Ectopic expression of a soybean phytase in developing seeds of Glycine max to improve phosphorus availability Joseph M. Chiera 1 , John J. Finer 2 and Elizabeth A. Grabau 1, * 1 Department of Plant Pathology, Physiology, and Weed Science, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA (*author for correspondence; e-mail egrabau@vt.edu); 2 Department of Horticulture and Crop Science, OARDC/The Ohio State University, Wooster, OH 44691, USA Received 24 May 2004; accepted in revised form 22 October 2004 Key words: Glycine max, phosphorus, phytase expression, phytate, soybean, transgenic Abstract A transgenic approach was used to alter soybean seed phytate content by expressing a soybean phytase gene (GmPhy) during seed development to degrade accumulating phytic acid (IP 6 ). An expression vector con- taining the soybean phytase cDNA controlled by the seed-specific b-conglycinin promoter (a 0 -subunit) was used to transform embryogenic soybean cultures. Plants from four independent transgenic lines were ana- lyzed for transgene integration and seed IP 6 levels. The reduction in IP 6 levels in transgenic seeds compared to control ‘Jack’ soybeans ranged from 12.6 to 24.8% as determined by HPLC. A low copy transformant was propagated to the T 4 generation and examined in more detail for phytase expression and enzyme activity during seed development. Expression of phytase mRNA and phytase activity increased during seed devel- opment, consistent with the use of an embryo-specific promoter. Ectopic phytase expression during seed development offers potential as an effective strategy for reducing phytate content in soybean seed. Abbreviations: HPLC, high performance liquid chromatography; IP 6 , myo-inositol hexakisphosphate, phytic acid; P, phosphorus Introduction Phosphorus (P) is stored in plant seeds as phytate (also known as phytic acid, myo-inositol hexakisphosphate or IP 6 ) and is degraded by the enzymatic activity of phytase during germination (Reddy et al., 1989). Similar to other agriculturally important crops, soybean contains 60–80% of total seed P in the form of phytate (Raboy et al., 1984). Soybeans are processed to produce meal, which is commonly used in animal feed because of its high protein content. Seed phytate in animal diets is not readily digested by non-ruminant ani- mals and is excreted in manure (Reddy et al., 1989; Ravindran et al., 1995a). Depending on meal treatment, a significant portion of phytate may remain undigested even in ruminant animals (Konishi et al., 1999; Park et al., 1999). Inefficient utilization of phytate P necessitates the addition of supplemental phosphate to animal rations to meet optimal growth requirements. Another anti-nutrient property of phytate is its ability to complex with mineral cations such as potassium, magnesium and calcium to form phy- tin, which accumulates in protein bodies in the seed (Prattley and Stanley, 1982). Mineral chela- tion by phytate reduces the availability of iron and zinc, leading to potential mineral deficiencies (Reddy et al., 1989). The widespread use of soybean meal in live- stock feed and the inability to utilize phytate P can lead to serious environmental consequences. Repeated applications of P-rich manure to pas- tures and cropland increase soil P levels and the Plant Molecular Biology 56: 895–904, 2004. Ó 2005 Springer. Printed in the Netherlands. 895