Synopsis
Chemical estimation of nucleic acid, essentially RNA, in fixed tissue from Jerusalem artichoke tubers, coupled with an examination of the types of RNA in the fixed tissue by gel electrophoresis, demonstrates that ribosomal and ‘soluble’ RNA are preserved in this tissue after various fixation procedures including methanol, ethanol-acetic acid and aqueous formaldehyde. Tissue fixed in ethanol-acetic acid or formaldehyde is resistant to loss of nucleic acid by aqueous extraction but tissue fixed in all three standard fixatives loses nucleic acid in citrate buffer under conditions used for Azure B staining. The presence of Azure B in the buffer does not wholly prevent this loss. Tissue fixed in formaldehyde or mixed fixatives containing formaldehyde is resistant to loss of nucleic acid during treatment with EDTA to obtain cell suspensions.
Preliminary experiments with Azure B, Gallocyanin chrome-alum and Methylene Blue showed that the Gallocyanin technique is the most practicable for demonstrating RNA cytochemically. Its specificity was confirmed by ribonuclease extraction of the tissue. Optimum staining conditions, requiring treatment of the tissue in Gallocyanin chrome-alum solution overnight at 40°C, are established for the artichoke tissue and for paraffin sections of pea shoot apices.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Alfert, M. (1952). Studies on the basophilia of nucleic acids. The methyl green stainability of nucleic acids.Biol. Bull. 103, 145–56.
Beswick, T. S. L. (1958). The Einarson gallocyanin-lake staining technique.J. Path. Bact. 76, 598–600.
de Boer, J. &Surnaker, R. (1956). The gallocyanin stain for Nissl bodies.Med. Proc. 2, 218–21.
Bucknall, R. A. &Sutcliffe, J. F. (1965). The nucleic acids and cellular differentiation in the root apex ofPisum sativum. I. A fluorometric method for the determination of nucleic acids in root cells.J. exp. Bot. 16, 423–32.
Chen, H-R. (1966). Gallocyanin chrome-alum stain for plant nucleic acids.Pl. Cell Physiol. 7, 489–91.
Deitch, A. D. (1964). A method for the cytophotometric estimation of nucleic acids using methylene blue.J. Histochem. Cytochem. 12, 451–61.
Evans, P. K. (1967). Studies on cell division during early callus development in tissue isolated from Jerusalem artichoke tubers. Ph.D. Thesis, University of Edinburgh.
Flax, M. H. &Himes, M. H. (1952). Microspectrophotometric analysis of metachromatic staining of nucleic acids.Physiol. Zoöl. 25, 297–311.
Jensen, W. A. (1962).Botanical Histochemistry, San Francisco: Freeman.
Kiefer, G., Kiefer, R. &Sandritter, W. (1967). Cytophotometric determination of nucleic acids in UV-light and after gallocyanin chrome-alum staining.Expl Cell Res. 45, 247–9.
Kupila, S., Bryan, A. M. &Stern, H. (1961). Extractability of DNA and its determination in tissues of higher plants.Pl. Physiol. Wash. 36, 212–15.
Loening, U. E. (1967). The fractionation of high molecular weight ribonucleic acid by polyacrylamide gel electrophoresis.Biochem. J. 102, 251–7.
loening, u. e. (1968). Personal communication.
Lyndon, R. F. (1968). Changes in volume and cell number in the different regions of the shoot apex ofPisum during a single plastochron.Ann. Bot. 32, 371–90.
Mcleish, J. (1963). Quantitative relationships between deoxyribonucleic acid and ribonucleic acid in isolated plant nuclei.Proc. R. Soc., Ser. B 158, 261–78.
Mitchell, J. P. (1968). The pattern of protein accumulation in relation to DNA replication in Jerusalem artichoke callus cultures.Ann. Bot. 32, 315–26.
mitchell, j. p. (1969). RNA accumulation in relation to DNA and protein accumulation in Jerusalem artichoke callus cultures.Ann. Bot. (In press.)
Miura, K.-I. (1967). Specificity in the structure of t-RNA. In:Progress in Nucleic Acid Research and Molecular Biology, Vol. 6. (ed. J. N. Davidson & W. E. Cohn). New York: Academic Press.
Pakkenberg, H. (1962). Gallocyanin chrome-alum staining. A quantitative evaluation.J. Histochem. Cytochem. 10, 367.
Pearse, A. G. E. (1960).Histochemistry; Theoretical and Applied. 2nd Ed. London: Churchill.
Richards, B. M. (1966). Cytochemistry of the nucleic acids.Protoplasmatologia V, part 3b. Vienna: Springer.
Sandritter, W., Diefenbach, H. &Kranz, F. (1954). Uber die quantitative Binding von Ribonucleinsaure mit Gallocyaninchromalaun.Experientia (Basel)10, 210–15.
Sandritter, W., Kiefer, G. &Rick, W. (1963). Uber die Stoichiometrie von Gallocyaninchromalaun mit Deoxyribonucleinsaure.Histochemie 3, 315–40.
Schmidt, G. &Thannhauser, S. J. (1945). A method for the determination of deoxyribonucleic acid, ribonucleic acid and phosphorprotein in animal tissue.J. biol. Chem. 161, 83–9.
Shires, T. K. (1967). Tests for nucleic acid selectivity of Einarson's gallocyanin chrome-alum stain on monolayers of strain L-929 mouse fibroblasts.Stain Technol. 42, 67–74.
Stenram, U. (1953). The specificity of the gallocyanin chrome-alum stain for nucleic acid as studied by the ribonuclease technique.Expl Cell Res. 4, 383–9.
Terner, J. Y. &Clark, G. (1960). Gallocyanin chrome-alum. I. Technique and specificity.Stain Technol. 35, 167–77.
den Tonkelaar, E. M. &van Duijn, P. (1964). Photographic colourimetry as a quantitative cytochemical method. II. Determination of relative amounts of DNA in cell nuclei.Histochemie 4, 10–15.
Woodard, J. W., Rasch, E. &Swift, H. (1961). Nucleic acid and protein metabolism during the mitotic cycle inVicia faba.J. biophys. biochem. Cytol. 9, 445–62.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Mitchell, J.P. Quantitative microspectrophotometry of RNA in plant tissue. Histochem J 1, 106–123 (1968). https://doi.org/10.1007/BF01002582
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF01002582