Over-expression of α-galactosidase in pea seeds to reduce raffinose oligosaccharide content

The raffinose family of oligosaccharides (RFO) is a series of complex carbohydrates stored in seeds of many plant families, especially in legumes. The digestive system of nonruminant animals, including that of humans, cannot break down all of the chemical bonds in these carbohydrates; therefore, catabolism is achieved anaerobically by intestinal flora. The resulting digestive problems reduce acceptance and limit the widespread consumption of these otherwise nutritious seeds. To demonstrate a solution to this problem, transgenic lines of pea (Pisum sativum L.) expressing the alpha-galactosidase gene from coffee (Coffea arabica L.) were developed. Plants with a single copy of the inserted gene were selected, and two of these lines showed significant reductions of up to 40% in oligosaccharide content (raffinose, stachyose). Quantitative RT-PCR confirmed the presence of the alpha-galactosidase RNA in both leaves and cotyledons. Sugars were analyzed using whole seeds or only a portion of a seed; in the latter case, germination rates for each of the seeds analyzed were determined. The reduced raffinose contents did not affect germination rates, which remained very high (96%). The relative oligosaccharide contents of tissues within a seed also were determined; these were highest in the embryo axis, lower in the cotyledon and lowest in the seed coat.