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. 2020 Feb 27;13(1):116.
doi: 10.1186/s13104-020-04968-9.

Agrobacterium-mediated transient transformation of sorghum leaves for accelerating functional genomics and genome editing studies

Rita Sharma  1   2   3 , Yan Liang  1   2 , Mi Yeon Lee  1   2 , Venkataramana R Pidatala  1   2 , Jenny C Mortimer  4   5 , Henrik V Scheller  6   7   8
Affiliations

Agrobacterium-mediated transient transformation of sorghum leaves for accelerating functional genomics and genome editing studies

Rita Sharma et al. BMC Res Notes. .

Abstract

Objectives: Sorghum is one of the most recalcitrant species for transformation. Considering the time and effort required for stable transformation in sorghum, establishing a transient system to screen the efficiency and full functionality of vector constructs is highly desirable.

Results: Here, we report an Agrobacterium-mediated transient transformation assay with intact sorghum leaves using green fluorescent protein as marker. It also provides a good monocot alternative to tobacco and protoplast assays with a direct, native and more reliable system for testing single guide RNA (sgRNA) expression construct efficiency. Given the simplicity and ease of transformation, high reproducibility, and ability to test large constructs, this method can be widely adopted to speed up functional genomic and genome editing studies.

Keywords: Agrobacterium; CRISPR; Sorghum; Transformation; Transient; sgRNA.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Image of sorghum seedling depicting the stage of sorghum plant required for efficient agroinfiltration. Leaves used for syringe-mediated infiltration on abaxial side are marked by white arrows
Fig. 2
Fig. 2
Results of agroinfiltration with Agrobacterium suspension in sorghum and tobacco leaves. Column A shows bright field images and column B depicts GFP expression detected using fluorescence microscope. Scale bar: 100 μm
Fig. 3
Fig. 3
Successful editing of GFP in tobacco and sorghum leaves using agroinfiltration. Column A presents bright field images, whereas, columns B and C present expression of GFP and DsRed, respectively. The C476 vector construct contained sgRNA required for editing, while C475 lacked the sgRNA and serves as negative control. Expression of GFP in leaves transformed with C476 demonstrates successful editing. Scale bar: 100 μm
See this image and copyright information in PMC

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