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Physiology and genetics of the kernel color of barley. Faris, Donald George 1955

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THE PHYSIOLOGY AND GENETICS OF THE KERNEL COLOR OF BARLEY by Donald George F a r i s A thesis submitted i n p a r t i a l f u l f i l m e n t of the requirements for the degree of MASTER OF SCIENCE IN AGRICULTURE i n the D i v i s i o n of PLANT SCIENCE We accept t h i s thesis as conforming to the standard required from candidates f o r the degree of MASTER OF SCIENCE IN AGRICULTURE Members of the D i v i s i o n of PLANT SCIENCE The University of B r i t i s h Columbia November, 1955 i i ' ABSTRACT The generally accepted trademark of Canadian malting barley v a r i e t i e s i s "blue" aleurone color i n the kernel. New Canadian feed barley v a r i e t i e s are preferably marked by a "white" aleurone color. In attempting to meet the color q u a l i f i c a t i o n s i n new introductions, Canadian barley breeders have often experienced d i f f i c u l t y i n separating genet-i c a l l y "blue" from genetically "white" seed. An attempt has been made i n t h i s thesis to extend the knowledge on the inheritance, physiology, and separation of "blue" and "white" barley v a r i e t i e s . "Purple" and "black" barley kernel colors were also studied. Crosses were made between twenty barley v a r i e t i e s i n an attempt to ascertain the inheritance patterns f o r kernel color. The study of color inheritance i n the kernel acted as a background fo r p h y s i o l o g i c a l , histo-chemical, and chromato-graphic experiments. The physiological studies of color development i n barley plants and caryopses made use of three barley v a r i e t i e s , "Smyrna" (white), "Awnless" (blue), and "Black Hulless" (purple). These three v a r i e t i e s were fed complete, low N, and low P solu-tions and were divided equally into the f i v e following treatment blocks: "control", " u l t r a - v i o l e t " , " i n f r a - r e d " , "dextrose" and i i i "low temperature". The most c o n s i s t e n t i n c r e a s e i n p l a n t and seed c o l o r development was a s s o c i a t e d w i t h the low N and low P n u t r i e n t l e v e l s . There was a ""barely s i g n i f i c a n t " d i f f e r e n c e i n c o l o r development between the f i v e treatment b l o c k s . Dry and s p r o u t i n g k e r n e l s of "white" and " b l u e " seeded b a r l e y v a r i e t i e s were examined under n a t u r a l , c o l o r e d , and u l t r a -v i o l e t l i g h t s . The most c o n s i s t e n t d i f f e r e n c e between seed o f the two c o l o r types was found when dry seed was observed under a d i s s e c t i n g microscope i n n a t u r a l l i g h t . H i s t o - c h e m i c a l examinations of c o l o r e d k e r n e l s showed t h a t the c o l o r i n g pigments were l o c a l i z e d i n the aleurone and p e r i -carp l a y e r s . The blue and the p u r p l e pigments, l o c a t e d i n the aleurone and p e r i c a r p l a y e r s r e s p e c t i v e l y turned red when p l a c e d i n c o l d c o n d i t i o n s . Under a l k a l i n e c o n d i t i o n s the pigments of the two k e r n e l l a y e r s g e n e r a l l y appeared a green c o l o r . These -c o l o r changes i n the aleurone and p e r i c a r p l a y e r s o f c o l o r e d b a r l e y k e r n e l s s t r o n g l y suggest the presence of anthocyanin pigments. B l a c k p e r i c a r p pigments appeared unchanged under a l k a l i n e and a c i d c o n d i t i o n s . B a r l e y k e r n e l s of many v a r i e t i e s were e x t r a c t e d w i t h a l -c o h o l and b o i l i n g 2N HC1 and the pigment f r a c t i o n s of the e x t r a c t s separated by paper p a r t i t i o n chromatography. Two pigment f r a c -t i o n s were obtained from e x t r a c t s of "blue" seeded b a r l e y v a r i e t i e s and at l e a s t f o u r from e x t r a c t s o f " p u r p l e " seeded v a r i e t i e s . The Rp v a l u e s of the pigment f r a c t i o n s obtained from e x t r a c t s o f i v "purple" seeded v a r i e t i e s were very si m i l a r to the Rp values of the pigment fr a c t i o n s separating from extracts of red colored barley plant t i s s u e s . A l l these spots g«ve the c h a r a c t e r i s t i c red to blue color change of anthocyanins i n acid and alkaline conditions. V TABLE OF CONTENTS Page v I. INTRODUCTION 1 I I . LITERATURE REVIEW 4 A. LINKAGE OF COLOR AND MALTING QUALITY 4 B. AGRONOMIC CHARACTERISTICS AND MALTING QUALITY 5 C. COLOR VARIANTS IN BARLEY: BLUE-RED-PURPLE- BLACK- . LOCATION IN SEED 6 D. COLOR VARIANTS IN OTHER CEREALS: MAIZE - WHEAT 9 E. INHERITANCE OF BARLEY KERNEL COLORS 11 1 . Blue and White Kernel Color: xenla - a monofaetorial hypothesis - a complementary fact o r hypothesis - nomenclature - genetic linkage 11 2 . Other Kernel Colors: Purple-Black-Red .... 18 3 . Barley Hybridization Techniques 21 F. RELEVANT CHEMISTRY OF PIGMENTS 23 1 . Anthocyanin Structure 24 2 . Co-pigments 27 3 . Leuco-anthocyanlns 29 4 . Extraction of Pigments 31 5. P u r i f i c a t i o n and Analysis of Anthocyanins. 33 6 . Chromatography 33 G. BIOCHEMISTRY AND PHYSIOLOGY OF ANTHOCYANINS AND RELATED PIGMENTS 47 1 . Biosynthesis 47 2 . Genetics Related to Biosynthesis 50 v i II. LITERATURE REVIEW - continued Page 3 . Comments on General P h y s i o l o g y 53 III. EXPERIMENTS 58 / 1 . CROSSING EXPERIMENTS TO RESOLVE THE GENETICAL CONSTITUTION OF THE FACTORS FOR KERNEL COLOR IN BARLEY 58 2 . EFFECT OF CERTAIN GROWING CONDITIONS ON THE DEVELOPMENT OF BARLEY PLANT PIGMENTS 6 l A. An Attempt to Produce C o l o r i n S e e d l i n g s . 61 B. Experiments to Produce C o l o r i n P l a n t , K e r n e l and Detached Leaves 62 (a) P l a n t C o l o r 62 D i s c u s s i o n 70 (b) K e r n e l C o l o r 71 D i s c u s s i o n 75 (c) Detached Leaves 77 3 . HISTO-CHEMICAL AND SUPERFICIAL EXAMINATIONS OF KERNEL FOR PIGMENTATION 79 A. S u p e r f i c i a l D i f f e r e n c e s Between Blue and White. B a r l e y K e r n e l s 79 B. The Appearance of M i c r o s c o p i c S e c t i o n s of the K e r n e l s of V a r i o u s B a r l e y V a r i e t i e s .. 82 4. EXTRACTION AND CHROMATOGRAPHIC EXAMINATION OF BARLEY KERNEL PIGMENTS ., 87 A. E x t r a c t i o n 87 B. Chromatography 92 IV. CONCLUDING REMARKS 104 V. LITERATURE CITED 107 APPENDICES 117 v i i Table of Contents - continued LIST OF ILLUSTRATIONS AND TABLES TABLE PAGE 1 . Counts of the Seed on the F i Heads of Cross Between Blue and White Barley V a r i e t i e s 12 2. Counts of the Seed on the Fo Heads of Cross Between Blue and White Barley V a r i e t i e s , Using the Classes (a) Colored or Blue plus Heterozygous Blue, and (b) Color- 14 less or White 3 . Counts of the Seed on the F 2 Heads of Cross Between Blue and White Barley V a r i e t i e s , Using the Classes (a) Homozygous Blue, and (b) Heterozygous Blue plus Colorless.... 14 4 . The Factors f o r Blue Aleurone Color i n Certain Barley Va r i e t i e s 15 5 . Symbols Used by Various Workers f o r the Genes of Blue Aleurone Color i n Barley 17 6 . Count Showing the Crossover Value of Genes f o r Blue (Bl) and Hooded (K), Both i n Linkage Group IV, Using a Non-Blue Variety with Awns X Blue Variety with Hoods.. 17 7 . Emax ^F Value of Some Anthocyanidins 42 8 . The Chi Square Values and Contingency C o e f f i c i e n t s of Contingency Table Data 74 9 . Rp Values of Anthocyanins Extracted from V a r i e t i e s of Barley with Blue Kernel Pigments 95 1 0 . Rp Values f o r Anthocyanidins Formed from V a r i e t i e s with Blue Kernel Pigmentation 96 1 1 . Rp Values f o r Pigments of Unknown, but Non-anthocyanin, Nature i n V a r i e t i e s with White Kernels 9 7 12. R F Values f o r Anthocyanins Extracted from V a r i e t i e s of Barley with Purple Kernel Pigmentation 98 1 3 . Rp Values f o r Anthocyanidins Formed from V a r i e t i e s of Barley with Purple Pigmentation 99 14. R F Values f o r Anthocyanins Extracted from Red Colored Tissues of Cereal Plants 100 v i i i LIST OF ILLUSTRATIONS Continued: FIGURE PAGE 1. More Hi g h l y Hydroxylated Members of a Homologous S e r i e s i n F l a v o n o l s , Flavones, Anthocyanidins and Chalcones G e n e r a l l y have a Lower Rp Value..... 46 2. With the A d d i t i o n of Each Sugar Residue to the A n t h o c y a n i d i n Nucleus the Rp Value of the Anthocyanin so Formed i s Decreased 46 3 . Diagram of a Lamp Rack Used i n the U l t r a - V i o l e t and Infra-Red R a d i a t i o n Treatments • 65 4. B a r l e y p l a n t s growing i n the Greenhouse Under U l t r a -V i o l e t Lamps 66 5 . Appearance of "Black H u l l e s s " B a r l e y P l a n t s Fed a Com-p l e t e N u t r i e n t S o l u t i o n . . . . . . . . . . . . . . . 66 6 . Development of Red Color i n Stems of "Black H u l l e s s " B a r l e y P l a n t s f e d Low P and Dextrose 66 7 . Color Classes of the Purple V a r i e t y "Black H u l l e s s " C l a s s e s 1 to 3 i n c r e a s e i n Brown Col o r , C l a s s e s 4 to 5 i n Blue Color and C l a s s e s 6 to 10 Increase i n P u r p l e . . 72 8. Color C l a s s e s of the White V a r i e t y "Smyrna". C l a s s 1 i s White, C l a s s 2 Somewhat Brown and C l a s s 3 Quite Weathered i n Appearance 73 9 . B a r l e y seeds are of Many c o l o r s . On the L g f t i s the White V a r i e t y "Vantage", i n the Center the Blue V a r i e t y "Kwan", and on the Right the Purple V a r i e t y "Black H u l l -e s s " . . . . .. .1 81 10. The Glume Coloring. V a r i e s Between V a r i e t i e s and during the Growing Season. Here i s seen P u r p l e , S t r i p e d and C l e a r Glumes . . . • • 31 11. A M i c r o s c o p i c S e c t i o n of the B a r l e y V a r i e t y "Kwan" showing the N a t u r a l Blue Color i n the Aleurone Layer 84 12. A M i c r o s c o p i c s e c t i o n of the B a r l e y V a r i e t y "Black H u l l -ess" with HC1 Added, Showing the R e s u l t i n g Red Color i n Both the Aleurone and P e r i c a r p 84 ix; ACKNOWLEDGEMENTS The writer wishes to express his appreciation to the many people who made t h i s study possible, and e s p e c i a l l y to the following: Dr. V. C. Brink of the D i v i s i o n of Plant Science for his encouraging i n t e r e s t and fo r the hours spent i n consultation on t h i s problem; Dr. C. A . Hornby of the Department of Horticulture f o r his inter e s t and counsel; Dr. J. J . R. Campbell of the Dairy Department f o r his advice on chromatography and fo r the use of the Department's chromato-graphic equipment; Mr. H. Vaartnou and other members of the Di v i s i o n of Plant Science f o r t h e i r aid i n conducting many of the experiments; Dr. N. S. Wright of the Dominion Plant Path-ology Laboratory f o r his help i n taking pictures; Dr. D. G. Hamilton of the Central Experimental Farm at Ottawa and h i s associates who proposed the problem and helped with suggestions and material; Mr. J . G. C. Fraser, also of the Central Experi-mental Farmi,. 3 1 1 ( 3 Mr. E. A. Hurd of the Experimental Substation, Regina, Saskatchewan, who obtained the blue wheat seed f o r t h i s project; Dr. G. A. Wiebe of the United States Department of Agriculture, B e l t s v i l l e , Maryland, who supplied many of the barley v a r i e t i e s used; Dr. T. J . Harrison and,the Barley Im-provement I n s t i t u t e , who made funds available f o r t h i s study. To a l l these the writer wishes to extend his thanks. THE PHYSIOLOGY AND GENETICS OF THE KERNEL COLOR OF BARLEY I. INTRODUCTION Although the i d e a l barley v a r i e t y would produce good malt and abundant feed, today outstanding feed barley v a r i e t i e s are r a r e l y ( i f ever) top-rated f o r malting. In recognition of th i s apparent antipode Canadian barley breeders are tending to accept blue seed genes to mark malting v a r i e t i e s and white seed genes to designate feed barleys. Relation of seed color to purpose was emphasized by American buyers of Canadian malting barleys during World War II who dismissed the fact that most of our good malting stocks were, by happenstance, blue. Although the idea of using marker genes has merit, i t i s not without d i f -f i c u l t y . Unfortunately blue barleys vary greatly i n color development from region to region and even within a farmer's f i e l d . In technical terms, the expressivity and penetrance of blue marker genes are not high. It i s regrettable, too, that there appears to be no certain basic association of color and qu a l i t y . Considerable s c i e n t i f i c i n t e r e s t might be attached to any findings concerning color physiology and to a detailed study of color inheritance i n the barley kernel to p a r a l l e l that i n maize (97) • - 2 -Some s t a t i s t i c s and a short statement of the h i s t o r y of Canadian b a r l e y breeding w i l l p o i n t up the s i g n i f i c a n c e o f s t u d i e s o f c o l o r i n b a r l e y k e r n e l s . B a r l e y i s one of the world's important c e r e a l c r o p s . I t i s exceeded i n tonnage p r o d u c t i o n per annum o n l y by wheat, r i c e , and maize ( 2 8 ) . There are few l a n d areas from the equator t o the c i r c l e s where b a r l e y i s not grown, and i t i s w i d e l y accepted as an important component i n the maintenance and f a t t e n i n g r a t i o n s of l i v e s t o c k . I n Canada i n r e c e n t years b a r l e y has i n c r e a s e d i n acreage and im-portance. I n the p e r i o d 1935-39 she ranked s i x t h among coun-t r i e s f o r which s t a t i s t i c s are a v a i l a b l e ; i n 1952 and 1953 she was f i r s t . About 75 per cent of the Canadian crop i s used f o r stock feed and 20 per cent i s used f o r m a l t i n g . The remaining 5 per cent i s used m i s c e l l a n e o u s l y , f o r purposes such as b a r l e y f l o u r , p e a r l e d b a r l e y and b r e a k f a s t foods ( 3 5 ) -O.A.C. 2 1 , r e l e a s e d as the f i r s t Canadian m a l t i n g b a r l e y i n 1 9 1 0 , was s e l e c t e d from the Manchurian b a r l e y v a r i e t y Mandscheuri, which had been obtained from R u s s i a ( 2 9 ) . At t h i s time the U n i t e d S t a t e s was a l s o i n t r o d u c i n g s i m i l a r Manchurian types from A s i a , and, i n a d d i t i o n , v a r i e t i e s from Europe. When one r e a l i z e s that the Manchurian types c o n t a i n e d both white and blue seeded p l a n t s , one can see how breeders would s e l e c t p l a n t s to produce e i t h e r pure breeding "blue" or pure breeding "white" seeded l i n e s . Thus i t happened t h a t i n Canada O.A.C. 21 was s e l e c t e d , -3-a blue seeded va r i e t y , while i n the United States and Europe the malting v a r i e t i e s such as Oderbrucker were selected white seeded (1). Therefore, i n the United States and Europe a preference fo r white malting barley grew up, which had a l l the s u p e r f i c i a l -i t y of the preference held by some people f o r brown or white eggs. American and European maltsters came to regard white barley v a r i e t i e s as the good malting barley. It was not u n t i l World War I I , when the American maltsters were short of t h e i r white malting barley, and were forced to use the Canadian blue v a r i e t i e s , that they came to regard blue malting barley as synon-ymous with Canadian malting barley. While i t i s possible, i n Canada, to license white malting v a r i e t i e s , i t might be d i f f i c u l t to license a white aleuroned malting barley i f i t could not be distinguished i n seed samples from the existing white feed v a r i e t i e s . With t h i s d i f -f i c u l t y i n l i c e n s i n g white malting barleys the Canadian barley breeders have found i t more s a t i s f a c t o r y to produce blue malting and white feed barleys (34). -4-I I . LITERATURE REVIEW One of the problems c o n f r o n t i n g the b a r l e y breeder, as s t a t e d above, i s to d i s t i n g u i s h g e n e t i c "white" seed from l a t e n t but g e n e t i c " b l u e " seed. The problem a r i s e s l a r g e l y because of the v a r i a t i o n i n a c t i o n of c l i m a t i c and edaphic f a c -t o r s on the f o r m a t i o n of c o l o r i n the aleurone l a y e r of the b a r l e y k e r n e l . I n s p i t e of t h i s v a r i a t i o n , most workers use the r easonably constant c h a r a c t e r of k e r n e l c o l o r i n c l a s s i f y -i n g and s e p a r a t i n g the c u l t i v a t e d v a r i e t i e s of b a r l e y , espec-i a l l y a f t e r the crop i s threshed (1, 21, 43). Aufhammer has even suggested t h a t c o l o r f o r m a t i o n can be used to d i f f e r e n t i a t e summer and w i n t e r b a r l e y (11). Hurd, at the Regina S u b - s t a t i o n , has proposed t h a t feed wheat v a r i e t i e s , too, might be marked by the blue f a c t o r (47). A. LINKAGE OF COLOR AND MALTING QUALITY I t would be u s e f u l to know i f good m a l t i n g q u a l i t y and k e r n e l c o l o r are a s s o c i a t e d . M a l t i n g b a r l e y must be a h i g h c l a s s b a r l e y and i s u s u a l l y purchased at a c o n s i d e r a b l e premium over feed b a r l e y (35)• I f the p l a n t breeder knew whether th e r e was any c o n n e c t i o n between c o l o r and m a l t i n g q u a l i t y he might save h i m s e l f years of work, as i t takes t e n to f i f t e e n years of p a i n s t a k i n g work to produce a new v a r i e t y (35> 92). A few workers ( 3 1 , 53) f e e l t h a t those who c u l t i v a t e b a r l e y f o r brewing purposes should pay more a t t e n t i o n to the f o r m a t i o n of pigments. The w r i t e r has not been able to secure o r i g i n a l w r i t i n g s , so t h e i r s i g n i f i c a n c e i s not c l e a r . S c h u l z (92) found more anthocyanin i n b a r l e y k e r n e l s having more pro-t e i n s . H a r l a n (40) i s of the o p i n i o n t h a t the development o f anthocyanin i n the seeds of b a r l e y i s p r o b a b l y a v e r y minor phase of metabolism, and i t might be i m p l i e d , t h e r e f o r e , t h a t he f e e l s there would be l i t t l e i n the a s s o c i a t i o n of c o l o r w i t h q u a l i t y . B. AGRONOMIC CHARACTERISTICS AND MALTING QUALITY To a s s o c i a t e v a r i o u s agronomic c h a r a c t e r s w i t h m a l t i n g q u a l i t y i s v e r y d i f f i c u l t , as m a l t i n g q u a l i t y i n b a r l e y i s not easy to d e f i n e . I t depends on the m a l t i n g method to be adopted, on the brewing process i n use, and on the type of beer r e q u i r e d . These c o n d i t i o n s a l l v a r y w i t h each country or area. Thus a b a r l e y used f o r m a l t i n g i n any s p e c i f i c c ountry i s probably adapted a g r o n o m i c a l l y to t h a t r e g i o n , and t h e r e f o r e m a l t i n g and brewing procedures, as w e l l as the t a s t e s of the customers, have developed around t h a t p a r t i c u l a r b a r l e y . I t f o l l o w s t h a t the p l a n t breeder must be c a r e f u l i n making marked changes i n the m a l t i n g q u a l i t i e s , because any a l t e r a t i o n might upset the produc-e r ' s methods and oppose the consumer's p r e f e r e n c e s . Thus he f i n d s i t best to work on improving o n l y the agronomic q u a l i t i e s ( 2 ) . -6-Canada's malting trade has developed around O.A.C. 21; under our conditions we can produce a mellow malt from t h i s barley at a low cost (2). To re c a p i t u l a t e , the blue O.A.C. 21 has been the malting v a r i e t y around which Canada's export trade, e s p e c i a l l y to the United States, has been b u i l t up. This v a r i e t y and i t s blue color has become Canada's trade-mark of malting q u a l i t y . I t i s very convenient i n grading to keep the malting v a r i e t i e s separate from the feed v a r i e t i e s . C. COLOR VARIANTS IN BARLEY Caryopsis colors i n barley are many; some barleys pro-duce blue grains, some black, some purple, and some red; other barleys are lacking well defined color, but nonetheless may not be designated as colorless — the greys, "•dirty" whites, etc.; others doubtless can be properly designated as white. Color may develop independently i n endosperm, aleurone, pericarp and chaff (38, 94). The blue color of barley i s caused by the presence of anthocyanin i n the aleurone layer of the barley kernel, and white barleys are characterized by i t s absence (38, 95). The aleurone or the outer layer of the endosperm, consists of from two to four c e l l s of varying thickness and depth. The v a r i a t i o n i n aleurone thickness i s associated with differences i n variety,'as well as i n -7-climate and s o i l (38, 45, 90). This l a y e r does not s t a r t to d i f f e r e n t i a t e u n t i l the embryo sac i s e n t i r e l y f i l l e d w i t h c e l l s , and anthocyanin i s not apparent u n t i l a few days before matur-i t y (40). The presence or absence of blue aleurone c o l o r may be masked by other caryopsis c o l o r s . I f anthocyanin i s present i n the p e r i c a r p , the caryopsis appears red (17)? or v i o l e t (38). I f anthocyanin i s present i n both the aleurone and p e r i c a r p , the caryopsis shows up as purple. The anthocyanin appears blue i n the aleurone because i t i s i n an a l k a l i n e c o n d i t i o n , but i n the pe r i c a r p i t i s i n an acid s t a t e and manifests as red or v i o l e t . The purple v a r i e s from deep c o l o r to an almost c o l o r -l e s s s t a t e . Sometimes i t i s evenly d i s t r i b u t e d ; sometimes i t appears as l o c a l i z e d patches, while at other times i t o n l y appears i n the veins of the lemma. I n the h u l l e s s v a r i e t i e s , or when a p o r t i o n of the seed i s exposed to s u n l i g h t , the purple p e r i c a r p c o l o r i s more i n t e n s e . Some spots i n a f i e l d of b a r l e y can be conspicuous f o r pronounced purple pigment development. This v a r i a t i o n i n c o l o r i s apparently not the r e s u l t of d i f f e r -ences i n f e r t i l i t y l e v e l , f o r the a d d i t i o n of various f e r t i l i z e r s , s i n g l y and i n combinations, d i d not seem to a f f e c t the purple c o l o r (103). Some v a r i e t i e s have a black m e l a n i n - l i k e substance i n the p e r i c a r p which causes the seed to appear black or grey. When t h i s b l a c k c o l o r occurs i t masks a l l the other seed c o l o r s . - 8 -To sum up: I f there i s no pigment i n the aleurone or p e r i c a r p l a y e r s , the c a r y o p s i s appears white or y e l l o w . I f the aleurone has anthocyanin present and the p e r i c a r p has no pigment, the c a r y o p s i s appears b l u e . When the aleurone l a y e r has no p i g -ment present and the p e r i c a r p c o n t a i n s anthocyanin, the c a r y o p s i s i s red or v i o l e t . Should both the aleurone l a y e r and the p e r i -carp c o n t a i n anthocyanin, the c a r y o p s i s emerges as p u r p l e . However, i f the p e r i c a r p should c o n t a i n the m e l a n i n - l i k e substance, the c a r y o p s i s w i l l range from b l a c k or grey to brown, depending on whether the pigment i s more or l e s s c o n c e n t r a t e d ( 1 , 1 7 , 3 8 , 4 5 , 9 4 ) . H a r l a n (38) used Mann's technique (63) to determine the pigments present i n b a r l e y k e r n e l s . The method i n v o l v e s making freehand s e c t i o n s of the dry b a r l e y caryopses to avoid m o d i f i c a -t i o n s from s o l v e n t s when embedding. A l s o the freehand s e c t i o n s which were around 50 to 100mu proved t o be j u s t as s a t i s f a c t o r y , or even more so, f o r o b s e r v i n g c o l o r s i n the outer l a y e r s of the seed than the t h i n n e r s e c t i o n s cut on a microtome. H a r l a n placed h i s s e c t i o n s on a dry s l i d e , covered them w i t h a cover s l i p , and s e a l e d two o p p o s i t e edges of the cover s l i p with p a r a f f i n . He was then able to study under the microscope the r e a c t i o n of the pigments t o s e v e r a l reagents which were run through the two open s i d e s of the cover s l i p . I n the case of the blue b a r l e y , the aleurone l a y e r q u i c k l y turned blue when he a p p l i e d 2 per cent c a u s t i c potash, and red when he used 2 per cent h y d r o c h l o r i c a c i d . These r e a c t i o n s are t y p i c a l f o r an anthocyanin; and as the -Sl-ant hoc yanin appeared blue i n the natural condition of the aleurone layer, he concluded that i t was alkaline i n reaction. Many have transcribed Harlan's findings to the ef f e c t that the color of blue seeded barley i s due to anthocyanin i n an alkaline aleurone, and white or yellow seeded barley results from lack of anthocyanin (1, 17, 38, 45, 47, 94, 95, 99). D. COLOR VARIANTS IN OTHER CEREAL'S Inasmuch as parellelisms may exist i t would be of value to mention the work done on the aleurone, endosperm, and pericarp colors of the other grains, p a r t i c u l a r l y corn and wheat. Much fine work has been done on the inheritance of the seed colors and plant colors of maize, much of which has been well reviewed by Emerson ejt al. (24). As early as 1918 the three complementary factors A, C, and R had been found to be necessary i n the dominant form before any color i s expressed i n the aleurone layer. Thus when any one of these factors i s present i n the homozygous reces-sive form, the aleurone i s c o l o r l e s s . I f homozygous recessive p_r p_r i s present with A C and R the aleurone i s red, but the aleurone i s purple when the dominant Pr i s present. In some corn strains gene I acts as an i n h i b i t i n g f a c t o r which, i n the dominant form, prevents the formation of color i n the aleurone layer. Many other factors are involved i n the kernel colors of maize. Bn i n i t s dominant form gives a brown aleurone. In -10-controls the i n t e n s i t y of the aleurone color ( 8 9 ) . Other factors cause dark cap, speckling, blotching and many other color effects of maize kernel color ( 8 9 ) . The reds, blues, and purples of the corn plant, as i n barley, results from the presence of anthocyanins (104). Mention should also be made of the multiple a l l e l o -morphic series at the R locus. This series controls plant color and seed color independently ( 2 3 ) . Recently Stadler has done considerable research on t h i s locus with re s u l t s which may cause us to revise many ideas on the nature of genes ( 9 7 ) . Using Blue 1 wheat from an Agropyron elongatum x Triticum vulgare cross, Hurd (47) has shown that the inheritance of t h i s endospermic blue seeded character was controlled by two complementary genes. In his Blue 1 the pigment he found was an anthoxanthin l y i n g throughout the endosperm but concentrated i n the aleurone layer. Despite possible difference i n gene dosage due to double f e r t i l i z a t i o n and the 3n endosperm, Hurd found that the i n t e n s i t y of blueness of Blue 1 did not indicate the genotype. Thus his dark blue seed segregated i n the same r a t i o as the l i g h t blue seed. Also any mottled blue seeds segregated i n a manner sim i l a r to the regular blue seeds. He discovered that drought resulted i n an increase of a c i d i t y and sugar content of c e l l sap, which i n turn caused the blue pigments to change to red and y e l -low. Red color i n wheat i s mainly due to three characters act-ing i n the t e s t a and purple color i s due to anthocyanin i n the -11-c r o s s l a y e r s of the p e r i c a r p . I t would appear from the above t h a t environmental c o n d i t i o n s p l a y an important p a r t i n c o l o r development i n wheat seed. Indeed, Mendelian se g r e g a t i o n s cannot be s a t i s f a c t o r i l y d i s c e r n e d i n some of these c h a r a c t e r s under c e r t a i n environmen-t a l c o n d i t i o n s . E. INHERITANCE OF BARLEY KERNEL COLORS There i s c o n s i d e r a b l e knowledge extant on the g e n e t i c s and l i n k a g e groups of b a r l e y (94-). T h i s i n l a r g e measure i s because b a r l e y has o n l y seven p a i r s o f chromosomes, seven l i n k -age groups, and i s t h e r e f o r e c o m p a r a t i v e l y easy to work w i t h g e n e t i c a l l y . Although s e v e r a l g e n e t i c i s t s have worked on the c h a r a c t e r of blue and white aleurone c o l o r , as w e l l as on other k e r n e l c o l o r s , the knowledge of the i n h e r i t a n c e of these charac-t e r s i s not complete. 1. Blue and White K e r n e l C o l o r X e n i a i s the immediate e f f e c t of p o l l e n on the endo-sperm, and i s one of the r e s u l t s of double f e r t i l i z a t i o n , a phenomenon which occurs i n seed p l a n t s ( 4 4 ) . As the aleurone i s p a r t of the endosperm, the secondary nucleus of the p o l l e n tube a f f e c t s the e x p r e s s i o n of blue c o l o r i n the aleurone l a y e r . I n 1914, So et a l . (95) demonstrated x e n i a i n white and b l u e seeded b a r l e y v a r i e t i e s . Whenever they cross a blue w i t h a -12-y e l l o w seeded v a r i e t y , the seeds are a l l b l u e . S i m i l a r l y , b l u i s h g r a i n s were c o l l e c t e d from the ears of a w h i t i s h - y e l l o w grained v a r i e t y which had been p o l l i n a t e d by a blue seeded v a r i e t y . On s e l f i n g the h y b r i d s from these c r o s s e s , So e_t a l . (ibicL) obtained the r e s u l t seen i n Table 1, which r e c o r d s the seeds from the F-j_ p l a n t s . TABLE I. COUNTS OF THE SEED ON THE F i BLUE AND WHITE BARLEY VARIE. 1 HEADS OF PIES (95). CROSS BETWEEN Item Blue Yellow T o t a l Grains D e v i a t i o n Probable D/PE E r r o r Observed Expected 7498 2504 10,002 7501.5 2500.5 10,002 = 3.50 = 43.31 0.08 So et a l . ( i b i d . ) r e p o r t s Miyazawa (67) as t h i n k i n g t h a t the b l u e g r a i n c o l o r was due to pigment present i n the c e l l s of the p e r i c a r p and " t e s t a " . Miyazawa f u r t h e r b e l i e v e d t h a t the count of 1:1, which he obtai n e d , was the r e s u l t of the vege-t a t i v e s e g r e g a t i o n of the genes j u s t before the f o r m a t i o n of the growing p o i n t of the ear. I n a review o f h i s paper of 1918 (68), i t i s r e p o r t e d that Miyazawa r e a l i z e d t h a t h i s r e s u l t s , u s i n g the F-j_ s e g r e g a t i o n of "b l a c k " and "white" g r a i n s , could be ex-p l a i n e d by x e n i a . So f u r t h e r r e p o r t s t h a t the c o l o r i n t e n s i t y may be connected w i t h the g e n e t i c composition o f the t r i p l e system of the endosperm nuc l e u s , as has been suggested by Hayes and East (42) f o r maize i n h e r i t a n c e . E x p r e s s i o n of t h i s - 1 3 -character i s also frequently modified by d i f f e r e n t environments ( 9 4 ) . Because of the 3 * 1 segregation, which he obtained i n the F 2 > B i f f e n ( 1 0 ) indicated i n 1 9 0 7 that the character f o r colored grain i n barley i s dominant over the colorless charac-t e r . Other early reports also point - to a single dominant gene for blue aleurone color (Smith ( 9 4 ) documents: 1 7 5 3 7 5 4 6 , 5 1 > 5 4 , 6 6 , 7 8 ) . So et a l . ( 9 5 ) examined about 1 0 , 0 0 0 grains on the heads of F-j_ plants r e s u l t i n g from ten crosses of blue x white, and obtained an almost perfect 3 * 1 r a t i o of blue to white seeds (Table I ) . Buckley ( 1 7 ) also reported on the inheritance of blue and white aleurone (BI, b l ) . He made crosses of Col-sess 17 and Minnesota 72-8, and his F]_ grains on the heads of the parent plants a l l had blue aleurones. S e l f i n g the F]_ plants from t h i s seed, he obtained a segregation of 3 blue : 1 white. Robertson et a l . ( 7 8 ) , on the other hand, found d i f f i c u l t y i n d i f f e r e n t i a t i n g the blue from the white aleurone i n the seed of the F]_ plants. They found i t more s a t i s f a c t o r y to separate, i n the f i e l d , the heads of the F 2 plants into the classes homozy-gous blue aleurone, heterozygous (blue and white on the same head) and homozygous white. They then set these counts up i n two classes: (a) colored or blue, plus heterozygous blue, and (b) colorless or white. Their re s u l t s can be seen i n Table 2 . They next recast t h e i r data, using the classes: (a) homozygous blue, and (b) heterozygous blue plus colorless (Table 3 ) . Robert-son et a l . (ibid . ) believed that t h i s count was more accurate, -14-TABLE 2. COUNTS OF THE SEED ON THE F 2 HEADS OF CROSS BETWEEN BLUE AND WHITE BARLEY VARIETIES, USING THE CLASSES (a) COLORED OR BLUE PLUS HETEROZYGOUS BLUE, AND (b) COLORLESS OR WHITE (78). Item Colored Colorless Deviation D/PE Observed count 4 ,553 1>395 Calculated segregation 3:1 4,46l 1,487 92 4 . 0 9 because a colorless head would become a heterozygous blue should any blue pollen land on i t ; on the other hand, the homozygous blue would not be affected i n t h i s way. Again, these r e s u l t s seem to indicate a single dominant gene f o r blue. TABLE 3 . COUNTS OF THE SEED ON THE F 2 HEADS OF CROSS BETWEEN BLUE AND WHITE BARLEY VARIETIES* USING THE CLASSES (a) HOMOZYGOUS BLUE, AND (b) HETEROZYGOUS BLUE PLUS COLORLESS (78). Item Homozygous Heterozygous Deviation D/PE Blue Blue + Colorless Observed count 1?528 Calculated segregation 3*1 1?487 4,420 4,461 -15-l Myler et a l . (69) were the f i r s t to recognize the presence of two complementary factors for blue aleurone. They found that when they crossed any of the three blue v a r i e t i e s — Kwan, Algerian, or &wnless — with the white v a r i e t y Nepal, they obtained a single factor difference, as reported above. However, when they crossed the two white v a r i e t i e s , G o l d f o i l x Nepal, the result was a count of 9 blue : 7 white on the heads of the F-^ plants. Hox-zever, when they crossed the two white v a r i e t i e s , Hanna x G o l d f o i l , they obtained a l l white on the heads of the parents and on the heads of the F i plants. The postulated geno-type for the v a r i e t i e s which they studied are given i n Table 4. TABLE 4. THE FACTORS FOR BLUE ALEURONE COLOR IN CERTAIN BARLEY VARIETIES ( 6 9 ) . Color Variety Factors Black Hulless C.I. No. 5628 B l B l B l B l B l i B l i B l i B l i Blue aleurone layer Algerian Kwan Awnless Bolsheviki B l B l B l B l B l B l ... B l B l ''Bll B l i 111 B H B l l B l i B l l 111 White aleurone layer G o l d f o i l Hanna Neoal C I . No. 5649 b l b l b l b l B l B l B l B l B l l Bin B l i B l l b l l b l l b l l b l i Briggs et a l . (16) also report two complementary fac-tors for blue aleurone. The designation of the factor pairs -16-was reversed, however, for G o l d f o i l was assigned the Bl fact o r , and Nepal the B l ^ fa c t o r f o r blue aleurone. The naming of the p a r t i c u l a r character pair of blue and white aleurone followed the rules set down by Emerson et a l . (24) i n t h e i r work on the genetics of maize. As restated f o r barley by Robertson et a l . (79)» they contain the following points: 1. The name of the character i s generally suggestive of one of the chief attributes of that character. 2. The symbol of the character consists of the i n i t i a l l e t t e r of the name, and i f necessary some other appro-priate l e t t e r i n the name. 3. Allelomorphic series of genes have a common basic symbol, and are d i f f e r e n t i a t e d by subscript l e t t e r s . 4. Phenotypically s i m i l a r characters are usually given the same name, and d i f f e r e n t i a t e d by subscript numerals or l e t t e r s . Robertson (80) c l a r i f i e d the discrepancy of the symbols f o r the complementary factors of blue aleurone c o l o r . He c a l l e d the factors B l , b l and B I 2 , b]_2 according to the rules he set down i n his e a r l i e r paper. Because of the d i f f i c u l t y i n pri n t i n g the subscript, B l , b l remains unchanged although i t i s actually B l - p b l j . The former B l ^ , ] ^ , of Myler now becomes Bl2«bl?. Smith (i b i d . ) also follows t h i s system. (See Table 5») - 1 7 -TABLE 5- SYMBOLS USED BY VARIOUS WORKERS FOR THE GENES OF BLUE ALEURONE COLOR IN BARLEY. Authority- Symbol of Genes Linkage Group IV I II Buckley (17) Robertson et a l . (78) Myler et a l . T&9) Briggs et a l . (16) Robertson et a l . (80) Smith (94) BI BI BI BI BI BI B l l Blx B l i B l l BI BI B l BI* B l 2 Bl2 BI B l * Bl2 Bl2 * BIJL (understood) There have been several studies on the linkage of the character aleurone color with other characters. Buckley (17)? with a segregating sample of 714, found a crossover value of 40-56 per cent between blue and colorless aleurone (Bl,bl) and hooded and awned heads (K,k) i n linkage group IV. Robertson et a l . (78) obtained a crossover percentage of 22.58 +0 . 82 per cent, as shown i n Table 6. Myler et a l . (69) obtained a cross-over value of 24 .72 +1.73 per cent, very s i m i l a r to Robertson's TABLE 6 . COUNT SHOWING THE CROSSOVER VALUE OF GENES FOR BLUE (Bl) AND HOODED (K), BOTH IN LINKAGE GROUP IV, USING A NON-BLUE VARIETY WITH AWNS X BLUE VARIETY WITH HOODS. (78) Item Non-blue Blue K k K k Observed count 30^6 1 , 3 3 4 1 ,455 73 Calculated segregation 2 2 . 5 8 ^ crossover 3 0 4 9 . 8 1,411.2 1,411.2 7 5 . 8 x 2 = 6 . 1157 P = 0 . 1 0 7 0 - 18 -value. Iramer et a l . (48) made a cross K K b l b l x k k B l B l , and using a phenotypic K k and a genotypic B l B l , B l b l , b l b l basis, secured a crossover value of 44.0 + 6.30 per cent. Also i n linkage group IV, Immer found the crossover value to be 36.0 +3*3 per cent between blue and white aleurone and normal and glossy seedling (.01,gj.) . Briggs et a l . (16) established a value of 26.3 jl 5.0 per cent between the blue and white aleurone and resistance and s u s c e p t i b i l i t y to the race of mildew (Mlg,mlg). From t h e i r findings they proposed the gene order Bl-K-Mlg. Hanson et, a l . (37) indicate some linkage of blue aleurone with one of the genes f o r s e m i - s t e r i l i t y , with which he was working. With the above findings as background, Immer et a l . (48) have suggested the order i - k - z d - l g i - g l - g ^ - h l f o r linkage group IV, while Smith (94) proposed K-lg4-z-gl-Ml g-Bl. When Myler et a l . (69) revealed the presence of com-plementary factors f o r blue aleurone, they found that Blp,bl? were linked with naked and hulled (N, n). He calculated a crossover value of 9*88 + 0.44 per cent. , Later, Briggs et a l . (16) estimated the crossover value between Blp,blp and red stem and green stem (Rs,rs) to be 9.07 + 1.24 per cent; and the crossover value between N, n and Rs_, rs to be 14.5 JL 1.06 per cent. From t h i s they deduced that the probable order of these three l o c i would be N-Blp-Rs f o r linkage group III (94). 2. Other Kernel Colors Black, red and purple pericarp are a l l inherited - 1 9 -independently of blue aleurone color. To make our study of. the color of barley more complete, '.these factors w i l l be b r i e f l y out-l i n e d , , B i f f e n i n 1 9 0 7 ( 1 0 ) concluded that "purple palea" was simply dominant over '.'white palea" and his experiments showed that palea and caryopsis colors were associated. Robertson et a l . ( 7 9 ) have subsequently shown that "purple" and " c o l o r l e s s grain" (P,jo) are i n the same linkage group as "purple" and "white veined" lemma (Pc,p_e) and "red" and "white" pericarp (Re,re) (Woodward and T h i e r e t ) . Myler et a l ( 6 9 ) were also able to c l a s s i f y most of the purple seeds without removing the lemma. When using only the naked kernels they obtained counts which were quite a close f i t f o r one factor difference between white and purple seed. It i s i n t e r e s t i n g to note that both the purple parents they worked with were of the genetic c o n s t i t u t i o n P P, B l B l , B i o B l 2 . I f Harlan's explanation of "purple" seed color held true, v i z . , that purple i s due to red pericarp underlain by blue aleurone, they expected to get a count of 9 purple : 3 v i o -l e t : 3 blue : 1 white. However, no attempt was made to separ-ate plants with purple seeds and the blue aleurone factor from plants carrying the purple f a c t o r and colorless aleurone. Therefore, they obtained a count of 12 purple : 3 blue : 1 white. But u n t i l Woodward and Thieret ( 1 0 3 ) published t h e i r study of purple kernel color, most investigators regarded "purple" seed color as a simple dominant over non-purple ( 1 0 3 ) . Certain workers had interpreted the results of F2 segregations on the - 2 0 -basis of two factor p a i r s . Buckley (17) reports that Kajanus and Berg (51) found a "yellow" x "dark v i o l e t " cross which gave a complicated F 2 segregation. They explained that t h e i r results were due to two factor p a i r s , A,a co n t r o l l i n g the blue,color of the aleurone and B,b the brown color of the pericarp. Without A the aleurone i s co l o r l e s s , and without B the pericarp i s y e l -low. When both A and B are present the kernel i s v i o l e t brown. Suggestions have also been made that one of the complementary genes f o r red pericarp may be necessary for the development of purple pigment ( 1 7 ) . Woodward and Thieret ( 103) made 28 crosses of "purple" x "non-purple" and obtained 4 , 3 7 5 seeds of which 3 ) 2 7 6 were pur-ple and 1 , 0 9 9 were non-purple. This segregation f i t s quite c l o s e l y the expected 3*1 r a t i o (P = 0 . 7 - 0 . 8 ) . Following t h i s work they made 24 crosses between non-purple parents and obtained 5 , 5 4 7 seeds. Of these 3 > l l 6 were purple and 2,431 non-purple, which i s close to a 9 - 7 r a t i o (P = 0 . 8 - 0 . 9 ) . The F 3 segrega-tions supported t h e i r findings. They have ca l l e d these two complementary genes for purple color P,p_ and C,c and they placed them i n linkage groups I and II respectively. There are also reports of purple F 2 seeded plants developing from crosses between black and white seeded v a r i e t i e s as well as from crosses between other non-purple seeded v a r i e t i e s ( 1 0 3 ) . As mentioned e a r l i e r , black barley seeds contain a melanin-like pigment i n the pericarp. Buckley (17) reports that "white" and "black" pericarp (Bk,bk) are simply inherited. Robertson et a l . (79) symbolizes the genes as B,b, and state 21-that they also control the black and white lemma color, and are i n linkage group II (103). Red pericarp color i s controlled by complementary factor pairs Re, re and Rei>Z£l i n linkage groups V and I res-pectively (17, 79). 3 . Barley Hybridization Techniques B i f f e n (10) made successful i n t r a - s p e c l f i c barley crosses as e a r l y as 1907. His method was to remove a l l but eight to twelve of the median f l o r e t s on the rachis, just as the awns were emerging from the boot. These were then emasculated by c l i p p i n g the end of the palea and removing the anthers. To p o l l i n a t e the emasculated heads, stamens just at the breaking point were inserted into the opening at the apex of the palea, and the heads were bagged. In 1918 2 e l i n c k (49) f i r s t introduced the idea of the approach method of hybridization, which was l a t e r modified by Rosenquist (87), Pope (74, 75), and Hamilton (33). The approach method of barley hybridization, now used at the Central Experi-mental Farm at Ottawa (33), i s characterized by the enclosure of male and female (emasculated) spikes under one bag. This allows the p o l l e n from the male spike to p o l l i n a t e the f l o r e t s i n the emasculated spike. With a minimum of e f f o r t , seed sets of 70-90 per cent are obtained. I f the procedure i s carried on i n a greenhouse i t i s -22-preferable to keep the plants i n a cool house, around 40 F. or s l i g h t l y above. I f the temperature i s much warmer the spikes must be emasculated at an e a r l i e r stage i n the plants' develop-ment. For f e r t i l i z a t i o n and bagging, the plants should be moved to a house where the temperature i s around 6 5 ° F. The spike chosen f o r the female parent should have no viable pollen present. The f l o r e t s i n the l a t e r a l spikelets are removed, as well as any over-mature or under-mature f l o r e t s at the basal or apical ends of the spike. About one t h i r d of the apex of the lemma and palea of each f l o r e t i s clipped o f f , leaving the male and female organs exposed. The stamens are now removed from each f l o r e t , Although the spikes used are s t i l l immature, they should be capable of being f e r t i l i z e d i n a few hours, or at most i n a day or two. Therefore they should i be bagged immediately. The spike used as the male parent should have the top of the lemma clipped to allow the pollen to escape from the dehiscing anthers. However, i t i s only necessary to c l i p o f f the awns i f the spike i s of the type i n which the an-thers do not extrude p r i o r to dehiscence. The male and female spikes thus prepared are then brought together under one bag. The male spike i s set s l i g h t l y higher, so that any mature pollen from i t w i l l have a chance to f a l l into the exposed styles i n the female spike. To encourage t h i s transfer of pollen the bag should be tapped at least three times a day over a period of two or three days, p a r t i c u l a r l y i f - 2 3 -the weather i s sunny. For greater success t h i s male spike should be replaced by another after two or three days, so that pollen w i l l be available over a six day period. For convenience the parent plants should be grown i n pots so that the spikes can be r e a d i l y placed together. An alternative method i s to cut the male parent about eight inches below the spike. It can then be placed i n a small bottle of water, which i n turn i s t i e d to a metal rod or similar support standing adjacent to the female plant ( 3 3 ) . F. RELEVANT CHEMISTRY OF PIGMENTS The topic of coloring matter i s an extremely involved one. However, since most of the experimental work involves blue and white barley v a r i e t i e s , most of the emphasis has been placed upon anthocyanin and i t s related pigments and co-pigments. The chemistry of the anthocyanin compounds i s reviewed and meth-ods related to the study of these compounds are examined. Some consideration i s also given l a t e r to the possible controls of these pigments i n plants. W i l l s t a t t e r (101), i t i s well known, undertook some of the early fundamental investigations on the chemistry of the anthocyanins. Karrer ( 5 2 ) and Robinson (81, 8 2 , 8 3 , 8 4 ) have continued his work, so that today the chemistry of these com-pounds i s quite well understood. Only the co n s t i t u t i o n of n i t r o -genous anthocyanins and leuco-anthocyanins needs much further i n v e s t i g a t i o n . - 24 -1. Anthocyanin Structure Anthocyanin i s a flavonoid compound which i s glyco-s i d i c i n nature. Flavonoid compounds are characterized "by the possession of a C^-C^-C^ carbon skeleton, which i s two aromatic rings linked by an a l i p h a t i c three-carbon chain. On the.basis of the oxidized state of the a l i p h a t i c part, the flavonoid com-pounds are sub-divided into well-known types, such as anthocyan-ins , flavones, chalcones, etc. (28a). The aglucone part of the anthocyanin moleculesis calle d anthocyanidin. It seldom occurs i n nature i n t h i s aglucone state. The anthocyanidin may also be regarded as a derivative of 2-phenylbenzopyrylium or flavylium chloride. 4-Each of the aromatic rings has c h a r a c t e r i s t i c types of hydroxyl-ation. The six most common anthocyanidins are: QO^> TX^"U X X ^ 5 Pelargonidw Cyamdm Deiphimdin -25-OH r l i r s a t i d t n G e n e r a l l y , an increase i n the number of hydroxyl groups tends to increase the b l u i s h t i n t i n anthocyanins and anthocyanidins, as i n the p e l a r g o n i d i n to d e l p h i n i d i n s e r i e s . A change from the 3- to the 3»5- sugar attachment also increases the blueness. The methylation of the OH groups, on the other hand, tends to increase the red c o l o r (11). The m a j o r i t y of anthocyanins are very s t a b l e at room temperature i n 1 per cent aqueous HC1. However, some complex anthocyanins are r e a d i l y hydrolyzed, e s p e c i a l l y during p u r i f i c a -t i o n . The anthocyanidins, on the other hand are a l l s t a b l e as s a l t s of strong a c i d s , f o r they a l l possess a p o s i t i v e charge. This p o s i t i v e charge i s due to the presence of a carbonium i o n , which i s the r e s u l t of the o s c i l l a t i o n of a p o s i t i v e charge between carbon atoms 2 and 4. Thus i t might be envisioned that the "double bond" o s c i l l a t e s between the two p o s i t i o n s , and two s t o i c h i o m e t r i c forms of the compound are present, v i z : -26-In the anthocyanins, the glucosidic residues are usually attached to p o s i t i o n 3 i n the monoglucosides and d i g l u -cosides, or i n positions 3 and 5 i n the dimonoglucosides (104). The most common sugar molecules which can make gly c o s i d i c OCc9 OCc£> C£cP monoglucoside diglucoside dimonoglucoside attachments to the anthocyanidins are glucose, galactose, rhamnose, cellobiosey and gentiobiose (11). The c o n s t i t u t i o n a l formulae of anthocyanins were devel-oped by the conversion of flavonols of known structure into anthocyanidins by fusion with potassium hydroxide (11). Robinson ( 8 5 ) gives a good review of t h i s and other methods. As mentioned e a r l i e r , anthocyanin-color varies consid-erably because of the differences i n the anthocyanidin molecule. More noticeable v a r i a t i o n i s due to differences i n pH of the solvent. Pure anthocyanins are generally red i n acid solutions, v i o l e t i n weakly alkaline solutions, and deep blue i n strongly alkaline solutions. This i s explained by the formation of a v i o l e t color base at pH 8 . 5 , and a blue s a l t color base at pH 11.0 (61), v i z . - 2 7 -oxonium s a l t colour base N a s a l t colour base pH 3 or less - red pH 8.5 - v i o l e t pH 1 1 . 0 - blue We can see now why a standard pH i s extremely neces-sary when dealing with anthocyanin color ( 2 6 ) . With t h i s standard pH the oxonium s a l t s of anthocyanins i n solutions can be distinguished by t h e i r shade of red ( 1 1 ) . Robertson and Robinson ( 7 7 ) were able to determine which anthocyanin was pres-ent by tests with buffered solutions of d e f i n i t e pH values. The c e l l sap pH of the tissues of a given botanical v a r i e t y i s often s l i g h t l y higher i n blue than i n red tiss u e of the same kind ( 1 1 ) . Histochemical tests for anthocyanin are also f a i r l y easy. The tissues with anthocyanin are red i n acid vapours, changing through v i o l e t to blue i n ammonia fumes. However, often i n alkaline vapours the tissue section becomes green due to the presence of other substances such as the yellow flavones referred to i n the next section. 2 . Co-pigments It would be an error to assume that the flavones merely blend with the anthocyanins. They are often a major feature of color v a r i a t i o n as a result of t h e i r tendency to form pigment - 2 8 -complexes with anthocyanins. It should be realized that many factors determine the color of anthocyanin i n a plant: (a) "inherent" differences i n the chamical c o n s t i t u t i o n of anthocy-anins, (b) changing amounts and proportions of mixtures of them, (c) a l t e r a t i o n i n the pH of the c e l l sap, (d) co-pigments, (e) c o l l o i d a l condition of c e r t a i n other components of sap ( 1 1 ) . The known co-pigments are anthoxanthins (flavones and f l a v o n o l s ) , tannins, and possible g a l l i c acid. Thus the sap of blue or purple flowers always contains a co-pigment, usually a colorless anthoxanthin, which forms a l a b i l e purple compound with anthocy-anin ( 3 2 ) . Reproduction of natural colors can be effected quite p r e c i s e l y i n v i t r o i f co-pigment associations are taken into account ( 8 2 ) . Co-pigment association with the anthocyanin molecule cannot be explained merely by s a l t formation, for they may be bonded even i n the presence of a large excess of mineral acids. It i s more l i k e l y that the two substances form a weak additive complex which may be dissociated by elevated tempera-tures or by the action of solvents, such as ethyl acetate, which are used to extract anthoxanthins ( 3 2 , 8 1 ) . Some Anthoxanthins Lutedln Apigerun - 2 9 -Qu-ercetitv Bu.t e i n F l a v o n e s , one of the group of the anthoxanthins, have formulae v e r y s i m i l a r to the a n t h o c y a n i d i n s . They appear u s u a l l y as y e l l o w c r y s t a l l i n e s o l i d s , s o l u b l e i n water, a l c o h o l , d i l u t e m i n e r a l a c i d s , and a l k a l i e s . However, u n l i k e anthocy-a n i d i n s , they do not form s t a b l e s a l t s w i t h a c i d s , but remain unchanged i n the presence of a l k a l i e s , which u s u a l l y i n t e n s i f y t h e i r y ellow c o l o r . Flavones are u s u a l l y g l u c o s i d i c i n n a t u r e , as are anthocyanins. They are thought to be s y n t h e s i z e d from a common s t a r t i n g p o i n t ( 3 2 ), v a r i a t i o n s being due to d i f f e r e n c e s i n paths of o x i d a t i o n . Flavones may even be converted i n t o antho-cyanins through the leuco-anthocyanins, which are of widespread occurrence i n p l a n t t i s s u e ( 6 0 , 8 3 ) . 3 . Leuco-anthocyanins Since l e u c o - a n t h o c y a n i n occurs i n b a r l e y ( 8 3 )» i t might be w e l l to review some of the l i t e r a t u r e r e l a t i v e to them. Rosenheim (86) found i n young grape-vine l e a v e s a c o l o r l e s s w a t e r - s o l u b l e substance which y i e l d e d a n t h o c y a n i d i n on - 3 0 -treatment with hot 20 per cent HC1. This reaction took place just as fast i n a stream of carbon dioxide as i n a i r when these gases were bubbled through the reaction mixture. The conversion of the leuco-anthocyanin to anthocyanidin apparently demands a leuco-anthocyanin cyanidin dehydration, not an oxidation. As the OH at the 4 p o s i t i o n i s to the carbonyl group, the reaction w i l l proceed i n the d i r e c t i o n leading to flavylium s a l t formation rather than to flavone ( 8 2 ) . It i s interest to note that almost a l l leuco-anthocyanins y i e l d only cyanidin and delphinidin when digested with HC1 ( 5 ) . For t h i s reason Bate-Smith (5) states 11 ... i t i s therefore a matter of some importance, i n connexion with the o r i g i n and function i n the plant of the leuco-anthocyanins and catechins, that the leuco-anthocyanins should be r e s t r i c t e d to the patterns of hydroxylation found i n the catechins." This does not preclude, however, the fac t that the leuco-anthocyanins have a "connection" with the anthocyanidins. But I t should be noted that "When anthocyanins and leuco-anthocyanins are both present i n the same part of the plant, the normal anthocyanin i s not i n every case ... derived from the same anthocyanidin as the leuco-anthocyanin. , , (11) - 3 1 -The wide d i s t r i b u t i o n of leuco-anthocyanins opens up many i n t e r e s t i n g q u e s t i o n s of b i o s y n t h e s i s and p h y s i o l o g i c a l f u n c t i o n . I t should be re-emphasized t h a t the r e l a t i o n between le u c o - a n t h o c y a n i n and anthocyanin pigments i s not as simple and d i r e c t as i t appears at f i r s t s i g h t ( 8 3 ) . 4. E x t r a c t i o n of Pigments Anthocyanin i s r e l a t i v e l y easy to e x t r a c t from p l a n t t i s s u e as i t i s s o l u b l e i n water ( 1 1 ) . The methods r e p o r t e d i n the l i t e r a t u r e are a l l " v a r i a t i o n s on the same theme". Robinson and Robinson ( 8 1 ) , and Bate-Smith (3) used a 1 per cent aqueous HC1 s o l u t i o n t o e x t r a c t anthocyanin from p e t a l s . Using much the same method, Kuroda ejt a l . (55) soaked the Kuro-mame type of soya bean i n c o l d water and obtained a r e d d i s h - p u r p l e s o l u t i o n of anthocyanin. However, they l a t e r r e p o r t e d (56) t h a t when these water-soaked seeds were t r e a t e d w i t h methyl a l c o h o l i c HC1 more of the same anthocyanin was ob-t a i n e d . I n a s i m i l a r manner, Blank (11) used 1-2 per cent HC1 i n methyl a l c o h o l to e x t r a c t anthocyanins from w e l l - d e s i c c a t e d p l a n t m a t e r i a l . Sando et, a l . ( 8 8 ) found i t more s a t i s f a c t o r y to e x t r a c t the anthocyanin w i t h 0 . 5 per cent HC1 i n methyl a l c o h o l , a f t e r f i r s t removing the f a t s and waxes by t r e a t i n g them w i t h e t h e r i n a l a r g e p e r c o l a t o r . And f i n a l l y , to round out the p i c -t u r e , Rosenheim (86) used 1 per cent HC1 i n e t h y l a l c o h o l t o e x t r a c t anthocyanin from p l a n t t i s s u e , while Eddy and Mapson ( 2 2 ) employed b o i l i n g e t h y l a l c o h o l . - 3 2 -The amount of anthocyanin i n flower petals varies considerably. In c e r t a i n blue v a r i e t i e s of corn flower the anthocyanin consists of 0.65 - 0.75 per cent of the dry weight, while i n c e r t a i n purple v a r i e t i e s i t goes as high as 14 per cent of the dry weight (11). In the blue barley t h i s percentage of anthocyanin i s much lower. Anthocyanidins are not soluble i n water. They may be removed from a water misture, where they have been produced by hydrolysis, through selective extraction with iso-amyl alcohol ( 5 , 83). It has been suggested that i f leuco-anthocyanin were insoluble i n iso-amyl alcohol, the anthocyanidins i n solution could be removed by selec t i v e extraction with the iso-amyl, and leuco-anthocyanin would remain i n the aqueous solution. Bate-Smith (5) was interested i n obtaining leuco-anthocyanin as antho-cyanidin. His problem was to obtain the anthocyanidin i n s u f f i -cient concentration to chromatogram without concentrating irrelevant substances. His method was to just cover 0.2 to 1.0 grams of tissue with 2N-HC1 i n a test tube. He then heated the test tube i n a b o i l i n g water bath for t\-/enty minutes and decanted off the aqueous solution into a small narrow test tube. This aqueous solution was then shaken with s u f f i c i e n t iso-amyl (3-methylbutan-l-ol) to give a supernatant layer just deep enough to be drawn cleanly into a c a p i l l a r y tube. From t h i s c a p i l l a r y tube the solution could be spotted on the st a r t i n g l i n e of the - 3 3 -chromatogram. The applications were repeated, with drying i n between, u n t i l the color was deep enough to ensure v i s i b i l i t y of the anthocyanidins. Leaves and other tissues pigmented with anthocyanin as well as leuco-anthocyanin are d i f f i c u l t to work with. I f the anthocyanin pigmentation i s not excessive, leuco-anthocyan-ins might be detected by comparison with a control prepared from an aqueous extract of the tissue hydrolysed with d i l u t e acid ( 5 ) « Otherwise the anthocyanin pigments tend to mask any color pro-duced by the leuco-anthocyanin when the extract i s hydrolysed. 5 . P u r i f i c a t i o n and Analysis of Anthocyanins P u r i f i c a t i o n of anthocyanins requires long and involved procedures, e s p e c i a l l y when the structure of the anthocyanin i s to be determined by chemical means, such as s p l i t t i n g o f f various sugar molecules ( 5 8 , 8 1 , 8 2 , 8 3 5 8 8 ) . Also of value are deter-minations using colorimetric readings ( 1 1 , 27? 59)? where stand-ard pH readings are extremely important ( 2 6 ) . Various color reactions i n buffered solutions are also reported ( 5 8 ) . 6. Chromatography The use of paper p a r t i t i o n chromatography has greatly reduced the work required to separate and i d e n t i f y the anthocyan-i d i n pigments. Chromatography was f i r s t introduced i n 1906 by the Russian botanist Ts\tfett. His work indicated the presence of two components i n chlorophyll. As t h i s was contrary to the -34-f.indings of the chemists of the day, Tswett's work f e l l into disrepute (62). In the type of chromatography developed by Tswett, there i s a s o l i d immobile par t i c u l a t e phase, over which moves the f l u i d solvent phase which carries the solute to be separated. P a r t i t i o n chromatography d i f f e r s from the above method, not i n the mechanism involved, but i n the media selected. . The chief difference i s that the " s o l i d " phase consists of a stationary l i q u i d , over which flows another immiscible mobile l i q u i d phase (64). The rate of migration of the solute i s a function of i t s p a r t i t i o n c o e f f i c i e n t i n the two l i q u i d s ( 6 2 ) . England and Cohn (25) show the method of actually determining t h i s c o e f f i c -ient i n amino acids by means of comparisons of t h e i r s o l u b i l i t y i n various solvents. The f i r s t workers to apply chromatography systematically to plant materials were Dent ejb a l . ( 2 0 ) . Paper p a r t i t i o n chromatography had i t s inception i n the work on proteins done by Consden et a l . (19). Partridge (73) did much i n applying the method to sugar separations, while G i l l e s jet a l . (39) used i t i n p a r t i t i o n i n g the carbohydrates of t h e i r barley f l o u r extracts. Wender and Gage (98) separated flavonoid pigments by the same method, and Bate-Smith (3) applied i t to the anthocyanins i n petals. Later, Bate-Smith and Westall (7) u t i l i z e d i t to d i f f e r e n t i a t e anthocyanidins, and Simmonds (93) was able to employ th i s method of Bate-Smith and Westall to d i f f e r e n t i a t e c l o n a l and c l i m a t i c differences on the - 3 5 -production of anthocyanin i n banana f l o w e r s . Bate-Smith ( 5 ) and Bate-Smith and Lerner ( 6 ) have more r e c e n t l y applied paper chromatography f o r the determination of leuco-anthocyanins. The Bp value i s the r a t i o of distance t r a v e l l e d by the band of pigment or substance under i n v e s t i g a t i o n to the d i s -tance t r a v e l l e d by the solvent or advancing f r o n t of l i q u i d ( 9 8 ) . The R F value i s r e l a t e d to the p a r t i t i o n c o e f f i c i e n t by the f o l l o w i n g formula: A L % = 1 L - aA s where: A L = cross s e c t i o n area of solvent phase (mobile phase). Ag = cross s e c t i o n area of water phase ( s t a t i o n a r y phase). a = p a r t i t i o n c o e f f i c i e n t of the pigment or substance between the water and the solvent phase. I t agrees w e l l w i t h the p a r t i t i o n c o e f f i c i e n t s reported by England and Conn ( 2 5 ) . Indeed, Bate-Smith and W e s t a l l (7 ) a c t u a l l y found that the use of chromatography to determine the p a r t i t i o n c o e f f i c i e n t of a substance had decided advantages. Although i t i s not as accu-r a t e , i t i s (a) simple and r a p i d , (b) r e q u i r e s extremely l i t t l e m a t e r i a l , and (c) there i s no need of a high degree of p u r i t y i n the m a j o r i t y of cases. S e v e r a l d i f f e r e n t arrangements of paper p a r t i t i o n chromatograms have been used by d i f f e r e n t workers. However, the - 3 6 -b a s i c p r i n c i p l e of a l l of them, as o u t l i n e d below, i s the same: 1. F i r s t , there i s a f i l t e r paper on which spots of the unknown substance are placed and d r i e d . D i f f e r e n t types of f i l t e r paper o f t e n have d i f f e r e n t a c t i o n s , but most workers have found Watman No. 1 q u i t e s a t i s -f a c t o r y (3, 13). 2. This paper i s then i r r i g a t e d w i t h a solvent while i n a saturated atmosphere. In order to o b t a i n t h i s c o n d i t i o n , i t i s necessar^ to c a r r y on the i r r i g a t i o n i n a sealed c o n t a i n e r . The solvents which have been found most s a t i s f a c t o r y c o n s i s t of three l i q u i d s . One of these l i q u i d s i s almost i n v a r i a b l y water, wh i l e the second i s immiscible w i t h the f i r s t . A t h i r d l i q u i d i s m i s c i b l e i n e i t h e r of the f i r s t two, and should have a low v i s c o s i t y to keep the solvent f r o n t moving more r a p i d l y , as w e l l as to allow i t , i f necessary, to d r i p o f f the end of the paper (50). 3. I r r i g a t i o n i s allowed to proceed u n t i l the solvent f r o n t has moved a s a t i s f a c t o r y d i s t a n c e along the paper — a distance which v a r i e s c o n s i d e r a b l y w i t h d i f f e r e n c e s i n paper, so l v e n t , temperatures, proced-ures, s o l u t e , e t c . Sometimes, however, i r r i g a t i o n of the paper must continue -- even a f t e r the solvent f r o n t has reached the end of the paper — u n t i l the v a r i o u s substances i n the spot of s o l u t e become s u f f i -c i e n t l y separated from each other. - 37 -4. The f i n a l step i s the removal and reading of the chromatogram. When the paper i s removed, the l i n e of the solvent front, i f present, i s marked. The paper i s then allowed to dry. I f necessary the chromatogram i s developed by spraying i t with some substance to bring out the spots; f o r example, nin-hydrin to indicate amino acids (18), or ammoniacal AgNO^ to indicate p-catechin (98). Also u l t r a - v i o l e t l i g h t can be used to separate several of the flavones, flavonols, flavonones, and chalcones (98). And f i n a l l y , to determine the Rp value, the distances the solute spots have moved are measured from t h e i r center of gravity, i n order that t h e i r measurements can be compared to the distance which the solvent front has t r a v e l l e d . These chromatograms are of two main types: ascending and descend-ing. The ascending type consists of a s t r i p of f i l t e r paper with the solute spots near the bottom. The s t r i p i s dipped into the solvent, and the l a t t e r then ascends by c a p i l l a r y action, carry-ing the spots with i t . The paper may be made into a cylinder and stapled with the edges not quite touching. Williams et. a l (100) claim that the ascending method gives greater consistency of r e s u l t , and allows f o r s i m p l i c i t y of apparatus and ease i n making a large number of runs. They also maintain that the s e n s i t i v i t y of t h i s method for determining the presence of various - 3 8 -amino acids i s double that of the descending type. In spite of t h i s report, the descending form of chromatography i s the one most generally used and accepted. Here the spots are placed near the upper end of the s t r i p of f i l t e r paper, the upper edge of which i s then dipped into a trough containing the solvent. The solvent then flows down the s t r i p carrying the spots with i t and separating the various components of the solute as i t descends. Two dimensional chromatograms are used for further separating c e r t a i n substances; that i s , when the spot contains several materials — some of which w i l l be separated by one s o l -vent and some by another — these two dimensional chromatograms are useful. E s s e n t i a l l y , t h i s method consists of placing a spot i n one corner of the f i l t e r paper, and i r r i g a t i n g i t with one of the solvents u n t i l the components of the solute are well separated. The paper i s then dried, turned so that the l i n e of separated materials i s next the solvent, and f i n a l l y i r r i g a t e d with t h i s second solvent ( 1 9 ) • As f o r the actual apparatus, many types have been used, such as the earthenware j a r , the glass cylinder ( 1 0 2 ) , the gasoline pump cylinder ( 9 8 ) , or the various manufactured types. Accurate chromatography can be used to determine the i d e n t i t y of an unknown, while at the same time giving some i n d i -cation of i t s molecular configuration. Several factors influence - 3 9 -t h i s accuracy, some of which are as follows: 1. Constant temperature, within + 0 . 5 ° C , during i r r i g a -t i o n . Most Rp values are determined at 20° C. 2. The solvent should he thoroughly mixed and allowed to stand f o r three days at the temperature at which i t i s to be used. It i s important that i t should never be used for a longer period than two weeks after i t s preparation. 3 . The paper s t r i p s with the test spots on them should be allowed to stand f o r 24 hours i n the vapour of the aqueous phase of the solvent before i r r i g a t i o n commen-ces. This i s necessary so that the whole system can come into equilibrium. 4 . A known control spot should always be run on the same s t r i p , and the s t r i p should be discarded i f . t h e Rp of that known i s wrong. 5 . The i r r i g a t i o n i s continued u n t i l the leading edge has t r a v e l l e d 30 - 35 cm. from the l i n e of spots ( 7 ) ' These above-mentioned factors are not e s s e n t i a l for ordinary runs. However, i n the case of usual runs, c e r t a i n points should be kept i n mind: (a) The paper needs to be of a constant type. (b) The distance between the spots and the source of the solvent should likewise be constant, for the greater the distance, the smaller the Rp value. (c) The quantity of substance and (d) the presence of extraneous materials also i n -fluence the Rp value ( 1 9 ) . (e) Certain substances are affected -40-by pH; for example, i t has been found that basic proteins are inc l i n e d to t r a v e l faster than the non-basic ones (18). On the other hand, a s l i g h t l y acid state of anthocyanins tends to cause les s diffuse spots than the aqueous condition, and when a small amount of acid i s added to plant extracts, pH v a r i a t i o n of the plant sap can also be cut down ( 3 ) . Bate-Smith ( 3 ) worked with the anthocyanins extracted from flower petals, using a 1 per cent HC1 solut i o n . He found that the butanol-acetic acid-water solvent (40:10:50 by volume) used by Partridge for separating sugars was the best of those that he t r i e d f o r p a r t i t i o n i n g these anthocyanins. However, he decided that the Rp value was not the only factor i n di s t i n g u i s h -ing the various anthocyanins, for he discovered that the color of the spots at diff e r e n t pH values, i n both ordinary and u l t r a -v i o l e t l i g h t , i s also important. On hiydrolyzing the anthocyan-ins , he further found that he was able to determine the glucosides present by the use of Partridge's method ( 7 3 ) . Bate-Smith and Westall (7) applied chromatography to anthocyanidins as well as to anthocyanins. To obtain some of the desired anthocyanidins, they hydrolyzed the appropriate anthocyanins f o r 15 minutes at 100° C. i n 5*5 N HC1. .Again they found the butanol-acetic acid-water (40:10:50 by volume) s a t i s -factory. However, they showed that a solvent consisting of m-cresol-acetic acid-water ( 5 0 : 2:48 by volume) tended to spread the d i f f e r e n t substances farther apart, while s t i l l keeping them -41-i n the same order. To prevent the anthocyanidins from fading during i r r i g a t i o n , they concluded i t was necessary to use a solvent consisting of equal volumes of butanol and 2N HC1. The F o r e s t a l laboratories have now developed a solvent composed of water-acetic acid-HCl ( 1 0 : 3 0 * 3 by volume), which has proved superior to the butanol - 2N HC1 solvent for chromatograming anthocyanidins ( 5 ) . For developing these chromatograms i t was discovered that the majority of the compounds studied gave c h a r a c t e r i s t i c spots when sprayed with a 2 per cent aqueous solution of f e r r i c chloride. Partridge's cold ammoniacal s i l -ver n i t r a t e reagent was also of use. This i s p a r t i c u l a r l y true because the rate of development of the s i l v e r on the spots i s dependent on the number of hydroxyl groups present; therefore, t h i s test can be diagnostic. They also placed the paper s t r i p s i n MH3 vapour to obtain the c h a r a c t e r i s t i c anthocyanin color changes, as well as the i n t e n s i f i c a t i o n of the anthoxanthin spots. The NH3 vapour likewise affects a number of other sub-stances i n a c h a r a c t e r i s t i c manner. F i n a l l y , to note further differences, they also observed the s t r i p s under an u l t r a - v i o l e t l i g h t . Simmonds (93) extracted the flower pigments i n eleven clones of bananas. After hydrolysis, the anthocyanidins present were determined by means of chromatography, using the F o r e s t a l and the butanol-2N HC1 solvents. These solvents were used separately, or j o i n t l y f o r a two dimensional separation. By these means he discovered excellent agreement between the scoring -42-of two observers as to the i n t e n s i t y of the various anthocyan-id i n s present. In his recent work with leuco-anthocyanins, Bate-Smith (5) converted them to anthocyanidins and chromatographed these v i s i b l e pigments. He maintained a low pH while running his chromatograms f i r s t by using the upper phase of the mixture n-butanol -2N HC1 (1:1 by volume). Later he switched to "Forestal Solvent", consisting of water-acetic acid-HCl ( 1 0 : 3 0 : 3 by volume) as well as m-cresol - 5 . 5 N HCl-acetic acid (1:1:1 by volume). His results can be seen i n Table 7» TABLE 7- E m a x AND Rp VALUE OF SOME ANTHOCYANIDINS (5). Anthocyanidin •^ max Solvent System (mu) n-Butanol-HC1 . Rp Value Acetic Acid-HCl Rp Value m-Cresol-HCl ""Acetic Acid Rp Value Pelargonidin 530 0 . 8 0 0 . 6 8 0 . 8 2 Cyanidin ) Paeonidin ) 545 0 . 6 9 0 . 7 2 0 . 5 0 O .63 O .69 0 . 8 7 Delphinidin ) Petunidin ) Malvidin ) 555 0 . 3 5 0 . 4 5 0 . 5 3 0.30 0 . 4 5 0 . 6 0 0 . 5 2 0 . 7 5 0 . 9 0 With these pink anthocyanidin spots, Bate-Smith ( 4 ) also obtained brown substances which either flowed with the solvent front or remained on the o r i g i n a l spot. These brown - 4 3 -impurities were from several sources, but were not from the de-composition of the anthocyanidin. When catechins are heated with HC1, they are converted to 'phlobaphenes•, which dissolve i n iso-amyl alcohol to give deep golden or brown solutions. There i s also a p o s s i b i l i t y that these leuco-anthocyanins under-go a co-condensation with the catechins. Sugars ( i n ripe f r u i t ) when heated with HC1 produce a golden-brown color which i s soluble i n iso-amyl alcohol to give a red-brown solution. On the chromatogram t h i s pigment appears as a brown zone near the solvent front, and i t appears cream colored i n u l t r a - v i o l e t l i g h t . To further confirm his chromatographic r e s u l t s , Bate-Smith (5) used spectrophotometer readings. Anthocyanidins have well-defined peaks i n the v i s i b l e region either i n ethanolic HC1 solution or when examined d i r e c t l y as a spot on the paper chromatogram (accurate + 2 mu), after Bradfield and Flood ( 1 5 ) . The p r e c i s i o n on c e l l u l o s e paper i s less than i n solutions but i t i s s u f f i c i e n t to recognize "band envelope's". &s the number of absorbent molecules i n the path of the beam is. unknown, i t i s best to eliminate the e f f e c t of t h i s unknown quantity on the shape of the absorption curve by p l o t t i n g log D rather than D against wave-length. The curves can then be compared d i r e c t l y by suitable v e r t i c a l transposition. They used Watman #1 f i l t e r paper with butanol-acetic acid-water. The measurements were made with a Unicam S.P. 500 -44-quartz spectrophotometer. S t r i p s 1 .2 cm. wide and 4-5 cm. long with the desired spot on them were cut from the chromato-gram. Two of the spring spacers supplied were inserted back to back i n one of the compartments of the c e l l c a r r i e r i n the two slot s closest together: i . e . s l o t s used with 2 mm. and 5 mm. c e l l s respectively, and the paper inserted between them. A s i m i l a r s t r i p from a blank chromatogram was also inserted i n the adjacent compartment of the c e l l c a r r i e r . Because of the opacity of the paper, modified methods were used. We s h a l l only review one of these. The results were not s i g n i f i c a n t below 225 mu. The H 2 lamp i s used and the s l i t width set at 1 .5 mm. for the range 400-270 mu; below 270 mu the s l i t i s increased to 2 mm. The instrument switch i s set at 0 . 1 and the normal procedure of the set i s followed. When strong absorption occurs at 320-400 mu i t may be necessary to use the tungsten lamp ( 1 5 ) . England and Cohn ( 2 5 ) showed that i n an homologous series of amino-acids the p a r t i t i o n c o e f f i c i e n t changed regu l a r l y with each additional carbon atom i n the chain. This would imply that the R-p value should vary with a corresponding degree of reg-u l a r i t y . One i s not surprised, then, to le a r n that Consden, Gordon, and Martin (19) were able to point out a discernable r a t i o n a l change i n Rjp value with changes i n c o n s t i t u t i o n of amino-acids. Although t h e i r series was too small for the p r i n -c i p l e to be f i r m l y established, Bate-Smith and Westall (7 ) were -45-able to report that Martin, through the use of anthocyanins, flavones, chalcones, etc., was able to add further weight to the t r u t h of t h i s p r i n c i p l e . A l a t e r work by B r a d f i e l d and Bate-Smith (14) showed that even variations i n configuration of cate-chins gave d e f i n i t e Bp patterns. Bate-Smith and Westall (7) show that the more highly hydroxylated substances generally have a lower Rp value i n an homologous s e r i e s . This they found to be true i n flavones, flavonols, anthocyanidins and chalcones (see Figure 1). Rp value also seems to r i s e with successive methylation of OH groups. This r i s e apparently depends very l i t t l e on the p o s i t i o n of the methylated hydroxyls, but rather on the degree of hydroxylation of the parent substance. As a general r u l e , the r i s e of Rp value per unit of hydroxyl which i s methylated i s only about one t h i r d as large as would r e s u l t from complete re-moval of the hydroxyl group. The glycosidation of OH groups with glucose i n any posi-t i o n usually causes a large decrease i n Rp value. Glycosidation of a second hydroxyl group has as great, or almost as great, an effect on the value as the replacement of the f i r s t group (see Figure 2 ) . However, the attachment of a second sugar molecule to the f i r s t has much less influence. With the addition of rhamnose the e f f e c t on the Rp value was found to be i r r e g u l a r . This was probably the re s u l t of the counterbalancing e f f e c t of terminal methyl groups on the sugar residues. -46-1.0 .6-X 2 0 . Pelargonidln. Delphi'nidin N' NuMBfcR OF OM GROUPS Figure 1. - More highly hydroxylated members of an homologous series i n Flavonols, Flavones, Anthocy-anidins and Chalcones generally have a lower R F value. A f t e r Bate-Smith and Westall (7) c Figure 2. - With the addition of each sugar residue to the anthocyanidin nucleus the Rp value of the anthocyanin so formed i s decreased. Afte r Bate-Smith and Westall (7). -47-G. BIOCHEMISTRY AND PHYSIOLOGY OF ANTHOCYANINS AND RELATED PIGMENTS Anthocyanins occur throughout the plant kingdom, except i n the Thallophytes. Because of t h e i r o p t i c a l proper-t i e s , the anthocyanins are probably the most prominent secondary plant substances. Chemists and botanists have always found them of considerable interest (11). Although the botanists have recorded a large number of observations on the occurrence of anthocyanins i n plants, there i s no clear picture of t h e i r function i n plants. In higher plants, anthocyanins are found i n many tissues - root, stem, flower, etc. Since they are water soluble they are c h i e f l y found i n the vacuoles, although they are also l o c a l i z e d i n both c e l l plasma and membrane. The presence or absence of anthocy-anins i s a useful attribute i n the c l a s s i f i c a t i o n of many c u l t i -vated plants, such as barley. Their use f o r taxonomic purposes i s despite the fa c t that t h e i r formation i s sometimes modified by external factors (11), arrested development, hastened maturity, or abnormal conditions due to malnutrition ( 9 9 ) • 1. Biosynthesis As early as 1914, W i l l s t a t t e r and Mallison (101) were able to demonstrate the intimate r e l a t i o n between anthocyanidins and other substances. Although anthocyanidins " l i e between" flavonols and catechins from the point of view of degree of o x i -dation, there i s no amount of experimental evidence pointing to - 4 8 -any one path of anthocyanin formation (11). Some paths which have been suggested are as follows: (a) from flavone-similar substances (b) from smaller s t r u c t u r a l elements (c) from the de-animation products of decarboxylated amino acids (d) from leuco-anthocyanins The f i r s t p a r t i a l synthesis of anthocyanidins was a transformation of flavonol when W i l l s t a t t e r and Mallison (ibid) changed the flavonol quercetin to cyanidin chloride by reduction with Mg i n aqueous methyl alcoholic HC1. S i m i l a r l y , other f l a v -onols i n nature were changed to flavylium s a l t s . Reactions from flavylium s a l t s to flavones were also effected by dir e c t and i n -di r e c t oxidation. In degree of oxidation then, anthocyanidins " l i e between" flavonols and catechins. However, i t i s now be-liev e d that anthocyanidins are not formed from flavonols i n the plant, and there appears to be no connection i n the plant between the flavones and anthocyanidins (11, 7 2 , 8 8 ) . Certain workers believe that anthocyanins are formed from oxy-flavonols i n the aleurone grains i n the Gramineae (11). Although the path of the formation of anthocyanins and t h e i r related C^-C^-C^, compounds i s s t i l l quite hypothetical, there are c e r t a i n genetic proofs which make i t appear f e a s i b l e . Both the C£'s are aromatic i n nature, ring A usually being a phloroglucinol and ring C a catechol. Both these rings could be - 4 9 -b u i l t of hexoses and bound together through aldol condensations to form a hypothetical intermediate product, v i z : , .OH 0 M r IT t G H O H - V C \\OM a j X L / A possible course of action i s that ring C condenses with a tr i o s e f i r s t (the C^-C^ framework i s wide-spread i n nature) and then adjuncts with ring A. This hypothetical intermediate can be e a s i l y trans-formed into various end-products through oxidation, followed by dehydration and ring closure. For example, oxidation at C-j_ would give cyanidin; at and the fl a v o n o l , quercetin. This then may be the reason f o r simultaneous occurrence of cor-responding flavonols and anthocyanins (11). There i s also a suggestion that flower flavone pigments result from de-animation connected with growth and development, while anthocyan pigments r e s u l t from de-animation connected with senescence. The precise reaction of t h i s condensation i s not known, but the phenyl ring may originate from tyrosine or phenyl-alanine (the phloroglucinol ring from short chain residues of a l i p h a t i c amino-acids or hexose). By oxidation, OH groups are introduced into the phenyl ring ortho to the existing group ( 7 2 ) . The role of leuco-anthocyanins i n t h i s cycle i s s t i l l f a r from c l e a r . As mentioned e a r l i e r , the r e l a t i o n of the leuco-- 5 0 -anthocyanins to the anthocyanidins i s not as simple as i t appears at f i r s t glance. It may be that they are both end products of p a r a l l e l synthesis from a common pigment precursor, but the i n v i t r o experiments which have been conducted do not confirm t h i s theory ( 1 1 ) . From what has been found so f a r i t seems probable that the oxidation processes seem to play an important part i n the synthesis of the anthocyanin compounds ( 1 1 ). The actual catalyst f o r t h i s oxidation process does not appear to have been located i n plants, but i t i s believed that the amount of oxidation i s con-trolled- by the reaction of the tissue before condensation ( 7 1 ) • Consideration of d i f f e r e n t degrees of oxidation as related to pigments produced might prove a good point of attack for the gen-e t i c i s t s ( 7 2 ) . 2 . Genetics Belated to Biosynthesis To achieve any given stage i n a series of biochemical reactions, a number of conditions must be favourable. Examples of such are the presence of the right raw materials i n proper con-centration, a suitable pH, the presence of some catalyst, and so on. In a plant these d i f f e r e n t conditions are governed by d i f -ferent genes., which are sometimes even thought of as having the properties of ordinary enzymes ( 3 2 ) . Even a short step of a biochemical reaction may be controlled by a number of genes. I f one of these factors i s lacking, some condition i s made unfavour-able f o r that p a r t i c u l a r reaction. Even with the small -51-differences that exist between anthocyanidins and anthocyanins, and between anthocyanins themselves, i t must not be assumed that only one gene i s necessary to convert an anthocyanidin into an anthocyanin or one anthocyanin into another, although the two pigments may segregate i n a 3*1 r a t i o ( 8 ) . Working with snap-dragons, Haney ( 3 6 ) has concluded that the formation of the dominant purple color may be interrupted at d i f f e r e n t points of development by lack of dominant color f a c t o r s . This then yields the seven observed phenotypic classes. In general, white or yellow flowers contain anthoxanthin but no anthocyanin. This lack of anthocyanin pigment i s usually due to a recessive gene blocking synthesis, but i t can also be due to a dominant i n h i b i -tor ( 3 2 ) . Actually, however, i t i s the whole set of chromosomes which i s responsible for the quantitative balance of the system of reactions. Thus i t i s that i n investigations of the heredity of anthocyanins i n flower petals, geneticists have been interested i n : (a) the genes c o n t r o l l i n g pigment production (b) those con-t r o l l i n g modification of the anthocyanin type, and (c) those co n t r o l l i n g the condition of the c e l l sap. Through th i s study of the inheritance of i n d i v i d u a l chemical substances, rather than of color as a whole, much important evidence concerning the bio-synthesis of anthocyanins has been found (11). The r e s u l t s of t h i s work indicate the following: (a) P l a s t i d pigment, co-pigment, anthoxanthins and both general and s p e c i f i c anthocyanin production are - 5 2 -generally dominant to t h e i r absence, but r a r e l y i s a dominant i n h i b i t i n g factor involved. (b) Modification involving more oxidized anthocyanin pigmentation i s dominant to less oxidized, and more methylated pigmentation to less methylated. (c) 3 - 5-diglycosidic and acylated (complex) anthocyanin pigmentation are dominant to 3-mono-glycosidic and normal anthocyanins respectively. (d) More acid petal pH i s dominant to less acid. (e) Uniform pigment d i s t r i b u t i o n i s generally dominant to fl e c k i n g or marbling. Evidence has been put forward which contradicts these r e s u l t s . In many cases i t has been demonstrated that only one gene controls pigment formation, c e l l sap reaction, etc. Mix-tures of anthocyanins occurred i n Verbena, due to incomplete dom-inance or to modifying factors (11). Also, a gene which alters the chemical nature of the anthocyanin i n one part of a plant possibly changes the nature of the anthocyanin i n the same way i n a l l of the plant ( 3 2 ) . Very l i t t l e work has been done i n the f i e l d of antho-cyanin genetics i n the past f i f t e e n years, and further study i s needed to bring t h i s f i e l d of i n v e s t i g a t i o n up to the l e v e l of the knowledge of genes c o n t r o l l i n g carbohydrate differences (Hal-dane 1954). - 5 3 -3 . Comments on General Physiology A very l a r g e l i t e r a t u r e of the physiology of antho-cyanins e x i s t s and no attempt w i l l be made to review i t . T h e i r importance i n many ways has been stressed by Onslow (70) Blank (11) M i l l e r (65) and many others. Although the g e n e t i c a l background of a pla n t determines that p l a n t ' s p o t e n t i a l i t y to develop anthocyanin, the a c t u a l pro-d u c t i o n of t h i s pigment i s l a r g e l y c o n t r o l l e d by the environment i n which the pla n t i s growing. The environment g e n e r a l l y i s more important i n governing formation of anthocyanin i n parts of the p l a n t other than the f l o w e r s , and i t i s to these p a r t s that a t t e n t i o n s h a l l be d i r e c t e d . I n b a r l e y seed, the i n t e n s i t y of the blue c o l o r i s con-t r o l l e d p a r t l y by the v a r i e t y and p a r t l y by the growing condi-t i o n s ( 1 0 , 4 5 ) . The c o l o r i s heightened by abundant moisture, e s p e c i a l l y when nearing m a t u r i t y . However, t h i s moisture must not be allowed to wet the seed; otherwise i t w i l l cause bleach-i n g . Thus, under humid or very dry c o n d i t i o n s , the l a c k of expression of anthocyanin i n the aleurone l a y e r of the blue v a r i e t i e s may be so complete the sepa r a t i o n of the blue from the white cannot be made w i t h c e r t a i n t y ( 1 , 38). A l s o there i s an increase of c o l o r i n the purple v a r i e t i e s when the seed i s ex-posed to s u n l i g h t or i s naked. G e n e r a l l y , however, c o l o r i s heightened under dry con-d i t i o n s , p o s s i b l y by the formation of anthocyanins from the -54-aromatic amino-acids after de-amination. Hurd (47) has found that drought increases the amount of acid and the amount of sugar i n the wheat seed, thus making the anthocyanin less blue. This may also be true i n barley, f o r bluer barley does have a greater percentage protein (11). E a r l i e r authors assumed that there was a connection between anthocyanin formation and nitrogen metabolism. Others believe that the appearance of red c e l l sap was i n close r e l a t i o n to sugar content of the c e l l sap (11). It i s conceivable that the presence of sugar i n the tissue may prevent the de-amination i n the types which normally do not produce pigment; or the excess of sugar may bring about condensation as i n sugar feeding, which brings on color formation. If so, the factors are correlated with some metabolic cycle other than that of pigmentation (11, 22, 72). Such ef f e c t s might be the destruction of chloroplasts by the added sugar, or the accumulation i n the tissue of assimilated sugar. The content of other aromatic compounds (naphthochinones, tannins and arbutin) can be increased by the use of n u t r i t i v e solu-tions r i c h i n sugar. It i s possible that anthocyanins might increase i n the same way (41). Quantitative investigations are almost e n t i r e l y lacking on these problems and u n t i l they are under-taken the questions of connections between sugar and nitrogen metabolism and anthocyanin formation w i l l not be cleared up (11). Plants which have a mineral nutrient deficiency often show increased anthocyanin formation. Some think that the i n -crease i n pigment formation under low nutrient le v e l s may be -55-explained by the effects of the amount of available carbohydrates. The young barley plants which were d e f i c i e n t i n N, P and K pro-duced more color. On the other hand, the red color of red cab-bage was promoted when K was added, but was reduced when N and P were added. Why the anthocyanin i s not used i n the hunger metabolism of the plant i s not understood. It would appear that the sugar of anthocyanin can hardly be s i g n i f i c a n t as a source of energy (11). As with chlorophyll, c e r t a i n plants do and c e r t a i n plants do not require l i g h t for the formation of anthocyanin. The rays of the solar spectrum most i n f l u e n t i a l i n coloring apples o were i n the range 3,600 A - 4,500 A. The best a r t i f i c i a l l i g h t f o r the same purpose was a mercury vapour arc i n Uviol glass. Plum color formed without i l l u m i n a t i o n but the color was more con-centrated i n l i g h t . U l t r a v i o l e t rays which injured the plants brought about anthocyanin formation. Some believe that plants containing anthocyanins have a greater resistance against u l t r a -v i o l e t rays. In a c e r t a i n case i t has been found that continuous il l u m i n a t i o n caused a stoppage i n the formation of the pigment. In t h i s case i t seems that a photochemical and a darkness reaction are necessary f o r the formation of anthocyanin (11). The numerous reports on the influence of temperature on the formation of anthocyanin are contradictory, because i n most cases the pigment was not extracted and quant i t a t i v e l y determined. Apparently a l l plants have an optimal temperature for anthocyanin formation, probably coinciding with the optimal temperature for -56-metabolism (11). Extreme temperatures probably have much the same effect as attacks by parasites or some other sort of i n j u r y . These tend to disturb the normal metabolism of the plants, f r e -quently causing an increase i n anthocyanin formation (11). There are so many i n t e r - r e l a t e d factors c o n t r o l l i n g anthocyanin formation i n the plant that i t i s very d i f f i c u l t to determine t h e i r r e l a t i v e importance. This i s es p e c i a l l y true as the place of anthocyanin i n plant metabolism i s not under-stood. * * * * * * * The above review i s a very inadequate coverage of the vast number of reports on the subjects discussed. It i s s u f f i ' cient for the purpose of forming a p a r t i a l background for the following experiments. - 5 7 -I I I . EXPERIMENTS The experiments on the i n h e r i t a n c e and p h y s i o l o g y o f k e r n e l and p l a n t c o l o r i n b a r l e y , r e p o r t e d here, were conducted on the campus at the U n i v e r s i t y of B r i t i s h Columbia from the autumn of 1953 to the autumn of 1955* Inasmuch as c o l o r expree-s i o n i s s u b j e c t to c o n s i d e r a b l e m o d i f i c a t i o n by environmental f a c t o r s , i t was f e l t t h a t some study of e x t e r n a l c o n d i t i o n s probably a s s o c i a t e d w i t h c o l o r e x p r e s s i o n should be made. I n other words, i t was hoped t h a t s t u d i e s of t h i s k i n d might l e a d to the establishment of s e t s of e x t e r n a l c o n d i t i o n s under which c o l o r development could be ensured. A prime purpose of the experiments, too, was to substan-t i a t e , i f p o s s i b l e , the p a t t e r n s of i n h e r i t a n c e f o r b a r l e y k e r n e l c o l o r s proposed by other workers ( 1 7 , 53? 9 4 ) . G e n e t i c a l s t u d i e s to date, although c a r e f u l l y and a c c u r a t e l y conducted, have not been ext e n s i v e enough to f i r m l y e s t a b l i s h confidence i n t h e i r g e n e r a l i t y . A l s o d i s p o s i t i o n of pigments i n t i s s u e s has r e c e i v e d o n l y the s k e t c h i e s t of study and t h a t was s e v e r a l decades ago ( 3 9 ) . Pigments i n a v a r i e t y of p l a n t s , but not b a r l e y , have been f a v o r i t e s u b j e c t s f o r s t u d i e s which attempt to r e l a t e g e n e t i c s and i n t e r m e d i a r y metabolism and f o r some of v e r y fundamental s t u d i e s on the nature of the gene. I t appeared t h e r e f o r e , t h a t b a r l e y pigments might be used f o r these purposes, - 5 8 -e s p e c i a l l y when one considers i t s d i p l o i d nature and the fact that i t i s , genetically, one of the best known plants. The experimentation, here reported, i s incomplete. Planting of seeds, now awaiting harvest, and study of t h e i r seed y i e l d , w i l l be needed to f u l l y c o l l a t e the data. Inasmuch as a large population must be planted, results w i l l not be to hand u n t i l after the harvest season of 1 9 5 6 . 1 . CROSSING EXPERIMENTS TO RESOLVE THE GENETICAL CONSTITUTION OF THE FACTORS FOR KERNEL COLOR IN BARLEY. cons t i t u t i o n of kernel color i n several barley v a r i e t i e s i n order to substantiate, i f possible, the results obtained at the Central Experimental Farm, Ottawa, as well as elsewhere, and to extend the l i s t of barley v a r i e t i e s studied f o r the, genetics of kernel color. The genetical information w i l l be of considerable value when related to the re s u l t s of the chromatographic and h i s t o l o g i -c a l experiments which are reported l a t e r . Seeds of the following v a r i e t i e s were used i n the crosses: The purpose of these crosses was to determine the genetic Algerian Awnless Black Hulless Carlsberg * Deficiens Ethiops Fort * G o l d f o i l Hanna Kitchen Kwan Lion Nepal Smyrna Trebi Velvon 11 * C L . 5628 4811-68-2 * 5 0 9 0 - 1 5 - 1 C 5 4 - 5 5 5425-8 * Crosses made i n 1953 and 1955--59-Three plantings were made i n six- i n c h clay pots f i l l e d with s t e r i l i z e d greenhouse s o i l which had 2 - 1 6 - 6 f e r t i l i z e r added. These plantings were made i n the greenhouse at about ten day i n t e r v a l s ; actually each seeding was made when the preceding planting had reached i t s second l e a f stage. A plant-ing consisted of two pots with f i v e plants per pot, v i z . ten plants per v a r i e t y . In 1953 there were s u f f i c i e n t pollen parents to make crosses using the approach method (see L i t e r a t u r e Review). In 1955) because of the shortage of ripe pollen, the following method was used: Before the pollen was r i p e , the glume t i p s of the head selected as the female parent were clipped with a small p a i r of sharp scissors and the immature anthers removed with pointed tweezers. Care was taken that there was no pollen on the anthers. The tweezers were immersed i n alcohol and dried between the emasculation of each f l o r e t i n order to k i l l any stray pollen. The emasculated head was then tagged and covered with a glassine bag. After 24- to 48 hours, mature anthers from the v a r i e t y selected to complete the cross, were broken into the open ends of the f l o r e t s and then the glassine bag was replaced. After the seed was r i p e , each head was harvested and threshed separately, and then the seed was examined for color. This seed was planted to produce the F i plants. When these plants are mature, the seed from each plant w i l l be harvested -60-and threshed separately, and counts made of the colored and un-colored seed. I f necessary, t h i s seed w i l l be planted i n the f i e l d and grown to produce the F 2 plants. The seed from each of these F 2 plants can then be bulked and c l a s s i f i e d f o r kernel color. The seeds from the crosses made i n 1953 were not mature enough* to show any color. They were planted i n the spring and grew very r a p i d l y i n the hot greenhouse. However, because of the high temperature, they did not mature properly and i t was impossible to di s t i n g u i s h the blue from the white seed with any degree of accuracy. Discerning the color of the kernel was made more d i f f i c u l t because moth larvae destroyed large portions of the seed. The color of the kernels on the female parents i n the 1955 crosses was noted where possible. As t h i s seed was not ready for planting u n t i l l a t e i n the summer, i t had to be planted outdoors i n pots. When the weather became too cold and stormy, these pots were moved into the greenhouse. This seed should be ready to harvest soon. It w i l l be necessary to grow the 1955 crosses f o r one more generation to give F 2 plants. The aleurone of the seed produced on the F 2 plants w i l l be F3 because i t i s part of the endosperm, and thus exhibits xenia. For t h i s reason i t i s pos-s i b l e to designate the whole F 2 plant as homozygous blue or white, * As e a r l i e r noted, aleurone colors develop just p r i o r to har-vest. E a r l y harvest may not permit t h e i r development. -61-or heterozygous f o r blue and white aleurone. Because i t i s part of the maternal t i s s u e , the pericarp w i l l be just s t a r t i n g to segregate i n the F 2 plants. The r e s u l t s of these counts w i l l be included l a t e r i n an appendix to th i s t h e s i s . The crosses which were made at Ottawa gave the following results i n the heads of the plants: Velvon 11 x 4811-68-2 — 3 blue : 1 white Carlesberg x Fort — 1 blue : 1 white The re s u l t s of the Ottawa crosses w i l l also be compared with the writer's F 2 r e s u l t s , i n an appendix to be added l a t e r . 2. EFFECT.>0F CERTAIN GROWING CONDITIONS ON THE DEVELOPMENT OF BARLEY PLANT PIGMENTS It i s d i f f i c u l t to d i f f e r e n t i a t e g e n e t i c a l l y blue seed from g e n e t i c a l l y white seed when they have been grown under cer-t a i n environmental conditions. For th i s reason an attempt was made to produce sets of growing conditions which would e f f e c t the formation of barley plant and kernel colo r . A. An Attempt to Produce Color i n Seedlings A large number of barley seedlings can be produced i n a r e l a t i v e l y small space and with a minimum of equipment. I f these barley seedlings would produce pigments, then they could be e a s i l y subjected to a large range of environmental conditions to ascertain which set of conditions produced the most pigment development. For these reasons an experiment was conducted to determine whether barley seedlings would produce color when -62-germinated i n petri-dishes. Seeds of the following barley v a r i e t i e s were used: Kwan O.A.C. 21 Vantage Fort Algerian Black Hulless Montcalm Velvon 11 F i l t e r paper was placed i n the bottom of eight p e t r i -dishes and then saturated with water. Twenty-five seeds of a va r i e t y were placed i n each p e t r i - d i s h . The dishes were then covered with t h e i r l i d s and placed i n a window. The tempera-ture was about 70° F. On a few scattered Algerian seeds there was a s l i g h t c oloring. The roots of "O.A.C. 21", "Fort" and "Black Hulless" showed a few pinkish spots. This coloring, which appeared to be either inconsistent or due to colored fungi, subsequently faded or turned brown. There was also a s l i g h t reddening i n the c o l e o p t i l e of the "Black Hulless" seedlings, but t h i s also faded and turned brown. It appeared that seedlings grown under these conditions do not produce s i g n i f i c a n t c o l o r i n g . Therefore i t was decided to abandon the use of seedlings and ran tests on the whole plant. B. Experiments to Produce Color i n Plant, Kernel and Detached Leaves. (a) Plant Color The following experiment was made i n an attempt to determine a nutrient l e v e l which, i n r e l a t i o n to certa i n - 6 3 -e n v i r o n m e n t a l c o n d i t i o n s , would i n d u c e t h e development o f c o l o r i n the b a r l e y p l a n t . The b a r l e y v a r i e t i e s "Smyrna", "Awnless" and " B l a c k H u l -l e s s " were used. They are w h i t e , b l u e and p u r p l e seeded r e s p e c -t i v e l y . E i g h t - i n c h c l a y p o t s , c o n t a i n i n g a l a y e r o f g r a v e l i n the bottom, were f i l l e d w i t h b u i l d e r s ' sand. F o r t y - f i v e p o t s o f each o f the t h r e e v a r i e t i e s were seeded on December 12, 1954, s i x seeds t o a p o t . These p o t s were t h e n p l a c e d on the c e n t r a l wooden bench i n the greenhouse, and watered w i t h t a p wa t e r u n t i l t he p l a n t s emerged. When t h e s e e d l i n g s emerged, t h e y were t h i n n e d t o f o u r p l a n t s p e r pot and watered w i t h m o d i f i e d Knop n u t r i e n t s o l u t i o n s (6la). Three t y p e s o f n u t r i e n t s o l u t i o n s o were p r e p a r e d by w e i g h i n g out s u f f i c i e n t s a l t t o make up 20 l i t r e s of each s o l u t i o n , as f o l l o w s : S a l t Complete -N -P KC1 5 gms. 6 gms. 7*64 gms. K H 2 P 0 4 5 gms. 3 gms. MgS0 4 .7H 20 5 gms. 9 gms. 9 gms. Ca (N0-j) 2.4H 20 20 gms. — 10 gms. CaS0 4.2H 20 — 7.28 gms. F e C l 3 0.2 gms. 0.2 gms. 0.2 gms. The q u a n t i t y o f s a l t s r e q u i r e d t o make t h e 20 l i t r e s o f n u t r i e n t s o l u t i o n was p l a c e d i n a b o t t l e and made up t o 19 l i t r e s -64-with d i s t i l l e d water. The FeCl-^ solution was made up separ-ately with 1 l i t r e of water and added to the nutrient solution just before i t was applied to the pots. Each pot received 450 cc. of solut i o n at each watering. Pea gravel was placed on the surface of the pots to prevent the sand from being gouged and the roots from being disturbed when the nutrient solution was poured on. It was also found necessary to n a i l two s t r i p s of wood about 1-jjr inches apart under the pots, to prevent the roots from growing down onto the bench where they might obtain extra nutrients. When the plants i n the pots receiving the -N and -P solutions appeared to need some extra nutrients i n order to mature seed, they were given an application of complete solution. They were thus designated "low nitrogen" (low N) and "low phos-phorus" (low P). The low nitrogen and low phosphorus plants received, respectively, three and two applications of the com-plete solution. The pots were set out into the following treatment blocks: "Control", " U l t r a - v i o l e t " , "Infra-red", "Dextrose", and "Outside 1! Each of these f i v e treatment blocks contained f o r t y - f i V e pots, 9 of each of the v a r i e t i e s "Awnless", "Black Hulless" and "Smyrna". Each of these v a r i e t i e s i n turn had three pots re-ceiving each of the nutrient l e v e l s , complete, low N, and low P. - 6 5 -The pots of each treatment block were grouped together but the v a r i e t i e s and nutrient l e v e l treatments were placed at random within each block. The centers of the pots were about 10 inches apart- and the blocks were 20 inches apart. Lamps f o r the " u l t r a - v i o l e t " and " i n f r a - r e d " r a d i a t i o n treatments were placed 3-^ feet above the sand l e v e l . When the u l t r a - v i o l e t rays appeared to injure the plants, the u l t r a -v i o l e t lamps were raised to 4^ feet above the sand l e v e l . The u l t r a - v i o l e t rays were produced by the six General E l e c t r i c sun lamps (275 watts) and the in f r a - r e d rays by the six General E l e c t r i c i n f r a - r e d heat r e f l e c t o r lamps (250 watts). Racks were made to hold these two sets of s i x lamps i n two rows of three (see Figures 3 and 4). The rows were 18 inches apart and the lamps were 24 inches apart i n the row. Removable blinders were set up to prevent the rays from s t r i k i n g adjacent treatment V^ ooAen Frame 16 -A h — Figure 3 . Diagram of a lamp rack used i n the u l t r a - v i o l e t and infra-red r a d i a t i o n treatments blocks. The l i g h t treatments were begun when the plants were about two months old. At f i r s t the period of treatment was -66-Figure 4-. Barley plants growing i n the greenhouse under u l t r a - v i o l e t lamps Figure 5. Appearance of Black Hulless barley plants fed a complete nutrient solu-t i o n Figure 6. Development of red color i n stems of Black Hulless barley plants fed low P and dextrose - 6 7 -about one hour per day, but the treatment time was soon increased to about six hours per day. The "dextrose" treatment was started when the plants were two months old. It consisted of applying five grams of dextrose per pot at weekly intervals. This dextrose was washed down into the sand by the nutrient solutions. When the plants were two months old the "outside" treat-ment was also commenced. These plants were moved into an open, unheated greenhouse un t i l they were nearly mature, and then they were moved outside. The temperature and relative humidity i n the main green-house were recorded on a hygro-thermograph. The instrument was placed at " s o i l level" near the side of the bench, between two treatment blocks. The seed from each plant was threshed separately and re-tained to be graded. The procedure and results of this grading are given in the next experiment. The seed which was planted on December 12, 1954, emerged rather irregularly after 5 to 7 days. By the end of December the stems of the "Smyrna" plants which were not receiving nitro-gen were beginning to turn red. Color appeared just above and' below a marked constriction which occurred in the stem at the top of the coleoptile. The red color continued to spread up and down the stem, and by the end of January had become particularly - 6 8 -noticeable just above each node. These colored plants appeared espe c i a l l y weak at this time. At the beginning of March the "Black Hulless" plants under the low N and low P treatments were showing red c o l o r a t i o n at the base of the stems. Also towards the end of the same month the "Black Hulless" plants under the u l t r a - v i o l e t lamps were beginning to show considerable i n j u r y . This i n j u r y con-sisted of a chlorosis and browning which only occurred on the upper surface of the leaves and only i f these leaves were at right angles to the u l t r a - v i o l e t sources. The "Black Hulless" plants which were receiving the complete solu t i o n were the most damaged. There appeared to be l i t t l e i l l e f f e c t to the "Awn-l e s s " and "Smyrna" v a r i e t i e s under u l t r a - v i o l e t treatment u n t i l the plants of these v a r i e t i e s were nearly mature. At that time the "Awnless" and "Smyrna" plants i n the center of the u l t r a -v i o l e t block showed considerable c h l o r o s i s . During the f i r s t week of A p r i l the plants were subject-i v e l y rated f o r color development: (See tabulated statement on following page.) At t h i s time the "outside" plants, which were grown at lower temperatures, were showing a more widespread but l e s s i n -tense red coloring. This coloring extended through the l e a f veins and into the glumes of "Black Hulless" and "Awnless". Towards the end of A p r i l a sun shade of whitewash was sprayed on the greenhouse. At t h i s time the plants i n the - 6 9 -Variety Black Hulless Smyrna Awnless Treatment  Block Control U l t r a - v i o l e t Infra-red Dextrose Control U l t r a - v i o l e t Infra-red Dextrose Control U l t r a - v i o l e t Infra-red Dextrose Nutrient Level  Complete Low N Low P +++ ++ ++ ++ + + + ++ ++ +++ +++ ++++ + + yellowing at le a f ends no color - see Figure 5» + very s l i g h t to l i g h t red at base of stem ++ red at base of stem or on nodes +++ heavy red color at base of stem - see Figure 6. ++++ Heavy red color on entire stem main greenhouse began to turn yellow and require much les s n u t r i -ent solution. Within two weeks the seed was nearly r i p e , although i n about f i f t y per cent of the plants no seed had been produced at a l l . The plants i n the cool greenhouse continued to grow for another eight weeks after t h i s whitewash application before they were ready to harvest. When the plants were young they required approximately the same amount of nutrient solution under each nutrient l e v e l . Toward maturity the pots receiving the complete solution required about twice as much of the nutrient s o l u t i o n as the pot receiving the low N and low P treatments. -70-The sand i n the pots of the block receiving dextrose became very caked by the end of the experiment. It even became d i f f i c u l t i n c e r t a i n instances to get the nutrient so l u t i o n to run into the sand. Discussion The purple seeded v a r i e t y "Black Hulless" showed consid-erably more red plant color than the non-purple seeded v a r i e t i e s "Awnless" and "Smyrna". The blue seeded v a r i e t y "Awnless" showed s l i g h t l y more color development than the white seeded v a r i e t y "Smyrna". These observations, however, cannot be i n t e r -preted to represent how a l l colored and c o l o r l e s s seeded v a r i e -t i e s would act under si m i l a r conditions. Of more importance i s the fact that, i n a l l v a r i e t i e s , certain sets of conditions caused much more color development than others. The most noticeable effects were caused by d i f f e r -ences i n nutrient l e v e l . The low N treatment gave the most general increase i n plant color except i n "Black Hulless", i n which the low P condition appeared to give the greatest color development. The marked increase i n color with the dextrose treatment may be due to the assimilation of sugar by the plant. A reduction i n essential mineral nutrients, caused by increased consumption of minerals by a sugar-activated s o i l f l o r a , might also r e s u l t i n increased color. Furthermore, the sugar may have caked the s o i l so that the sand was poorly aerated. It would be i n t e r e s t i n g to note any p o s i t i v e color -71-c o r r e l a t i o n between the plant color development and the forma-t i o n of color i n the seed. This w i l l be considered i n the next part of this experiment. The ,only possible explanation f o r the sudden ripening of the plants i n the greenhouse before they were mature seems to be the reduction i n l i g h t i n t e n s i t y when the whitewash shade was sprayed on. (b) Kernel Color Each plant used i n the previous experiment was harvested and threshed separately. In order to remove the h u l l s so that the kernel color could be observed, the seed of the "Awnless" and "Smyrna" v a r i e t i e s were pearled i n a small hand pearler. Since "Black Hulless" i s a hulless v a r i e t y , the caryopsis color can be observed d i r e c t l y . The prepared seed of each plant was placed on f i l t e r paper i n a separate p e t r i - d i s h . Each v a r i e t y was placed on a separate bench;. Three observers were asked to rank t&e p e t r i -dishes as to i n t e n s i t y of seed color within each variety., The time of day when the ranking was conducted was not taken into consideration. I t was found possible to divide the ranked seeds into d e f i n i t e color classes: The white seeded v a r i e t y "Smyrna" f e l l into three classes (see Figure 8); the blue seeded v a r i e t y "Awnless" into seven classes; and the purple seeded v a r i e t y "Black Hulless into ten classes (see Figure 7). The class of the seed i n each plate was recorded by each observer. Figure 7 . Color classes of the purple v a r i e t y "Black Hulless". Classes 1 to 3 increase i n brown color, classes 4 to 5 i n blue color and classes 6 to 10 i n -crease i n purple color Figure 8 . Color classes of the white v a r i e t y "Smyrna". Class 1 i s white, class 2 somewhat brown and class 3 quite weath-ered i n appearance -73-Observations of the pane l members are not easy to t r e a t s t a t i s t i c a l l y f o r a number of reasons, one of which i s the l a r g e numbers of m i s s i n g v a r i a t e s . An a n a l y s i s of v a r i a n c e was made but was d i s c a r d e d . F i n a l l y contingency t a b l e s were e s t a b l i s h e d and t e s t s f o r independence of c e r t a i n of the v a r i a b l e s were undertaken. The v a r i a b l e s were considered i n what appeared t o be l o g i c a l a s s o c i a t i o n s and contingency c o e f f i c i e n t s were ob-t a i n e d f o r comparative purposes ( 3 1 a ) . The a s s o c i a t i o n s o f v a r i a b l e s , C h i Square v a l u e s , and contingency c o e f f i c i e n t s are g i v e n i n Table 8. Pl e a s e see the Appendix f o r the da t a and the contingency t a b l e s . Frequency d i s t r i b u t i o n s of the v a r i a b l e s were s t u d i e d and were found t o be badly skewed. Very few p l a n t s produced f u l l heads. Many produced l i t t l e seed and over 50 per cent produced none at a l l . Fo r . t h e purposes of s t a n d a r d i z i n g the judgments of the panel members, cards w i t h r e p r e s e n t a t i v e seeds of the v a r i o u s c o l o r c l a s s e s , p l a c e d i n s e r i e s , were made up. " C o l o r " c l a s s e s f o r the "white" v a r i e t y "Smyrna" were thr e e — ( 1 ) c l e a r white, (2) d i s c o l o r e d , and (3) weathered. Seed of the v a r i e t y "Awnless" was set out i n a s e r i e s of f i v e c l a s s e s of b l u e ; seed of c l a s s 1 was v e r y l i g h t and seed o f c l a s s 7 was v e r y dark and, i n f a c t , l o o ked, t o the o b s e r v e r s 1 eyes, to be much l i k e seed o f c l a s s 5 of the B l a c k H u l l e s s -74-v a r i e t y . The "Black Hulless" seed a c t u a l l y f e l l i nto three series of color classes. The f i r s t s e r i e s , classes 1 to 3» recorded an increasing brown development i n the seed. The second s e r i e s , classes 4 to 5> showed an increasing blue devel-opment i n the aleurone layer, while the t h i r d series exhibited increasing purple development i n the pericarp of the kernel. TABLE 8. THE CHI SQUARE VALUES AND CONTINGENCY COEFFICIENTS OF CONTINGENCY TABLE DATA (See Appendix III) Variety Appendix Treatments Compared Table No. df x C o e f f i c i e n t of Contingency Awnless Black Hulless Smyrna Complete-Low P 5 29.48** .3424 Complete-Low N 5 59.65** .4028 Low P - Low N 5 6.84 .1626 C o n t r o l - U l t r a - v i o l e t 5. 12.75* .2790 Control-Infra-red 5 7.12 .2005 U l t r a - v i o l e t - I n f r a - r e d 5 3.74 .1439 Control-Dextrose 5 6.31 .2159 Control-Outside 5 14.01* .2862 Complete-Low P 3 4.44 .2238 Complete-Low N 3 87.86** .5459 Low P-Low N 3 69.67** .5190 C o n t r o l - U l t r a - v i o l e t 4 11.01* • 3193 Control-Infra-red 2 29.97** .4998 U l t r a - v i o l e t - I n f r a - r e d 2 10.10** .3367 Control-Dextrose 4 14.01** .3567 Control-Outside 4 8.12 .2518 C o n t r o l - U l t r a - v i o l e t 2 1.05 .1280 Control-Infra-red 2 8.60* .3466 U l t r a - v i o l e t - I n f r a - r e d 2 5.32 .2724 Control-Dextrose 2 0.36 .0837 Control-Outside 2 1.18 .1389 Complete-Low P 2 12.34** .2144 Complete-Low N 2 89.22** .4751 C o n t r o l - U l t r a - v i o l e t 2 4.93 .1594 Control-Infra-red 2 9.27** .2146 U l t r a - v i o l e t - I n f r a - r e d 2 5.93 .1669 Control-Dextrose 2 3.42 .1572 Control-Out side 2 22.33** .3322 s i g n i f i c a n t at the % l e v e l s i g n i f i c a n t at the 1% l e v e l -75-Discussion "Nutrient l e v e l s " had a much more marked e f f e c t on the development of color than did the "block" treatments. The most marked effect obtained under "low N" which caused a marked increase i n color of "Awnless" and "Black Hulless"seed. "Smyrna" seeds, on the other hand, appeared to be more d e f i n i t e l y "white" under the "low N". The "low P" had a s i m i l a r though much l e s s marked e f f e c t on the kernel color of "Awnless" and "Smyrna" but had no s i g n i f i c a n t e f f e c t on the kernel color of "Black Hulless". There was no consistent s i g n i f i c a n t difference i n kernel color between the various block treatments. The u l t r a -v i o l e t treatment caused an observable difference i n the darkening of "Awnless" and "Black Hulless" seed. The i n f r a - r e d treatment caused a substantial increase of color i n the kernels of "Black Hulless" and "Smyrna". "Awnless" seed showed a s i g n i f i c a n t i n -crease of color, and "Smyrna" kernels were considerably darker when obtained from plants placed "outside". Only "Black Hul-l e s s " seed showed more color compared to the "control" when pro-duced on plants grown with added dextrose. Only the kernels of the "Black Hulless" plants which 2 received "low N" treatment were used i n determining the x and c o e f f i c i e n t of contingency (Table 8). This was deemed necessary because of the complete lack of seed on "Black Hulless" plants receiving low P and complete nutrient l e v e l s when given c e r t a i n of the block treatments. On the other hand, of the "Black Hulless" plants which received low N almost a l l developed seed - 7 6 -and the seed color data were nearly complete. When only the 2 low N plants of Black Hulless were used i n ca l c u l a t i n g the x and c o e f f i c i e n c y of contingency, there was found to be no s i g -n i f i c a n t difference between the various block treatments and the control, except i n the infr a - r e d treatment. Missing data from the other v a r i e t i e s are so extensive that differences reported as s i g n i f i c a n t between c e r t a i n t r e a t -ments and "co n t r o l " may not be as s i g n i f i c a n t as reported. Although the experiment was planned to permit t h e i r determination, fa c t o r interactions could not be assessed properly from the data obtained. Of the "main e f f e c t s " reported, those associated with nutrient l e v e l s give r e s u l t s which are c l e a r l y p o s i t i v e . "Low N", es p e c i a l l y , increased plant and kernel color i n the colored v a r i e t i e s and gave "purer white" i n the kernels of the white v a r i e t i e s . Limiting nitrogen might, then, make fo r more color i n the production of colored barleys of com-merce and might make for easier separation of "g e n e t i c a l l y blue" from " g e n e t i c a l l y white" barleys. Limiting phosphorus did not y i e l d r e s u l t s as c l e a r l y p o s i t i v e as those obtained when nitrogen was l i m i t e d . Plant color was quite uniformly modified but seed color, apparently, was not. The results with seed color are, however, not trustworthy f o r very l i t t l e seed was produced on plants of the "Black Hulless" v a r i e t y receiving "low phosphorus". Also much of the seed produced under the .limited phosphorus treatments was immature. - 7 7 -I n g e n e r a l , too, i t can be s a i d that treatments which increased p l a n t c o l o r tended a l s o to increase k e r n e l c o l o r . (c) Detached Leaves Zarudnaya (104) was able to cause maize leaves w i t h genes Pr and r 0 * 1 to develop anthocyanin c o l o r i n g when they were f l o a t e d on sugar s o l u t i o n s . I t would be of value to have a s i m i l a r quick method f o r developing pigments i n b a r l e y leaves f o r use i n the study of b a r l e y p l a n t pigments. On J u l y 25) 1955) t h e r e f o r e , an experiment was s t a r t e d to observe the e f f e c t s of f l o a t i n g b a r l e y leaves i n s e v e r a l sugar s o l u t i o n s . Leaves of the f o l l o w i n g b a r l e y v a r i e t i e s were used: A l g e r i a n K i t c h e n Awnless C.I. 5628 Black H u l l e s s C54-55 G o l d f o i l 54-25-8 and of the blue seeded wheat v a r i e t y , "Regina 551". Leaves were t o r n i n t o two-inch pieces and f l o a t e d on the f o l l o w i n g sugar s o l u t i o n s i n p e t r i - d i s h e s : Lactose 2$ and 10$; Dextrose 2$, 10% and 30$; Sucrose 2$, 10$ and 30$; and Levulose 2$. The covered p e t r i - p l a t e s were placed at a window which faced south. Four days l a t e r , the leaves of a " d i l u t e sun red" corn p l a n t were placed on s i m i l a r dextrose and sucrose s o l u t i o n s . The leaves were t o r n i n t o s m a l l p i e c e s , as above. A l s o , c i r c u -l a r pieces were cut out w i t h a cork borer and t r e a t e d i n a manner s i m i l a r to the t o r n p i e c e s . - 7 8 -Certain of the barley leaves produced considerable red color. The red color was p a r t i c u l a r l y noticeable i n the leaves of the "Black Hulless" v a r i e t y floated on sucrose solu-t i o n . The leaves of the v a r i e t y "C.I.5628" also produced color, though not to the extent of the "Black Hulless" leaves. The leaves of the v a r i e t i e s "Algerian", "Awnless" and " 5 4 2 5 - 8 " produced only a s l i g h t trace df color, while the v a r i e t i e s " G o l d f o i l " , "Kitchen" and ,tC5^-59i developed none. The maximum red color development occurred after about four or f i v e days. After t h i s time the color faded into c h l o r e t i c yellow. Certain sections of the corn leaves developed considerable red color, while other pieces of the same l e a f i n the same solution formed no color. The wheat leaves showed no red. Instead they soon became brown and water soaked. The sugar which appeared to cause most red color to develop i n a l l these l e a f samples was sucrose; lactose, dextrose and levulose followed i n that order. The concentration of the sugar seemed of l i t t l e consequence i n developing the red color, although the sugars i n higher concentration held back the growth of the fungi. The value of t h i s method of securing colored barley leaves f o r study of t h e i r pigments was somewhat limit e d , since large quantities df highly colored barley leaves could be picked i n the f i e l d . I t might be usef u l , with c e r t a i n modifications, for developing barley plant color where i t w i l l not develop natur a l l y . -79-3. HISTO-CHEMICAL AND SUPERFICIAL EXAMINATION OF KERNEL FOR PIGMENTATION The purpose of these examinations was to determine d i f f e r e n c e s i n appearance and i n c o l o r i n g matter i n the seed of v a r i o u s b a r l e y v a r i e t i e s . A. S u p e r f i c i a l D i f f e r e n c e s between Blue and White Barley K e r n e l s . I t has been pointed out i n the " L i t e r a t u r e Review" that i t i s sometimes d i f f i c u l t to d i f f e r e n t i a t e "genetic blue" from "genetic white" seed. This experiment has been set up i n an attempt to f i n d any c o n s i s t e n t s u p e r f i c i a l d i f f e r e n c e s between blue and white b a r l e y k e r n e l s . Seeds of the f o l l o w i n g v a r i e t i e s were used; A l g e r i a n G o l d f o i l Montcalm Velvon 11 Black H u l l e s s Hanna Nepal 4811-68-2 Byng Husky O.A.C. 21 Blue 1 (wheat) Ca r l s b e r g Kwan Vantage R. Blue (wheat) F o r t A l s o used was seed from the F-^  p l a n t s of the f o l l o w i n g crosses: F o r t x Velvon 11 Velvon 11 x C a r l s b e r g F o r t x 4811 - 6 8 - 2 4811 - 6 8 - 2 x Carlsberg 4811 - 6 8 - 2 x Velvon 11 F o r t x C a r l s b e r g Seed w i t h h u l l s had the h u l l s peeled o f f . The seeds of the v a r i e t i e s were examined dry i n day-l i g h t under a d i s s e c t i n g microscope and then under an u l t r a -v i o l e t l i g h t (maximum emission 2650 ft). These same seed were then placed i n a p e t r i - d i s h w i t h moistened f i l t e r paper and - 8 0 -t h e y were o b s e r v e d at 24 h o u r i n t e r v a l s , i n o r d i n a r y l i g h t and u l t r a - v i o l e t l i g h t , f o r two weeks. The o n l y c o n s i s t e n t d i f f e r e n c e s n o t i c e d between the w h i t e and b l u e seeds were t h o s e observed i n o r d i n a r y l i g h t under the d i s s e c t i n g m i c r o s c o p e . H e r e , even i f t h e c o l o r o f t h e b l u e v a r i e t i e s was not f u l l y e x p r e s s e d , th e k e r n e l s t i l l u s u a l l y appeared s l i g h t l y d arkened, e s p e c i a l l y toward th e a p i c a l end. The s p r o u t i n g seeds showed no c o l o r development, e i t h e r i n t h e seed o r i n the s h o o t . Under the u l t r a - v i o l e t l i g h t t h e r e were no c o n s i s t e n t and d e f i n i t e s i g n s o f f l u o r e s c e n c e w h i c h c o u l d i n any way be used t o s e p a r a t e t h e b l u e f r om the w h i t e s e e d s . The F^ k e r n e l s o f the c r o s s e d m a t e r i a l were examined i n d a y l i g h t and an attempt was made t o s e p a r a t e the " b l u e " f r o m the " w h i t e " seed. As most of the seed was immature o r b a d l y e a t e n by moth l a r v a e , the s e p a r a t i o n proved v e r y u n s a t i s f a c t o r y . I t had been n o t i c e d t h a t when the v a r i e t i e s " F o r t " , "4811-68-2", " C a r l s b e r g " , and " V e l v o n 11" were b o i l e d i n 2N HC1 f o r one min-u t e , the b l u e v a r i e t i e s " F o r t " and "4811 -68-2" t u r n e d q u i t e r e d , and the w h i t e v a r i e t i e s " C a r l s b e r g " and " V e l v o n 11" showed l i t t l e c o l o r i n g . The c r o s s e d m a t e r i a l was a l s o b o i l e d i n 2N HC1 f o r one minute and t h e n o b s e r v e d . There was a c o n s i d e r a b l e d i f f e r -ence i n t h e amount o f c o l o r produced i n t h e seed by t h i s t r e a t -ment. The d i f f e r e n c e o c c u r r e d even between heads w i t h t h e same c r o s s . However, t h e r e appeared t o be no s e g r e g a t i o n o f k e r n e l -81-F i g u r e 9 . B a r l e y seeds are o f many c o l o r s . On the l e f t i s the white v a r i e t y "Vantage", i n the center the blue v a r i e t y "Kwan", and on the r i g h t the pu r p l e v a r i e t y "Black H u l -l e s s " . F i g u r e 10. The glume c o l o r i n g v a r i e s between v a r i e t i e s and du r i n g the growing season. Here we see p u r p l e , s t r i p e d and c l e a r glumes -82-color within any of the heads. This was contrary to expecta-t i o n , f o r i t had been hoped that t h i s reddening of the seed which had been boiled i n 2N HC1 was due to the presence of • anthocyanin i n the blue aleurone. Had t h i s been the case, then the color of the seeds would have shown segregation within t h i s seed, inasmuch as xenia for blue aleurone occurs. From these tests i t would appear that the most promis-ing method f o r separating blue from white seed i s the use of the dissecting microscope i n natural l i g h t . However, th i s procedure requires a f a i r amount of practice before any degree of accuracy can be obtained, and even then i t i s not i n f a l l i b l e . The tests reported do not exhaust the possible approaches to t h i s problem of separation. It may s t i l l be possible to develop a simple procedure. B. The Appearance of Microscopic Sections of the Kernels of Various Barley V a r i e t i e s No study of the kernel color of barley would be complete without a microscopic examination of the l o c a t i o n of the pigments i n the kernel. Microscopic examinations of the kernels have therefore been made of the following v a r i e t i e s : (See page 83 f o r tabulated l i s t } The seed of these v a r i e t i e s was treated and examined i n a manner sim i l a r to that reported by Harlan (see "Literature Review"). After the seed was sectioned freehand with a razor-blade, the sections were placed on dry s l i d e s and cover s l i p s - 8 3 -V a r i e t y Seed C o l o r V a r i e t y Seed C o l o r A l g e r i a n Blue K i t c h e n B l a c k Awnless #1* Blue L i o n B l a c k Awnless #7 Blue Montcalm Blue B l a c k H u l l e s s P u r p l e Nepal White B l a c k H u l l e s s #1 P u r p l e O.A.C. 21 Blue B l a c k H u l l e s s #5 P u r p l e Smyrna #1 White B l a c k H u l l e s s #10 P u r p l e Smyrna #3 White Byng White T r e b i Blue C.I. 5628 P u r p l e Vantage White F o r t Blue V e l v o n 11 White Gat ami B l a c k 3 3 - b l b l - 1 3 White G o l d f o i l White 33-B1 B l - 1 3 Blue Gopal P u r p l e 36-B1 B'l-21 Blue Hanna White 71-pr pr - 1 0 White Husky White 71-Pr P r - 1 0 P u r p l e I r a s a k a P u r p l e * The number r e f e r s to the c l a s s d e s i g n a t i o n i n the experiment "Kernel C o l o r " . were fa s t e n e d i n p l a c e by s e a l i n g two o p p o s i t e edges w i t h p a r a f -f i n . Each s e c t i o n was f i r s t observed under low power ( c a . lOOx) on the microscope. Then i t was observed under the same power while 2 per cent HC1 was run under the cover s l i p . F i n a l l y a new s e c t i o n of the same seed was observed m i c r o s c o p i c a l l y w h i l e 2 per cent NH4OH was run under the cover s l i p . A complete l i s t of the observed changes can be seen i n Appendix V. In the " p u r p l e " v a r i e t i e s the p e r i c a r p and aleurone both showed a reddening when the a c i d was added. On the ot h e r hand,,when the a l k a l i n e s o l u t i o n was added, the aleurone u s u a l l y went green, w h i l e the p e r i c a r p took on an orange or y e l l o w c o l o r i n most cases. I t i s i n t e r e s t i n g to note the d i f f e r e n c e s between the t h r e e c o l o r c l a s s e s o f "Black H u l l e s s " . I n the a c i d -84-F i g u r e 1 1 . A microscopic s e c t i o n of the b a r l e y v a r i e t y "Kwan" showing the n a t u r a l blue c o l o r i n the aleurone l a y e r . F i g u r e 1 2 . A microscopic s e c t i o n of the b a r l e y v a r i e t y "Black H u l l e s s " w i t h H C 1 added, showing the r e s u l t i n g red c o l o r i n both the aleurone and p e r i c a r p . - 8 5 -c o n d i t i o n the l i g h t c o l o r e d " B l a c k H u l l e s s #1" had an orange-p i n k aleurone and a c o l o r l e s s p e r i c a r p . Under the same c o n d i -t i o n the blue c o l o r e d "Black H u l l e s #5" had a red aleurone and a yellow-orange p e r i c a r p . The dark purple " B l a c k H u l l e s s #10" showed on l y a l i g h t pink aleurone, while the p e r i c a r p was a dark red w i t h the a c i d added. I t appeared i n a l l cases t h a t i n the Jtourple" v a r i e t i e s an i n c r e a s e o f c o l o r i n the p e r i c a r p was accom-panied by a decrease o f c o l o r i n the aleurone, while a l i g h t p e r i c a r p was o f t e n present w i t h a dark a l e u r o n e . Throughout the " b l u e " v a r i e t i e s , the aleurone showed a development o f a pin k c o l o r , and the p e r i c a r p a y e l l o w or orange c o l o r when a c i d was i n t r o d u c e d under the cover s l i p . When NH4OH was placed on the s e c t i o n the aleurone became a green o r yellow-green c o l o r , but i n most i n s t a n c e s the c o l o r of the p e r i -carp changed v e r y l i t t l e . I t i s important t h a t there appeared to be v e r y l i t t l e d i f f e r e n c e i n appearance between the l i g h t c o l o r e d '•Awnless #1" and the dark c o l o r e d "Awnless #7" when sec-t i o n s of these two c o l o r c l a s s e s were observed under the micro-scope. G e n e r a l l y the "white" v a r i e t i e s showed an orange or orange-pink c o l o r i n the aleurone when a c i d was added. Under the same c o n d i t i o n s the p e r i c a r p appeared c o l o r l e s s , w i t h p o s s i b l y a dark l i n e of c e l l s i n i t . The aleurone l a y e r showed some range of c o l o r when the a l k a l i n e s o l u t i o n was i n t r o d u c e d . The c o l o r s v a r i e d from orange through y e l l o w to green. The p e r i c a r p became -86-v a r y i n g shades of green when the NH4OH s o l u t i o n was added. The main d i f f e r e n c e between the l i g h t "Smyrna #1" and the "dark" "Smyrna #3" was i n the p e r i c a r p ; under the a c i d c o n d i t i o n s the p e r i c a r p s of the " l i g h t " c l a s s appeared c o l o r l e s s , w h i l e the p e r i c a r p s of the "dark" c l a s s appeared dark. I n the a l k a l i n e and dry c o n d i t i o n s t h i s d i f f e r e n c e was a p p a r e n t l y not so s i g n i f -i c a n t . D i f f e r e n c e s i n aleurone c o l o r between the " b l a c k " v a r -i e t i e s showed up when the HC1 was a p p l i e d . Thus the v a r i e t y "Gatami" had a p i n k aleurone, the v a r i e t y " K i t c h e n " a l i g h t pink aleurone, while the v a r i e t y " L i o n " had a c o l o r l e s s aleurone i n the a c i d c o n d i t i o n . These d i f f e r e n c e s were not apparent i n the a l k a l i n e s t a t e . S i n c e the b l a c k c o l o r i s s i t u a t e d i n the p e r i -carp and covers up any blue development i n the aleurone l a y e r o f the seed, the d i f f e r e n c e i n the aleurone c o l o r among the three v a r i e t i e s i s to be expected. T h e r e f o r e , i n order to determine whether a b l a c k v a r i e t y has a blue aleurone, one must e i t h e r examine s e c t i o n s of th a t v a r i e t y m i c r o s c o p i c a l l y , or e l s e s e a r c h f o r the presence of blue c o l o r development i n the segregates of a c r o s s o f a white b a r l e y w i t h the b l a c k v a r i e t y . Perhaps the q u i c k e s t way to determine t h i s c h a r a c t e r by h y b r i d i z a t i o n would be t o cross the " b l a c k " v a r i e t y onto a "white" v a r i e t y and observe the seed on the female parent of t h i s c r o s s . The white v a r i e t y would show x e n i a and the seed on the "white" female parent would appear blue i f the "b l a c k " v a r i e t y had a blue a l e u r -one. However, the t e s t would o n l y be completely d i a g n o s t i c i f -87-two " w h i t e " v a r i e t i e s w h i c h c o n t a i n e d d i f f e r e n t s e t s o f t h e two c o m p l e m e n t a r y f a c t o r s f o r b l u e a l e u r o n e were u s e d , o r i f a " w h i t e " v a r i e t y w h i c h c o n t a i n e d n e i t h e r o f t h e c o m p l e m e n t a r y f a c t o r s was u s e d . F rom a c o n s i d e r a t i o n o f t h e r e s u l t s o f t h e m i c r o s c o p i c e x a m i n a t i o n s , i t w o u l d appear t h a t t h e o b s e r v a t i o n o f the s e c t i o n s t r e a t e d w i t h H C 1 was g e n e r a l l y more d i a g n o s t i c t h a n o b s e r v a t i o n s o f t h e d r y o r N H ^ O H - t r e a t e d s e c t i o n s . The o n l y e x c e p t i o n t o t h i s seemed t o be the d i f f e r e n c e i n p e r i c a r p c o l o r o f w h i t e v a r -i e t i e s between t h e a c i d and a l k a l i n e c o n d i t i o n s . The r e s u l t s r e p o r t e d i n t h i s e x p e r i m e n t do n o t c o m p l e t e -l y ag ree w i t h H a r l a n ' s f i n d i n g s ( 3 8 ) , f o r he r e p o r t s t h a t t h e a l e u r o n e o f " b l u e " b a r l e y v a r i e t i e s t u r n e d b l u e when 2% NH^OH was a p p l i e d t o t h e s e c t i o n s . The d e v e l o p m e n t o f t h i s b l u e c o l o r may be masked by o t h e r p i g m e n t s w h i c h a r e y e l l o w i n NH^OH, o r by c o - p i g m e n t s w h i c h f o r m a g r e e n o r y e l l o w c o l o r complex i n t h e a l k a l i n e s t a t e . Whatever t h e s i t u a t i o n , t h e s e p i g m e n t s o r c o -p i g m e n t s a re n o t a p p a r e n t l y h i g h l y c o l o r e d i n t h e a c i d s t a t e . I n t h e " w h i t e " v a r i e t i e s t h e r e a p p e a r s t o be some s u b s t a n c e i n t h e a l e u r o n e l a y e r , p r o b a b l y n o t an a n t h o c y a n i n , w h i c h p r d d u c e s a p i n k i s h c o l o r i n a c i d . 4. EXTRACTION AND CHROMATOGRAPHIC EXAMINATION OF BARLEY KERNEL PIGMENTS A . E x t r a c t i o n I t was n e c e s s a r y t o d e v e l o p t e c h n i q u e s whereby t h e p i g m e n t s i n t h e b a r l e y k e r n e l c o u l d be e x t r a c t e d f o r c h r o m a t o g r a m -- 8 8 -ing. A f t e r many t r i a l s , the most promising method of extrac-t i o n was applied to a l l of the material at hand. In the f i r s t methods a l l the barley samples were ground i n a m i l l to pass through a 20 mesh screen. This coarser material was found better than f i n e r material passed through a 40 or a 60 mesh screen. In l a t e r extractions even the whole seed was used. The, f i r s t method consisted of placing ten grams of the ground barley i n an extracting thimble, and then lowering the thimble into a soxhlet extractor containing 200 ec. of ethyl alcohol. In some instances t h i s alcohol was a c i d i f i e d to make a 2 per cent solution of HC1 while i n other cases no acid was added. A f t e r the barley had undergone about ten hours of ex-tr a c t i o n , the alcohol was evaporated to about 20 cc. by gentle heating. The color of the extract from "blue" or "purple" barley seed was a marked red, deepening considerably when reduced i n volume by evaporation. The red color of the extract i n a c i d i -f i e d alcohol was much deeper than the red of the extract i n neutral alcohol. The white v a r i e t i e s produced a green or yellow extract under the same conditions. Next, ten grams of the ground grains were refluxed f o r about an hour i n 1% HC1. The "blue" and "purple" seeded v a r i e -t i e s produced a red tinge i n the a c i d i f i e d water. However, the l i q u i d contained too much c o l l o i d a l material and debris from the grain to be of any p r a c t i c a l value. -89-I n an attempt to minimize changes i n the chemical nature of the e x t r a c t caused by e x c e s s i v e h e a t i n g , a m o d i f i c a -t i o n of the s o h x l e t method was t r i e d . I n t h i s method the s o x h l e t apparatus was eet up on a clamp stand so t h a t c o l d methyl or e t h y l a l c o h o l c o u l d d r i p from a s e p a r a t o r y f u n n e l i n t o the s o x h l e t e x t r a c t o r . The a l c o h o l which c o l l e c t e d i n the Erlenmeyer f l a s k at the bottom of the apparatus was r e t u r n e d to the s e p a r a t o r y f u n n e l when the l a t t e r became empty. T h i s e x t r a c t i o n was continued f o r about f o u r hours. The 200 c c . o f a l c o h o l used f o r t h i s e x t r a c t i o n was then evaporated to about 20 c c . e i t h e r by g e n t l e h e a t i n g i n the a i r or i n vacuo, or by l e t t i n g i t stand i n an e v a p o r a t i n g d i s h . The e x t r a c t from the m o d i f i e d s o x h l e t method d i d not appear to be as deep a c o l o r as the e x t r a c t i n the r e g u l a r soxh-l e t method above. On h e a t i n g i t darkened somewhat. Reducing the volume of the e x t r a c t i n vacuo was i n e f f e c t i v e because i t was d i f f i c u l t to develop a good enough vacuum. Even when vacuum c o n d i t i o n s f o r t h i s o p e r a t i o n were so i d e a l t h a t h e a t i n g was unnecessary, the e x t r a c t s t i l l changed hue c o n s i d e r a b l y when reduced i n volume. T h i s may be p a r t l y due to the pigment con-c e n t r a t i o n . One cannot, however, d i s c o u n t the f a c t t h a t the a c i d c o n c e n t r a t i o n i n c r e a s e d and t h i s may have caused changes i n the chemical composition of the pigments. As i n the e v a p o r a t i o n i n vacuo, the c o l o r of the e x t r a c t i n the e v a p o r a t i n g d i s h i n c r e a s e d to a g r e a t e r extent than would be caused by i t s reduc-t i o n i n volume. The l i m i t a t i o n s of t h i s m o d i f i e d s o x h l e t system - 9 0 -are the amount of equipment and the length of time required to make each extraction. Also the amount of hand-work involved necessitated a large expenditure of the operator's time. In order to reduce the labour and equipment required fo r each extraction a method was developed whereby several ex-tractions could be made simultaneously. Ten grams of ground barley of each v a r i e t y to be extracted were weighed into a 125 cc. Erlenmeyer f l a s k . Twenty-five cc. of alcohol were run into the f l a s k , the stopper was inserted, and the f l a s k was placed on a shaker f o r four hours. At the end of t h i s time the l i q u i d was decanted and centrifuged, and the clear portion saved. This method proved quite s a t i s f a c t o r y , since twelve extractions could be made at once. The centrifuged extract was quite clear and the red color from "blue" and ™purple" v a r i e t i e s was r e a d i l y v i s i b l e i n the 1% HCl-alchhol extractions. This meant that the pigments were of s u f f i c i e n t concentration that the extract did not have to be increased by evaporation. One of t h i s method's main drawbacks i s that ten grams of seed are required to make e f f e c t i v e extractions — a quantity greater than that available i n some of the seed samples. Also, consid-erable labour was s t i l l entailed when a large number of v a r i e t i e s was to be extracted. Consequently, to reduce the amount of labour required per extraction and also the amount of seed required, the follow-ing method was devised. One or two grams of the whole barley -91-kernel were placed i n a test tube. Any hulled v a r i e t i e s were f i r s t pearled long enough i n a small hand pearler to remove most of the h u l l s . Enough alcohol was run into the t e s t tube to just cover the kernels. The test tubes were then plugged and allowed to stand f o r four to seven days. The extract was then ready f o r d i r e c t use. The length of time required f o r the extraction meant that the extracting process had to be started long before the extract would be required. The long duration of the extraction also meant that many substances other than the pigments might appear i n the extract. Furthermore, the extract might contain many breakdown products. However, thi s was the method selected f o r the extraction of the bulk of the v a r i e t i e s which were chromatogrammed. In a l l of the methods reported above, a much more sa t i s f a c t o r y extraction of the pigments was obtained by the use of a s l i g h t l y a c i d i f i e d medium, a r e s u l t s i m i l a r to that reported i n the l i t e r a t u r e . Since barley kernels have been reported to contain leuco-anthocyanins ( 83 ) , the writer used the method developed by Bate-fSmith (5) to extract leuco-anthocyanins as well as anthocy-anins from plant tissue i n the form of anthocyanidins. About one gram of the seed was covered with 2N to 2.4N HC1 i n a test tube. Afte r t h i s mixture had been heated for twenty minutes i n a b o i l i n g water bath, the aqueous solution was decanted into a small narrow test tube. In order to clear t h i s solution, i t -92-was found necessary to f i l t e r the l i q u i d i n most cases. S u f f i -cient iso amyl alcohol (3-methylbutan-l-ol) was run into the small t e s t tubes to give a supernatant layer just deep enough to be drawn cleanly into a c a p i l l a r y tube. The tube containing the aqueous solution and alcohol was shaken and allowed to stand to l e t the alcohol form an upper la y e r . This layer was then drawn into a c a p i l l a r y tube, from which i t was spotted onto the chromatogram. Several applications were usually necessary and a hot current of a i r was employed to accelerate drying between between application. The extractions from a l l of the above procedures were investigated on chromatograms. The barley kernel pigments were compared to pigments i n the tissues of several plants. The plant material was dried and the pigments extracted with alcohol and by Bate-Smith's (ibid.) method. B. Chromatography Paper p a r t i t i o n chromatography was used to determine the number and the Rp values of barley kernel pigments. The action of the barley kernel pigments on the chromatograms was also compared with the action of pigments from other plant mater-i a l . The ascending type of chromatogram was used f o r the preliminary investigations of method of application, types of - 93-solvents and types of extract. In t h i s type of chromatogram, half an inch of the desired solvent was placed i n the bottom of a si x - i n c h glass cylinder which had one open end. In the e a r l i e r experiments the chromatogram was hung inside the c y l i n -der from a glass rod i n such a way that the paper just touched the cylinder bottom. The glass rod was s l i g h t l y shorter than the diameter of the cylinder, so that with a short piece of rubber hose on each end i t would remain i n place i n the cylinder. This method was a l i t t l e slow because the f i l t e r paper had to be cut into f i v e - i n c h s t r i p s and only one s t r i p could be run at a time. Therefore, i n order to accommodate more spots, the f i l t e r paper used i n l a t e r experiments was cut f i f t e e n inches wide and eleven inches high, and was made into cylinders. The edges of the f i l t e r paper were stapled so that they did not quite touch. The glass cylinders were then made a i r t i g h t by covering them with a piece of glass sealed with petroleum j e l l y . The ascending chromatograms proved very s a t i s f a c t o r y for they were easy to handle and gave clear and consistent r e s u l t s . These chromatograms were set up away from heat sources and windows, and were run at night because uneven l i g h t and heat cause an uneven r i s e of the solvent i n the f i l t e r paper. The Rp values reported i n the l i t e r a t u r e are usually calculated on the basis of the results of descending chromato-grams. Thus descending chromatograms were set up. The appar-atus was of a manufactured type and consisted of a sta i n l e s s -94-s t e e l frame which held the solvent troughs. This frame stood i n a 12-inch glass cylinder which had a glass l i d sealed with petroleum j e l l y . These chromatograms were run i n a chamber which was kept at a constant 30° C. The f i l t e r paper used f o r the chromatograms was Watman No. 1, i n sheets of 22 inches by 18 inches. The sheets were cut into the appropriate size to f i t the apparatus i n which they were to be used. The extracts to be investigated were spotted i n a l i n e about one inch apart. Gn the ascending chromatograms the extracts were spotted one inch from the bottom of the paper, and on the descending chromatograms they were located one inch down from the uppermost f o l d of the paper. Large spots were found to be more e f f e c t i v e than small spots, and about ten app l i -cations of two drops each were usually made to each spot, with drying between each application. This amount of extract was found necessary to make the color of the advancing spots v i s i b l e . The f i r s t solvent t r i e d i n running the chromatograms was butanol-acetic acid-water (40:10:50 by volume). It was found to be the most s a t i s f a c t o r y solvent f o r separating the var-ious anthocyanin f r a c t i o n s . In order to substantiate the R F values obtained by the f i r s t solvent, a solvent of m-cfesol-acetic acid-water (50:2:48 by volume) was used. There was i n s u f f i c i e n t color i n the chromatographed anthocyanin pigments to penetrate the yellow color of the m-cresol, so t h i s solvent had to be abandoned. In order to prevent the anthocyanidins from fading, i t i s necessary to maintain a low pH during chromatography. - 9 5 -TABLE 9 t Rp VALUES OF ANTHOCYANINS EXTRACTED FROM VARIETIES OF BARLEY WITH BLUE KERNEL PIGMENTS Variety Water-Acetic-Hydro-chloric Acid Solvent (10:30:3) 1 2 *3 Algerian . 5 2 7 8 . 6 8 7 4 Atlas .6924 Atlas Hooded .5391 . 6 6 5 2 Atlas (U.B.C.) .1411 . 5 5 8 7 .6924 Awnless . 5 2 7 8 . 6 6 7 9 Bolsheviki .1467 .4721 . 6 7 3 4 C54-22 . 4 9 9 2 .6965 Fort .2062 .4413 .6341 Gatami .5144 . 7013 Kitchen .4940 Kwan .5186 . 6 7 7 4 Montcalm .1561 .5054 . 6 8 7 8 O.A.C.21 .5573 . 6 9 8 0 O l l i . 1519 .5314 .7158 Sonalta . 2 3 1 6 .5712 .7140 Tennessee . 2 0 2 6 . 6 3 2 3 .7408 Trebi .5140 . 7 2 3 8 22-B1 Bl - 1 3 . 5 0 6 6 . 6 9 6 7 36-B1 Bl-21 .4710 . 6 6 0 2 4811-68-2 . 2 2 0 5 .515? . 6 5 0 3 5090-2-3 .2118 . 5 9 3 0 .6731 5423-4 .2948 .5663 .6957 5424-7 . 1 8 6 3 .5463 .6938 5425T8 .2042 . 5 2 5 5 . 6 7 6 0 5428-2 .5610 . 6 8 0 6 5430-1 . 1 6 7 7 . 5 5 8 1 . 6 8 2 5 Average .1965 . 5 2 6 0 . 6 8 7 1 Butanol-Acetic-Water Solvent (40:10:50) 4 5 6 *7 8 . 1279 .2890 .4195 . 6 2 0 2 . 7 0 2 9 . 1 0 7 6 . 2 8 7 2 . 4 3 4 9 . 5 1 2 6 .1101 . 2 5 7 5 . 4 0 3 6 . 5 6 9 1 . 7 0 5 0 . 1 0 2 7 .2684 .4137 .5775 .7137 .1104 .2627 .4693 .1063 .2742 .4390 . 5 1 2 2 .1364 .3231 . 6 1 6 0 .1372 . 3 1 7 1 .6172 .7555 . 1206 .2941 . 6 2 9 6 .1297 .3411 . 6 3 6 3 .1540 .3106 .4670 . 7017 .1258 .2929 .6040 . 6 6 7 1 .1024 .2041 .3840 .5815 . 7375 .1111 . 2 5 9 4 . 3 9 3 5 . 5 7 2 9 . 6 7 4 7 .1440 .2348 .3842 . 5 9 0 7 . 6 7 2 1 .1068 .2908 .4660 .5816 . 6 9 3 8 .1408 .3326 .6145 .1190 . 2 9 3 9 .4503 .1011 .2824 .4414 .1435 •2975 .6224 . 6 9 2 6 .1241 . 3 0 3 5 .6064 .1242 . 3 2 6 5 .6424 .1189 .2898 .6423 .1299 .2913 .6184 . 6 7 6 5 . 1 2 2 5 . 3 0 2 5 .6362 .1396 . 3 2 8 2 .6491 . 1233 . 2 9 0 6 .4282 .6023 .6994 * 3 probably not anthocyanin; * 7 probably not anthocyanin; red with acid and red with base red with acid and red with base -96-TABLE 1 0 : % VALUES FOR ANTHOCYANIDINS FORMED FROM VARIETIES WITH BLUE KERNEL PIGMENTATION Va r i e t i e s H 20-HA c-HC 2 Large Cylinder H 2 0-HA c-HCl Small Cylinder Butanol Water -Acetic-Solvent 1 2 3 4 5 6 Algerian .3812 .5504 . 3 3 4 6 .5071 . 3 6 5 3 .4953 Atlas . 2 9 5 0 .4500 .4031 .5482 Atlas Hooded . 3 7 9 1 .5578 . 2 7 1 8 .4408 . 3 5 2 0 .4918 Atlas U.B.C. . 3 9 0 6 . 5 6 7 8 . 2 7 0 0 .4255 . 3 6 3 3 . 5 0 6 1 Awnless .4166 .5841 . 3 4 6 6 .5428 . 2 8 0 0 . 5 0 3 2 Bolsheviki . 2 9 9 0 .4615 .4053 .5599 C-54-22 .3754 . 5 5 0 5 .3140 .4864 Fort . 3 4 7 9 . 5 0 7 1 . 2 8 8 5 . 4 4 8 8 . 3 5 5 5 . 4 8 6 6 Gat ami . 3 9 4 6 . 5 6 2 3 .2894 . 4 5 3 2 .3411 Kitchen . 4 0 3 8 .5686 Kwan .4044 . 5 7 5 3 . 3 1 7 6 . 4 8 7 7 . 3 7 8 8 .5111 Montcalm . 3 7 2 2 .5519 . 3 1 8 7 . 4 5 7 7 .3483 . 5 2 5 9 O.A.C. 21 . 3 5 3 0 .5130 .2913 . 4 6 2 7 . 3 6 1 9 .4193 O l l i . 3 9 1 0 . 5 7 0 6 . 2 9 5 9 . 4 6 3 6 . 3 5 6 0 . 4 9 8 7 Sohalta . 3 8 6 4 . 5 6 6 9 . 3 9 3 0 Tennessee . 3 4 0 5 .5219 .4144 Trebi . 3 8 4 5 .5711 . 3 1 9 0 .4804 2 2 - B 1 Bl-13 . 3 9 2 6 . 5 6 0 0 . 3 8 7 4 . 5 6 5 1 3 6 - B 1 B l - 2 1 . 3 9 2 2 . 5 7 8 8 .3418 . 5 2 8 6 . 3 1 3 0 . 4 8 6 7 4811-68-2 . 3 9 8 2 . 5 7 4 9 .3513 . 5 3 9 4 . 3 6 6 6 . 5 0 1 0 5090-2-3 •3770 . 5 4 3 2 .4215 . 5 7 6 2 ... . .^ 5423-4 . 3 7 6 1 .5614 . 3 3 3 6 .5145 5424-7 . 3 7 0 4 . 5 5 1 4 . 3 4 8 7 . 5 4 8 7 5424-8 . 3 7 2 7 . 5 5 8 1 . 3 0 9 4 . 4 9 0 0 . 3 8 9 4 .6480 5428-2 .3459 .5194 .3416 .5126 .3441 5430-1 . 3 4 3 0 .5181 .2923 . 4 6 6 0 . 3 3 6 2 Average . 3 7 8 2 .5535 .3178 . 4 8 1 7 . 3 5 8 2 . 5 0 9 8 - 9 7 -TABLE 1 1 : R F VALUES FOR PIGMENTS OF UNKNOWN, BUT NON-ANTHOCYANIN, NATURE IN VARIETIES WITH WHITE KERNELS Variety Butanol-Acetic Water Solvent Water-HCl-HAc Solvent Abyssinian . 6 9 8 2 Andie . 5 1 2 8 Carlsberg . 6 5 2 1 . 6 3 6 4 Compana . 5 7 6 0 . 7 2 0 2 c - 5 4 - 5 5 .6110 .6770 Deficiens . 4 3 5 2 . 6 8 9 2 Ethiops . 6 2 6 6 .7155 . Golden Pheasant . 4 7 6 7 . 7 5 0 5 Goldfoil . 6 2 8 7 Hanna . 6 3 8 6 . 6 9 6 3 Kama-Ore . 7 0 2 2 Lion .4361 . 6 2 8 9 . 6 7 3 9 Orange-Lemma .4434 . 7 3 6 6 Plush . 5 9 4 6 . 7 3 2 2 Smyrna . 4 3 9 7 . 6 6 8 3 Titan . 5 6 7 5 .7304 Velvon II . 7331 3 3-bl bl - 1 3 .4431 . 6 5 6 0 3 6-bl bl - 2 1 .4492 . .6924 71-pr pr - 1 0 , ^ - " . ' 4 5 1 9 5 0 9 0 - 9 0 - 1 . 5 8 9 7 . 6 9 2 2 5 0 9 0 - 1 0 - 4 . 6 2 5 1 .6842 5 0 9 0 - 1 5 - 1 . 6 4 2 7 . 6 7 5 0 Average . 4 4 6 9 . 6 1 3 9 . 6 8 6 4 TABLE 1 2 : Rp VALUES FOR ANTHOCYANINS EXTRACTED FROM VARIETIES OF BARLEY WITH PURPLE KERNEL PIGMENTATION Variety Water-Acetie-Hydrochlorie Acid Solvent (10:30:3) Butanol-Acetic-Water Solvent (40:10:30) 1 2 3 4 6 7 2 4 5 6 7 Black Hulless > . 0 2 5 8 .7148 . 8 3 6 4 * .9004 .1783 . 2 3 3 9 .4049 * * * C.I. 5628 .5410 .6173 . 6 9 9 4 . 7 5 7 7 .8143 * . 0 9 1 8 . 1767 . 3 8 2 1 * . 7479 * Gepal .5123 .6163 . 6 9 9 8 . 7 8 5 6 * .8604 * .1014 .1748 .2453 . 3 8 9 8 .5984 * . 8 0 4 3 Irasaka . 5 7 5 9 . 6 3 9 4 .7166 * .8241 . 8 6 8 4 .9244 . 0 9 9 3 . 1 9 0 0 . 2 3 2 3 . 3 6 5 7 .5473 * .8315 71-Pr-Pr-10 . 5 5 5 6 . 6 6 9 6 .7593 . 8 3 2 7 . 8 7 9 2 * . 1746 . 2 2 5 6 . 3 8 8 2 . 5 6 9 8 * . 8 5 3 7 Average . 5 4 6 2 . 6 2 4 7 . 7 0 0 0 . 7 6 7 5 . 8 2 6 9 . 8 6 9 3 .9124 .0975 . 1789 . 2 3 4 3 . 3 8 6 1 .5715 . 7 4 7 9 . 8 2 9 8 Column 1 . Noted only with M b Vapor " 2 , 3» 4 - Appears as a compound spot with two foci represented by columns 2 and 4 «' 7 - Often associated with a non-anthocyanin brown spot * - Streaks, not spots " 5» 6 , 7 - Appears as a compound streak usually with two foci represented by columns 5 & 7 TABLE 1 3 : % VALUES FOR ANTHOCYANIDINS FORMED FROM VARIETIES OF BARLEY WITH PURPLE PIGMENTATION Variety Water-Acetic-Hydrochloric Acid Solvent ( 1 0 : 3 0 : 3 ) Butanol-Acetic-Water Solvent (40 : 10:30) 1 2 3 4 5 6 7 8 9 10 Black Hulless . 3 3 5 9 . 5 2 8 6 .6816 . 8 6 9 0 . 1 0 9 7 . 2 3 0 3 . 4 4 2 5 .5440 . 6 1 9 4 C.I. 5628 . 3 3 7 6 . 5 1 6 5 . 6 7 8 0 . 8 3 5 5 . 1 0 3 5 . 2 5 5 4 . 4 2 7 9 . 6 0 3 5 Gopal . 2 8 8 6 .4935 . 6 3 2 4 . 7 1 3 4 .8149 . 1 0 7 8 . 3 4 3 4 . 4 3 0 1 .5626 .6404 Irasaka .3155 . 5 0 2 9 .6512 .7193 .8153 .1273 .2750 . 4 2 9 7 . 5 5 5 2 . 6 2 8 3 71-Pr Pr-klO .3710 . 5 0 6 8 . 6 2 3 5 . 7 1 7 8 . 8 5 0 9 .1407 . 2 5 9 8 . 4 3 7 5 . 5 6 2 6 . 6 3 5 8 Average . 3 2 9 7 . 5 0 9 3 . 6 5 3 3 . 7 1 6 8 . 8 3 6 7 .1178 . 2 5 2 8 . 4 3 3 6 . 5 5 6 1 . 6 3 1 0 7 - streaked 8 - compound spot with, possibly, several foci TABLE 14: VALUES FOR ANTHOCYANINS EXTRACTED FROM RED COLORED TISSUES OF CEREAL PLANTS Plant and Tissue Water-Acetic-Hydrochloric Acid Solvent Butanol-Acetic-Water Solvent Black Hulless Leaves from Low P plants receiving u l t r a - v i o l e t treatment . 5 6 0 9 .6335 .7361 .8922 . 1 5 0 0 .4166 . 5 2 2 9 .6843 Black Hulless Leaves from Low P plants receiving Dextrose .5642 .6578 .7389 .8020 . 8 8 2 7 .1737 .3171 .6121 . 7 2 9 5 .8216 Black Hulless Hulls . 6 6 2 9 . 7 5 6 6 . 8 6 6 7 .1771 .3653 .4759 . 5 9 5 1 .7898 .8802 C.I . 5628 Hulls .8302 .1248 .3128 V i c t o r y Oats Leaves .5714 .6742 .7703 .1840 .3545 .6314 .7738 Purple Kernels .5462 .6247 .7675 .8269 .8693 .1789 .2343 .3871 .5713 .7479 .8298 -101-Thus a solvent of water-acetic acid-concentrated HC1 (10 :30:3 by-volume) was used to chromatogram the barley extracts. It was found necessary to run the ascending chromato-grams about eight to ten hours, while four to seven hours s u f f i c e d f o r the descending type. The chromatograms were ready to read as soon as they had dried. At t h i s time they were observed under ordinary and u l t r a - v i o l e t l i g h t (maximum emission 2650 A). The reaction of the spots to alkaline and acid conditions was tested by placing the chromatograms i n an atmosphere saturated by either NH4OH or HC1 f o r a short time. They were then immed-i a t e l y observed. The differences were noted right on the chrom-atogram with colored p e n c i l . The R F values of these various spots were calculated by d i v i d i n g the distance "the center of gravity" of the spots had moved from the l i n e where they were f i r s t spotted on the chromatogram, by the distance the solvent front had t r a v e l l e d from this same l i n e (see Figures 11 and 12). A l i s t of these Rp values i s given i n Table 12. Often i t was very d i f f i c u l t to locate the anthocyanin pigment spots i n the blue barleys. However, by placing the chromatogram over bright diffused l i g h t the spots could usually be seen. They gave the c h a r a c t e r i s t i c red color i n the acid state and blue color i n the basic state. On the descending chromatograms i r r i g a t e d with the solvent butanol-acetic acid-water (40:10 :50 by volume) there appeared a "water" l i n e with an Rp of 0.5114. This l i n e was marked by a band of fluorescence. - 1 0 2 -The color i n t e n s i t y of the pigment f r a c t i o n s which separated when the extracts of "blue" seeded v a r i e t i e s were chromatogrammed was very weak. It i s d i f f i c u l t to be c e r t a i n whether there were one, two or three anthocyanin pigment spots appearing from the "blue" extracts. On the chromatograms there were two consistent anthocyanidin pigment spots. Their Rp values appeared very similar to the Rp values f o r the anthocyani-dins cyanidin and delphinidin or peonidin ( 6 ) when the chromato-grams were i r r i g a t e d with the solvent water-acetic acid-hydrochlor-i c acid ( 1 0 : 3 0 : 3 by volume). From the chromatographic result (see Table 1 0 ) , there appears to be a large number of pigment fr a c t i o n s i n the extracts from "purple" seeded barley v a r i e t i e s . Many of these fract i o n s appeared as merely pigment concentration within a continuous streak of color. Time and equipment were not available to sep-arate these spots further by two-dimensional chromatography. The r e s u l t s of the ascending chromatograms indicate two d e f i n i t e anthocyanin spots from extracts of "blue" seeded v a r i e -t i e s . The average Rp values of these spots, when i r r i g a t e d with the butanol-acetic acid-water solvent, were 0 . 1 8 1 5 and 0 . 2 3 7 9 * The average Rp of the "water" l i n e on these chromatograms was 0 . 5 2 5 9 . There were also two anthocyanidin spots on the ascending chromatograms. When the chromatogram was i r r i g a t e d with the water-acetic acid-hydrochloric acid solvent the average Rp values of these spots were 0 . 2 6 7 0 and 0.4400. -103-Fluorescent spots separated out i n a l l the chromaton grains of b a r l e y e x t r a c t s . These spots were most n o t i c e a b l e i n the chromatograms i r r i g a t e d w i t h b u t a n o l - a c e t i c acid-water s o l v e n t . There were some d i f f e r e n c e s n o t i c e d between v a r i e t i e s but these d i f f e r e n c e s d i d not c o i n c i d e w i t h seed c o l o r d i f f e r -ences. The Rp values of the pigment spots obtained from the e x t r a c t s of d r i e d flower p e t a l s are given i n Appendix V. Only s u f f i c i e n t chromatograms were run to i n d i c a t e whether the p l a n t contained pigments of s i m i l a r Rp value to the pigments i n the b a r l e y k e r n e l s . I f the pigment f r a c t i o n s - o f these flowers are known, then by a comparison of t h e i r Rp values w i t h those of the b a r l e y pigments the p o s s i b l e nature of the b a r l e y pigments can be a s c e r t a i n e d . However, f u r t h e r work needs to be done to more ac c u r a t e l y determine the Rp values of the f l o w e r and b a r l e y p i g -ments . I n f u t u r e s t u d i e s , c e r t a i n parts of the chromatographic techniques w i l l need to be improved. Such improvements might i n c l u d e p l a c i n g the f i l t e r paper i n the chromatographic c y l i n d e r and a l l o w i n g i t to stand twenty-four hours i n the fumes before i r r i g a t i n g solvent was introduced. This would a l l o w the paper to become"saturated" f i r s t , and v a r y i n g lengths of i r r i g a t i o n would not give such widely v a r y i n g r e s u l t s . A l s o , a pigment of known c o n s t i t u t i o n and Rp value should be placed on each f i l t e r paper. I f the Rp value of t h i s "known" spot i s i n a c c u r a t e , the whole chromatogram should be discarded. -104-IV CONCLUDING REMARKS The establishment of d i s t i n c t i v e c o l o r grades f o r Canadian export m a l t i n g b a r l e y s would be d e s i r a b l e . However, c o l o r genes f o r markers present some d i f f i c u l t i e s i n use as t h i s study a t t e s t s , and i t may be t h a t the hope of e s t a b l i s h i n g "blue genes" i n Canadian m a l t i n g b a r l e y s and "white genes" i n feed b a r l e y s may not be r e a l i z e d . C o l o r appears to p l a y a minor r o l e i n b a r l e y matabolism and m a l t i n g q u a l i t y . T h i s may make the t a s k of the breeder e a s i e r but i t a l s o makes c o l o r o f l e s s s i g n i f i c a n c e to the t r a d e . I t i s too e a r l y to say whether the i n h e r i t a n c e p a t t e r n of the c o l o r genes w i l l present any r e a l d i f f i c u l t y to the p l a n t breeder to whom the trade would be l a r g e l y dependent f o r the i n -c o r p o r a t i o n of the genes i n Canadian b a r l e y s . Myler's work (69) e s t a b l i s h e d a f a i r l y simple Mendelian p a t t e r n i n v o l v i n g some com-plementary i n t e r a c t i o n but the e x i s t e n c e of f a c t o r s other than those p o s t u l a t e d by Myler may yet be shown. R e s u l t s from c r o s s e s made at U.B.C. are, as i n d i c a t e d e a r l i e r , incomplete but i t does seem p o s s i b l e t h a t f o r most v a r i e t i e s c o l o r i n h e r i t a n c e w i l l ap-proximate or f o l l o w the Myler p a t t e r n . The c o l o r genes of b a r l e y f a l l s h o r t of being i d e a l markers. I n g e n e t i c a l terms they l a c k "penetrance" and "expres-s i v i t y " o r , i n oth e r words, cannot be r e l i e d upon to express themselves under a wide range of genotypic background and e n v i r o n -ment. Attempts to d i s c e r n the e c o l o g i c a l f a c t o r s which were - 1 0 5 -most i n f l u e n t i a l i n aiding expression have been p a r t i a l l y suc-ce s s f u l but appear to take unfortunate trends. I t was shown that low l e v e l s of nitrogen and phosphorus i n the s o i l s o l u t i o n favour color development but the useful l e v e l s are so low as to seriously reduce y i e l d . There would, of course, be no producer-i n t e r e s t i n such means of securing color. The knowledge gained, however, might be exploited to secure better color expression i n genetical and physiological studies. It was surprising, i n view of reports i n the l i t e r a t u r e , that u l t r a - v i o l e t l i g h t and dextrose feeding did not favour more pigment development. Although the p r i n c i p a l ecological factors associated with anthocyanin pigment production may not have been discerned i n the tests at U.B.C., the results of the tests follow c l o s e l y those of Emerson (24) f o r maize. L o c a l i z a t i o n of pigments i n aleurone and pericarp had been demonstrated by Harlan i n 1914 (38) but apparently no studies have been published on the subject since then. Histo-chemical tests described on preceding pages confirmed and extended Harlan's observations. I t Is suggested, however, that freehand sections leave much to be desired f o r color characterization of large numbers of barley v a r i e t i e s . Perhaps a vibratory microtome might be employed to advantage. The r e s u l t s from chromatography experiments provide some most i n t e r e s t i n g speculation. It should be r e c a l l e d that genes B l and B l 2 are both required f o r blue color expression i n -106-the aleurone layer. According to a "one gene-one enzyme" hypothesis one might expect, through complementary action, the production of one blue pigment. The chromatogram, however, shows that there are two blue pigments, probably quite c l o s e l y rela t e d . Their Rp values are s i m i l a r to those determined f o r the well known anthocyanins, cyanidin, d e l p h i n i d i n or peonidin. More study w i l l be necessary to determine the "pathways" by which the two pigments are produced and a r e c o n c i l i a t i o n with complementary gene action w i l l be effected. It i s also i n t e r -esting to note that the Rp values f o r two of the several pigments obtained from "purple" barleys are nearly i d e n t i c a l with the two from the "blue" barleys. This i s confirmation of the genetic postulation of Myler (ibid . ) who believed that the Bl and B l 2 genes were present both i n the "purple" and "blue" barleys. 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Genetics 3 1 : 3 7 7 - 3 9 4 . 9 7 . . 1 9 5 4 . The gene. Science 120 : 8 1 1 - 8 1 8 . 9 8 . Wender, S. H., and Gage, T. B. 1949. Paper chromatography of flavonoid pigments. Sci. 109 : 2 8 7 - 2 8 9 . 9 9 . Wiggans, R. G. 1 9 2 1 . A classification of the cultivated varieties of barley. Cornell Agric. Exp. Sta. Mem. 4 6 . 1 0 0 . Williams, R. H., and Kirby, Helen. 1948. Paper chromatography using capillary ascent. Sei. 107 : 481-483. 1 0 1 . Wilstatter, R., and Mallison, H. 1914. Ueber die Verwandschaft der Anthocyane und Flavone. Sitz Ber. Ak. Wiss. Berlin, pp. 7 6 9 -7 7 7 . [Cited after Zarudnaya, 1 9 5 0 . ] - 1 1 6 -1 0 2 . Winston, W. A. 194-8. A s i m p l i f i e d apparatus f o r one-dimensional paper p a r t i t i o n chromatography. S c i . 107 • 6 0 5 • 1 0 3 . Woodward, R. W., and Thieret, J . W. 1953. A genetic study of complementary genes f o r purple lemma, palea, and pericarp i n barley (Hordeum  vulgare L.) Agron. Jour. 4£ : 182-185". 104. Zarudnaya, K. I. 1 9 5 0 . A chromatographic study of anthocyanins and related substances i n various genotypes of Maize. Doctorial thesis at Univ. of Missouri. [Also cited i n Mich. Univ. Microfilm Abst. 10 : 3 2 - 3 2 . ] -117-APPENDIX I TABLE GIVING LIST OF PLANT MATERIAL USED IN THE EXPERIMENTS WITH THEIR COLOR AND SOURCE Designation Color of Kernel Source Barley Abyssinian white U.S.D.A. Algerian blue 2 Ottawa Andie C.I.728 white U.S.D.A. Atlas C.I.4118 blue blue U.S.D.A. U.B.C. Atlas hooded blue U.B.C. Awnless C.I.5631 blue U.S.D.A. Black Hulless purple Ottawa Bolsheviki C.1.2523 blue U.S.D.A. Byng white and dirty white Ottawa Carlsberg white Ottawa Compana white U.B.C. C.I.5628 purple U.S.D.A. C54-22 pale blue Ottawa C54-55 white and dirty white Ottawa 3 1 Obtained from G.A.Wiebe, U.S.D.A., Bettsville, Maryland. 2 Obtained from D.G.Hamilton, Central Experimental Farm, Ottawa.' 3 Material grown at the University of Brit i s h Columbia. -118-Continued Designation Color of Kernel Source Barley - continued Deficiens C.I. 2225 white U.S.D.A. Ethiops white and d i r t y white Ottawa Fort blue Ottawa Gat ami black Ottawa Golden Pheasant C.I. 24-88 white U.S.D.A. G o l d f o i l white Ottawa Gopal C.I. 1091 bright purple U.S.D.A. Hanna white Ottawa Husky white Ottawa Irasaka C.I. 1083 purple U.S.D.A. Kama-ore C.I. 694 d i r t y white U.S.D.A. Kitchen black Ottawa Kwan blue Ottawa Lion black Ottawa Montcalm blue Ottawa Nepal white Ottawa O.A.C. 21 blue Ottawa O l l i blue U.B.C. Orange Lemma C.I. 5649 white and d i r t y white U.S.D.A. Plush white U.B.C. Smyrna C.I. 910 white U.S.D.A. Sonalta white U.B.C. -119-Continued Designation C o l o r of K e r n e l Source B a r l e y - continued Tennessee Blue U.B.C. T i t a n white U.B.C. Tr e b i P e c u l i a r blue Ottawa Vantage white Ottawa Velvon 11 white Ottawa 33-bl bl-13 white U.S.D.A. 33-B1 Bl-13 blue U.S.D.A. 3 6 - b l b l - 2 1 white U.S.D.A. 36-B1 B l - 2 1 blue U.S.D.A. 71-pr pr - 1 0 white U.S.D.A. 71-Pr Pr - 1 0 purple U.S.D.A. 4811-68-2 blue Ottawa 5090-2-3 grey and d i r t y white Ottawa 5 0 9 0 - 9-I grey and d i r t y white Ottawa 5 0 9 0 - 1 0 - 4 white and d i r t y white Ottawa 5 0 9 0 - 1 5 - 1 white Ottawa 5 4 2 3 - 4 dark blue or grey Ottawa 5424-7 dark blue or black Ottawa 5425-8 grey Ottawa 5 4 2 8 - 2 black Ottawa 5 4 3 0 - 1 black Ottawa - 1 2 0 -continued D e s i g n a t i o n C o l o r of K e r n e l Source Wheat j Blue 1 blue Ottawa R. Blue blue Ottawa Regina 551 blue 4 Regina Regina 552 blue Regina Maize N.W.Dent red U.B.C. 4 Obtained from E. A. Saskatchewan. Hurd, Experimental Sub-s t a t i o n , Regina, D e s i g n a t i o n C o l o r of F r e s h M a t e r i a l Source D r i e d M a t e r i a l . P l a n t s B lack H u l l e s s l e a v e s red U.B.C. Black H u l l e s h u l l s red U.B.C. V i c t o r y oat l e a v e s red U.B.C. Flowers A s t e r K i r k w e l l Dwf salmon pi n k 5 McKenzie Queen of Market Crimson McKenzie Snowden Giant white McKenzie Triumph Giant rose p i n k McKenzie 5 Grown at U.B.C. - seed obtained from A.E.McKenzie Seed Co. Continued -121-Designation Color of Fresh Source Material Flowers Calendula Exquisite Mixed Orange Blake 6 Rays of Sunshine orange Robinson'7 Carnation dark red U.B.C. Centaurea Double Blue Boy blue Robinson Geranium Maxine red U.B.C. Lobelia C r y s t a l Palace Imp. blue Robinson Nemesia Superbissima Triumph bright red Robinson Triumph Mixed (cool) coral orange Blake Nicotiana Crimson red pink Blake Daylight Sensation bright red Robinson Daylight Sensation purple Robinson Pansy Swiss Giant dark blue Robinson Phlox Gigantea Art shades blue purple Blake T a l l Grandiflora s c a r l e t Blake T a l l Grandiflora mauve Blake Scabiosa Annual T a l l purple Blake Zinnia C a l i f o r n i a Giant dark red McKenzie Dahlia flowered Chinese red Blake Super Giant red McKenzie 6 Grown at U.B.C. - seed obtained from F. 0. Blake Seed Co. 7 Grown at U.B.C. - seed obtained from G. A. Robinson and Son Seed Co. APPENDIX I I , TABLE SHOWING BARLEY CROSSES MADE IN THE SPRING OF 1955 at U.B.C. Data on the P i seed includes the following: The cross number, the appearance of the crossed seed produced on the female parent, color of seed of similar crosses reported i n the l i t e r a t u r e , the possible blue or black color of crosses not reported i n the l i t e r a t u r e , the maturity of the harvested crosses, and the int e n s i t y of color of the crossed seed compared to the seed of the female parent produced under similar conditions. Cross Cross No. Actual Color of Seed Color of Seed i n Literature Possible Color of Seed Maturity of Seed* Color c f . £ Parent # Algerian x Black Hulless 1 Blue Blue +++ 2 ++ 5 + " x Black Hulless 2 Blue Blue ++ 0 " x Carlsberg 3 Blue Blue +++ -" x Carlsberg 4 Blue Blue ++ 0 " x Deficiens 5 Blue -" x Lion 6 Blue Blue +++ -M x Smyrna 7 Blue Blue ++ — - no seed # - l i g h t e r than parent + immature (seeds needle-like) 0 the same as parent ++ p a r t i a l l y mature ( p a r t i a l l y f i l l e d seeds) + s l i g h t l y darker than parent +++ p a r t i a l l y mature to mature ++ markedly darker than parent ++++ mature (completely f i l l e d seeds) APPENDIX II - continued Cross Cross No. Actual Color of Seed Color of Seed i n L i t e r a t u r e Possible Color of Seed Maturity of Seed* Color of* 0 Parent # Awnless x Ethiops 8 Blue Blue ++++ + II x Fort 9 Blue Blue ++++ 0 it x Kitchen 10 Blue 1 Blue ++++ 5++ 4 -n x Kwan 11 Blue Blue ++++ ++ ii x Smyrna 12 Blue Blue +++-; 6++ 4 -ti x C54-55 13 Blue Blue +++ 5+ 1 0 -II x 5 0 9 0 - 1 5 - 1 14 Blue Blue ++ 0 Bolshevik! x Black Hulless 15 Blue Blue ++++ + 11 x Defidiens 16 Blue -II x Deficiens 17 Blue Blue +++ + ti x Ethiops 18 Light Purple +++ Purple II x Kitchen 19 Blue Blue +++ 0 II x Kitchen 20 Blue Blue +++ 0 it x Kwan 21 Purple Hulless Blue ++ Dark Purple APPENDIX II - continued Cross Cross Actual Color Color of Seed Possible Maturity Color of. No. of Seed i n Literature Color of of Seed* ?Parent # Seed  Bolshevik! X 5 4 2 5 - 8 22 Blue -11 X 5 4 2 5 - 8 23 Blue Blue ++++ ++ Carlsberg X Black Hulless 24 Blue ++ 0 ti X Deficiens 25 ++ 0 11 X Hanna 26 +++ -ti X Hanna 27 +++ 0 it X Kitchen 28 ++ 0 u X C 5 4 - 5 5 29 ++ -u X c 5 4 - 5 5 30 +++ - . ti X Trebi 31 Blue ++ -Deficiens X Awnless 32 10 Blue 2 white Blue ++++ 10++ 3 0 u X Fort 33 Blue -ti X G o l d f o i l 3 4 -it X Hanna 35 White ++++ 0 APPENDIX II - continued Cross Cross No. Actual Color of Seed Color of Seed i n Literature Possible Color of Seed Maturity of Seed £ Color of. Parent # Deficiens X Kitchen 36 Blue ? ++ + H X Lion 37 Blue ++++ ++ II X Nepal 38 White ++++ 0 i t X C54-55 39 -II X 5425-8 40 4 Blue 8 white ++++ 4++ 8 0 Ethiops X Algerian 41 ? Blue ++ 0 II X Awnless 42 Blue Blue +++ 8 ++ 10 +.. i t X Awnless 43 Blue Blue +++ ++ M X Bolsheviki 44 Blue + + II X Deficiens 45 Blue ? 1 ++ 1 + t l X Deficiens 46 Blue ++ + II X Fort 47 Blue -II X Fort 48 Blue ? Blue ++ + It X Kitchen 49 Blue ++++ 6+ 3 0 APPENDIX II - continued Cross Cross No. Actual Color of Seed Color of Seed i n Literature Possible Color of Seed Maturity of Seed Color c f . $ Parent # Ethiops x Lion 50 White ? ++++ 0 '« x Smyrna 51 White ? +++ 0 M x Smyrna 52 White ? ++ 0 x C.I. 5628 53 White ? Blue ++ 0 x C.I. 5628 54 White ? Blue ++ 0 x 5 4 2 5 - 8 55 Blue ++++ 10 ++ 5 + Fort x Awnless 56 Blue Blue ++ — x Deficiens 57 Blue Blue ++++ + x Kitchen 58 Blue Blue ++++ 1 + 1 -x C54-55 59 Blue Blue ++ -x 5 0 9 0 - 1 5 - 1 60 Blue Blue +++ 0 x 5 0 9 0 - 1 5 - 1 61 Blue 1 APPENDIX II - continued Cross Cross No. Actual Color of Seed Color of Seed i n L iterature Possible Color of Seed Maturity of Seed Color c f . 0 Parent # G o l d f o i l x Algerian 62 Blue ? Blue ++ + n x Algerian 63 Blue ? Blue +++ + II x Black Hulless 64 Blue ? Blue +++ + II x Black Hulless 65 -II x Carlsberg 66 ? ++ + it x Carlsberg 67 ? ++ + » x Kitchen 6 8 ++ + II x Nepal 69 Blue ? Blue ++++ + ti x Nepal 70 Blue ++ + II x Smyrna 71 Poor Seed + 0 II x Trebi 72 Blue -it x 5 0 9 0 - 1 5 - 1 73 Poor Seed + + Hanna x Algerian 74 Blue -ti x Algerian 75 Blue ++ + it x Awnless 76 Blue Blue +++ + II x Awnless 77 Blue Blue ++ APPENDIX II - continued Cross Cross No. Actual Color of Seed Color of Seed i n L iterature Possible Color of Seed Maturity of Seed Color c f . £ Parent # Hanna X Ethiops 78 ++++ ' + it X Ethiops 79 White ? +++ 0 it X Fort 80 Blue +++ + it X G o l d f o i l 81 White ++++ 0 ti X Kitchen 82 Blue ? ++ + ii X Lion 83 White ++++ + ti X Nepal 84 Blue ++ + it X Smyrna 85 ++++ + it X Trebi 86 Blue Blue ++++ + ti X Velvon II 87 ++ + II X C 5 4 - 5 5 88 Blue ? ++++ + tt X C . I . 5628 89 Blue +++ + it X 5 0 9 0 - 1 5 - 1 90 ++ ii X 5 0 9 0 - 1 5 - 1 91 -Kitchen X A l g e r i a n 92 Black Black ++ 0 II X Awnless 93 Black Black ++ mm APPENDIX II - continued Cross Cross No. Actual Color of Seed Color of Seed i n Literature Possible Color of Seed Maturity of Seed Color c f . | Parent # Kitchen x Hanna 9 4 Black Black ++ 0 it X Smyrna 95 Black Black ++++ 0 it X C.I. 5628 96 Black Black ++ 0 Kwan X Awnless 97 Blue Blue ++++ 2 + 9 0 II X Deficiens 98 Blue Blue ++ ++ II X Fort 99 Blue Blue ++++ + II X Fort 100 Blue Blue ++ -it X Hanna 101 Blue Blue ++++ -it X C 5 4 - 5 5 102 Blue -II X 5 0 9 0 - 1 5 - 1 103 Blue Blue +++ -it X 5 4 2 5 - 8 104 Blue - 13 ++ 6 0 II X 5 4 2 5 - 8 105 Blue Blue +++ Lion X Awnless 106 Black Black +++ + II X Awnless 107 Black Black +++ + it X Black Hulless 108 Black Black +++ 0 it X Carlsberg 109 Black Black +++ 0 APPENDIX I I - continued Cross Cross No. A c t u a l Color of Seed Color of Seed i n L i t e r a t u r e P o s s i b l e C olor of Seed M a t u r i t y of Seed Color c f . o Parent # L i o n X F o r t 110 Black Black +++ 0 II X K i t c h e n 111 Black Black +++ it X T r e b i 112 Black Black ++++ + II X Velvon I I 113 Black Black +++ + II X C54-55 114 Black Black ++++ 7 + 3 0 II X 4811 - 6 8 - 2 115 B l a c k -it X 5 0 9 0 - 1 5 - 1 116 Black Black ++ 0 Nepal X Black H u l l e s s 117 White Blue +++ + II X Ethiops 118 White ++++ + ti X Ethiops 119 White ++++ + ti X K i t c h e n 120 White ++++ 0 it X L i o n 121 White ++++ + ti X Smyrna 122 ++ + it X Smyrna 123 Blue ? ++ + II X T r e b i 124 Blue ++ II X T r e b i 125 Blue ++ + APPENDIX II - continued Cross Cross Actual Color Color of Seed Possible Maturity Color c f . No. of Seed i n Literature Color of of Seed £ Parent # Seed Nepal x Velvon II 126 2 Blue + ti x C.I. 5628 127 Blue — Smyrna it ti x Algerian „ Black x Hulless x Deficiens 128 129 130 White ? Blue ? Blue Blue ++ ++++ ++ + + ++ II x Deficiens 131 ++ + ti x Hanna 132 ++ + ti x Kitchen 133 White ? +++ + ti x Nepal 134 +++ II x Trebi 135 Blue ++ + II x Velvon II 136 ++ + II x Velvon I I 137 +++ + it x C.I. 5628 138 Blue +++ it x 5 0 9 0 - 1 5 - 1 139 +++ + Trebi II x Algerian x Algerian 140 141 Blue ? Blue ? Blue Blue ++ ++ 2 0 1 2 -APPENDIX II - continued Cross Cross No. Actual Color of Seed Color of Seed i n Literature Possible Color of Seed Maturity of Seed Color c f . $ Parent # Trebi x Black Hulless 142 Blue Blue ++ 0 ti x G o l d f o i l 143 White Blue ++ -II x Hanna 144 Blue ? Blue ++ -ti x Kitchen 145 Blue ? Blue ++ -tt x Smyrna 146 Blue Blue ++ 5 o 5-Velvon I I x Algerian 14? Blue ++ + it x Carlsberg 148 -it x Carlsberg 149 +++ +-.-ti x Deficiens 150 Blue ? +++ ++ ti x G o l d f o i l 151 ++ + it x G o l d f o i l 152 ++ + it x Kitchen 153 Blue ? ++ + tt x Lion 154 ++ + it x Nepal 155 mm ii x Trebi 156 Blue ++ it x C.I. 5628 157 Blue ? Blue ++ ++ APPENDIX II - continued Cross Cross No. Actual Color Color of Seed of Seed i n Li t e r a t u r e Possible Color of of Seed Maturity of Seed Color c f . £ Parent # Velvon II X 4811-68-2 158 Blue ++ 11 X 5090-15-1 159 -ti X 5 4 2 5 - 8 160 ++ 0 C 5 4 - 5 5 X Deficiens 161 Blue ? +++ ++ ti X Ethiops 162 White ? +++ 0 ti X Ethiops 163 +++* + H X Fort 164 Blue -n X Kitchen 165 ++ + II X Smyrna 166 ++++ + II X Smyrna 167 ++++ + ti X 5 0 9 0 - 1 5 - 1 168 ++++ 0 n X 5 4 2 5 - 8 169 ++++ + C . l . 5628 X Ethiops 170 Purple (Purple) Blue +++ + it X Smyrna 171 Purple (Purple) Blue ++++ + 4811-68-2 X Awnless 172 Blue Blue ++++ 0 APPENDIX II - continued Cross Cross No. Actual Color of Seed Color of Seed in Literature Possible Color of Seed Maturity of Seed Color cf. $ Parent # 4811-68-2 x Carlsberg 173 Blue ++ -M x Deficiens 174 Blue Blue ++ 0 " x Ethiops 175 Blue ++ -»« x Kitchen 176 Blue ++ -" x Nepal 177 Blue -•' " x C54-55 178 Blue - . » x C54-55 179 Blue ++ 0 " x 5425-8 180 Blue Blue ++ 0 5090-15-1 x Awnless 181 Blue ? Blue ++++ 0 " x Deficiens 182 ++ -" x Ethiops 183 ++ -" x Fort 184 Blue ++ -11 x Kwan 185 Blue ++ -" x Smyrna 186 White ? ++ -5 0 9 0 - 1 5-I x C.I. 5628 187 Blue — APPENDIX II - continued Cross Cross Actual Color No. of Seed Color of Seed Possible Maturity Color c f . i n Literature Color of of Seed £ Parent # Seed 5090-15-1 x 4811-68-2 " x 5 4 2 5 - 8 188 189 Blue +++ 5425^8 X Awnless 190 Blue Blue +++ 0 II X Awnless 191 Blue • Blue +++ 0 II X Deficiens 192 -it X Fort 193 Blue -ti X Hanna 194 -it X Smyrna 195 +++ -ti X Smyrna 196 Blue ? ++++ 0 it X C54-55 197 Blue ? ++ 0 tt X 5090-15-1 198 -- 1 3 5 -APPENDIX III. TABLES COMPARING THE COLOR SCORES OF SEEDS DEVELOPED UNDER VARIOUS NUTRIENT LEVELS AND BLOCK TREATMENTS TABLE 1 . CONTINGENCY TABLE COMPARING THE COLOR SCORE OF EACH AWNLESS" PLANT TO THE NUTRIENT TREATMENTS Nutrient level Light Color Scale Dark 1 2 3 4 5 6 7 Complete 6 37 29 33 12 16 6 Low P 2 10 13 9 15 19 15 Low N 1 9 22 28 38 43 28 TABLE 2 . CONTINGENCY TABLE COMPARING THE COLOR SCORE OF EACH "AWNLESS" PLANT TO THE BLOCK TREATMENTS Block Light Color Scale Dark Treatment 1 2 3 4 5 6 7 Control 4 12 11 13 11 13 8 Ultra-violet 1 6 7 15 7 28 15 Infra-red 1 10 10 16 17 30 14 Dextrose 6 13 12 14 6 6 Cool 3 22 24 13 16 1 6 - 1 3 6 -APPENDIX III - continued TABLE 3 - CONTINGENCY TABLE COMPARING THE COLOR SCORE OF EACH "BLACK HULLESS" PLANT TO THE NUTRIENT TREATMENTS Nutrient Level Light Color Scale Dark 1 2 3 4 5 6 7 8 9 10 Complete 5 10 14 4 3 13 2 Low P 2 12 5 3 7 3 1 Low N 3 1 6 5 18 10 22 33 41 17 i TABLE 4. CONTINGENCY TABLE COMPARING THE COLOR SCORE OF EACH "BLACK HULLESS" PLANT TO THE BLOCK TREATMENTS Block Treatments Light Color Scale Dark 1 2 3 4 5 6 7 8 9 10 Control 4 12 2 2 6 8 6 3 11 U l t r a -v i o l e t 1 2 1 2 9 8 7 11 1 Infra-red 1 1 3 15 15 1 Dextrose 3 1 7 7 4 4 4 5 4 3 Cool 2 8 15 1 1 17 6 3 1 12 - 1 3 7 -APPENDIX III - continued TABLE 5. CONTINGENCY TABLE COMPARING THE COLOR SCORE OF EACH "BLACK HULLESS" PLANT RECEIVING THE LOW N TREATMENT TO THE BLOCK TREATMENTS Block Treatments Light Color Scale Dark 1 2 3 4 5 6 7 8 9 10 Control 2 4 3 5 3 10 [Jltra-violet 9 8 7 11 1 Infra-red 1 1 3 15 15 1 Dextrose 3 3 3 3 5 4 3 Cool 1 6 1 6 3 3 1 12 TABLE 6. CONTINGENCY TABLE COMPARING THE COLOR SCORE OF EACH " SMYRNA4.' PLANT TO THE NUTRIENT TREATMENTS Nutrient Light Color Scale Dark Level 1 2 3 Complete 8 39 81 Low P 3 66 60 Low N 65 89 24 TABLE 7 . CONTINGENCY TABLE COMPARING THE COLOR SCORE OF EACH "SMYRNA" PLANT TO THE BLOCK TREATMENTS Block Light Color Scale Dark Treatments 1 2 3 Control 24 37 26 U l t r a - v i o l e t 15 54 33 Infra-red 14 40 51 Dextrose 20 19 9 Cool 3 44 46 - 1 3 8 -APPENDLX IV TABLE RECORDING THE MICROSCOPIC APPEARANCE OF THE ALEURONE AND PERICARP LAYERS OF BARLEY UNDER DRY, ACID AND ALKALINE CONDITIONS Legend: B B l C Dk Black Blue Colorless Dark G - Green Lt - Light 0 - Orange P - Purple Pk - Pink R - Red Y - Yellow Variety- Condition of Section Dry 2% HC1 2% Na OH Aleurone Pericarp Aleurone Pericarp Aleurone Pericarp Purple Black Hulless B l Dk Dk R-Pr R-0 G O-P » No.l* Lt B l 0 O-Pk C G Y-G •« No.5 B l Y^O R Y - 0 G-Bl Y C Dk Lt Pk Dk R G 0 C.I. 5628 Dk 0+P Lt Pk Dk R G 0 Gopal C P -0 Pk O+Dk R G G-Y+O Irasaka C Pk-0 O-Pk Pk + Dk Bl-G Y+0 71-Pr Pr-10 C Dk Pk Dk R Bl-G Bl+O+Y Blue Algerian C 0-R Pk G-O-Y Awnless #1 LtBl+Pk 0 Pk Dk+Y O-G Y-G Awnless #7 Bl+O. 0 Pk Y G Y-G Fort Dk O+Lt Pk Dk 0 Y-G 0 Montcalm Lt B l O+Dk Pk Y O.A.C. 21 Lt B l Pk 0 G-Y-0 Bl-G O-Y Trebi Lt B l Dk Pk 0-Y Y-G O-Y 33-B1B1-I3 Lt B l Dk Pk Dk+C Y-G O-G 36-HLHL-21 Bl Lt 0 Pk C+O G 0 White Byng Lt 0 Y -0 G G o l d f o i l C+Lt 0 O-Y+GY Hanna C O-Pk Y G-Y+O Husky C C O-Pk G-Y G-Y Nepal C Lt Pk Y-G Y-G Smyrna #1 c+o Dk 0 Pk 0 C Y-G G Smyrna #3 c 0 Pk 0 Dk G+0 O-G Vantage C+Lt 0 Pk 0 Dk+O G-Y G Velvon II Lt 0+C O-Y+G 3 3-blbl - I 3 - C+0 Lt O-Pk C Y-G O-G 71-pr pr-10 C Dk B l O-Pk Dk+C G Dk+G Black Gat ami C Dk Pk Dk O+B Y-G O-B Kitchen c O-B Lt Pk O-B G O-B Lion c Dk C Dk+O Y-G O-B * The number refers to the seed color class (see seed color experi-ment ). APPENDIX V. R F VALUES OF DRIED FLOWER PETAL PIGMENTS TABLE 1. R w VALUES OF ANTHOCYANINS EXTRACTED FROM DRIED FLOWER PETALS Flower and S o l v e n t V a r i e t y * Water-Acetic a c i d - H y d r o c h l o r i c (10:30:35 B u t a n o l - a c e t i c acid (40:10:50) - water A s t e r K i r k w e l l .6318 .7667 .1799 .3307 Queen of Mk. .5827 .6829 .7169 .8719 .0404 .1139 .1645 .2181 Triumph .5877 .6690 .7606 .0406 .1044 .1541 .1948 .2319 Carnation .3544 .4846 .6428 .1317 .2573 Centaurea Blue Boy .7403 .0295 .1218 Geranium Maxine .5846 .7118 .0513 .1140 .2593 .3264 L o b e l i a X a l Palace .6343 .6794 .7385 .0219 .0685 .1091 .2169 .5331 Nemesia Superbissma .6113 .7088 -7777 .0434 .1139 N.C.Triumph .6181 .6473 .7224 .7495 .0243 .0946 .2394 .2814 .5324 N i c o t i a n a Crimson .4946 .5993 .7200 .0311 .0754 Daylight .5635 .0215 .0586 Daylight .4706 .6034 .7418 .0120 .0566 .0784 Pansy Swiss .5899 .6461 .7223 i .0279 .0867 .1586 Phlox Gigantea .4847 .6010 .6861 .7430 .0329 .131$ G r a n d i f l o r a .4993 .6292 .7300 .8182 .9213 .0371 .1239 G r a n d i f l o r a .4684 .6141 .7339 .0375 .1138 .1592 .1952 Scabiosa Annual .6335 .7835 .8807 1 .0158 .0487 .1688 .2818 Z i n n i a C a l . Giant .5717 .6452 .7403 .0554 .1302 .3041 D a h l i a .6560 .7240 .0278 .1041 .1603 Super .6178 .6724 .7338 .0510 .1219 * More d e t a i l s on v a r i e t y , c o l o r , and source of these flowers given i n Appendix I . APPENDIX V. TABLE 2. Rp VALUES OF ANTHOCYANIDINS EXTRACTED FROM DRIED FLOWER PETALS Flower and S 0 1 v e n t Variety- Water-Acetic Acid-Hydro-c h l o r i c (10:30:3) Large C y l i n d e r Water-ace t i c - H y d r o -c h l o r i c Small C y l i n d e r B u t a n o l - A c e t i c acid-Water (40:10:50) Aster K i r k w a l l .5532 .6989 .5231 .6961 .2076 .3522 .5240 Queen of Mk. .5703 .7224 .8195 .4775 .6340 .7523 .2139 .3450 .5234 Triumph .5639 .7178 .8151 .4599 .6120 .7206 .2163 .3556 .5252 Carnation .6122 .7907 .2961 .5423 Centaurea Blue Boy .6213 .7927 Geranium Maxine .6843 .7752 .1691 .3529 .4241 .5314 .8272 L o b e l i a X a l Palace .3669 .5279 .7032 .2909 .4515 .6257 .0735 .2031 .3550 .4490 Nemesia Superbissma .3635 .5408 .6611 .2962 .4870 .6212 .3520 .4512 N.C.Triumph .4126 .5976 .7276 .2922 .4587 .5776 • 3997 .4704 .5539 .7478 N i c o t i a n a Crimson .4038 .5731 .7635 .3500 .5268 .7422 .0881 .4110 Daylight .3736 .5257 .7079 .3633 .5408 • 7837 .1037 .3843 -Daylight .3706 .5214 .7120 .3527 .5426 .7617 .0977 .3868 Pansy — Swiss .3654 .5704 .6821 .0535 .2996 .4542 .5691 .7910 Phlox Gigantea .4008 . 5786 . 7117 .7968" .4176 .6484 .7820 .-• .4803 .5418 • 7195 G r a n d i f l o r a .3984 .5749 .7012 .7833 .4126 .6435 .7883 .3906 .4690 .5546 G r a n d i f l o r a .5684 .6969 .7750 .6058 .7639 - .5372 .6748 • Scabiosa Annual .5517 .6984 .8252 .4750 .6293 .7625 .1050 . 2 4 2 4 .4689 .7063 Z i n n i a C a l Giant .5482 .6941 .5168 .6779 .0962 .2205 .4816 .7238 D a h l i a .5483 .7017 .4902 .6536 .0858 .2025 .4736 Super .5463 .7001 .4863 .6495 .1640 .4762 APPENDIX; VI Some Typical Chromatograms Obtained From From Petals of Common Flowers And From Tissues of Cereal Kernels. (Film Record of Color not Adequate; Further Details on Chromatograms Unwarranted) 

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