Downregulating the expression of a lignin biosynthetic gene by antisense technology to improve forage quality

Lignin is a complex aromatic polymer in plant cell walls. It limits cell wall degradability by ruminants, reducing the nutritional value of forages. Genetic engineering using an antisense gene offers the potential to modulate enzymes in the lignin biosynthetic pathway to reduce and or modify lignin and thereby improve forage quality and animal performance.; Maize (Zea mays L.) Hi-II was used as a model system to investigate the effectiveness of sorghum [Sorghum bicolor (L.) Moench] caffeic acid O-methyltransferase (OMT) antisense constructs to down regulate maize OMT and improve forage quality. Sorghum OMT antisense constructs were assembled with the bar selectable gene, which confers glufosinate herbicide resistance, along with the appropriate promoters and terminators. Embryogenic maize callus tissue was transformed by biolistic particle bombardment and 350 plants were regenerated.; These plants were characterized using herbicide resistance, PCR, Southern and northern analyses. About 55% of the 350 plants were resistant to 1% glufosinate and 85.3% of the 204 PCR-tested Plants were PCR positive for bar gene. About 10% of the transgenic plants expressed the brown midrib phenotype at the 4- to 6-leaf stage and vascular tissue discoloration in mature stems similar to the natural bm3 maize mutants, which have improved digestibility. Southern analysis of OMT indicated that transgene copy number in transgenic plants varied from 1 to 20, with full-length and rearranged transgenes, present. The bar gene was more stably transmitted to progeny than the antisense OMT, which exhibited multiple rearrangements.; OMT activity of the transgenic plants was significantly (p < 0.05) reduced compared to the controls, with some transgenic plants showing 60% reduction. The distribution of OMT activity and OMT mRNA steady state level showed OMT activity was highly correlated to the OMT mRNA level.; Transgenic plants with down-regulated OMT activity had improved (p < 0.05) in vitro organic matter digestibility (IVOMD) as compared to controls, with the stems being most improved (7.4%), followed by sheaths (5.2%) and leaves (1.9%). Lignin content was significantly reduced (stems –19.8%, sheaths –18.8%, leaves –11.8%). On a whole plant basis, the transgenic plants increased IVOMD an average of 5.0% with lignin reduction of 16.5%. The highest IVOMD increase was 8.0% with lignin reduction of 30.6%.