Three anthocyanin regulatory genes of maize (Zea mays; Lc, B-Peru, and C1) were introduced into alfalfa (Medicago sativa) in a strategy designed to stimulate the flavonoid pathway and alter the composition of flavonoids produced in forage. Lc constructs included a full-length gene and a gene with a shortened 5Ј-untranslated region. Lc RNA was strongly expressed in Lc transgenic alfalfa foliage, but accumulation of red-purple anthocyanin was observed only under conditions of high light intensity or low temperature. These stress conditions induced chalcone synthase and flavanone 3-hydroxylase expression in Lc transgenic alfalfa foliage compared with non-transformed plants. Genotypes containing the Lc transgene construct with a full-length 5Ј-untranslated region responded more quickly to stress conditions and with a more extreme phenotype. High-performance liquid chromatography analysis of field-grown tissue indicated that flavone content was reduced in forage of the Lc transgenic plants. Leucocyanidin reductase, the enzyme that controls entry of metabolites into the proanthocyanidin pathway, was activated both in foliage and in developing seeds of the Lc transgenic alfalfa genotypes. Proanthocyanidin polymer was accumulated in the forage, but (ϩ)-catechin monomers were not detected. B-Peru transgenic and C1 transgenic populations displayed no visible phenotypic changes, although these transgenes were expressed at detectable levels. These results support the emerging picture of Lc transgene-specific patterns of expression in different recipient species. These results demonstrate that proanthocyanidin biosynthesis can be stimulated in alfalfa forage using an myc-like transgene, and they pave the way for the development of high quality, bloat-safe cultivars with ruminal protein bypass.The ability to manipulate flavonoid biosynthesis in crop plants is gaining rapidly in importance as new economically important uses are found in the areas of nutraceuticals, food quality, and feed quality. The introduction of proanthocyanidin (PA, a flavonoid polymer) into alfalfa (Medicago sativa) forage is particularly important to ruminant livestock producers. Proanthocyanidins eliminate pasture bloat, improve the efficiency of conversion of plant protein into animal protein (ruminal protein bypass), reduce greenhouse gases, reduce gastrointestinal parasites, and inhibit insect feeding (Waghorn, 1990; Neizen et al., 1995 Neizen et al., , 1998 Aerts et al., 1999; Muir et al., 1999; McMahon et al., 2000). Alfalfa forage (leaf and stem tissues) accumulate anthocyanins only at senescence or locally under some stress conditions such as insect feeding (Goplen et al., 1980). No known conditions induce proanthocyanidins in alfalfa forage, although they are structurally related to anthocyanins. However, these compounds do accumulate in seed coats (Koupai-Abyazani et al., 1993).Anthocyanins and proanthocyanidins share early and middle steps of the flavonoid biosynthetic pathway, including chalcone synthase (CHS), chalcone isomerase, flavan...

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