Alfalfa was transformed with a CaMV 35S promoter::resveratrol synthase expression construct, resulting in the accumulation of resveratrol glucoside (RGluc) in leaves and stems (Hipskind and Paiva, 2000). Resveratrol is well known as an antifungal agent in many plants, but does not naturally accumulate in alfalfa. When inoculated with the alfalfa pathogen Phoma medicaginis, leaves of these transgenic plants showed dramatically lower levels of mycelia growth and sporulation, compared to wild-type or empty vector control plants under laboratory conditions. This suggests that these plants may be resistant to this foliar pathogen in the field, and would dramatically reduce pathogen spread. In addition to protecting forage yield and quality, large amounts of transgenic alfalfa biomass could be harvested and fed to livestock directly, or processed for purified resveratrol glucoside, for use as a dietary supplement. Resveratrol and its glucosides are thought to be beneficial to human and animal health, preventing heart disease and cancer initiation, perhaps by serving as a strong antioxidant.

Small scale field studies with independent primary trangenic lines were initiated to

(1) compare the levels of resveratrol observed in the greenhouse conditions with those observed in the field, (2) compare the relative levels of RGluc between lines under field and greenhouse conditions, and (3) compare the forage yields between high and low accumulating lines, to determine if RGluc accumulation has a negative impact on forage production. Ten single transgene copy transgenic lines representing high, medium, and low RGluc-accumulating and two control lines (untransformed RegenSY parental line) were planted in a randomized block design (5 replicates). Biomass (fresh weight and dry weight) production and RGluc content (in both fresh and dried samples) were evaluated. The relative levels of RGluc was the same in both environments, although the variations in absolute RGluc levels varied more in the field enviroment. Forage yield was independent of the level of RGluc accumulation.

Progeny of crosses between two high-accumulating primary transgenics and a clone of an improved cultivar (Cimarron) are also being evaluated under field and greenhouse conditions. The two transgenic parents, their untransformed RegenSY parent, the Cimarron parent, and three high-producing progeny of each cross were planted in a randomized block design (5 replicates).
Preliminary analysis indicated that some of the progeny lines accumulated less RGluc in the field than in the greenhouse. We suspect this may be due to differences in the growth and maturation rates of the lines; the Cimarron parent regrows much faster than the RegenSY parent, while the RegenSY parent blooms earlier, and the progeny vary in these traits. In primary transgenics, RGluc concentrations were higher in younger leaves than in older leaves, and may decrease as the shoots mature and bloom. Samples are now being taken at different stages of maturity for each line, to check for age-dependent variations in RGluc concentrations.

Hipskind, J.D. and Paiva, N.L. 2000. Constitutive accumulation of a resveratrol-glucoside in transgenic alfalfa increases resistance to Phoma medicaginis. Mol. Plant-Microbe Interact. 13: 551-562.

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