Ensifer-mediated transformation: an efficient non-Agrobacterium protocol for the genetic modification of rice.

Evelyn Zuniga-Soto,Ewen Mullins,Beata Dedicova

doi:10.1186/s40064-015-1369-9

Abstract

While Agrobacterium-mediated transformation (AMT) remains the most widely used technique for gene transfer in plants, interest exists for the use of non-Agrobacterium gene delivery systems due to freedom-to-operate issues that remain with AMT across several jurisdictions. In addition, the plant pathogenic mode of action of Agrobacterium tumefaciens significantly increases the costs to passage engineered cultivars through the regulatory process. Ensifer adhaerens (OV14) is a soil-related bacterium with the proven ability to genetically modify the model plant A. thaliana and the staple crop S. tuberosum (Wendt et al., Trans Res 21:567–578, 2012). While previous work was relevant for dicotyledonous species, in this study, the efficacy of Ensifer adhaerens (OV14)-mediated transformation (EMT) was determined on two japonica rice varieties, Curinga and Nipponbare, and the recalcitrant indica variety, IR64. The results indicated that E. adhaerens (OV14) exhibits infection efficiencies ranging between 50–70 %, 90–100 % and 90–95 % for Curinga, Nipponbare and IR64 respectively. Curinga and Nipponbare plants transformed with E. adhaerens (OV14) and A. tumefaciens (LBA4404 and EHA105) were regenerated achieving transformation efficiencies of 16 % and 26–32 % for Curinga and 7 and 4 % for Nipponbare respectively. Separately, the transformation of IR64 was only recorded via EMT (transformation efficiency ~1 %). Integration analyses conducted on 24 transgenic rice lines illustrated that T-DNA insertion occurred randomly throughout the rice genome for EMT (and AMT), with similar integration patterns in the rice genomic DNA observed for both bacterial species.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-015-1369-9) contains supplementary material, which is available to authorized users.

Highlights

Rice (Oryza sativa L.,) remains a stable food for more than half of the world’s population but entered the new millennium facing a series of significant challenges relevant to a/biotic stress
We evaluated the T-DNA integration patterns found in rice plants transformed via E. adhaerens (OV14) and A. tumefaciens (LBA44404 and EHA105 with the pCAMBIA 1305.2 plasmid) in order to evaluate (1) propensity for backbone sequence integration, and (2) insertion patterns within the post-integration genome
Nipponbare was more amenable for infection with E. adhaerens (OV14), exhibiting transient GUS activity in more than 90 % of treated calli, only 50–70 % of treated Curinga-derived calli reported to be positive for GUS staining following Ensifer adhaerens (OV14)-mediated transformation (EMT)

Summary

Introduction

Rice (Oryza sativa L.,) remains a stable food for more than half of the world’s population but entered the new millennium facing a series of significant challenges relevant to a/biotic stress. The cost of securing regulatory approval for AMT-derived lines is significant primarily due to the plant pathogenic nature of A. tumefaciens (CropLife International 2011), which in turn ensures that bringing engineered crop lines through to commercialisation is typically an option exclusive to the largest ag-biotech companies. Addressing this would require a gene delivery system that is based on a non-pathogenic organism with rates of transformation equivalent to that of AMT for both dicot and monocot species. In 2005 Broothaerts and collaborators demonstrated the efficiency of different Rhizobia

Methods

Results

Conclusion