Optimization and standardization of transient expression assays for gene functional analyses in strawberry fruits

Abstract

Strawberry is increasingly used as a model plant for research on fruit growth and development. The transient gene manipulation (TGM) technique is widely used to determine the function of plant genes, including those in strawberry fruits. However, its reliable application for the precise identification of gene function has been difficult owing to the lack of conditional optimization. In this study, we found that successful transient gene manipulation requires optimization, with the vector type, temperature, and fruit developmental stage being three major factors determining success. Notably, we found that transient gene manipulation was feasible only from the large green fruit stage onwards, making it especially suitable for identifying genes involved in strawberry fruit ripening. Furthermore, we established a method called percentage difference of phenotype (PDP), in which the functional effect of a gene could be precisely and efficiently identified in strawberry fruits. This method can be used to estimate the functional effect of a gene as a value from 0 to 100%, such that different genes can be quantitatively compared for their relative abilities to regulate fruit ripening. This study provides a useful tool for accelerating research on the molecular basis of strawberry fruit ripening.

Rapid Alkalinization Factor (RALF) gene family genomic structure and transcriptional regulation during host-pathogen crosstalk in Fragaria vesca and Fragaria x ananassa strawberry

“…Indeed Agrobacterium can be perceived as a pathogen by the fruit and stimulate similar responses as C. acutatum , including those leading to FaRALF3 expression. The variability affecting strawberry Agrobacterium -mediated transformation depending on technical and environmental conditions has recently been described [64]. It was shown that the expression level of a reporter gene is normally distributed in an population of 30 treated fruits, with huge variation among different fruits.…”

Genomic structure and transcript analysis of the Rapid Alkalinization Factor (RALF) gene family during host-pathogen crosstalk in Fragaria vesca and Fragaria x ananassa strawberry

“…Indeed Agrobacterium can be perceived as a pathogen by the fruit and stimulate similar responses as C. acutatum, including those leading to FanRALF3 expression. The variability affecting strawberry Agrobacterium-mediated transformation depending on technical and environmental conditions has recently been described [76]. It was shown that the expression level of a reporter gene is normally distributed in a population of 30 treated fruits, with huge variation among different fruits.…”

Establishment of a Rapid and Stable Transgenic System by Agrobacterium-Mediated Transformation of the Germination Seeds in Diploid Strawberry.

“…Strawberry (Fragaria), as a distinct member of Rosaceae, is not only an important fruit crop worldwidely, but is also regarded as a model for studying non-climacteric fruit ripening [1]. In the past years, a large of genes related to strawberry fruit ripening have been identi ed via traditional stable and transient transgenic systems in strawberry; however, more or less, the limitation is existed [2][3][4]. Thus, development of a rapid, e cient, and stable transformation system is required for strawberry research and breeding.…”

“…Such as, it takes at least 15 months from transformation to gaining transgenic fruits for gene function analysis in ripening [17,18]. In addition, although the transient transformation technology in strawberry fruits provides a rapid tool to study gene function in ripening, some limits are existent in fruit transient transgenic system [2,4].…”