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Microscopical observations showed that two types of bluing exist in the petals of red rose, the one being due to the presence of bluish spherule in epidermal cells, and the other to the cell sap of bluish tinge. The present paper deals with some cytochemical tests for the bluing of the former type. The color of the spherule changed from blue into red upon treatment with hydrochloric acid, showing that the anthocyanin is its pigment component. In epidermal cells decolorized by 1% methanolic hydrochloric acid or Kaiser's solution, the spherule was stained brown with chromic acid, greenish blue with ferric chloride, blue with methylen blue or toluidine blue. These color reactions are indicative of a tannin-like ground substance in the spherule. This colorless substance may be stained red with cyanin, and blue with a mixture of cyanin and ferric salt. As a consequence, it becomes quite plausible that the blueness of the spherule may be brought about by synergistic effect of at least three components, cyanin, tannin-like substance and iron. The petals of certain cultivars of red rose sometimes become bluish at a later stage of flowering. This phenomenon has been called "bluing" by rose breeders. In this investigation, the term "bluing" will be used in this sense. Curreyl' has described that the bluing in red rose petals is caused by the lack of tannic matter in their tissue cells, whereas Weinstein2' has attributed the same phenomenon to a rise in pH value of the cell sap. However, a question remains still to be answered : How does the bluing proceed in red rose petals? Of course, it is necessary to study the mechanism of bluing phenomenon not only for floricultural purpose but also for physiological importance of the bluing of flower colors. The present paper describes the result of some cytochemical observations on the bluing of red rose petals. Materials and Methods The petals, in which the buling had been fully attained, were harvested from a red rose cultivar, cl-Crimson Glory, which had been grown outdoors. For the purpose of cytochemical observation, the following three kinds of microscopic specimens were prepared : 1) Cross sections-Fresh petals were cut at 30,i in thickness using a freezing microtome. * Department of Biology , Faculty of Science, Shinshu University, Matsumoto, Japan. 234 YASUDA, H. Vol. 83 2) Peeled epidermis-An upper epidermis was peeled off from the petals. 3) Decolorized epidermis-Peeled epidermis was soaked in 1°o methanolic hydrochloric acid for about four hours, and washed with water. In some cases, peeled epidermis was fixed with Kaiser's solution [mercuric chloride (10 g) + glacial acetic acid (3 ml) +distilled water (300 ml)]. By these treatments, anthocyanin was completely removed from tissue cells. The cytochemical tests examined are indicated in Tables 1-3 and Fig. 2. The test solutions shown in Table 3 were prepared by dissolving the reagents in the 1 : 1 mixture of 0.1 N phosphoric acid and 0.1 N malic acid, both being the acids inherent to the rose petals3'. As described in the previous paper4' , the pH of the cell sap of rose petal epidermis is near to 3, so that the pH of the above reagents was adjusted to this value with aqueous sodium hydroxide. Results and Discussion

Studies on ^|^ldquo;Bluing Effect^|^rdquo; in the Petals of Red Rose, I. Some Cytochemical Observations on Epidermal Cells Having a Bluish Tinge