Antisense suppression of the lignin biosynthetic enzyme, caffeate O-methyltransferase, improves in vitro digestibility of the tropical pasture legume, Stylosanthes humilis

Abstract

The high lignin content of tropical forage plants reduces digestibility and voluntary feed intake by ruminants. We have used antisense technology to suppress caffeate O-methyltransferase (COMT EC, 2.1.1.68), a lignin biosynthetic enzyme in the tropical forage legume, Stylosanthes humilis Kunth. Plants were transformed using a Ti binary vector containing an antisense COMT construct under the control of the CaMV 35S promoter. From 50 transgenic plants, five were selected on the basis of normal morphology, high levels of antisense gene expression and altered lignin histochemistry. No plants with altered lignin were observed in a population of 20 transgenic plants derived using a binary vector that lacked the COMT cDNA insert. The progeny of lignin-altered plants were analysed for COMT enzyme activity and lignin histochemistry. A variety of COMT and lignin phenotypes was observed. In several T₁ plants, COMT activity was specifically suppressed by more than 95% compared to controls. In these plants, expression of antisense mRNA was high while sense mRNA could not be detected on northern blots. The overall lignin content of these plants was unchanged but histochemical tests showed abnormally low levels of the syringyl component, mimicking the pattern of young tissue. Digestibility of these transgenic plants was assessed by incubation of stem material with rumen fluid and acid pepsin in vitro. The digestibility of the antisense material was increased dramatically compared to that of equivalent samples from control transformed plants (72 vs 62%).