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Efficient Agrobacterium-mediated transformation and genome editing of Fagopyrum tataricum.

ABSTRACT: Fagopyrum tataricum (L.) Gaertn. is an exceptional crop known for its remarkable health benefits, high levels of beneficial polyphenols and gluten-free properties, making it highly sought-after as a functional food. Its self-fertilisation capability and adaptability to challenging environments further contribute to its potential as a sustainable agricultural option. To harness its unique traits, genetic transformation in F. tataricum is crucial. In this study, we optimised the Agrobacterium-mediated transformation protocol for F. tataricum callus, resulting in a transformation rate of regenerated plants of approximately 20%. The protocol's effectiveness was confirmed through successful GUS staining, GFP expression, and the generation of albino plants via FtPDS gene inactivation. These results validate the feasibility of genetic manipulation and highlight the potential for trait enhancement in F. tataricum.

SUBMITTER: Pinski A

PROVIDER: S-EPMC10515086 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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Efficient Agrobacterium-mediated transformation and genome editing of Fagopyrum tataricum.

Pinski Artur A Betekhtin Alexander A

Frontiers in plant science 20230908

Fagopyrum tataricum (L.) Gaertn. is an exceptional crop known for its remarkable health benefits, high levels of beneficial polyphenols and gluten-free properties, making it highly sought-after as a functional food. Its self-fertilisation capability and adaptability to challenging environments further contribute to its potential as a sustainable agricultural option. To harness its unique traits, genetic transformation in F. tataricum is crucial. In this study, we optimised the Agro ...[more]

PMID: 37746024

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Project description:BackgroundAgrobacterium-mediated genetic transformation is a widely used and efficient technique for gene functional research in crop breeding and plant biology. While in some plant species, including soybean, genetic transformation is still recalcitrant and time-consuming, hampering the high-throughput functional analysis of soybean genes. Thus we pursue to develop a rapid, simple, and highly efficient hairy root system induced by Agrobacterium rhizogenes (A. rhizogenes) to analyze soybean gene function.ResultsIn this report, a rapid, simple, and highly efficient hairy root transformation system for soybean was described. Only sixteen days were required for the whole workflow and the system was suitable for various soybean genotypes, with an average transformation frequency of 58-64%. Higher transformation frequency was observed when wounded cotyledons from 1-day-germination seeds were inoculated and co-cultivated with A. rhizogenes in 1/2 B5 (Gamborg' B-5) medium. The addition of herbicide selection to root production medium increased the transformation frequency to 69%. To test the applicability of the hairy root system for gene functional analysis, we evaluated the protein expression and subcellular localization in transformed hairy roots. Transgenic hairy roots exhibited significantly increased GFP fluorescence and appropriate protein subcellular localization. Protein-protein interactions by BiFC (Bimolecular Fluorescent Complimentary) were also explored using the hairy root system. Fluorescence observations showed that protein interactions could be observed in the root cells. Additionally, hairy root transformation allowed soybean target sgRNA screening for CRISPR/Cas9 gene editing. Therefore, the protocol here enables high-throughput functional characterization of candidate genes in soybean.ConclusionA rapid, simple, and highly efficient A. rhizogenes-mediated hairy root transformation system was established for soybean gene functional analysis, including protein expression, subcellular localization, protein-protein interactions and gene editing system evaluation.

Dataset Information

Efficient Agrobacterium-mediated transformation and genome editing of Fagopyrum tataricum.

Publications

Efficient Agrobacterium-mediated transformation and genome editing of <i>Fagopyrum tataricum</i>.

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