Virus-induced gene silencing (VIGS) for functional analysis of genes involved in the regulation of anthocyanin biosynthesis in the perianth of Phalaenopsis-type Dendrobium hybrids

Abstract

Virus-induced gene silencing (VIGS) is an efficient way to study floral gene functions of orchids that have long life cycle and inefficient genetic transformation. However, reports of the use of the VIGS technique for Dendrobium spp. are scarce. To explore the applicability of Cymbidium mosaic virus (CymMV)-based VIGS in Phalaenopsis-type Dendrobium hybrids (Den-Phals), the regulator gene DhMYB2 of anthocyanin biosynthesis was used as a target gene. Several variables including the length of DNA insertion, the location of the cDNA, and the developmental state of inflorescences were examined. The results suggested that a CymMV-based VIGS system could be used with Den-Phals to silence DhMYB2 up to 98%, resulting in the loss of anthocyanin coloration in sepals, petals and mid-lobes of labella and down regulation of the expression levels of major genes in the anthocyanin biosynthetic pathway (ABP). The sizes of the DNA insert for silencing were between 120 and 200 bp. The most obvious phenotype was obtained when using the 5′ terminus of the coding region of DhMYB2 cDNA. The plants of a younger development stage of inflorescence with no more than four visible floral buds were suitable for Agro-infiltration. The present study demonstrated the feasibility of this CymMV-based VIGS system using DhMYB2 as the visible marker gene for the functional analysis of floral genes and illuminated the regulatory effects of DhMYB2 on the floral pigmentation in Den-Phals.