Agrobacterium tumefaciens mediated genetic transformation of selected elite clone(s) of Eucalyptus tereticornis
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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.
KeywordsAcetosyringone Co-cultivation Plantation forestry 16S rRNA GUS expression
35S promoter of the cauliflower mosaic virus
Murashige and Skoog (1962)
Optical density at 600 nm
Polymerase chain reaction
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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