The modification of Anthocyanin biosynthesis in dicotyledonous plants
The aim of this study was to modify the pattern of anthocyanin biosynthesis in dicotyledonous plants, in particular legumes, using four different anthocyanin regulatory genes and two promoters. The anthocyanin regulatory genes consisted of a matched pair from maize and a mismatched pair from two dicotyledonous species of plants. In order to accomplish this aim, a series of vectors were made that enabled expression of the genes following both transient and stable transformation experiments in plants. A novel method was devised to overcome the problems associated with the insertion of multiple chimeric genes into a binary transformation vector. A total of fourteen intermediate expression plasmids and sbrteen transformation plasmids were produced. Microparticle bombardment of monocotyledonous and dicotyledonous cell suspensions with constructs made for transient expression, confirmed that the coding regions of various constructs were functional. Microparticle bombardment with the maize anthocyanin regulatory genes stimulated anthocyanin accumulation when a member from each myc-type and myb-typc family was delivered with a the particle inflow gun (PIG) into monocotyledonous cell lines. Microparticle bombardment of dicotyledonous ceU suspensions with the maize anthocyanin regulatory genes did not stimulate anthocyanin accumulation. The efficiencies of microparticle bombardment with two types of microparticle delivery systems, the BioRad and PIG, were compared and it was determined that the BioRad helium gun was more proficient than the PIG for transient anthocyanin production in a monocotyledonous ceU suspension. Microparticle bombardment of maize suspension cultures with the maize anthocyanin regulatory genes using the BioRad helium gun, provided an understanding of the stoichiometry between the two families of regulatory genes. Delivering maize anthocyanin regulatory genes into a cell suspension of Trifolium subterraneum with the BioRad helium gun induced anthocyanin accumulation in cells. However, the dicotyledonous anthocyanin regulatory genes did not induce anthocyanin production in any of the cell suspensions assayed with either delivery system. Transient expression experiments in whole plant tissues of pea and white clover confirmed that maize anthocyanin regulatory genes could cause anthocyanin accumulation in legumes. This was achieved by either co-bombardment of myc and myb anthocyanin regulatory genes or by a single myc gene complementing an endogenous anthocyanin regulatory gene. Microparticle bombardment of the maize anthocyanin regulatory genes displayed tissue specificity in the production of both GUS and anthocyanin. Microparticle bombardment of various cell suspensions, as well as plant tissues, with the dicotyledonous anthocyanin regulatory genes did not result in anthocyanin accumulation. Tobacco and white clover plants were stably transfonned and regenerated with fifteen different plasmid constructs containing combinations of anthocyanin regulatory genes. In tobacco, the only tissues found to accumulate anthocyanin were the corolla and the anther filaments of the flowers. Transformation of tobacco with the dicotyledonous and monocotyledonous myb type anthocyanin regulatory genes on their own had no visible effect on anthocyanin accumulation. In contrast, transformation of tobacco with the dicotyledonous and monocotyledonous myc type anthocyanin regulatory genes on their own enhanced anthocyanin accumulation. The most intense anthocyanin was observed in the corolla of the tobacco plants transformed with the vector containing both of the maize anthocyanin regulatory genes B-Peru and Cl, each driven by the 35S promoter. Tobacco plants that had their anthocyanin enhanced often displayed various aberrant petal pigmentation patterns, presumably associated with the phenomenon of 'silencing'. In white clover plants transformed with either the dicotyledonous or monocotyledonous anthocyanin regulatory genes on their own or in combmation, a large range of different tissues exhibited enhanced anthocyanin accumulation. One plant, transformed with the B-Peru gene driven by the 35S promoter, displayed a unique pattern of anthocyanin accumulation m the leaf. The accumulation of anthocyanin in this plant was closely associated with the leaf crescent and disappeared in the oldest leaf stage. The tobacco and white clover anthocyanin patterns were shown to be heritable. Plants transformed with both a myb and a myc anthocyanin regulatory gene seemed to have a much lower transformation efficiency than those transformed with only a myc or a myb gene. The anthocyanin regulatory genes used in this study had no detectable effect on tannins or other flavonoids. This study presents the first evidence of anthocyanin regulatory genes altering anthocyanin expression in various legummous plants using transient and stable transformation experiments.
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