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‘AGRODATE’: a rapid Agrobacterium-mediated transient expression tool for gene function analysis in leaf discs

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Abstract

Transient gene expression utilizing syringe mediated agro infiltration offers a simple yet efficient technique for various transgenic applications. Although soybean is commonly used as a model plant for functional genomic studies, till date there have been no reports available on a high throughput agro infiltration protocol for transient gene expression. In the present study, we developed a simple transient expression system, named Agrobacterium-mediated disc assay for transient expression (AGRO DATE) in mature soybean leaves involving the use of an optimized infiltration buffer [10 mM 2-(N-morpholino) ethane sulfonic acid sodium salt, 10 mM MgCl2, 0.2 mM acetosyringone, 400 mg L−1, l-cysteine, 0.5 mM dithiothreitol and 0.01% Tween 20] under vacuum using a needle-less syringe. This not only limits the genetic pollution but can also be conducted without the intervention of any specialized equipments. Model protein β-glucuronidase (GUS) was used for optimizing various parameters and the synergized composition delivered 58% transformation efficiency within 4 days of infiltration. We demonstrated the versatile applicability of the method for examining reporter genes (GUS and bar), over expression (GmIPK1) and antisense suppression of GmMIPS in soybean. In addition, AGRODATE offers a viable approach for application of agro infiltration in other recalcitrant plant species and may become a useful tool for the research community.

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Abbreviations

AGRO DATE:

Agrobacterium-mediated disc assay for transient expression

DTT:

Dithiothreitol

GUS-β:

Glucuronidase

IIP:

Initial infiltration protocol

SAAT:

Sonication-assisted Agrobacterium-mediated transformation

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Acknowledgements

Authors are grateful to Indian Council of Agricultural Research-National Agricultural Science Fund (Grant-RNAi-2011) for supporting with financial assistance. We are thankful to Division of Genetics, Indian Agricultural Research Institute, New Delhi, India for soybean samples (DS-9712 cv) which were utilized for the agro infiltration studies.

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KV conceived the experiments. KV, JM and JJ performed the experiments and analyzed data together with SA and MM. KV wrote the paper and editing was carried out by SA, RK, VT and SP. All authors discussed the results and commented on the manuscript.

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Correspondence to Archana Sachdev.

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The authors declare that they do not have any conflict of interest.

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Krishnan, V., Jose, J., Jolly, M. et al. ‘AGRODATE’: a rapid Agrobacterium-mediated transient expression tool for gene function analysis in leaf discs. J. Plant Biochem. Biotechnol. 29, 294–304 (2020). https://doi.org/10.1007/s13562-019-00536-w

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  • DOI: https://doi.org/10.1007/s13562-019-00536-w

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