Drought stress enhances the efficiency of floral dip method of Agrobacterium-mediated transformation in Arabidopsis thaliana

Abstract

Abstract The Agrobacterium-mediated floral dip protocol is the most extensively used transformation method for a model plant Arabidopsis thaliana. Several useful methods for Agrobacterium tumefaciens–mediated transformations of Arabidopsis are existing, but they are time consuming and with low transformation efficiency. Here, we developed a transgenic Arabidopsis lines TET12p::TET12-RFP in a short period of time and enhanced transformation efficiency by using a modified transformation method by applying drought stress after floral dip. In this protocol, Agrobacterium cells carrying TET12p::TET12-RFP recombinant vectors were resuspended in a solution of 5% sucrose, 0.05% (v/v) silwet L-77 to transform female gametes of developing Arabidopsis inflorescences. Treated Arabidopsis were then applied with different levels of drought stresses to stimulate plants for the utilization of maximum plant energy in seed maturation process. The applied stresses achieved the fast maturation of already treated inflorescences while stopped the growing of newly arising untreated inflorescence, thus decreased the chances of wrong collection of untransformed seeds. Consequently, the collected seeds were mostly transgenic with a transformation frequency of at least 10%, thus the screening for positive transformants selection was more advantageous on a selective medium as compared to a classical floral dip method. Within 2-3 months, two hundred of individual transgenic plants were produced from just 10 infiltrated plants. This study concludes that application of drought stresses in a specific stage of plant is a beneficial strategy for achieving the transgenic Arabidopsis in a short period of time with high transformation efficiency.

Keywords

• Arabidopsis thaliana
• drought stress
• Agrobacterium tumefaciens
• floral dip
• transformation