Journal of Food, Agriculture and Environment

Transformation of Nicotiana tabacum with dehE gene

Author(s):

Yilmaz Kaya ^{1, 2}, Sibel Yilmaz ², Sevgi Marakli ², Nermin Gozukirmizi ², Fahrul Huyop ¹

Recieved Date: 2013-06-20, Accepted Date: 2013-10-18

Abstract:

In this study, a constructed vector pCAMBIA1301a carries dehalogenase E (dehE) gene was transferred to Nicotiana tabacum cv. TAPM24 via Agrobacterium tumefaciens method to analyse dehalogenase activity in transgenic plants. dehE gene had been originally characterized in Rhizobium sp. and it produces an enzyme which degrades the Dalapon herbicide. Totally 176 regenerated shoots were obtained from 130 transformed explants on selective media. The shoots were transferred to rooting MS medium. Thirty of them were able to be transferred to soil. Stable integration and expression of the dehE gene were confirmed in randomly selected 10 putative transformants with PCR and qPCR analysis. In addition, dalapon tolerance of confirmed transgenic plants was determined by leaf painting method. Three non-transformed tobacco plants were used as control in all analyses. PCR analysis demonstrated that 9 of 10 tested putative transgenic plants carry dehE gene in their genomes, but 2 of these 9 plants did not have dehE expression by reverse transcriptase PCR analysis. qPCR analysis showed that total dehE transcript level was significantly higher in 7 plants unlike three non-transformed control plants lacking from dehE expression. Importantly, dehalogenase activity was proved with higher dalapon tolerance in transgenic plants by leaf painting assay. Dalapon herbicide with different concentrations was applied to tobacco leaves for one week. The results showed that transformed tobacco plants have more tolerance to dalapon herbicide then the non-transformed ones. Dalapon effects started being in the transformed and non-transformed plants at 100 and 60 mg/l concentration, respectively. In our knowledge, this is the first experimental data of transgenic N. tabacum engineered with dehE gene originated Rhizobium sp.

Keywords:

Nicotiana tabacum, Agrobacterium tumefaciens, Dalapon, dehalogenase E, Gene expression, herbicide, Rhizobium sp., qPCR, transformation, transgenics

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Journal: Journal of Food, Agriculture and Environment
Year: 2013
Volume: 11
Issue: 3&4
Category: Agriculture
Pages: 777-780

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