13. Establishing Agrobacterium mediated transient gene expression assay in Nicotiana tabacum

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Muhammad Rafique Rind Aneela Yasmin Muharam Ali Saboohi Raza

Abstract

Preliminary functional characterization of a gene within a short time period is only possible through transient gene expression assays. Transient gene expression assays are simple, cost effective and easy to perform as compared to generation of stable transformants. Here we report an Agro-infiltration based transient gene expression assay, optimized in Nicotiana tabacum using β-glucuronidase (GUS) intron gene as an indicator to evaluate various factors effecting its expression. The factors evaluated include bacterial concentration and its growth mode, infiltration medium, concentration of acetosyringone and surfactant, co-cultivation time of Agrobacterium and host cells and temperature during co-cultivation. When Agrobacteria were grown in/ on YEP liquid and solid media, both mode of growth did not affect the transformation efficiency of bacteria and it was recorded 100% in both cases. However, the transient gene expression efficiency found optimal when fully expended true leaves of tobacco infiltrated with Agrobacterium strain EHA-101 carrying GUS intron marker gene in 0.3% glucose medium with an optical density (OD600) of 0.3 without acetosyringone and surfactant. After infiltration samples were stored at 22ºC for two days and evaluations were carried out after histochemical staining of agro-infiltrated discs by visual inspection. This optimized protocol gave the highest level of GUS expression- 3 in 66.7% of samples, followed by 33.0% (GUS expression level – 2) and 0% (GUS expression level – 1) of samples. The simplicity of this method suggests its utility for the production of pharmaceutical proteins in native plants of Pakistan and can help to promote biotechnology awareness at schools and colleges.


Keywords: Agrobacterium mediated transient expression; β-glucuronidase; Nicotiana tabacum


http://dx.doi.org/10.19045/bspab.2018.700149

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