Betalain synthesis in alfalfa impacts fiber digestion and enteric methane production

Authors: Heuschele, Deborah; NIMPOENO, JASON - University Of Minnesota; Curtin, Shaun; Samac, Deborah

Submitted to: Plant Biotechnology Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/4/2025
Publication Date: 1/26/2026
Citation: Heuschele, D.J., Nimpoeno, J., Curtin, S.J., Samac, D.A. 2026. Betalain synthesis in alfalfa impacts fiber digestion and enteric methane production. Plant Biotechnology Journal. 66(1):e70226. https://doi.org/10.1002/csc2.70226.
DOI: https://doi.org/10.1002/csc2.70226

Interpretive Summary: Genetic transformation is difficult to obtain in most alfalfa cultivars but is needed for advancing gene editing techniques for alfalfa improvement. A novel transformation vector with additional virulence genes from the bacterium Agrobacterium was found to increase transformation efficiency five-fold compared to a vector lacking these genes. To assist with identification of transformed cells, the RUBY marker was added as part of the transferred DNA. Expression of the RUBY marker results in production of a bright red pigment, betalain, the same pigment that is found in beet root, which is used in nutritional supplements and as a natural dye in foods and cosmetics. The RUBY marker was expressed in all parts of the alfalfa plants with concentrations of betalain in the herbage equal to or greater than in beets, or other plants expressing the RUBY marker. Alfalfa plants containing the RUBY marker could be used as a sustainable and inexpensive source of betalain.

Technical Abstract: Transformation of most plant species is limited to cultivars or genotypes that regenerate easily in tissue culture. We used the reporter construct RUBY, a noninvasive marker, to monitor transformation of the perennial forage legume alfalfa (Medicago sativa). Expression of RUBY, consisting of three concatenated genes, converts tyrosine into the stable red compound betalain, resulting in purple-red plant cells. The ternary helper vector pRiA4-VIR increased RUBY expressing somatic embryos by ~5-fold. Regenerated alfalfa plants with the RUBY construct controlled by the constitutive cauliflower mosaic virus 35S promoter expressed betacyanin in leaves, stems, roots, flowers, and seeds with the intensity of coloration varying with RUBY expression as measured by qRT-PCR. Concentrations of betacyanin ranged from 50 to 650 mg/g and betaxanthin from 0.28 to 2.55mg/g, significantly higher on a dry weight basis than the concentrations of these compounds in beet root. Alfalfa plants expressing RUBY could provide a low cost and sustainable source of betalain.