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The cytochemical localization of peroxidase in root tip tissue of Alaska PEA (Pisum sativum )
THE CYTOCHEMICALLOCALIZATION OF PEROXIDASEIN ROOTTIP TISSUE OF ALASKA PEA (Pisum sativum). By: Bradford O. Bratton, Nels Baver I I , James F. Russo and Egbert W. Henry~ Department of Biological Sciences - Oakland University - Rochester, MI 48063
Electron microscopic fixation methods were utilized to prepare "thick sections" for viewing by light microscopy. The terminal 3mm sections of four week old dark-grown Alaska pea (Pisum sativum) seedlings were excised and prefixed for 2 hours in 2.5% g l u ~ d e - ~ i s s o l v e d in O.SM potassium phosphate buffer at pH 5.8. Subsequently, the glutaraldehyde fixed tissues were repeatedly rinsed in O.05M potassium phosphate buffer, pH 5.8. The tissues were incubated in a medium containing 0.5% p-phenylene diamine and 0.5% H202, for 30 minutes. Tissue incubated in O.05M potassium phosphate buffer, pH 5.8 only were used as controls. After staining in the incubation medium, the tissues were rinsed several times in O°05M potassium phosphate buffer, pH 5.8, dehydrated in a graded ethanol series, transferred to propylene oxide and finally embedded in Epon 812 (4 parts of mixture A and l part of mixture B, accelerated with 1.5% DMP 30). Thick sections were cut with a Porter-Blum ultramicrotome, using glass knives. The present study indicates that peroxidase activity is localized in the cell walls, chromosomes, nucleus and nucleolus of root tip tissue of Alaska pea (Fig. I-3). Peroxidase activity is known to be associated with chromosomes, particularly during cell division (Raa, 1973). Tissue incubation in buffer only reveals an absence of peroxidase localization sites (Fig. 4). I t is suggested that only those peroxidase isoenzymes capable of association with the internal cellular structures may participate in the modulation of endogenous IAA levels and in cellular differentiation. REFERENCES: Raa, J., 1973. Physiol. Plant., 25, 132. *To whom correspondence should be directed.
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FIGURE I: Unstained control tissue showing absence of peroxidase (P) in the nucleus (N) and cell walls (CW). X3500. FIGURE2: Low power view of tissue incubated in p-phenylenediamine (PPD) shows intense P staining in the nuclei (arrows) and cell walls. Xl300. FIGURE3: PPD incubated tissue showing P staining in the nuclear (N) and cell walls (CW). X3500. FIGURE4: PPD incubated tissue showing P ~taining in the nucleus (N), cell walls (CW) and chromosomes (arrows). X4300.
Electron microscopic fixation methods were utilized to prepare "thick sections" for viewing by light microscopy. The terminal 3mm sections of four week old dark-grown Alaska pea (Pisum sativum) seedlings were excised and prefixed for 2 hours in 2.5% g l u ~ d e - ~ i s s o l v e d in O.SM potassium phosphate buffer at pH 5.8. Subsequently, the glutaraldehyde fixed tissues were repeatedly rinsed in O.05M potassium phosphate buffer, pH 5.8. The tissues were incubated in a medium containing 0.5% p-phenylene diamine and 0.5% H202, for 30 minutes. Tissue incubated in O.05M potassium phosphate buffer, pH 5.8 only were used as controls. After staining in the incubation medium, the tissues were rinsed several times in O°05M potassium phosphate buffer, pH 5.8, dehydrated in a graded ethanol series, transferred to propylene oxide and finally embedded in Epon 812 (4 parts of mixture A and l part of mixture B, accelerated with 1.5% DMP 30). Thick sections were cut with a Porter-Blum ultramicrotome, using glass knives. The present study indicates that peroxidase activity is localized in the cell walls, chromosomes, nucleus and nucleolus of root tip tissue of Alaska pea (Fig. I-3). Peroxidase activity is known to be associated with chromosomes, particularly during cell division (Raa, 1973). Tissue incubation in buffer only reveals an absence of peroxidase localization sites (Fig. 4). I t is suggested that only those peroxidase isoenzymes capable of association with the internal cellular structures may participate in the modulation of endogenous IAA levels and in cellular differentiation. REFERENCES: Raa, J., 1973. Physiol. Plant., 25, 132. *To whom correspondence should be directed.
161
162
B.O. Bratton et al.
FIGURE I: Unstained control tissue showing absence of peroxidase (P) in the nucleus (N) and cell walls (CW). X3500. FIGURE2: Low power view of tissue incubated in p-phenylenediamine (PPD) shows intense P staining in the nuclei (arrows) and cell walls. Xl300. FIGURE3: PPD incubated tissue showing P staining in the nuclear (N) and cell walls (CW). X3500. FIGURE4: PPD incubated tissue showing P ~taining in the nucleus (N), cell walls (CW) and chromosomes (arrows). X4300.