Anthocyanin Genes as Visual Markers for Wheat Transformation

Abstract

Regulatory genes of the maize anthocyanin biosynthetic pathway have proven useful as scorable markers for transformation because of their high sensitivity, ease of visualization, cell-autonomous expression and lack of requirement for exogenous substrates. This is particularly advantageous when using particle bombardment as a DNA delivery system, since cells expressing genes can be counted easily and unambiguously (Bowen 1992). Furthermore, the cells that have been damaged by microprojectile bombardment can still express GUS (giving false positive), but they will not accumulate anthocyanin since the latter requires an intact vacuole and coordinate expression of many genes. Expression of anthocyanin genes in plant tissues will be useful where false positive results have been reported due to intrinsic GUS-like activity in several plant parts (Hu et al. 1990) and GUS production by endophytic microorganisms (Tor et al. 1992). The anthocyanin markers permit visualization of transgenic tissue from the beginning and throughout development without sacrificing the tissues. In addition, expression of anthocyanin genes in transformed tissues can provide a system for investigating the regulation of gene expression in plants, which is difficult with GUS (Ben-fey et al. 1990). Transient and stable expression of anthocyanin genes in various corn intact tissues (Klein et al. 1988; Ludwig et al. 1990; Wong et al. 1991; Bowen 1992; Dunder et al. 1993) and transient expression in wheat seedling tissues (Wong et al. 1991) has been reported previously. We have used anthocyanin as a visual marker for optimization of bombardment conditions using different target tissues (transiently) and evaluation of different selectable markers (stably) for wheat transformation by selecting the transformants visually in the early stage of development.