Acta Physiologiae Plantarum

, Volume 33, Issue 5, pp 1603–1611 | Cite as

Agrobacterium tumefaciens mediated genetic transformation of selected elite clone(s) of Eucalyptus tereticornis

Original Paper


Procedure for the Agrobacterium tumefaciens mediated T-DNA delivery into the elite clone(s) of Eucalyptus tereticornis using leaf explants from microshoots has been developed. Amongst two strains of A. tumefaciens namely, EHA105 and LBA4404 (harbouring pBI121 plasmid), strain EHA105 was found to be more efficient. Pre-culturing of tissue (2 days) on medium supplemented with 100 μM acetosyringone, before bacterial infection significantly increased transient expression of reporter gene (GUS). Co-cultivation period of 2 days and a bacterial density of 0.8 OD600 resulted in higher transient GUS expression. Method of injury to tissue, presence of acetosyringone in co-cultivation medium and photoperiod during co-cultivation also influenced the expression of transient GUS activity. Amongst the three clones tested, maximum transient GUS activity was recorded in clone ‘CE2’ followed by clone ‘T1’. Regeneration of transformed shoots was achieved on modified Murashige and Skoog medium (potassium nitrate was replaced with 990 mg/l potassium sulphate and ammonium nitrate with 392 mg/l ammonium sulphate, and mesoinositol concentration was increased to 200 mg/l). Stable transformation was confirmed on the basis of GUS activity and PCR amplification of DNA fragments specific to uidA and nptII genes. The absence of bacteria in the stable transformed tissues was confirmed by PCR amplification of fragment specific to 16S rRNA of bacteria.


Acetosyringone Co-cultivation Plantation forestry 16S rRNA GUS expression 



2,4-Dichlorophenoxyacetic acid




35S promoter of the cauliflower mosaic virus




Murashige and Skoog (1962)


α-Naphthaleneacetic acid


Nopaline synthase


Neomycin phosphotransferase


Optical density at 600 nm


Polymerase chain reaction


5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid



The authors would like to thank Prof. S.B. Gelvin, Purdae University, Purdae, USA for providing A. tumefaciens strain EHA105 and Dr. N. Das, Thapar University, Patiala for providing A. tumefaciens strain LBA4404. The authors are also thankful to Council of Scientific and Industrial Research (CSIR), Govt. of India, New Delhi for providing financial support (Scheme no. 38(1158)/07/EMR-II). TIFAC—CORE, Thapar University, Patiala is thanked for providing facilities.


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Copyright information

© Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2011

Authors and Affiliations

  • Diwakar Aggarwal
    • 1
  • Anil Kumar
    • 1
  • M. Sudhakara Reddy
    • 1
  1. 1.Department of Biotechnology and Environmental Sciences, TIFAC-Center of Relevance and Excellence in Agro and Industrial BiotechnologyThapar UniversityPatialaIndia

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