Highly efficient Agrobacterium rhizogenes-mediated transformation for functional analysis in woodland strawberry

Abstract

Background The diploid woodland strawberry (Fragaria vesca) is an excellent model plant for investigating economically significant traits and several genetic resources within the Rosaceae family. Agrobacterium rhizogenes-mediated hairy root transformation is an alternative for exploring gene functions, especially the genes specifically expressed in roots. However, the hairy root transformation has not been established in strawberry. Results Here, we described an efficient and rapid hairy root transgenic system for strawberry using A. rhizogenes. Strain of A. rhizogenes MSU440 or C58C1 was the most suitable for hairy root transformation. The transformation efficiency was highest when tissues contained hypocotyls as explants. The optimal procedure involves A. rhizogenes at an optical density (OD600) of 0.7 for 10 min and co-cultivation duration for four days, achieving a transgenic efficiency of up to 71.43%. An auxin responsive promoter DR5ver2 carrying an enhanced green fluorescent protein (eGFP) marker was transformed by A. rhizogenes MSU440, thereby generating transgenic hairy roots capable of high eGFP expression in root tip and meristem of strawberry where auxin accumulated. Finally, this system was applied for functional analysis using jGCaMP7c, which could sense calcium signals. A significant upsurge in eGFP expression in the transgenic hairy roots was displayed after adding calcium chloride. The results suggested that this approach was feasible for studying specific promoters and could be a tool to analyze gene functions in the roots of strawberries. Conclusion We established a rapid and efficient hairy root transformation in strawberry by optimizing parameters, which was adequate for promoter analysis and functional characterization of candidate genes in strawberry and other rosaceous plants.

Advances in genomics and genome editing for improving strawberry (Fragaria ×ananassa)

“…Protocols using agrobacterium have undergone significant optimization to improve transformation efficiency. Thus far, protocols have been established for Agrobacterium-mediated transformation and subsequent regeneration of a range of tissues (Li et al, 2018a;Feng et al, 2019;Feng et al, 2021b;Wilson et al, 2019;Duan et al, 2021;Mao et al, 2022;Yan et al, 2023). In strawberry, agrobacterium-mediated transformation most commonly uses Agrobacterium tumefaciens strains LBA4404, GV3101, and MP90.…”

“…Transformation of strawberry has also been performed using the hairy root system (Yan et al, 2023). Hairy roots are valuable for functional analysis of root traits as well as for validation of transgenic and genome editing methods due to the relatively short period of root development (Ozyigit et al, 2013).…”