| Abstract: | A simple, rapid and reproducible procedure for the identification of extracellular cucumber (Cucumis sativus L.) α‐galactosidase is described using callus cultures of seedlings from the tested plant, hairy roots of 2‐day‐old seedlings of cucumber germinating on agar plates as well as cell suspension cultures derived from callus cultures. For the determination of the intracellular and extracellular activities of α‐galactosidase, 6‐bromo‐2‐naphthyl‐α‐D‐galactopyranoside and p‐nitrophenyl‐α‐D‐galactopyranoside, respectively, were used as synthetic substrates. The extracellular α‐galactosidase activity was identified by evaluating the dye‐zones in agar medium. The enzyme from cucumber callus cultures and seedling roots, cultivated on agar plates supplemented with 6‐bromo‐2‐naphthyl‐α‐D‐galactopyranoside, hydrolyzed this substrate releasing 6‐bromo‐2‐naphthol. By simultaneous coupling with hexazonium p‐rosaniline the corresponding azodye was formed. Thus, the extracellular enzyme was detected by the presence of reddish‐brown zones on the agar plates around the plant material. The parallel extracellular and intracellular activities were determined in cell suspension cultures derived from callus cultures. The results show a 44.6% intracellular and 55.4% extracellular distribution of α‐galactosidase activity. The described agar plate method enables a rapid, simple and specific detection of plant producers of extracellular α‐galactosidase. |
