A simple method for the identification and assay of extracellular plant beta-galactosidase

A simple, rapid and reproducible procedure for the identification of extracellular Californian poppy (Eschscholzia californica Cham.) beta-galactosidase is described using callus cultures of seedlings from the tested plant, roots of 4-days-old seedlings of Californian poppy germinating on agar plates and cell suspension cultures cultivated from callus cultures. 6-Bromo-2-naphthyl-beta-D-galactopyranoside and p-nitrophenyl-beta-D-galactopyranoside were used as substrates for the determination of the intracellular and extracellular activities of beta-galactosidase. The extracellular beta-galactosidase activity was identified by evaluating the dye-zones in an agar medium. The enzyme from Californian poppy callus cultures or from seedling roots cultivated on agar plates supplemented with 6-bromo-2-naphthyl-galactopyranoside hydrolyzed this substrate releasing 6-bromo-2-naphthol. By simultaneous coupling with hexazonium p-rosaniline the corresponding (reddish-brown) azo-dye was formed. The agar plate method described permits rapid, simple and specific detection of plant producers of extracellular beta-galactosidase.