J. Japan. Soc. Hort. Sci. 43(4) : 443-448. 1975. Studies on the Coloration of Carnation Flowers III. The Effect of Light Quality on the Anthocyanin Formation in Detached Petals Susumu MAEKAWA Faculty of Agriculture, Kobe University, Nada-ku, Kobe Summary Effect of light quality was investigated on the anthocyanin formation in detached carnation petals cultured using 4% sucrose liquid medium in vitro for 18 days. The cultures were treated with artificial light in all cases, and the results obtained are summarized in the following. 1. When the detached petals were irradiated with various pure colored fluorescent lamps, anthocyanin formation in the petals was more stimulated in the irradiation with fluorescent lamps than in darkness, and its content was highest under either red or blue light, becoming gradually lower in the order of orange, yellow and green lights. 2. The irradiation of far-red light at either short or long period gave rise to a markedly increased content of anthocyanin as compared with darkness. 3. The petals showed the formation of anthocyanin at a higher level when irradiated not only with light from white fluorescent lamp but also with a mixed light from red and blue ones than when irradiated alone with red or blue fluorescent lamp. 4. As to the irradiation with ultraviolet ray, anthocyanin formation in the petals was suppressed by its shorter wavelength region, and not promoted even by its longer wavelength region. Introduction On the relation between light quality and anthocyanin formation of garden crops, it has been manifested through a series of experiments by SIEGELMAN et al. (3, 9, 10, 11) that the available wavelength of visible light for anthocyanin formation differs among various plants under high intensity condition and that photoreversible reaction for anthocyanin f ormation between far-red and red light is found in some plants under low light intensity condition. On the other hand, only a few detailed reports on the effects of ultraviolet ray on anthocyanin formation have been so far published(1, 7), in spite of investigations having been done for a long time. The present author has been sometimes informed from some carnation's growers that coloration of carnation flowers may be influ- Received for publication June 4, 1974. enced by the kinds of film material covering the structure. If this is true, it is due probably to transmission rates varying at different wavelength regions in addition to various degrees of light intensity. The present investigation was, thus, planned as a f oundamental one to solve analytically these practical problems. In this paper an account is described about the effects of light quality on anthocyanin formation using the detached carnation petals as in the previous paper. Meterials and Methods The material used is carnation plant, Dianthus caryophyllus L. culttvar `Coral' and the uncolored petals detached were dealt with for the experiments. In all experiments, they were cultured on glass wool containing 4% sucrose solution for 18 days, incubation temperature being regulated at 20 C. 443
444 JOURNAL OF THE JAPANESE SOCIETY FOR HORTICULTURAL SCIENCE Procedures of the petal culture have been described in detail in the previous paper (6). Anthocyanin in petals was extracted immediately from five petals of each plot with 10 ml of methanolic 0.2% hydrochloric acid, and the optical density of the crude extracts was measured with colorimeter at 510 nm. The amount of anthocyanin which was increased by light irradiation was evaluated in comparison with that in the dark-grown petals prepared as control. Radiant energies were controlled by changing the distance between lamp and petals or! and by winding an aluminium foil tape of 2cm width around each fluorescent lamp and were measured with a compensated thermopile (Kipp & Zonen) for ultraviolet lamps and with a spectroradiometer (ISCO) for other lamps. Details of light treatments will be described in each of the items in the results and discussion. Results and Discussion 1. Effect of irradiation with visible light. i) Irradiation with monochromatic light from pure colored fluorescent lamp. Five 20 W pure colored fluorescent lamps (Mitsubishi) - red, orange, yellow, green and blue - were used as light sources, and they had such wavelength distributions as shown in Figure 1. Light intensity was about 50,aW/cm2 in every case. The results obtained under various lights are given in Table 1. Significant differences in the anthocyanin content were recognized between the petals under red light and those under each of orange, yellow and green lights and likewise between blue light and green light. The petals irradiated with red or blue light formed large amount of anthocyanin. Although the petals under green light showed the lowest value in anthocyanin content, it being 0.468 in optical density, they formed more anthocyanin than the dark-grown petals with the value of 0.262. ii) Irradiation with far-red light. The light sources used were incandescent and white fluorescent lamps. Far-red light from the former was obtained by passing of two sheets of each of red and blue cellophanes and a 5 cm layer of water. The culture under full light through a transparent cellophane and the layer of water was prepared as light control. The procedures to provide light conditions of the latter were essentially the same as those in incandescent lamp. Light intensity was about 2, 000 lux under full light condition in either case. The results obtained under continuous irradiation with two different lamps are shown in Table 2. When the petals were irradiated with incandescent lamp, the formation of anthocyanin was highest under full light and was prominently enhanced by the irradiation of far-red Table 1. The effect of irradiation with lights from various pure colored fluorescent lamps on the anthocyanin formation of detached carnation petals Fig. 1. Wavelength distributions for the from various pure colored fluorescent lights lamps.
MAEKAWA : The coloration of carnation flowers 445 Table 2. The effect of incandescent and white fluorescent light filtered with red and blue cellophane filters on the anthocyanin formation of detached carnation petals. Table 3. The effect of single and alternate irradiations with red and far-red light at short period on the anthocyanin formation of detached carnation petals. light as compared with that in darkness. Under the irradiation of white fluorescent light, on the other hand, a significant difference was not found in anthocyanin formation between the petals irradiated using red and blue cellophane filters and the dark-grown petals. These seem to be due to different amount of energy in far-red light region between two lamps. In another experiment, a short period irradiation with red or far-red light, as shown in Table 3, was given to examine for the involvement of phytochrome pigment. The degree of anthocyanin formation under short and interrupted irradiation with far-red light alone was higher than in darkness, and moreover, under alternate irradiation with red and far-red lights, the reversible reaction for anthocyanin formation was not recognized. iii) Irradiation with mixed light. For comparing the effects of mixed and monochromatic lights on anthocyanin formation, the following light conditions were provided using white and pure colored fluorescent lamps : a) red light, b) blue light, c) a mixture of red and blue lights and d) white light, and their radiant energies were equally adjusted to about 50 u W /cm2. As shown in Table 4, the petals grown under mixed or white light had approximately 1.5 times as large amount of anthocyanin as the petals grown under red or blue monochromatic light, i. e. the optical density of the former, which exceeds that of dark grown Table 4. The effect of irradiation with monochromatic and mixed lights from red, blue and white fluorescent lamps on the anthocyanin formation of detached carnation petals. control, was about 0.450 as compared with about 0.300 of the latter. STICKLAND et al. (12) have studied the influence of light quality on anthocyanin synthesis in the cultures of dark-grown Haplopappus gracilis callus and pointed out that anthocyanins are formed at higher lever in the order of green, red, white and blue lights, though not either in darkness or under far-red light. SIEGELMAN et al. (3, 10, 11) have reported that the action maximum for anthocyanin formation under high light intensity is found at the red light region near 650 nm for apple
skin, at the blue light region of 470 nm for milo seedlings, at 690nm and near 450nm for red cabbage seedlings and at near 725, 620 and 450 nm for turnip seedlings, and that the phytochrome action in anthocyanin formation is exhibited under relatively low light intensity in red cabbage and milo seedlings. With respect to the phytochrome effect, similar results have been also obtained by PIATTELLI et al. (8) for amaranthin synthesis in Amaranthus tricolor and by BELLINI et al. (2) for anthocyanin synthesis in radish seedlings. Though a definite conclusion cannot be drawn from the present experiments using light of rather broad wavelength region from fluorescent lamps in comparison with light from spectrograph, it may be considered that the anthocyanin formation in detached carnation petals is stimulated at two or more light regions as seen in the action spectra of red cabbage and turnip seedlings. Based on the results of alternate irradiation of red and far-red lights, it is assumed that anthocyanin formation in carnation is not controlled by phytochrome pigment. As to mixed light, it has been known that the growth response varies in different plants and is influenced by the mixture of different light intensities in each light source (4, 5,13). No detailed reports, however, have been so far published on the effect of mixed light on the anthocyanin formation in petals of flowering plants. In view of the fact that the plants have evolved under sun light it is worth noting that white light or mixture of red and blue lights were effective for anthocyanin formation in detached carnation petals. 2. Effect o f irradiation with ultraviolet ray. Transmission spectra of the various films and wavelength distribution of the various ultraviolet lamps (National) used are summarized in Figure 2. i) Irradiation at far-ultraviolet region from GL-15 lamp (Max. 253.7nm). Polyethylene (PE) film (0.03mm thich) transmitting ultraviolet ray in a high degree and glass (3.0 mm thick) cutting off this region were used as covers of the petri-dishes. The detached petals were irradiated through these covers at Fig, 2. Transmission spectra of various filters and wavelength distributions for the ultraviolet rays from various lamps. PE : Polyethylene film. PVC : Polyvinylchloride film. Table 5. The effect of irradiation with ultraviolet rays from various lamps on the anthocyanin formation of detached carnation petals.
MAEKAWA : The colorati on of carnation flowers 447 a distance of 70cm from the lamp. The irradiated petals changed to brown on and after about the 5th day of irradiation and most of them withered at the end of culture. Anthocyanin was, therefore, formed in a little amount, showing the value of 0.014 in optical density as shown in Table 5, and its value was remarkably lower than 0.284 in darkness. When the far-ultraviolet region was cutted off by the glass, the anthocyanin content was considerably increased, showing 0.472 in optical density without any retarded growth of the petals. ii) Irradiation at middle-ultraviolet region from FL 20E lamp (Max. 305nm). Petridishes were covered with the same two filters as mentioned above and the detached petals were irradiated at distances of 40 and 70cm from the lamp. Table 5 notes the results obtained in this experiment. When the petals were set at 40cm distance and covered with the PE film, though the same damage as GL-15 lamp was not recognized on the petals, the irradiated surface of the petals exhibited mosaic-like red stripes in their red-colored parts and the anthocyanin content was nearly as low as in darkness. At a distance of 70cm, the retardation for anthocyanin formation in the petals was remarkably reduced. On the other hand, the petals under the light filtered with the glass produced large amount of anthocyanin at both 40 and 70cm distances. An additional experiment was conducted under a lower intensity of the ultraviolet ray (one-sixth of the case at 70cm distance) and 1000 lux of white fluorescent light. The anthocyanin formation was not promoted even at such a light condition as compared with the light excluding the ultraviolet ray, though the data are not presented in Table 5. iii) Irradiation at near-ultraviolet region from FL 20 BL-B and FL 20 BA-37 (Max. 352 and 370 nm). All petri-dishes were covered with 0.03mm PE film to prevent a contamination and then kept either under 0.1 mm polyvinylchloride (PVC) film transmitting large amount of this wavelength region or under 0.1 mm BONSET film transmitting small amount of it. The amounts of energy irradiated onto the detached petals through PVC and BONSET films were 82 and 32,uW/cm2 respectively under light from FL 20BL-B lamp and 117 and 78,ci W/cm2 respectively under light from FL 20BA-37 lamp. No significant difference between the PVC and BONSET films, as shown in Table 5, was recognized for anthocyanin formation under both FL 20 BL-B and FL 20 BA-37 lamps. As to the difference between two lamps, the petals under FL 20BA-37 lamp formed large amount of anthocyanin as compared with those under FL 20BL-B lamp. Such differences appear to depend on amount of radiant energy of visible light from each lamp transmitting through BONSET film. From the results obtained with ultraviolet ray, it can be seen that the shorter the wavelength, the greater the retardation both in petal growth and in anthocyanin formation in petals becomes. Moreover, even relatively long wavelength region, which is known as a stimulative one to anthocyanin formation in several plant (1, 7), had unexpectedly little or no effect in detached carnation petals. The results obtained here may not be applicable immediately to flower coloration in carnation plants grown in green house, because these were obtained in the petal cultures in vitro. For making the present results applicable practically, further experiments should be carried out on the pigmentation of intact carnation petals. Literatures Cited 1. ARTHUR, J. M. 1932. Red pigment production in apples by mean of artificial light sources. Contrib. Boyce. Thompson Inst. 4 : 1-18. 2. BELLINI, E., and M. MARTELLI. 1973. Anthocyanin synthesis in radish seedlings : Effect of continuous far red irradiation and phytochrome transformations. Z. Pflanzenphysiol. 70 : 12-21. 3. DOWNS, R. J., and H. W. SIEGELMAN. 1963. Photocontrol of arthocyanin synthesis in milo seedlings. Plant Physiol. 38 : 25-30. 4. HORIGUCHI, I. 1972. The effects of mixture of red and blue light on the growth of Cucu-
448 JOURNAL OF THE JAPANESE SOCIETY FOR HORTICULTURAL SCIENCE mber, Spanish-paprika and Radish plants. Environ. Control in Biol. 10: 12-17. 5. KLEIN, R. M., P. C. EDSALL, and A. C. GEN- TILE. 1965. Effect of near ultraviolet and green radiation on plant growth. Plant Physiol. 40: 903-906. 6. MAEKAWA, S. 1974. Studies on the coloration flowers. II. The effect of light quantity on the growth and the anthocyanin formation of detached petals. J. Japan. Soc. Hort. Sci. 42: 347-352. 7. MATSUMARU, K., T. KAMIHARA, and K. INADA. 1971. Effect of covering materials with different transmission properties on anthocyanin content of Eggplant. pericarp. Environ. Control in Biol. 9 : 91-97. 8. PIATTELLI, M., M. G. DENICOLA, and V. CASTROGIOVANNI. 1969. Photocontrol of amaranthin synthesis in Amaranthus tricolor. Phytochemistry. 8 : 731-736. 9. SIEGELMAN, H. W. 1964. Physiological studies on phenolic biosynthesis. Biochemistry of Phenolic Compounds (Ed. Harborne, J. B.) 437-456. Academic Press. London and New York. 10.., and S. B. HENDRICKS. 1957. Photocontrol of anthocyanin formation in turnip and red cabbage seedlings. Plant Physiol. 32 393-398. 11.., and. 1958. Photocontrol of anthocyanin synthesis in apple skin. Ibid. 33: 185-190. 12. STICKLAND, R. G., and N. SUNDERLAND. 1972. Rhotocontrol of growth, and of anthocyanin and chlorogenic acid production in cultured callus tissues of Haplopappus gracilis. Ann. Bot. 36:671-685. 13. TERABUN, M. 1970. Studies on the bulb formation in onion plants. V. Effect of mixed light of blue, red and far-red light on bulb formation. J. Japan. Soc. Hort. Sci~ 39 : 325-330.