Abstract
Herbicides are important constituents of modern integrated weed management system. However, the continuous use of a single herbicide leads to the frequent evolution of resistant weeds which further challenges their management. To overcome this situation, alternating use of multiple herbicides along with conventional weed-management practices is suitable and recommended. The development of multiple herbicide-tolerant crops is still in its infancy, and only a few crops with herbicide tolerance traits have been reported and commercialized. In this study, we developed transgenic rice plants that were tolerant to both bensulfuron methyl (BM) and glufosinate herbicides. The herbicide tolerant mutant variant of rice AHAS (Acetohydroxyacid synthase) was overexpressed along with codon optimized bacterial bar gene. The developed transgenic lines showed significant tolerance to both herbicides at various stages of plant development. The selected transgenic lines displayed an increased tolerance against 100 μM BM and 30 mg/L phosphinothricin during seed germination stage. Foliar applications further confirmed the dual tolerance to 300 μM BM and 2% basta herbicides without any significant growth and yield penalties. The development of dual-herbicide-tolerant transgenic plants adds further information to the knowledge of crop herbicide tolerance for sustainable weed management in modern agricultural system.
Highlights
IntroductionIntroduction ofP171S tolerant mutation in rice AHAS. The mutation P171S in the rice AHAS gene was introduced via PCR based site directed mutagenesis (refer to supplementary information for details).Preparation of gene construct and generation of transgenic plants.The final gene construct with mutated AHAS and bar genes was cloned into the pMDC99 Gateway compatible cloning vector and transformed into Agrobacterium strain EHA 105, which was further used for transformation in rice calli (refer to supplementary information for details) (Fig. 1B, 1C).Molecular and expression analyses of putative transgenic plants.The tissue culture generated putative transgenic plants were confirmed by PCR and southern blotting, and the expression analysis of the confirmed transgenic plants was performed by northern blotting and semi-quantitative RT-PCR
Introduction ofP171S tolerant mutation in rice AHAS
The amino acid proline was highly conserved in all the tested plant species at 197 with reference to Arabidopsis; this residue was located at position 171 in the rice AHAS protein
Summary
Introduction ofP171S tolerant mutation in rice AHAS. The mutation P171S in the rice AHAS gene was introduced via PCR based site directed mutagenesis (refer to supplementary information for details).Preparation of gene construct and generation of transgenic plants.The final gene construct with mutated AHAS and bar genes was cloned into the pMDC99 Gateway compatible cloning vector and transformed into Agrobacterium strain EHA 105, which was further used for transformation in rice calli (refer to supplementary information for details) (Fig. 1B, 1C).Molecular and expression analyses of putative transgenic plants.The tissue culture generated putative transgenic plants were confirmed by PCR and southern blotting, and the expression analysis of the confirmed transgenic plants was performed by northern blotting and semi-quantitative RT-PCR. The mutation P171S in the rice AHAS gene was introduced via PCR based site directed mutagenesis (refer to supplementary information for details). Preparation of gene construct and generation of transgenic plants. The final gene construct with mutated AHAS and bar genes was cloned into the pMDC99 Gateway compatible cloning vector and transformed into Agrobacterium strain EHA 105, which was further used for transformation in rice calli (refer to supplementary information for details) (Fig. 1B, 1C). Molecular and expression analyses of putative transgenic plants. The tissue culture generated putative transgenic plants were confirmed by PCR and southern blotting, and the expression analysis of the confirmed transgenic plants was performed by northern blotting and semi-quantitative RT-PCR. The presence and expression of the mutaned AHAS gene was confirmed by restriction digestion analysis (refer to supplementary information for details)
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