Transformation and isoflavonoid analyses of suspensioncultured cells of soybean [Glycine max (L.) Merr. cv. Enrei]

We established cell suspension cultures of soybean [Glycine max (L.) Merr.] (cv. Enrei). Calli were induced from cotyledon explants on callus induction medium containing 1 mg l⁻¹ kinetin and 0.1 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and then successfully suspension-cultured in Murashige and Skoog basal medium containing 1 mg l⁻¹ kinetin and 0.05 mg l⁻¹ 2,4-D. The resulting suspension-cultured cell line, designated as ECW1, has been maintained for 3 years by subculturing at 8-day intervals. Flavonoids that accumulated in ECW1 cells during cultivation were analyzed by high performance liquid chromatography. These analyses showed that 6″-O-malonyldaidzin was the most abundant flavonoid until day 8 of cultivation, followed by 6″-O-malonylgenistin, daidzin, and genistin. Trace amounts of isoflavone aglycons, glyceollin, and other flavonoids were detected during cell cultivation. We established an Agrobacterium-mediated method to transform suspension-cultured soybean cells using the ECW1 cell line as the host. A gene encoding green fluorescent protein was heterologously expressed under the control of the CaMV 35S promoter in ECW1 cells. The recombinant cells were stably maintained for 2.5 years via repeated subculturing. This is the first example of the stable transformation of suspension-cultured soybean cells using the Agrobacterium method. The cell line ECW1 is a promising tool for research on the biochemistry, molecular biology, and physiology of soybean.