High-Efficiency Agrobacterium-Mediated Transformation of Tobacco (Nicotiana tabacum)

Document Type : Original research paper


1 Department of Plant Breeding and Biotechnology, Faculty of Agriculture, Tabriz University, Tabriz, Iran

2 Department of Agricultural Biotechnology, Faculty of Agriculture and Natural Resources, Imam Khomeini International University, Qazvin, Iran

3 Department of Plant Breeding, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran


To improve Agrobacterium-mediated transformation of tobacco, factors influencing gene delivery, including genotype of the plant, bacterial strain, and Agrobacterium transformation procedure, were tested via direct somatic embryogenesis. Leaf tissue of three different tobacco genotypes (Nicotiana tabacum L. cvs. Samsun, and Xanthi, and N. benthamiana) were used as explant. Leaf explants were transformed using three Agrobacterium tumefaciens strains (EHA105, GV3101, and LBA4404) harboring the binary vector pCAMBIA1304 using three different types of transformation methods as named Agro-inoculation, Agro-infection and Agro-injection. Selection of hygromycin resistant shoots was conducted on MS medium containing 3.0 mgL-1 BAP and 0.2 mgL-1 IAA, 250 mgL-1 cefotaxime and 30 mgL-1 hygromycin. Hygromycin resistant shoots were then rooted on MS medium supplemented with 250 mgL-1 cefotaxime and 15 mgL-1 hygromycin. The results indicated that A. tumefaciens strain LBA4404 was more effective in gene delivery than EHA105 and GV3101 and Agro-infection method proved to be significantly better than two other methods. The highest transformation rate was obtained with the Agrobacterium strain LBA4404 and Agro-infection method with approximately 72.80%, 84.57%, and 93.33% for N. benthamiana, Samsun and Xanthi, respectively. Histochemical GUS assay confirmed the expression of gusA gene in putatively transformed plantlets. PCR and RT-PCR analysis using gene-specific primers confirmed the integration of the gusA and hpt genes and the expression of the gusA and hpt genes, respectively. Furthermore, Southern blot analysis confirmed stable integration of the gusA gene in selected T0 transformants.


[1] Ahmad Jan, S., Shinwari, Z.K., Hussain Shah, S., Shahzad, A., Zia, M.A. and Ahmad, N. 2016. In-planta transformation: recent advances. Rom. Biotechnol. Lett, 21:11085–11091.
[2] Bakhsh, A., Anayol, E. and Ozcan, S.F. 2014. Comparison of transformation efficiency of five Agrobacterium tumefaciens strains in Nicotiana Tabacum L. Emir. J. Food Agric, 26:259–264.
[3] Deo, P.C., Tyagi, A.P., Taylor, M., Harding, R. and Becker, D. 2010. Factors affecting somatic embryogenesis and transformation in modern plant breeding. South Pac. J. Nat. App. Sci, 28:27–40.
[4] Dhumale, D.R., Shingote, P.R., Dudhare, M.S., Jadhav, P.V. and Kale, P.B. 2016. Parameters influencing Agrobacterium-mediated transformation system in safflower genotypes AKS-207 and PKV Pink. 3 Biotech, 6:181.
[5] Duan, W., Wang, L. and Song, G. 2016. Agrobacterium tumefaciens-mediated transformation of wild tobacco species Nicotiana debneyi, Nicotiana clevelandii, and Nicotiana glutinosa. Am. J. Plant Sci, 7:1–7.
[6] Heidari-Japelaghi, R., Haddad, R. and Garoosi, G.A. 2011. Rapid and efficient isolation of high quality nucleic acids from plant tissues rich in polyphenols and polysaccharides. Mol. Biotechnol, 49:129–137.
[7] Hoekema, A., Hirsch, P.R., Hooykaas, P.J.J. and Schilperoort, R.A. 1983. A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature, 303:179–180.
[8] Holsters, M., Silva, B., van Vliet, F., Genetello, C., de Block, M., Dhaese, P., Depicker, A., Inze, D., Engler, G., Villarroel, R., Van Montagu, M. and Schell, J. 1980. The functional organization of the nopaline Agrobacterium tumefaciens plasmid pTiC58. Plasmid 3:212–230.
[9] Hood, E., Gelvin, S., Melchers, S. and Hoekema, A. 1993. New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Res, 2:208–218.
[10] Indurker, S., Misra, H.S. and Eapen, S. 2010. Agrobacterium-mediated transformation in chickpea (Cicer arietinum L.) with an insecticidal protein gene: optimization of different factors. Physiol. Mol. Biol. Plants, 16:273–284.
[11] Jefferson, R.A. 1987. Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol. Biol, 5:387–405.
[12] Jeong, J.H., Jung, S.J., Murthy, H.N., Yu, K.W., Paek, K.Y., Moon, H.K., Choi, Y.E. 2005. Production of eleutherosides in in vitro regenerated embryos and plantlets of Eleutherococcus chiisanensis.Biotechnol. Lett, 27:701–704.
[13] Jimenez, V.M. 2001. Regulation of in vitro somatic embryogenesis with emphasis on the role of endogenous hormones. Rev. Bras. Fisiol. Vegetal, 13:196–223.
[14] Khanna, H.K., Paul, J.Y., Harding, R.M., Dickman, M.B. and Dale, J.L. 2007. Inhibition of Agrobacterium-induced cell death by antiapoptotic gene expression leads to very high transformation efficiency of banana. Mol. Plant-Microbe Interact, 20:1048–1054.
[15] Koetle, M.J., Baskaran, P., Finnie, J.F., Soos, V., Balázs, E. and van Staden, J. 2017. Optimization of transient GUS expression of Agrobacterium-mediated transformation in Dierama erectum Hilliard using sonication and Agrobacterium. South Afr. J. Bot, 111:307–312.
[16] Kumar, V. and Chandra, S. 2014. High frequency somatic embryogenesis and synthetic seed production of the endangered species Swertia chirayita. Biologia 69:186–192.
[17] Lin, G.Z., Zhao, X.M., Hong, S.K. and Lian, Y.J. 2011. Somatic embryogenesis and shoot organogenesis in the medicinal plant Pulsatilla koreana Nakai. Plant Cell Tiss. Organ Cult, 106:93–103.
[18] Mahendran, G. and Bai, V.N. 2016. Direct somatic embryogenesis of Malaxis densiflora (A. Rich.) Kuntze. J. Gen. Eng. Biotechnol, 14:77–81.
[19] Maleki, S.S., Mohammadi, K. and Ji, K.S. 2018. Study on factors influencing transformation efficiency in Pinus massoniana using Agrobacterium tumefaciens.Plant Cell Tiss. Organ Cult. https://doi.org/10.1007/s11240-018-1388-7.
[20] Martin, K.P. 2004. Plant regeneration protocol of medicinally important andrographis paniculata (burm. f.) wallich ex nees via somatic embryogenesis. In Vitro Cell Dev. Biol. –Plant, 40:586–591.
[21] Michalczuk, L., Cooke, T.J. and Cohen, J.D. 1992. Auxin levels at different stages of carrot somatic embryogenesis. Phytochem, 31:1097–1103.
[22] Möller, B. and Weijers, D. 2009. Auxin control of embryo patterning. Cold Spring Harb Perspect Biol. 1:a001545. doi:10.1101/cshperspect. a001545.
[23] Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant, 15:473–497.
[24] Nyaboga, E.N., Njiru, J.M. and Tripathi, L. 2015. Factors influencing somatic embryogenesis, regeneration, and Agrobacterium-mediated transformation of cassava (Manihot esculenta Crantz) cultivar TME14. Front. Plant Sci. doi: 10.3389/fpls.2015.00411.
[25] Pathi, K.M., Tula, S. and Tuteja, N. 2013. High frequency regeneration via direct somatic embryogenesis and efficient Agrobacterium- mediated genetic transformation of tobacco. Plant Signal. Behav, 8:6, e24354.
[26] Prakash, M.G. and Gurumurthi, K. 2010. Effects of type of explant and age, plant growth regulators and medium strength on somatic embryogenesis and plant regeneration in Eucalyptus camaldulensis. Plant Cell Tiss. Organ Cult, 100:13–20.
[27] Rai, G.K., Rai, N.P., Kumar, S., Yadav, A., Rathaur, S. and Singh, M. 2012. Effects of explant age, germination medium, pre-culture parameters, inoculation medium, pH, washing medium, and selection regime on Agrobacterium-mediated transformation of tomato. In Vitro Cell Dev. Biol. Plant, 48:565–578.
[28] Rossin, C.B. and Rey, M.E.C. 2011. Effect of explant source and auxins on somatic embryogenesis of selected cassava (Manihot esculenta Crantz) cultivars. South Afr. J. Bot, 77:59–65.
[29] Song, G.Q., Walworth, A. and Hancock, J.F. 2012. Factors influencing Agrobacterium-mediated transformation of switchgrass cultivars. Plant Cell Tiss. Organ Cult, 108:445–453.
[30] Stam, M., Mol, J.M. and Kooter, J.M. 1997. The silence of genes in transgenic plants. Ann. Bot, 79:3–12.
[31] Stolarz, A., Macewicz, E. and Lorz, H. 1991. Direct somatic embryogenesis and plant regeneration from leaf explants of Nicotiana tabacum L. J. Plant Physiol, 137:347–357.
[32] Terzi, M. and Lo Schiavo, F. 1990. Somatic embryogenesis. In: Bhajwani, S.S., (Eds.), Plant tissue culture: applications and limitations. Elsevier Publishing Inc, Amsterdam, Netherlands, pp. 54–66.
[33] Trick, H.N. and Finer, J.J. 1997. SAAT: sonication-assisted Agrobacterium-mediated transformation. Transgenic Res, 6:329–336.
[34] Vancanneyt, G., Schmidt, R., O’Connor-Sanchez, A., Willmitzer, L. and Rocha-Sosa, M. 1990. Construction of an intron-containing marker gene: splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium-mediated plant transformation. Mol. Gen. Genet, 220:245–250.
[35] Wang, K. 2006. Model plants. In: Clements, T., (Eds.), Methods in molecular biology, Agrobacterium protocols. Humana Press, New York, USA, pp. 143-154.
[36] Xu, J., Dolan, M.C., Medrano, G., Cramer, C.L. and Weathers, P.J. 2012. Green factory: plants as bioproduction platforms for recombinant proteins. Biotechnol. Adv, 30:1171–1184.
[37] Yang, L., Hu, W., Xie ,Y., Li, Y. and Deng, Z. 2016. Factors affecting Agrobacterium-mediated transformation efficiency of kumquat seedling internodal stem segments. Sci. Hort, 209:105–112.
[38] Zhang, Z., Coyne, D.P. and Mitra, A. 1997. Factor affecting Agrobacterium-mediated transformation of common bean. J. Am. Soc. Hortic. Sci, 122:300–305.
[39] Zhu, Y.J., Nam, J., Humara, J.M., Mysore, K.S., Lee, L.Y., Cao, H., Valentine, L., et al. 2003. Identification of Arabidopsis rat mutants. Plant Physiol, 132:494–505.
[40] Ziemienowicz, A. 2013. Agrobacterium-mediated plant transformation: factors, applications and recent advances. Biocatal. Agric. Biotechnol. http://dx.doi.org/10.1016/j.bcab.2013.10.004i.
Volume 6, Issue 2
December 2018
Pages 38-50
  • Receive Date: 20 August 2018
  • Revise Date: 08 October 2019
  • Accept Date: 26 October 2019
  • First Publish Date: 26 October 2019