Abstract
Transformation approach is a useful tool for the study of gene function, the mechanism of molecular regulation, and increase usefulness of components by reverse genetic approach in plants. In this study, we developed a stable and rapid method for Agrobacterium-mediated transformation of a medicinal plant Chelone glabra L. using leaf explants. Stable transformants were obtained using Agrobacterium tumefaciens strains GV2260 and GV3101 that harbored the binary vector pBI121 and contained the neomycin phosphotransferase gene (NPT II) as a selectable marker and a reporter gene β-glucuronidase (GUS). Putative transformants were identified by kanamycin selection and a histochemical assay. PCR and Southern blot analysis confirmed the integration of the GUS gene into transformed genomes as well as detected stable expression of the β-glucuronidase gene (GUS) by RT-PCR. Resulting transformed plants had morphologically normal phenotypes. This method requires two changes of medium and few leaf explants as well as the transformation efficiency of 2–8 % after 2–3 months of inoculation. This method can provide a quick and economical transformation method for reverse genetic approach to change the secondary metabolic pathway to increase useful components in C. glabra.
Similar content being viewed by others
References
Albach, D. C., Meudt, H. M., & Oxelman, B. (2005). American Journal Botany, 92, 297–315.
Nelson, A. D., & Elisens, W. J. (1999). American Journal Botany, 86, 1487–1501.
Cooperrider, T. S., & McCready, G. A. (1970). Brittonia, 22, 175–183.
Bergeron, C., Marston, A., Gauthier, R., & Hostettmann, K. (1996). Pharmaceutical Biology, 34, 233–242.
Konoshima, T., Takasaki, M., Tokuda, H., & Nishino, H. (2000). Cancer Letters, 157, 87–92.
Park, K. S., & Chang, I. M. (2004). Planta Medica, 70, 778–779.
Franzyk, H., Olsen, C. E., & Jensen, S. R. (2004). Journal of Natural Products, 67, 1052–1054.
Menger, L., Vacchelli, E., Kepp, O., Eggermont, A., Tartour, E., Zitvogel, L., Kroemer, G., & Galluzzi, L. (2013). Oncoimmunology, 2, e23082.
Vanisree, M., & Tsay, H. S. (2004). International Journal of Applied Science and Engineering, 2, 29–48.
Verpoorte, A., & Memelink, J. (2002). Phytochemistry Reviews, 1, 13–25.
Sparks, C. A., & Jones, H. D. (2014). Methods in Molecular Biology, 1099, 201–218.
Brew-Appiah, R. A., Ankrah, N., Liu, W., Konzak, C. F., von Wettstein, D., & Rustgi, S. (2013). PloS One, 18, e80155.
Mano, H., Fujii, T., Sumikawa, N., Hiwatashi, Y., & Hasebe, M. (2014). PloS One, 9, e97211.
Zambryski, P., Joos, H., Genetell, C., Leemans, J., Van Montagu, M., & Schell, J. (1983). EMBO Journal, 2, 2143–2150.
Frame, B. R., Shou, H., Chikwamba, R. K., Zhang, Z., Xiang, C., Fonger, T. M., Pegg, S. E., Li, B., Nettleton, D. S., Pei, D., & Wang, K. (2002). Plant Physiology, 129, 13–22.
Wang, G., & Xu, Y. (2008). Plant Cell Reports, 27, 1177–1184.
Leelavashi, S., Sunnichan, V. G., Kumria, R., Vijaykanth, G. P., & Bhathagar, R. K. (2004). Plant Cell Reports, 22, 465–470.
Ozawa, K. (2009). Plant Science, 176, 522–527.
Tsuda, K., Qi, Y., Nguyenle, V., Bethke, G., Tsuda, Y., Glazebrook, J., & Katagiri, F. (2012). Plant Journal, 69, 713–719.
Li, Y., Gao, Z., Piao, C., Lu, K., Wang, Z., & Cui, M. L. (2014). Applied Biochemistry and Biotechnology, 172, 1807–1817.
Cui, M., Ezura, H., Nishimura, S., Kamada, H., & Handa, T. (2004). Plant Science, 166, 873–879.
Murashige, T., & Skoog, F. (1962). Physiologia Plantarum, 15, 81–84.
Koncz, C., & Shell, J. (1986). Molecular Genetics and Genomics, 204, 383–396.
Deblaere, R., Bytebier, B., De Greve, H., Deboeck, F., Schell, J., Van Montagu, M., & Leemans, J. (1985). Nucleic Acids Research, 13, 4777–4785.
Shen, W. J., & Forde, B. G. (1989). Nucleic Acids Research, 17, 8385.
Jefferson, R. A., Kavanaghm, T. A., & Bevan, M. W. (1987). EMBO Journal, 6, 3901–3907.
Rogers, S. O., & Bendich, A. J. (1985). Plant Molecular Biology, 5, 69–76.
Bevan, M. W., Flavell, R. B., & Chilton, M. D. (1983). Naturem, 304, 184–187.
Waldronm, A., Murphy, E. B., Roberts, J. L., Gustafson, G. D., Armour, S. L., & Malcolm, S. K. (1985). Plant Molecular Biology, 5, 103–108.
Akama, K., Puchta, H., & Hohn, B. (1995). Plant Cell Reports, 14, 450–454.
Shirgukar, M. V., Naik, V. B., Von-Arnold, S., Nadgauda, R. S., & Clapham, D. (2006). Plant Cell Reports, 25, 112–116.
Gao, C., Liu, J., & Nielsen, K. K. (2009). Plant Cell Repirts, 28, 1431–1437.
Catlin, D. W. (1990). Plant Cell Repots, 9, 285–288.
Mathias, R. J., & Mukasa, C. (1987). Plant Cell Reports, 6, 454–457.
Diemer, F., Jullien, F., Faure, O., Moja, S., Colson, M., Matthys-Rochon, E., & Caissard, J. C. (1998). Plant Science, 136, 101–108.
Ye, G. N., Stone, D., Pang, S. Z., Creely, W., Gonzalez, K., & Hinchee, M. (1999). Plant Journal, 19, 249–257.
Cui, M., Handa, T., & Ezura, H. (2003). Molecular Genetics and Genomics, 270, 296–302.
Acknowledgments
We thank Karen Lee in John Innes Centre (UK) for critical comments on the manuscript. This work was supported by a grant from Yunnan provincial Science and Technology Department (no. 2012IB011) to MLC and ZXC.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Gao, Z., Li, Y., Chen, J. et al. A Rapid and Stable Agrobacterium-Mediated Transformation Method of a Medicinal Plant Chelone glabra L.. Appl Biochem Biotechnol 175, 2390–2398 (2015). https://doi.org/10.1007/s12010-014-1414-0
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12010-014-1414-0