A simple and efficient genetic transformation protocol for Eucalyptus camaldulensis using internodal explants

Venkatachalam Rathinavel, Boby Vattekkattu Unnikrishnan, Suribabu Thaliyakula, Gurumurthy Demalapura Shankaranarayana


A simple and efficient genetic transformation protocol was developed for Eucalyptus camaldulensis. Selected line of eucalyptus was established under in vitro conditions by using nodal cuttings.Assessment of hygromycin sensitivity on explants collected from in vitro established cultures showed that hygromycin concentration of 2.5 mg/L was optimum for transformation. Transformation procedure was standardized with internodal explants through indirect organogenesis. Transformation efficiency of intermodal explants was assessed by plasmids pCAMBIA1305.1 and pRNAiLIC-Hyg carrying GA2ox gene. The transformation efficiency of 20 % was achieved with the present protocol and regeneration of transgenic shoot was obtained in two months of culture compared to three to four months in previous studies.Confirmation of gene integration in eucalyptus was confirmed by histochemical staining and PCR based methods.


Eucalyptus, transformation, internodes, efficient, Protocol

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Aggarwal, D., Kumar, A., and Reddy, M.S. 2010. Shoot organogenesis in elite clones of Eucalyptus tereticornis. Plant Cell Tissue Organ Cult., 102: 45–52.

Dibax, R., Deschamps, C., Bespalhok Filho, J.C., Vieira, L.G.E., Molinari, H.B.C., De Campos, M.K.F., and Quoirin, M. 2010. Organogenesis and Agrobacterium tumefaciens-mediated transformation of Eucalyptus saligna with P5CS gene. Biol. Plant, 54: 6–12.

Eldridge, K., Davidson, J., Hardwood, C., and vanWyk, G. 1993. Eucalypt domestication and breeding. Clarendon press, Oxford.

Fernando, C.M., Goodger, J.Q.D., Gutierrez, S.S., Johnson, A. and Woodrow, I.E. 2016. Plant regeneration through indirect organogenesis and genetic transformation of Eucalyptus polybractea RT Baker. Ind. crop and prod., 86:73-78.

Girijashankar, V. 2011. Genetic transformation of Eucalyptus. Physiol. Mol. Biol. Plants, 17: 9–23.

Ho, C.K., Chang, S.H., Tsay, J.Y., Tsai, C.J., Chiang, V.L., and Chen, Z.Z. 1998. Agrobacterium tumefaciens-mediated transformation of Eucalyptus camaldulensis and production of transgenic plants. Plant Cell Rep., 17:675–680.

Holsters, M., de Waele, D., Depicker, A., Messens, E., van Montagu, M., and Schell, J. 1978. Transfection and transformation of Agrobacterium tumefaciens. Mol. Gen. Genet., 163:181-187.

Jefferson, R.A., Kavanagh, T.A., and Bevan, M.W. 1987. GUS fusions: betaglucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J., 6: 3901.

Ma, C., Deepika, R., Myburg, A.A., Ranik, M., and Strauss, S.H. 2011. Development of eucalyptus tissue culture conditions for improved in vitro plant health and transformability. BMC Proc., 5: 153.

MacRae, S., and Van Staden, J. 1999. Transgenic Eucalyptus. In Biotechnology in Agriculture and Forestry; Transgenic trees. Edited by Bajaj, Y.P.S. Springer-Verlag, Berlin- Heidelberg vol. 44, pp. 88-114.

Matsunaga, E., Nanto, K., Oishi, M., Ebinuma, H., Morishita, Y., Sakurai, N., Suzuki, H., Shibata, D., and Shimada, T. 2012. Agrobacterium-mediated transformation of Eucalyptus globulus using explants with shoot apex with introduction of bacterial choline oxidase gene to enhance salt tolerance. Plant Cell Rep., 31: 225–235.

Mullins, K.V., Llewellyn, D.J., Hartney, V.J., Strauss, S., and Dennis, E.S. 1997. Regeneration and transformation of Eucalyptus camaldulensis. Plant Cell Rep., 16:787–791.

Prakash, M.G. and Gurumurthi, K. 2009. Genetic transformation and regeneration of transgenic plants from precultured cotyledon and hypocotyl explants of Eucalyptus tereticornis Sm. using Agrobacterium tumefaciens. In Vitro Cell. Dev. Biol. Plant, 45: 429-34.

Rezende, G.D.S., de Resende, M.D.V., and de Assis, T.F. 2014. Eucalyptus breeding for clonal forestry. In Challenges and opportunities for the world’s forests in the 21st century. Edited by Fenning, T. Springer, Dordrecht. pp. 393–424.

Teulieres, C., Marque, C. and Boude, A.M. 1994. Genetic transformation of Eucalyptus. In Biotechnology in agriculture and forestry. Edited by Bajaj, Y.P.S. Springer, Berlin Heidelberg. pp. 289-307.

Thanananta, N., Vuttipongchaikij and Apistvanich, S. 2018. Agrobacterium mediated transformation of a Eucalyptus camaldulensis x E. tereticornis hybrid using peeled nodal stem segments with yeast HAL2 for improving salt tolerance. New Forests, 49: 311-327.

Unnikrishnan, B. and Gurumurthy, D.S. 2015. Progress on genetic modification of pulp wood tree species relevance to India-A review. Agri. Review, 36: 265-276.

Xu, G., Sui, N., Tang, Y., Xie, K., Lai, Y. and Liu, Y. 2010. One step zero background ligation independent cloning intron containing hairpin RNA constructs for RNAi in plants. New Phytol., 187: 240-250.


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