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 A degrees C day/18 A degrees 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.