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GENETICS, GENOMICS, AND MOLECULAR EVOLUTION

Expression of Anthocyanins and Proanthocyanidins after Transformation of Alfalfa with Maize Lc^1,2

Saskatoon Research Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, Saskatchewan, Canada S7N 0X2 (H.R., M.Y., P.A., L.B.-B., N.W. B.C., M.Y.G.); Plant Biotechnology Institute, 110 Gymnasium Place, Saskatoon, Saskatchewan, Canada S7N 0W9 (H.R.); Department of Plant Agriculture, University of Guelph, Guelph, Ontario, Canada N1G 241 (S.B., B.M.); and Department of Botany, Institute for Cellular and Molecular Biology, University of Texas, Austin, Texas 78713 (A.L.)

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 5'-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 5'-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 (+)-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.

¹ This work was supported in part by grants from the Alberta Agriculture Research Institute, the Alberta Cattle Commission, and the Saskatchewan Agricultural Development Fund. This is Institute paper no. 1547.

² This paper is dedicated to the memory of Dr. Bernie Goplen and Dr. Ron Howarth. Together, they had a vision for improving alfalfa forage quality by introducing proanthocyanidins and faith that it could be accomplished.

³ These authors contributed equally to the paper and to the assessment of the transgenic plants.

⁴ P.A. has retired from Agriculture and Agri-Food Canada.

⁵ Present address: BASF Plant Science, 26 Davis Drive, Research Triangle Park, Raleigh-Durham, North Carolina 27709-3528.

^* Corresponding author; e-mail gruberm{at}agr.gc.ca; fax 306–956–7247.

Received December 27, 2002; returned for revision February 20, 2003; accepted April 15, 2003.

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