Localization of acid phosphatase in rice calli

Abstract

Acid phosphatase is an important hydrolytic enzyme and widely distributed in plants. The role of acid phosphatase in plant tissues has been suggested involving in mobilization of nutrient reserves, sugar transport, differentiation of plastid, cell autolysis and senescence. In culture, the differentiating calli show the changes in isozyme patterns and activities, and anatomical localization. In this study, subcellular localization of acid phosphatase in regenerating callis is emphasized.Sterized rice seeds(Oryza sativa Tainong 67) were grown on modified MS medium containing 2 ppm 2,4-D. After 2 weeks, calli 2 to 3 mm in size were transfered on differentiation medium containing 1 ppm NAA and 2 ppm kinetin. Different stages of calli were collected and cut into thin slice with razor blade. Slices were fixed in 2.5% glutaraldehyde in 0.1M phosphate buffer(pH 7.0) for 2h and then washed, overnight . Specimens were incubated in Gomori solution containing 3mM p- nitrophenyl phosphate as substrate at 37°C for 90 min, postfixed with 1% OsO4 for lh, dehydrated in ethanol series and embedded in Spurr's resin. Control experiments were done as follows: l), incubation medium without substrate; 2),incubation medium without lead nitrate; 3), incubation medium containing 0.01% NaF. Sections were doubly stained with uranyl acetate and lead citrate, and finally observed under Hitachi H-600 at 75 KV.