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The physiology and genetics of the pigments of barley (Hordeum volgare L) Mullick, Dharam Bir 1959

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THE PHYSIOLOGY AND GENETICS OF THE PIGMENTS OF BARLEY (Hordeum vulgare L). II. STUDIES OF THE ANTHOCYANINS PIGMENTS by Dharam Bir Mullick B. Sc. (Hons.) Agr., Delhi University, 1954. A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCINCE IN AGRICULTURE in the Division of PLANT SCIENCE We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA Apr i l , 1959 ABSTRACT 1. The extraction and purification of anthocyanins from barley kernels presents problems not commonly encountered in other plant tissues. Special techniques using, for example, a pearler, sonic oscillator and alternate freezing and thaw-ing of extracts have aided in the production of reasonably complete and pure anthocyanin and anthocyanidin extracts from chaff, pericarp and perisperm-spermoderm. Partial extraction of anthocyanin from the very proteinaceous aleurone layer of the grain was made possible by removing the dilute acid-water soluble fraction from the pearled aleurone tissue followed by acid-alcohol extraction. Extraction of anthocyanins from plant tissues presents l i t t l e d i f f i c u l t y . 2. Paper chromatography has proven to be an excellent means of separating and partially characterizing anthocyanins and anthocyanidins in the barley soma and grain. Special techniques, such as the chromatostripe technique was developed, which greatly assisted the resolution of large quantities of anthocyanins. The Whatman No. 7 and No. 3 chromatographic paper greatly improved the resolution and reduced the t r a i l i n g in comparison to Whatman No. 1 and No. 3 nu&» commonly used in anthocyanin chromatography. Electrophoresis, too provided an excellent means for separating the yellow flavanoids from the anthocyanins. 3. A peeling technique was developed which greatly extended the possibilities of studying the anthocyanin develop-ment in the separate tissues of the caryopsis. This technique may also be of help to breeders and taxonomists in the accurate classification of barley. h. Exploratory studies were undertaken on the pattern of distribution of anthocyanins in barley kernels of one white, three blue, two purple and two black verieties. Two anthocya-nins "B" and "C" (probably cyanidin -3-glucoside) occured in one black (Gatami) and in the blues (Kwan, Montcalm and Trebi) and the purples (Gopal and Black Hulless). Additionally, of three anthocyanins found in the purple varieties, two "D" and "E" were common to both and one "F" was found in the variety, Gopal. Also found in the purple varieties was a poorly resol-ved group of "slow-moving" anthocyanins. The possible agly-cones of these anthocyanins have been discussed. 5. These studies which represent the pigments of mature kernels (as a whole) were followed by the investigations on anthocyanins and anthocyanidins present in the separate tissues of the caryopsis viz. awns, hulls, pericarp, perisperm-spermo-derm and aleurone, at different stages of growth. Broad conclusions have emerged from these studies. Some anthocya-nins, "fast-moving" under the conditions of chromatography of these investigations, hitherto, not detected in the mature tissues of the caryopsis, were present, in quantity, in the iv early stages of caryopsis development. In addition, the "slow-moving" anthocyanins, present in the mature kernels, could not he dectected in the early stages of caryopsis development. During later stages of caryopsis development, the "fast-moving" anthocyanins gradually disappeared and the "slow-moving appeared. In mature kernels of a l l the varieties studied, two antho-cyanidins, viz. delphinidin and cyanidin were present. Addi-tionally, pelargonidin was present in the kernels of the purple varieties. During the developing stages of the kernel, however, only two anthocyanidins viz. cyanidin and pelargonidin were definitely present. Delphinidin could not be recovered. Simi-l a r l y pelargonidin and cyanidin, hut not delphinidin, have been obtained from the maternal tissues such as the leaf sheath, awns, and pericarp of the barley plant. Delphinidin has been obtained only from the grain and may originate in the aleurone tissues or may come from leuco-anthocyanins; in colorless varieties, i t is certain that the delphinidin comes from leuco-compounds but there i s some reason to believe that in colored varieties some delphinidin may come from aleurone tissues. In the grain of blue varieties, delphinidin i s relatively more abundant than cyanidin but, in purple varieties, the reverse appears to be true. Pelargonidin appears only in the purple varieties. 7. Leuco-anthocyanins which yield, on hydrolysis, cyanidin and delphinidin occur in the white barleys, such as Golden V Pheasant, and in black barleys, such as Lion, which contain no anthocyanin. They may well occur with anthocyanins in the blue and purple barley varieties, but methods for their segre-gation have not been f u l l y worked out. 8. Ocular studies on anthocyanins showed that color appeared in the awn tips about the time of meiosis. Other characteristic changes occurred in the pigment content during the transition from vegetative to reproductive stages. Field observations', then, led to the belief that there i s some asso-ciation of sexuality and anthocyanin development in barley. In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of th i s thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It i s understood that copying or publication of th i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of PLANT SCIENCE The University of B r i t i s h Columbia, Vancouver 8, Canada. Date May 5« 1959 v i TABLE OF CONTENTS Page INTRODUCTION I THE REVIEW OF LITERATURE 3 SOME GENERAL COMMENTS ON THE BARLEY PLANT. 3 COLOR IN THE BARLEY PLANT. 4 THE HISTOLOGY OF COLOR IN BARLEY PLANT. 11 THE GENETICS OF THE BARLEY COLORS. 12 DISTRIBUTION OF ANTHOCYANINS IN NATURE. 17 CELLULAR LOCALIZATION OF ANTHOCYANINS. 18 OBSERVATIONS ON FLAVONOID CHEMISTRY WITH PARTICULAR REFERENCE TO ANTHOCYANINS. 22 SPECIAL PROPERTIES OF ANTHOCYANINS AND ANTHOC YANIDINS. 2? EXTRACTION AND PURIFICATION PROCEDURES. 30 IDENTIFICATION METHODS. 33 a. Color reactions. 33 b. Paper-chromatography. 33 c. Two-dimensional chromatography. 37 d. Spectrographic analysis. . 38 e. Column chromatography. 4l f. Enzymatic identification. 44 g. Other methods. 45" THE LEUCO-ANTHOCYANINS. 45 THE ORIGIN AND RELATIONSHIPS OF ANTHOCYANINS. 48 CHEMICAL THEORIES OF THE ORIGIN OF ANTHOCYANINS AND 53 AND OTHER FLAVONOIDS. RECENT APPROACHES TO THE "RECONSTRUCTIVE THEORY" OF BIOGENESIS. 66 a. Biochemical studies. 67 b. The influence of light on biogenesis. 77 v i i Page e. The influence of nutritional factors on biogenesis. 82 1. Sugars 82 2. Temperature 85 3. Menerals 86 h. Chemicals and growth substances. 91 d. The influence of antiboitics on biogenesis. 92 PHYSIOLOGICAL IMPORTANCE OF ANTHOCYANINS AND RELATED FLAVONOIDS. 92 Pharmacological activity. 92 Vitamin P activity. 94 Protective action of vitamin C. 95 Protective action of tannins on anthocyanins. 95 Fungistatic and bactericidal activity. 96 Anthocyanins as natural f i l t e r s . 96 Sex-hormonal activity. 97 As an index of the developmental phase of plants. 97 Aroma in cacao beans. 98 MELANINS 99 EXPERIMENTAL STUDIES WITH COLOR IN BARLEY. 103 MATERIALS 104 METHODS 106 A. EXTRACTION OF ANTHOCYANINS. 106 1. Extraction from mature kernels. 106 2. Extraction from aleurone layers and other f l o r a l tissues. 112 3. Extraction with acetone. 113 h. Extraction from plant materials. 113 B. METHODS OF VOLUME REDUCTION AND PURIFICATION. iXh C. EXTRACTION OF ANTHOCYANIDINS. 117 D. A METHOD TO DETERMINE THE LOCALIZATION AND ANALYSIS OF ANTHOCYANINS IN SEPARATE TISSUES OF THE CARYOPSIS. 118 E. PAPER CHROMATOGRAPHY. 120 1. The chromatostripe-an automatic banding v i i i Page technique for paper chromatography (see appendix III for details.) 120 2. Spotting. 121 3. Variations in Rf values. 121 k. Effect of different grades of paper, on resolution. 122 5. Effect of different chromatographic solvents. 123 F. ELECTROPHORETIC STUDIES WITH ANTHOCYANINS. 124 OBSERVATIONS AND RESULTS. 125 A. THE ANTHOCYANINS AND ANTHOCYANIDINS OF BARLEY KERNELS. 125 B. OCCULAR STUDIES ON COLOR IN THE DEVELOP-ING VEGETATIVE AND FLORAL STRUCTURES. C. OBSERVATIONS AND RESULTS FROM PEELING TISSUES AT THE HARD DOUGH STAGE OF THE GRAIN. 129 D. THE ANTHOCYANINS AND ANTHOCYANIDINS IN THE SEPARATE TISSUES OF THE CARYOPSIS. 130 a. The anthocyanins of Gopal in Awns, Hulls, perisperm-spermoderm tissues during early stages of development. 132 b. The anthocyanins of Gopal and Black Hulless in awns, hulls, pericarp and perisperm-spermoderm tissues during later stages of development. 134 c. The anthocyanidins of Gopal in awns, . hulls and perisperm-spermoderm tissues during early stages of development. d. The anthocyanidins of Gopal and Black Hulless in awns hulls pericarp, and perisperm-spermoderm tissues during later stages of development. 138 e. The anthocyanidins of Black Hulless aleurone. 140 f. Behaviour of anthocyanin in aqueous and alcoholic solvents. l4 l ix Page DISCUSSION 144 General comments on anthocyanin literature. 144 Extraction of anthocyanins from barley kernels. 146 Methods of volume reduction and purification. 147 Occular studies on color in the developing vegetative or f l o r a l structures. 148 The peeling technique. 150 Analysis of the anthocyanins and anthocyanidins in the separate tissues of the caryopsis. 150 SUMMARY AND CONCLUSIONS. 19+ APPENDICES I. COLOR REACTIONS OF ANTHOCYANINS. 157 II. ANTHOCYANINS AND ANTHOCYANIDINS OF THE BARLEY PERICARP AND ALEURONE TISSUES. 159 III. THE .CHROMATOSTRIPE - AN AUTOMATIC STRIPING TECHNIQUE FOR PAPER CHROMATOGRAPHY. 171 IV. ALPHABETICAL LIST OF THE COLOR GENES IN BARLEY. 182 LITERATURE CITED. I83 X Table of Contents - continued LIST OF TABLES TABLE PAGE I. Alphabetical l i s t of color genes in barley. 182 II. Some naturally occurring anthocyanins. 26 III. Important properties of the anthocyanidins occurr-ing in barley. 30 IV. Color reactions of some anthocyanidins. 3*+ V. Effl of anthocyanidins derived from leuco-antnocyanins. 39 VI. Spectral maxima of anthocyanidins. 41 VII. Oxidation state of flavonoid compounds. 51 VIII. List of varieties most commonly used. 104 IX. Alphabetical l i s t of varieties additionally used. 105 X. Observations on color development around transition from vegetative to reproductive stage. 127 XI. Observations on barley kernel colors in different varieties. 131 LIST OF ILLUSTRATIONS FIGURE PAGE 1. Semi-diagrammatic representation of the antho-cyanins in awns, hulls, pericarp and perisperm-spermoderm tissues of Gopal in early stages of development. 133 2. Semi-diagrammatic representation of the anthocyanins of the grain tissues of Gopal and Black Hulless barley in later stages of development. 13? 3. Semi-diagrammatic representation of the anthocya-nidins of Gopal barley grain tissues in early stages of development. 137 4. Semi-diagrammatic representation of the anthocyanidins of Gopal and Black Hulless grain tissues in later PAGE stages of development. 5. Showing the effect of extractants on the st a b i l i t y of • • the anthocyanins from the Gopal grain. 142 x i i ACKNOWLEDGMENTS The writer i s indebted to Dr. V. C. Brink of the Division of Plant Science for his deep interest in the problem and for his help, his constant encouragement and guidance in this study. The writer is grateful to the Brewing and Malting Research Institute, Winnipeg, Manitoba, especially to Dr. T. J. Harrison and Mr. M. G. Madden of that organization and to the National Research Council of Canada who made funds available for this study. In addition, the writer wishes to heartily thank Dr. J. J. R. Cambell, Division of Animal Science, for permitting the li b e r a l use of laboratory f a c i l i t i e s , such as the Sonic Oscillator and other equipment, Dr. J. C. Sawyer, Division of Plant Science for discussion and helpful suggestions, Dr. R. M. Acheson, Department of Biochemistry, Oxford, England, for helping in i n i t i a l stages of the work. The writer i s also deeply indebted to his wife for her extensive help in many ways. THE PHYSIOLOGY AND GENETICS OF THE PIGMENTS OF BARLEY (Hordeum vulgare L). II. STUDIES OF THE ANTHOCYANIN PIGMENTS. INTRODUCTION A biochemical basis of gene action has been sought for five decades. The pioneer studies of Wright, Haldane, Wheldale, Garrod and Goldschmidt in the f i r s t two decades of this century are well known. However, following their work, substantial progress in the quest for a better understanding of gene action did not occur u n t i l recent years when Beadle and Tatum initiated their studies with the fungus Neurospora  crassa. Very intensive work followed with many other micro-organisms. The results are impressive. In higher plants and animals comparable progress in the biochemistry of gene action has not been made despite the fact that rewards might be a better understanding of the basic physiological and biochemi-cal processes of these organisms. With a view to enlarging our knowledge of one series of gene controlled processes, viz. certain processes of pigmentation in a plant of considerable economic significance, barley, the studies elaborated in this essay were undertaken. The formal genetics of pigment inheritance in barley has been the subject of several investigations, most of -2-which have been reviewed by Smith (1951). However, the physiological and biochemical information associated with the formal genetics i s very meagre. To augment this information became the prime objective of this study. To guide i t s pro-gress were the now classical studies of Onslow, Scott-Moncrieff, Haldane and Lawrence of the John Innes Horticul-tural Institution, England and Robinsons of Oxford University, England. Although, the prime objective of this study i s to relate the genetics of certain pigments of barley to their physiology and biochemistry, which may seem abstruse to some, yet values may accrue from the work to the plant breeder and the maltster. Variation in the color expression of presumably stable geno-types has troubled barley breeders for many years. In a crop where color has been a hallmark of quality, i t is easy to see the problems color in s t a b i l i t y creates. Also maltsters have some evidence that the flavonoid pigments including the antho-cyanins are related in several ways to malt and beer quality, although the relationships are not yet determined to be beyond doubt the information obtained in this study could materially assist in their establishment. -3-• l . THE REVIEW OF LITERATURE SOME GENERAL COMMENTS ON THE BARLEY PLANT. Barley, the oldest of the common cereals has also the widest habitat range. It grows well beyond the Arctic Circle where, in summer, the s o i l thaws no more than a few inches below the surface and also on the tropical plains of India. High on Ethiopian mountain slopes, barley may ripen beside pools of water which freeze nightly and i t matures on the lower delta of the Nile where salt water is found at depths of a l i t t l e more than a foot. In Tibet i t occupies large acreages at an altitude of 15,000 feet and may grow occasionally even higher on adjacent Himalayan slopes. It is an important crop in nearly every agricultural region whether It be on the plains of the American Midwest, on the high plateaus of Bolivia and Peru, on the alkaline soils of Australia or the Saharan Oases. Barley, as has been mentioned, is a very old crop and has played an important role in the development of neolithic culture in the Old World. The problem of i t s origin interests archaeologists and anthropologists as i t does the agronomists and the geneticists. Barley, i t may be gathered, Is one of the most important plants from the stand-point of history of agriculture, and -4-industry. Its importance to the human race has grown steadily and i t has emerged as one of the leading cereals of the world. Barley is the subject of a number of books, monographs, and extensive reviews; famous amongst these are works by the "great barleyman" Harlan, H. V. (1957), Smith (195D, Weaver (1943), Aberg and Wiebe (1946, 1948), Derr (1911), Beaven (1947), Hunter (1952), and Takahashi (1955). Thousands of scholars have made contributions to the sum of knowledge about barley. Takahashi (1943) states that about 1300 articles had been published by 194l dealing with general morphology and classification, genetics, cytology and plant breeding, dis-eases (including the genetics of disease resistance), physi-ology and cultivation, livestock feeding, brewing, and chemical and physical properties of the grain. Smith (195D brought certain sections of the bibliography up to date u n t i l 1950, but directed his attention primarily to the works on cytology, genetics and breeding. So far, there has been no serious attempt to bring to-gether the relevant literature on colors and pigments of barley and, in particular, on the water-soluble anthocyanin pigments. A brief review appears appropriate before discussing the practi-cal studies on barley pigments. COLOR IN THE BARLEY PLANT. Several colors largely attributable to anthocyanins viz. -5-white, brown, black, violet, purple, red, pink and blue-grey may develop in various parts of barley plant. The pigment-ation is usually restricted in the case of barley to basal leaf sheath, upper leaf sheath, leaf t i p , node, stem, glume awn, lemma awn, h u l l , pericarp, aleurone and occasionally endo-sperm. The pigment may be present in a l l the organs at the same time or in a few organs at one time or other organs at a different time. (Harlan 191h, Lewicki 1929). Harlan (191*+) pointed out that differences in the quantity of pigment deposited from year to year may occur. Part of this may be due to conditions of growth and part to the conditions of ripening. The anthocyanins, like the melanins, are often formed during the later stages of growth. It may be that an abbreviation of the ripening period due to heat or drought would result in a reduction of pigment in the plant. Harlan (ibid) thought that minor phases of pigment development in the foliage and in the nerves of the glumes and awns, may be due to variations in the nutritional status of the plant, especially during heat waves and drought. Woodward (1953) reported that portions of the pericarp of certain varieties exposed to sun-light developed intense purple coloration. It i s now well-known that the development of anthocyanins in other species is also influenced considerably in certain organs of the plant by photoperiod, rate of respiration, minerals, metals, sugars, etc. As general features these w i l l be referred to again. As early as 1885, Kornlcke emphasized the value of color as a taxonomic character in cultivated barley. Beaven i n 1902, using color, differentiated a large number of a r t i f i c i a l hy-brids produced by Kornlcke and Karl Hansen. That the anthocya-nins were the colorants of barley was f i r s t demonstrated by Harlan in 1914. He also suggested that melanin-like compounds were present in black barley. Aufhammer (1933) suggested that anthocyanin formation can be used to differentiate winter and summer barleys. Takahashi et. a l . (1950) have discussed the importance of pigmentation of plant parts with regard to the classification and geographical distribution of barley v a r i -eties. Goldner (1923) and Kiessling (193D point out the impor-tance of pigments for brewing. Harlan (1914) states that maltsters have a preference for blue color which appears after steeping i.e. when the coverings have become transparent. Harris (1956) and McFarlane et. a l . (1955) found cyanidin and delphinidin and several unidentified anthocyanidins in barley-malt . Color, even though, i t is one of the most easily deter-mined characters of barley, i s sometimes a source of uncertain-ty in classification. Harlan (1914) found that the occurrence of pigments in certain cases and in certain tissues (presum-ably, Harlan, in general, meant kernel tissues) i s undoubtedly hereditary and i s transmitted unfailingly from generation to generation. In other cases (presumably, plant tissues other -7-than kernel) color appears intermittently and sporadically i n strains and tissues ordinarly free from pigments. This broad observation of Harlan regarding the developmental aspects of plant colors (as distinct from kernel colors ) has been amply confirmed recently by the systematic studies of Aberg and WIebe (19^6, 1948). a. Color in the Leaf Sheath and Auricles. The purple color in the leaf sheath at the base of the plant, in the upper leaf sheaths and auricles i s caused by presence of anthocyanins. Color seems more pronounced follow-ing periods of prologned cold weather than following periods of moderate temperature, and under some environmental condi-tions and during certain stages of plant development, the color may be more nearly red (Harlan 1957). Aberg et. a l . (1946) in using color in varietal classification appreciated the variations introduced by environment and state that no attempt should be made to distinguish between red and purple coloration. It i s possible merely to distinguish three groups on the basis of pigment quantity, namely 'absent1, 'present' and 'strong 1. The absent group i s well defined but the dividing line between the "present and the strong" groups i s indefinite. The character can be used advantageously in classification when the anthocyanin is completely absent or i s strongly expressed. -8-b. Color in the Nodes. Aberg et. a l . (1948) reported that anthocyanins appear in nodes only when they are exposed i.e. when not enclosed by the leaf-sheaths. A variety may develop anthocyanins in one geographical l o c a l i t y and not in another. This again empha-sizes the need of observations at several locations and in more than a single year i f the character is to be used generally. No conclusive results were obtained regarding i t s taxonomic value in experimental t r i a l s of 119 spring varieties and 69 winter varieties. c. Color in the Stem. The absence or presence of anthocyanins in the stem i s a character of limited taxonomic use. In certain varieties, the stem color i s evident under nearly a l l conditions but in most varieties i t occurs only under very favourable, dry, cool, sunny climates (Aberg et. a l . 1948). d. Color in the Glume Awns and Lemma Awns. The purple color in these organs is similar to those found in the leaf sheath and auricles and is undoubtedly due to anthocyanin pigment (Aberg et. a l . 1946). There is a close association of the anthocyanin color in the glume awns and lemma awns. (Aberg et. a l . 1948). Only occasionally do vari-eties completely lack anthocyanin. A moderate number of -9-varieties are strongly colored while the majority of the varieties are intermediate. The paucity of varieties in the 'color absent' group makes this easily recognised character of limited value. e. Color in the Hulls (Lemma and Palea or Chaff). Harlan (1914) stressed the value of grain pigmentation because of i t s lesser var iab i l i ty compared to plant colors and to the existence of well defined kernel color classes. He followed Mann's (1914) technique used in the identif icat ion and location of the pigments in cowpeas. The color in various ways may occur in the barley hul l s , pericarp, aleurone and occasionally in the starch endosperm. The resulting color patterns of the grain are, therefore, quite complicated. To add to the complexity, Harlan (1914) found that a heavy deposit of the melanin-like compound produces a masking "black" a l ight melanin brown coloration; and anthocyanin alone may confer, a violet coloration to the hul l s . Lewicki (1929) made a valuable contribution to the knowledge of pigments in chaff and grain by histological and biochemical studies. He recognised four ear (presumably he meant hull) colors; a) white or yellow; b) black without anthocyanin; c) black with much anthocyanin, some of which remained at maturity and d) black with much anthocyanin in immature stages only. Aberg et. a l . (1946) report that black is the only color which remains constant and has a high taxonomic value. The -10-anthocyanlns in lemma palea and pericarp on the other hand are transient and fade out at maturity or disappear i f the kernels are weathered excessively. Buckley (1930) observed color in the veins of certain variet ies . Aberg et. a l . (1948) concluded from a study of 284 varieties that the anthocyanin pigmentation In the nerves (veins), whenever present, was highly variable and has no taxonomic value. f. Color in the Pericarp and Aleurone. The color of caryopsis may be the combination or blend of colors in the aleurone and colors in the pericarp. In hulless forms, Harlan (1914) states the melanin-like compounds in the pericarp result in black kernels; the anthocyanins in a violet ones. Harlan (ibid) states the following observations on kernel colors: "the anthocyanin is always violet in hulls and pericarp indicating that the inert tissues are in acid condition, and always blue in the aleurone, indicating that resting condi-t ion of the protoplasm was alkaline. The acid condition of anthocyanins in pericarp superimposed upon the alkaline con-dit ion in aleurone layer gives the effect of a purple color, while a blue aleurone beneath a colorless pericarp i s blue-grey. White hulls over a blue aleurone cause the grain to appear bluish or bluish-grey. Black hulls over a blue aleu-rone give, of course, a black appearance." The aleurone layer consists of from two to four cel ls of varying thickness and depth depending upon the variety, climate and s o i l (Harlan 1914 j Sawicki 1950). This1"'layer starts d i f f er -entiating only when the embryo sac is entirely f i l l e d with the c e l l s , and anthocyanin development in i t takes place only a few days before maturity (Harlan 1920). The color of -11-aleurone is influenced to a very high degree by climatic conditions. It is very d i f f i c u l t to separate blue aleurone from white in kernels produced under humid conditions since the weathering affects the transparency of glumes (Harlan 1914). Under arid conditions, however, separations of blue and white colors are easily made and here i t i s even possible to distinguish three or four shades of blue (Aberg et. a l . 1948). The taxonomic value of aleurone color i s , therefore, restricted except for barley grown in dry areas where the taxonomic value is very high. THE HISTOLOGY OF COLOR TS BARLEY. The only works on the site of anthocyanin formation at the cellular level in barley i s that of Harlan (1914) and Faris (1956). Harlan mainly worked on the kernels, however, the differences of awn colors viz. purple and red in certain varieties, led him to histological investigations. In the spikes of certain selections, the awn was marked with two parallel stripes of red extending from i t s base to i t s t i p and in other selections the same stripes were deep purple. On examination he found that the difference in color was due to two bright-red stripes in the epidermis, running the f u l l length of awn, below which were two chlorophyll-bearing parenchymatous areas. As long as the chlorophyll was present, the color effect was deep purple but i t became light red as soon as the chlorophyll disappeared. -12-Farls (1956) following Mann's technique, studied exten-sively the distribution of pigments in various layers of barley kernels of different varieties. Owing to the presence of other pigments and similar compounds, the results obtained could not be very clear cut though certainly they were helpful to confirm some of his paper-chromatographic findings. THE GENETICS OF THE BARLEY COLORS. Barley has been examined most thoroughly genetically because i t Is diploid, because i t has a low chromosome number, i t has well-determined linkage groups, is almost completely s e l f - f e r t i l i z e d , i s relatively easily a r t i f i c i a l l y hybridized, has a wealth of easily classifiable hereditary characters, an abundance of spontaneous mutants and hundreds of a r t i f i c i a l l y induced mutants. As a consequence of these features and of the world-wide distribution and economic importance of this plant, hundreds of workers have contributed to a better under-standing of the science of heredity by selecting barley as an experimental plant. The formal genetics of barley pigments has received ample prominence from the time of Tschermak (1901), in the dawn of the mendelian era to the present. Color, apart from an undoub-ted inherent value in barley, has been considered as a most agreeable character to work with. It i s , therefore, not sur-prising that out of 1019 articles documented by Smith (-1951) that bear directly on the cytology and genetics of barley, over -13-100 are concerned with the color variants other than chloro-phyll. Despite the many studies on color inheritance in barley, the precise results of some workers have been shadowed in doubt due to inabi l i t y ofother workers to confirm them with similar materials in other environments. The classification of several color classes adopted by different workers lack unanimity. Explanation of the differences in results, proba-bly, l i e s in (a) the differences in genotypes used by di f f e r -ent workers, (b) the variations induced by environment, (c) the failure to adopt universal color standards. The broad color inheritance patterns seem well established and Smith (195D points out that an effort has been made by barley geneticists, particularly D. W. Robertson and G. A. Wiebe, to establish a uniform system of gene symbols through a Committee on nomenclature (Robertson et. a l . 1941, 1947). The approved gene symbols for color characters and their linkage groups (as extracted from Smith 1951, pp. 298 - 304) are given in table I, submitted as Appendix IV. The linkage groups of almost a l l the major color genes l i s t e d , have been worked out and most of these have been substantiated by a number of independent workers. Smith (195D and Faris (1956) have reviewed the genetics of barley color variants. Since no additional information appears to have been reported after the publication of Smith's review (ibid), the author, for economy, concludes discussion -14-by summarizing the salient features and behaviour of known color genes li s t e d in the table I, submitted as Appendix IV. CHAFF COLORS. Black Chaff (B; BPb; B g - II). Black glume color is dominant to non-black and segregates in a 3:1 ratio In many crosses (30)*. Black in the glume and pericarp appears to be conditioned by the same gene ( 2 ) . A cross between a purple-chaffed variety and black-chaffed vari-ty, in F 2, gave 12 black: 3 purple: 1 white indicating digenic inheritance with black dominant to purple and purple dominant to white ( 1 ) * . Orange Chaff (o - V). Orange chaff color is caused by a single gene which i s recessive to the factor for white chaff (4) . Purple Chaff (P - 1, P2 - P3 - ? ) . Purple chaff color is dominant to white (7)*; however, some believe, that this character is conditioned by a single gene pair, others report that two incompletely dominant gene pairs are involved, while s t i l l others find that two comple-mentary gene pairs are involved as has been clearly shown by Waddoups (1949). He observed a 3:1 ratio in the F 2 of a cross * No attempt w i l l be made to cite the bibliography, since that has been dealt with by Smith (195D, however, the number of publications bearing on the same issue w i l l be stated in parenthesis. -15-between two white-chaffed parents gave 9:7 ratio of purple to white in F 2 . Purple Veins in Lemma (P c - 1, P e - ?, Pf- ?). Purple veins in lemma are determined by three dominant gene pairs (1) . GRAM COLORS. Apparently, more grain colors have been studied than were described by Harlan (1914) and Lewicki (1929). Some of these can probably be accounted for by assuming that some investi-gators gave the same genotype different phenotypic names. Black Pericarp (Approved gene symbol not assigned). Black color in the hulls i s associated with black color i n the pericarp (2)*. However, sometimes colored pericarp may be enclosed in colorless hulls (1) . In crosses between black and white grained (pericarp) type, black was dominant to white and segregated i n 3:1 ratio in F 2 . Black was also dominant to red-pericarp (2)*. Certain crosses with black and white grain gave odd results which according to the authors were due to unfavourable environmental conditions for the development of pigment (2) . Several crosses between black and yellow or white seeded types, gave i n F 2 , black, purple, white-yellow seeds in varying proportions (2) . Smith (195D thinks that the results lack uniformity * see foot-note on page 14. -16-probably because a careful distinction was not always made between glume and pericarp colors and recommends the regular use of naked varieties for obtaining clear cut results. Blue Aleurone (Bl - IV5 B I 2 - III). Most of the reports indicate that blue aleurone is caused by a single dominant gene (8)*. Genes for black chaff color and bluish-black aleurone are not only different genes but are also unlinked (1)*. Xenia in white and blue seeded barley varieties has been clearly demonstrated by So and Imai (1918). The present concept, that two complementary factors are involved in color expression of the aleurone i s due to Myler and Stanford (1942) who In crosses between two white varieties, clearly demonstrated a 9:7 ratio in F2. Purple Pericarp (Approved gene symbol ambiguous). The purple color is dominant to white or yellow and i s conditioned by a single gene(3)*. Red Pericarp (Re - V, Re 2 - 1, 3 - ?, - 52 -Red has been reported to be dominant to white pericarp and caused by one gene in some crosses (4) , and two genes in others (1)*. Red i s recessive to black pericarp and is condi-tioned by two genes in some crosses and two additional comple-mentary factors that affect the expression of the two primary genes in other crosses (2 )* . The modern concept i s that of * see foot-note on page 14. ' ~~ -17-"complementary-factor hypothesis" put forward by Robertson. PLANT COLORS. Purple Straw (Pr - I). Purple straw i s mono-factorially determined. (1) . Red Stem (Rs - III). Red stem color has been reported to be determined by a single gene pair in one cross (1)*. Auricles (Approved gene symbol not assigned ). Presence and absence of red pigment in auricles i s usually determined by a single gene pair, though occasionally erratic behaviour has been reported, which may, in a l l proba-b i l i t y , be due to environment. (2)*. DISTRIBUTION OF ANTHOCYANINS IN NATURE Anthocyanins are known to occur in higher plants, conifers, mosses and ferns. For some time, they were thought to be absent in micro-organisms (Blank 19*+7), but they have now been detected in green alga the Chlamydomonas (Moewus 195D, and in fungi. * See foot-note on page 14. -18-Brown et. a l . (1952) have isolated numerous pigments from insects of the Aphididae group; they are^not as once thought (Palmer and Knight, 1929), flavone-like or anthocyanin-l i k e . Thompson (1926) and Ford (19W showed that certain yellow flavone pigments occurring in some plants occur in the wings of butter-flies which eat them. It would appear then that anthocyanins are exclusively, or nearly so, of the plant origin. CELLULAR LOCALIZATION OF ANTHOCYANINS. Cytologists have divided plant pigments into two groups; (a) the chymochromen i.e. water-soluble vacuole pigments and (b) the plasmochromen or plastid bound pigments (Reznik 1956). The anthocyanins belong to the chymochromen. A few attempts appear to have been made to trace the biogenesis of pigments cytologically and cyto-chemically. In barley this does not appear to have been done. Wada (1950) followed the biogenesis of chrysanthemin (one of the various types of anthocyanins) cytologically in withered petals of Oenothera and found that this anthocyanin developed in the c e l l when the cytoplasm was highly viscous from glucose, developed under conditions of very active carbon assimilation. Pardatscher (1953) observed that when the cells of the buds of Iris germanlca are strongly contracted (presumably, f a i r l y dehydrated), anthocyanin is often found in the form of drops. After these drops are formed, the c e l l dies. -19-In his studies on the site of carotenoid and anthocyanin synthesis in sweet potato, Kehr (1955) observed that by r e c i -procal grafting techniques, the anthocyanins and carotenoid are synthesized in the various tissues In situ, and that the synthesis of these pigments seems to be governed by the genetic factors found in the storage organs. In his studies on the special forms and types of localiza-tion of anthocyanins in the tissues of Splanum, Tradescantia. Atropa belladonna etc., Oztig (1956) observed that anthocyanins may be present as droplets, granules and crystals, in the c e l l -wall and c e l l nucleus. Matlack (193D observed crystallized anthocyanins in the juice of blood orange. With aid of such crystals Schorr (1935) was able to follow the plasma streaming in Allium cepa. P o l i t i s (19^7) reports two types of anthocyanin formation in the epidermal cells of the anthers. In Pyrus communis, Convolvulus tenuissimus, and Linum usitatissimum etc., the anthocyanins are elaborated in the only cyanoplast of the c e l l , whereas in other species they are elaborated in numerous elements which take the form of spherules or long filaments. He believes that a gene leaves the nucleus and becomes a cyanoplast (1st type) or after active division, the numerous elaborating elements (2nd type). In Chenopodium amarantlcolor t Forni (1953), demonstrated that microscopically, the color was localized in thin layer -20-composed of rounded fatty globules about 60u in diameter. The pigment was red in acid but yellow in alkaline solution and did not give Robinson's tests for anthocyanins. However, paper chromatography showed one anthocyanin, which on account of i t s yellow color, was expected to contain nitrogen. Forsyth et. a l . (1952 a), found that the anthocyanins appear in the vacuoles of cacao beans, from where they migrate into the other sections of the tissue and are retained by adsorption. K i l l i n g of the dehydrated tissue, by high or low temperature, prevents migration and enables alcholic extrac-tion of the pigment. Anthocyanins are commonly stored in the c e l l walls of higher and lower plants (Blank 1947). For details the reader is referred to the studies of Herzfelder (1921) on mosses, Bodmer (1927) and Schoch (1938) on pollen exines, and Molisch (1930) on the membranes of root, stems and leaves of higher plants. Guillermond (193D holds the opinion that anthocyanin formation in the plant cells :,has some relation to the chondriosomes. Reznik (1957) believes that the nitrogenous anthocyanins (betanine and flavocyanine ) are the specific pigments for the centromeres of Chenopodiaceae. Gertz (1906) has been reported to have done a comprehen-sive and fundamental work on the topographical distribution -21-of anthocyanins in many families of vegetable kingdom. Even today, his researches are considered highly useful for any one intending to work on the histology and physiology of anthocyanins. Mann's technique (1914) for determination of presence of anthocyanins which Harlan (1914) and Faris (1956) used on barley tissues has since been slightly modified by Molisch (1923) and Tunmann (1931). The technique is f a i r l y simple. The tissues containing anthocyanins change red in acid, violet to blue in ammonia vapours, and green in alkaline solutions, the green color resulting from blue anthocyanin and yellow flavones. Molisch (1905) brought about the crystallization of oxo-nium salts from tissues rich in anthocyanins under the cover-glass by covering the tissue with HC1 or CH3COOH and allowing i t to evaporate very slowly under a cover of the be l l jar. Chaze (1933 and 193*0 has discussed the development of anthocyanins in the aleurone layer of the grain of certain Gramineae during maturation. Fischer (1930) described the localization of anthocyanins in the guard cells of the stomata in Hyoscyamus niger. In roots of certain families, Molisch (1930) found that pigment i s exclusively restricted to the cellular walls of the epidermis and sub-epidermal cortex layers. -22-In stem of Monotrona sp., Funk (1937) found that epidermis and cortex are slightly colored whereas the greatest quantity of anthocyanins is stored in the phloem and parenchymatous cells surrounding i t . In his histology text (Mobius 1927) beautifully describes the relationship between the histological location of anthocya-nins and morphological appearance of leaves. In general the pigment Is distributed in epidermis and occasionally in hypo-derm and rarely in spongy parenchyma of the leaf. The histological data on flower coloration appears to be considerable and the interested reader is referred to Blank (19^7). During the last decade, no literature could be found on general anthocyanin histology and i t appears that very l i t t l e importance is being currently attached to this useful f i e l d . OBSERVATIONS ON FLAVONOID CHEMISTRY WITH PARTICULAR REFERENCE TO ANTHOCYANINS. The chemistry of the natural coloring-matter of higher plants has, received considerable attention over the past two decades. In flavonoid chemistry many fundamental advances have been made, particularly, in methods for detection, separation, recognition and structure determination; in addi-tion a number of new pigments have been discovered. The -23-review which follows is broad and of general value to one specifically interested in the physiology and genetics of barley pigments. No attempt has been made to touch on any but special features of pigment chemistry. Nor does the review attempt to note a l l papers relating to anthocyanin physiology; many of them are quite superficial. Accordingly the attempt rather has been made to review the literature which seems to contribute points In our more fundamental understanding of the role of anthocyanins and related pigments. Other comprehensive reviews are ALink (1938), Blank (19^7, 1958), Dyson (1950), Wawzonek (195D and Geissman (1952, 1955). The term "flavonoid" is used in this discussion to include a l l of the water-soluble pigments that possess structures based upon the C£ - C3 - C D carbon skeleton found in anthocyanins, flavones, chalcones, aurones etc. The numbering system used in the following discussion is as follows: Anthocyanins, Flavones etc. Chalcones. Aurones. -24-The parent rings of the whole family of flavonoid pigments are the 'pyran', believed to be much as follows: 0 <* -pyran J-Pyran oC-pyrone #-pyrone From X-pyran are "derived" the anthocyanins and from the pyrone structures, the flavones, chromones etc. The anthocyanins* belong to a group of glycosides, the sugar-free parts or aglycones of which are called anthocya-nidins. The fundamental parent substance of the entire group may be viewed as 2-phenylbenzopyrylium chloride and the various classes of anthocyanidins may be named from this structure by presence of additional hydroxyl and raethoxyl group as * The term "anthocyan" i s derived from the Greek roots signifying respectively "flower" and "blue". It was introduced by the botanist Marquart in 1835 to designate the blue pigments of flowers. Shortly thereafter the belief developed that the red and blue pigments were merely different forms of the same substance and that their different colors were due to variations in the character of the c e l l sap; consequently, the use of the term was extended to include a l l the soluble pigments of this group. When i t was learned that these pigments are always combined with sugars, and thus occur as glycosides, the ending "in" was attached. (Link 1938). indicated below:-cl 2- phenylbenzo pyrylium chloride 3,5,7 ,4'-tetrahy-droxy-3,5,7,3'4'-pentahy-droxy-3,5,7,3'4 '5*-hexa-hydroxy- • 3,5 ,7 ,4'-tetrahy-droxy-3--methoxy-3,5,7,4»5'-penta-hydroxy-3 methoxy-3,5,7,4'-tetrahy-droxy-3',5'-dimethoxy-3,5,4'-trihydroxy-7 , 3 ' ,5'-trimethoxy-5,7,4'-t r ihydroxy-5,7 ,4 '-trihydroxy-*.3-(R=H)-5,7 ,3 ' ,4 '-tetrahydroxy-Pelargonidin Cyanidin Delphinidin Peonidin Petunidin Malvidin Hirsutidin Gesneridin Apigeninidin Luteolinidin In anthocyanidins, the methylation is restricted in a l l but one known case (hirsutidin) to the 3'-and 5 ' - hydroxyl groups. Anthocyanidins containing 4 ' - methoxyal group are unknown. The anthocyanins appearing in nature are partly mono-, partly di-glycosides. The glycosidic attachment is either at the 3-position or 5-position or both. The principle members are listed in table II. A new class of anthocyanins has recently been found in - 2 6 -TABLE IT-SOME NATURALLY OCCURING ANTHOCYANINS • NAME AGLYCONE Callistephin Pelargonidin Fragarin Pelargonidin Gloxinin Pelargonidin Punicin Pelargonidin Pelargonin Pelargonidin Salvianin ) Pelargonidin Monardoein) Chrysanthemin) Asterin ) Cyanidin Sambucin ) Idaein Cyanidin Antirhinin ) Keracyanin ) Cyanidin Prunicyanin ) Mecocyanin Cyanidin Cyanin Cyanidin Oxycoccicyanin Peonidin (Cyanidin 13-methyl ether) Peonin Peonidin Vicin land II Delphinidin Delphinidin Gentianin Delphinidin Violanin Delphinidin Delphinin Delphinidin Ampelopsin Ampelopsidind^-Methyl-delphinidin) Myrtillin M y r t i l l i d i n (? Methyl-delphinidin) CEnin (Primulin) Malvidin. (Delphinidin 13, 15-dimethyl ether) Malvin Malvidin Hirsutin Hirsutidin (7, 13, 15-Tri-methyl delphinidin) Gesnerin Apigenidin (5, 7, 14-tri-hydroxy-2-phenylbenz pyrylium chloride). -27-SPlanum where two sugar residues are present in 3-position and one in 5-position. (Dodd 1957). A number of plants contain anthocyanins In ester combi-nation with organic acids. So far malonic, p-hydroxybenzoic, p-hydroxycinnamic and h-hydroxy, 3, 5, -dimethoxycinnamic acids have been obtained from the degradation of these acylated antho-cyanins. The acid radicals either can be in ester combination with one of the hydroxyl groups of anthocyanidins or can be attached to an hydroxyl group of a sugar component. A large number of plants contain nitrogenous anthocyanins usually known as Betanin, because i t was originally discovered in red beets. The structure of betanin has not been worked out so far. Recently however, Reznik (1957) has found that nitrogenous anthocyanin is specifically present in the centro-meres of Chenopodiaceae etc. has been discussed the role of nitrogenous anthocyanins with regard to general anthocyanin metabolism. SPECIAL PROPERTIES OF ANTHOCYANINS AND ANTHOCYANIDINS. Since these pigments usually occur i n the c e l l sap, they Salvianin or Monordaein O H Violanin. -28-are soluble In water and other hydroxylic solvents such as methanol, ethanol etc., and insoluble in such non-hydroxylic solvents as ether, benzene, chloroform etc. Because of their amphoteric nature, they can form true oxonium salts with acids; the salts are remarkably stable and have extraordinary crystallizing properties. Consequently, in the f i n a l stages of the isolation, the pigment is usually con-verted into i t s hydrochloric or picric acid salt. The cyanin, in vitro, is red in solutions of pH 3.0 or less, violet at pH 8 .5, and blue at pH 11.0. Suggestions have been made that the quadrivalent oxygen ring is responsible for these peculiarities of color in. anthocyanins. The red form of the cyanin is the oxonium salt. The violet form is represented by the color base, to which a quinoid structure, may be attributed. The conception that blue and violet forms of anthocyanins have a quinone similar structure, is supported from chemical consi-derations, especially by the fact that a l l anthocyanins and Oxonium salt (red) Color base (violet) No salt of the color base (blue). - 2 9 -anthocyanidins appearing i n nature show an unclosed hydroxyl group i n the 4'-position. The blue form of the cyanin i s pre-sent as the salt of the color base. Numerous anthocyanins and anthocyanidins change into a colorless modification, the pseudo-base, in very weakly acid, neutral and esp e c i a l l y a l k a l i n e solutions. The pseudo-base of cyanidin may have the following s t r u c t u r a l formula: Pseudo-base. The anthocyanins show increased blueness with increase i n the hydroxyl groups and change from 3 - to 3, 5-sugar type. Methylation of one or more hydroxyl groups on the other hand increases the redness of these pigments. Some of the anthocyanins and nitrogenous anthocyanins are reducing agents i n plant tissues as revealed recently by t h e i r interference i n the determination of vitamin C (ascorbic acid) reducing a b i l i t y (Somers et. a l . 1949, 1 9 5 D . The important properties of anthocyanidins are summed up in the table III. (extracted from Link (1938) pp. 1128). -30-TABLE III IMPORTANT PROPERTIES OF THE ANTHOCYANIDINS OCCURRING IN BARLEY Pelargonidin Cyanidin Delphinidin Color of aqueous solution Solubility of chloride in water Ferric chloride reaction Behavior toward Fehling's solu-t ion Color change in soda solution Behavior In aque-ous solution Red Violet red Bluish red Readily sol- Only slightly Very soluble uble soluble Not definite Intense blue Intense blue Reduces when warmed Violet then blue Color fades on standing Reduces in the cold Violet then blue Color dis-appears on heating Reduces in the cold Violet then blue Slow fading in the cold; when heated, rapid fading EXTRACTION AND PURIFICATION PROCEDURES. The relative quantities of flavonoids in a given plant sample may be considerably affected by the manner of collec-tion and the conditions of i t s storage owing to the enzymatic autolysis. This may result in the production of corresponding aglycones from the glycosides present within the c e l l and sub-sequent isolation of these hydrolytic artefacts may lead to - 3 1 -an erroneous description of the constitution of the plant. (Geissman 1955) reports that immediate and rapid drying of plant material usually preserves i t in a form substantially equivalent to fresh material; and thoroughly dried and properly stored material may be kept without change for extended periods of time. In general the flavonoid compounds and particularly the anthocyanins can be completely extracted with acidified ethyl and methyl alcohol and water; but i t is often advantageous, especially when dried material is used, to carry out a system-atic series of extractions with the use of three or more solvents of increasing polarity. Since most of the flavonoid glycosides are rather readily hydrolysed by acid, care must be taken, especially, when fresh material is used, to prevent the decomposition of glycosides during extraction with boiling solvents. Rapid exposure of plant material to boiling alcohol is effective in inactivating hydrolytic enzymes but the materials in the extract are s t i l l exposed to the danger of hydrolysis by accompanying plant acids. (Geissman 1 9 5 5 ) . Sadow (1953) employed the countercurrent distribution method (Craig and Craig 1950) for extracting large volumes of anthocyanin pigments. L i (1956) has described a quicker method for reducing the large volume of alcoholic extracts of anthocya-nins. The alcoholic extracts are transferred to aqueous solu-tion by shaking with equal parts of light petroleum ether and -32-and a few mis. of HC1. This step may reduce the volume by 7 0 % . Kazui T. et. a l . (1948) extracted anthocyanins from the sweet potato peelings with aqueous N a 2 C 0 3 ; the dye is taken up with acid clay and liberated by MeOH-HCl to obtain the oxonium salt of the dye. Reznik (1957) tried extraction with water, buffer solutions, Water: MeOH (1:1), Water: MeOH (1:2) and pure MeOH. He pre-ferred extraction with water and buffer mixtures. Thimann et. a l . (1949) have described detailed methods for quantitative extraction of anthocyanins from Spirodela oligorrhiza. Hayashi (1953) has been reported to have outlined a general scheme for analysis of anthocyanins and crude plant material. The crude extract of the anthocyanins may be somewhat purified in a number of ways (Robinson and Robinson 193D. 1. "Solutions of diglycosides can be repeatedly extracted with amyl.alcohol. Occasionally this i s sufficient. 2. The pigment is taken up in a mixure of amyl alcohol (2 parts) and acetophenone (1 part) containing picric acid. The organic layer i s separated, f i l t e r e d , diluted with ether and shaken with 1% hydrochloric acid. The aqueous solution is completely freed of picric acid by repeated extraction with ether. 3. Monoglycosides can be purified as in 2. by extraction with cyclohexanonepicric acid or ethyl acetate-picric acid followed by dilution of the organic phase with light petroleum ether and extraction of the pigment with 1% hydrochloric acid. The aqueous solution is then extracted with benzene, cyclohexa-none and again with benzene.*; Occasionally the process has to be repeated." -33-The selective precipitation of anthocyanins with aid of lead acetate has been employed. Sadow (1953) used one molar lead acetate solution to accomplish the precipitation of lead-anthocyanin complex. The anthocyanins were recovered as the chlorides, by treating the lead complex with 2% hydrogen chloride in methyl alcohol. Geissman (1955) has reviewed the use of lead acetate precipitations In other flavonoids. L i (1956) precipitated the MeOH 1% HC1 extract of antho-cyanins as lead salts and regenerated the anthocyanins by dissolving the precipitate in methanol containing dry HC1. This solution was treated with dry Et20 to precipitate the pigment as a red powder. IDENTIFICATION METHODS a) COLOR REACTION Prior to the modern methods of chromatographic and spect-rophometric analysis, the only means of identifying these pigments were the detailed color reactions (see Appendix I) developed by Robinson and Robinson (1931). A few of the color reactions of some anthocyanidins are, however, set out in table IV. b) PAPER CHROMATOGRAPHY. The use of filter-paper chromatography in analysis of T A B L E TV COLOR REACTIONS OF SOME ANTHOCYANIDINS Add 10% NaOH and shake i n a i r Extract with amyl alcohol; add sodium acetate and trace of f e r r i c chlo-ride to extract D i s t r i b u t i o n between 1% HC1. aq. and % p i c r i c acid in anisole/ethyl i s o -amyl ether (5:1) Di s t r i b u t i o n between 1% HC1. aq. and cyclohexanol/ toluene (1:5) Petunidin Delphinidin Cyanidin Pelargonidin Peonidin Malvidin Destroyed Destroyed Stable Stable Stable Stable Pure blue Pure blue Pure blue S l i g h t l y extracted Not extracted Some extracted Completely extracted Completely extracted Completely extracted Not extracted Not extracted Pale rose Extracted Extracted Faint blue -35-sap-soluble pigments has become wide-spread since Bate-Smith's description (1948) of i t s application to identification and separation of flavones and anthocyanins. The recognition of anthocyanins and other flavonoid pigments has proved f a i r l y easy using this simple technique. By reason of the widely differing solubilities of flavonoids and by reason of differences which can be created in partition characteristics by hydrolyzing" their glycosides, distinctive Rf values obtain. The color of subs-tances themselves in the visible and or ultra-violet spectrum and the colors produced by spray reagents on the chromatograms a l l help to make easy identifications. Inspite of these advan-tages, in unidimensional chromatography, reliance cannot be placed as to the absolute identification of the compounds under study owing to variations in the chromatographic paper, Rf values etc. As such the chromatographic evidence usually needs further confirmation through other channels. Procedural details pertinent to anthocyanins have been reviewed by Faris (1956). Hayashi (1952, 1953, 1957) has revi -ewed anthocyanin research in Japan with special reference to paper chromatography. Though originally, the choice of solvents has, more or less, been on empirical basis, nonetheless the following generalizations have been made in recent years. In general, the selection of the solvent used for the development of the chromatograms depends upon the solubility characteristics of the substances to be separated. -36-1. "WATER has the very useful property of moving glyco-sides but.leaving aglycones at or very near the origin (Roberts and Wood, 1953). While water gives relatively poor separations, so far as measurements of Rf are concerned, i t s a b i l i t y to bring about gross separations of groups of compound types and to remove by virtue of their rapid movement the very water-soluble sugars, makes i t a valuable solvent to be used in combi-nation with organic solvents. 2. BUT AN 0 L -WAT ER - OR GM IC ACID mixtures of varying proportions move most flavonoid compounds with an excellent range of Rf values and with good separations and definition of spots. The most widely used solvent of this type i s the organic phase of a n-butanol-water-acetic acid mixture in the propor-tions of 40:50: 10, respectively. Compounds containing numerous hydroxyl groups or sugar residues (myricetin 0.43, delphinin 0. 11) run slowly with this solvent; less highly hydroxylated or glycosylated compounds (isoquercitrin 0.68, callistephin 0.59) run more rapidly; and gglycones such as quercetin (0.74), luteo-l i n (0.88) and apigenin (0.92) run nearer to the front the fewer hydroxyl groups they contain. (Geissman 1955 a)." Over the past few years, a number of irrigation solvents for anthocyanin chromatography have been reported and some are discussed below:-L i (1956) used phenol : water (73:27) W/W for anthocyanins and n-BuOH : 2N HC1 (1:1) V/V for anthocyanidins. Axston (1955) used t-BuOH : AcOH :H20 (15:2:5), and m-cresol : AcOH : H2O (25:1:24) for anthocyanidins derived from leuco-anthocyanins. Hayashi et. a l . (1953, 1952) have been reported to have tabulated Rf values of twenty naturally occuring anthocyanins and their anthocyanidins, developed separately with the follow-ing solvents; iso-AmOH (36%) -HC1-H20; BuOH-AcOH-H20; iso-BuOH (36%) -HC1-H20 AcOH-AcOH-H20; AcOAm-AcOEt-H20; AcOEt-AcOH-H20 -37-PhOH-HgO; and a few other solvents. A newer attack, the use of moving pictures to study antho-cyanin chromatography has been launched by Dixmier (1956). Anthocyanoside separations, using AcOH and BuOH systems with and without water, are photographed in descending chromato-graphic apparatus over a period of hours by taking one picture per minute. He found that the solvent movement i s not constant; separations do not start at the beginning but rates of movement change as the composition of the solvent changes on the paper. One of the interesting findings is that i f the spots are placed further away from the source of irrigation solvent, the solvent does not*'take up low Rf value substances. Since the anthocya-nins are red in acid and blue in a l k a l i , he noted that with 15$ AcOH as irrigation solvent, the spots 15cm. from the so l -vent source, originally red due to acid vapours, turn blue on f i r s t contact with the solvent front indicating the presence of a l k a l i , possibly due to the degradation of the paper. The influence of evaporation and the effect of concentration of the material can also be studied by this method. Additional useful information derived from these studies was that the folds in the paper affect the solvent-front movement and that the spots can be kept from widening by placing them between two folds. c) TWO-DIMENSIONAL CHROMATOGRAPHY. "With any one irrigating solvent there w i l l be more than one compound with the same, or nearly the same, Rf value. -38 It is usually possible, however, to find a different solvent that wil l separate such groups. Such a pair of solvents are most effectively used in conjunction when mixtures are being studied. This may be done by preparing separate one-dimen-sional chromatograms with each of the solvents; or by prepar-ing a two-dimensional chromatogram using a single sheet of paper in which the solvents travel at right angles to each other. The preparation of individual one-dimensional chromatograms with two or more solvents offers a means of absolute indenti-fication in which the possiblity of error diminishes, the greater the number of solvents used. This method is most effective when the individual chromatograms are prepared with mixtures of the unknown substance and an. authentic sample of the compound it is suspected to be. The appearance of single well-defined spot on each of the several chromatograms, coupled with the observation of appropriately selected color reactions may be regarded as absolure inentification." (Geissman 1955). d)SPECTROGRAPHIC ANALYSIS. Although absorption spectra of anthocyanins and anthocyani-dins has been examined in the past, by Link (1938), Blank ( 1 9 4 7 ) , Robinson (1954) and Geissman (1955)? l i t t l e attempt was made to apply these data to the characterization of these pigments. Bate-Smith (1954) for the first time undertook studies on spec-tra l methods of characterization and reported that anthocyanidins have well defined peaks in the visible region, either in -39-ethanolic HC1 solution or when examined direct ly as a spot on a paper chromatogram by the method of Bradfield at. a l . (1953)• By the later method, the values are accurate to no more than Hr 2 mu. Values for six anthocyanidins are given below i n table V. TABLE V AnTHOCYANIDIN E m a x (mu) Pelargonidin 530 Cyanidin) ^ Peonidin) w Delphinidin) Petunidin ) 555 Malvidin ) The work of Bate-Smith (1954) and later that of by Roux (1957 a, b) has been mainly connected with anthocyanidins derived from plant leuco-anthocyanins. Geissman et. a l . (1953, 1955 b) found that whereas the addition of AICI3 solution to the solution of cyanidin and i t s glycosides causes a color change from red to blue and a shift in the absorption maxima of 16.35 mu. the pelargonidin deriva-tives are not affected. From these and subsequent studies on other anthocyanins, they concluded that aluminum chloride shifts the absorption maxima only in the case of pigments possessing orthodihydroxyl groups. Wolf (1956) studied the effect of pH on the absorption spectra of anthocyanins in red cabbage. The absorption spectra -40-of aqueous solutions were determine! at pH 1. 0 - 10.0. With the change in pH, the pigment varies in color from red to purple to blue to green to yellow. At pH 1.0 and 2.0 there was an absorption peak at 530 nip:. As the pH increased, maximal absorp-tion shifted to longer wave lengths. At pH 9.0, maximal absorp-tion occured at 615 mu. At s t i l l higher pH values, this peak was obliterated, and maximal absorption in the visible then occured below 420 mu. These spectral properties indicated that the red cabbage anthocyanins have great potential usefulness as a naturally occurring pH indicators which cover a very wide range. Harborne (1958) has reported a comprehensively on spectral methods of characterizing naturally occurring anthocyanins and anthocyanidins. The existing ^ methods of anthocyanin character-ization e.g. the well-known color and distribution tests (Robinson and Robinson 1931, 1932), although of value, provide only limited information about the glycosidic nature of antho-cyanins. The three most valuable features of Harborne's study are that the position of the attachement of sugar residues in the anthocyanin molecule may be deduced from the spectral data; secondly the spectral characteristic of a particular anthocya-nin are related to^the anthocyanidin from which i t is derived and thirdly the cyanidin, delphinidin and petunidin glycosides can be distinguished from the other anthocyanins by the use of aluminum chloride. The spectral data for both the common and rare naturally occurring anthocyanidins in the visible region are given in table VI. -41-TABLE VI SPECTRAL MAXIMA OF ANTHOCYANIDINS Amax. (mu) Pigment MeOH-HCl EtOH-HCl AICI3 shift A A Cmu) Hirsutidin 536 545 0 Malvidin 542 554 0 Petunidin 543 558 24 Delphinidin 546 557 23 Rosinidin 524 534 0 Peonidin 432 542 0 Cyanidin 53 5 545 18 Pelargonidin 520 530 0 Luteolinidin 493 503 52 Apigeninidin 476 483 0 Spectral maxima for the characterization of 30 anthocya-nins in the visible region are also reported but are not given here because of large space requirement. 3 -and 3*5 -diglyci-sides have very characteristic absorption properties in the 400-450 mu. region and other glycosides have similarly distinc-tive absorptions. Further more, measurements of the absorption spectra in the ultra-violet region of anthocyanins acylated with hydroxy-aromatic acids can be used to determine a valuable hydroxy-aromatic acid : pigment ratio. e)C0LUMN CHROMATOGRAPHY. The use of packed columns for the separation and isolation of anthocyanins has not been exploited extensively mainly for -42-lack of column f i l t e r s that w i l l give good separations of macro amounts of structurally diversified compounds. The survey of the literature of the last decade indicate that progress is being made in this f i e l d and that now the techni-que is useful in anthocyanin isolations. It is usually nece-ssary to supplement the separations carried out on a column by paper chromatographic procedures in order to establish the homogeneity, purity and identity of the fractions obtained from the columns. The combination of ion-exchange resins, fat-free cotton-cellulose columns (discussed below) and paper chromato-graphy promises to be very useful in the study of anthocyanins and related flavonoids. Spaeth and Rosenblatt (1950) used partition chromatography ( s i l i c i c acid) for separating synthetic anthocyanidins. The solvent was phenol : toluene : phosphoric acid. No anthocyanins appear to have been separated by this method so far. Ice et. a l . (195D on their preliminary studies of antho-cyanins, used "Amberlite IRC 50" in i t s "H" form and isolated about 30 grams of anthocyanins from aqueous extract of 1 kg. of berries. They feel the method is quite useful. Vil'yam et. a l . (1951) separated and characterised the coloring substance of wines, the 'enin' and 'enidin' by using a 170 mm. long and 8-10 mm. diameter glass -tube tightly packed with fat free cotton cellulose wool to the height of 140mm. One to three ml. wine samples were acidified by addition of a -43-drop of concentrated HC1, diluted 1:2 with d i s t i l l e d water, and transferred to the chromatographic column. 'Enin 1 was eluted from the column f i r s t with d i s t i l l e d H20 acidified by HC1 to pH 1-2; 'enidin' was eluted next with acidified (pH 1-2) 50% EtOH. The elution rate was 15-20 drops/min. The eluate containing enin was diluted 1:1 with EtOH and that containing enidin brought to certain volume by addition of 50$ EtOH; aliquots of the solutions were then taken for spectroscopic determination (Pulfrich spectrophotometer 530 mu f i l t e r s-53) with a pure preparation of enidin as a standard. To obtain the amount of enin the value found on the standard curve was multiplied by 1.44. The authors report that the method can be successfully used for the separation of the aglucon from any glucoside of any anthocyanins pigment. Endo (1954) separated the anthocyanins from Viola t r i c o l o r on a Whatman B. cellulose powder column using the organic phase of BuOH (36$) -HC1-H20 (5:1:4. V A ) as the eluting solvent. The cellulose powder column was packed b using the aqueous phase of the same solvent system. The column was then extruded, cut into bands, and the anthocyanins were eluted with MeOH. After the methanolic solutions were concentrated, they were rechromatographed as before. The anthocyanins violanin and karacyanin were isolated in pure form by use of the procedure described. L i . (1956) separated on a large scale two distinct crude components (Fraction A and B) of anthocyanins from sour cherries - 4 4 -in three hours with a flow rate of 6.5 ml/min. on 250g. coarse s i l i c i c acid column using the same solvent as employed by Endo (1954). Further resolution of these fractions was achi-eved by repeated chromatography on s i l i c i c acid columns. The fraction A. ultimately resolved i t s e l f into three distinct' bands which after elution and subsequent paper chromatography were identified as mecocyanin, chrysanthemin and cyanin (proba-bly). The fraction B. was identified as antirrhinin. f) ENZYMATIC IDENTIFICATIONS. Huang (1955) found that several crude fungal enzyme prepa-rations derived from Aspergllli exert a significant decoloriz-ing effect on extracts of pigments derived from berry f r u i t s . The over-all decolorization process involves an enzymic hydro-lys i s of the anthocyanins to anthocyanidins and sugar, and a spontaneous transformation of the aglucone into a colorless compound. Later on Huang (1956) observed that the fungal enzyme "CN; 558" possessed/glucosidase activity and this fact he u t i l i z e d in establishing the configuration of cyanidin -3-^monogluco-side which was in dispute. The enzyme hydrolyzes only the -glucoside of the anthocyanins and simultaneously converts the aglucone into colorless form. Sherrat and Harborne (1957) investigated the specificity of enzyme "CN 558" towards a wide range of anthocyanidin -45-glycoside and found that most simple anthocyanins were quickly hydrolysed, 3-rhamnoglucosides however were split more slowly and aeylated anthocyanins and 3:5 -trimonosides appeared to be resistant. g) OTHER METHODS. Zuman (1952) has reported on the polarographic behavior of anthocyanins in aqueous and alcoholic solutions at various pH values. This method appears to be useful for purposes of iden-t i f i c a t i o n s . On his paper-chromatographic studies of anthocyanin and other colored materials from the sap in relation to breeding for flower color, Werckmeister (1954) used the Horticultural Color Chart for defining the pigments. (Royal Horticultural Society Color Chart ? ). L i (1956) used the infra-red spectrophotometry and made i n i t i a l attempts to determine the infra-red absorption spectra of anthocyanins with Perkin-Elmer model 21 recording infra-red spectrophotometer, employing KBr pellet technique (Ingebrigtson and Smith 1954). Reznik (1957) successfully employed high and low tension ionophoresis to resolve the various components of nitrogenous anthocyanins. LEUC 0*AHTB0C YANIHS. The occurrence of leuco-anthocyanins in barley has been reported by Robinson (1933). The leuco-anthocyanins usually - 4 6 -appear in the plant in colorless form, and on hydrolysis with 2N HC1, generally yield cyanidin and delphinidin and, occasion-a l l y pelargonidin (Bate-Smith 1954). In some woody plants, they may appear light-brown in sectioned heartwood, but they change, characteristically, to purplish-red on exposure to air and light. H i l l i s (1956) has reported acetone-soluble leuco-anthocyanins. Robinson (1931, 1932, 1933) classified the leuco compounds into 3 classes : viz. "a) those that are insoluble in water and the usual orga-nic solvents, or which give only colloidal solutions. b) those readily soluble in water and which cannot be extracted from the solution by means of ethyl acetate c) those capable of extraction from aqueous solution by means of ethyl acetate. Class (b) probably consists of relatively simple glycoside whereas members of class (c) are sugar-free and should be regarded as leuco-anthocyanidins". Because of varying behaviour of these colorless substances no single constitutional formaula has been assigned them and, in a l l probability, a number of leueo-compounds with- different chemical constitutions occur in nature. In the literature whether leuco-anthocyanin, leuco-compound, pseudo-base, and colorless base are used interchangeably, i s not certain. For the purposes of this discussion, the "leuco-anthocya-nin" is defined as a colorless compound which, on hydrolysis with HC1, in a i r , yields colored anthocyanidins. Pigman (1953), Swain (1954) and Roux (1958) have reported work on the chemical constitution of the leuco-anthocyanins of -47-woody plants. Bauer, et. a l . (1954), after attempting a number of syntheses in vitro, hypothesized that natural leuco-anthocya-nin may he derived of 3, 4-dihydroxy-flavanan. Pigman (ibid) has reported a quantitative method for the estimation of leuco-anthocyanins present in woody plants. It has been widely adop-ted. That leuco-anthocyanins are related, probably in origin, to tannins has been amply demonstrated by the systematic chemical studies of Bate-Smith (1954). Kieser et. a l (1953) believe that the major component of pear tannin Is a complex leuco-anthocyanin of high molecular weight similar to cyanindin. Roux et. a l . (1958 a, 1958 b) report that complex leuco-antho-cyanins are present in condensed tannins and are probably their main precursors. H i l l i s (1955) brought about the conversion of leuco-anthocyanins to anthocyanidins, anthoxanthidins, and catechins in in vitro systems. The extent of conversion depend-ed upon the extent of oxidation. H i l l i s (1954) also made quantitative determinations of anthocyanidins obtained from leuco-anthocyanins present in certain species of Eucalyptus and Pinus and suggested that the leuco-anthocyanins, from which the anthocyanidins originated, are the precursors of the red color of leather tanned with the bark extracts of these ; trees. H i l l i s (1955) has made additionally excellent studies on the developmental aspects of leuco-anthocyanins. He found that s - 4 8 -the extreme t i p (particularly the colorless one) of the young leaves i.e. the areas of most active cell-division, contain the greatest amount of leuco-anthocyanin. Later (1956), he reported that the amount of leuco-anthocyanin decreased with increasing maturity of leaves. The amount of leuco-anthocyanin in the bark decreases during the growing season, but the most rapid decline occurs in the cambium as i t begins to differentiate. The systematic distribution of leuco-anthocyanins in plants has been surveyed by Bate-Smith (1953, 1954, 1956). He reports that the presence or absence of leuco-anthocyanins in the leaves of vascular plants is related to their systematic position. In monocotyledons, their occurrence is scattered but in dicotyledons the occurrence is general and is related to the woody habit. THE ORIGIN AND RELATIONSHIPS OF ANTHOCYANINS. As a result of the combined chemical and genetical studies on pigments, there has been a growing realization that a close relationship exists between anthocyanins and other naturally occuring acyanic C^ -C-^ -C^  flavonoids and C<$- and C^-C^- poly-phenolic compounds present in the plant. Geissman (1952, 1955a) has given an excellent comprehensive review of the known chemis-try of biogenesis. In his opinion, the following compounds are inter-related in the overall biogenesis of flavonoid compounds. A. Benzene derivatives, without side-chains. B. Cg-Ci-, C6-C 2-, C6-C3- compounds, including those which _ u 9 -may be regarded as being dimers of these (e.g. lignanes as (C5-C3) 2). C. Cg-Cn, where n ^ 3 . D. Chromones. E. Flavonoid compounds (C^-C^-Cg). F. Isoflavones (Cg-^-Cg). G. Xanthones and benzophenone derivatives (Cg-C-Cg). H. Stilbenes (C^-C^-Cg). I. Brazilin and hematoxylin (C5-C3- (C) -Cg) and variation of other kinds. (See Geissman 1952 pp. 80). It is appropriate to consider the chemical constitutions of some of these substances which appear to be involved in the biogenesis of anthocyanins i.e. naturally occurring substances which have either been recovered after the chemical degradation of anthocyanins and which have shown close systematic correla-tions with them, or which have been shown to be precursors of anthocyanins. NATURALLY OCCURING C6-COMPOUNDS. H O PYROCATECHOL (1,2-dihydroxybenzene) PHLOROGLUCINOL GUAIACOL (l-hydroxy-2- ) (1,3,5-trihy- ) (methoxy benzene.) (droxybenzene.) - 5 0 -HO Ch\0 R0< COOH \COoU VANILLIN PROTOCATECHUIC ACID (3-methoxy-4-hydr oxy) (3,4-d ihydroxy-) benzaldehyde. (benzoic acid. ) GALLIC ACID (3)4,5-trihydroxy-) (benzoic acid. ) H O - COOH >C0CH, H0< >COCH* p-HYDROXY BENZOIC (4-hydroxy-benzoic acid) ACEIOPHENOHE PHLORO-ACETO-PHENONE (2,k,6-t rihydroxy-) (acetophenone. ) NATURALLY OCCIJRING C6-C3- COMPOUNDS. >CH - CH. CooH H0< ,CH=CH.COOH CINNAMIC ACID CAFFEIC ACID O C M 3 H0< CH=CH. COOH FERULIC ACID BASIC STRUCTURE OF CHROMONES. -51-FLAVONOID COMPOUNDS. Geissman (1955 a) states that the range of structural variation found in the known compounds of the flavonoid type is associated primarily with variation in the oxidation level of the C^-portion of the molecule (see table VII). The range of oxidation level extends from the highly reduced catechin type ( A - CH2 -CHOH-CHOH- B) to the highly oxidized flovonol ( A - CO-CO-CHOH- B) ll 0 T A B L E VII Compound type Catechins Dihydrochalcones Chalcones Flavanones Flavanonols Flavones Anthocyanins Aurones Flavonols Oxidation state of C3 A - CHpCHOHCHOH - B ) A - C0-CH 2-CH 2- B (Iso: A - COCH- B CRo A - CO-CH-CH - B A - CO-CR2-CHOH - B A - CO-CHOH-CHOH - B A - C 0 C H 2 C 0 - B A - CH 2C0C0 - B A - C 0 C 0 C H 2 - B A - COCOCHOH - B -52-The great majority of the naturally-occurring flavonoid substances possess a phloroglucinol-derived r i n g A and a cate-chol derived ring B, as i n the following widely-distributed compounds. ' BASIC STRUCTURE OF FLAVONE AND FLAVONOL Apigenin Luteolin Kaempferol Quercitin Rutin Isorhamnetin H-' ,5,7-trihydroxy flavone 3 -,4',5,7-tetrahydroxy flavone 3 , 4 - ' , 5,7-tetrahydroxy flavonol 3,3 1 >4' ,5,7-pentahydroxy flavonol 3-rutinoside of q u e r c i t i n flavonol 3 '-methoxy-3, H-', 5,7-tetrahydroxy fl a v o n o l . BASIC STRUCTURE OF FLAVANONE BASIC STRUCTURE OF FLAVONOL sr0 II o BASIC STRUCTURE OF CHALC ONES BASIC STRUCTURE OF DIHYDRO-CHALC ONES -53-BASIC STRUCTURE OF ISOFLAVONES From the foregoing i t seems apparent that the aromatic rings and side-chains of numerous groupd of naturally occurring substances are related on the basis of certain characteristic structural features to various fragments of the C6(A)-C3-C,$(B) flavonoid skeleton (Geissman 1952). CHEMICAL THEORIES OF THE ORIGIN OF ANTHOCYANINS AND OTHER FLAVONOIDS. Many chemical theories have been put forward to explain the origin, in the plant, of the flavonoid skeleton. Most of these are highly conjectural. Those proposed prior to 1952 have been comprehensively reviewed by Geissman. Some chemists have tended towards the view, that i f the anthocyanins can be synthesized in vitro from certain of the Cg- and C6-C3- compounds earlier l i s t e d , then these compounds - 5 > + -might a l s o be i n v o l v e d i n i n v i v o s y n t h e s i s as w e l l . S i m i l a r l y , t h e v i e w i s h e l d t h a t c e r t a i n d e g r a d a t i o n compounds, f o u n d as a n t h o c y a n i n s a r e broke n down, r e p r e s e n t s t a g e s i n s y n t h e s i s . Thus i t was h e l d by t h e w e l l known c h e m i s t s , K a r r e r , W i l l s t a t t e r and t h e Robinsons t h a t p - h y d r o x y b e n z o i c a c i d , p r o t o e a t e c h u i c a c i d and g a l l i c a c i d , o b t a i n e d d u r i n g t h e d e g r a d a t i o n o f p e l a r -g o n i d i n , c y a n i d i n and d e l p h i n i d i n , r e s p e c t i v e l y , were t h e s m a l l e r s t r u c t u r a l u n i t s , t h e b u i l d i n g b l o c k s so t o speak, o f a n t h o c y a n i n m o l e c u l e . To su p p o r t t h e i r < ; v i e w t h e y a c h i e v e d t h e s y n t h e s i s o f a n t h o c y a n i n s t h r o u g h t h e c o n d e n s a t i o n o f t h e s e s m a l l e r s t r u c t u r a l c o n s t i t u e n t s . B l a n k (1947) and Geissman (1952) have commented more e x t e n s i v e l y on t h i s v i e w . A second t h e o r y d e a l s w i t h t h e i n t e r - c o n v e r t i b i l i t y of t h e C5-C3-C6 compounds. A number o f cases i n v o l v i n g i n t e r - c o n v e r t -i b i l i t y have been r e v i e w e d by Geissman (1952) and B l a n k (1947) . The s i m u l t a n e o u s o c c u r r e n c e o f a number o f f l a v o n o i d s i n n a t u r e and t h e i r I n t e r - c o n v e r t i b i l i t y , i n v i t r o T has been c o n -s i d e r e d as a p r o o f t h a t such t r a n s f o r m a t i o n s c a n account f o r t h e c o - e x i s t e n c e o f two or more r e l a t e d compounds i n a s i n g l e p l a n t o r r e l a t e d s p e c i e s . The c o n v e r s i o n o f a n t h o c y a n i n s t o c a t e c h i n s , and o f q u e r c i t i n t o a n t h o c y a n i n s and t h e s i m u l t a n e o u s p r e s e n c e o f t h e s e compounds i n n a t u r e has been made much o f by B l a n k (1947). R e c e n t l y , t h e r e l a t i o n s h i p between a n t h o c y a n i n s and f l a -v o n o l s w h i c h d i f f e r o n l y by one s t e p o f o x i d a t i o n , has been -55-discussed by Mirza and Robinson (1951) and Robinson ( 1 9 5 D . They point out that the conversion of anthocyanins or anthocya-nidins to flavonols has never been accomplished in the test-tube. The reverse process is recorded in one case only with certainty, viz. the reduction of quercitin to cyanidin by Willstatter. The same authors have reported an improved method for the conversion of kaempferol to pelargonidin chloride and quercitin to cyanidin chloride by the use of lithium aluminum hydride in a tetrahydrofuran solution. Bauer, Birch and H i l l i s (1954) brought about the reduction of rutin acetate to cyanidin-3-rhamnoglucoside with lithium aluminum hydride followed by treatment with cold dilute HC1. Using this method they demonstrated the reduction of other f l a -vanones. Kozlowski (1953? 1954) approached the conversion of flavonols in another way. He observed that when aqueous extracts of certain yellow or white flowers containing flavonols are mixed with anti-oxidants e.g. ascorbic acid or mannose sugars and exposed to active H, the flavonols became decolorised; on subsequent oxidation in a i r , the colorless flavonoid deria-tives are converted to anthocyanins. •Stansbury (1950), refluxed the catechol-type tannins (extracted from the red skins of peanut kernels) with alcoholic HC1, and obtained .a red pigment which behaved, in certain res-pects, like anthocyanin. -56-There are a number of other structurally related flavonoid compounds which exist together in nature. The mere coexistence of such compounds, their structural similarities and their speculative interconversions in vivo by certain plausible reac-tions, are the basis of a third theory. To cite the more recent cases, Stansbury (1950) and Bate-Smith (1954) showed a close relationship between tannins, leuco-anthocyanic-chromogen, phlobaphenes and anthocyanins. Bate-Smith (1954) after studying a large number of plant species reached the conclusion that the systematic distribution of leuco-antho-cyanins closely follows the recorded distribution of tannins in the botanical literature and that leuco-anthocyanins, in fact, have the properties of tannins and are probably the substances most commonly responsible for the reactions in plant tissues attributed to tannins. Bate-Smith (1956) using special methods for the separation and identification of phenolic compounds, conducted a systematic survey of the phenolic constituents of leaves of vascular plants. He found that caffeic acid i s most prominent in leaves closely followed by quercetln and then by kaempferol, p-coumarie acid, and ferulic acid. The overall picture for monocotyledons Is not very different from that for dicotyledons; however, they do seem to have fewer trihydroxy compounds, e.g. flavonols and caffeic acid and more sinapic acid and fe r u l i c acid. A striking find-ing is the high incidence of the hydroxy-cinnamic acids in the monocotyledons and flavonols in the dicotyledons. He emphasised -57-that particular phenolie compounds tend to be associated with certain systematic groups. Endo (1954) on his investigations of the inter-relationships between pigment constituents of 10 varieties of Viola t r i c o l o r concluded that pigments of the same kind tended to occur in groups. Forsyth (1952) demonstrated that anthocyanins may be pre-sent as free-color bases, free colorless pseudo-bases and as ethanol-insoluble color complexes with other polyphenols. The same author (1952 b, 1955 a) also reports the occurrence of 11 different polyphenolic compounds present with anthocyanins in cacao beans. An interesting case has been reported by Asen (1957) where-in he found that anthocyanins as well as other polyphenolic com-pounds in red and blue sepals of Hydrangea were identical. Alston (1955, 1958), Kieser (1953), Goodman (1954), Sastry (1952) and a number of other workers have pointed out similar close relationships of anthocyanins and other polyphenols. In a fourth theory, which may be called "Reconstructive Theory", emphasis is placed upon the processes and starting materials known to be available in the c e l l . A number of chemists in the past have ventured to explain the "reconstruc-tive" approach to plant biosynthesis. Robinson (see Blank 1947, pp. 267) put forward a highly presumptive hypothesis that the ring A of anthocyanin structure H° HYPOTHETICAL LUTEOLIN INTERMEDIATE OH ^ QUERETIN usually occurs as phloroglticinol and Ring B in the form of catechol. Both rings are built of hexoses and are bound together by a triose by means of aldol condensations, with for-mation of a hypothetical intermediate product, which can be converted to various end-products by oxidation, subsequent deny dration and ring closure as shown above. According to Frey-Wyssling, 1938 (see Geissman 1952 p. 170), the genesis of flavonoids could take place by the condensation of 2 ethylenes (from alanine) and 1 isoprene (from leucine) as shown on the next page. -59-CH<r = C f l i = . c « a o- - a H CO 0 H Inasmuch as the starting material, from which these complex Cg-C^-Cg substances are synthesised, is believed to be the "pool of carbon compounds", comprised of sugars, amino acids and compounds arising through the inter-related processes of photo-synthesis and glycolytic metabolism, these theories from the point of view of structural p l a u s i b i l i t i e s envisaged, appear very intersting yet probably many of the reactions postulated lack convincing biochemical and biological analogy. The chemi-cal approach, apparently, has not resulted in any definite theory so far. The biological trends on the line of "recons-tructive theory" have more recently been initiated by a few biochemists notably, Thimann, Moewus, Birch and Forsyth. Their -60-approaches are very impressive and shall be discussed under biochemical studies. A fifthe theory though somewhat, analogus to the "recons-tructive theory", is a l i t t l e at higher level inasmuch as i t deals with biogenesis at precursor level rather than the smaller structural level envisaged in the "reconstructive" theory. This has drawn support from the combined chemical and genetical studies. Some of the recent findings are discussed below:-It appears Dr. Geissman and his coworkers are the poineers in attempting a thorough systematic examination of the chemical relationships of both the cyanic and acyanic flavonoids of a genetically known population. From their earlier studies on carnations, (Geissman and Mehlquist 19^ 7) an indication was obtained that a genetic fac-tor which controls the degree of hydroxylation (oxidation) of anthocyanin, exercises the same kind of control upon the oxida-tion level of the structurally related flavone pigments found in the same flowers. Recent extension of this has added sup-port to this earlier finding and has shown that the factor (R) which determines whether the anthocyanin is a cyanidin (Rr, RR) or pelargonidin (rr) glycoside, also determines the degree of oxidation of the flavonols found in these flowers: Quercitin * Geissman T. A. and Hinreiner E. H., unpublished results (see Geissman et. a l . (1954). -61-and kaempferol are found in R flowers but the only flavonol that has been found in rr flowers is kaempferol (It may be recalled that in vitro, quercitin can be converted to cyanidin and kaempferol to pelargonidin). These observations indicated that the control imposed by the genetic factors, responsible for the structures of the anthocyanin pigments, is not an effect upon oxidation reactions occurring directly on an anthocyanin (or anthocyanidin) molecule, but i s , at some point, in the synthetic sequence prior to the completion of the f i n a l pigment molecule. It was made clear as a result of such studies that as more individual, genetically-controlled chemical differences can be described, more information w i l l be gained concerning the pat-tern of chemical transformations on the path , to pigment syn-thesis. Geissman, Jorgensen and Johnson (1954) therefore, under-took further chemical studies of the homozygous P,M,Y color types in Antirrhinum ma .jus, with respect to the aglycones of the flavone, flavonol, aurone and anthocyanin glycosides found to be presnt in this plant. The most conspicuous findings are that: a. Genetic factors which effect the hydroxylation pattern of one pigment type exert a corresponding effect upon other types; b. The factor P, which appears to control the presence or absence of anthocyanins, controls the presence or absence of - 6 2 -flavonols as well. This suggests that this factor controls a specific kind of oxidation of the C-^-fragment that joins the two 6-carbon rings (A and B), because the flavonol structure varies from anthocyanin structure only in the oxidation level of C^- portion of the molecule (see table VII). c. The M factor controls the oxidation of the C6(B)-ring in the flavones, flavonols and anthocyanidins : quercitin, apigenin luteolin and cyanidin are formed in the presence of M; kaempferol, apigenin and pelargonidin in i t s absence. The oxidation of the B ring of aurone pigment is not controlled by M : aureusidin (an aurone) glycosides are present in both M-and mm flowers. d. There is no observable effect of the Y - and yy factors upon the basic pattern of pigmentation. The phenotypic effect of the Y factor is mainly upon the concentration of the aurone pigment, yy flowers being yellow or orange, Y flowers having aurone pigmentation in restricted areas only. The qualitative data thus offer no clue as to the role of Y factor in the scheme of pigment biosyntheis. However, two striking observation are: 1. aurone pigment is confined to the flower petals while the other flavonoid pigments are present in the leaves and stems as well; and 2 . the aurone pigment concentration in the flower increases from the unopened flower buds to the f u l l y mature flowers, although no comparably conspicuous changes occur in the concentration of non-anthocyanin flavonoids, Y may be considered as a factor of flower color only in contrast to P and M which affect the structure of pig-ments found in other parts of the plant as well. -63-Jorgensen and Geissman (1955) continued the study to determine the concentration of anthocyanin and aurone pigment in the P, M, Y, color types and found that high anthocyanin concentration is related to low aurone concentration or to state i t otherwise, the increased dominant factors result in lower aurone production and increased recessive factors result in lower anthocyanin production. The other finding of interest is that the chemical consequence of heterozygosity is the alteration in the magnitude of the effects produced by the dominant alle l e s . A possible explanation for this action could be a reduction i n the rate of a reaction step, caused by a reduction in the a v a i l -a b i l i t y (or effective concentration) of the enzyme, controlling the step. Geissman and Harborne (1955 c) further undertook the anal-ysis of the albino flowers (-mm-nn) and found that they contain no pigments that can be recognized as belonging to the Cg-C^-Cg class of flavonoid pigments. They do, however, contain deriva-tives (probably esters) of both p-coumaric and caffeic acids. This discovery of what represents the C-^ -CgCB) portion of the two kinds of flavonoid structures (typified by apigenin and luteolin) of the colored genotypes suggests that the albino factor effects a blockage in the pigment synthesis at a point at which the Cg(A) and C^-CgtB) fragments are combined to give the carbon skeleton of the f i n a l flavonoid pigments, or blocks the elaboration of the Cg(A) precursor. -64-Coe ( 1 9 5 5 ) pointed out that a l l the known genetically acyanic kernels in maize accumulate substances (presumably other flavonoids or polyphenols) which can be converted to an-thocyanidins, and suggested that glycosidation was very close to the f i n a l step in anthocyanin production. However, Bockian et. ai. ( 1 9 5 5 ) , i t may be mentioned, found the following order of appearance of anthocyanin pigments in the maturing grapes, viz. malvidin diglucoside, malvidin monoglucoside, delphinidin glucoside, petunidin glucoside and free malvidin, contrary to the general belief, that in the developing fr u i t or flowers, an-thocyanins are formed at the expense of the corresponding anthocyanidins. Leuco-Compounds as the Possible Precursors of Flavonoids. In their studies on the color inheritance in Rudbeckia. Stephens and Blakeslee (1948) and Stephens (19^9) have presented convincing evidence to support the contention that the leuco-sub-stance present in young flower buds is a common precursor of both anthoxanthins and anthocyanin pigments in the flower petals and that i t i s convertible to either quercitln or cyanidin by a single genetically-controlled chemical step. It is briefly discussed below. The following flower cone types are known in R. hirta: Purple (BY -RY), Black yellow (by - RY), so called because florets turn black in strong a l k a l i and Red Yellow (BY * ry and by - ry), which turn red in a l k a l i . Chemical studies of pigments involved, have shown that purple cone contains an -65-anthocyanin pigment, cyanidin; Black Yellow a lueco-anthocyanin convertible to cyanidin in in vitro; and Red Yellow contains neither leuco-anthocyanin nor cyanidin. The flower cones of a l l types contain a yellow anthoxanthin pigment. Genetic and chemical findings are consistent with the hypothesis that the gene, RY, is responsible for the production of the leuco-antho-cyanin, and the gene, BY, for i t s further, conversion to cyanidin. The f i r s t step is blocked when RY is replaced by ry, and the 2nd step is blocked when BY is replaced by by,. Seyffert (1955) has furnished genetic evidence that leuco-anthocyanins are the possible precursors of anthocyanins and flavonols. Alston et. a l . (1955) during their investigation on the genetic control of anthocyanin pigmentation in Impatiens  balsamina observed that leuco-anthocyanin from stem yields cyanidin and from sepals, delphinidin. The buds (sepals and petals) collected before appearance of red pigments yielded cyanidin and delphinidin from leuco-anthocyanins. Further analysis showed that both the leuco-anthocyanins disappear before the purple flowers mature. These results point to the fact that those leuco-anthocyanins which do not correspond direct-ly with the ultimate pigment disappear as anthocyanins develop. Leuco-anthocyanins do not disappear during the maturation of white flowers, hence the disappearance of leuco-anthocyanins . and appearance of anthocyanins are the same genes. -66-Simmonds (1954) has reported a somewhat similar situation in developing buds of the inflorescence of bananas where non-methylated leuco-anthocyanins disappear as mehylated anthocya-nins appear. RECENT APPROACHES TO THE "RECONSTRUCTIVE THEORY" OF BIOGENESIS. The combined chemical and genetical studies, as we have seen, have clearly indicated that the biogenesis of anthocyanins is intimately related to the biogenesis of other flavonoids. Though these studies have clearly demonstrated the nature of gene-controlled processes involved, and that the anthocyanins and flavonoids have common precursors, they have shed no light on the sequence of metabolic steps and other points of constitu-tional details e.g. how the various states of oxidation are attained or whether flavonoid formation precedes anthocyanin formation during biogenesis. The chief d i f f i c u l t i e s have been the extreme complexity of both genetic factors and pigment mix-tures. The nomenclature lacks uniformity and some of the older work, for want of proper techniques, may have to be repeated with modern techniques to be of real value. The recent approaches to the "reconstructive theory" of biogenesis are discussed under the following headings; a. Biochemical studies. b. Influence of light on biogenesis. -67-c. Influence of n u t r i t i o n a l factors on biogenesis. 1. Relationship of sugars to biogenesis. 2. Relationship of temperature to biogenesis. 3. Relationship of minerals to biogenesis. 4. Relationship of chemicals and growth substances to biogenesis. d. Influence of a n t i b i o t i c s on biogenesis. a) BIOCHEMICAL STUDIES. A useful organism for biochemical studies i s the green alga Chlamydomonas, since i t uses the flavonoids, isorhamnetin and the anthocyanin, peonin as sex-hormones. B r i e f l y , a dioe-cious culture transferred from agar to water, i f illuminated, undergoes a series of changes, developing f l a g e l l a e , m o t i l i t y , and a b i l i t y to copulate. A l l these stages are hormone cont-r o l l e d , the hormones required f o r sex-determination and there-fore ultimately f o r copulation being, for the male, peonin, fo r the female, isorhamnetin (Moewus 1939-19515 Birch, Donovan and Moewus 1953). Raper (1952) i n his review on the "Chemical regulation of sexual processes i n Thallophytes", has summarised the several d i s t i n c t and sequential stages through which c e l l s of Chlamydomonas progress i n passing from the vegetative condi-tions to zygote formation (see pp. 479). The s p e c i f i c chemical compounds (isorhamnetin, peonin e t c . ) , which regulate the sexual processes, are the successive products of enzymatic degradation and / or synthetic reactions comprising two complex biochemical systems, one involving carotenoids and the other flavonols. -68-One of the three schemes, published by Moewus (1951) to account for the biogenesis of the flavonoids which i n i t i a t e and control the sexual determination and gametic copulation i s given below:-ISORHAMNETIfl^ (gynotermoner :irha J hydrolysis) PEONlNi (androtermone) PRECURSOR (probably a diglycoside) (of Isorhamnetin. ) -pae (reduction) — c --b X — a QUERCETIN —qu Scheme of the biosynthetic reactions leading to production of the various hormones involved i n the sexual reaction of Chlamydomonas (adapted from Moewus 1951 with s l i g h t modifications by the author). The s p e c i f i c biochemical reactions known to be controlled by single genes are indicated. The common precursor of isorhamnetin and peonin i s thought to be a diglycoside of i s o -rhamnet i n which i n the presence of the irha-controlled enzyme -69-(female) is hydrolysed to isorhamnetin, while in the presence of the pea enzyme (male) is reduced to peonin. These two enzymes exhibit maximal activity at different hydrogen ion concentrations, irha in acid medium and pae in alkaline medium, respectively. This difference in pH optima of the two enzymes would account for the sex-determining effects of acid and alka-line conditions in certain monoecious races in which cells grown or suspended in a medium at pH 9.5 produce only male gametes, while at pH 4.5 only female gametes. After carrying out a number of substitution experiments, Birch, Donovan and Moewus (ibid) have outlined the following scheme for the biogenesis of quercitin in Chlamydomonas. The steps involved in the transformation of quercitin to peonin have already been outlined-on page 68. The nature of "X" (.see page 70) i s , at present, unknown, but the authors think, i t behaves like a chalcone. Frey (1954) in his review on "the activity and localiza-tion of acid phosphatase in the vegetative parts of some angio-sperms and in some seeds", i t is reported, has discussed the role of this enzyme on the synthesis of anthocyanins. The studies of Mosiman (1947) indicate the presence of enzyme system in the cacao beans which hydrolyzes cyanidin -3-glycoside during fermentation. The catechins and tannins are oxidized to red-brown materials and a part of the anthocyanin -70-Precursors pha-Phenylalanine + ty-tyrosine dipha-3.4-d.ihydroxy phenylalanine dipro-3 .^--dihydroxyphenyl-propionic acid dicin-i 3:4-dihydroxy-cinnamic acid Precursor's - m o meso-inositol -phlo Phloroglucinol -x Quercitin Scheme of the biosynthetic reactions leading to the production of Quercitin (adapted from Birch. Donovan and Moewus, 1953 with slight modifications.;. becomes insoluble. Forsyth (1952 c) studied the changes in polyphenolic constituents of cacao cotyledons during commercial fermentation. .The main change, he observed, was the conversion -71-of simple cyanidin compounds to more complex leuco-cyanidins. Although oxidases are present, they do not act during fermen-tation due to the anaerobie condition prevailing in the coty-ledons. Forsyth (1953) reports that one of the anthocyanin pigments i s destroyed rapidly during fermentation while the other slowly. Most of the destruction i s enzymic; though the slowness of the reaction suggests that the enzyme may be pre-sent only in trace amounts. A more f r u i t f u l approach to unravel the metabolic proces-ses that take place in vivo is contained systematically in a series of six publications (1949-1958) by Dr. K.V. Thimann and coworkers.of Harvard University, and indeed this b r i l l i a n t work is a major step forward in our quest towards better understand-ing of the biogenesis of anthocyanins. Thimann and Edmondson (19^9) during their studies on the general nutritional conditions leading to anthocyanin formation in the duckweed Spirodela oligorrhiza. observed that amounts of copper above the optimum favors increased anthocyanin formation by inhibiting the growth. Further evidence for the mediation of Cu in anthocyanin formation was gathered by systematic in-hibitory studies (Edmondson and Thimann 1950). Cultures of Spirodela were raised in the presence of certain reagents like phenylthiocarbamide (PTC), which would combine with Cu thereby inhibiting i t s action. It was found that in presence of PTC, anthocyanin formation was inhibited by some 70$, while the -72-growth of the plant was not affected; when non-growing plants were floated on PTC solution, under illumination, similar inhi-bition was obtained. Addition of copper to PTC medium served, in part, to reverse the inhibition. Other copper combining reagents, salicylaldoxime and diethyldithiocarbamate, also inhibited pigment formation, but interfered with growth. The pigment in PTC-inhibited plants appeared to be changed qualita-tively, as judged by i t s absorption spectrum. PTC does not act through sugar depletion because sugars are actually accumulated within the inhibited plants. They concluded that PTC acts by combining with copper already within the plant c e l l s , and hence that copper, probably, in the form of a copper-containing enzyme, must participate in the formation of anthocyanin pigments. Further studies indicated that this enzyme is possibly identical with the polyphenol oxidase. (Polyphenol oxidases are copper proteins and convert o- and p-polyhydroxyphenols to corresponding quinones. Several polyphenol oxidases have been reported; some of them are highly specific for catechols and pyrogallols). They adduced two pieces of evidence from this. In the f i r s t place a number of phenolic compounds which can be oxidised inhibit pigment production, because tyrosine 10 M, _5 or catechol 2 x 10 M, reduces the amount of pigment formed by -U-non-nutrient cultures to 50% of that of the control. At 10 M, no pigment is formed at a l l and a black precipitate appears i n -dicating the probable fate of these compounds and suggests that in the formation of anthocyanins, an oxidation of phenolic -73-groups takes place which may be prevented by supplying alter-native substrates. In the second place, i t was realised that PTC which inhi -• bits anthocyanin formation i s also known to inhibit tyrosinase and thyroxin. In view of the obvious relationship between thyroxin formation and tyrosinase, It was considered worth-while to survey the inhibiting properties of other known antithyroids like methionine and other sulfur-containing compounds. Thimann and Radner (1955 a) found that most potent inhibitors of antho-cyanin formation were ethionine, methionine, sulpha-diazine and 2-thiouracil but that the inhibition cannot be ascribed to sul-fhydral group or the sulfur atom of these compounds. Though many other known antithyroid compounds inhibit pigment forma-tion, yet there is no correlation between the antithyroid and antianthocyanin activity. However, among these substances, a case of special interest was provided by 2-thiouracil. Not only is i t of high activity against anthocyanin formation, and one of the most potent of antithyroids, but i t has also been shown to inhibit melanin formation in the tyrosinase-tyrosine system (Paschkis et. a l . 1944). Since there is no correlation between antithyroid and anti-anthocyanin activity, Thimann et. a l . (1955 b), thought that the inhibiting action of thiouracil must rest on some other property. For this there are at least two po s s i b i l i t i e s . In the f i r s t place agents which combine with copper are known to inhibit anthocyanin formation by interfering with a copper--7h-containing enzyme. Since thiouracil is a copper-chelator, i t could act by inhibiting the copper-enzyme system. In the second place, thiouracil is known to inhibit the bacterial growth and metabolism, and the synthesis of tobacco mosaic virus and since uracil (one of the pyrimidines) reverses the inhibition, i t was deduced that thiouracil might act by interfering with pyrimidine metabolism. These suggestions led Thimann and Radner (1955 b) to inves-tigate whether thiouracil, (which is the only agent known to possess both goitrogenic and antipyrimidine activity) inhibits anthocyanin formation because of i t s copper-combining property, or by functioning as an antimetabolite for pyrimidine, or both. In these studies they found that various compounds, which interfere with purine and pyrimidine metabolism, inhibit antho-cyanin formation. These include (in order of increasing effect-iveness) benzimidazole, 2, 6-diaminopurine, quinine, azaadenine and azaguanine, the last-named compound being 650 times stron-ger as an inhibitor than t h i o r a c i l . With the exception of that due to benzimidazole, these inhibitions of pigment formation are partially or largely reversed by certain pyrimidines and purines. In the case of thiouracil, the inhibition is exerted in the light, but not in the dark. The inhibition in the light is completely reversed by copper ions or by uracil or thymine, gnd partially reversed by adenine and hypoxanthine. However, plants -75-preilluminated i n a t h i o u r a c i l solution produce no pigment i n the dark even i n the presence of u r a c i l . This evidence, together with that previously presented, supports the idea that there are at least two stages i n antho-cyanin production: "a. a l i g h t reaction i n which a copper enzyme participates and which probably involves the synthesis either of a nucleo-t i d e , or of one or more pyrimidines or purines, and b. a dark reaction u t i l i z i n g the products of t h i s l i g h t reaction for formation of anthocyanin." In seeking an explanation f o r these diverse i n h i b i t i o n s , they noted the observations of Goodwin et. a l . (1954) that methionine and some other amino acids i n h i b i t the synthesis of r i b o f l a v i n e and that r i b o f l a v i n e formation i n certain cases i s stimulated by purines. That r i b o f l a v i n e formation i s i n h i b i t e d by azaadenine and azaguanine, and that possibly a purine mole-cule may be incorporated lar g e l y unchanged into the r i b o f l a -vine molecule was reported by Brown et. a l . (1956) and MclTutt (1954). A l l these observations suggested that i t might be the formation of r i b o f l a v i n e which was being i n h i b i t e d i n Spirodela by these various agents, and therefore, that r i b o -f l a v i n e might function i n some way i n the production of anthocyanin. This formed the basis of t h e i r recent paper (Thimann and Radner 1958) wherein they found that the i n h i b i -t i o n of anthocyanin formation, brought about by methionine, ethionine, t h i o u r a c i l , azaguanine, etc. can be completely reversed by r i b o f l a v i n e . The amount of r i b o f l a v i n e needed for - 7 6 -reversal is approximately constant and independent of the concentration of inhibitor. Riboflavine alone has a small promoting effect on antho-cyanin formation, but only after the linear phase of pigment increase has been passed. When the plants are preilluminated, riboflavine increases anthocyanin production in a subsequent dark period; this action is enhanced by sucrose and diminished by the absence of C 0 2 . Yields in the dark almost as high as those in the light can thus be obtained. They deduced that riboflavine acts, not as a photoreceptor but as a dark catalyst to produce anthocyanin from sucrose or other precursors. It is calculated that eadh molecule of ribo-flavine leads to the formation of 30 to 60 molecules of antho-cyanin. The above deduction is confirmed by direct riboflavine determinations, which show that the riboflavine content of the plant varies parallel to their anthocyanin content. When aza'guanine inhibits the formation of anthocyanin (in the li g h t ) , riboflavine formation is also prevented, and the addition of riboflavine solution restores both the riboflavine and the anthocyanin content. Similarly, azaguanine and ethionine, which do not inhibit anthocyanin formation in the dark, do not affect riboflavine formation in the dark either. -77-The data suggest that the light reaction in the formation of anthocyanins is probably, at least in the main, the syn-thesis of riboflavine. This contention gains further support from the independent studies of Siegelman (1957 a, b) which w i l l be discussed under the influence of light on the formation of anthocyanins. Another encouraging approach to the resolution of meta-bolic processes in vivo, is that of Forsyth (1957) who investi-gated the oxidation of catechol in the presence of a polyphenol oxidase using manometric and paper chromatographic techniques. He found that under conditions of very low substrate concentra-tion and optimum uptake, the only intermediate which could be detected was a purplish-red pigment. b) THE INFLUENCE OF LIGHT ON BIOGENESIS. Earlier notions about the role of light have been reviewed by Blank (1947). The recent studies of Dr. Thimann and his colleagues, discussed above, aire indeed illuminating in regard to the influence of photoperiod and lay a foundation for a newer approach to the biogenetic aspect of anthocyanins. The other investigations over the last decade are discussed below:-Eddy and Mapson (1951) observed that low light intensities stimulate anthocyanin production in cress seedlings grown on water. The curve relating the amount of pigment produced to the amount of light received forms a rectangular hyperbola. He -78-clearly showed that the effect of light may manifest i t s e l f , after i t has been removed. The attention of the reader here is drawn to the findings of Thimann et. a l . (1955 b) who pointed out that, in the presence of light, a copper enzyme participated in the synthesis of a nucleotide and similar com-pound, which were ut i l i z e d in the dark period for the formation of anthocyanins. Siegelman (1957 a) found that red illumination accelerted anthocyanin development in early harvested apples stored at 32° F. He believes that a light-absorbing pigment is associ-ated with copper, forming a copper-flavoprotein, responsible for the development of anthocyanins. These independent find-ings of Dr. Siegelman, then further support the more recent studies conducted by Thimann et. a l . (1958) on the role of riboflavine in anthocyanin synthesis. That the synthesis of anthocyanin i s , at times, phot©peri-odically controlled has been well demonstrated by Gemisi (1952 b). The seedlings of Amaranthus caudatus. germinated in dark-ness and placed in light after 42 hours, developed as much anthocyanin as those kept in sunlight during germination. The anthocyanins were, however completely absent in seedlings, germinated in darkness for 72 hours. The light sensitive period seemed to vary according to season; i t s duration in July was 72 hours, against 48 hours in March. The duration of exposure required for anthocyanin.formation seemed to depend on -79-the age of seedlings. In July, a 2 minute exposure to sunlight was quite effective in producing anthocyanins in seedlings aged 48-51 hours; where as 120 minutes exposure was required for seedlings aged 27 hours and 69 hours, respectively. Light pre-sumably influences either the formation or activation of an enzyme catalyzing the reduction process, from flavonol antho-cyanin. The same author (1952 a) reports that, in absence of light, flavonols are formed instead of anthocyanins and he, thereby, suggests a biogenetic relationship between anthocyanins and flavonols. Siegelman (1957 b) showed that two distinct phases depend-ent on light are required for anthocyanin formation in apple hypodermis. The f i r s t phase is an induction period of about 20 hours, and the second is a period of linear formation of antho-cyanins at constant radiant energy. Pol i t i s (1947), in his studies on the genes elaborating anthocyanins in anthers of certain plants, found that antho-cyanin formation was closely related to light. Poton (1955) reported that reduction in light intensity rediiced both the anthocyanin pigmentation and growth. Thimann and Edmondson (19^9)5 showed that light is essential for continued pigment formation, and varies directly with the intensity, but that growth phenomenon is independent of pigment formation. This enabled pigment to accumulate when the rate of growth is slowed down. Withrow et. a l . (1953) have done a comprehensive work on the influence of visible and near infra-red radiant energy -80-on organ development and pigment synthesis i n corn and bean. A l l i r r a d i a t i o n treatments caused large increases i n antho-cyanins. The maximum synthesis occured under the f a r red i . e . same conditions which produced the maximum photomorphogenic e f f e c t s . They concluded that synthesis of the anthocyanins i s dependent on a photo-process which does not involve photo-synthesis, as shown by marked formation of anthocyanins i n the far red where photosynthesis, could not occur because no chloro-p h y l l was formed. Slabecka-Szweylowska (1955) observed that anthocyanin formation i n the tissues of V i t i s v i n i f e r a , grown i n l i g h t of dif f e r e n t wave-lengths, i n a medium with a high concentration of saccharose, greatly accelerated anthocyanin formation. He found that blue, v i o l e t and green l i g h t had the strongest, and red, the least influence on the anthocyanin synthesis. K l e i n et. a l . (1957), working under somewhat diff e r e n t conditions than the above, found that the increase i n antho-cyanin synthesis i s proportional to the log of irradiance. Red wave-lengths are more e f f e c t i v e than the blue and markedly more so than the far-red. Terrien, Truffaut and Carles (1957, pp. 86) i n t h e i r book on "Light, Vegetation and Chlorophyll" reported that the reddening of Mcintosh apples could be produced i n natural or colored l i g h t intercepted by o p t i c a l f i l t e r s , under the action of a l l the v i s i b l e radiations of \rave-length shorter than -81-6000 A. But the blue and violet rays were the most efficacious o and the optimum was situated around H-100 A i.e in the extreme violet. Similarly, anthocyanins of certain flowers, lost their color, proportionately, as the rays of short wave-length were suppressed. Similar studies on other plants clearly•demonstra-ted the importance of the action of ultra-violet, near the visible, in the formation of anthocyanins. The role of light on the synthesis of leuco-anthocyanins in Victoria plums was studied by H i l l i s (1957). The tabulated figures clearly illustrated the differences that existed in the content of methanol-soluble leuco-anthocyanin and total phenols between leaves taken from the sunny and shady sides of the tree; shading experiments showed that, the higher leuco-anthocyanin content on the sunny-side was due to more intense illumination. The dependence of leuco-anthocyanin synthesis, on illumination, was paralleled by lignin formation in the stone (endocarp) of the f r u i t ; stones on the shady-side showed slower hardening than those from the sunny side. Alston (1958) followed the leuco-anthocyanin synthesis in cotyledon and hypoeotyl of etiolated seedlings of Impatiens  balsamina upto the 13th day after germination. Leuco-antho-cyanins yielding pelargonidin and cyanidin began to appear in both regions at about the 3rd day. In hypocotyls, leuco-anthocyanin content increased steadily throughout the course of the experiment. The relative concentration of pelargonidin -82-and cyanidin derived from leuco-anthocyanins were essentially unchanged in a l l extracts and followed closely those of antho-cyanins formed upon exposure to light. Consideration of these implications suggested two possibilites: "a. The light might be indirectly involved i n the con-version of leuco-anthocyanins to anthocyanins or b. the light affected directly or indirectly the oxida-tion level of the C5-C3-units furnishing the B ring so that anthocyanin was formed". The author interpreted, "though i t seems the former explanation faces more obstacles, practically a l l evidence garnered to this time on the problem of leuco-anthocyanins-anthocyanin relationship is circumstantial and the question is s t i l l open". c) THE :ilFLUENCE OF NUTRITIONAL FACTORS ON BIOGENESIS. Probably, the most important and best documented conclu-sion, from literature beginning in 1897 (Thimann et. a l . 1949 p. 34), is the marked promotion of anthocyanins due to sugar feeding and sugar accumulation. Blank (1947) has examined the past investigations bearing on the role of sugars, nitrogen and minerals on anthocyanin formation. The recent developments in this direction are discussed below: 1. SUGARS. There is now a growing realization that anthocyanin formation and carbohydrate metabolism are somehow closely related, particularly so because of the glycosidic nature of -83-anthocyanin molecule. Thimann and Edmondson (1949) found, in Spirodela, that the addition of sucrose to the medium stimulated pigment production. Fructose and some other sugars were active but to a lesser extent; glucose was inactive. Sucrose reached i t s maximum effectiveness at a concentration of about 0.015 M. The different behaviour of sucrose and reducing sugars led Thimann, Edmondson and Radner (195D to further systematic studies connected with the relationship between sugar and pig-ment within the plant tissues; modification of the effects of sugars by growth; the effect of added glycolytic intermediates and the effect of cyclohexanol derivatives. They found that sucrose, glucose and fructose were about equally effective in promoting anthocyanin formation in non-growing cultures. In growing cultures, the anthocyanin formation was promoted by sucrose but not by glucose; the reason being that glucose was used-up for growth. Fructose was intermediate in both respects. Further, a number of treatments, which decreased or increased anthocyanin content, had parallel effects on the reducing sugar content. A graph of anthocyanin content plotted against re-ducing sugar content showed simple relationship. Phosphate apparently did not participate in the formation of anthocyanins; nontheless, i f the process did take place dir-ectly from the sugars, then, probably, the usual glycolytic pathway was not used, (since none of the-glycolytic interme-diates tried, gave rise to any anthocyanin). The author how-ever, did find some evidence for the participation of meso--84-inositol in the biogenesis of anthocyanins. The effect of other cyclohexanol derivatives (quinic and shikimic acids) appeared to be too small to be considered as intermediates. Eddy and Mapson (195D« as mentioned before, made compre-hensive studies of some factors affecting anthocyanin synthesis in cress seedling. They found that the normal anthocyanin accumulation of seedlings grown on water, in dark, could be increased by feeding 1$ solutions of glucose, fructose, sucrose, invert sugar, galactose, sorbose and arabinose. Their observations, in regards to the behaviour of glucose, are very different from those of Thimann et. a l . (1951). They found that whereas 1$ glucose caused a stimulation in the anthocyanin production of 50$ above normal, 2% glucose caused a stimulation of 150$ and 4$ glucose, a stimulation of 450$. They however, concluded that anthocyanin production was related more closely to total sugar content than to the con-tent of any one sugar. Poton and Goodman (1955) found that addition of sugars slightly retarded growth and greatly increased anthocyanin in Sphagnum. Blank (1951) found that the additions of fructose, dextrose or sucrose increased the anthocyanin formation in red cabbage. Paech and Eberhardt (1952) have reported that the increase of anthocyanins observed in germinating seeds of red cabbage, after feeding with sugar, could be further augmented by addition of J2>-indolylacetic acid. -85-Maleic-hydrazide sprayed plants produce abundant anthocya-nin coloration. A l l tomato plants treated with 2000 ppm of maleic hydrazide spray, formed abundant anthocyanins on the underside of their leaves within 10 days after treatment, though the branches developed later did not contain anthocya-nin (Greulach 1950). In corn, Nylor et. a l . (1950) observed that droplets of sugar appeared on the underside of leaves, eight days after the maleic-hydrazide spray and anthocyanins appeared later on. This contention has been confirmed by Craft (1950), Greulach (195D and many others, who believe the antho-cyanin formation to be due to sugar accumulation. The latter author has supported the view that maleic hydrazide causes physiological derangement and, specifically, affects the trans-location of sugars from leaves of treated plants. See Zukel (1957) for a literature summary on maleic hydrazide. 2. TEMPERATURE Stiles and Leach (1952), in their book on "Respiration in Plant pp. 27-8", have reported an interesting fact that at low temperature, sugars and organic acids tend to accumulate. Poton and Goodman (1955) observed that anthocyanin formation in Sphagnum, in fields situated in the South of England, was associated with the lowest temperatures and have pointed out that this may be due to growth retardation. De Capite (1955) has reported that anthocyanin formation within the c e l l i s associated both with the presence of sugar and the action of -86-low temperature. But this is operative only under aerobic condition; In anaerobiosis, however, even the combined action of cold and sugar proves Ineffective. The effect of temperature on anthocyanin formation in Mcintosh apples was studied by Uota (1950) by enclosing one limb of tree In four separate plastic chambers where the tem-perature was held at different levels. The tabulated results show that anthocyanin synthesis proceeds most rapidly in the cold i.e. at an average temperature of 4-6.5°F during night and 60.5°F during the day. Uota (1952) further reported that mean temperatures around 8l°F prevented synthesis of the red pigment while low temperatures increased the red color. There was a good correlation between night temperature and red color forma-tion; however, temperature did not affect the sugar content of the f r u i t . Joslyn and Peterson (1957) observed that in freshly harvested early onions, pigment formation occured i n macerated bulb tissue stored at a temperature range of 20°C to 50°C. Pigment formation was optimal at pH 3-3.5 but did not occur at pH levels below 2.8 or above 5.2. Upon storage at 0°C for a month, pigmentation occurred at a l l levels of pH. The red pig-ment formed was similar in general properties to the nitro-genous anthocyanin of red beets. 3. MINERALS. The earlier investigations on the relationship of - 8 7 -anthocyanins to minerals have been reviewed by Blank (1947). More recently, the demonstration by Dr. Thimann and his co-workers, (already discussed), that phenylthiocarbamide acts by combining with copper already within the plant c e l l s , and hence that copper, probably in the form of a copper-containing enzyme, must participate In the formation of anthocyanins, has paved the path to highly f r u i t f u l avenues of sci e n t i f i c inquiry. A survey of literature, past and present, points out that, in general, the deficiency of minerals, other than copper (and special cases of potassium and aluminum to be discussed later), causes the formation of anthocyanins. The author finds i t useful, for the purposes of this discussion, to split the several mineral.' elements according to their action into two groups; (I) is that, in which, the anthocyanin formation i s related to a deficiency of the element and the second (II) i s that in which i t i s related to an excess. "Group I" As is well known, there is an intimate relationship between phosphorus and nitrogen metabolism. Synthesis of proteins does not occur in phosphate deficient plants; cor-related with a decrease in protein synthesis, is often, an accumulation of sugars. Calcium also is known to play a role -88-in the nitrogen metabolism of the plants. In i t s absence, some species are unable to absorb or assimilate nitrates. As such, when calcium is deficient, sugars may accumulate in the plant. During deficiency conditions of magnesium, the phosphate meta-bolism is defective; therefore, the sugars w i l l accumulate. The exact role of boron is not known though i t s deficiency causes accumulation of carbohydrates and break-down of protein synthesis (Briggs 1943, Scripture and McHargue 1954). Molyb-denum is involved in the reduction of nitrates (Arnon and Stout 1939, Hewitt 1951), as such , i t s deficiency would cause sugar accumulation. Deficiency of a l l the above minerals is associated with sugar accumulation. Theoretically, therefore, i t may logically be argued that their deficiency should promote anthocyanin for-mation. That this i s so, has been confirmed by independent attempts of a number of investigators in case of phosphorus, calcium and nitrogen (Blank 1947). More recently Heller (1948), Behrens (1952) and Garrigues (1956) have shown that nitrogen deficiency causes excessive anthocyanin development. Thimann and Edmondson (1949) have shown similar relationship in case of molybdenum and boron. Potassium seems to occupy a special position in 'Group I', since i t has been reported that, in certain cases (particularly barley), i t s deficiency causesanthocyanin formation; in others -89-i t s excess causes anthocyanin formation (see Blank 1947 PP' 276). Though potassium i s not d e f i n i t e l y known to be b u i l t in-to organic compounds esse n t i a l f o r the continued existence of the plant; sometimes i t s deficiency causes an accumulation of amino acids and amides, thereby protein synthesis i s affected and at other times the carbohydrate metabolism i s disturbed, photosynthesis i s checked and r e s p i r a t i o n i s increased. The effects of potassium are usually f i r s t apparent i n disturbed nitrogen metabolism because of f a i l u r e of protein synthesis. In that event, the anthocyanins may appear to start with. Later on, as potassium deficiency continues, carbohydrates rapid l y decrease i n quantity as a result of decreased photo-synthesis and increased r e s p i r a t i o n . In that event, the addi-t i o n of a potassium may restore the l a t t e r phenomenon and anthocyanins may appear once again. It i s , however, a moot question whether the anthocyanins w i l l disappear when the f a c -t o r s a f f e c t i n g the f a i l u r e of protein synthesis, are corrected by further additions of potassium. There appears to be a c o r r e l a t i o n between minerals that cause chlorosis and anthocyanin formation. In t h i s category, the case of i r o n and magnesium may be c i t e d . F u j i (1950) observed anthocyanin formation due to iron de f ic iency . Meyer and Anderson (1952, pp. 48l) report similar r e l a t i o n s h i p i n case of magnesium. - 9 0 -"Group II" This group includes minerals that are either catalysts for "oxidation-reductions" or, are essential constituents of certain oxidizing-reducing enzymes (since there i s an increas-ing evidence from recent studies that such enzymes or catalysts may be involved in anthocyanin formation in vivo). The role of copper (which is an essential part of certain polyphenol oxidases and ascorbic acid oxidase etc. has already been dis-cussed) feeding in anthocyanin formation is highly suggestive that further experiments to' assess the roles of manganese, zinc, cobalt etc. are l i k e l y to yield further informations on the oxidising-reducing enzymes involved in anthocyanin formation. The role of aluminum occupies a special consideration under this group. Aluminum, in as low concentrations as 1 p.p.m. may be toxic to barley and corn particularly in acid soils (pH 5 or less). In acid s o i l s , aluminum precipitates phosphorus and hence decreases i t s availability to plants, the formation of nitrogenous compounds is also slowed down and sugars may accumulate when this element is in excess (Meyer and Anderson 1952, pp. 488). The flower color of Hydrangea  macrophylla is related to aluminum content of the f l o r a l tissues. Addition of soluble aluminum compounds to the s o i l , in which, hydrangea plants are growing induces a shift in flower color from pink to blue (Allen 1943). -91-4. RELATIONSHIP OF CHEMICALS AND GROWTH SUBSTANCES TO BIOGENESIS Uota (1950) found that coloring of Mcintosh apples was greatly improved by several applications of lfo-0.5% sodium diethyldithiocarbamate spray. This chemical proved more effec-tive and less injurious than soluble thiocyanate salts, reported by Dustman et. a l . (1940). Applications of 20-25 ppm of 2,4,5-trichlorophenoxypro-pionic acid to apples, about 6 weeks before the normal picking date, greatly accelerated fr u i t ripening and higher anthocyanin content, though the storage quality of the fr u i t was impaired (Abbott. 1954). It is however, a moot question whether the storage quality is affected by higher anthocyanin content, or the spray. Solacolu T. et. a l . (1937)> report that when indole - 3 -acetic acid was injected into Ricinus plants, formation of anthocyanins was increased not only at the point of injection, but also in the tissues of the medulla. Paech et. a l . (1952) demonstrated that formation of anthocyanins observed in the germinating seeds of red cabbage after feeding with sugar can be further augmented by addition of 10~7g/ml J3-indolylacetic acid. Methoxy acetic acid caused excessive anthocyanin develop-ment at the second internode of beans (Mitchell et. a l . 1953). - 9 2 -d. THE INFLUENCE OF ANTIBIOTICS ON BIOGENESIS. The French worker Netien Georges (1955 a, b), in his studies, on the action of antibiotics on formation of pigments, in radish plantlets, demonstrated that when germinating radish seeds were soaked in terramycin, streptomycin, aureomycin or Chloromycetin solutions (10~^-10~"!+), formation of anthocyanins and flavones was increased to considerably above normal; chloro-phyll content decreased, while there was no change in the xanthophyll and carotene content. The author (ibid.) concluded that the antibiotics, probably, interfere with the activity of chlorophyllogenase. PHYSIOLOGICAL IMPORTANCE OF ANTHOCYANINS AND RELATED FLAVONOIDS Previously not much was known about the role of anthocya-nins except that they were thought to be present in flowers for attracting insects to secure pollination. Considerable attention was focused on such studies and Blank (194-7) has re-viewed this aspect f u l l y . PHARMACOLOGICAL ACTIVITY. Lately, the therapeutic and physiological importance of anthocyanins, and other flavonoids has been the subject of comprehensive investigations. Almost a l l of the work has been -93-done in Europe. Otto (1947) in his review, reported in detail, the therapeutic and physiological effects of pigments like caro-tenoids, lactoflavine, capsanthin, quercitin and the large class of anthocyanins. Zicavo (1953, 1955) administered purified anthocyanin extract intraperitoneally and found that i t increased ca p i l -lary resistance in the guinea pig (maximum at 36-48 hours). The plasma flow in the small vessels i s decreased. On the frog mesentarium, i t causes prolonged vasoconstriction, followed by transcient vasodilation. Administration of 1200-2400 H/day to human subjects had a beneficial effect on edema of the legs and peresthesias of the extremities after prolonged rest. Zicavo (ibid) suggests that anthocyanins improve the u t i l i s a -tion of certain sugars in human and animal organisms, as well as, in plants. The use of Crataegus (hawthorn) extracts in cardiac diseases i s rather well-known. Ullsperger (1953 a), believes that anthocyanins are, apparently, responsible for the well-known sedative action of Crataegus extracts. With isolated frog hearts, the anthocyanin of Crateagus and myrtillin, violanin and cyanin, show minimum volume increase without change in rate. However, others like peonin pelargonidin and monardaein decrease cardiac output. Quinidine injury to frog heart is prevented by myrtillin and anthocyanins of Crataegus. - 9 4 -The same author (1953 b) has published a review on the develop-ment of Crataegus research. Jeney and Uri (1954) have reviewed the pharmacological action of flavone dyes. Seel (1954, 1957). reports that from clinico-pharmacolo-gical studies on approximately 220 patients, i t transpired that triterpene carboxylic acids, and several anthocyanin and flavone dyestuffs of Crataegus oxycantha. produced similar results on coronary, and myocardial insufficiency, as well as, in compen-sated or recompensated heart disease. The anthocyanins and other Crataegus compounds improve coronary flow and thereby regulate the general circulation and are superior to d i g i t a l i s in that they favor assimilation of sugars from the blood; the glycogen metabolism of the heart is thus improved. Anthocya-nins oppose hypoxemia as well, and there are other numerous Indications where they can be used in cardiac diseases. VITAMIN P ACTIVITY. Certain leuco-anthocyanin derivatives and flavonic pigments present in wines and cider have been reported to possess vitamin P activity (Masquelier 1955, 1956). Vitamin P is involved in raising the capillary resistance and alleviates the tendency towards hemorrhage. The findings of Zicavo (1953) wherein he reported that anthocyanin injections increased the capillary resistance in guinea pigs are highly suggestive that the antho-cyanins may also possess vitamin P activity (author's comments). -95-PROTECTIVE ACTION ON VITAMIN C. Masquelier (195D reported that leuco-anthocyanins from peanut p e l l i c l e s do afford protection against the copper-catalysed a i r oxidation of ascorbic acid. Sondheimer (1953) has presented evidence that ascorbic acid (vitamin C), i n d i r e c t l y , induces the destruction of pelargonidin-3-mono-gluside - an anthocyanin present i n strawberries. These findings are highly suggestive that anthocyanins, leuco-antho-cyanins and, perhaps, other related flavonoids may maintain the vitamin C (ascorbic acid) i n reduced state at the expense of t h e i r own oxidation and degradation. This i n t e r p r e t a t i o n gains further support from Sondheimer's (ibid) observations that factors which decrease oxidation of ascorbic acid i . e . lack of oxygen or addition of thio-urea, also decelerate the rate of destruction of anthocyanins. Pratt (1954) has also confirmed the above findings. PROTECTIVE ACTION OF TANNINS ON ANTHOCYANINS Sastry (1952) on his studies on the s t a b i l i t y of anthocya-nin pigments i n Concord grape j u i c e , found that exposure of juices and p u r i f i e d pigments to u l t r a - v i o l e t radiations i n d i -cated that tannins, e s p e c i a l l y those found i n grapes and grape stems, had an important protective action on the anthocyanins. Tannins, decrease the rate of loss of anthocyanins. -96-FUNGISTATIC AND BACTERICIDAL ACTIVITY. •Schneider (1953) obtained a f u n g i s t a t i c substance from the seed coat of certa i n pea v a r i e t i e s resistant to Ascochyta  p i s i . The chemical examination revealed that the f u n g i s t a t i c properties were due to a condensation product of anthocyanidins and probably tannins. A similar substance was contained i n the seed coat of the peanut. The a n t i b a c t e r i a l action of anthocyanin pigments of higher plants has been reviewed by Mandrik (1953). ANTHOCYANINS AS NATURAL FILTERS. In his studies on the effect of anthocyanin f i l t e r s on plant behaviour and development, Manning (1950) observed tha t red l i g h t , from a f i l t e r of anthocyanins, extracted from beets, resulted i n a plant respohse similar to that r e s u l t i n g from, red l i g h t from red glass. Bean plants exposed to red l i g h t f o r 30 days, were heavier than controls, even though, the l a t e r showed 33.3$ greater leaf surface. Plants exposed to red l i g h t , yielded s t r i k i n g modification i n leaf size and shapes. A 7-days exposure to red l i g h t , followed by growth i n white l i g h t , gave best results i n respect to, stem length and diameter, number and area of leaves, weight of pods and t o t a l plant. Exposure to red lig h t for 55 days, was l e t h a l , while root development was retarded by a l l periods of exposure. - 9 7 -Nystrom ( 1 9 5 6 ) observed, "the rate of C 0 2 f i x a t i o n , found i n an anthocyanin containing section of Coleus l e a f , i n red, and green l i g h t , supported the f i l t e r effect theory of Gabrielsen. The rate of C-^C^ f i x a t i o n was d i r e c t l y propor-t i o n a l , i n red l i g h t , to the chlorophyll content of the two sections. In green l i g h t , the rate was much lower i n the antho-cyanin section, than i n the anthocyanin-free section of the l e a f . Studies u t i l i z i n g the same Coleus l e a f , a f t e r treatment with u l t r a - v i o l e t l i g h t , f a i l e d to reveal any protective action of anthocyanin against u l t r a - v i o l e t r a d i a t i o n . In f a c t , the antho-cyanin section appeared to be more sensitive to the effect of u l t r a - v i o l e t l i g h t . " SEX - HORMONAL ACTIVITY. The b r i l l i a n t work of Moewus (195D on Chlamydomonas has already been discussed under biochemical studies. The same author ( 1 9 5 4 ) has summed up the findings, that two groups of compounds are active i n the sexual reproduction, the flavonoids and the carotenoids. Out of the 50 flavonoids and anthocyanins tested, peonin was active as a male sex-determining hormone; r u t i n , on the other hand, induced s t e r i l i t y . He further reports that cis-cinnamic acid and i n d o l e a c r y l i c acids are the hormones which are responsible for the induction of meiosis. AS AN INDEX OF THE DEVELOPMENTAL PHASE OF PLANTS. The investigations of Ermolaeva ( 1 9 4 8 ) indicate that, as - 9 8 -plants pass from vegetative to reproductive phase, there is a sharp drop in the amount of anthocyanin, resulting in almost complete disappearance in certain cases. He concluded that the appearance and disappearance of anthocyanins in certain plants or their quantitative changes in others, may serve, in some degree, as a criterion (or an index) for judging the develop-mental phase of the plant. The attention of the reader may be brought to the findings of Moewus ( 1 9 5 1 + ) which, in this case, are highly suggestive that this sharp drop in anthocyanins may be connected with the utilization of anthocyanins, as sex-hormones, during the period of transition from vegetative to the reproductive stage (author's comments). Bottazzi ( 1 9 5 0 ) , during a statistical investigation of 1000 sunflowers, showed that distribution of anthocyanins in different parts is not random but that a correlation exists between presence of the pigments in the stems and in various parts of the reproductive organs. AROMA IN CACAO BEANS. Forsyth (1952 b) has suggested, that "aroma" in cacao beans is due to the conversion of simple cyanidin compounds to complex leuco-cyanidins during the anaeorbic fermentation of these beans with yeast or lactic-acid bacteria. The same author (1955 b) has discussed the part played, in cacao fermentation, by the products of microbial action and the role of leuco-cyanidins -99-in cacao aroma. MELANINS Malanins are, often, intimately related with anthocyanins and are generally associated with a dark brown or black pig-mentation, which accumulates in certain parts of plant and animal. The information available in literature regards the chemical nature of the naturally occurring melanins, is largely based upon the melanins obtained from animal tissues. Previously i t was considered that an enzyme, dopa oxidase, catalyzes the formation of melanins from dopa (dihydroxy-phenylalanine). Lerner (1953), in his review, considers that the enzyme tyrosinase is a copper protein, and in the oxidation of tyrosine to dopa and dopa quinone, the copper goes through a cycle, Cu—>Cu—*Cu during which i t acts as a catalyst of oxidations. Most polyhydroxyphenyl and aminophenyl compounds, having ortho or para groups, can be oxidized to pigmented polymers, and the type of melanin is best shown by Indicating the subs-tance from which the melanin i s formed. Thus we may have dopa-melanin, adrenalin-melanin, homogentisic acid-melanin etc. Thomas (1955), in his review on melanins, has distinguished - 1 0 0 -between the black pigments derived from the polyhydroxy-phenyl compounds and the amino-phenyl compounds; so to say, he has differentiated between the black pigments derived from non-nitrogenous and nitrogenous polyphenyl compounds. According to him, even the jet-black pigmentation, derived from non-nitrogenous compounds is not; an authentic melanin. Melanin, he considers, may be defined as "dark polymeric indole derivative of high molecular weight, which are produced by a series of reactions involving tyrosinase, oxygen and organic substrates, either tyrosine or dopa." Lerner (1953)? has reviewed the formation of melanins. The general behaviour of melanins is briefly discussed below?-a. The melanins are very complex substances of high mole-cular weight and are insoluble in most solvents. In tissues, they are generally combined with proteins. b. The production of melanins is lacking in albinism. c. Melanins form a reversible oxidation-reduction system, in which the reduced form is tan and the oxidized form is black. reduced melanin (tan)^===* oxidized melanin(black) On the identification of pigments as melanins, Jacobson and Millott (1953)? in their report on melanogenesis in the coelomic fluid of an echinoid, state, "Although chemical tests specific for melanins are non-existent, it is possible to identify, at least, provisionally, a naturally occurring brown or black pigment as melanin, provided that it shows certain characteristics, namely, resistance to solvents, bleaching when -101-sutgected to the action of oxidants, and the capacity to reduce, d i r e c t l y , ammoniacal solutions of s i l v e r n i t r a t e . " The occurrence and taxonomic value of melanins, i n barley, (see pp. 9) was pointed out by Harlan (1914), Lewicki (1929) and Aberg and Wiebe (1946, 1948). A good number of genetical studies were carr i e d out (see pp. 14, 16) but no attempt has so f a r been made to study t h e i r biogenetic relationships to other polyphenolic compounds not only i n barley but also i n other vascular plants. However, the thought-provoking observa-tions of Lewicki (1929) i n regard to the simultaneous presence of anthocyanins and melanins; and melanins with much anthocya-nin i n immature stages only, i n barley, and that of Thimann and Radner (1955 a, b) on the action of phenyl thiocarbamide, 2 - t h i o u r a c i l , and other sulphur containing compounds, which i n h i b i t tyrosinase a c t i v i t y and anthocyanin formation i n Spirodela, are suggestive of a close biogenetic r e l a t i o n s h i p between anthocyanin and melanin formation. The extension of such studies i n barley may, therefore, be highly f r u i t f u l . A n a l y t i c a l methods for melanins have been reviewed by Thomas (1955); most of these r e l a t e to melanins occurring i n animals. Comparable a n a l y t i c a l studies of plant-melanins are p a i n f u l l y lacking; i n general, i t would seem that there i s l i t t l e interest In them because no fuction has been assigned to them. So f a r , no major study on inheritance of melanic pig-ments invasular plants has been undertaken. In contrast, as reported by Ginsburg (1944), "the melanic pigmentation i n the -102-mammal has been subjected to a more extensive genetic analysis than any other single character yet studied i n t h i s group." The only other recent work on plants, that the author came across, i s given below:-Wickberg (1956) has been reported to have conducted a systematic study of melanin-forming substances i n sugar beet. He attempted a paper chromatographic separation of the phenolic compounds i n an e f f o r t to establish t h e i r role i n melanin f o r -mation. His technique, however, detected tyrosine but not catechol or chlorogenic acid which are known to be present i n t h i s material. Gimesi (1952 a) reported that the seedlings, and white c o r o l l a of M i r a b i l i s turned black when placed i n 96% or absolute ethanol. The color was interpreted as due to melanins a r i s i n g from tyrosine by enzymatic action. -lo3-'•\ EXPERIMENTAL STUDIES WITH COLOR IN BARLEY. C o l o r has.been c o n s i d e r e d as a co n v e n i e n t c h a r a c t e r , i n g e n e t i c a l s t u d i e s , because of t h e ease i t a f f o r d s p h e n o t y p i c d i s t i n c t i o n s . C o l o r c h a r a c t e r s a r e , however, not as s i m p l e as t h e y might, at f i r s t , appear. D i f f i c u l t i e s a r e o f t e n e x p e r i -enced, i n c l a s s i f y i n g s e g r e g a t e s and, not i n f r e q u e n t l y , v a r i -a t i o n s i n e n v i r o n m e n t s , may m o d i f y e x p r e s s i o n . B a r l e y c o l o r s have a t t r a c t e d t h e a t t e n t i o n of over 100 i n v e s t i g a t o r s . D e s p i t e t h i s c o n s i d e r a b l e a t t e n t i o n , o n l y t h e broad p a t t e r n s of c o l o r i n h e r i t a n c e , i n t h e c r o p , a r e known and v e r y l i t t l e has been done on t h e c h e m i s t r y o f r e s p o n s i b l e pigments. To extend t h e knowledge of t h e c o l o r p h y s i o l o g y and g e n e t i c s i s t h e purpose of a s e r i e s of s t u d i e s , t h e second of which i s r e p o r t e d here (see F a r i s , D.G., M.S.A. T h e s i s 1956 : B a r l e y S t u d i e s I . ). I t was r e a l i z e d a f t e r t h e i n i t i a l work of F a r i s ( i b i d ) t h a t i f a r e a l p r o g r e s s was t o be made on t h e p h y s i o l o g y and g e n e t i c s of t h e b a r l e y pigments, much more a t t e n t i o n would have t o be p a i d t o t h e pigments per s e , t h e i r e x t r a c t i o n , d i s -t r i b u t i o n and c h e m i s t r y . I n o t h e r words, l e s s e f f o r t would be d i r e c t e d towards t h e i r p h y s i o l o g y and g e n e t i c s and more e f f o r t would be d i r e c t e d towards t h e methodology o f t h e i r i s o l a t i o n and c h a r a c t e r i z a t i o n . -104-MATERIALS From some oO v a r i e t i e s of barley grown on U.B.C. farm and displaying a wide range of colors, a few were selected to provide suitable d i v e r s i t y for the purposes of t h i s study. For most of the work, the v a r i e t i e s mentioned i n table VIII were used. TABLE VIII. LIST OF VARIETIES MOST COMMONLY USED NAME COLOR ORIGINAL SOURCE Gatami Lion Kwan Montcalm Tre b i Black Hulles Gopal C.I. 1091 Golden pheasant C.I. 2488. Hanna G-54-55 black black blue blue peculiar blue purple bright purple whit e Ottawa U.S.D.A. white Ottawa, white and d i r t y white " -105-Some f i e l d work involved the use of additi o n a l v a r i e t i e s given i n table IX. TABLE IX ALPHABETICAL LIST OF VARIETIES ADDITIONALLY USED. NAME COLOR ORIGINAL SOURCE Atlas C.I. 4118 Compana C.I. 5628 C-54-22 Deficiens C.I. 2225 Ethiops Kama-Ore. C.I. 694 Kitchen Orange lemma C.I. 5649. Orange lemma. 00 57-AB-1390 Normal Lemma.00 57-Ab-1394. Vantage 33-bl bl -13 33-B1 Bl -13 36-bl bl - 2 1 36-B1 Bl - 2 1 71-pr pr -10 blue white purple pale blue white white and d i r t y white d i r t y white black white and d i r t y white white white blue white blue whit e U.S.D.A. U.B.C. U.S.D.A. Ottawa U.S.D.A. Ottawa U.S.D.A. Ottawa U. S.D.A. U.S.D.A, U.S.D.A, -106-71-Pr Pr-10 purple U.S.D.A. 5090-2-3 grey and d i r t y white Ottawa 5090-10-U- white and d i r t y white 5090-15-1 white 5423- 4 dark blue or grey 5424- 7 dark blue or black 5425- 8 grey 5428-2 black METHODS A. EXTRACTION OF ANTHOCYANINS. 1. EXTRACTION FROM MATURE KERNELS. The extraction of anthocyanins from barley kernels i s not as easy as i t i s i n f l o r a l or other plant t i s s u e s . Paris (1956), t r i e d a number of methods of extraction namelys-a. refluxing the ground grains f o r one hour i n 10% HC1. This brought out a lot of concomitant c o l l o i d a l mate-r i a l alongwith the anthocyanins; b. using the ground kernels i n soxhlet extractor for 10 hours. This considerably affected the color of solu-tions when the volume was reduced on heating; c. shaking the ground kernels i n ethanol on the shaker proved sat i s f a c t o r y . d. the last method consisted of extraction i n ethyl alcohol from the whole kernels placed i n a test tube. The kernels of the hulled v a r i e t i e s were pearled -107-long enough i n small hand pearler to remove most of of the h u l l s . The test tubes were plugged and allowed to stand for four to seven days. This method was selected by him for the extraction of a large number of v a r i e t i e s chromatographed by him. The author, i n order to standaridise the extraction technique, selected 'Gopal' and 1 Black Hulless', the purple v a r i e t i e s , which contained, comparatively, large amount of anthocyanins. I n i t i a l l y , a few grams of ground kernels were placed i n erlenmeyers containing about H-0-50 ml. of 1% HC1-EtOH. The extraction was hastened by shaking f o r a period extending from 2k hours to 72 hours. Whenever the color appear-ed deep enough, the extract was f i l t e r e d and the volume reduced on a f l a s h evaporator at 30°C. It was observed that most of the anthocyanin color remained adsorbed on the starchy and pro-teinaceous materials of the seed. The recovery of the antho-cyanins was further reduced i n the process of f i l t e r a t i o n , f o r the f i l t e r paper adsorbed large quantities of pigment when the extract was i n concentrated form. Later extractions, carried out i n 1% aqueous HC1, also gave similar results as reported above and, i n addition, the aqueous solvent extracted a large amount of c o l l o i d a l material which made chromatography d i f f i c u l t . It was, at that time, considered that the aqueous solvent, though of value i n flower color extraction, could not be used i n case of seeds. As reported above, the starchy material present i n the endosperm tended to adsorb color during extraction. It was found that the endosperm of the v a r i e t i e s , on hand, was white -108-and i f i t could be removed, the extraction would be made much easier. Consequently, a hand pearler (which now runs on a motor) was used to dust off the outer layers v i z . h u l l s , p e r i -carp and aleurone and the white endosperm was discarded. About 3 to 5 grams of t h i s powder was placed i n an erlenmeyer con-tai n i n g 50 ml. of 1% HCl-EtOH. The mixture was put on the shaker and after every 12 hours or so, the extract was c e n t r i -fuged off (instead of f i l t r a t i o n ) , and the p e l l e t re-dissolved i n about 40-50 ml. of 1% HCl-EtOH and re-extracted as above. Usually by repeating t h i s process over a period of 4-5 days, large quantities of anthocyanins were extracted and were quite s a t i s f a c t o r y i n so f a r as the quantitative y i e l d of anthocyanin was concerned, though the length of the period of extraction always cast doubts with regard to the s t a b i l i t y of the extracts and .again the extraction was not complete since the color of the p e l l e t remained deep red, even, after ten days of repeated extraction. A more polar solvent methyl alcohol was substi-tuted i n place of ethyl alcohol. Though i t brought out larger quantities of anthocyanins, the p e l l e t s t i l l remained red and every time, the solvent was added, more anthocyanins came out. Two possible reasons were considered responsible f o r t h i s behaviour. Either the c e l l - w a l l s donot permit the anthocyanins to diffuse out or i f they do, the rate of d i f f u s i o n may be very slow. Secondly, i t was equally possible that the remaining anthocyanins may be too strongly bound to the tissues of the kernel to be released by the action of the solvents employed. -109-Accordingly, an effort was made, f i r s t to break the c e l l walls of the fragments of dust obtained after pearling with the help of a 10K.C. Ratheon Sonic O s c i l l a t o r . The procedure has been b r i e f l y described i n the e a r l i e r work (Mullick, F a r i s , Brink and Acheson 1958). and i s being elaborated i n l i g h t of the knowledge gained i n subsequent studies. About 3 to 5 grams of the powdered pericarp and aleurone was thoroughly scanned with a magnet and 1% HCl-MeOH was added at the rate of 10 ml/gm powder. The material was then extracted i n the sonic o s c i l l a -tor for 30 minutes, with-drawn with a pro-pipette and c e n t r i -fuged i n polyethylene tubes at 14,000 rpm for 5 to 7 minutes. The p e l l e t was re-extracted 2 to 3 times s i m i l a r l y . These operations, however, extracted large quantities of anthocyanins i n comparatively much shorter time. The number of days, previously taken f o r extraction on a shaker, were reduced to about the same number of hours with ultrasonic extraction. After about k to 5 extractions on the sonic o s c i l l a t o r , the color of the p e l l e t , after centrifugation, was s t i l l s l i g h t l y red and extended extractions did not remove any appre-ciable quantities of color. The pell e t remaining af t e r these extactions, appeared to be largely proteinaceous. This suggested that the proteins eof the barley kernel, might be "holding" the anthocyanins. The known proteins i n barley kernels are hordein, one of the -110-glut e l i n s . The former i s alcohol soluble, and may not be involved. Therefore, a p o s s i b i l i t y that g l u t e l i n s , which are insoluble i n alcohol but soluble i n d i l u t e a c i d i c and alk a l i n e solutions, may be involved, was examined. As such, the p e l l e t , a f t e r extraction with a c i d i f i e d methanol, as described above, was dissolved i n 1% aqueous HC1 and extracted i n sonic o s c i l l a -tor as before. After centrifuging, the pe l l e t was found to be s t i l l reddish and the supernatent l i q u i d , was highly c o l l o i d a l , also contained some color. An aliquot from the c o l l o i d a l solution was taken and to i t was added concentrated n i t r i c acid. On warming, a yellow color was obtained. This turned to orange when the solution was cooled and was made alkaline with ammonium hydroxide. This,,then, i s a positive xanthoproteic test for proteins. To another aliquot from the c o l l o i d a l solution was added sodium hydroxide solution and a.drop of 2.% copper sulphate solution; a v i o l e t coloration appeared; t h i s , then i s a pos i t i v e biuret test for proteins. These two tests are considered to be s p e c i f i c for glute-l i n s (hordenin i n case of barley) by Steele (1949). In an effort to remove t h i s proteinaceous coagulum from the c o l l o i d a l supernatent l i q u i d without a f f e c t i n g the nature of anthocyanins, the supernatent f l u i d was placed i n a freezer at ca. 26°F for 24 hours (better results were obtained by keeping i t i n the freezer for over hQ hours), and on thawing, the proteinaceous - I l l -mass coalesced i n the form of a gelatinous lump, leaving the supernatent f l u i d free of any v i s i b l e t u r b i d i t y . A s l i g h t centrifugation gave a very clear extract containing a s l i g h t amount of anthocyanin. The behaviour of the pe l l e t which was obtained a f t e r ext-ta c t i o n with 1% HC1 aqueous was rather i n t e r e s t i n g . When t h i s p e l l e t was dissolved i n alco h o l i c - HC1 and subjected to sonic o s c i l l a t o r and centrifuged, the supernatent contained large qua-n t i t i e s of anthocyanins. The p e l l e t , though s t i l l containing color was a much l i g h t e r i n shade than before. Alternate extractions with acid-water and acid-alcohol as explained above, almost completely extracted the anthocyanins. It may be stated that the author could not obtain a p e l l e t , absolutely free of red color. A l l t h i s was suggestive that towards the end of the extrac-t i o n , some of the anthocyanins might be conjugated with some materials, soluble i n a c i d i f i e d water but insoluble i n a c i d i -f i e d alcohol, and soon as t h i s a c i d i f i e d water-soluble material was removed, the anthocyanins could be extracted with an alco-hol i c solution. In view of the r e s u l t s , reported above, i t appears that towards the end of the extraction, alternation with aqueous and alcoholic solvents may be advantageously employed. -112-2. EXTRACTION FROM ALEURONE LAYER AND OTHER FLORAL TISSUES. A method (to be described l a t e r ) was worked out, whereby the pigments could be extracted separately from pericarp* perisperm-spermoderm and aleurone layers around hard dough stage of seed maturation. The extraction from the h u l l s , awn t i p s , pericarp and perisperm-spermoderm tissues was carried out by the usual method of extraction used i n f l o r a l or vegetative tissues i . e . by placing the tissues i n 1% HCl-ethanol or metha-nol. This gave complete extraction over a period of 24 hours. However, when the aleurone, either intact on the endosperm or separated from the endosperm, was placed i n the solvent, no color was extracted even when the seeds were kept for over two months and as long as six months i n the solvent. Aleurone tissues containing anthocyanins -were homogenised i n a homogenizer using the aqueous or a l c o h o l i c solvents. The proteins were precipitated with k to 10$ t r i c h l o r o a c e t i c acid and centrifuged. No color could be recovered i n the supema-tent f l u i d and the p e l l e t s t i l l was deep red. Later on, butyl alcohol, which also precipitates the pro-t e i n s , was employed as an extractant but without good r e s u l t s . The only method that did extract color was that of a l t e r -nate extraction with alcoholic and aqueous solvents as des-cribed before when pearled dust from mature kernels was used. The problem of extraction of color from aleurone, therefore, as yet remains pa r t l y unsolved. The quantity of color present i n the aleurone layer of i n d i v i d u a l kernels i s rather scanty and large scale extraction cannot he employed becaiise of the eff o r t i t takes to obtain seeds consisting of aleurone alone. 3. EXTRACTION WITH ACETONE. Since a c i d i f i e d alcohols and water did not give a complete extraction of anthocyanins, as already reported, acetone ext-ractions were t r i e d both on a shaker and sonic o s c i l l a t o r . It was found that 3 to h repeated extractions with 1% HCl-acetone completely extracted the color from the powder obtained af t e r pearling. The p e l l e t was l e f t absolutely c o l o r - f r e e . On chromatographing these extracts, a number of different, spots, with different Rf values appeared. Some of these were b r i l l i -ant-red spots and are after many months preserved on the paper-grams. However, further work was suspended since the extracts were not stable. Most probably, hydrochloric acid a f f e c t s the s t a b i l i t y of acetone since the acetone solution containing hydrocloric acid turns brown and dark brown on standing. These complications, i t was considered, can be most e f f e c t i v e l y tackled by a chemist. h. EXTRACTION FROM PLANT MATERIAL. Large quantities of anthocyanins develop i n basal leaf sheath, nodes and a u r i c l e s . The anthocyanins from these -114-organs were extracted from fresh plant tissues at di f f e r e n t seasons and di f f e r e n t growth stages. The fresh material was thoroughly washed under the running tap water, and the excess water was blotted out as completely as possible. The tissue s , where the anthocyanins development was profuse, were cut off with scissors and immersed i n absolute alcohol caontaining 1% HC1. In about 3 to 4 hours, large quantities of anthocyanins were i n the solvent. B. METHODS OF VOLUME REDUCTION AND PURIFICATION. When the anthocyanins are extracted from a powder obtained a f t e r pearling, the extract, i n addition to anthocyanins, con-tains other materials soluble i n a c i d i f i e d alcohol or a c i d i f i e d water depending upon the solvent used. In case of aqueous extraction, most of the concomitant material i s c o l l o i d a l i n nature, whereas i n case of alco h o l i c extraction, i t i s i n solu-ble form. Unless the concomitant materials are removed, the chromatographic resolution of these compounds i s greatly a f f e c -ted owing to overloading of the paper. The c o l l o i d a l material from aqueous extraction i s best removed by freezing and thawing and centrifugation as explained before. Usually, a very clear supernatent f l u i d i s obtained after centrifugation. Since i t appeared to be an easy method of removing the accompanying c o l l o i d s , t h i s method of aqueous extraction was recommended i n the e a r l i e r work (Mullick, F a r i s , -115-Brink and Acheson 1958). However, l a t e r on, i t was found that comparatively fewer application of concentrated aqueous extract caused over-loading during chromatography. This indicated that other materials, soluble i n a c i d i f i e d aqueous solutions of the extract were responsible of overloading. As such alcoholic extraction appeared to be more sa t i s f a c t o r y . Most of the soluble material, i n a l c o h o l i c and aqueous extractions i s removed during the process of volume reduction i n vacuo. As the volume of the solution i s reduced, the saturation point of the solution with respect to certain solu-tes i s reached and the l a t t e r p r e c i p i t a t e out. As such during volume reduction, intermittent.centrifugation helps eliminate the precipitate as i t i s formed. If the centrifugation i s delayed u n t i l the volume i s f i n a l l y reduced to 2 ml or so, the author observed that large amounts of o i l y material, dark red i n color (due to adsorption of anthocyanins) f l o a t s at the top of the micro-centrifuge tubes and removal of the clear antho-cyanin extract below t h i s o i l y layer becomes very d i f f i c u l t . Occasional centrifugation at high speed during the process of volume reduction not only helps keep the accumulation of o i l y material to the minimum but also reduces the loss of pigment due to adsorption on i t . As has been reported i n the e a r l i e r work, the alcoholic extract of anthocyanins i s reduced to 10 ml. i n vacuo and 20 ml. of 1% HC1 aqueous i s added. Owing to t h i s change i n the medium, a large quanitity of c o l l o i d a l matter precipitates out which i s removed by centrifugation at high speed. The volume -116-i s further reduced i n vacuo and when the alcohol completely evaporates, large quantities of c o l l o i d a l matter again p r e c i -p i t a t e , which may be centrifuged, before the f i n a l centrifuga-t i o n , when the volume i s reduced to about 2 to 5 ml. The clear supernatent may s t i l l contain dissolved materials and co-pigments which may cause over-loading during chromatography. Some of the co-pigments p a r t i c u l a r l y flavones are removed by scrubbing the extract with ethyl acetate followed by benzene washing to remove the ethyl acetate. For chromatography, a highly concentrated extract contain-ing no c o l l o i d a l or other concomitant matter i s considered i d e a l . However, i n vacuo, i t i s rather d i f f i c u l t to reduce the volume beyond 2 ml. or so. As such further reduction of volume, i n e a r l i e r stages of the work, was achieved by placing the small tubes containing the extract i n a desiccator contain-ing calcium chloride or sulphuric acid. This process was time-consuming, during which some degradation of anthocyanins could take place. A method was l a t e r worked out which, i n broad terms, i s similar to the one reported by L i (1956). The volume of the extract was reduced i n vacuo to about 5 ml. This was washed with about 3 to h times the quantity of ethyl acetate twice or t h r i c e . The ethyl acetate extracted most of the aqueous por-t i o n of the extract and simultaneously removed cert a i n flavones etc. In t h i s \^ ay the volume could be reduced to a few drops i n a highly concentrated form. -117-A point of caution may be recorded here, about ethyl acetate. It should be made absolutely certain that the ethyl acetate does not contain acetic acid. The anthocyanins are readily soluble in acetic acid and when the ethyl acetate con-taining traces of acetic acid, i s used for washing the aqueous anthocyanin extract, some of the color i s displaced in the ethyl acetate. C. EXTRACTION OF ANTHOCYANIDINS. The anthocyanidins were, in general, obtained by the hydrolysis of anthocyanin extract with about 3 N HC1 solution at temperature of boiling water for a period between 20 to 45 minutes. The procedure after the hydrolysis, is similar to the one reported in the earlier work (please see the folder at the end), and gives quite satisfactory results. The only difference in the procedure mentioned above and the one already reported is the stage, at which the hydrolysis is carried out. In the former, the hydrolysis i s carried out on the anthocyanins extract whereas in the later, i t is carried on the tissue i t s e l f . This procedure i s , In general, employed for the extraction of anthocyanidins from leuco-anthocyanins. (Bate-Smith 1954). This procedure (Mu.llick et. a l . , 1958) was used for two -118-reasons. F i r s t l y , when the anthocyanin extracts of Black Hulless and Gopal kernels wer hydrolysed., three anthocyanidins were obtained. S i m i l a r l y , when the Black Hulless and Gopal kernel tis s u e s , without prior extraction of anthocyanins, were hydrolysed d i r e c t l y , the same three anthocyanidins were pro-duced. Since the l a t t e r procedure was much easier, i t was adopted in hydrolysis of a l l kernels t i s s u e s . The other reason was that the amount of anthocyanins extracted from certain blue v a r i e t i e s was too l i t t l e and could not be e f f e c t i v e l y hydrolysed d i r e c t l y with the former procedure. D . A METHOD TO DETERMINE THE LOCALIZATION AND ANALYSIS OF THE ANTHOCYANINS IN THE SEPARATE TISSUES OF THE CARYOPSIS. A method to determine the l o c a l i z a t i o n and analysis of anthocyanins i n in d i v i d u a l components of caryopsis was consi-dered essential since pericarp, perisperm-spermoderm and h u l l s are genetically d i f f e r e n t from aleurone and endosperm. During developmental studies of color i n barley kernels, i t was observed that the color develops aft e r the milk 'stage. It was found that an easy way to persue the development of color In dif f e r e n t layers of barley kernel was through peeling. Individual layers of the kernel were peeled off manually one after the other to f i n d out i n which layers the color develops. - 1 1 9 -The microscopic examination of peelings revealed that the f i r s t layer s o l e l y consisted of pericarp. A f t e r the pericarp, were found two membranous layers, perisperm and spermoderm. These two layers could not be peeled apart unless great care was exercised and t h e i r separation became rather more d i f f i c u l t around the hard dough stage. As such, since both the layers are the remains of nucellus and genetically s i m i l a r , have been grouped as a single "perisperm-spermoderm" layer f o r a l l prac-t i c a l purposes. Afte r the perisperm-spermoderm are peeled, o f f , the embryo and the aleurone appears. It i s impossible to peel off the aleurone since i t grows ( at least 3 to h c e l l u l a r layers) deep into the endosperm. The surface examination of the peelings with regard to t h e i r i d e n t i f i c a t i o n under the microscope, was further con-firmed by examination of t h e i r cross-sections under the micro-scope. For t h i s , the kernel as a whole was embedded i n the p a r a f f i n at the beginning of hard dough stage and sectioned on the s l i d i n g microtome ( free-hand sections were also quite good for t h i s purpose), and a l l the layers were i d e n t i f i e d . Next, the outer layer (pericarp) was peeled off and the remain-ing kernel was sectioned as pointed out above. This kernel showed endosperm, aleurone, perisperm-spermoderm and no p e r i -carp. S i m i l a r l y , the next layer was peeled off and the remain-ing kernel was examined as before. This only revealed the pre-sence of aleurone. Obviously t h i s meant, that the method to separate the -120-various layers had been found when the kernel was r e l a t i v e l y In immature stages. It was observed that these layers (which develop plenty of color) could e a s i l y be separated, u n t i l f a i r l y l a t e hard dough stage, but c e r t a i n l y before the f l i n t y stage. A method for the extraction of anthocyanins from these peelings i s s i m i l a r to the one used i n case of f l o r a l tissues and has already been discussed. E. PAPER-CHROMAT OGRAPHY. The general procedural d e t a i l s of chromatography followed i n these studies, have been described elsewhere (please see the reprint submitted as appendix I I ) . However, some of the aspects which were not be covered f u l l y are described below; 1. THE C HR OM AT 0 ST RI PE - AN AUTOMATIC STRIPING TECHNIQUE FOR PAPER CHROMATOGRAPHY. It has already been pointed out that d i l u t e a c i d i f i e d a lcoholic and aqueous solutions, extract, i n addition to antho-cyanins, large quantities of soluble proteins, copigments, and other concomitant materials. These materials af f e c t the reso-l u t i o n and rate of movement of anthocyanins considerably. Rechromatography of the eluates and banding large volumes of extracts became necessary. However, i t was d i f f i c u l t to apply large amounts of extract' manually i n such a way that -121-l o c a l l z e d over-loading was avoided. Good resolution of the bands of anthocyanins on the paper was obtained with a tech-nique designated as the "Chromato-stripe technique", the de t a i l s of which are described i n Appendix I I I . 2. SPOTTING. Spotting i n our e a r l i e r work, proved to be a highly time-consuming process. An automatic method for drying the spots quickly has been reported by Paterson (1956) but that could not be used owing to u n a v a i l a b i l i t y of compressed a i r . How-ever, a wooden stand 30 X 30 J. 6 i n . f i t t e d with a thermosta-t i c a l l y controlled stainless s t e e l s t r i p 22 X 3 i i n . at a d i s -tance of 9 inches from an edge, proved very s a t i s f a c t o r y . The temperature of the s t e e l s t r i p was regulated at 30°C and a stream of a i r through a small blower, was directed on the surface of the chromatographic paper while applying the spots. This enabled a much faster drying of spots. With respects to the size of spots, i t was found that the resolution of the spots was inversely related to i t s si z e . Usually spots around 0.5 to 0.6 mm i n diameter were found to be the best. 3. VARIATIONS IN Rf VALUES. In ascending chromatography, i t was observed that the -122-spots placed nearer to the edges of a paper, always moved a greater distance than those placed around the centre of the •same paper, even though they were applied from the same extract. In other words, the Rf values of similar spots were higher around the edges and lower i n the centre, though there was no change i n the number of spots and t h e i r r e l a t i v e positions and concentration r a t i o s to one another. To add to these v a r i a t i o n s , were the variations due to the constitution of the paper i t s e l f i . e . the d i r e c t i o n of the paper c a p i l l a r i e s and the uniformity of thickness etc. These may cause occasional e r r a t i c behaviour. These d i f f i c u l t i e s were partly over come by running authentic anthocyanins and anthocyanidins i n mixture with the extract containing unknown compounds. The respective positions of the other spots were determined i n r e l a t i o n to these spots and Rf values measured. The Rf values reported are always averaged out from a number of similar "runs". Paper-chromatography, though generally a good t o o l for securing separation of compounds i n a pure form and evaluating of the number compounds present i n a given extract, i s not alone s u f f i c i e n t for dependable i d e n t i f i c a t i o n s . h. EFFECT OF DIFFERENT GRADES OF CHROMATOGRAPHIC PAPER ON RESOLUTION. The Rf values of anthocyanin chromatography that have been reported i n l i t e r a t u r e are based upon Whatman No. I. paper. -123-During our studies on the plant colors, i t was found that when Whatman No. 7 paper was used i n place of Whatman No. I, spots with much better resolution and d e f i n i t i o n were obtained. In addition, the t r a i l i n g was considerably reduced. Si m i l a r l y , Whatman No. 3 was much better than Whatman No. 3mm when large quantities of extracts were required to be spotted. Therefore, i n our l a t e r studies, Whatman papers Nos. 7 and 3 have been regularly used. 5. EFFECT OF DIFFERENT CHROMATOGRAPHIC SOLVENTS. In general the solvents used during t h i s work consisted of the organic phase of n-butanol: acetic a c i d : water ( 4 : 1 : 5 v/v) for anthocyanins and water: hydrochloric a c i d : acetic acid (10 : 3 : 30 v/v) for anthocyanidins. Lesins (1958) reported that a 5% solution of ortho-phosphoric acid gave good separations of anthocyanins. Accordingly t h i s solvent was t r i e d but i n our extract, i t resolved only two major streaked compounds as against eight or more resolved by the butanol-acetic acid-water. The f e r r i c chloride reactions of the spots developed i n these ti^o solvents were widely d i f f e r e n t . This suggested that the solvents used, may effect the chemical nature of anthocyanins. Ortho-phosphoric acid was t r i e d with butanol and water i n varying proportions and the chromatograms were developed i n both the organic and aqueous phases. None of these solvents proved good and almost i n every case, a pronounced -124-streaklng was observed. F. ELECTROPHORETIC STUDIES WITH AHTHOCYANINS. During chromatography, some of the anthocyanins do not resolve and a long streak i s observed on the paper-gram. It was considered that electrophoresis might be of help. To start with 0.4- N acetic acid was t r i e d as solvent but i t f a i l e d to give any e f f e c t i v e resolution. However, 2 N acetic acid did separate c l e a r l y , the yellow colored compounds from the red colored anthocyanins. I t , however, f a i l e d to separate the various constituent anthocyanins present i n the extract since a l l the three tubes In which the anthocyanin solutions were collected gave a maxima at 54-0 mu. This method, i t appears, can be e f f e c t i v e l y used for securing the separation of anthocyanins from other co-pigments and c o l l o i d s . It i s suggested that the charge on the anthocyanin molecule was weak and the concomitant yellow colored compounds probably possessed no charge because they just " t r a i l e d s t r a -ight" on the paper. Probably, i f a stronger acid l i k e 2 N formic acid i s t r i e d instead of 2 B acetic acid, the res o l u t i o n of anthocyanins may be considerably improved. -125-O B S E R V A T I O N S AND R E S U L T S . A. THE ANTHOCYANINS AND ANTHOCYANIDINS OF MATURE BARLEY KERNEL. A q u a l i t a t i v e exploration of the d i s t r i b u t i o n of antho-cyanins i n the kernels of several, blue, black, purple and white v a r i e t i e s of barley had f i r s t to be undertaken. No r e a l guide was available from the l i t e r a t u r e on barley or the related species. This work culminated i n the publication of "anthocya-nins and anthocyanidins of the barley pericarp and aleurone t i s s u e s " (Mullick, F a r i s , Brink and Acheson, 1958), a reprint of which i s appended i n a folder for ready reference. It seems unnecessary to repeat the contents of t h i s paper here. However, as under "methods" cert a i n extensions and additions of our obs-ervations since the publication of the paper have already been discussed. B. OCCULAR STUDIES ON COLOR IN THE DEVELOPING VEGETATIVE AND FLORAL STRUCTURES. A c h a r a c t e r i s t i c c y c l i c a l appearance of anthocyanin colors i n various plant parts was observed during the h y b r i d i -zation work. A most s t r i k i n g feature of color development, was of the i n d i v i d u a l t i l l e r from vegetative to reproductive stage. The top a u r i c l e was always bright-red compared to the lower a u r i c l e s , before the anthocyanin-free awn t i p s emerged from the boot. Later on, the anthocyanins started developing In the awn -126-t i p s . As the color development i n the awn t i p s increased, the in t e n s i t y of color i n and around the top a u r i c l e decreased. Again, i n almost a l l the v a r i e t i e s , i t was observed that the best stage for selection of a female parent f o r hybridiza-t i o n was when the top a u r i c l e was deeply colored with anthocya-nins and the color development i n the t i p s of the awns had just commenced. 1 In most of the v a r i e t i e s , the anthocyanins i n the-awn t i p s faded ax^ay around the maturity of the f l o r e t s . In most cases, i t was also observed that i n a young plant an anthocyanin coloration develops i n the basal leaf sheath f i r s t , and the a u r i c l e s are c o l o r l e s s . Then i t develops i n the a u r i c l e s , at a maximum In lowermost and least i n the uppermost. Then afte r a c e r t a i n stage of growth, the color development process appears to reverse probably,"in a u r i c l e s . Any d e f i n i t e conclusion, however, w i l l await further investigations since the phenomenon involved appears to be complicated. Again i t was observed that c e r t a i n v a r i e t i e s either did not develop color i n plant parts or i f they did, i t was almost n e g l i g i b l e for a l l p r a c t i c a l purposes.- But the awn-tips did develop anthocyanins, when the t i p s had. emerged out of the boot. In mature t i l l e r s the basal leaf sheath i s sloughed off or the sheath turns yellow and anthocyanins disappear. However, the basal l t a f sheath of young t i l l e r s , a r i s i n g i n the centre -127-of the plant do show anthocyanin coloration. One of the inte r e s t i n g features-of color development was observed i n nodes of cert a i n v a r i e t i e s ( p a r t i c u l a r l y C-54-55). In general, i t has already been reported that the nodes do not develop color unless they have elongated out of the leaf sheath (Aberg and Wiebe 19^8). The feature that the author observed was that only that part of the exserted node develops color which i s exposed to sunlight (or being more cautious, exposed to the open environments). If a part of the node i s encircled by a leaf sheath then, the part covered by the leaf sheath does not develop anthocyanin color. However, i f the sheath i s re-moved, a complete r i n g of anthocyanin color develops around the node. The broad pattern and salient features of anthocyanin color development based upon f i e l d observations have been stated above. Below are described the salient observations on some of the v a r i e t i e s studied i n a tabular form. TABLE X OBSERVATIONS ON COLOR* DEVELOPMENT AROUND TRANSITION FORM VEGETATIVE, TO REPRODUCTIVE STAGE. "VARIETY BASAL LEAF SHEATH AURICLES AWN-TIPS Golden colored Pheasant. top a u r i c l e shows bright-red color which gradually awn-tips deve-lop color i n early stages - 1 2 8 -VARIETY BASAL LEAF SHEATH AURICLES A W U - T I P S Hanna C - 5 4 - 5 5 Lion •Gopal Black Hulless Montcalm Trebi Kwan Kitchen Gatami deeply colored deeply colored colored fades on maturity only veins show color only veins show color at maturity no color very l i t t l e color 5 0 9 0 - 2 - 3 very l i t t l e no color color colored s l i g h t l y colored no color no color 3 6 - B 1 B l - 2 1 colored 3 6 - b l b l - 2 1 colored no color of growth which st a r t s fading a f t e r soft dough stage. same as i n # 1 , how-ever, the color changes to dark brown l a t e r on. same as i n # 1 , the color remains for a longer time. same as i n #1 , some times the color was present, some times i t could not be observed. no color observed. hooded variety, awns not present. color develops i n the awn-tips but changes to dark brown. as i n case of # 1 . not d e f i n i t e . * Color means anthocyanin color. -129-C. OBSERVATIONS AND RESULTS FROM P E E L I N G TISSUES AT THE HARD DOUGH STAGE OF THE GRAIN. It i s well-known that color i n the barley kernel may develop independently i n endosperm, aleurone, pericarp and chaff (Harlan, 191L(-, Miyazawa, 1918 and Smith, 1 9 5 D . The chaff i s often cemented over the pericarp around the f l i n t y stage. As such the color of the chaff masks the color of the underlyaing pericarp. Sandwiched between the aleurone and pericarp, are two membranous layers-the perisperm and spermo-derm-the remains of the nucellus, which as well, develop color i n purple v a r i e t i e s (author's observations). However, whether, t h i s color i s independently determined, i s yet to be studied. As such the actual color of these two layers may be masked by the colored pericarp. S i m i l a r l y the color of the aleurone may be masked by three layers, the perisperm-spermoderm and p e r i -carp together, and the color of the endosperm by four layers. By the peeling technique already described, i t was possible to observe and analyse the developmental aspects of color. The color of the pericarp u n t i l the milky stage i s green. In purple v a r i e t i e s , the color f i r s t of a l l develops i n the perisperm-spermoderm tis s u e s . The anthocyanin development, i n general, starts at the end of the milky stage or the beginning of soft dough stage and continues u n t i l maturity. However, i t was observed at U.B.C. that i f the ripened crop i s not harvested -130-and i s allowed to stand i n the f i e l d , sunlight and possibly-other environmental factors, cause even the brightest colors of the stable genotypes to fade. The observations on color development i n various tissues of barley kernels, upto end of the hard dough stage, are re-corded i n table XI. .lone of the v a r i e t i e s developed color i n endosperm. A l l the blue v a r i e t i e s l i s t e d contain anthocyanins only i n aleurone. Trebi has been c l a s s i f i e d as a peculiar blue variety. The reason becomes apparent'because the pericarp and perisperm-spermoderm tissues develop excessive yellow colored pigments compared to the other two v a r i e t i e s , Kwan and Mont,calm. S i m i l a r l y , 5090-2-3 has been classed as d i r t y white or grey be-cause the aleurone contains a melanic pigment. D. THE ANTHOCYANINS AND ANTHOCYANIDINS IN THE SEPARATE TISSUES OF THE CARYOPSIS. The peeling technique, made possible the study of the developmental features of anthocyanins and anthocyanidins pre-sent i n paricarp, perisperm-spermoderm and aleurone t i s s u e s . A perusal of table XI would indicate that no anthocyanin pigment develops i n the white variety, Golden Pheasant, the black variety, Lion and d i r t y white or grey variety, 5090-2-3. Simi-l a r l y , the pericarp, perisperm-spermoderm tissues of the blue v a r i e t i e s , Kwan, Montcalm and T r e b i , the black v a r i e t i e s , Lion and Gatami are without anthocyanins, or are melanic. Therefore, the only.tissues that are to be analysed for anthocyanins and TABLE XI OBSERVATIONS ON BARLEY KERNEL COLORS* IN DIFFERENT VARIETIES VARIETAL NAME VARIETAL COLOR** ALEURONE PERISPERM-SPERMODERM PERICARP LEMMA AND PALEA Golden Pheasant white No. A. No. A. No. A. No. A. Kv/an blue blue 11 i t some of the veins are i n i t i a l l y colored. Montcalm blue 11 11 i t 11 Trebi peciiliar blue 11 11 n 11 Gopal bright purple No. A. deep-red red streaky red Black Hulless purple deep blue purple purple streaky purple Lion black No. A. brown (M) very l i g h t brown mostly transparent (M). bluish powdery tinge over the brown color (M). Gatami black l i g h t blue streaky brown (M) mostly transparent with a few streaks of brown (M). outer surface black inner surface brownish-black (M). 5090-2-3 grey and d i r t y white brownish-grey (M) No. A. No. A. ? * Color means anthocyanin color. ** Color as given by-plant breeders. No. A. means no anthocyanin. (M) means melanin. -132-anthocyanidins are the pericarp and perisperm-spermoderm tissues of the purple v a r i e t i e s , Gopal and Black Hulless and the aleurone tissues of the blues, Kwan, Montcalm and Tr e b i , the black, Gatami and the purple Black Hulless, the only purple variety which develops color i n the aleurone. The anthocyanins and anthocyanidins (obtained from anthocya-nins) of aleurone tiss u e could not be determined because with the present technique of extraction, large quantities of grain with pericarp and perisperm-spermoderm peeled o f f , would be re-quired for analysis at successive intervals of development. The analysis of anthocyanins i n aleurone t i s s u e s , therefore, cannot be e f f e c t i v e l y undertaken u n t i l d i f f e r e n t techniques are available or, using the present technique, a great deal of labor i s made available. Below are described the re s u l t s obtained aft e r analysis of the pericarp and perisperm-spermoderm tissues of the two purple v a r i e t i e s . In order to determine the pattern of pigment deve-lopment, analysis was carried out at two successive i n t e r v a l s . In one case, the pigment development had just started whereas i n the other, the pigment had developed a l l over the ti s s u e s . It was observed that during l a t e r stages, the pigment develops profusely at the t i p s of the kernel tissues. a. THE ANTHOCYANINS OF GOPAL IN AWNS, HULLS, PERISPERM-SPERMODERM TISSUES DURING EARLY STAGES OF DEVELOPMENT. In Gopal, i t was found that the pigment development i s -133-Fig. 1. Semi-diagrammatic representation of the anthocyanins in awns, hulls, pericarp and perisperm-spermoderm tissues of Gopal in early stages of develop-ment. Authentic cyanidin-3-glucoside as "control"; Rf = Rf approximations x 100; butanol : acetic acid : water solvent. I H to i g i Fco r UJ o E < o CL tO 98 94 74 66 62 56 C4-44 t t t t AWNS / HULL -135-Fig. 2. Semi-diagrammatic representation of the anthocyanins of the grain tissues of Gopal and Black Hulless barley in later stages of development. Authentic cyanidin-3-glucoside as "control"5 Rf = Rf approximations x 100; butanol : acetic acid : water solvent. AWNS HULLS PERICARP PrZRISPERM AWNS HULLS PERICARP PERISPERM CYANIDIN GLUCOSIDE F « C l 3 REACTION SPOT AND Rf DESIGNATIONS CD o o m o X — TO i 1 i i cn i • 1 to ro o ro 00 ro ro CD 03 • • • • • O •» • • • 4 > • • • -\» • • O 03 00 O) rjj 03 JO 03 -136-altered during- l a t e r stages of development. and gradually, the spot C becomes a major spot. The matured kernels, as already reported, also show that the spot C i s a major spot. No attempt was made to elute and hydrolyze the anthocyanins of i n d i v i d u a l spots to t h e i r respective anthocyanidins, because large scale peeling operations could not be undertaken for reasons already stated. The spot C, as already reported, however, showed color reactions and Rf values of cyanidin - 3 - glucoside and the focus of resolution of authentic cyanidin - 3 - glucoside and the spot C on the paper-gram remained the same, when they were run i n mixtures. c. THE. ANTHOCYANIDINS OF GOPAL IN AWNS, HULLS, AND PERISPERM-SPERMODERM TISSUES DURING EARLY STAGES OF DEVELOPMENT. There are tv/o anthocyanidins v i z . pelargonidin and cyanidins which are present during early stages of development ( f i g . 3 ) . The authentic samples of cyanidin and pelargonidin were run i n mixtures with the extract and the focus of t h e i r r e s olution on the paper-gram remained the same. The f e r r i c chloride test did not a l t e r the pelargonidin color and the color of cyanidin was changed to blue as usual. Further confirmation was obtained through u l t r a v i o l e t observations. Under the long wave "S L 366O" u l t r a v i o l e t lamps, i t was found i n t h i s laboratory, that a l l , t h e anthocyanidins of barley, so far detected v i z . delphi-n i d i n , cyanidin and pelargonidin fluoresce. S i m i l a r l y the authentic samples of cyanidin and pelargonidin, available at -137-Fig. 3. Semi-diagrammatic representation of the anthocyanidins of Gopal barley grain tissues in early stages of development. Authentic cyanidin and pelar-gonidin as "controls"; Rf - Rf approximations x 100; forrestal solvent. -138-hand, also fluoresce. It was further observed that the antho-cyanins do not fluoresce and rather absorb the ultraviolet. As such, ultraviolet observations have been used as further confirmatory evidence in distinguishing between anthocyanins and anthocyanidins. The fluorescence in ease of anthocyanidins is bluish-red for delphinidin, bright red for cyanidin and orange red for pelargonidin. The ultraviolet color observations further con-firmed that the spots observed were cyanidin and pelargonidin. It may be noted that delphinidin is not present in any of the tissues though i t has been found to be present in mature kernels as reported in the earlier studies. (see pp. 165). d. THE ANTHOCYANIDINS OP GOPAL AND BLACK HULLESS IN AWNS, HULLS, PERICARP AND PERISPERM-SPERMODERM TISSUES DURING LATER STAGES OP DEVELOPMENT. In addition to the two anthocyanidins, cyanidin and pelar-gonidin (which were identified as reported above), a third fast moving (see Fig. 4) red colored pigment was detected (Rf 0.84), in a l l the tissues. It did not give a distinct f e r r i c chloride reaction and also did not fluoresce under ultra-violet. The nature of this pigment, however, is doubtful. It may again be pointed out that delphinidin s t i l l was not present. - 1 3 9 -Fig. 4 . Semi-diagrammatic representation of the anthocyanidins of Gopal and Black Hulless grain tissues in later stages of development. Authentic cyanidin and pelargonidin as "controls"; Rf approximations x 1 0 0 ; ferrestal solvent. 100 CD O 5 LTJ I -> O m c o CO -141-In absence of a dependable technique for the extraction of anthocyanins from aleurone layer, remaining half of the aleurone powder was d i r e c t l y hydrolyzed with 3 N HC1. This gave three d e f i n i t e spots with Rf values and f e r r i c chloride color reac-t i o n corresponding to delphinidin, cyanidin and polargonidin. In addition to these three spots, a very fast moving spot (Rf 0 . 8 8 ) , as reported above was also present. It was instantane-ously decolorised with f e r r i c chloride and no d e f i n i t e conclu-sions could be drawn with regard to Its nature. The r a t i o of pigment concentration of these spots as examined under the u l t r a v i o l e t was roughly 2 : 10 : 1 : 2 respectively. The spot (Rf 0 .88) did not fluoresce under the u l t r a v i o l e t l i g h t . BEHAVIOUR OF A NTII OC YAN I NS IN AQUEOUS AND ALCOHOLIC SOLVENTS An inter e s t i n g behaviour of anthocyanins In aqueous and alcoholic solvents was observed. Normally anthocyanins were extracted i n a c i d i f i e d alcohol. A f t e r reduction of t h e i r volume when the anthocyanins i n al c o h o l i c extract were brought into 1% HCl-aqueous a part of the pigment remained afloat and did not dissolve i n the aqueous solvent. This p r e c i p i t a t e was r e d i s s -olved i n \% HCl-EtOH and chromatographed along with the a c i d i -f i e d alcoholic extracts. The following r e s u l t s were obtained. -14-2-Fig. 5. Showing the effect of extractants on the stability of the anthocyanins from the Gopal grain. X 100 - I — 01 o 1 1 — ro oo -i— ~\— ro ro ro 10 <£> <D 00 r - H U L L S h PERISPERM • E T C . i- H U L L S P E R I S P E R M E T C . U H U L L S h PERISPERM <XZs> ETC. O O CYANIDIN 3- GLUCOSIDE -143-A glance at the figure 5 would reveal that the fast moving anthocyanins disappeared when the alcoholic extract was trans-ferred to aqueous solvent. It was, hoirever, observed l a t e r on that these anthocyanins conspicuously show up on the paper-gram upto about 6 hours of development and af t e r that they start de-grading and f i n a l l y disappear. The disappearance of the fast moving spots was repeatedly observed, and consequently the transfer of al c o h o l i c extracts to aqueous extracts was discontinued. -144-D I S C U S S I O N GENERAL COMMENTS ON THE, ANTHOCYANIN. LITERATURE. The anthocyanins are a frequent subject of study by gene t i c i s t s , chemists and others. In the cacao bean industry, anthocyanins are i n t e r e s t i n g l y Involved i n aromaticity. The tanners study them because the leuco-relatives i n wood extracts are responsible for the tanning properties. In the f r u i t pre-servation and canning industry they are involved i n the a t t r a c t -iveness or unattractiveness of f i n i s h e d products. The a p i a r i s t wants to get r i d of anthocyanin colors i n his products. On the other hand, the man storing apples, strawberries and ce r t a i n other products may be anxious to r e s t r i c t or encourage the anthocyanin development. The pharmacologist i s interested because they, l i k e d i g i t a l i s , are useful i n heart therapy. As vitamin P, capable of contracting blood vessels, and as substances involved i n the browning of certa i n wines and beers, the anthocyanins have attracted d i s t i l l e r s and brewers. That they may be involved as natural f i l t e r s of solar r a d i a t i o n has been suggested by botanists. Physiologists are interested i n t h e i r sex hormonal and optico-physiological a c t i v i t y . Plant breeders and agrono- . mists view t h e i r presence and absence as a c r i t e r i o n of the developmental phase of plants. Their f u n g i s t a t i c and b a c t e r i -c i d a l a c t i v i t y has attracted the attention of pathologists. The biochemists are interested i n t h e i r biogenesis; t h e i r role -145-i n redox system and consequent protective action on vitamin C and related compounds. The plant taxonomists consider them as a character of c l a s s i f i c a t i o n . The evolutionists are interested i n t h e i r systematic and geographical d i s t r i b u t i o n . No wonder then, that these secondary plant products, which primarily vrere studied for t h e i r ocular properties, have emerged out as a major f i e l d of inquiry, p a r t i c u l a r l y , over the l a s t decade. The l i t e r a t u r e i s now very large. The chemistry of anthocyanins and the broad pattern of t h e i r inheritance have been comprehensively worked out. Not much i s known of t h e i r formation i n i n vivo, though genetical studies i n t h i s d i r e c t i o n have been very h e l p f u l . The series of biochemical approaches of Dr. Thimann and his co-workers i n i t i a t e d i n 1947 are notable and t h e i r future work should be very f r u i t f u l . The physiological role of anthocyanins s t i l l l i e s i n obscurity. However, detailed and systematic study of the time of appearance of anthocyanins i n various plant parts, i . e . t h e i r development and disappearance i n these organs during ontogeny, w i l l greatly help construe t h e i r physiological r o l e . Observa-t i o n a l studies of t h i s kind may be adventageously supplemented with c y t o l o g i c a l and histochemical studies. More should be undertaken. E f f o r t s made here may not only aid i n understanding the orgns of these pigments but may give some indications as to why are they present i n the plasma, the vacuoles, the membranes, the centromeres and the chondriosomes. - 1 4 6 -EXTRACTION OF ANTHOCYANINS FROM BARLEY KERNELS. From most plant organs, anthocyanins may be obtained quite e a s i l y using the extractants of the Robinsons, Kerrer and others. Few studies of anthocyanins i n grain or seeds have been made. The extraction of anthocyanins from aleurone tissues of barley i s s t i l l a problem and that from other grain tissues not easy. Forsyth (1952 a) encountered a similar problem i n the extraction of anthocyanins from cacao beans. He found that the anthocyanins which o r i g i n a l l y occured i n vacuoles of the cacao bean l a t e r migrated into other sections of the tiss u e and were retained by adsorption. He observed that k i l l i n g of the dehy-drated tissue by high or low temperatures prevented the migra-t i o n and enabled the alcoholic extraction of the pigment. It may be pointed out that a precise method of extraction f o r aleurone tissues i s highly important In barley studies. A perusal of table XI indicates that anthocyanins i n the three blue v a r i e t i e s , and one black variety are present only i n the aleurone ti s s u e s . The accurate analysis of these v a r i e t i e s , then, would depend upon a suitable method of extraction. It may be pointed out here that i n the e a r l i e r work (Mullick, F a r i s , Brink and Acheson 1958), d i f f i c u l t i e s were experienced i n the extraction of anthocyanins from blue and the black v a r i e t i e s . The extrac-tions were incomplete. Owing to poor y i e l d of anthocyanins i n the extract from blue and black v a r i e t i e s they could not be hydrolysed e f f e c t i v e l y for anthocyanidins. Because of these -14-7-d i f f i c u l t l e s , the authors had to resort to the method of hydro-l y s i s commonly used i n the hydrolysis of leuco-anthocyanins. It has been already reported that a c i d i f i e d acetone gave a complete extraction and l e f t the pel l e t c o l o r l e s s . However, the extracts were not stable over a period of time. These d i f f i c u l t i e s might be overcome by studying the chromatographic and spectrographs behaviour of authentic anthocyanins dissolved i n a c i d i f i e d acetone. This however, would involve the synthesis or procurement of a number of authentic anthocyanins. METHODS OF VOLUME REDUCTION AND PURIFICATION. After a preliminary reduction of the anthocyanin extracts i n vacuo, they were usually p u r i f i e d with large quantities of ethyl acetate and benzene. This procedure, as generally con-ducted, concentrated a reasonably pure extract of anthocyanins to droplet proportions. This, however, can only be done i n case of aqueous extracts of anthocyanins. Because of the variations observed i n the behaviour of anthocyanins when they are trans-ferred from alcoholic to aqueous solutions, the aqueous extrac-t i o n has been discontinued as fa r as possible. For reducing the volume and purifying the alcoholic extracts, the p r e c i p i -tations by lead acetate as reported by L i (1956) and Sadow (1953) should, perhaps, be employed i n future work. Yellow pigments (probably flavones ), extract with antho-cyanins and are d i f f i c u l t to separate from them, probably -lH - 8 -because of the great s i m i l a r i t y of t h e i r chemical structure and properties. Preliminary attempts with paper electropho-r e s i s indicate the p o t e n t i a l usefulness of t h i s t o o l i n sep-arating the yellow pigments from the anthocyanins. This would be h e l p f u l i f p a r a l l e l studies on other flavonoid constituents of barley are undertaken to ascertain t h e i r biogenetic r e l a t i o n -ships to anthocyanins, OCCULAR STUDIES ON COLOR IN THE; DEVELOPING VEGETATIVE OR FLORAL STRUCTURES. The flavonoid and carotenoid colors, c h a r a c t e r i s t i c a l l y appear i n most of the flowering plants, at the time when the reproductive organs develop. Insects s i m i l a r l y develop varied colorations, i n many cases, a f t e r t r a n s i t i o n from feeding to reproductive stage. In birds, the plumage often develops d i s t i n c t i v e coloration during the mating period. In c e r t a i n f i s h , pigment variations are known to occur during spawning, so much so, that the water looks red. A question arises then, why these pigments develop or i n t e n s i f y so generally i n plants and animals during the t r a n s i -t i o n from vegetative or growth stages to reproductive stages. That the carotenoids and flavonoids are involved as sex hormones i n the green alga Chlamydomonas has been amply demons-tra t e d by Moewus (195V) . The observations i n barley ( see table X) that the awn t i p s invariably develop anthocyanins -14-9-even i f the variety i s colorless and the appearance of color i n the awn t i p s at a time when f l o r e t s are a c t i v e l y involved i n the reproductive process, are suggestive that the anthocyanin pig.--ments may be involved, i n seme way, i n the reproductive process. That there i s a c o r r e l a t i o n between the presence of anthocyanins i n vegetative and reproductive organs of plants has been reported by Bottazzi ( 1 9 5 0 ) . The suggestion becomes, more plausible i n l i g h t of the findings of Moewus (1954-) that cis-cinnamic acid, one of the degradation products of anthocyanins, i s involved as a hormone which regulates the meiotic a c t i v i t y . The author's observation that anthocyanin colors i n the top au r i c l e s fade and that, c o i n c i -dently, color i n the awn t i p s accentuates at the time when the reproductive organs are ac t i v e l y developing, may suggest that anthocyanins or t h e i r degradation products may be translocated to reproductive organs. In barley, three anthocyanidins v i z . pelargonidin, cyanidin and delphinidin, have been detected and i t might be of interest to see If there i s a preponderance of any or a l l of the degradation products of these anthocyanidins v i z . p-hydroxybenzoic acid, protocatechuic acid and g a l l i c acid respectively i n the reproductive organs. The observations on the behaviour of color i n the au r i c l e s may obtain further sup-port from Ermolaeva's findings (194-8) v i z . reduction i n the content of pigments or t h e i r complete disappearance may occur i n plants during t r a n s i t i o n from vegetative to reproductive stages. -150-THE PEELING TECHNIQUE. The peeling technique has contributed greatly to the study of anthocyanin development i n the d i f f e r e n t tissues of the barley caryopsis. Also i t appears to have a potential i n c l a s s i f y i n g barley v a r i e t i e s . The c l a s s i f i c a t i o n of barley v a r i e t i e s i s d i f f i c u l t p r i -marily because the h u l l s and pericarp lose t h e i r transparency a f t e r maturity ( p e r t i c u l a r l y so i n humid climates), thereby masking the color of the aleurone or perisperm-spermoderm layers. The problem of c l a s s i f i c a t i o n i s made further d i f f i c u l t by environmental influences which may cause color to fade af t e r a crop matures. The fading of color may have a temporal r e l a -tionship with environments. The time of harvesting, for example, may cause variations i n the kernel colors of the most stable genotypes. It i s , therefore, f e l t that c l a s s i f i c a t i o n based sol e l y on the matured kernels, may be misleading. Inasmuch as some of the d i f f i c u l t i e s referred to may be obviated, i t i s f e l t , the peeling technique can be advantageous-l y used by breeders i n c l a s s i f y i n g barley v a r i e t i e s . Using t h i s technique a number of " d i f f i c u l t " v a r i e t i e s were c l a s s i f i e d (table XI ). ANALYSIS OF THE ANTHOCYANINS AND ANTHOCYANIDINS IN THE SEPAP.TE TISSUES OF THE CARYOPSIS. A s t r i k i n g feature of anthocyanin development i n barley -151-i s p r e s e n t e d i n F i g s . 1 and 2 , w h i c h show t h e number o f a n t h o -c y a n i n s p r e s e n t i n t h e e a r l i e r s t a g e s of development. On comparing t h e s e two f i g u r e s , i t becomes apparent t h a t some o f t h e a n t h o c y a n i n s w h i c h move s l o w l y under t h e c h r o m a t o g r a p h i c c o n d i t i o n s , s t a r t d e v e l o p i n g d u r i n g t h e l a t e r s t a g e s of growth of t h e pigment. I t i s a l s o apparent t h a t some o f t h e " f a s t -moving" a n t h o c y a n i n s s t a r t d i s a p p e a r i n g d u r i n g l a t e r s t a g e s of growth. B e h a v i o u r s i m i l a r t o t h e " f a s t - m o v i n g " and "slow-moving" a n t h o c y a n i n s of t h e g r a i n has been o b s e r v e d i n t h e d e v e l o p m e n t a l s t u d i e s on t h e p l a n t t i s s u e s (not r e p o r t e d i n t h i s t h e s i s ) . The g e n e r a l d e v e l o p m e n t a l f e a t u r e s of a n t h o c y a n i n s i n a l l t h e mater-n a l t i s s u e s (2n) t h e n i s b r o a d l y s i m i l a r i . e . i n t h e e a r l i e r s t a g e s of growth, when some o f t h e " f a s t - m o v i n g compounds" a r e p r e s e n t . L a t e r on, t h e s e compounds may g r a d u a l l y d i s a p p e a r and some of the"slow-moving" ones appear. There a r e c o n s p i c u o u s changes i n t h e i r r e l a t i v e i n t e n s i t y though t h e " f a s t - m o v i n g " compounds a r e much g r e a t e r i n q u a n t i t y . L a t e r on t h e spot C ( p r o b a b l y c y a n i d i n - 3 - g l u c o s i d e ) emerges as t h e major spot w i t h much g r e a t e r i n t e n s i t y and q u a n t i t y t h a n a l l t h e o t h e r s . The p i c t u r e o f t h e s e d e v e l o p m e n t a l s t u d i e s a r e s u p p o r t e d by t h o s e r e p o r t e d e a r l i e r (see pp. M+9 of t h e paper s u b m i t t e d as appendix I I ) . I t may be n o t e d t h a t no " f a s t - m o v i n g " a n t h o -c y a n i n s a r e p r e s e n t i n mature k e r n e l t i s s u e s , t h e spot C i s prominent and t h e "slow-moving" compounds a r e more i n number and g r e a t e r i n q u a n t i t y t h a n t h o s e r e c o v e r e d from r e l a t i v e l y immature s t a g e s . T h i s i n d i c a t e s t h a t d u r i n g f l i n t y s t a g e s , "slow-moving" compounds are being elaborated. The anthocyanidins present an interesting pattern of development. A comparison of figures 3 and 4 with the figure on page 4 5 1 of the paper (submitted as appendix II) reveals that delphinidin was not present u n t i l the hard dough stage. Delphi-n i d i n could not be recovered from plant tissues at any stage (the d e t a i l s of t h i s work are not included). The question arises then as to how, when, and where does the delphinidin, recorded for mature grains, appear. There are two p o s s i b i l i t i e s . F i r s t l y i t may again be noted here that the method of hydrolysis used i n our e a r l i e r work was based, upon the method usually used in obtaining anthocyanidins from leuco-anthocyanins. Because of t h i s method, we could recover the two anthocyanidins, del p h i n i d i n and cyanidin from leuco-anthocyanins of the white variety, Golden Pheasant and the black variety, Kwan. This suggests that the delphinidin i n a l l the v a r i e t i e s might have been recovered from leuco-anthocyanins. The appearance of delphinidin, however, cannot be merely explained on t h i s basis, because, when the anthocyanin extracts of mature purple v a r i e t i e s were hydrolysed, the delphinidin was recovered i n addition to cyanidin and pelargonidin. This suggest that the delphinidin might be formed during the f l i n t y stage or else, some of the leuco-anthocyanins giving r i s e to delphinidin on hydrolysis might be soluble i n the aqueous and alcoholic s o l -vents used for anthocyanins. Because of t h e i r s o l u b i l i t i e s , they might have appeared i n the anthocyanin extracts and on hydrolysis -1 ? J > -yielded delphinidin. This can however, be checked by the extrac-t i o n of v a r i e t i e s containing leuco-anthocyanins with a l c o h o l i c solvents normally used for anthocyanin extraction. If the hydrolysis of these extracts, y i e l d s delphinidin, i t w i l l be established, doubtlessly, that the delphinidin i s derived from leuco-anthocyanins. A further interesting picture i n t h i s connection i s provided by the purple variety, Black Hulless. The pericarp, and p e r i -sperm-spermoderm do not contain delphinidin u n t i l hard dough stage. However, during the hard dough stage, when these outer layers are peeled off and the aleurone i s hydrolysed by the "method of leuco-anthocyanins", delphinidin i s recovered, there by indicating that delphinidin i s not formed during the f l i n t y stage. The res u l t s obtained af t e r hydrolysis of anthocyanins •• extracted from aleurone t i s s u e s , did indicate the presence of a weak delphinidin spot, though no d e f i n i t e conclusions could be drawn whether.the delphinidin i s present only i n the aleurone tis s u e s . -154-SDMMARY AND CONCLUSIONS. 1. It i s clear, from a survey of an extensive literature on anthocyanins, that very l i t t l e is known, as yet, of the role of these flavonoid pigments in plant physiology. Nonetheless, as common secondary substances, they are important economically and their chemistry is well known. 2. It i s also evident from the literature that some of the most interesting studies of gene controlled reactions i n higher plants have been demonstrated with anthocyanins. Bar-ley, i t would appear, possesses a suitable range of genes con-t r o l l i n g anthocyanin produc tion in soma and grain and Is good material for extending our knowledge of gene controlled reac-tion in higher plants. 3. Extraction and purification of anthocyanins from barley grain presents problems not encountered, by and large, in other plant tissues. Special techniques using, for example, a sonic oscillator, and alternate freezing and thawing of extracts have aided in the production of reasonably pure anthocyanin and an-thocyanidin extracts. Extraction from the very proteinaceous aleurone layer of the grain has, however, not been complete and presents d i f f i c u l t i e s in extraction yet to be surmounted. Extractions of anthocyanins from plant tissues presents l i t t l e d i f f i c u l t y . 4. Careful dissection of the barley grain at the early -155-dough stage of hull from pericarp, and pericarp from perisperm-periderm, and perisperm-periderm from aleurone tissues has greatly extended the possibilities in the study of anthocyanin development in the caryopsis. 5. Paper chromatography has proven to be an excellent means of separating anthocyanins and anthocyanidins in the barley soma and grain. It has also assisted in partially characterizing them. Special techniques, such as the chromato-stripe technique, have made the chromatography of the "nins" and "dins" more precise. 6 . Anthocyanidins in twelve barley varieties were studied. It is reasonably certain that cyanidin, delphinidin, and pe-largonidin occur in barley; apparently other anthocyanidins are present but are not stable under the conditions of our i n -vestigation. Pelargonidin and cyanidin, but not delphinidin, have been obtained from the maternal tissues such as the leaf sheath, awns, and pericarp of the barley plant. Delphinidin has been obtained only from the grain and may originate in the aleurone tissues or may come from leucoanthocyanins; in color-less varieties, i t i s certain that the delphinidin comes from leuco compounds but there is some reason to believe that in colored varieties some delphinidin may come from aleurone t i s -sues. In the grain of blue varieties, delphinidin i s relatively more abundant than cyanidin but, in purple varieties, the -156-reverse appears to be true. Pelargonidin appears only in the purple varieties. 7. Leucoanthocyanins which yield, on hydrolysis, cyanidin and delphinidin occur in the white barleys, such as Golden Pheasant, and in black barleys, such as Lion, which contain no anthocyanin. They may well occur with anthocyanins in the blue and purple barley varieties, but methods for their segre-gation have not been f u l l y worked out. 8. The anthocyanins of barley have been characterized only generally. In the f i r s t place, i t i s apparent from ocu-lar study of pigment occurrence and disappearance and from the chromatographic study of anthocyanin-containing extracts that these compounds are intimately related to phasic development in the barley plant. Given genetical competence in the caryo-psis tissues, for example, anthocyanins develop f i r s t in chaff, then in perisperm and spermoderm, then in pericarp, and f i n a l l y in aleurone. 9 . The peeling technique can greatly assist the breeder for accurate classification of the barley kernels. -157-APPENDIX I COLOR REACTIONS OF ANTHOCYANINS* On addition of sodium acetate to the o r i g i n a l solution, the following colors are observed: C a l l i s t e p h i n : d u l l browninsh V i o l e t - r e d . Pelargonin: bright b l u i s h red. Peonidin-3-glycosides: similar to c a l l i s t e p h i n but brighter. Peonin: r e d - v i o l e t . Cyanin;: v i o l e t . Mecocyanin, chrysanthemin: v i o l e t - r e d . Oenin: d u l l v i o l e t . Delphinidin glycosides: blue-violet to blue. This reaction i s subject to interference from iron s a l t s and tannins. Pelargonin (3 , 5-diglycoside). V i o l e t c o l o r a t i o n with aqueous N a 2 C 0 3 $ t h i s becomes greenish-blue on addition of ace-tone. Decisive confirmation i s obtained by adding one-quarter the volume of concentrated HC1 to the solution and b o i l i n g f o r about a minute. On extraction with AmOH a green fluorescence due to pelargonenin w i l l be observed. Pelargonidin - 3-glycosides (e.g., c a l l i s t e p h i n ) . Red-violet color with N a 2 C 0 3 that i s rather stable towards NaOH. No other anthocyanin type behaves s i m i l a r l y . Peonin (3,5-diglycoside). Blue coloration with N a 2 C 0 3 # Cyanin (3 , 5-diglycoside). Rich, pure blue with N a 2 C 0 3 , unstable to NaOH. Malvin (3o 5-diglycoside). Bright greenish-blue with Na2C03» Oenin (malvidin-3-glucoside). Blue-violet with N a 2 C 0 3 , unchanged by NaOH. When new anthocyanins are encountered the tests described -158-above are useful in allowing an estimate to be made of the extent and pattern of hydroxylation, but are incapable of establishing the details of new structures. In such cases the isolation of the crystalline pigment in amounts sufficient for degradation experiments (Kerrer et. a l . , 1927, 1929, 1932), i s necessary. The color tests are likewise useless in elucidating the nature of the sugars present in the glycosides. The subtle differences in the behavior in such tests between glucosides and galacto-sides, for example, would not permit their positive identifica-tion by this means. Distinctions between glycosides containing different sugar types - hexosides, pentosides, methylpentosides, dihexo-sides or pentosehexosides - can be made with reasonable cer-tainty by careful measurements of the distribution of anthocya-nins between amyl alcohol and dilute hydrochloric acid. This procedure, extensively used by Robinson and his co-workers, i s described in detail by Robinson and Todd (1932). APPENDIX II Reprinted from Canadian Journal of Plant Science 38 : 445-456, October, 1958 A N T H O C Y A N I N S A N D A N T H O C Y A N I D I N S O F T H E B A R L E Y P E R I C A R P A N D A L E U R O N E T I S S U E S D . B . MULLICK 1 , D . G. FARIS 2, V . C . BRINK 3 AND R. M . ACHESON 4 [ R e c e i v e d f o r p u b l i c a t i o n M a y 2 6 , 1958] ABSTRACT The anthocyanins and anthocyanidins of the pericarp and aleurone tissues of one white, three blue, two purple, and two black barley varieties were isolated by paper chromatography. Two anthocyanins, " B " and " C " (per-haps cyanidin-3-glucoside), occurred in one black and in the blue and purple varieties. Additionally, of three anthocyanins found in the two purple varieties, two, " D " and " E " , were common to both and one, " F " , was found only in the variety Gopal. Also found in the purple varieties was a poorly re-solved group of anthocyanins designated as "A". Two anthocyanidins, delphinidin and cyanidin, were found in all varieties and one, pelargonidin, was found only in the purple varieties. It is probable that anthocyanins A and Ai are delphinidin derivatives; anthocyanins C and D, cyanidin deriva-tives; and anthocyanins E and F , pelargonidin derivatives. The relation-ships of the anthocyanins to colour inheritance patterns were discussed. Colour may develop independently in the endosperm, aleurone, pericarp, and chaff of the barley grain (11, 17). The grain at maturity may be black, blue, purple, red, yellow, grey, white, or an intergrade of these. The inheritance of the principal colour genes has been reviewed by Smith (17). The expression of the colour genes may be modified considerably by variations in climate and soil (7, 15, 18) and may occur late in development of the plant (8). Inheritance in pericarp and chaff is, of course, maternal and diploid; in aleurone and endosperm, triploid and maternal-paternal. Colour in pericarp may mask colour in inner tissues. The pigments largely responsible for grain colours are flavonoid; purple, blue, and red are given by anthocyanins (7, 8, 4, 1) and other flavonoids give brown and yellow and act as co-pigments of anthocyanins. Melanin-like pigments occur in black and possibly in brown and grey grains (7, 4, 1). Colour in barley is useful, superficially, to distinguish one variety from another (1, 5) and may serve as a hallmark of quality, viz., Canadian malting barley is commonly blue. A more fundamental role in feed and malt quality has been suggested (6, 10, 16) for the barley pigments. The present study, restricted though it is to anthocyanins and anthocyanidins of the pericarp and aleurone, was undertaken to provide a chemical basis for studies of barley colour inheritance and, secondly, to further biogenetic and quality studies. MATERIALS Eight barley varieties were selected from fifty-seven immediately available for pigment extraction, viz., the black barley varieties, Lion and Gatami; the purple, Black Hulless and Gopal; the blue, Kwan, Mont-1 G r a d u a t e A s s i s t a n t , D i v i s i o n of P l a n t S c i e n c e , U n i v e r s i t y o f B r i t i s h C o l u m b i a , V a n c o u v e r , B . C . 2 F o r m e r G r a d u a t e A s s i s t a n t , D i v i s i o n of P l a n t S c i e n c e , U n i v e r s i t y o f B r i t i s h C o l u m b i a ; n o w w i t h t h e M i n i s t r y of A g r i c u l t u r e , N i g e r i a . 1 P r o f e s s o r , D i v i s i o n of P l a n t S c i e n c e , U n i v e r s i t y of B r i t i s h C o l u m b i a , V a n c o u v e r , B . C . 4 D e p a r t m e n t of - B i o c h e m i s t r y , O x f o r d U n i v e r s i t y , O x f o r d , E n g l a n d . 445 -160-446 CANADIAN JOURNAL OF PLANT SCIENCE ' [Vol. 38 calm, and Trebi ; and the white, Golden Pheasant. The varieties were chosen for the uniformity of their colour development in the University plots. The seed stocks of the varieties Gopal and Golden Pheasant were given to the authors by G . A . Wiebe, Bureau of Plant Industry, United States Department of Agriculture; the seed stocks of the other varieties were provided by D . G . Hamilton, Chief, Cereal Division, Canada Depart-ment of Agriculture, Ottawa. METHODS (a) Extraction of Anthocyanins T o obtain material with as much aleurone and pericarp tissue as possible, barley grains were pearled well into the endosperm. Five to eight grams of the powder from the pearling operation were collected, scanned with a magnet to remove any metallic bristles, and 1 per cent hydro-chloric acid was added at the rate of 8 to 10 ml. for each gram of dust. The material was then extracted in a 1 0 K C Raytheon sonic oscillator for 20 to 30 minutes, withdrawn with a pro-pipette, and centrifuged in polythene tubes at 14,000 r.p.m. for 5 to 7 minutes. The centrifugate was re-ex-tracted with 1 per cent hydrochloric acid and again centrifuged. The combined supernatants were placed in the refrigerator overnight. After thawing, a proteinaceous coagulum formed and was removed by centri-fuging at 14,000 r.p.m. for 5 to 7 minutes. Volume was reduced in vacuo at 30°C. and further centrifugation, freezing, and volume reduction was undertaken as needed to obtain a clear anthocyanin-containing extract of 1 or 2 ml. volume. E t h y l acetate (free from ethanol and acetic acid), added slowly and in quantity, was vigorously shaken with the extract. The mixture was allowed to stand for 10 minutes. The ethyl acetate removed water and some interfering substances from. the anthocyanin extract. Repeated scrubbing with ethyl acetate reduced the pigment extract to 0.2 to 0.3 ml . E t h y l acetate remaining in the extract was removed in benzene. In some instances, where the anthocyanin concentrations in aleurone and pericarp were low, rapid extraction was obtained with 1 per cent hydrochloric acid in methyl alcohol rather than in water. After extraction with the methanolic solvent, volume was reduced in vacuo to about 10 ml . and then about 20 ml. of 1 per cent aqueous hydrochloric acid was added. Ten minutes' centrifugation at 14,000 r.p.m. then brought down a large amount of colloidal material which was discarded. Volume was reduced in vacuo to about 2 ml. and again any colloidal material which appeared was removed by centrifugation. Purification of the extract with ethyl acetate and benzene was carried out as previously. (b) Paper Chromatography of the anthocyanins Ascending paper chromatography at 30°C. was employed in most cases. Whatman No. 1 filter paper was used generally but, for the banding of large volumes, Whatman 3-mm. was preferred. The developing solvent which gave best results was the organic phase of a butanol-acetic acid-water in the proportions of 4:1:5 by volume. October, 1958] M U L L I C K E T A L . C O L O U R I N B A R L E Y 447 In those instances where spots were difficult to resolve, as in the case of the A and D - E groups of the purple barleys, re-chromatography was necessary. Two or three applications of the anthocyanin extract were applied to Whatman 3-mm. paper on a kymograph drum with an auto-matic pipette designed by the senior author. The solvent was allowed to run off the chromatogram for 3 days during which time a reasonable resolution of the anthocyanin bands was obtained. The bands were eluted with 1 per cent hydrochloric acid in methanol or ethanol and re-chromato-graphed on Whatman No. 1 paper to obtain Rf values. The time required for good resolution with ascending chromatography on No. 1 paper was usually 24 hours. Some slow-moving anthocyanins found in the purple varieties tended to be unstable during solvent develop-ment and could not be assigned definite Rf values. The ferric chloride test was carefully applied to the anthocyanin spots; concentration was important and was varied to suit the circumstance from 0.05 to 1.0 per cent. Spots to be tested were cut out and slipped slowly sideways into the ferric chloride solution. Crit ical trials were run with cyanidiri-3-glucoside alone and in combination with extracts from the eight varieties. Concentrations of the anthocyanins (and anthocyanidins) were ocularly estimated from the chromatospots. The ratios reported were always the result of the observations of two or more of the authors. Attempts at more objective presentation were not successful. (c) Hydrolysis of Anthocyanins and the Extraction of the Anthocyanidins The following procedure gave the best extracts for the chromatography of the anthocyanidins. To 3 grams of the powder from the purple barley pearling and 8 grams from blue, black, or white barley pearlings, 3 N hydrochloric acid was added at 15 ml. per gram. Hydrolysis for 30 minutes at near boiling temperatures was followed by centrifugation at 8000 r.p.m. for 15 minutes. T o the supernatant fluid, iso-amyl alcohol was added at 1 ml. per 5 ml. of extract. The passage of the anthocyanidins from water to alcohol was best achieved by adding at one time a small amount of iso-amyl alcohol, shaking vigorously, and drawing off the alcoholic phase with a pro-pipette. The procedure was repeated to leave the hydrochloric acid free of anthocyanidins. The combined alcoholic fractions to which 1 or 2 ml. of 1 per cent aqueous hydrochloric acid had been added were mixed with petroleum ether in generous amounts (five to ten times the total volume of acid-alcohol solution) with vigorous shaking. Pelargonidi" particularly was difficult to move from the iso-amyl alcohol-petroleum ether to the acid-water phase. Accordingly, the procedure had to be repeated to be sure of complete displacement. After separation of the phases, 2 to 3 drops of iso-amyl alcohol were added to the acid-water phase which contained the anthocyanidins and again the anthocyanidins moved into the alcohol. Usually the iso-amyl alcohol extract was pure enough for chromatography. (d) Paper Chromatography of the Anthocyanidins The anthocyanidins from the barleys were chromatographed on Whatman No. 1 paper by the ascending technique, using the Forrestal -162-448 CANADIAN JOURNAL OF PLANT SCIENCE [Vol. 38 solvent (water-hydro-chloric acid-acetic acid, 10:3:30 by volume). Authentic cyanidin and pelar-gonidin were chromatographed alone and mixed with extracts as controls. To relate anthocyanins to "parent" anthrocyanidins, stripes of anthocyanin extract were applied to Whatman 3-mm. paper and bands eluted as described before. After hydrolysis of the eluates, the anthocyanidins were collected and chromatographed in the usual way. (e) Histochemical Tests for Anthocyanin in the Aleurone and Pericarp Tissues The caryopses of the eight barley varieties selected for the special study of anthocyanidins and anthocyanins by paper chromatography were, with others, sectioned freehand and with a sliding microtome in a manner T A B L E 1.—ANTHOCYANINS IN THE PERICARP AND ALEURONE OF EIGHT VARIETIES OF BARLEY P l a n t b r e e d e r s ' c o l o u r c l a s s i f i c a t i o n B l u e P u r p l e P u r p l e B l a c k B l a c k C h a r a c t e r i s t i c s R f X 100 c o l o u r F e C h r e a c t i o n c o n c e n t r a t i o n r a t i o R f X 100 c o l o u r F e C l a r e a c t i o n c o n c e n t r a t i o n r a t i o R f X 1 0 0 c o l o u r F e C l a r e a c t i o n c o n c e n t r a t i o n r a t i o R f X 100 c o l o u r F e C l s r e a c t i o n c o n c e n t r a t i o n r a t i o R f X 1 0 0 c o l o u r F e C l a r e a c t i o n c o n c e n t r a t i o n r a t i o R f X 1 0 0 c o l o u r F e C l a r e a c t i o n c o n c e n t r a t i o n r a t i o R f X 1 0 0 c o l o u r F e C l a r e a c t i o n c o n c e n t r a t i o n r a t i o R f X 100 c o l o u r F e C l a r e a c t i o n c o n c e n t r a t i o n r a t i o R f X 100 c o l o u r F e C l a r e a c t i o n c o n c e n t r a t i o n r a t i o C h r o m a t o - s p o t s ; s o l v e n t f r o n t -B D < 1 3 < 6 < 1 3 < 6 N o a n t h o c y a n i n p r e s e n t 0 2 1 - 2 2 B - R B - B l 3 2 8 - 3 0 R B 1 0 0 0 2 1 - 2 2 B - R B - B l 3 2 8 - 3 0 R B 1 0 0 0 2 1 - 2 2 B - R B - B l 3 2 8 - 3 0 R B 1 0 0 1 3 - 1 6 B > 1 2 1 - 2 2 R - B r B 2 2 8 - 3 0 R B 15 3 6 - 3 7 L - R 3 4 1 L - R n o 6 > 1 2 1 - 2 2 B - R B - B l 4 2 8 - 3 0 R B 5 3 6 - 3 7 L - R 2 4 1 L - R n o 2 N o a n t h o c y a n i n p r e s e n t 2 1 - 2 2 2 8 - 3 0 0 0 0 R - B R B - B l B 3 1 0 2 8 - 3 0 0 0 0 R B B M o r e p i g m e n t t h a n i n T r e b i F l a v o n o i d s o t h e r t h a n a n t h o c y a n i n s a b u n d a n t B r B r o w n R R e d B B l u e L - R L i g h t R e d V - L - R V e r y l i g h t r e d B l B l a c k -163-October, 1958] <?4 M U L L I C K E T A L . — C O L O U R I N B A R L E Y 449 RF x 100 ro o TRAILING SPOTS r s f— O CT) O DISTINCT SPOTS A > oo o o m LEUCO-ANTHOCYANINS LEUCO - ANTHOCYANINS CD o m FIGURE 1. Diagrammatic representation of the anthocyanin chromatogram for eight barley varieties and authentic cyanidin-3-glucoside. similar to that reported by Harlan (7). Sections were placed on dry slides and cover slips were fastened over them by sealing two opposite sides with paraffin. Sections were observed dry and, in the presence of 2 per cent hydrochloric acid or 2 per cent ammonium hydroxide, run under the cover slip. Some of the reactions were unsatisfactory because of the presence of coloured concomitant substances and because of variations within the caryopses of a given barley variety. 450 C A N A D I A N J O U R N A L O F P L A N T S C I E N C E [Vol. 38 RESULTS (a) Distribution, Number, Nature, and Concentration Ratios of the Antho-cyanins Some of the data on the anthocyanins in the aleurone and pericarp tissues of the eight selected barley varieties are given in Table I and Figure I. Anthocyanins, it can be seen, are not present in the white barley Golden Pheasant although the presence of leucoanthocyanins can be confirmed. Two distinct anthocyanins, spots B and C, are present in the three blue varieties, K w a n , Trebi , Montcalm, and the black variety Gatami. The anthocyanin C gives the colour reactions and Rf values of cyanidin-3-glucoside. The other anthocyanin B is probably a delphinidin glycoside. The concentration ratios, given in Table I, are to be applied only within varieties not between varieties. However, it may be noted that total pigment extracted from Montcalm was much less than from Trebi or Kwan . Trebi contained much flavonoid co-pigment which modified the anthocyanin colour in vivo. In all the blue varieties, there was about three times as much delphinidin glycoside as cyanidin glycoside. In the purple varieties, Black Hulless and Gopal , anthocyanins corresponding to spots B , C , D , and E are distinct. Anthocyanins B and 'C in Black Hulless are chromatographically the same as those in the blue varieties. In the variety Gopal, anthocyanin C is chromatographi-cally identical with anthocyanin C of Black Hulless and of the blue varieties, but anthocyanin B may differ in the glycosidic part of the molecule. It is rusty red rather than bluish red in colour. The concentration ratio of the B and C anthocyanins in the two purple varieties is very different. In both purple varieties, there occurred a mixture of red-brown compounds designated in Table I as Ai and A which could not be readily resolved in the chromatograms. The components of the group did not separate on elution and re-chromatography, and they oxidized rapidly to blue-black materials. Delphinidin derivatives occur in the mixture but whethei only these are present is uncertain. Anthocyanins D and E were found only in the purple varieties. Anthocyanin E is a pelargonidin derivative and its colour is not altered by ferric chloride. Distinctive ferric chloride reactions could not be obtained with D . The spot F was always given by extracts from Gopal but not from Black Hulless; it was perhaps an anthocyanin, but too little was present to give a reliable ferric chloride reaction. In Gopal the major anthocyanins contributing to colour were C, E , and D ; in Black Hulless, B and C. Gopal yielded more pigment than Black Hulless but both gave more than any of the blue or black varieties. L ion , a black variety, yielded no anthocyanin but Gatami, the other black variety, gave two anthocyanins, B and C, which appeared to be identical to and in about the same concentration ratio as the B and C anthocyanins of the three blue varieties. (b) Distribution, Number, and Concentration Ratio of Anthocyanidins The data for the anthocyanidins of the aleurone and pericarp tissues of the eight barley varieties are given in Table 2 and Figure 2. Cyanidin October, 1958] MULLICK ET AL.—COLOUR IN BARLEY 451 s 4 > 9ot Rp X 100 o CD o ~~I— CO o o FIGURE- 2 . Diagrammatic representation of the anthocyanidin chromatogram for eight barley varieties and authentic pelargonidin and cyanidin. and delphinidin were obtained from the caryopses of all varieties and, in all but the purple varieties, the delphinidin was more plentiful than the cyanidin. In the purple varieties, cyanidin occurred in relatively larger amounts than delphinidin and, in addition, pelargonidin was present. 452 CANADIAN JOURNAL OF PLANT SCIENCE [Vol. 38 T A B L E 2.—ANTHOCYANIDINS IN THE PERICARP AND ALEURONE TISSUES OF EIGHT VARIETIES OF BARLEY Variety Breeders' colour class Characteristics Chron sob D lato-spot /ent fron C s; t-> P 1 Rf X 100 34 54 Golden Pheasant White colour B-R R FeCI3 reaction B B concentration ratio 3 1 U.V. B-R R 2 Rf X 100 34 54 Kwan Blue colour B-R R FeCl3 reaction B B concentration ratio 3 1 U.V. B-R R 3 Rf X 100 34 54 Trebi Blue colour B-R R FeCl 3 reaction B B concentration ratio 3 1 U.V. B-R R 4 Rf X 100 34 54 Montcalm Blue colour B-R R FeCl3 reaction B B concentration ratio 3 1 U.V. B-R R 5 Rf X 100 34 54 74 Gopal Purple colour B-R R OR FeCU reaction B B OR concentration ratio 1 15 5 6 U.V. B-R R O-R Black Hulless Purple Rf X 100 34 54 74 colour B-R R OR FeCl3 reaction B B OR concentration ratio 5 15 1 U.V. B-R R O-R 7 Rf X 100 34 54 Lion Black colour B-R R FeCl3 reaction B B concentration ratio 3 1 U.V. B-R R 8 Rf X 100 34 54 Gatami Black colour B-R R FeCl3 reaction B B concentration ratio 3 1 U.V. B-R R Rf X 100 74 Pelargonidin colour OR FeCl3 reaction OR U.V. OR Cyanidin Rf X 100 54 colour R FeCl3 reaction B U.V. R B - R B l u i s h r e d R D a r k r e d B B l u e O R O r a n g e r e d O c t o b e r , 1958] MULLICK ET AL.—COLOUR IN BARLEY 4 5 3 T A B L E 3 .—HISTOCHEMICAL OBSERVATIONS FOR ANTHOCYANINS V a r i e t y D r y M o u n t 2 % H C L 2 % N H 4 O H A l e u r o n e P e r i c a r p A l e u r o n e P e r i c a r p A l e u r o n e P e r i c a r p White G o l d e n P h e a s a n t C C C C Y - 0 Y Blue + + + K w a n B B - B l R O - Y B B r - 0 T r e b i + + + B B r R O - Y B O - Y M o n t c a l m + + + B 0 R 0 Y - G 0 Purple + + + + + + G o p a l B R - B R R - B G G - B B l a c k H u l l e s s _ + + + + + C R R R G O - G Black + + + _ G a t a m i B B l R - B B l Y - G B l L i o n C B l C B l Y B l + Anthocyanin probably present C Colourless — Anthocyanin not likely present or masked Y Yellow O Orange B Blue R Red or pink G Green Bl Black Br Brown N o differences in the behaviour of the barley anthocyanidins and the available authentic anthocyanidins were observed when they were studied singly, in mixtures, in the ferric chloride test, or under short-wave ultra-violet fluorescence. The relationships of specific anthocyanins to possible "parent" antho-cyanidins were well established by hydrolysis of eluted anthocyanins and rechromatography. Thus anthocyanin C is, beyond doubt, a cyanidin derivative; B , a delphinidin derivative, and E , a pelargonidin derivative. D , which may be a cyanidin or pelargonidin derivative, gave weak reactions which could not be called distinctive. The Ai-A group of anthocyanins found in the purple varieties gave delphinidin reactions but the members could not be well separated because of their apparent instability. (c) The Histochemical Tests M a n y compounds other than anthocyanins appear to contribute to some extent to the colour of barley aleurone and pericarp tissues. It is, therefore, not surprising that the histochemical tests were somewhat unsatisfactory. Some results nonetheless are given (Table 3) for, in some instances, they confirm and extend other observations. For example, -168-454 CANADIAN JOURNAL OF PLANT SCIENCE [Vol. 38 histological differences in black varieties confirm the observations of extraction and chromatography, viz., L ion from which anthocyanins were not extracted had a colourless aleurone and a melanin-black pericarp, while Gatami which gave two anthocyanins had a coloured aleurone and a black pericarp. Purple varieties showed colour in both pericarp and aleurone, but increased colour in the pericarp was accompanied by de-creased colour in the aleurone and vice versa. In blue varieties, most of the pigment occurred in the aleurone tissue and gave the typical acid-base reaction of anthocyanins. Orange or yellow flavonoid pigments occurring in the pericarp did not give anthocyanin reactions. They tended to mask blue anthocyanins and, in their presence, to give a green tissue colour in alkali . White varieties showed orange or pink colours in aleurone and pericarp and did not give good anthocyanin reactions. DISCUSSION Difficulties are encountered in the study of the anthocyanins of the barley caryopsis which are not met in other parts of the plant. In the first place, the aleurone and pericarp tissues, which contain anthocyanins, differ genetically and cannot be separated from one another or from the chaff and endosperm. A t best the tissues can be concentrated as dust from pearling the whole barley grain. Pigment develops late in the cary-opsis and appears to be singularly subject to influence by changing environ-mental conditions. Accompanying the anthocyanins in aleurone and pericarp are other related flavonoid substances and large amounts of reserve protein and carbohydrate. Before the anthocyanins can be resolved chromatographically, concentrated extracts of reasonable purity must be obtained. The amount of extract spotted on chromatogram loci must be determined within very narrow limits to prevent overloading and trailing on the one hand or weak development on the other. This diffi-culty may be associated with the variation in glycosidation of the parent anthocyanidins which in turn may reflect the physiological state of the plant on a given day or week. The anthocyanin chemistry as so far developed can be related to some extent to the colour genetics of the caryopsis. Myle r and Stanford (13) and later Briggs and Stanford (3) stated that blue barley colour was im-portantly concerned with two complementary, independently assorting gene pairs B l and B l 2 . Thus the genotype of pure breeding K w a n , a barley with blue aleurone, would be designated as B l B l , B l 2 B l 2 ; Goldfoil , a white barley, as bl bl , B l 2 B l 2 ; and Napal , another white barley, as B l B l , b l 2 b l 2 . Leucoanthocyanin, in the only white barley studied, yielded two anthocyanidins, delphinidin and cyanidin, in the approximate concen-tration ratio of 3:1. In the three blue varieties and in one of the black varieties examined, two anthocyanins were found: one a delphinidin, and one a cyanidin derivative. Superficially it would appear that two gene-controlled steps are required to develop the two anthocyanins from two leucoanthocyanins. Robinson and Robinson (14) reported leucoantho-cyanins in barley. Bate-Smith (2) noted that almost all leucoanthocyanins yield cyanidin and/or delphinidin; Harris (9) and McFarlane, Wye, and October, 1958] M U L L I C K E T A L . - — C O L O U R I N B A R L E Y 455 Grant (12) found cyanidin, delphinidin, and several unidentified antho-cyanidins in barley malt. To this point, then, investigations form a consistent pattern. Very little can be said of the relationships of the chemistry and the genetics of the purple factors. Harlan (8) believed that purple caryopsis was attributable to a red pericarp underlain by a blue aleurone but this statement, although generally true, may prove to be too simple. Until the work of Woodward and Thieret (18), most barley specialists believed "purple seed" to be simply dominant over "colourless seed". However, they were able to show that two independently assorting, complementary gene pairs, P p and C c , were involved. Our studies would support the involvement of at least two gene pairs in the two purple varieties Gopal and Black Hulless. The inheritance pattern, however, is not a simple imposition of "the pattern for purple" on "the pattern for blue". In the purple grains, perlargonidin derivatives, which do not appear in the blue grains, occur and the delphinidin and cyanidin derivatives, which appear in the blue grains, are in very different concentration ratios. Moreover, there is a decided difference between the two purple varieties in the distri-bution and number of their anthocyanins. It might be reasonable to assume that determination involves precursors of the anthocyanidins. Harlan (7), as mentioned earlier, noted the melanin-like pigment in the pericarp of the black barleys. Buckley (4), studying the inheritance of "black" and "white", reported monofactorial patterns. It seems probable that the chemistry of melanin pigments is quite unrelated to that of the anthocyanins. Nevertheless it would be of interest to know if a full range of anthocyanins is found in the black varieties. Our variety, Gatami, was "blue" masked by "black"; perhaps other varieties are "purple" masked by "black". ACKNOWLEDGEMENTS This work was largely supported by a grant from the Brewing and Malting Barley Research Institute, Winnipeg, Manitoba, for which the authors are grateful. Our appreciation of the loan of equipment from J. J. R. Campbell, Division of Animal Science, The University of British Columbia, is also recorded. REFERENCES 1. Aberg, E., and G. A. Wiebe. Classification of the barley varieties grown in the United States and Canada. U.S. Dept. Agr. Tech. Bull. 907. 1946. 2. Bate-Smith, E. C. Leuco-anthocyanins. 1. Detection and identification of antho-cyanins from leuco-anthocyanins in plant tissue. Biochem. J. 58:122-125. 1954. 3. Briggs, F. N., and E. N. Stanford. Linkage relations of the Goldfoil factor for resis-tance to mildew in barley. J. Agr. Research 66:1-5. 1943. 4. Buckley, G. F. H. Inheritance in barley with special reference to the colour of cary-opsis and lemma. Sci. Agr. 10:460-492. 1930. 5. Dollery, A. F., O. H. Owen, and U. Martens. Identification of barley and wheat varieties by kernel characters. Board of Grain Commissioners for Canada. Queen's Printer, Ottawa. 1950. 6. Goldner, H. Die Gerstengramme. Diss. Univ. Breslau. 1923. 7. Harlan, H. V. Some distinctions in our cultivated barleys with reference to their use in plant breeding. U.S. Dept. Agr. Bull. 137. 1914. 456 C A N A D I A N J O U R N A L O F P L A N T S C I E N C E [Vol. 38 8. Harlan, H. V. Daily development of kernels of Hannchen barley from flowering to maturity at Aberdeen, Idaho. J. Agr. Research 19:393-430. 1920. 9. Harris, G. General composition of non-biological hazes of beer and some factors in their formation. II. Chromatographic separation of hop and malt tannins. J. Inst. Brewing 62:390-406. 1956. 10. Kiessling, L., and G. Aufhammer. Bilderatlas zur Braugerstentunde. Veroff ver Ford. Dent. Braugerstendbaus. 1931. 11. Miyazawa, D. On the inheritance of fruit colour of barley. Botan. Mag. (Tokyo) 32:308-310. 1918. 12. McFarlane, W. D., E. Wye, and H. L. Grant. Proc. European Brewing Conf., Baden-Baden, p. 298. 1955. 13. Myler, J. L., and E. H. Stanford. Colour inheritance in barley, J. Amer. Soc. Agrbn. 34:427-436. 1942. 14. Robinson, G. M., and R. Robinson. A survey of anthocyanins. III. Notes on the distribution of leuco-anthocyanins. Biochem. J. 27:206. 1933. 15. Sawicki, J. Studies on the structure of the aleurone layer in varieties of the culti-vated barley Hordeum sativum Jess. Cracovie. Imprimerie de l'Universite. 1950. 16. Schulz, K. G. Uber Anthozyan-Verfarbungen an Braugersten, Wochens, Brau. 52:33-36, 41-45, 51-54. 1935. 17. Smith, L. Cytology and genetics of barley. Botan. Rev. 17:1-355. 1951. 18. Woodward, R. W., and J. W. Thieret. A genetic study of complementary genes for purple lemma palea and pericarp in barley Hordeum vulgare L. Agron. J. 45: 182-185. 1953. P R I N T E D E V 7 H E Q U E E N ' S P R I N T E R , O T T A W A , li)5ft -171-APPEIDIX III THE CHROMATO-STRIPE - A l AUROMATIC STRIPING TECHNIQUE FOR PAPER CHROMATOGRAPHY. D.B. Mullick and V.C. Brink D i v i s i o n of Plant Science The University of B r i t i s h Columbia, Vancouver. "Many who are banding large volumes i n paper chroma-tography have experienced d i f f i c u l t i e s i n resolving stripes applied manually, p a r t i c u l a r l y when i n t e r -f e r i n g concomitant compounds are contained i n the extract. The "chromatostripe" i s a simple automatic technique by which extracts can be adsorbed uniformly on the paper i n the form of a s t r i p e , thereby f a c i l i -t a t i n g the development of the components of the ex-tract into well defined p a r a l l e l bands. This technique solves the problem of l o c a l i z e d overloading encountered when a st r i p e i s l a i d on manually. In addition, the quantity of extract adsorbed can be e a s i l y determined i f required." " During our chromatographic studies on anthocyanins i n barley kernels (3) ? i t was found that d i l u t e a c i d i f i e d aqueous and alcoholic solvents, i n addition to anthocyanins, extracted large quantities of co-pigments, proteins l i k e g l u t e l i n s and prolamins (which are soluble i n d i l u t e a c i d i f i e d aqueous and alcoholic solvents r e s p e c t i v e l y ) , and other concomitant materi-a l s . The presence of these soluble materials affected the resolution and rate of movement of the anthocyanins consider-ably, thereby necessitating re-chromatography of the eluates for obtaining well-defined spots. In addition, the spectro-graphic and other a n a l y t i c a l determinations, f o r which large quantities of eluates were required, made i t necessary to band large volumes of extracts on the paper. - 1 7 2 -In general, the adsorption of the solute on paper i n quantity was secured either by one application of a concen-trated extract on the starting l i n e or repeated applications over the same str i p e i f the color was not deep enough. Obviously, owing to lack of uniformity i n the rate of app l i c a t i o n of the extracts manually, the stripe was overloaded at ce r t a i n points. The overloading may very e a s i l y be caused when the extract contains large quantities of soluble proteinaceous and other concomitant materials. This, usually, gave a semi-lunar shaped resolution of c e r t a i n fast-moving spots and " s t r a i g h t - l i n e t r a i l i n g " i n a d i r e c t i o n perpendicular to the str i p e (that i s i n the d i r e c t i o n of solvent flow) of certa i n slow moving spots, at the point of over loading. This queer behaviour could be advantageously seen owing to the colored nature of extracts 5 i n colorless extracts, probably, t h i s could have passed unnoticed. Striping by hand, therefore, appeared unsatisfactory and to overcome these d i f f i c u l t i e s , the "chromatostripe" technique was developed and i s described below. "CHROMATOSTRIPE APPARATUS" Pig. (1) shows a photograph of the chromatostripe apparatus which i s a sl i g h t modification of a "Kymograph". A cylinder (12 i n . diameter by 12 i n . high) i s mounted on the v e r t i c a l spindle of E l e c t r i c 12 Recording Drum (•+) controlled by 12 fixed, speed gears with a separate control lever f o r f i n e -173-Flg. 1. Chromatostripe Apparatus. -174-adjustment of each giving a continuously variable surface speed of from one revolurion i n 0.8 seconds to one revolution i n 13 hours. Special c a p i l l a r i e s , usually 12 inches long, are drawn out from 1 mm. barometer tubing so that about 1/2 inch i s bent at an angle ( <fi ) of 150° or so with the rest of the tubing as shown below: The c a p i l l a r y surface i s ground at _L to i t s axis and i s 0.09 sq. cm. i n area. The bore of the c a p i l l a r y i s kept around 0.18 mm. The c a p i l l a r y i s fastened i n the clamp by a cork and moun-ted on the uprights f i t t e d on a double jointed arm which pro-vides a most s a t i s f a c t o r y method of adjusting the c a p i l l a r y surface around the recording cylinder. At the base of the up-right i s f i t t e d "the adjustable "x" block" (5) which i s a -175-very useful device for returning the c a p i l l a r y back to i t s o r i g i n a l p o sition f o r multiple applications of the extract. In addition, a blower may be placed at the proper p o s i t i o n fo r drying the s t r i p e . METHODOLOGY The cylinder i s covered uniformly with a cellophane paper. The Whatman #3 mm paper i s then wrapped around the cylinder and fastened by a tape. The screw knob on the "x" Block i s comp-l e t e l y screwed i n . The c a p i l l a r y i s f i l l e d with the extract and clamped on the upright so that when the double arm i s pro-perly adjusted, the following conditions r e s u l t : i ) the c a p i l l a r y surface i s at tengent to the rotating face of the cylinder and axis of the c a p i l l a r y i s normal to the cylinder diameter, i i ) the angle Q which the c a p i l l a r y makes with the ver-t i c a l i s kept at 90° to start with. (This angle can be adjusted through the brass wing nut provided on the clamp). i i i ) the surface of the c a p i l l a r y i s just opposite the start i n g l i n e on the paper, iv) the distance between the c a p i l l a r y surface and rota t -ing surface of the paper i s about 0 . 5 cm. Both of the fly-nuts of adjustable double arm are now screwed t i g h t l y i n position. The e l e c t r i c drum may now be -176-engaged* in. the gear selected for desired speed. The choice of speed gear depends upon the various considerations (see discus-sions,). Usually gear #8 gives optimum results. When the desired pojnt from where the striping i s to start approaches, the knob on the "x Block" is screwed out so that the capillary surface just makes a clean contact with the surface of the paper without any pressure, otherwise the fibre arrangement of the paper would be distrubed. The air from the blower i s directed towards the stripe. When the desired length of the strips has been l a i d , the arm of the "x Block" (on which the screw i s located) is pulled back promptly to break off the point of contact. After the f i r s t application, the portion of the paper where the stripe is laid swells up a l i t t l e . A.s such for restriping, before returning the arm, the screw knob on the "x Block" is screwed in by about a half rotation, so that the surface of the capillary is pulled back slightly. If this i s not done, a streaking perpendicular to the direction of the stripe w i l l be observed. RESULTS The results obtained by using the chromotostripe technique are shown in Fig. (2). Due to the limited color response of the film and the presence of a diffused background gn the chromatographic paper, the distinctive nature of the * see the manufacturer's directions for operation. -177-the bands could not be completely reproduced on black and white printing paper. Nevertheless, this technique gave well defined parallel bands which could, conveniently, be cut off and eluted, Starting line (applied by the chromato-stripe technique) Band No. 1 Band No. 2 Band No. Band No. Band No. 3 h 5 Band No. 6 Fig. 2. Showing the chromatographic resolution of a stripe applied by the chromatostripe technique, DISCUSSION It w i l l be opportune to discuss the relationships among the factors affecting the rate of flow from glass capillary to the paper and the amount of solute adsorbed per unit area of the - 1 7 8 -s t r i p e . In the former case, the permeability- of the paper to f l u i d under the action of a pressure difference may arise i n a va r i e t y of ways ( 1 ) . i . In paper, the f l u i d may be adsorbed at the i n t e r n a l walls of the c a p i l l a r y structure by p a r t i t i o n i n g between the solute and the solvent and be transported by d i f f u s i o n along a concentration gradient produced by the pressure difference. i i . The f l u i d may flow through c a p i l l a r i e s of the paper at a rate limited by i t s v i s c o s i t y and surface tension. Another factor which further affects the above considera-ti o n s , Is the r e l a t i v e size of the bore of the c a p i l l a r y as compared to the mean-free-path of the molecules and t h e i r s i z e . In the present case, however, i t was observed that when the c a p i l l a r y was horizontal and i t s surface i n contact with the paper on the cylinder, the rate of suction by the paper c a p i l l a r i e s was quite s i g n i f i c a n t and compared reasonably with direct viscous flow through the glass c a p i l l a r y held v e r t i c a l l y , under the pressure difference of i t s own weight. As such, the rate of flow of f l u i d from the glass c a p i l l a r y and rate of d i f f u -sion of the f l u i d on the paper i s almost of the same magnitude. Theoretical treatment of the rate of flow i s thus made more complex since the calculations involved must take into account the relationships between the forces of d i f f u s i o n on the paper on one hand and the forces of c a p i l l a r y flow on the other. In view of these, no vigorous t h e o r e t i c a l treatment w i l l be attem-ted. However, the discussion of these two factors with regard to p r a c t i c a l aspects of the method appear i n order. -179-a) Viscous Flow Average volume (V) of a l i q u i d which flows through a c a p i l -l a r y i n a unit time i s given by the r e l a t i o n : L -r e 1 where h i s the average height of a column of l i q u i d which provi-des the pressure difference across the horizontal c a p i l l a r y (L) d i s the density of f l u i d g is the acceleration due to gravity \ i s the v i s c o s i t y of the f l u i d 6 i s the angle which the c a p i l l a r y makes with the v e r t i c a l axis r i s the average radius of the c a p i l l a r y tube L i s the length of horiz o n t a l c a p i l l a r y tube Evidently by adjusting any of the above fac t o r s , the rate of flow can be controlled. However, for chromatographic pur-poses, the most convenient factor that can be adjusted i s angle 0 .' We have pointed out (see methodology) that the c a p i l l a r y may be kept horizontal. In that event, the ' 0' w i l l be zero -180-and therefore there w i l l be no flow from the c a p i l l a r y tube under the pressure difference of i t s own weight. And we have found that the suction forces of the paper are usually s u f f i c i e n t to lay a stripe of uniform width. b) Diffus i o n through the Paper It may primarily be determined, among other factors by the following; i . Surface tension of the f l u i d , i i . The accommodation c o e f f i c i e n t of the paper (defined as a measure of free space i n the paper available to the l i q u i d ( 2 ) . i i i . Diameter of paper c a p i l l a r i e s i v . Density, v i s c o s i t y and mean-free-path of l i q u i d mole-cules. The rate of adsorption or the rate of d i f f u s i o n can be controlled by adjusting the speed of the cylinder because the former depends upon the time, the unit area of the paper Is i n contact with the glass c a p i l l a r y containing the f l u i d . The slower the speed, the greater the adsorption and t h i s w i l l again affect the width of stripe as discussed below. c) Area of C a n i l l a r y Surface This i s another factor which to some extent determines the width of the s t r i p e . When the capillary-surface comes i n contact with the paper, a t h i n f i l m of the f l u i d i s formed on the -181-c a p i l l a r y surface due to surface tension and may feed the paper f i b r e s coming i n touch w i t h . i t . As such the feeding surface i s f a r greater than the actual'bore of the c a p i l l a r y . Width, how-ever, depends upon the speed of the cylinder as well. Too slow a speed would give a much wider st r i p e than the diameter of the c a p i l l a r y surface and comparatively fewer applications may cause overloading at slow speed. Rate of Adsorption of Solute It i s cumbersome to determine the c o e f f i c i e n t of accommoda-t i o n of the paper since i t w i l l change after every repeated s t r i p i n g , proportionate to the amount of solute adsorbed and the solvent evaporated. Moreover, i n absence of a suitable measure to determine the load* of the paper whether i t i s not-, under-, or overloaded, no simple t h e o r e t i c a l basis can be worked out for the c a l c u l a t i o n of the amount of solute adsorbed on the paper. Nevertheless, the amount of solute adsorbed can be deter-mined empirically by f i x i n g a graduated reservoir on the opposite end of the glass c a p i l l a r y which i n turn, may also be graduated. If the concentration of the solute i s known, the amount of solute adsorbed can be calculated. * Whether the f l u i d has been soaked into the t o t a l thickness of the paper. -182-APPENDIX IV "TABLE I. ALPHABETICAL LIST OF THE COLOR GENES IN BARLEY. GENE CHARACTER LINKAGE SYMBOL GROUP B Black lemma and caryopsis II B g Grey lemma and caryopsis II info B Medium black lemma and caryopsis II Bl Blue aleurone (Complementary factor) IV Big Blue aleurone( Complementary factor) III j Complementary factors inhibiting red ? pericarp color. 2 j Complementary factors inhibiting red ? pericarp color. o Orange lemma V or Orange seedling I P Purple lemma I P 2 Purple lemma ? P3 Purple lemma ? P c Purple veined lemma I P e Purple veined lemma ? Pf Purple veined lemma ? Re Red pericarp V R e 2 Red pericarp I Rs Red stem III -183-LITEMTURE CITED 1. Abbott, D. L. 1954. Recent applications of grov/th substances i n f r u i t growing. Ann. Appl. B i o l , 4 l : 215-20. 2. Aberg, E. and Wiebe, G. 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