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Development of glyphosate-resistant alfalfa (Medicago sativa L.) upon transformation with the GR79Ms gene encoding 5-enolpyruvylshikimate-3-phosphate synthase

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Abstract

Main conclusion

The glyphosate-resistant gene, GR79Ms, was successfully introduced into the genome of alfalfa. The transgenic events may serve as novel germplasm resources in alfalfa breeding.

Weed competition can reduce the alfalfa yield, generating new alfalfa germplasm with herbicide resistance is essential. To obtain transgenic alfalfa lines with glyphosate resistance, a new synthetic glyphosate-resistant gene GR79Ms encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) was introduced into alfalfa germplasm by Agrobacterium tumefaciens-mediated transformation. In total, 67 transformants were obtained. PCR and Southern blot analyses confirmed that GR79Ms was successfully inserted into the genome of alfalfa. Reverse transcription-PCR and western blot analyses further demonstrated the expression of GR79Ms and its product, GR79Ms EPSPS. Moreover, two homozygous transgenic lines were developed in the T2 generation by means of molecular-assisted selection. Herbicide tolerance spray tests showed that the transgenic plants T0-GR1, T0-GR2, T0-GR3 and two homozygous lines were able to tolerate fourfold higher commercial usage of glyphosate than non-transgenic plants.

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Abbreviations

A. tumefaciens :

Agrobacterium tumefaciens

6-BAP:

6-Benzylaminopurine

CAAS:

Chinese Academy of Agricultural Sciences

2,4-D:

2,4-Dichlorophenoxyacetic acid

DIG:

Digoxigenin

eCaMV35S :

Enhanced cauliflower mosaic virus 35s

EPSPS:

5-Enolpyruvylshikimate-3-phosphate synthase

GM:

Genetically modified

IAA:

Indole-3-acetic acid

ISAAA:

International Service for the Acquisition of Agri-Biotech Applications

NAFA:

National Alfalfa and Forage Alliance

PCR:

Polymerase Chain Reaction

PEP:

Phosphoenolpyruvic acid

RT-PCR:

Reverse transcription polymerase chain reaction

S3P:

3-Phosphoshikimic acid

Tnos:

Nopaline synthase terminator

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Acknowledgements

This work was supported by Beijing Natural Science Foundation (6184047), the Modern Agricultural Industry Technology Research System (CARS34), the Fundamental Research Funds for Central Non-profit Scientific Institution (2018-YWF-YB-4; 2016ywf-yb-10), the National Natural Science Foundation of China (31372362), and the Agricultural Science and Technology Innovation Program (ASTIP-IAS10) of China. We thank Prof. Min Lin and Wei Lu for providing AM79 gene. The authors particularly grateful to Prof. Zan Wang for his assistance in improving the manuscript.

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  1. Dengxia Yi and Lin Ma contributed equally to this work.

Authors and Affiliations

  1. Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, 100193, China

    Dengxia Yi, Lin Ma & Cong Li

  2. Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, China

    Min Lin

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  1. Dengxia Yi
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  2. Lin Ma
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Correspondence to Cong Li.

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Yi, D., Ma, L., Lin, M. et al. Development of glyphosate-resistant alfalfa (Medicago sativa L.) upon transformation with the GR79Ms gene encoding 5-enolpyruvylshikimate-3-phosphate synthase. Planta 248, 211–219 (2018). https://doi.org/10.1007/s00425-018-2898-6

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