Studies on "Bluing Effect" in the Petals of Red Rose
Hitoshi Yasuda et al.

Bot. Mag. Tokyo 83(985): 233-236 (July 25, 1970)
I. Some cytochemical observations on epidermal cells having a bluish tinge
Hitoshi Yasuda

Abstract
    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.

Cytologia 39: 107-112, 1974
II. Observation on the development of the tannin body in the upper epidermal cells of bluing petals
Hitoshi Yasuda

In a previous paper (Yasuda 1970), it was indicated that the bluing effect of red rose petals is mainly due to the blue spherule presented in the upper epidermal cells of fresh petals, and it was also suggested that some tannic substance is a basic component of this spherule. At the same time, a possibility was foreseen that the bluish color of this spherule may be exhibited essentially through a combination of the following three component: anthocyanin, tannic substance and iron. A question arises how the spherule is formed. As a first step in resolving this question, the present study was designed to make observations on the development of the blue spherule which will be called "tannin body" in this paper.

Jour. Fac. Sci., Shinshu Univ. 8(1): 91-94. (1973)
III. The histochemical detection of iron in the bluing petals of rose.
Hitoshi Yasuda

Abstract
    
The bluing petals of red rose cultivar, cl-Crimson Glory, were fixed with 10% neutral buffered formalin. In the paraffin sections, the region corresponding to the blue spherule present in the upper epidermal cells, was clearly stained blue with the Prussian blue reaction. The same regions showed a brownish red color of ferric oxide with incineration.
    These facts are suggestive of that iron exists within the blue spherule, making the previous possibility more likely.

Jour. Fac. Sci., Shinshu University 11(1): 41-46 (June 1976)
IV. Calcium in the blue spherical body.
Hitoshi Yasuda

Summary
    Both the localization of calcium within the blue spherical body presented in the upper epidermal cells, and the role of the element in this body were studied. The upper epidermis of the bluing petals of a rose cultivar, cl-Crimson Glory, was fixed with 10% neutral buffered formalin, and paraffin sections were prepared. The region corresponding to the central layer of the spherical body was clearly stained red with alizarin red sulfonate under pH 5.5. In the bluing petals treated with EDTA, the central layers of the bodies were not found and the body looked somewhat ring-like. These results show that the calcium localizes within the central body, (a) making a salt or being weakly bound with a tannic substance within the body, and (b) playing a role in making the layer rather rigid.

Jour. Fac. Sci., Shinshu University 13(1): 79-86 (June 1978)
V. A Survey of the Various Bluing Types
Hitoshi Yasuda and M. Kikuchi

Abstract
    One hundred and forty five rose cultivars, including old roses, were used to determine the various bluing patterns of red rose petals. The upper epidermis of fresh petals, which had exhibited the bluing phenomenon, were peeled and examined microscopically.
    The bluing patterns observed were grouped into three types. The first was the cell sap-type where the central vacuole of upper epidermal cells was uniformly blue without any apparent blue structures. The second was a tannin body-type in which blue spherical tannin bodies appeared in the vacuoles. The third was a miscellaneous-type which included blue structures in the vacuole other than tannin bodies.
    Old roses derived from Rosa chinensis usually exhibited the cell sap-type of bluing while those derived from Rosa gallica had a tendency to exhibit a combination of the tannin body-miscellaneous bluing types. Combinations of two or even all three of the bluing types were found in some cultivars. With the long history of rose breeding, the various bluing types and their combination could have evolved through the segregation and recombination of the bluing factors, specifically when Rosa chinensis or Rosa gallica were used as parents.

Cytologia 47: 717-723 (1982)
Vl. Further observations on the development of blue color of the spherule
Hitoshi Yasuda

In the previous papers (Yasuda 1970, Yasuda et al. 1978), it was reported that the blue spherule appeared in the upper epidermal cells of petals could be adduced as one of the causes of bluing effects in red roses. The histochemical observations provided that the spherule was composed of tannic substances, anthocyanin, iron and calcium for the most part (Yasuda 1970, 1973, 1974, 1976). At the same time, it was suggested that the blue color of this spherule could be displayed by the co-operation of the following three components: tannic substances, anthocyanin, and iron.
    The present study was designed to gain a better understanding about this cooperation of three components.

Jour. Fac. Sci., Shinshu University 20(1): 15-20 (1985)
VII. Cytological observation on the epidermal cells of bluing petals incorporated into the miscellaneous-type.
Hitoshi Yasuda and Akio Yoneda

Summary
    The miscellaneous-type of bluing in rose petals (YASUDA and KIKUCHI, 1978) was re-examined using the following five cultivars: Cardinal de Richelieu, Blue Boy, Reine des Violettes, Shigyoku, and Samurai. The massive structures appearing in the epidermal cells of their petals showed striking similarity to the anthocyanophore-like structure in some rose petals which was reported by YASUDA in 1974b, 1976 and 1979 at three points: 1) "staining features both with ruthenium red and safranine", 2) "developmental process", 3) "behaviors against weak acids".
    From these results, it was proposed that the bluing pattern exhibited in such roses as five cultivars mentioned above should be separated from the miscellaneous-type as a new group of bluing, an anthocyanophore-type.

Introduction
    In the previous paper YASUDA et al (1978) grouped various bluing patterns of red rose petals into three types, on the basis of the survey which was performed on one hundred and forty five cultivars including some old roses.
    1. Cell sap-type: Central vacuole of the upper epidermal cells is uniformly blue without any apparent blue structures.
    2. Tannin body-type: The tannin bodies, which are spherical in shape and blue in color, appeared in the central vacuole of the upper epidermal cells.
    3. Miscellaneous-type: Some blue structures other than tannin bodies are recognized in the central vacuole of the upper epidermal cells.
In the same paper YASUDA et al explained developmental mechanism of cell sap-type, applying the theory that ASEN et al (1971) gave to the cause of bluing in Better Times rose. That is, the bluing of cell sap-type is due to the color action of the anthocyanin-flavonol co-pigment complex and to the effects of the decrease in acidity associated with aging of the petals.
    YASUDA in 1970 and 1974a presented an opinion that the formation of tannin body is one of the important mechanism of bluing effects in the petals of a rose cultivar, cl-Crimson Glory. Same opinion was adopted as general explanation about the cause of bluing effect of the tannin body-type by YASUDA et al in 1978.
    As indicated in the previous paper, multitudes of bluing patterns, in which the epidermal cells having some structures other than tannin body, are lumped together into miscellaneous-type. The evidence to be presented in this paper shows that a pattern, in which the massive structure similar to the anthocyanophore-like body as reported by YASUDA in 1976 and 1979 appears in the upper epidermal cells, separated from the miscellaneous-type of bluing, brought up for new one type, anthocyanophore-type.

Literature cited

  • Blank, F. 1946. The anthocyanin pigments of plants. Bot. Rev. 13: 241-317.
  • Weber, F. 1936. Vakuolenkontraktion und Anthicyanophoren in Pulmonaria Blütenzellen. Protoplasma 22: 100-107.
  • Yasuda, H. 1965. Studies on the expression of color tone in rose petals II. Changes of the epidermal structure in the velvety dark red petals as flower develops. Jour. Fac. Lib. Art. Sci. Shinshu Univ. 15: 15-21.
  • ——— 1970. Studies on "bluing effect" in the petals of red rose I. Some cytochemical observations on epidermal cells having a bluish tinge. Bot. Mag. Tokyo 83: 233-236.
  • ——— 1974. Studies on the insoluble states of anthocyanin in rose petals I. The insoluble state of anthocyanìn and its relationship to petal color, together with a new instance of this relationship. Jour. Fac. Sci. Shinshu Univ. 9: 63-69.
  • ——— 1974. Studies on "bluing effect" in the petals of red rose II. Observation on the development of the tannin body in the upper epidermal cells of bluing petals. Cytologia 39: 107-112.
  • ——— 1976. Studies in the insoluble states of anthocyanin in rose petals II. Histochemical observation on its basal substance. Cytologia 41: 487-492.