| Alfalfa (Medicago sativa L.) clone N4-4-2 was bio-engineered for the synthesis of fructans in order to generate genetic variability to improve its winter hardiness, yield stability, and forage quality. The sacB gene from Bacillus amyloliquefaciens encoding levansucrase was expressed and targeted to the cytosol. T0 genotypes accumulated fructan in crown tissue but winter survival was reduced. Three expression vectors were constructed to deliver the levansucrase protein to the vacuole. Two constitutive promoters (CaMV 35S and Super promoter) or a root specific promoter (Mas2') were used. A group of 25 T0 plants per construct were produced by Agrobacterium -mediated transformation. PCR and Southern hybridisation confirmed insertion of the sacB gene. In a field trial established at Elora, Ontario, in 1999, transgenic alfalfa plants exhibited significantly higher dry matter accumulation than the wild type. Crown tissue accumulated fructan up to 13 mg/g dw and also contained more starch than the wild type. |
