Betalain synthesis in alfalfa impacts fiber digestion and enteric methane production

Abstract Betalain pigments, betacyanins, and betaxanthins are widely used as coloring agents as well as in pharmaceutical and functional foods. However, significant betacyanin production occurs in only a few species of plants, which limits their wider application. We used the reporter gene RUBY controlled by CaMV 35S promoter to produce betalain in alfalfa ( Medicago sativa L.). Betalain pigments were produced throughout the plant with the intensity of coloration varying with RUBY expression. Concentrations of betacyanin in herbage ranged from 50 to 650 mg/100 g dry wt and betaxanthin from 0.28 to 2.55 mg/100 g dry wt, significantly higher than in roots of table beet. Also, alfalfa plants expressing RUBY reduced methane production in in vitro enteric methane assays and had increased stem cell wall fiber digestibility compared to wild‐type plants. Alfalfa plants expressing RUBY would provide a low cost and sustainable source of betalain for food and feed applications. When RUBY alfalfa is incorporated into rations, it could be advantageous in cattle production systems to increase forage digestibility and protein synthesis.