An optimized TRV-based virus-induced gene silencing protocol for Malus crabapple

Virus-induced gene silencing (VIGS) is a valuable reverse genetics tool to study gene function in a broad range of plants. As the efficiency of apple stable transformation is very low and it is still not easy to produce a large number of apple transgenic plants for most laboratories. Here, we developed an easy and effective transient transformation method of agro-inoculation for virus-induced gene silencing based on vacuum infiltration in genus of apples, the crabapple. The entire young tissue cultured crabapple plantlets were used as the plant material for infiltration, and a −90 kPa vacuum pressure and 19 °C day/18 °C night cultivation temperature were shown to result in highest VIGS efficiency. Infiltration with tobacco rattle virus containing a fragment of the transcription factor gene McMYB10 , which regulates the synthesis of anthocyanin pigments, resulted in low levels of McMYB10 transcripts and characteristic faded red leaves. In addition, the expression of several anthocyanin biosynthetic genes was down-regulated in the transformed leaves. The results of this study indicate that the MYB10 gene represents a useful reporter gene for VIGS in crabapple and that the VIGS technique can be successfully applied to crabapple, thereby providing a platform for functional genetic studies in this and likely other Malus species.