, V. K., & Rao, K. S. (2001). Plant Science, 160, 889–898.
Datta, K., Tu, J., Oliva, N., Ona, I., Velazhahan, R., & Mew, T. W. (2001). Plant Science, 160, 405–414.
Mondal, K. K., Chatterjee, S. C., Viswakarma, N., Bhattacharya, R. C., & Grover, A. (2003). Current Microbiology, 47, 171–173.
Mondal, K. K., Bhattacharya, R. C., Koundal, K. R., & Chatterjee, S. C. (2007). Plant Cell Report, 26, 247–252.
Shin, S., Mackintosh, C. A., Lewis, J., Heinen, S. J., Radmer, L., Dill-Macky, R., Baldridge, G. D., Zeyen, R. J., & Muehlbauer, G. J. (2008). Journal of Experimental Botany, 59, 2371–2378.
Girhepuje, P. V., & Shinde, G. B. (2011). Plant Cell Tissue and Organ Culture, 105, 243–251.
Ignacimuthu, S., & Ceasar, S. A. (2012). Journal of Bioscience, 37, 135–147.
Jach, G., Gornhardt, B., Mundy, J., Logemann, J., Pinsdorf, E., Leah, R., Schell, J., & Maas, C. (1995). Plant Journal, 8, 97–109.
Kim, J. K., Jang, I. C., Wu, R., Zuo, W. N., Boston, R. S., Lee, Y. H., Ahn, P., & Nahm, B. H. (2003). Transgenic Research, 12, 475–484.
Bieri, S., Potrykus, I., & Futterer, J. (2003). Molecular Breeding, 11, 37–48.
Li, H. Y., Zhu, Y. M., Chen, Q., Conner, R. L., Ding, X. D., & Zhang, B. B. (2004). Biologia Plantarum, 48, 367–374.
Sridevi, G., Parameswari, C., Sabapathi, N., Raghupathy, V., & Veluthambi, K. (2008). Plant Science, 175, 283–290.
Wally, O., Jayaraj, J., & Punja, Z. (2009). European Journal of Plant Pathology, 123, 331–342.
Eapen, S. (2008). Biotechnology Advances, 26, 162–168.
Atif, R. M., Patat-Ochatt, E. M., Svabova, L., Ondrej, V., Klenoticova, H., Jacas, L., Griga, M., & Ochatt, S. J. (2013). In U. Lüttge, W. Beyschlag, D. Francis, & J. Cushman (Eds.), Progress in Botany (Vol. 74, pp. 37–100). Berlin Heidelberg: Springer-Verlag.
Saini, R., Sonia, & Jaiwal, P. K. (2003). Plant Cell Reports, 21, 851–859.
Saini, R., & Jaiwal, P. K. (2005). Plant Cell Reports, 24, 164–171.
Saini, R., & Jaiwal, P. K. (2007). Biologia Plantarum, 51, 69–74.
Muruganantham, M., Amutha, S., Selvaraj, N., Vengadesan, G., & Ganapathi, A. (2007). In Vitro Cellular and Developmental Biology –Plant, 43, 550–557.
Bhomkar, P., Chandrama, P., Upadhyay, P., Saxena, M., Muthusamy, A., Shiva Prakash, N., Pooggin, M., Hohn, T., & Sarin, N. B. (2008). Molecular Breeding, 22, 169–181.
Murashige, T., & Skoog, F. (1962). Physiology Plant, 15, 473–497.
Gamborg, O. L., Miller, R. A., & Ojima, K. (1968). Experimental Cell Research, 50, 151–158.
Rogers, S. O., & Bendich, A. J. (1988). In S. B. Gelvin & R. A. Schilperoot (Eds.), Plant molbiol manual (pp. 1–11). Dordrecht: Kluwer.
Sambrook, J., Fritsch, E. F., & Maniatis, T. (1989). Molecular cloning: a laboratory manual (2nd ed.). Cold Spring Harbor: Cold Spring Harbor Laboratory Press.
Zhu, H., Xu, X., Xiao, G., Yuan, L., & Li, B. (2007). Science in China Series C: Life Sciences, 50, 31–39.
Logemann, J., Jach, G., Tommerup, H., Mundy, J., & Schell, J. (1992). Nature Biotechnology, 10, 305–308.
Leah, R., Tommerup, H., Svendsen, I., & Mundy, J. (1991). Journal of Biological Chemistry, 266, 1564–1573.
Broglie, K., Chet, I., Holliday, M., Cressman, R., Biddle, P., Knowlton, S., Mauvais, C. J., & Broglie, R. (1991). Science, 254, 1194–1197.
Acknowledgments
We are grateful to University Grant Commission, New Delhi for financial support to our research program. We are also thankful to the Max Planck Institute fur Zuchtungsforschung, Germany and Prof. P. K. Jaiwal, India for providing the gene construct.
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Chopra, R., Saini, R. Transformation of Blackgram (Vigna mungo (L.) Hepper) by Barley Chitinase and Ribosome-Inactivating Protein Genes Towards Improving Resistance to Corynespora Leaf Spot Fungal Disease. Appl Biochem Biotechnol 174, 2791–2800 (2014). https://doi.org/10.1007/s12010-014-1226-2
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DOI: https://doi.org/10.1007/s12010-014-1226-2