Stable expression of Phytase (phyA) in canola (Brassica napus) seeds:: towards a commercial product

The Aspergillus niger gene encoding phytase (phyA) was expressed in canola (Brassica napus). Phytase expression is controlled by the seed-specific cruciferin (CruA) promoter. Secretion of the enzyme was aimed for by incorporating the cruciferin signal peptide in the expression construct. Transgenic canola lines were generated by Agrobacterium mediated transformation using nptII as the selectable marker. Ninety-five independent transgenic events were generated. Phytase expression in the T1 seeds ranged from 0 to 600 U/g seed. Single-copy lines were selected (based on segregation for kanamycin resistance, phytase expression and Southern analyses) from originally multi-copy transgenic lines. Phytase was expressed in these sub-lines up to 103 U/g. Expression levels were monitored through an additional 3-4 generations (in the greenhouse and in the field) and the accumulation of phytase appeared to be fairly stable. In the expression range studied, phytase expression was gene-dosage dependent.