Background
Up to 80% of the phosphorus in the cereal grain is stored in the form of phytic acid (phytate, myo-inositol 1,2,3,4,5,6-hexakisphosphate) which is deposited during seed development as phytin, a mixed salt with counterions including Ca ²⁺, Fe²⁺, K ⁺, Mg²⁺ and Zn²⁺. At germination the phosphate is liberated due to the action of the hydrolytic enzyme phytase. However, in dry seeds used for feeding non-ruminant animals, there is a limited degradation of the phytin compound. Digestible phosphate compounds are added to compensate for the low digestibility of phytin, leaving large amounts of phosphorus to pass through the gastrointestinal tract into the environment.

Aim
It is the aim of the project to study the opportunity of generating transgenic wheat with an increased phytase potential in the mature seed.

The project includes the establishment of a wheat transformation system, construction of phytase expression vectors, the study of heterologous phytase accumulation in the developing grain controlled by different promotors, processing of heterologous phytase and examinations of the influence of the phytase on myo-inositolphosphate composition.

Research Outline
The Aspergillus niger phytase-encoding gene (phyA) has been introduced into wheat. Transformation of wheat was achieved by particle bombardment of immature zygotic embryos, using the bar - Bialaphos selection system. Western blotting revealed the presence of an immuno-reacting protein of the expected size in leaves of the transformed plants.

The immunureacting protein was purified and N-terminal sequencing revealed that the recombinant protein, influenced by an ??amylase signal peptide, had been processed.

Phytase activity assays on transgenic single seeds showed an up to 4-fold increased phytase activity. A segregating population of transgenic seeds exhibited up to 2-fold increase of the phytase activity.

Metal-dye-detection HPLC showed that the processed protein did not affect the myo-inositolphosphate composition in the transgenic seed. However in plants accumulating a non-processed protein, the myo-inositolphosphate composition was clearly affected by the fungal enzyme.

The results show that a fully functional heterologous phytase can be synthesised in transgenic wheat. The transgenic seeds exhibit an elevated phytase potential when compared to the wild type.

Publications
Brinch-Pedersen H, Hatzack F, Holm PB, Olesen A & Rasmussen SK (1997). Transgen hvede med reduceret fytinindhold i kernen. Cerealienetværkets Årsmøde, 9-10 september , 27.

Brinch-Pedersen, H., Hatzack, F., Holm, P.B., Olesen, A., and Rasmussen, S.K. (1998). Transgen hvede med forøget fytaseaktivtet i kernen. Cerealienetværkets årsmøde, 5-6 november, 1998, Slagelse, Denmark