Background

Cultivated strawberry (Fragaria × ananassa, 8x) is an economically important fruit crop grown worldwide with lovely appearance and rich nutrition. The wild diploid strawberry Fragaria vesca has emerged as a model plant for cultivated strawberry as well as other fleshy fruit species. Moreover, strawberry is a typical non-climacteric fruit, studies on the ripening of which lack a nice model system, like tomato as a model of the climacteric fruit. Therefore, strawberry is also frequently used for studying fruit ripening.

Stable transformation is doable in both woodland and cultivated strawberry; however, the entire process is labor-intensive and time-consuming. In contrast, the transient expression assay in fruit is much faster and more efficient. There are two transient gene expression approaches in strawberry, namely Virus-Induced Gene Silencing (VIGS) and Agrobacterium-mediated transformation (Spolaore et al., 2001). Some studies obtained nice results by using VIGS (Jia et al., 2011; Li et al., 2019). However, we utilize the Agrobacterium-mediated transformation more frequently as it can achieve more experimental aims, including gene knock-down (Hoffmann et al., 2006), overexpression (Huang et al., 2018), and knock-out (Tang et al., 2018).

The F. vesca fruit takes 25-30 days from being pollinated to ripen under our growth conditions. The entire process is divided into seven stages: flower/anthesis, small green, medium green, large green, white, turning, and red. Fruit receptacle (the juicy flesh) at the large green stage, when the firmness starts to decline rapidly (before the achenes turn red for the red strawberry fruit), is suitable for agrobacterium injection. Fruit receptacles at prior stages are recalcitrant to the injection owing to the texture. Thus, Agrobacterium-mediated transformation in strawberry fruit delimits functional studies on genes acting at late developmental stages.

The bright red color of strawberry fruit is caused by the accumulation of anthocyanin compounds, which is a visible phenotype to check. In strawberry, the R2R3-MYB transcription factor FveMYB10 is a master regulator of anthocyanin synthesis (Lin-Wang et al., 2014). The woodland strawberry includes both red-fruited and white-fruited varieties, owing to a natural SNP occurred in FveMYB10; overexpression of FveMYB10 results in accumulation of red pigments in fruit of the white-fruited accession Yellow wonder (YW) (Hawkins et al., 2016). Recently, we identified one anthocyanin transporter encoding gene Reduced Anthocyanin in Petioles (RAP) through chemical mutagenesis, knock-down of which greatly reduced fruit pigmentation (Luo et al., 2018).

In this study, as an example, we describe the procedures of transiently overexpressing FveMYB10 in fruit of YW. Moreover, we exhibit the results of knocking down RAP in fruit of the strawberry cultivar ‘Sweet Charlie’.