UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

A. -The selection of male-sterile lines in alfalfa. B. -The witches' broom virus disease of alfalfa in.. 1951

You don't seem to have a PDF reader installed, try download the pdf

Item Metadata

Download

Media
UBC_1951_A4 P3 S3.pdf [ 10.11MB ]
UBC_1951_A4 P3 S3.pdf
Metadata
JSON: 1.0106547.json
JSON-LD: 1.0106547+ld.json
RDF/XML (Pretty): 1.0106547.xml
RDF/JSON: 1.0106547+rdf.json
Turtle: 1.0106547+rdf-turtle.txt
N-Triples: 1.0106547+rdf-ntriples.txt
Citation
1.0106547.ris

Full Text

P 3 5 3 A - THE SELECTION OF MALE-STERILE LINES IN ALFALFA. B - THE WITCHES' BROOM VIRUS DISEASE OF ALFALFA IN BRITISH COLUMBIA. by FREDERICK DOUGLAS .PETTEM A Thesis Submitted i n P a r t i a l Fulfilment of the Requirements for the Degree of Master of Science i n Agriculture in The Department of Agronomy We accept t h i s thesis as conforming to the standard required for candidates for the degree of MASTER OF SCIENCE IN AGRICULTURE Members^ of %\ie Department of Agronomy THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1951. A B S T R J - 3 T I n t h e h o p e o f f i n d i n g m a l e - s t e r i l e a l f a l f a s t r a i n s f o r u s e i n t h e p r o d u c t i o n o f h y b r i d a l f a l f a , a m i c r o s c o p i c s t u d y o f t h e p o l l e n p r o d u c e d b y a p p r o x i m a t e l y 1 0 0 0 l i n e s o f a l f a l f a g r o w n a t t h e U n i v e r s i t y o f B r i t i s h - C o l u m b i a w a s c o n d u c t e d . T h i s s t u d y r e v e a l e d 4 l i n e s t h a t c o n s i s t e n t l y s h o w e d a n a b s e n c e o f v i a b l e p o l l e n u n d e r d i f f e r e n t e n v i r o n m e n t s . 2 o f t h e m a l e - s t e r i l e l i n e s p r o d u c e d n o s e e d o n s e l f i n g a n d 2 p r o d u c e d v e r y s m a l l q u a n t i t i e s o f s e e d . I n p l a n t s g r o w n f r o m o p e n - p o l l i n a t e d s e e d o f t h e 4 m a l e - s t e r i l e l i n e s , t h e ¥ ± s e g r e g a t i o n f o r m a l e - s t e r i l e t o m a l e - f e r t i l e w e r e a s f o l l o w s : 1 : 7 . 9 , 1 : 7 . 3 , 1 : 6 . 8 , a n d 1 : 8 . 3 , f o r a n i n c o m p l e t e c o u n t . T h e s e r a t i o s s u g g e s t t h a t t h e m a l e - s t e r i l i t y i s c o n t r o l l e d b y c y t o p l a s i n a i c f a c t o r s i n a d d i t i o n t o r e c e s s i v e g e n e s . H o w e v e r , t h e m o d e o f i n h e r i t a n c e w i l l o n l y b e f a c t o r i a l l y i n t e r p r e t e d b y ]?2 and- b a c k c r o s s d a t a . I t a p p e a r s t h a t t h e s t e r i l i t y i s c a u s e d b y a b r e a k d o w n i n m e i o s i s , a s t h e p o l l e n s a c s a r e f u l l o f a n a m o r p h o u s m a t e r i a l s u g g e s t i v e o f a r r e s t e d d e v e l o p m e n t o f t h e p o l l e n g r a i n s . T h e m a l e - s t e r i l i t y s h o u l d e v e n t u a l l y p r o v e o f g r e a t e c o n o m i c v a l u e i n t h e p r o d u c t i o n o f h y b r i d a l f a l f a s e e d . M a l e - s t e r i l i t y h a s n o t p r e v i o u s l y b e e n i s o l a t e d i n a l f a l f a , a l t h o u g h i t i s c o m m o n i n t h e p l a n t k i n g d . o m . O v e r t h e p a s t 1 0 y e a r s t h e W i t c h e s ' B r o o m v i r u s d i s e a s e o f a l f a l f a h a s d e v e l o p e d i n t o s e r i o u s p r o p o r t i o n s i n t h e I n t e r i o r o f B . C . - t h i s w a s f i r s t p o i n t e d o u t b y q u a d r a t r e s u l t s o b t a i n e d - i n 1 9 4 2 - 4 4 . T h e d i s e a s e i s k n o w n t o b e d i s t r i b u t e d t h r o u g h o u t t h e l o w r a i n f a l l a r e a s o f t h e p r o v i n c e w h e r e i t i s w i d e s p r e a d a l t h o u g h s p o r a d i c . T h e d i s e a s e i s s h o w n t o b e t h e s a m e d i s e a s e t h a t o c c u r s i n W a s h i n g t o n a n d i n A u s t r a l i a . W i t c h e s ' B r o o m o f A l f a l f a c a u s e s s e v e r e d w a r f i n g o f t h e a f f e c t e d p l a n t s a n d a d e c i m a t i o n o f a l f a l f a s t a n d s . D r a s t i c s h o r t e n i n g o f i n t e r n o d e s a n d r e d u c t i o n i n s i z e o f l e a v e s i s a c c o m p a n i e d "by p r o l i f e r a t i o n o f c r o w n s a n d n o d . e s . U p t o 3 0 0 0 t h i n s p i n d l y s h o o t s a r e c o m m o n l y p r o d u c e d "by a s i n g l e d i s e a s e d ' c r o w n . T y p i c a l l y , l e a v e s a r e m a r g i n a l l y c h l o r o t i c ; i n f l o r e s c e n c e s a r e r e d u c e d t o 3 - 4 f l o r e t s p e r r a c e m e ; a n d , v e r y l i t t l e , i f a n y , s e e d i s p r o d u c e d . C r o w n s a n d r o o t s a r e s y m p t o m l e s s u n t i l l a t e s t a g e s o f t h e d i s e a s e a r e r e a c h e d , w h e n t h e y s h o w s e v e r e r o t t i n g . A f f e c t e d p l a n t s g r a d u a l l y d i e i n a p e r i o d o f 3 m o n t h s t o 2 y e a r s . H o w e v e r , 2 p l a n t s w e r e s h o w n t o a p p a r e n t l y r e c o v e r f r o m t h e d i s e a s e w h e n " b r o u g h t i n t o t h e U . B . C . g r e e n h o u s e f r o m t h e N i c o l a V a l l e y . A h i s t o l o g i c a l c o m p a r i s o n o f t h e h e a l t h y a n d d i s e a s e d p l a n t s s h o w e d g u m m o s i s o f t h e x y l e m v e s s e l s , a " b r e a k d o w n a n d d e g e n e r a t i o n o f t h e c h l o r o p a s t s o f t h e a f f e c t e d l e a v e s , a n d a m e c h a n i c a l " b r e a k d o w n o f t h e p a l i s a d e l a y e r i n t h e o u t e r e d g e o f d i s e a s e d l e a v e s . S t o r a g e o f s t a r c h i n t h e c r o w n s o f p l a n t s w a s f o u n d t o "be d e p l e t e d a c c o r d i n g t o t h e s t a g e o f t h e d i s e a s e , w i t h n o s t o r a g e s t a r c h p r e s e n t i n s e v e r e l y d i s e a s e d c r o w n s . H o w e v e r , s u c r o s e w a s f o u n d t o b e p r e s e n t i n s t o r a g e i n d i s e a s e d c r o w n s b u t n o t i n h e a l t h y c r o w n s . T h e d i s e a s e w a s f o u n d t o b e e a s i l y t r a n s m i t t e d b y c r o w n g r a f t s . O u t o f 1 4 2 a t t e m p t e d g r a f t s , u n i o n o f s c i o n a n d s t o c k w a s a c h i e v e d i n 3 1 c a s e s w i t h p o s i t i v e t r a n s m i s s i o n i n 2 7 c a s e s . S e e d t r a n s m i s s i o n a n d i n o c u l a t i o n s b y e x p r e s s e d c r o w n j u i c e h a v e g i v e n n e g a t i v e r e s u l t s t o d a t e . Q u a d r a t s w e r e p l o t t e d i n 1 9 5 0 i n t h e i n t e r i o r o f B . C . t o f u r t h e r s t u d y t h e d i s e a s e . T w i n n i n g e x p e r i m e n t s w e r e s e t u p t o s t u d y n a t u r a l r e s i s t a n c e o f t h e m e m b e r s o f t h e g e n u s M e d i c a ^ o . R e s u l t s f r o m b o t h o f t h e s e s t u d i e s w i l l n o t b e r e a d y f o r p u b l i c a t i o n f o r a t l e a s t 2 m o r e y e a r s . A n u r s e r y p l o t w a s e s t a b l i s h e d a t U . B . C . a n d a r e p l i c a t e p l o t a t K a m l o o p s , B . C . O n e y e a r a f t e r p l a n t i n g t h e K a m l o o p s p l o t , s e v e r a l o f t h e a l f a l f a p l a n t s w e r e f o u n d p r o d u c i n g s y m p t o m s o f t h e d i s e a s e , a n d t o b e b a d l y d i s e a s e d i n 1 5 m o n t h s t i m e . N o n e o f t h e p l a n t s a t U . B . C . s h o w e d a n y s i g n s o f t h e d i s e a s e . F r o m a p o t t e d p l a n t y i e l d t r i a l c o n d u c t e d a t U . B . C . t h e d i s e a s e d p l a n t s w e r e s h o w n t o h a v e a s t a t i s t i c a l l y s i g n i f i c a n t r e d u c t i o n i n y i e l d s a s c o m p a r e d t o h e a l t h y p l a n t s . AG_-T0Vyj_3D GMEHTS It is with real pleasure that I take this opportunity in thanking a l l those persons who helped in any way with my research and in the preparation of this thesis. My especial thanks are extended to Dr. V.C.Brink, Associate Professor of Agronomy, University of B r i t i s h Columbia, under whose direction the research was conducted and this thesis prepared. To him, I am indebted for much of the data included in this paper. . His ready and helpful advice, his sympathetic understanding of the other person, and his many kindnesses to my wife, my son and me, have contributed greatly to making the two years spent in graduate studies at the University of B r i t i s h Columbia an informative and happy period. My thanks are also extended to Dr. G.G.Moe, Professor and Head, Department of Agronomy, University of B r i t i s h Columbia, for the information that he kindly placed at my disposal regarding Rhizoma al fa l fa . An expression of sincere appreciation is also extended to Dr. H.S.Wright and Dr. JTitzpatrick of the Dominion Plant Pathology Laboratory, University of Br i t i sh C o l u m b i a , t o the former for his help in grafting studies, f i e l d observations and helpful advice on virus research; and to the latter for generously making available books, periodicals and laborat- ory equipment. I would also l i ke to thank the members of the Dominion Experiment Range Station at Kamioops, especially Mr. _ . Wi l l i s and Mr. A. McLean, for providing the space for a nursery,, and for their observations and maintenance of this plot . My thanks also go to Dr. J.D.Menzies in Washington for providing a l fa l fa stocks containing some measure of resist- ance to Witches' Broom disease for inclusion in our f i e l d t r i a l s ; to Mr. G-. Setterf ield of the Department of Botany, University of-B.C. for results of a micro chemical test oh diseased alfalfa ; to Dr. B.T.Dickson and associates of the Divis ion of Botany, Australian Dep't of Agriculture for information on the Australian Witches' Broom disease; to Mr. A. Richman, Dep't. of Horticulture, U.B.C. for his advice on, and help i n , greenhouse practices. Last, but by no means least, I would l ike to tender my sincere thanks to my wife for her wi l l ing help in plot work, greenhouse work,, and for her unwavering moral support in my undertaking of graduate studies. TABLE OF CONTENTS INTRODUCTION 1 A * THE SELECTION OF MALE/STERILE LINES IN ALFALFA 2 INTRODUCTION 2 REVIEW OF LITERATURE 7 A - Some General Remarks on S t e r i l i t y and Incompatibility i n plants 7 (a) Defining s t e r i l i t y and incompatibility 7 (b) Causes of s t e r i l i t y 9 (c) Incompatibility 12 B - M a l e - s t e r i l i t y i n the higher plants 17 (a) Some general remarks 17 (b) M a l e - s t e r i l i t y governed by one or a few recessive Mendelian factors 18 (c) M a l e - s t e r i l i t y governed by a single dominant gene 24 (d) M a l e - s t e r i l i t y governed by cytoplasmic factors i n addition to Mendelian factors 24 (•) M a l e - s t e r i l i t y reported, but mode of inheritance unknown at present 29 C - Observations r e l a t i v e to possible male- s t e r i l i t y i n a l f a l f a 31 (a) S e l f - versus C r o s s - f e r t i l i z a t i o n i n a l f a l f a 31 (b) Is t r i p p i n g necessary for a l f a l f a seed production? 32 STUDIES IN FIELD AND LABORATORY A - Materials and Methods (a) Pollen studies 36 (b) S e l f - p o l l i n a t i o n studies 39 (c) Open-pollination studies 40 B - Observations 41 C - Discussion 51 SUMMARY 56 LITERATURE CITED 57 B - THE WITCHES' BROOM VIRUS DISEASE OF ALFALFA IN B.C. INTRODUCTION 64 LITERATURE REVIEW 65 A - History and Di s t r i b u t i o n 65 B - Nature of the losses due to Witches' Broom Disease of A l f a l f a 70 C - Other a l f a l f a viruses and virus diseases 71 1. Common A l f a l f a Mosaic 72 2. A l f a l f a Mosaic 73 3. A l f a l f a Dwarf Disease 74 D - Other A l f a l f a Diseases Resembling Witches' Broom 78 WITCHES' BROOM OF ALFALFA SYMPT0M0LOGY 79 INVESTIGATIONAL WORK 83 I - Transmission Experiments A • Grafting studies (a) Literature review 83 (b) Experiments 85 B - Mechanical Inoculation Studies (a) Literature 89 (b) Experiments 90 C - Seed transmission of Witches' Broom of A l f a l f a 91 D - Plant m u l t i p l i c a t i o n through cuttings 92 E - Insect transmission studies ( l i t e r a t u r e only)93 II - Y i e l d T r i a l (a) Literature 96 (b) Experiment 96 III-» F i e l d Studies of the Witches' Broom disease of a l f a l f a A - Quadrat studies 1. Introduction lO0t 2. Study of quadrat data taken by Dr. V. C. Brink i n 1942-44. 101 3. Quadrat studies i n i t i a t e d i n 1950 i n the i n t e r i o r of B.C. 102* B - D i s t r i b u t i o n of A l f a l f a Witches' Broom i n B r i t i s h Columbia 104 C - Notes on a l f a l f a grown at the Dominion nange Station, Kamloops, B.C. on Sept. 17, 1951. 106 IV - H i s t o l o g i c a l studies of Witches' Broom diseased a l f a l f a (a) Literature review 108 (b) Experimental studies 1. Comparison of the c e l l u l a r structure of healthy a l f a l f a with a l f a l f a affected Witches' Broom disease a) Materials and Mebhods 110 b) Observations 111 2, Comparison of food reserves of healthy and diseased plants. 112 DISCUSSION 114 SUMMARY 1*7 120 LITERATURE CITED 1A# ^ A - THE SELECTION OF ILALE-STERILE LUES III ALFALFA. B - THE WITCHES1 BROOM VIRUS DISEASE OF ALFALFA IN BRITISH COLUMBIA. INTR QDUC TION An a l f a l f a breeding program has been c a r r i e d on at the University of B r i t i s h Columbia since 1918 and has constituted a major a c t i v i t y of the Department of Agronomy. Work had pro- gressed to such a point that i n 1949 a new v a r i e t y of a l f a l f a designated as Ehizoma (Iledicago f a l c a t a x M. sativa) was registered i n Canada. An a l f a l f a breeding program should be continuing. Although rated "by Horrison ( 6 4 ) as the " i d e a l " hay crop, an a l f a l f a suitable for a l l agronomic purposes has yet to be developed. New diseases and problems that can be co n t r o l l e d only through breeding methods are continually presenting themselves. There i s also i n a l f a l f a much vigour attributable to hybridit y (heterosis) y e t to be tapped. One aspect i n breeding a l f a l f a which s e e u s worthy of special attention at t h i s time i s the p o s s i b i l i t y of u t i l i z i n g heterosis or hybrid vigour. Harked heterosis i s exhibited by certain plants r e s u l t i n g from c r o s s - f e r t i l i z a t i o n . Now, a necessary factor i n u t i l i z i n g the heterosis i n a p r a c t i c a l way by agronomists involves c e r t a i n p o l l i n a t i o n controls. In a l f a l f a , which possesses perfect flowers, containing both male and female parts, t h i 3 necessitates the rendering of the 1 -2- male parts of the female parent plant nonfunctional. However, hand-emasculation of the male parts of the very small flowers, a very slow, tedious and expensive process, i s not practicable for large scale operations. One way i n which, t h i s obstacle can "be overcome i s through the use of male-sterile, female- f e r t i l e plants. In the hope that such plants could be found and u t i l i z e d , an i n v e s t i g a t i o n was i n i t i a t e d to determine their presence i n our B.C. a l f a l f a stocks. The f i r s t section of t h i s d i s s e r t a t i o n then i s a report upon our search f o r , and study of, male-sterile l i n e s . (Since two growing seasons only have been available f o r t h i s study, and, since only one seed set has been obtained as a r e s u l t , a report on the inheritance of the putative m a l e - s t e r i l i t y i n c e r t a i n l i n e s cannot yet be made.) While si m i l a r investigations are being taken by other interested i n s t i t u t i o n s ( i n Nebraska, Saskatchewan, Sweden, among others), a l f a l f a i s such an important crop i n B r i t i s h Columbia that an attempt to produce hybrid a l f a l f a would be worthwhile here. Although production i n B r i t i s h Columbia i s not extensive, a l f a l f a i s nevertheless this province's most important forage crop. This crop i s grown i n a l l parts of the province and i s the p r i n c i p a l hay species. While t o t a l acreage figures, although available are inaccurate, an est- imate of i t s importance to the p r o v i n c i a l economy can be determined by the following f a c t s . A l f a l f a i s the prime source of winter hay for the range l i v e s t o c k industry which has an annual production value of $19,000,000. One and one- -3' half tons of hay are required for each animal unit on the range each winter. In addition, seed production i s an important industry i n the province, p r i n c i p a l l y i n the leace River area and, to a minor extent, i n parts of the Okanagon and Upper Eraser V a l l e y s . There was an estimated seed acreage in 1950 i n B.C. of 8,985 acres with a production of 363,000 l b s . clean seed, 700,000 l b s . of seed was produced i n 1948 and 220,000 l b s . i n 1949. (2) A second section of th i s d i s s e r t a t i o n concerns a disease, f i r s t p o s i t i v e l y i d e n t i f i e d i n B.C. by Brink (17) i n 1942, which i s now becoming rather destructive to a l f a l f a i n parts of the province. The disease, 'Witches' Broom of A l f a l f a , i s Attributed by Kenzies (63) to a virus pathogen. Very few additional f a c t s , however, respecting the disease are known. Its seriousness, geographical d i s t r i b u t i o n , vectors, or rate of spread are unknown. I t i s not known, furthermore, whether any natural resistance occurs i n a l f a l f a s (Me die ago spp.). To obtain much information i n respect to these features w i l l require several years of research. Some hope that t h i s dis- ease might be co n t r o l l e d by plant breeding methods i s offered by the recent production (49) of an a l f a l f a r e s i s t a n t to another virus disease ("Alfalf a Dwarf") i n C a l i f o r n i a . 14 years of research, with ample support, were required to produce this variety. Accordingly, our studies into A l f a l f a Witches' Broom disease i n B.C. must be of a preliminary nature. -4 A - TIL. SELECTION Off HALS-STERILE LINES IN ALFALFA. IIIlliGDUGIION S t r i k i n g advances were obtained i n corn breeding by the development of hybrid s t r a i n s . Inbred l i n e s , developed at considerable cost, were l a t e r combined i n suitable outcrosses to produce hybrids superior i n y i e l d , disease and insect resistance, and uniformity. Advances were so s t r i k i n g that in the U.S.A. alone 90^ of the corn acreage i s now of hybrid sorts. Acreage has been r e t i r e d f o r soybeans and other crops, and yet national corn y i e l d s have increased 25$. Uniformity i s so marked i n hybrid corn that mechanical harvesting techniques are now eminently successful. A l f a l f a breeders, i t i s not surprising, therefore viewing the progress i n hybrid corn breeding have wondered i f similar successes might not/\realized i n a l f a l f a breeding. (1) Like corn, a l f a l f a i s heterozygous and open-pollinated, and marked heterosis i s demonstrated i n hybrids. (2) In addition, a l f a l f a , a perennial, can r e a d i l y be propagated vegetatively, and as such the maintenance and retention of a given genotype i s e a s i l y accomplished. (3) Unlike corn, which i s monecious, a l f a l f a flowers are small, hermaphroditic, and gathered i n tight racemes. (4) Unlike corn, a l f a l f a i s insect p o l l i n a t e d . (5) Unlike corn, i n a l f a l f a only a few seeds are produced per p o l l i n a t i o n . (6) A l f a l f a i s polyploidy i n nature which complicates inheritance, and melees transfer of characters d i f f i c u l t . In addition, there i s probably cytoplasmic influences. The fact that only a few seeds are produced from a single poll ination rules out the production of hybrid a l fa l fa by physical means. As in other plants of similar nature where the advantage of a hybrid program is being investigated (flax, tomatoes, onions, e tc . ) , considerable interest has been drawn to the poss ib i l i ty of avoiding the morphological d i f f i cu l t ie s inherent in the flower structure by the selection of male-sterile, but female-fertile, l ines which are capable of setting seed but not of producing pollen. Such plants, i f of agronomic value, would serve as female parents for seed produced by natural crossing in an isolat ion bloclc. The obvious necessity of simple inheritance of male- s t e r i l i t y becomes apparent i f we want to transmit this char- acter from one l ine to another. Hope for the existance of such l ines has been indicated by Tysdal and Kiesselbach (91), who in 1944 stated "male-steri l i ty, as such, has not been identif ied in a l fa l fa , but i t s possible occurrence and use should not be ignored", and by Armstrong and White (3) in 1935, Tome (89) in 1947 in Argentina, and Clarice and Pryer (27) among other workers. In our work, attention was directed to both male and female s ter i le l ines but the emphasis was directed to male-s ter i l i ty . Kany d i f f i cu l t i e s are inherent in a l fa l fa in working out the inheritance of male-ster i l i ty , or of any character. The common al fa l fa is shown by Pryer (44a), Ledingham (57), Hilan (65), Julen (54), Hilsson and Andersson (66) to "be a polyploid and as such the inheritance i s rarely siiaple. As Brink, Jones and Albrecht (16) pointed out in discussing Bacterial Wilt disease of a l fa l fa , a factor ia l interpretation of the inheritance of disease resistance is at present impossible. Duplicate genes are often involved in inheritance which w i l l obviously add to the d i f f i cu l t i e s . Atwood (5) wrote that a l fa l fa is highly heterozygous and as such i s using hybrid vigor naturally to a considerable extent. Inbreeding is a very costly procedure in al fal fa , requiring a great amount of tedious and slow hand labour, and the plants soon become weak and s ter i l e , and as such outcrossing may result in very l i t t l e , i f any, seed set. For this reason, and the foregoing ones, a program cannot be translated from corn to a l fa l fa . However, as Tysdal and Kiesselbach (91) pointed out in 1944 in a crop such as a l fal fa , which is perennial and easily propagated by clones, a given genotype can be maint- ained indefinitely, thus eliminating the need of self-fert- i l i z a t ion for i t s continuance. In the past few years, prompted to a large extent by Tysdal and associates work (91) ( 9 3 ) , attention has become directed to a general assessment of combining ab i l i ty . This is evidenced by the general theme of such papers as Tome's (89) discussion of a l fa l fa breeding in Argentina, Bolton's (10), and Hayes' (48). Due to the foregoing reasons, and especially due to the d i f f i cu l t ie s inherent in polyploidy, breeders on this 7- continent have largely given up the idea of the production of hybrid a l fa l fa . The lack of l ines showing complete male- s t e r i l i t y has been responsible as well for the trend away from hybrid seed production. However, studies at the University of B .C . are being conducted with the real i sat ion that the application of the work may b e some time away. It is f e l t that there is a pos- sible use of hybrid vigor even though there are many d i f f i c - u l t i e s . In some respects a l fa l fa lends i t s e l f to a "hybrid" program: i t propagates readily by vegetative means. It i s fe l t that in a few years a large proportion of a l fa l fa seed producing f ields w i l l D e propagated by cuttings, and as such the interplanting of rows of male-sterile plants with male- fer t i l e plc?nts with a selective harvest could become a method of production of hybrid seed. REVIEW OF LITERATURE A. Some general remarks on s t e r i l i t y and incompatibility in higher plants. (a) Defining s t e r i l i t y and incompatibility. S t e r i l i t y is not easily defined as i t may take several forms. East (38) in writing of s t e r i l i t y seems to incliide any phenomenon that results in reduced or no seed set, and considers male-ster i l i ty to be a special case of " s e l f - s t e r i l i t y " . Armstrong and White (3) write of pollen s t e r i l i t y in a l fa l fa as "a very definite factor in seed- production - both in determining the pod-setting and in the number of seeds per pod". (Incompatibility?). Crane and Lawrence (33) draw a d i s t i n c t i o n between incompatibility and s t e r i l i t y , confining the terms 1 s e l f - s t e r i l e ' and 1cross- s t e r i l e ' to s t e r i l i t y which arises from defective pollen and ovules and other aberrations. They would r e s t r i c t the term 'incompatibility' to the f a i l u r e of the pollen to grow down the s t y l e and e f f e c t f e r t i l i z a t i o n due to some ph y s i o l o g i c a l hindrance. They point out that although s e l f - i n c o m p a t i b i l i t y i s frequently r e f e r r e d to as s e l f - s t e r i l i t y and cross-incompat- i b i l i t y as c r o s s - s t e r i l i t y the terms are misnomers as i n incompatibility the pol l e n and ovules are functional at l e a s t on c e r t a i n plants. Beadle (7) divides s t e r i l i t y into two types: 'impotence* - due to morphological or physiological derangement of the sexual organs r e s u l t i n g i n an i n a b i l i t y to produce viable germ c e l l s ; and, ' incompatibility' wherein normal gametes are formed, but f e r t i l i z a t i o n may be impossible i n c e r t a i n matings while i n other matings are normally pro- ductive of offspring. R i l e y (78) i n 1932 admits that the terminology employed by various investigators i n t h i s f i e l d has not "been consistent. He writes that the term ' s e l f - s t e r i l i t y ' a much older term than ' s e l f - i n c o m p a t i b i l i t y ' or ' self-para- s t e r i l i t y ' , i s applied to the i n a b i l i t y of an hermaphrodite with functional gametes to s e l f - f e r t i l i z e when there are no mechanical b a r r i e r s to prevent s e l f - p o l l i n a t i o n . He thinks that there should be no confusion between the use of ' s e l f - s t e r i l i t y ' and true ' s t e r i l i t y ' as the p r e f i x ' s e l f q u a l i f i e s the expression and d i f f e r e n t i a t e s between the two. j?or the purposes of t h i s essay, 1 w i l l r e s t r i c t the term ' s t e r i l i t y ' to denote those cases where pollen (or female gametophyte) i s incapable of f e r t i l i z i n g (or being fert i l ized) in any case; and the term "incompatibility" to denote those cases where f e r t i l i z a t ion is prevented by genetical differences between normal functional pollen and normal functional ovules and styles, (b) Causes of s t e r i l i t y S t e r i l i t y in plants, as has previously been noted, may stem from a variety of causes and i t i s not surprising that many classi f ications have been proposed to aid in i t s description. S t e r i l i ty is c lass i f ied by Crane and Lawrence (33) into a) "Generational s t e r i l i t y , due to the fai lure of any of the processes concerned with the normal alternation of generations, namely, development of pollen, embryo-sac, embryo, and endo- sperm, and the relations of these to one another and their parents, regardless of the cross made, and b) morphological s t e r i l i t y due to suppression or abortion of the sex organs". Dohbzans_y (36), on the other hand, classes s t e r i l i t y into genie and chromosomal s t e r i l i t y , within species and within species hybrids. He defines "genie s t e r i l i t y " as s t e r i l i t y due to changes in the individual genes of the organism, and not due to diss imi lar i t ies in the gross structure of the chromosomes; and, "chromosomal s t e r i l i t y " due to structural diss imilar i t ies of the chromosome bivalents. Genie s t e r i l i t y in his sense rarely involves any interference'with the chromosomal mechanism as shown by the fact that meiotic chromosome pairing and subsequent divisions may occur before any disturbances leading to s t e r i l i t y are noted. Some genie s t e r i l i t y m a y , h o w e v e r , i n v o l v e f a i l u r e o f p a i r i n g o f h o m o - l o g o u s c h r o m o s o m e s ( r e f . " s y n a p t i c " g e n e m u t a t i o n i n c o r n ) . C h r o m o s o m a l s t e r i l i t y , i n D o b b z a n s k y * s ( i b i d ) u s a g e , h a s a t i t s b a s e , t r a n s l o c a t i o n s , i n v e r s i o n s , d e l e t i o n s , a n d o t h e r g r o s s m o r p h o l o g i c a l c h a n g e s i n t h e c h r o m o s o m e s . A n o t h e r m e t h o d o f c l a s s i f y i n g s t e r i l i t y , a c c o r d i n g t o D o b z h a n s k y ( i b i d ) , ( a t t r i b u t a b l e t o F e & e r l y a n d i i e n n e r e n d t o I l u n t z i n g ) , d i v i d e s s t e r i l i t y i n t o g a m e t i c ( h a p l o n t i c ) a n d z y g o t i c ( d i p l o n t i c ) . T h e f o r m e r r e f e r s t o s t e r i l i t y o f p o l l e n o r f e m a l e g a m e t o - p h y t e ( o r f o r a l l p r a c t i c a l p u r p o s e s , t h e u n f e r t i l i z e d e g g ) a n d t h e l a t t e r t o f a i l u r e o f t h e z y g o t e t o g r o w a f t e r f e r t - i l i z a t i o n h a s b e e n a c c o m p l i s h e d . W h a t e v e r t h e c l a s s i f i c a t i o n o f t h e c a u s e s o f s t e r i l i t y , m a n y p h e n o m e n a m a y b e r e s p o n s i b l e f o r t h e s t e r i l i t y o f a n o r g a n i s m . D e a l i n g f i r s t w i t h t h e m a l e p a r t s o f a f l o w e r , b r e a k d o w n s r e s u l t i n g i n s t e r i l i t y m a y o c c u r a t a n y s t a g e o f t h e l i f e h i s t o r y . A n t h e r s m a y b e r e p l a c e d b y p e t a l s t h u s r e n d e r i n g t h e p l a n t m a l e - s t e r i l e ( v e r y c o m m o n i n h o r t i c u l t u r a l f l o w e r s , s u c h a s t h e d o u b l e g e r a n i u m , o r t h e c h r y s a n t h e m u m ) . F a i l u r e o f t h e a n t h e r s t o d e v e l o p w h a t s o e v e r m a y o c c u r ( 7 3 ) ( 8 8 ) ( 9 0 ) ; ( I t i s p r o b a b l e t h a t m a n y p l a n t s r e c o r d e d a s g y n o - d i o e c i o u s , a s f o r e x a m p l e m a n y L a b i a t e , a r e i n e f f e c t s p e c i e s i n w h i c h a c e r t a i n p o p u l a t i o n o f i n d i v i d u a l s s h o w m a l e - s t e r - i l i t y o f t h i s t y p e ) . A n t h e r s m a y d e v e l o p p a r t i a l l y a n d t h e n a b o r t e n t i r e l y ( 8 2 ) ( 8 3 ) . A n t h e r s m a y d e v e l o p b u t p r o d u c e n o p o l l e n t h r o u g h f a i l u r e s o f r e d u c t i o n - d i v i s i o n ( 4 ) ( 7 ) ( 9 ) ( 4 0 ) ( 4 2 ) ( 4 3 ) ( 4 4 ) ( 4 5 ) ( 7 2 ) ( 7 3 ) ; o r f a i l u r e s i n m e i o t i c a c t i v i t y d u e t o s t i c k y g e n e s ( 4 2 , 4 3 ) , a s y n a p t i c g e n e s ( 7 3 ) , c h r o m o - -11- somal rearrangements (43). Pollen grains may Toe affected "before the thickening of the wall , or at the beginning of thickening, or after thickening but before the f i r s t d ivis ion. Part ia l or complete s t e r i l i t y may be caused by translocations, inversions, deletions, duplications etc. The production of sex cel l s carrying abnormal gene complements in individuals heterozygous for various chromosomal aberrations leads to a certain degree of s t e r i l i t y . ITon-dis junction of chromosomes may lead to part ia l s t e r i l i t y . Another i rregular i ty in meiosis may arise from allopoly- ploidy: mechanical d i f f i cu l t i e s may be set up to pairing of chromosomes due to excess of "choice" of partners. Although anthers may develop and pollen be produced, the anthers may f a i l to dehisce and thus the plant may remain male-sterile (4, 9, 81). Pollen may be produced but due to some innate characteristic i t w i l l not germinate (59). (Dwyer (37) in 1932 in an extensive test i i i Austral ia found that only 80/(> of normal pollen germinated). Pollen may not germinate on stigmas i f ^proper moisture conditions are not met (62). Pollen may be fu l ly viable but not be able to germinate due to the stigmatic membrane being too well developed (3) or unruptured (60). The stigmatic membrane may be ruptured but the pollen-tube may f a i l to grow down the style (38, 39) j or, i f grown down the style f a i l to reach the ovary (38, 39, 3, 15, 31, 50, 60 etc); or bypass the ovary (31), or f a i l to f e r t i l i z e any ovules (31) or f a i l to f e r t i l i z e the basal ovules of the ovary (31, 13) result ing in part ia l s t e r i l i t y . Hie ovules themselves may be nnn-f unction al and even i f the p o l l e n - t u b e r e a c h e s t h e o v u l e , f e r t i l i z a t i o n c a n n o t b e e f f e c t e d . I l a n y o f t h e i r r e g u l a r i t i e s o f m e i o s i s a f f e c t i n g t h e m a l e - g a m e t o p h y t e m a y a l s o a f f e c t , b u t n o t n e c e s s a r i l y , t h e f e m a l e g a m e t o p h y t e d e v e l o p m e n t . O v u l e s m a y r e m a i n i n f e r t i l e e v e n t h o u g h t h e p o l l e n - t u b e s a r e p r e s e n t a n d t h e g e n e r a t i v e n u c l e u s o f t h e p o l l e n t u b e a n d t h e e g g c e l l o f t h e o v u l e a x e i n c o n j u g a t i o n ( 3 6 ) . E v e n i f f e r t i l i z a t i o n i s e f f e c t e d t h e o v u l e s m a y a b o r t . C o l l a p s e o f f e r t i l i z e d o v u l e s , o r s o m a t o p l a s t i c s t e r i l i t y , m a y o c c u r f o l l o w i n g a b n o r m a l g r o w t h o f t h e s o m a t i c t i s s u e s a d j a c e n t t o t h e e m b r y o s a c ( 1 2 , 1 3 , 1 4 ) . . ' i l l o f t h e f o r e g o i n g m a y o c c u r i n t h e o r d i n a r y d i p l o i d 211 p l a n t s . I n t r i p l o i d s , p e n t a p l o i d s o r o t h e r u n b a l a n c e d p o l y p l o i d s t h e p i c t u r e i s s o m e w n a t m o r e c o m p l i c a t e d . S t e r i l i t y m a y b e d u e t o u n b a l a n c e i n c h r o m o s o m e c o m p l e m e n t s a s a n o d d n u m b e r c a n n o t d i v i d e e v e n l y . ( c ) I n c o m p a t i b i l i t y . T h e r e h a s b e e n a g r e a t a m o u n t o f l i t e r a t u r e p u b l i s h e d i n r e c e n t y e a r s o n i n c o m p a t i b i l i t y i n p l a n t s , m u c h o f w h i c h h a s b e e n d i s c u s s e d u n d e r t h e h e a d i n g o f s t e r i l i t y . T h e f o l l o w i n g r e v i e w h i g h l i g h t s s o m e o f t h e n o t a b l e l i t e r a t u r e . J o n e s ( 5 0 ) c r e d i t s K o l r e u t e r w i t h r e i ^ o r t i n g t h e f i r s t c a s e o f i n c o m p a t i b i l i t y i n 1 7 6 4 i n V e r b a s c u m . S i n c e t h a t t i m e m a n y i n s t a n c e s h a v e b e e n r e c o r d e d , J o n e s ( i b i d ) i n 1 9 2 8 e s t i m a t e d t h a t a b o u t 1 0 0 d i f f e r e n t s p e c i e s s c a t t e r e d a m o n g 5 0 p l a n t f a m i l i e s s h o w i n c o m p a t i b i l i t y . C r a n e a n d L a w r e n c e ( 3 4 ) i n 1 9 3 4 s t a t e t h a t s e x u a l i n c o m p a t i b i l i t y i s w i d e - s p r e a d -13- throughout the plant kingdom; a view substantiated by many authors. The best known, and most satisfactory explanation of incompatibility was presented by East (39) with his opposit- ional-factor hypothesis. By this postulation, incompatibility is determined by genes usually designated by the letter 3 and commonly forming a multiple allelomorphic series: S i , S2, . . . . . . . . S i 5 , any 2 of which may be carried by a given plant. Pollen cannot function, or shows very l i t t l e growth in the style of a plant carrying the same incompatibility factors as the pollen; and normal pollen tube growth occurs in stylar tissue carrying a different genetic factor for self-incompat- i b i l i t y . There was also discovered by East (ibid) a self- f e r t i l i t y factor, 3f, functional with any of the above series and dominant in crosses. Ri ley (78) in 1932, working with the genus Capsella at Princeton University found that the inheritance of self- s t e r i l i t y in C. grandiflora could not be explained by the theory of oppositional factors. This species is completely se l f - s ter i le , and had remained so through 18 generations. The 3?̂  from a cross between 3 other se l f - fer t i l e species and the se l f-s ter i le G. grandiflora were a l l f e r t i l e ; the j?2 sp l i t into 3 se l f - fer t i les to one se l f - s ter i le . 1/3 of the self- fer t i l e s breed true to s e l f - f e r t i l i t y ; while the other 2/3 spl i t into a 3:1 rat io of fer t i l e s to s ter i les . A l l the sel f-s ter i le segregates of any ̂ 2 family are cross-sterile among themselves. Ri ley (ibid) in 1932 attributed self- -14- s t e r i l i t y to a gene simply recessive to s e l f - f e r t i l i t y . In later papers (79, 80) in 1934 and 1936, he explained this s e l f - s t e r i l i ty on the basis of the sporophytic nature of the parent plants and on the interaction of two pairs of genes, a dominant gene T which is epistatic to S c and their recessive homologies. The incompatibility reaction can be modified by several factors. Crane and Lawrence (34) state that the incompatibility reaction is greatly affected within a species by temperature. Higher temperature increases the growth rate of compatible tubes whereas i t decreases the growth rate of incompatible tubes. This shows that the incompatibility reaction is due to a positive chemical reaction between the pollen and the style, and not merely due to a lack of some growth substance. Self- pollinations of flowers late in the season of growth and also of flowers in the bud stage in certain incompatible forms of Hicotiania has resulted in some seed set. Such seeds give rise to plants either of the same genetic constitution as the parent or homozygous plants. The former f a i l reciprocally with the parent. The latter are effective when used as females, but f a i l when used as males in pollinations with the parent. This phenomenon is known as "pseudo-fertil ity" or"end-season" f e r t i l i t y . Crane and Lawrence (ibid) further point out that the incompatibility reaction can be modified by allopolyploidy. In tetraploids of pear, Petunia, and Oenothera a breakdown of incompatibility ha3 been found to be due to the competitive i n t e r a c t i o n o f t w o d i f f e r e n t 3 a l l e l e s i n t h e s a m e p o l l e n g r a i n . 3 _ S 2 p o l l e n i s c o m p a t i b l e , w h i l e 3 _ 3 _ a n d o g S g p o l l e n i s i n c o m p a t i b l e i n a 3 1 S 1 S 2 S 2 s t y l e . H o w e v e r t h i s m a y b r e a k d o w n i f o n e a l l e l e i s d o m i n a n t o v e r t h e o t h e r . B r i n k a n d C o o p e r h a v e p u b l i s h e d a t l e a s t 4 p a p e r s ( 1 2 , 1 3 , 1 4 , 3 1 ) d e a l i n g w i t h s e l f - i n c o m p a t i b i l i t y a n d s o m a t o - p l a s t i c s t e r i l i t y i n a l f a l f a a n d o t h e r p l a n t s . T h e y r e p o r t t h a t s e l f - i n c o m p a t i b i l i t y i n a l f a l f a i s o n l y p a r t i a l a n d v a r i e s c o n s i d e r a b l y f r o m p l a n t t o p l a n t . T h e y a r e o f t h e o p i n i o n t h a t o v u l e a b o r t i o n o r s o m a t o p l a s t i c s t e r i l i t y m a y b e u n r e l a t e d t o s e l f - i n c o m p a t i b i l i t y a n d m a y v a r y i n d e p e n d e n t l y o f i t . F o l l o w i n g s e l f - f e r t i l i z a t i o n t h e y f o u n d t h a t o n e o f t h r e e t h i n g s m a y h a p p e n : p o l l e n t u b e s f r e q u e n t l y f a i l t o r e a c h t h e b a s a l o v u l e s , m a n y o v u l e s r e m a i n i n f e r t i l e e v e n t h o u g h p o l l e n t u b e s a r e p r e s e n t , a n d t h e a b o r t i o n o f f e r t i l e o v u l e s a r e o f c o m m o n o c c u r r e n c e . C r o s s i n g w a s f o u n d t o r a i s e t h e p r o p o r t i o n o f o v u l e s b e c o m i n g f e r t i l e f r o m 1 5 / £ t o 65 ; a n d t o r e d u c e t h e a b o r t i o n o f f e r t i l e o v u l e s f r o m 34,.j t o 7£» . T h e h i g h e r o v u l e f e r t i l i t y r e s u l t s f r o m m o r e e x t e n s i v e g r o v t h o f t h e p o l l e n t u b e s w i t h i n t h e c a v i t y o f t h e o v a r y a n d a n i n c r e a s e d t e n d e n c y f o r p o l l e n t u b e s w h i c h r e a c h t h e o v u l e s t o e n t e r t h e m i c r o p y l e s a n d a c c o m p l i s h f e r t i l i z a t i o n . T h e r e a p p e a r s t o b e n o c o r r e l a t i o n b e t v / e e n t h e p e r c e n t a g e o f t h e o v u l e s b e c o m i n g f e r t i l e a f t e r s e l f - f e r t i l i s a t i o n a n d t h e f r e q u e n c y o f f e r t i l e o v u l e s c o l l a p s i n g i n t h e s a m e i n d i v i d u a l . C o o p e r ( 3 0 ) d e s c r i b e d t h e n o r m a l e m b r y o l o g y o f a l f a l f a . A n o v a r y o f a l f a l f a c o n t a i n s 1 0 - 1 2 o v u l e s a r r a n g e d a l t e r n a t e l y - 1 6 - a l o n g t h e v e n t r a l s u t u r e i n s e r i a l o r d e r . T h e n e c e s s a r y c o n d i t i o n f o r the d e v e l o p m e n t o f the s e e d i s t h a t f e r t i l i z a t i o n o f t h e e g g w i t h i n the o v u l e o c c u r s . A l a r g e d i s p a r i t y i n t h e n u m b e r o f s e e d w h i c h d e v e l o p a f t e r s e l f a n d c r o s s - f e r t i l i z a t i o n i s c a u s e d b y t h e d i f f e r e n c e i n t h e p r o p o r t i o n o f o v u l e s t h a t b e c o m e f e r t i l e f o l l o w i n g t h e 2 t y p e s o f m a t i n g s . C o o p e r a n d A l b r e c h t ( 3 2 ) s t u d i e d , h i s t o l o g i c a l l y , a l f a l f a p i s t i l s f o l l o w i n g s e l f - a n d c r o s s - p o l l i n a t i o n a n d f o u n d a d e c l i n i n g g r a d i e n t i n f e r t i l i t y o f o v u l e s f r o m t h e a p e x t o t h e b a s e o f t h e o v a r y . I n s e l f e d s e r i e s a b o u t 1 / 3 o f t h e o v u l e s i n t h e t o p p o s i t i o n a t t h e a p e x o f t h e o v a r y b e c a m e f e r t i l e w i t h t h e f r e q u e n c y d e c l i n i n g t o z e r o t o w a r d s t h e b a s e . I n t h e c r o s s e d s e r i e s the f e r t i l i t y g r a d i e n t i s s o m e w h a t e q u a l t o t h i s , a l t h o u g h t h e p r o p o r t i o n o f o v u l e s b e c o m i n g f e r t i l e i n e a c h p o s i t i o n i s a l i t t l e h i g h e r t h a n a f t e r s e l f i n g - 85% o f t h e o v u l e s i n p o s i t i o n 1 d o w n t o a p p r o x i m a t e l y 33 ;£ a t t h e b a s e . B r i n k a n d C o o p e r ( i b i d ) a l s o d e s c r i b e d s o m a t o p l a s t i c s t e r i l i t y i n w h i c h a h i g h p e r c e n t a g e o f f e r t i l e o v u l e s f a i l t o d e v e l o p i n t o m a t u r e s e e d s : t h e o v u l e s c o l l a p s e f r e q u e n t l y d u r i n g t h e e a r l y s t a g e s o f p o s t - f e r t i l i z a t i o n p a r t i c u l a r l y a f t e r s e l f - p o l l i n a t i o n . T h i s c o l l a p s e f o l l o w s a b n o r m a l g r o w t h o f t h e s o m a t i c t i s s u e a d j a c e n t t o t h e e m b r y o s a c . S h o r t l y a f t e r f e r t i l i z a t i o n a c t i v e c e l l d i v i s i o n i s i n i t i a t e d i n t h e i n t e g u m e n t s a s w e l l a s i n t h e e n d o s p e r m m o t h e r c e l l a n d t h e z y g o t e . T h e c r i t i c a l f a c t o r f o r s u r v i v a l i n t h i s s t a g e i s t h e m a n n e r i n w h i c h t r a n s l o c a t e d f o o d i s s h a r e d b e t w e e n t h e - 1 7 - endosperm and the integument and this part i t ion depends on the rate of growth or c e l l divis ion inside and outside the embryo sac. The endosperm and embryo of hybridized ovules grows very quickly and can compete with the integuments for food supply; but the selfed ovules, growing slower, f a i l in competition and the balance shifts in favor of the integuments. Growth may continue for several hours, using breakdown of the endosperm cel ls as nutrients, before collapse. The chalazal portion of the endosperm is ruptured, the cel l s of the embryo become starved, and is followed by a collapse, with the resultant abortion of the remaining endosperm and embryo. B. Male-ster i l i ty in the higher plants. (a) Some general remarks. A litex-ature search of rdcent work on male-steri l i ty in plants brings to l ight 3 main modes of inheritance of this character. Male-s ter i l i ty may be governed by a) one or a few recessive llJendelian factors, or b) by dominant factors, and c) by cytoplasmic factors in addition to Hendelian factors. In some cases, the phenomenon is reported but the mode of inheritance is unknown at present. Cytoplasmic male-steri l i ty was reported, apparently erroneously, by Rick (75) in 1948, and by Lewis (61) in 1941, in l ight of present day findings, to be much less common in cultivated plants than genie male- s t e r i l i t y . Lev/is (ibid) writing in 1941 on male-ster i l i ty in natural populations of hermaphrodite plants, stated that male s t e r i l i t y due to a recessive gene is common as a mutant in moreu plants. Single examples are reported by Lewis (ibid) in ;\ntirrhinura. Capsella spp., Lathvrus odoratus. Lyeonersicum esculentum, Oryza sativa. and Rubus idaeus. More than 15 types of male- s t e r i l i t y were known in Zea mays at that time, a l l being due to the segregation of a single recessive gene. He reported two other cases of male-steri l i ty in maize due to a single dominant gene, but which since have been shown to be in rea l i ty due to an interaction of cytoplasmic factors and Mendelian factors. In 1931, male-steri l i ty had not been reported in wild populations of these plants, or in species naturally polymorphic for male-ster i l i ty . In a l l the gyno- dioecious species f u l l y investigated, according to Lev/is (ibid) male-steri l i ty is inherited through the cytoplasm. (b) Male-s ter i l i ty governed by one or a few recessive Mendelian factors. (If male-steri l i ty is inherited as a simple recessive, inbreeding would favor the production of the phenomenon.) Bohn and Uhitaker (9) in 1949 writing on the male- s t e r i l i t y in andromonecious muskmellon report that staminate flowers from male-sterile plants are normal in size and shape of a l l parts except the anther*. These are small, f a i l to dehisce and contain empty microspore walls. In the male- ster i le plants meiosis is apparently normal and follows the usual steps up to the formation of the tetrads; at this stage the development ceases and the contents of the young pollen grains disintegrate. The units of the tetrad remain cemented together. They conclude that in muskmellon male-steri l i ty -19- i s diplontic and is governed by a single recessive gene. Crane (33) in 1915 is credited with, the f i r s t report of male-steri l i ty in the tomato. He found that male-steri l i ty "behaved as a recessive character and "believed that i t depended on a mutant gene. Rick (74) in 1948, reported on a search conducted for male-sterile tomato mutants, potentially useful in producing _*X hybrid seed and in cross-breeding. Among 150 unfruitful plants, 12 were found to be genetically male-sterile. Breeding results indicate that the male-steri l i ty of each mutant is determined by a single recessive gene. One gene ms5 was recovered 4 times and 8 other non-al le l ic genes mss to msi3 were demonstrated. Only one mutant produced any pollen that was functional, and by using this pollen in s e l f - f e r t i l i z a t ion pure breeding male-sterile populations for this gene was obtained. Rick in a previous paper (73) in 1945, reported on a survey of cytogenetic causes of unfruitfillness in the tomato. Gametic s t e r i l i t y is largely responsible for the unfruitful- ness of 66 plants discovered in approximately 55,000 f i e l d plants of 3 tomato var iet ies . Failure of normal gamete formation was resolved to a cytogenetic basis in every case investigated. Rick (ibid) found in the 14 diploids discovered in this population that 3 plants were aberrant in gross morphology. S t e r i l i t y of these plants is probably conditioned either by a pleiotropic effect of the gene determining the deformity, or by the deficiency of a chromosomal segment which in c l u d e s a gene a f f e c t i n g the morphological character and another a f f e c t i n g s t e r i l i t y . R i c k ( i b i d ) found i n 3 other p l a n t s that the m a l e - s t e r i l i t y i s determined by a s i n g l e r e c e s s i v e gene. 5 p l a n t s showed complete p o l l e n and ovule s t e r i l i t y r e s u l t i n g i n one case from asynaptic meiosis. Roever (81) i n 1948, w r i t i n g on a promising type of m a l e - s t e r i l i t y f o r use i n h y b r i d tomato seed production, r e p o r t s a mutant i n which n a t u r a l s e l f i n g was prevented . because the anthers f a i l e d to dehisce. The mutant can be s e l f e d by hand but does not s e l f i n the f i e l d . He concludes that the character appears to behave as a simple r e c e s s i v e . L e s l e y and L e s l e y (59) working at R i v e r s i d e , C a l i f o r n i a , , r e p o r t e d i n 1939 on m a l e - s t e r i l i t y i n the tomato. They found that m a l e - s t e r i l i t y i s completely r e c e s s i v e and depends on -at l e a s t 2 r e c e s s i v e genes, msi and ms2» both of which are necessary f o r m a l e - s t e r i l i t y . M a l e - s t e r i l e p l a n t s are homozygous f o r both of these r e c e s s i v e characters .and produced non-viable p o l l e n . Segregation of s t e r i l e and v i a b l e pollen- was accomplished by means of germination t e s t s i n van Tiegliem c e l l s u s i n g 15^ cane sugar media plus, ground up stigmas. Scott and Rin e r (82) i n 1946, r e p o r t on the i n h e r i t a n c e of m a l e - s t e r i l i t y i n winter squash i n which the male flo w e r s produce no p o l l e n . Bo d i f f e r e n c e s were observed i n the s i z e or shape of the m a l e - s t e r i l e versus the normal fl o w e r , but there i s a marked d i f f e r e n c e i n the appearance of the androecium of the m a l e - s t e r i l e and normal f l o w e r s . In the m a l e - s t e r i l e f l o w e r s the androecium aborts i n the bud stage - 2 1 - b e f o r e t h e s t a m i n a / b e f l o w e r s o p e n s o t h a t no p o l l e n i s p r o d u c e d . T h e f a c t o r f o r m a l e - s t e r i l i t y i s i n h e r i t e d a s a s i m p l e r e c e s s i v e . I n the b a c k e r o s s p r o g e n i e s a 1 : 1 s e g r e g a t i o n for m a l e - s t e r i l e : m a l e - f e r t i l e f l o w e r s w a s o b s e r v e d . I n t h e F g p r o g e n i e s a 3 : 1 s e g r e g a t i o n o c c u r r e d . M a l e - s t e r i l e p l a n t s w e r e f u l l y f e m a l e - f e r t i l e , p r o d u c i n g a b u n d a n t s e e d a n d f r u i t w h e n f o r e i g n p o l l e n i s a d d e d . B e a d l e ( 7 ) r e p o r t e d i n 1 9 3 2 o n a s t u d y of 1 5 g e n e s f o r p o l l e n s t e r i l i t y in m a i z e . I n a l l c a s e s d e v e l o p m e n t , of m e g a - s p o r e s a n d f e m a l e g a m e t o p h y t e s was . . : n o r m a l . C y t o l o g i c a l l y , t h e maleriSterility w a s c h a r a c t e r i z e d b y d e g e n e r a t i o n o f t h e m i c r o s p o r o c y t e s o r o f t h e m i c r o s p o r e c e l l s . T h e t i m e o f d e g e n e r a t i o n v a r i e d w i t h t h e d i f f e r e n t m a l e - s t e r i l e s r a n g i n g f r o m t h e ^ ' s y n i z e t i e ' s t a g e s - o f m e i o s i s a l m o s t t o p o l l e n m a t u r - i t y . T h e m s g e n e s w e r e r e c e s s i v e a n d n o n - a l l e l o m o r p h i c . I n a p r e v i o u s p a p e r i n 1 9 2 1 , E r y s t e r ( 4 1 ) s t a t e s t h a t r e c e s s i v e M e n d e l i a n g e n e s a r e t h e c a u s e o f m a l e - s t e r i l i t y i n m a i z e . E m e r s o n , B e a d l e a n d F r a s e r ( 4 0 ) i n 1 9 3 5 r e p o r t e d 2 0 g e n e s h a d b e e n d e s c r i b e d t o d a t e f o r m a l e - s t e r i l i t y i n m a i z e . A l l o f t h e s e g e n e - s t e r i l e s a r e o r w e r e i n d e p e n d e n t r e c e s s i v e s , e a c h c a p a b l e o f b r i n g i n g a b o u t e i t h e r c o m p l e t e o r a h i g h d e g r e e o f s t e r i l i t y i n t h e m a l e i n f l o r e s c e n c e . M e i o s i s o c c u r s n o r m a l l y a n d t h e p o l l e n g r a i n s a r e f o r m e d b u t b e f o r e a n t h e s i s t h e s e g r a i n s a b o r t . B u r n h a m ( 2 0 ) r e p o r t i n g o n c y t o g e n e t i c s t u d i e s o f a c a s e o f p o l l e n a b o r t i o n i n m a i z e i n 1 9 4 1 f o u n d a g e n e f o r p o l l e n a b o r t i o n ( p a ) i n a n e s t a b l i s h e d i n b r e d l i n e . P l a n t s h e t e r o - -22- zygous for this gene had serai-sterile pollen but normal ears. The gene was l e tha l , or nearly so, to pollen carrying i t j but had no lethal effect on the ovules. As a result pa i s trans- mitted mainly, i f not entirely, through the ovules, and is located on the f i r s t linkage group. Shifriss (83) writing in 1945 on male-ster i l i t ies in cucurbits, found 3 l ines among 200 one-generation inbreds of Cucurbit a pepo L . which segregated for male-sterile plants. 3?̂ , ^2 ^ao^cross populations showed that the male-steri l i ty i 3 inherited as a recessive character whose expression is due to a single gene. This morphological male-steri l i ty is ex- pressed in complete abortion of the androecium at the bud stage before the staminate flowers open. Gregory (45) reported in 1905 on the abortive development of the pollen in certain sweet peas whose anthers were conta- escent. These peas were produced by se l f - f e r t i l i za t ion by Bateson. S t e r i l i t y was found to be correlated with a somatic character, the s ter i le plants possessing a green leaf ax i l , while f e r t i l e plants had red axi ls . A cytologic al invest- igation revealed tha,t the whole nucleus showed i rregular i t ies . Reduction-division followed the normal course, and young pollen grains were formed. In the contabescent anther's, growth ceases at this point, the pollen grains remain small and enclosed within the walls of the pollen mother-cells. Development of spindle fibres in the cytoplasm was weak or absent. Gregory (ibid) believed the male-steri l i ty character was inherited as a simple recessive. P u n n e t t ( 7 0 ) ( 7 1 ) , w o r k i n g w i t h G r e g o r y ' s ' ( i b i d ) p l a n t s , s h o w e d t h a t g e n e t i c a l l y t h e f e m a l e g a m e t o g e n e s i s o f t h e m a l e - s t e r i l e s w e e t p e a p l a n t s w a s n o r m a l , a n d t h a t s t e r i l i t y o f t h e p o l l e n w a s c o m p l e t e . H e a l s o d e m o n s t r a t e d t h a t t h e g e n e f o r s t e r i l e a n t h e r s w a s i n h e r i t e d a s a s i m p l e r e c e s s i v e . I n 1 9 3 7 , P a b e r g e ( 4 2 ) a t t h e J o h n I n n e s H o r t i c u l t u r a l I n s t i t u t i o n r e p o r t e d t h e r e s u l t s o f a d e t a i l e d c y t o l o g i c a l a n d g e n e t i c a l s t u d y o f m a l e - s t e r i l i t y i n L a t h y r u s o d o r a t u s . T h e r e c e s s i v e g e n e f o r m a l e - s t e r i l i t y w a s l i n k e d w i t h l i g h t a x i l c o l o r . O n a c y t o l o g i c a l b a s i s h e f o u n d t h a t t h e f o l l o w i n g o c c u r r e d ! ( 1 ) O n l y o c c a s s i o n a l l y a m e t a p h a s e p l a t e o f n o r m a l a s p e c t w a s f o r m e d ; g e n e r a l l y t h e c h r o m o s o m e s i n m e i o s i s r e m a i n e d s c a t t e r e d i n c e l l s . ' ( 2 ) V e r y f e w s e c o n d d i v i s i o n s o f m e i o s i s w e r e s e e n . O f t h o s e t h a t o c c u r r e d , m a n y h a d c e l l w a l l s e x t e n d i n g o n l y 2 / 3 o f t h e d i s t a n c e a c r o s s t h e c e l l . H e a l s o f o u n d t h a t o f t e n m o r e t h a n 4 c h a m b e r s w e r e p a r t i t i o n e d o f f w i t h s o m e c h a m b e r s b e i n g l e f t w i t h o u t a n y n u c l e a r m a t e r i a l , w h a t s o e v e r . ( 3 ) M e i o s i s m a y b e a r r e s t e d b y a c h a n g e i n t h e c o l l o i d a l s t a t e o f t h e c y t o p l a s m a s e a r l y a s d i a k e n a s i s o r a s l a , t e a s s e c o n d a n a p h a s e . T h e c y t o p l a s m b e corner., g r a n u l a r v e r y r a p i d l y a r r e s t i n g m e i o t i c a c t i v i t y . S h u l l ( 8 4 ) i n 1 9 2 7 r e p o r t e d o n f i n d i n g p o l l e n - s t e r i l i t y i n c r o s s e s b e t w e e n s p e c i e s o f s h e p h e r d ' s p u r s e . T h e m a l e - s t e r i l i t y i n t h e i n t e r s p e c i f i c G a p s e l l a h y b r i d w a s f o u n d t o b e s i m p l y r e c e s s i v e t o m a l e - f e r t i l i t y . S u n e s o n ( 8 8 ) r e p o r t e d i n 1 9 4 0 o n a 4 y e a r t e s t t h a t h e - 2 4 - p e r f o r m e d o n a m a l e - s t e r i l e m u t a n t o f b a r l e y . R e s u l t s o f 3?^ a n d _ , 2 s h o w e d t h a t t h e m u t a n t w a s m a l e - s t e r i l e ; a n d t h a t t h e m a l e - s t e r i l i t y w a s i n h e r i t e d a s a . s i m p l e r e c e s s i v e . A n t h e r s r e m a i n e d s h r u n k e n , r u d i m e n t a r y w h i l e p i s t i l l a t e p a r t s w e r e n o r m a l . (fe*) M a l e - s t e r i l i t y g o v e r n e d b y a s i n g l e d o m i n a n t g e n e . R i f e ( 7 6 ) s t u d y i n g t h e g e n e t i c s o f c e r a i n c o m m o n v a r i a t i o n s i n G o l e u s , f o u n d f o u r g e n e s a t a p p r o x i m a t e l y t h e s a m e c h r o m o s o m e l o c u s w h i c h p r o d u c e v a r y i n g c o m b i n a t i o n s o f d e e p v e r s u s s h a l l o w l o b e d l e a v e s a n d m a l e - s t e r i l e v e r s u s m a l e - f e r t i l e f l o w e r s ; b o t h d e e p l o b e s a n d m a l e - s t e r i l i t y b e i n g d o m i n a n t . I n a l a t e r p a p e r , R i f e ( 7 7 ) p o i n t s o u t t h a t m a l e - s t e r i l i t y ( p ) a n d d e e p l o b e s ( L ) a r e c l o s e l y l i n k e d a n d b o t h d o m i n a n t , ( d ) M a l e - s t e r i l i t y g o v e r n e d b y c y t o p l a s m i c f a c t o r s i n a d d i t i o n t o M e n d e l i a n f a c t o r s . T o e x p l a i n t h e o b s e r v e d d a t a o n c e r t a i n t y p e s o f m a l e - s t e r i l i t y b o t h c y t o p l a s m i c f a c t o r s a n d M e n d e l i a n f a c t o r s a r e r e q u i r e d . O w e n ( 6 7 ) s t a t e s t h a t i f a c h a r a c t e r i s t o ^ e x p l a i n e d b y M e n d e l i a n , o r g e n i e i n h e r i t a n c e t h e m a l e a n d f e m a l e p a r e n t s b o t h i n f l u e n c e t h e c h a r a c t e r i s t i c s o f t h e o f f s p r i n g , a n d p o p u l a t i o n s o b t a i n e d f r o m r e c i p r o c a l c r o s s e s ( e x c e p t i n g s e x - l i n k e d c h a r a c t e r s ) s h o u l d . b e i d e n t i c a l . I f . a c h a r a c t e r , o n t h e o t h e r h a n d , i s t o b e e x p l a i n e d b y c y t o p l a s m i c , o r m a t e r n a l i n h e r i t a n c e , , t h e m a l e p a r e n t s h o u l d h a v e n o i n f l u e n c e o n t h e c h a r a c t e r i s t i c s o f t h e o f f s p r i n g ; a n d p o p u l a t i o n s o b t a i n e d f r o m r e c i p r o c a l c r o s s e s s h o u l d n o t b e i d e n t i c a l . I f c y t o - p l a s m i c a n d g e n i e i n h e r i t a n c e a r e " b o t h i n v o l v e d , t h e r e s u l t s a r e m o r e c o m p l i c a t e d . T h e f e m a l e p a r e n t s h o u l d , i n t h i s c a s e , e x e r t m o r e i n f l u e n c e t h a n t h e m a l e p a r e n t i n t h e c h a r a c t e r i s t i c s o f t h e o f f s p r i n g , a n d p o p u l a t i o n s o b t a i n e d f r o m r e c i p r o c a l c r o s s e s s h o u l d n o t b e i d e n t i c a l . J o n e s ( 5 0 ) i n 1 9 5 0 , p r o p o s e s a s n e w t e r m i n o l o g y t h e t e r m , " c h r o m o g e n e s " t o d e s i g n a t e g e n e s i n t h e c h r o m o s o m e s , a n d t h e t e r m " p l a s m a g e n e s " t o d e s i g n a t e t h e d e t e r m i n e r s i n t h e c y t o - p l a s m . P o l l e n a b o r t i o n i s b r o u g h t a b o u t b y c h r o m o g e n e s a n d a l s o p l a s m a g e n e s , b o t h o f w h i c h a r e v a r i a b l e i n t h e i r c o n t r o l o f p o l l e n p r o d u c t i o n . P l a s m a g e n e s t h a t c o n d i t i o n p o l l e n s t e r i l i t y h a v e n o o t h e r e f f e c t u p o n t h e g r o w t h a n d s t r u c t u r a l d e t a i l s o f t h e p l a n t s . P l a s m a g e n e s a n d c h r o m o g e n e s f o r p o l l e n s t e r i l i t y , w h e n w o r k i n g t o g e t h e r i n t h e s a m e p l a n t s , a p p a r e n t l y h a v e n o e f f e c t o n e a c h o t h e r a n d a r e i n d e p e n d e n t i n t h e i r a c t i o n . R h o a d e s i n 1 9 3 1 a n d 1 9 3 3 ( 7 2 ) ( 7 3 ) s t u d y i n g m a l e - s t e r i l i t y i n Z e a m a v s c o n c l u d e d t h a t p o l l e n s t e r i l i t y i s i n h e r i t e d t h r o u g h t h e m a t e r n a l c y t o p l a s m . R h o a d e s ( i b i d ) r e p o r t e d t h a t t h e e g g c y t o p l a s m p l a y s t h e c h i e f r o l e i n t h e e x p r e s s i o n o f m a l e - s t e r i l i t y . T h e r e w a s n o t r a n s m i s s i o n o f t h e c h a r a c t e r t h r o u g h t h e p o l l e n o f p a r t i a l l y s t e r i l e p l a n t s . C y t o l o g i c a l l y , m i c r o s p o r o g e n e s i s i s n o r m a l w i t h d e g e n e r a t i o n o f p o l l e n u s u a l l y o c c u r r i n g b e f o r e t h e f i r s t v e g e t a t i v e d i v i s i o n . T h e r e i s a p r o n o u n c e d d i f f e r e n c e b e t w e e n t h e c y t o p l a s m i c e l e m e n t s o f m i c r o s p o r e s i n n o r m a l r a c e s a n d t h o s e o f t h e m a l e - s t e r i l e l i n e s . - 2 6 - J o s e p h s o n a n d J e n k i n s ( 5 3 ) r e p o r t e d i n 1 9 4 8 o n m a l e - s t e r i l i t y i n c o r n h y b r i d s . S e e k i n g a n e x p l a n a t i o n f o r s e r i o u s l y l o w s e e d s e t i n a n u m b e r o f f i e l d s o f w h i t e h y b r i d c o r n , m a l e - s t e r i l i t y w a s f o u n d t o b e t h e p r i m a r y c a u s e o f t h e d i f f i c u l t y . E x a m i n a t i o n o f t h e t a s s e l s d u r i n g p o l l e n s h e d d i n g ' t i m e r e v e a l e d t h e p r e s e n c e o f m a l e - s t e r i l e p l a n t s . H i g h s t e r i l i t y r e s u l t e d o n l y w h e n t h e h y b r i d s h a d c y t o p l a s m i c c o n t r i b u t i o n s f r o m o n e s t r a i n . S t e r i l i t y w a s a l s o f o u n d t o b e i n f l u e n c e d b y c e r t a i n c o n t r i b u t i o n s f r o m t h e m a l e p a r e n t , a m i n i m u m o f 2 g e n e s a p p a r e n t l y b e i n g r e q u i r e d . T h e a u t h o r s p o i n t o u t t h a t t h e e x p r e s s i o n o f s t e r i l i t y i s s u b j e c t t o e n v i r o n m e n t a l i n f l u e n c e . O w e n ( 6 7 ) w o r k i n g w i t h m a l e - s t e r i l i t y i n s u g a r - b e e t s i n 1 9 4 5 f o u n d t h a t s e v e r a l p a i r s o f M e n d e l i a n f a c t o r s m a y i n f l - . u e n c e p o l l e n d e v e l o p m e n t w h e n c a r r i e d b y p l a n t s w i t h s t e r i l e c y t o p l a s m , b u t s a m e f a c t o r s h a v e n o e f f e c t w h e n c a r r i e d b y p l a n t s w i t h n o r m a l c y t o p l a s m . C o m p l e t e m a l e - s t e r i l i t y w a s c h a r a c t e r i z e d b y e m p t y w h i t e a n t h e r s . O w e n c o n c l u d e d t h a t i n s u g a r - b e e t s , m a l e - s t e r i l i t y i s p r o d u c e d b y c o m b i n e d c y t o - p l a s m i c a n d g e n i e i n h e r i t a n c e . A r t s c h w a g e r ( 4 ) r e p o r t e d i n 1 9 4 7 o n t h e r e s u l t s o f a . c y t o l o g i c a l i n v e s t i g a t i o n i n t o t h e c a u s e o f p o l l e n a b o r t i o n i n O w e n ' s ( . i b i d ) p l a n t s . H e o b s e r v e d t h a t w h e n m a l e - s t e r i l i t y i n s u g a r - b e e t s i s c y t o p l a s m i c a l l y i n h e r i t e d , c o m p l e t e l y m a l e - s t e r i l e p l a n t s b e a r w h i t e , e m p t y a n t h e r s . N o r m a l p o l l e n m o t h e r c e l l s a n d n o r m a l m i c r o s p o r e s a r e p r o d u c e d , b u t t h e m i c r o s p o r e s f a i l t o d e v e l o p f u l l y a n d d i s i n t e g r a t e b y t h e t i m e t h e f l o w e r s - 2 7 - o p e n . I n t h e @ e & i - m a l e - s t e r i l e t y p e s , s m a l l , n o n - v i a b l e p o l l e n g r a i n s a r e f o r m e d h u t t h e a n t h e r s d o n o t d e h i s c e . O w e n ( i b i d ) h a d o b s e r v e d t h a t s o m e t i m e s v i a b l e p o l l e n i s p r o d u c e d b y s o m e b r a n c h e s o f t h e i n f l o r e s c e n c e , a n d o c c a s i o n - a l l y w h i t e a n t h e r s a n d y e l l o w o n e s a r e b o r n e w i t h i n t h e s a m e f l o w e r . P o l l e n a b o r t i o n , e s p e c i a l l y i n h y b r i d s o f p l a n t s w i t h - d i f f e r e n t c h r o m o s o m e n u m b e r s , u s u a l l y i s t h e r e s u l t o f a b n o r m a l m e i o t i c d i v i s i o n s ; i n s u g a r - b e e t s , h o w e v e r , A r t s c h w a g e r ( i b i d ) o b s e r v e d a d i f f e r e n t t y p e o f p o l l e n d e g e n e r a t i o n i n w h i c h t h e a n t h e r t a p e t u m , t h r o u g h t h e d e v e l o p m e n t o f a P l a s m o d i u m , p l a y s a n i m p o r t a n t r o l e . H e f o u n d t h a t p o l l e n a b o r t i o n i n a n t h e r s o f s u g a r - b e e t s w i t h c y t o p l a s m i c a l l y i n h e r i t e d m a l e - s t e r i l i t y i s a s s o c i a t e d w i t h e i t h e r a p e r i p l a s m o d i u m o r a c e l l u l a r t a p e t u m - b o t h o f w h i c h m a y o c c u r i n a f l o w e r c l u s t e r b u t n o t w i t h i n a s i n g l e f l o w e r . T h e P l a s m o d i u m ' s p r e s e n c e s o m e w h a t d e l a y s p o l l e n a b o r t i o n ; b u t w h e r e t h e t a p e t u m r e m a i n s c e l l u l a r s o m e m i c r o s p o r e s a r e d e s t r o y e d w h i l e s t i l l i n t e t r a d s . H e c o u l d , n o t o f f e r a n e x p l a n a t i o n o f t h e c a u s e o f t h e d e v e l o p m e n t o f t h e p e r i p l a s m o d i u m w h i c h i s r e s t r i c t e d t o s u g a r - b e e t s e x h i b i t i n g c y t o p l a s m i c m a l e - s t e r i l i t y . C y t o l o g y o f t h e y o u n g P l a s m o d i u m s u g g e s t s h y p e r m e t a b o l i c a c t i v i t y t o t h e d e t r i m e n t o f t h e d e v e l o p i n g m i c r o s p o r e s . Jone3 a n d C l a r k e ( 5 2 ) s t u d i e d t h e i n h e r i t a n c e o f m a l e - s t e r i l i t y i n t h e o n i o n w i t h t h e h o p e o f t h e p r o d u c t i o n o f h y b r i d s e e d . T h e m a l e - s t e r i l e o n i o n s p r o d u c e d n o v i a b l e p o l l e n b u t s e t s e e d r e a d i l y w h e n h y b r i d i z e d w i t h p o l l e n -28- from male-fertile plants. They found 3 types of inheritance which they explained by assuming that the male-3terile condition results from an interaction between a recessive gene and a non-nuclear or cytoplasmic factor , . A l l plants with normal cytoplasm (IT) produce viable seed. A l l male-sterile plants possess the s ter i le type of cytoplasm (s). Ho l ight was shed on the nature of the cytoplasmic factor which differs in the 2 types. A gene for male-steri l i ty (ms) also influences pollen development when carried by plants with S cytoplasm but has no effect when carried by plants with N cytoplasm. Clarke and Pollard (28) found in 1949 that male-steri l i ty in the onion was not complete and that the amount of self ing variew largely from one male-sterile plant to another, but the average of self ing for a l l l ines tested was 4.1%. Pineman (43) found complete pollen s t e r i l i t y in the potato to be a result of the failure of normal microspore formation during meiosis. This view is supported by numerous meiotic irregulari t ies she observed in steri le-pollen plants. The most common meiotic i rregular i t ies she observed were: (l) f i lure of the chromosomes to pair , (2) lagging of the chromosomes on the spindle and (3) fai lure to complete the normal reduction-division process. Par t ia l s t e r i l i t y of the fe r t i l e plants is conditioned, after what appears to be a normal meiosis, by microspore abortion, which frequently occurs after the spores have reached normal pollen grain size. Pineman (ibid) concluded that female parents transmit more pollen s t e r i l i t y than male parents. - 2 9 - B a t e s o n a n d G a i r d n e r ( 6 ) i n 1 9 2 1 c r o s s e d c o m m o n t a l l a n d a p r o c u m b e n t f l a x a n d f o u n d n o r m a l a n d m a l e - s t e r i l e i n d - i v i d u a l s i n a 3 : 1 r a t i o i n t h e P 2 » S t e r i l e p l a n t s w e r e o b t a i n e d o n l y w h e n t h e p r o c u m b e n t r a c e w a s u s e d a s a f e m a l e p a r e n t . T h e y a t t r i b u t e d t h e r e s u l t s t o " a n i s o g e n y " . C h i t t e n d e n a n d P e l l e w ( 2 6 ) o f f e r e d a s u g g e s t e d i n t e r - • p r e t a t i o n o f B a t e s o n a n d G a i r d n e r ' s ( i b i d ) " a n i s o g e n y " . T h e y i n t e r p r e t e d t h e s t e r i l i t y a s d u e t o t h e i n t e r a c t i o n o f a s p e c i f i c g e n e i n t h e c y t o p l a s m o f t h e p r o c u m b e n t r a c e . I n a n o t h e r p a p e r ( 2 5 ) C h i t t e n d e n i n 1 9 2 7 w r o t e t h a t a s i n g l e g e n e a p p a r e n t l y d e t e r m i n e s m a l e - s t e r i l i t y w i t h o n e t y p e o f c y t o - p l a s m ( p r o c u m b e n t ) b u t n o t w i t h a n o t h e r ( e r e c t ) . I n 1 9 3 2 , E a s t ( 3 8 ) f o u n d t h a t c e r t a i n S f a c t o r s f r o m flfcootiania s a n d e r a e p r o d u c e d m a l e - s t e r i l i t y i n t h e p r e s e n c e o f c y t o p l a s m d e r i v e d f r o m IT. l a n g s d o r f i i b u t p r o d u c e d v i a b l e p o l l e n i n t h e p r e s e n c e o f H . s a n d e r a e c y t o p l a s m . M a l e - s t e r i l e p l a n t s , p r o d u c e d n o p o l l e n a t a l l . . - H e f o u n d t h a t t h e r a t i o o f s e l f - f e r t i l e s t o m a l e - s t e r i l e s w a s 2 6 6 : 2 3 2 . e - ( J & ) M a l e - s t e r i l i t y r e p o r t e d , b u t m o d e o f i n h e r i t a n c e u n k n o w n a t p r e s e n t . W e l c h a n d G r i m b a l l ( 9 0 ) d i s c o v e r e d a m a l e - s t e r i l e b u t f e m a l e - f e r t i l e c a r r o t i n 1 9 4 7 . M a l e - s t e r i l i t y c o n s i s t e d o f a n t h e r s b e i n g s h r i v e l l e d a n d b r o w n i n c o l o r b e f o r e a n y p e t a l s u n f o l d e d . H y b r i d s e e d p r o d u c e d o n m a l e - s t e r i l e p l a n t h a d p l a n t s t h a t w e r e c l a s s i f i e d 3 7 m a l e - s t e r i l e a n d 1 5 n o r m a l . F r a n k e l ( 4 4 ) w o r k i n g i n H e w Z e a l a n d , o b s e r v e d m a l e - - 3 0 ' s t e r i l i t y w i t h f e m a l e - f e r t i l i t y i n n a t u r e i n 8 f o r m s o f t h e g e n u s H e h e , c o m p r i s i n g 5 s p e c i e s a n d 1 s p e c i e s - h y b r i d . A s H e b e o n l y f l o w e r s e v e r y 2 o r 3 y e a r s i t i s u n s u i t e d t o t h e s t u d y o f m a l e - s t e r i l i t y i n h e r i t a n c e . H o w e v e r , F r a n k e l ( i b i d ) c o n c l u d e s t h a t t h e m a l e - s t e r i l i t y i s d u e t o g e n e t i c c a u s e s a n d n o t t o e n v i r o n m e n t a l c a u s e s a s t h e p h e n o m e n o n i s r e g u l a r f r o m y e a r t o y e a r a n d i n an* n e w e n v i r o n m e n t a f t e r t r a n s p l a n t i n g . A c y t o l o g i c a l s t u d y o f t h e " b r e a k d o w n o f t h e p o l l e n s h o w e d d e g e n e r a t i o n o c c u r r e d r a p i d l y a n d w i t h r e g u l a r i t y , e i t h e r i n : ( 1 ) P a c h y t e n e o f p r o p h a s e - u p t o t h i s m e i o s i s i s n o r m a l . D i p l o t e n e l o o p s a r e n o t f o r m e d . " T h e p a c h y t e n e t h r e a d s c o - a g u l a t e i n d i v i d u a l l y , a p p r o x i m a t e l y t o t h e s i z e o f d i a k e n e s i s b i v a l e n t s , a n d c o l l a p s e i n t o a n a m o r p h o u s m a s s w h i c h i s r a p i d l y d i s s o l v e d " . S u b s e q u e n t l y t h e e m p t y c e l l w a l l s c o l l a p s e , r e m a i n i n g i n t h i s s t a t e i n t h e a n t h e r c a v i t y . T a p e t u m c e l l s d e g e n e r a t e s i m u l t a n e o u s l y . ( 2 ) T e t r a d d e g e n e r a t i o n - a l l m e i o t i c s t a g e s u p t o s e c o n d t e l o p h a s e a r e n o r m a l . T e t r a d s a r e f o r m e d b u t c o l l a p s e r a p i d l y . H o p o l l e n g r a i n s s e e n . ( 3 ) P o l l e n d e g e n e r a t i o n - a ) T e t r a d c e l l s r o u n d o f f , a n d a b o u t a d a y l a t e r , n o t h a v i n g u n d e r g o n e a c h a n g e e i t h e r i n d i a m e t e r o r i n w a l l s t r u c t u r e , t h e p o l l e n g r a i n s s h r i v e l , c o l l a p s e , , a n d g r a d u a l l y d i s i n t e g r a t e . T h e p r o c e s s i s r e g u l a r a n d r a p i d a n d o c c u r s w i t h c o n c u r r e n t d e g e n e r a t i o n o f t a p e t a l w a l l s o f t h e p o l l e n m o t h e r c e l l s . h ) P o l l e n g r a i n s f o r m e d , i n c r e a s e i n s i z e a n d c e l l w a l l s - 3 1 t h i c k e n e d ; i m m e d i a t e l y f o l l o w e d b y s e t t i n g i n o f s h r i v e l l i n g l e a d i n g r a p i d l y t o a c o l l a p s e o f p o l l e n g r a i n s a n d t a p e t u m . c ) . P o l l e n g r a i n s f o r m e d , p a r t i a l l y c o l l a p s e a n d r e m a i n w i t h o u t a n y f u r t h e r d e g e n e r a t i o n u p t o t h e o p e n i n g o f t h e f l o w e r s . H o w e v e r , t h e a n t h e r s f a i l t o d e h i s c e . G . O b s e r v a t i o n s r e l a t i v e t o p o s s i b l e m a l e - s t e r i l i t y i n a l f a l f a . S e v e r a l n o t a b l e r e v i e w a r t i c l e s o n a l f a l f a b r e e d i n g a n d i t s i n h e r e n t d i f f i c u l t i e s h a v e b e e n p u b l i s h e d i n - r e c e n t y e a r s . E x t e n s i v e p a p e r s b y W h i t e {92), A t w o o d ( 5 ) , a n d T y s d a l a n d K i e s s e l b a c h ( 9 2 ) c o v e r t h e f i e l d v e r y c o m p l e t e l y . I t i s n o t t h e p u r p o s e o f t h i s t h e s i s t o a t t e m p t a s u m m a r y - r e p e a t i n g t h i s w o r k . H o w e v e r , s e v e r a l p e c u l i a r i t i e s o f t h e a l f a l f a s e e d p r o d u c i n g m e c h a n i s m , w h i c h m a y c o m p l i c a t e a s t u d y o f m a l e - s t e r i l i t y , a r e d i s c u s s e d b r i e f l y i n t h i s p a p e r . ( a ) S e l f - v e r s u s G r o s s - f e r t i l i z a t i o n i n a l f a l f a . A l f a l f a i s n o r m a l l y a c r o s s - f e r t i l i z e d c r o p , a l t h o u g h s e l f - f e r t i l i z a t i o n m a y a l s o o c c u r e x c e p t w h e r e l i m i t e d b y s e l f - i n c o m p a t i b i l i t y . T h e a m o u n t o f n a t u r a l c r o s s - p o l l i n a t i o n i n a l f a l f a h a s b e e n s t u d i e d b y s e v e r a l i n v e s t i g a t o r s . T y s d a l a n d K i e s s e l b a c h ( 9 1 ) r e p o r t t h a t 8 9 % o f t h e s e e d p r o d u c e d i n N e b r a s k a r e s u l t e d f r o m c r o s s - f e r t i l i z a t i o n ; K n o w l e s ( 5 6 ) i n S a s k a t c h e w a n r e p o r t e d 9 4 . 2 % , B u r k a r t ( 1 8 ) f o u n d 8 4 . 5 % c r o s s i n g i n A r g e n t i n a , a n d H a d f i e l d a n d G a l d e r ( 4 6 ) r e p o r t e d 44% i n N e w Z e a l a n d . C r o s s - p o l l i n a t i o n p r o d u c e s a m u c h h i g h e r a v e r a g e n u m b e r o f s e e d s p e r f l o w e r p o l l i n a t e d t h a n s e l f - f e r t i l i z a t i o n ; i n f a c t , C o o p e r a n d B r i n k ( 3 1 ) r e p o r t a n e t f e r t i l i t y 6 t i m e s - 3 2 - a s h i g h p e r f l o w e r p o l l i n a t e d a f t e r c r o s s i n g a s a f t e r s e l f i n g . T y s d a l a n d K i e s s e l b a c h ( 9 1 ) s t a t e t h a t a c o n s i d e r a t i o n o f s e l f - f e r t i l i t y r e l a t i o n s h i p s i n a l f a l f a i s o f p a r a m o u n t i m p o r t a n c e , a n d p l a n t b r e e d e r s s h o u l d s e l e c t h i g h l y s e l f - s t e r i l e p l a n t s . M o t o n l y a x e m o r e p o d s s e t f o l l o w i n g c r o s s - t h a n s e l f - f e r t i l - i z a t i o n " b u t m o r e s e e d s a r e f o r m e d p e r p o d . T h i s i s h o m e o u t "by m a n y w o r k e r s : L . e s i n s ( 6 0 ) i n S w e d e n , A r m s t r o n g a n d w h i t e ( 3 ) i n C a n a d a , H a d f i e l d a n d C a l d e r ( 4 6 ) i n H e w Z e a l a n d , T y s d a l a n d a s s o c i a t e s ( 9 1 , 9 2 ) , C a r l s o n ( 2 2 ) , C o o p e r a n d B r i n k ( 3 1 , 3 2 ) a m o n g o t h e r s i n t h e U . S . A . S e l f - f e r t i l i z a t i o n n o t o n l y r e s u l t s i n a m a r k e d d e c r e a s e i n s e e d p r o d u c t i o n , b u t a l s o t o a l e s s e r d e g r e e i n v e g e t a t i v e v i g o u r ( 4 6 ) , w h e r e a s c r o s s - f e r t i l i z a t i o n r e s u l t s i n a m e a s u r e o f h e t e r o s i s . H a d f i e l d a n d C a l d e r ( i b i d ) f o u n d t h a t 6 5 s e e d s w e r e f o r m e d p e r 1 0 0 f l o r e t s t r i p p e d a n d o p e n - p o l l i n a t e d , a n d w h e n t h e f l o r e t s h a n d - t r i p p e d a n d t h e p l a n t s c o v e r e d 4 3 . 0 s e e d s w e r e f o r m e d p e r 1 0 0 f l o r e t s t r i p p e d . P i p e r a n d a s s o c i a t e s ( 6 9 ) i n a s e r i e s o f e x p e r i m e n t s i n 1 9 1 2 - 1 9 1 4 o b s e r v e d t h a t t o t a l s e e d p r o d - u c t i o n a n d n u m b e r o f s e e d s p e r p o d wa,s m o r e t h a n d o u b l e d o n o p e n - p o l l i n a t i o n o v e r s e l f - p o l l i n a t i o n . ( I f 1 0 0 ^ c r o s s i n g w e r e t h e n p o s s i b l e , a l f a l f a s e e d p r o d u c t i o n w o u l d b e i n c x - e a s e d b y 5 0 - 1 0 0 # . ( b ) I s t r i p p i n g ; n e c e s s a r y f o r a l f a l f a s e e d p r o d u c t i o n ? A m o r p h o l o g i c a l p e c u l i a r i t y o f a l f a l f a f l o w e r s t h a t f u r t h e r c o m p l i c a t e s t h e p i c t u r e o f s t e r i l i t y i s t h e n e c e s s i t y o f " t r i p p i n g " o f t h e f l o w e r b e f o r e f e r t i l i z a t i o n c a n b e , e f f e c t e d . A c c o r d i n g t o C o f f m a n ( 2 9 ) , D e C a n d o l i e ( 3 5 ) i n 1 8 3 2 -33- gave the f i r s t explanation of the tripping process in which he stated that the explosion of the flower takes place when a certain stage of i t s maturity is reached. Hildebrand (again quoting Gof f man (ibid)) believed as early as 1866 that fert- i l i z a t i o n may take place in untripped flowers. Brink and Cooper (15) observed pollen germinating within untripped flowers and pollen tubes entering the styles even in the late bud stage. Carlson (22) found 27% of the flowers producing seed without tripping in Utah in 1930. Hay (47) in 1925 found that 5.9% of untripped flowers set seed. On the other hand, Armstrong and White (3) write that pollen tubes only penetrate the stigma when the stigmatic surface is ruptured. However, they did f ind that 0.6% of the untripped flowers showed pollen germination. They observed a thin membrane covering the stigmatic surface which is suff iciently thick and impermeable to prevent penetration of pollen tubes and which, i f unruptured, w i l l prevent growth of pollen tubes down the style. Stigma scarif icat ion, and rupture of this membrane, with the release of the stigmatic content which ini t iates pollen germination, is normally achieved when the stamina! column is released from the keel or "tripped" str iking -the standard with, consid- erable force. Lesins (60) confirms this theory with observ- ations of the stigmatic membrane, and reports the presence in one ca,se of a broken membrane in an untripped flower. Hadfield and Calder (46) write that tripping i s a prerequisite to seed production. Bl inn (8) in Colorado reporting on the results of an experiment on "tripping" wrote -34- in 1920 that "there wa,s no clear evidence that bee3 or other- insects were essential to alfalfa, seed production. Fer t i l i za t ion can and does take place without insects", and that tripping is not necessary for seed production. Brand and v/estgate (11) in 1909 stated that poll ination is not effective u n t i l flowers are tripped. B u r k i l l (19) wrote that a l fa l fa pollen was shed, in the bud stages, to which Coffman (ibid) agrees and adds that tripping is not necessary for seed production. The question has aroused many papers and is s t i l l un- answered. It appears to this writer that the presence or absence of a stigmatic membrane may rest upon a genetic factor or upon a physiological response. The presence or absence of this membrane appears to be the determining factor in whether or not pollen can f e r t i l i z e the ovules or grow in the styles whether tripped or not tripped. The tripping c-f flowers i« ' normally performed by bees, and Peck and Bolton (68) in 1946 showed that the native bee population was often the l imit ing factor in a l fa l fa seed production in Northern Saskatchewan and Alberta. This view although contrary to that of Blinn ( ibid) , is generally agreed upon by the scientists working in this f i e l d . An idea of the importance attached to the role of insects in relat ion to tripping and deed sett ing. in al fal fa , can be gleaned by the report of the Twefth Al fa l f a Improvement Conference (l) wherein 9 out of the total of 23 papers read dealt with this phase of seed production. Dwyer (37) in 1932 reported instantaneous tripping of -35- a l l flowers at 104°P under most diverse moisture conditions providing the flowers are in a fresh turgid condition. Armstrong and White (3) state that the control l ing factor appears to he duration and intensity of hours of sunshine. Dwyer (ibid) also believed wind to be an important agent of pollen dissemination and insects to play a minor role in pol l inat ion. However, this view is certainly not agreed to by the majority of workers. Kirk (55) reported on the finding of an autogamous a l fa l fa which, is sel f-tr ipping, Automatic tripping of auto- gamous alfalfas occur when "the dynamic force present -in the staminal column is suff iciently greater than the static force present in the keel to cause the tripping mechanism to explode simultaneously" to quote Armstrong and White (3). Southworth (85) attempted to raise a high seed producing l ine by hybridizing M. lupulina (self-tripping) with M. sativa (not sel f-tr ipping) . He hoped to produce a self-tripping high seed setting alfalfa,, and achieved a considerable measure of success in experiments running over 20 years. However, use of highly se l f - f e r t i l e , self-tr ipping lines is undesirable in developing synthetics, because the seed yields from open progenies from such l ines is similar to the seed yields from their selfed progenies (87). In the past few years the u t i l i z a t ion of hybrid vigour to obtain maximum productivity of the concerned plants has assumed importance to many scientists . As Bonn and Whitaker (9) point out, male-steri l i ty enables seedsmen to produce -36- hybrid seed comparatively easily and cheaply. Clarke and Fryer (27) writing -on seed setting of a l fa l fa in 1930. found that many plants produced high percentages of sterile (empty) pollen grains. The percent varied among plants but remained constant for particular plants even when the pollen was produced under different conditions. In 1929 counts were -obtained from a number of a l fa l fa plants which, were setting pract ical ly no seed under f i e l d conditions: empty grains ranged from 50 to 90^. However, plants from a normal f i e l d cont ained ^from 3 to 48^ s ter i le pollen. Armstrong and White (abid) add to this in 1935 that plants having a high percent of s ter i le pollen were deficient in amount of pollen as well ; a high proportion of shrunken pollen grains seems "associated with faulty dehiscence of the anthers". Tysdal and Kiesselbach (ibid) (91) stated in 1944 that male s t e r i l i t y had not been identif ied in al fal fa , but that i t s possible use and occurrence should not be ignored. STUDIES IH FIELD AM) LABORATORY. A - Material and methods (.«•) P© WtA~ s^u.dr.«-s During the summer of 1949 investigations were begun to determine the presence of male-sterile l ines of a l fa l fa at the University of B r i t i s h Columbia farm. A microscopic examination of the pollen, produced by the 910 clonal l ines of Rhizoma a l fa l fa maintained by the Dep't. of Agronomy, was carried out. Several racemes representative of these lines were clipped while in a fresh turgid state and ( carried, with their excised ends in fresh water to the labor- - 3 7 a t o r y . H e r e 3 f r e s h f l o r e t s w e r e s e l e c t e d a t r a n d o m f r o m e a c h s a m p l e a n d t r i p p e d o n a m i c r o s c o p i c s l i d e . T h e p o l l e n w a s m o u n t e d i n m i n e r a l o i l a n d c o v e r s l i p s a f f i x e d . T h e s l i d e s w e r e e x a m i n e d u n d e r t h e 1 2 5 x p o w e r o f a R e i c h e r t r e s e a r c h " b i n o c u l a r - m i c r o s c o p e f o r q u a n t i t y a n d q u a l i t y o f p o l l e n g r a i n s . S e v e r a l s a m p l e s w e r e o b s e r v e d o f t h e l i n e s a t e i t h e r e n d s o f t h e q u a l i t y d i s t r i b u t i o n c u r v e . F o l l o w i n g - t h e m e t h o d o u t l i n e d b y B u r t o n ( 2 1 ) i n 1 9 4 4 , t h e p o l l e n w a s a s s e s s e d a n u m e r i c a l v a l u e o f 1 t o 5 b o t h f o r q u a n t i t y a n d q u a l i t y . A p r o d u c t i o n o f v e r y f e w p o l l e n g r a i n s r e c e i v e d a n u m e r i c a l r a t i n g o f l j l a r g e q u a n t i t i e s o f p o l l e n w a s a s s e s s e d a n u m e r i c a l r a t i n g o f 5 . I n t h e c a s e o f q u a l i t y , a n u m e r i c a l r a t i n g o f 5 r e p r e s e n t e d p o l l e n o f u n i f o r m l a r g e s s i z e a n d p r o p e r s h a p e w i t h a n a b s e n c e o f m i c r o - p o l l e n o r - s h r i v e l l e d d r i e d - u p p o l l e n . ( N o r m a l p r o p e r s h a p e i s r o u n d e d , s e m i - h y a l i n e a n d f i n e l y p i t t e d . ) A v a l u e o f 1 r e p r e s e n t e d " p o l l e n t h a t w a s s h r u n k e n , s h r i v e l l e d o r m i c r o - p o l l e n . 4 4 c l o n a l l i n e s p r o d u c e d n o f l o w e r s d u r i n g t h e s u m m e r s o c o u l d n o t b e c a t e g o r i z e d . O n t h e b a s i s o f t h e f i r s t e x a m - i n a t i o n , 4 4 l i n e s w e r e r a t e d 5 f o r q u a l i t y o f p o l l e n ; a n d 5 l i n e s w e r e t e n t a t i v e l y d e s i g n a t e d a s " s t e r i l e " a s a l l p o l l e n t h a t w a s p r o d u c e d w a s e i t h e r s h r i v e l l e d o r a m o r p h i c . Hew s l i d e s w e r e m a d e u p f o r t h e s e 4 9 l i n e s , a n d t h e p o l l e n w a s r e c a t e g o r i z e d . T h i s t i m e , 2 0 o f t h e 4 4 l i n e s r a t e d 5 , w e r e a g a i n r a t e d 5 b o t h f o r q u a n t i t y o f p o l l e n a n d q u a l i t y o f p o l l e n . O n e o f t h e s o - c a l l e d s t e r i l e l i n e s , s h o w e d a p r o d - u c t i o n o f p o l l e n , s o w a s g i v e n a h i g h e r c l a s s i f i c a t i o n . T h e -38- remaining 4 " s ter i le" l ines showed on a l l observations, in the'cases of any pollen production at a l l , only shrunken, shrivelled pollen. Table I shows the categorization at this date, 2 clonal cuttings of each of the 20 l ines designated as "high pollen producing l ines" (See Tables I and III), and 5 cuttings of each of the 4 l ines designated " s ter i le " (See Tables I and II) were made, and planted in the greenhouse November 4 and October 31, 1949 respectively. The cuttings grew well in the greenhouse and through the use of a r t i f i c i a l l ight were "forced" into flower in A p r i l , 1950. Pollen from the different l ines was. again examined under, 125x with results comparable to the previous summer's observations. (See Table I ) . Hone of the l ines designated as "male-sterile" produced .viable pollen, . The plants were transplanted from the greenhouse to the Al fa l fa Hursery plots in May 1950. Pollen was examined on the 18th of September, 1950, under 125x, with results as shown in Table I. A pollen examination of the following varieties of a l fa l fa , contained in the Alf alf a Hursery plots at U . B . C . , was also undertaken in 1949 and 1950 for the presence of male-steri l i ty. In 1949 Viking, Ranger, Grimm, Ladak, Per ax, Buffalo, Don (creeping M . f ale at a), and M. lupulina pollen was examined along with .pollen from 4 strains of Rhizoma: H71P27, H7, H68, H190, which were not included in the - previous pollen examination. In September 1950 an examination - 3 9 - o f t h e p o l l e n f r o m t h e a b o v e p l a n t s w a s c o n d u c t e d p l u s t h e f o l l o w i n g l i n e s : G r i m m S u m m e r l a n d S 2 7 4 , G r i m m V i d a r s h o v , G r i m m S a s k a t c h e w a n 6 6 6 , G r i m m S a s k a t c h e w a n 4 5 1 , e r e c t M . f a l - c a t a , H e m a s t o n , K a n s a s C o m m o n , A t l a n t i c , O r e g o n C r e e p e r I a n d I I , M . r u t h e n i c a , M . g l u t i n o s a , H u n t e r R i v e r l u c e r n e , B o o b o r - o o g i e l u c e r n e , a n d A u s t r a l i a n c r e e p i n g l u c e r n e . I n n o n e o f t h e c a s e s e x a m i n e d w a s a n y m a l e - s t e r i l i t y o b s e r v e d , ( b ) S e l f - p o l l i n a t i o n s t u d i e s A c o u n t e d n u m b e r o f f l o r e t s o f e a c h o f t h e s e l e c t e d l i n e s ( T a b l e I I ) w e r e s e l f - p o l l i n a t e d b y t h e f o l l o w i n g p r o c - e d u r e d u r i n g t h e l a t t e r p a r t o f J u l y a n d t h e f i r s t p a r t o f A u g u s t , 1 9 5 0 . R a c e m e s c o n t a i n i n g f r e s h u n t r i p p e d f l o r e t s w e r e s e l e c t e d a n d a l l l e a v e s , b u d s , t e r m i n a l g r o w t h , a n d o p e n e d f l o w e r s w e r e c u t a w a y w i t h s c i s s o r s l e a v i n g a m a x i m u m o f 1 0 u n t r i p p e d f l o r e t s p e r r a c e m e . T h e r a c e m e w a s w a s h e d t o r e m o v e a n y a d h e r i n g f o r e i g n p o l l e n o n t h e s t a n d a r d s b y d i p p i n g i n c l e a n w a t e r . W h e n d r y , t h e f l o r e t s w e r e a r t i f i c i a l l y t r i p p e d b y i n s e r t i n g t h e p o i n t s o f a c l o s e d p a i r o f s c i s s o r s ( s t e r i l i z e d b e t w e e n l i n e s w i t h e t h y l a l c o h o l f o l l o w e d b y a w a s h ) b e t w e e n t h e s t a n d a r d a n d t h e k e e l , a n d o p e n i n g t h e s c i s s o r s . T h i s c a u s e d t h e s t a m i n a l c o l u m n t o b e r e l e a s e d f r o m t h e k e e l a n d s t i k e t h e c o l u m n w i t h s o m e f o r c e . A f t e r t h e f l o r e t s o f a r a c e m e w e r e t r i p p e d , a c e l l u l o s e b a g w a s s e c u r e d o v e r t h e r a c e m e t o e x c l u d e a n y i n s e c t s t h a t m i g h t e f f e c t c r o s s - p o l l i n a t i o n . ( S t e v e n s o n a n d B o l t o n ( 8 7 ) f o u n d t h a t c r o s s - p o l l i n a t i o n o c c u r r e d i n a l f a l f a i f f o r e i g n -#o- p o l l e n , w a s a p p l i e d o n e h o u r o r p o s s i b l y l o n g e r a f t e r t r i p p i n g w h e r e f l o w e r s h a d n o t b e e n e m a s c u l a t e d . T y s d a l a n d G a r l ( 9 4 ) f o u n d t h a t i f f o r e i g n p o l l e n w e r e a d d e d t o u n t r e a t e d s t i g m a s i n a d d i t i o n t o i t s o w n , t h e f o r e i g n p o l l e n w o u l d b e t h e a c t i v e a g e n t i n f e r t i l i z a t i o n 7 0 - 9 8 ^ o f t h e t i m e , ) T h e c e l l u l o s e b a g s w e r e r e m o v e d i n a w e e k s t i m e t o p r e v e n t m i l d e w i n g a n d t o a l l o w a n y s e e d f o r m e d t o r i p e n . T h e r a c e m e s h a d b e e n t a g g e d p r e v i o u s l y s o t h a t t h e y c o u l d b e i d e n t i f i e d . T h e d e v e l o p e d p o d s w e r e h a r v e s t e d p e r i o d i c a l l y w h e n n e a r l y r i p e a n d a l l o w e d t o r i p e n i n t h e l a b o r a t o r y . W h e n - r i p e , t h e s e e d w a s t h r e s h e d b y h a n d a n d c o u n t e d . ( S e e T a b l e I I ) . T h e s e e d s w e r e p l a n t e d i n f l a t s i n t h e g r e e n h o u s e i n O c t o b e r 1 9 5 0 , a n d t r a n s p l a n t e d i n D e c e m b e r t o o t h e r f l a t s ( 4 0 p e r f l a t ) , a n d o v e r w i n t e r e d i n t h e s e c o n t a i n e r s . T h e y w i l l b e t r a n s p l a n t e d t o t h e A l f a l f a N u r s e r y i n M a y 1 9 5 1 f o r p o s s i b l e u s e i n f u t u r e g e n e t i c a l s t u d i e s . 1 p l a n t r e s u l t e d f r o m t h e 5 d e e d s o b t a i n e d b y s e l f i n g " s t e r i l e " l i n e 1 4 4 - 1 1 A , a n d 1 p l a n t f r o m t h e 1 s e e d o b t a i n e d f r o m " s t e r i l e " l i n e 9 I - 1 0 B . O n l y t h e l a t t e r p l a n t w a s i n f l o w e r b y A p r i l 2 5 , 1 9 5 1 , a n d a, p o l l e n e x a m i n a t i o n o f t h e s e f l o w e r s w a s c a r r i e d o u t . ( c ) O p e n - p o l l i n a t i o n s t u d i e s A l l s e e d p o d s , w h e n n e a r l y r i p e , w e r e c o l l e c t e d a t i n t e r v a l s o f a f e w d a y s f r o m A u g u s t 1 s t t o t h e f i r s t p a r t o f O c t o b e r 1 9 5 0 , f r o m b o t h t h e " m a l e - s t e r i l e " a n d h i g h p o l l e n p r o d u c i n g l i n e s . T h e s e e d s , w h e n r i p e , w e r e t h r e s h e d -41- and counted with results as shown in Table III. The open- pollinated seed from the "male-sterile" plants was planted in f lats in the greenhouse in November 1950, and 100 plants of each l ine were transplanted to 5" pots in late December. The plants began to flower on Apr i l 11, and microscopic slides were prepared daily of the pollen from the flowers as soon as they unfolded from the bud. By Apri l 25, 71 plants of the 20-DRC l ine had flowered, 12 plants of 142-1033, 41 plants of 91-10B, and 44 plants of the l ine 144-11A had come in£o flower. Microscopic slides of the pollen from these plants was examined under 125x on Apri l 26, with results as shown in Table 6. B - Observations Pollen showed a great amount of diversity both in shape and size. Pollen varied from normal, rounded,semi-hyaline, and f inely pitted; through e l l i p t i c a l , striated, and with dense cytoplasm; to irregular, angular and of large s i ze . Also observed was very small pollen of normal shape and density (designated 'micro-pollen') , and dark, amorphous, very small granular bodies (designated 'aborted' pollen). On the basis of pollen examination 20 l ines were selected as being superior pollen producers, and 4 l ines were selected as possessing "male-ster i l i ty" . The high pollen producing l ines a l l produced a large quantity of pollen (with a few exceptions) of uniform excellent quality except 3 l ines which produced,,the second year, some micro-pollen and shrunken pollen. The results -42/ are tabulated in Table I. The examination of the varieties other than Rhizoma was carried out as a check: most produced very good pollen and none showed any male-ster i l i ty . However, we had only 6 plants of each variety in the nursery plot to make observations on, so the examination would hardly be significant. The lines designated as "male-sterile" were observed in the main production f i e l d in August and September 1949 with the following observations recorded. The original 20-DRC l ine was complete and showed superior vegetative growth. Only 2 plants were l i v i n g in the original clonal row of 91-10B: both being healthy, vigorous plants. Clonal row 144-11A had only one poor unvigorous plant lef t in the row. Clonal row 142-10B had only one plant, - although vigorous and healthy, remaining. The original observations on the pollen taken in 1949 of a l l the selected "male-sterile" and " high-pollen" l ines are contained in Table I. In a c r i t i c a l examination of the pollen of the "male- s ter i le" l ines on 18 Sept. 1950 the following observations were made. 20-DRC: No loose pollen. The pollen sacs appear f u l l of a dark amorphous substance. 142-10B: Very l i t t l e good pollen, mush aborted. Pollen sacs f u l l of granular dark substance. 144-11A: Approximately equal amounts of good pollen with misshapen, irregular, shrivelled pollen; however there was not very much of either. -43- 91-10B: Very l i t t l e pollen was produced, Tout i t seemed normal pollen although very small in size (micro). On se l f - f e r t i l i za t ion 20-DRC and 142-10B produced no seeds; whereas 144-11A produced .0427 seeds per f loret selfed, •and 91-1OB produced .008 seeds per f loret selfed. These amounts of seed setting agree very well with the results ag, oh served on the amount of pollen produced "by these l ines . (See Table I) Of the normal or high pollen producing l ines 0.654 seeds .were set per f loret selfed, with a range from 0.009 to 1.877 seeds produced per f loret selfed. (Clarice and Fryer (27) found 0.44 seeds set per f loret selfed in Saskatchewan in 1930. Cooper and Brink (31) found that an average of 0.80 seeds 1 v/ere set per f loret selfed with a range of 0.16 to 1.76. This agrees f a i r l y weifel with my findiggs.) Shrivelled, small, and discolored seed constituted 34.84$ of the seed produced on self pol l inat ion, and 16.19$ of the seed produced on open-pollination. (See Tables II and III) The amount of seed produced on sel f-pol l inat ion is show in Table II, and on open-pollination of the same lines in Table III. An examination of the pollen from the 71,>]?i plants of the 20-DRC line that had. flowered at time of reporting showed 2 plants that produced no pollen at a l l , and 7 plants that produced only very shrivelled, misshapen, amorphic, apparently s ter i le , pollen. The 2 plants producing no pollen at a l l had empty pollen sacs in abundance, but no signs of pollen, excepting a very fine dust l ike debris which may be the remains -44- of early aborted'pollen. An examination of the pollen from the 44 3?]_ Plants of the 144-11A l ine showed 4 plants that produced empty pollen sacs without a vestige of free pollen, and 2 plants that produced the shrivelled, amorphic, apparently s ter i le pollen as'observed in the 20-DRC segregates. The pollen from the 41 plants of 91-10B showed 1 plant that fa i led to produce pollen, and 5 plants that produced only misshapen ' s t e r i l e ' pollen. Line 142-10B had only 12 3?̂ . segregates in flower by Apri l 26, 1951, and of these, only 1 plant appeared, to produce pollen that could be designated ' s t e r i l e ' . Table VT shows the frequency distribution of the pollen of the 3?]_ segregates of the 4 male-sterile l ines as they were observed on Apri l 26, 1951. As previously outlined, a value of 5 indicates pollen of uniform, semi-hyaline, rounded, shape. 1 indicates pollen that is misshapen and probably steri le or incapable of germination. A value of 0 indicates an absence of pollen. The intermediate grades indicates pollen of intermediate quality. That i s , a value of 3 would indicate the presence of rectangular, striated pollen. A value of 4 would designate the presence of normal pollen and also the abnormal pollen of 3. A value of 2 indicates the presence of presence of aborted pollen along with both the normal and abnormal. TABLE I - RESULTS OF POLLEN EXAMINATIONS. LINE OBS. AUOUST 1949 OBS. 18-24 APRIL 1950 OBS. 18. SEPT. 1950. Qnan. Qual. Quantity Quality Quantity Quality 20-DRC 0 0 - © 0 0 91-10B 2- 0 1 1 1+ 1 142-10B 0 0 0 0 0 0 144-11A 2- 1- 1 1 3 2 106-DEC 5- 5 5 5+ 5 4+ 57-10B 5 5+ 4+ 4+ 3 4+ 68-10B 4 5- 4+ 3- 4+ 4- 83-10B 5 5- 5 4 5- 5-102-10B 5- 5 5 4 5 3 103-10B 4+ 5- 5 4+ 5 3 128-10B 5- 5- 5 4+ 5 5 144-10B 5+ 5- 5 5- 5 4+ 15-11A 5- 5- 5 5 4+ 3-JO-llA 5- 5- 5 4+ 5- 5 34-11A 5- 5- 5 5* 5 3- 35-11A 5- 5- 5 5+ 5 4-59-11A 5- 5- 3 4- 5+ 4+ 7 1-llA 5- 5- 5 4+ 4+ 4+ 73-11A 5- 5- 5 5* 5- 4* 81-11A 4+ 5- 4+ 4+ 5 4+ 94-11A 5- 5- 5- 5- 5 4+ 95-HA 5 5* 4 5- 5 5-90-11B 5+ 5- 5- 4+ 5- 5 115-11B 5+ 5*» 4+ 4- 5 5 Note: # indicates presence of micro -pollen or aborted pollen. I I TABLE II - SEEDS PRODUCED UPON SELF-POLLINATION Line F u l l seeds Small brown seeds No. % t o t a l Total seeds No. f l o r e t s selfed Seeds/floret Male-sterile l i n e s 20-DBC 0 0 0 217 0 142-10B 0 0 - 0 142 0 144-11A 5 0 - 5 117 0.0427 91-10B 1 0 - 1 118 0.0085 High pollen producing l i n e s • 106-DRG 118 20 14.49 138 106 1.302 37-10B 0 1 100. 106 0.009 68-10B 8 10 55.56 18 105 0.171 83-10B 172 27 13.57 199 106 1.877 102-10B 10 41 80.39 51 106 0.481 103-10B 24 14 36.84 38 107 0.355 128-10B 44 15 25.42 59 99 0.596 144-10B 125 12 8.76 137 100 1.370 15-11A 27 31 " 53.44 58 108 0.537 30-11A 25 11 30.56 36 103 0.350 34-11A 85 8 8.60 93 103 0.903 60 33 35.48 93 110 0.845 59-11A 106 20 15.87 126 105 1.200 71-11A 5 1 16.6? 6 103 0.058 73-11A 25 13 ' 34.21 38 106 0.358 81-11A 12 0 12 100 0.120 94-11A 39 7 1 110 109 1.009 95-11A 70 16 18.60 86 103 0.835 90-11B 5 12 70 .59 17 48 0.354 115-11B 33 5 13 .16 38 107 0.355 Average 34.84^ Average 0.654 seeds per f l o r e t s e l f e d . TABLE III SEEDS PRODUCED UPON OPEN-POLLINATION Line P u l l seeds Small brown seeds Total seeds No. of plants Seeds/Plant No, % t o t a l M ale-sterile l i n e s 20-DRC 491 89 15.34 580 5 116 142-10B 258 139 35.01 397 5 79.4 144-11A 528 111 17.37 639 5 127.8 91-10B 909 60 6.19 969 5 193.8 High pollen produeing l i n e s . 106-DRC 295 44 12.98 37-10B 697 54 7.19 68-10B 74 20 21.28 83-lOB 1314 376 22.25 102-10B 654 58 8.15 103-10B 773 31 3.86 128-10B 41 54 56.84 144-10B 354 78 18.06 15-11A 400 54 11.89 30-11A 86 . 46 34.85 34-11A 622 36 5.47 3 5 - l l A 569 108 15.95 59-11A . 470 69 12.80 71-11A 106 6 5.36 7 3-HA 1165 118 9.20 81-11A 565 148 20.76 94-11A 368 53 12.59 95-HA 309 52 14.40 9 0 - l l B 550 61 9.99 339 2 169.5 751 2 375.5 94 2 47.0 1690 2 845.0 712 2 356.0 804 2 402.0 95 2 47.5 432 2 216.0 454 2 227.5 132 2 66.0 658 2 329.0 677 2 338.5 539 2 269.5 112 2 56.0 1283 2 641.5 713 2 356.5 421 2 210.5 361 2 180.5 611 2 305.5 Average 16.19^ Average 287.8 seeds per plant. -48- TABLB IV CORRELATION OF SEED SET UNDER OPEN & SELF-POLLINATION. Line Seed set Open-pollinated S e l f - p o l l i n a t e a # seeds Rank # seeds. Rank 68-10B 47 1 0.171 4 128-10B 47 .5 2 0.596 12 71-11A 56 3 0.058 2 30-11A 66 4 0.350 5 106-DRG 169.5 5 1.302 18 95-HA 180.5 6 0.835 13 94-11A 210 .5 7 1.009 16 144-10B 216 8 1.370 19 15-11A 227.5 9 0.537 11 59-11A 269 .5 10 1.200 17 90-11B 305.5 11 0.354 6 113-1IB 316 12 0.355 7.5 34-11A 329 13 0.903 15 33-11A 338.5 14 0.845 14 102-lOB 356 15 0.461 10 81-11A 356.5 16 0.120 3 .37-1GB 375.5 17 0.00? 1 1I03-10B 402 18 0.355 7.5 7 3 - 11A 641.5 19 0.358 9 83-lOB 845 20 1.877 20 Using ranking correlation '.£"*' - 210 ir 210 i& 2581.0 £ y 2 -- 2581.5 (ix)2- 4 4 , 1 0 0 (*.y>2- 44,100 S.S. X = 376 S « S « y - 376.5 txj - 2223 r : +.0478 t « .209 At n-r2 ( l 8 ) degrees of freedom, tabled t equals 2.101 at pr . 0 5 . Calculated t i s smaller than tabled t so no s i g n i f - icance can be asserted to the corr e l a t i o n c o e f f i c i e n t . That i s , there i s no co r r e l a t i o n . TABLE V RANK CORRELATION OJ? SEED SET UNDER OPEN & SELF-POLLINATION . USING WILCOXON'S (98) APPROXIMATE STATISTICAL PROCEDURE. (Ranks as assigned i n Table IV) Open-noil. S e l f - p o l l . gftflk, t o t a l s (Rftfik tQtftlg) 1 4 5 25 2 12 14 196 3 2 5 25 4 5 9 81 5 18 23 529 6 13 19 361 7 16 23 529 8 19 2? 729 9 11 20 400 10 17 27 729 11 6 17 289 12 7.5 19.5 380 13 15 28 784 14 14 - 28 784 15 10 25 625 16 3 19 361 17 1 18 324 18 7.5 25.5 650 19 9 28 784 20 20 40 1600 10, 185 r x r - 1. P-l 0 _ 12 Sum (rank t o t a l s ) 2 - 3n(p+l) 1 r " np (p+l) a a 20 p = 20 (rank t o t a l s ) = 10, 185 X2r * 10. 185 - 126 70 = 145.5 - 126 = 1 9 . 5 r ~ - 1 = 1.03 - 1 = +.03 To test whether t h i s d i f f e r s s i g n i f i c a n t l y from zero i t majr_be compared with i t s standard error which i s l/J~ p-T or l/v/19 = 1/4.66 • . 2 2 7 . The r a t i o . 0 3 / . 2 2 7 equals .132, and there- fore the correlation cannot be considered s i g n i f i c a n t as i t s value (+.03) i s less than one quarter of i t s standard error ( . 2 2 7 ) . TABLE VI - FREQUENCY DISTRIBUTION OF THE POLLEN CLASSES OF THE E l SEGREGATES OF THE 4 'MALE-STERILE1 ALFALFA LINES ON APRIL 26. 1951 Line No. of plants Category for qual i t y of pollen examined 5 4 3 2 1 0 20-DEC 71 2 10 22 28 7 2 144-11A 44 0 2 8 28 2 4 91-10B 41 0 3 12 21 5 1? 142-10B 12 0 0 7 4 1 0 Totals 168 2 15 39 81 15 7 Note Quality category 1 indicates that no viable appearing pollen i s present Quality category 0 indicates that no pollen i s produced. i O I -51- G - D i s c u s s i o n . „ Lin e s 20-DRG and 142-10B show c o n s i s t e n t l a c k of v i a b l e p o l l e n and produced no seed on s e l f i n g , and as such can be considered m a l e - s t e r i l e . 20-DRG being extremely vigorous and a f a i r seed s e t t e r upon o p e n - p o l l i n a t i o n i s worthy of f u r t h e r study. 142-1033., although showing m a l e - s t e r i l i t y , i s weakly veget a t i v e and low i n seed s e t t i n g on o p e n - p o l l i n at i o n . Due to these reasons 142-1033 does not possess the same value agronomic a l l y as the 20-DRG l i n e ; but even c o n s i d e r i n g these f a c t o r s i t i s f e l t t hat i t i s also worthy of f u r t h e r study. I t was noted that the F x segregates of 20-DRG were much f u r t h e r advanced than those of the l a t t e r l i n e r i g h t from the s e e d l i n g stage. This i s f u r t h e r evidenced by the f a c t that 71 p l a n t s of the 20-DRG F-j_ were i n flower by the time 12 142-1033 F i were at the same stage. L i n e s 144-11A and 91-1033 appear to possess p a r t i a l male- s t e r i l i t y . 91-1033-, i n s p i t e of there being only 2 p l a n t s l e f t i n the main c l o n a l row, and the f a c t that i t produced seed on s e l f i n g , i s worthy of f u r t h e r study due to i t s e x c e l l e n t seed set on op e n - p o l l i n at i o n and i t s very low percentage of sma l l , brown d i s c o l o r e d seed. Although the same objec t i o n s can be app l i e d to l i n e 144-11A, i t i s f e l t that i t i s espec- i a l l y worthy of f u r t h e r study as 9.1% of the F x segregates showed a complete absence of p o l l e n when examined on A p r i l , 26, 1951. L i n e 57-1033 appears to possess a s e l f - i n c o m p a t i b i l i t y f a c t o r . This l i n e appeared w e l l above the average i n seed -52- ^ Z s e t upon o p e n - p o l l i n a t i o n , - w h i l e o n l y 0.009 s e e d s were s e t p e r f l o r e t s e l f e d on s e l f - p o l l i n a t i o n . The p r o d u c t i o n o f s m a l l , brown, d i s c o l o r e d and w r i n k l e d s e e d was g r e a t l y i n c r e a s e d b y s e l f - p o l l i n a t i o n (34.84$) o v e r o p e n - p o l l i n a t i o n ( 1 6 . 1 9 $ ) . ( C a r l s o n and S t e w a r t (24) r e p o r t e d i n 1931 t h a t s h r i v e l l e d , d i c o l o r e d s e e d r a n g e d f r o m 7.7$ t o 27$ f o r a 4 y e a r a v e r a g e . They f o u n d t h a t an i n c r e a s e i n s h r i v e l l e d d i c o l o r e d s e e d was e x p e c t e d l a t e i n t h e s e a s o n due t o i n s u f f i c i e n t t i m e , b e f o r e f r e e z i n g w e a t h e r , t o p r o d u c e f u l l s e e d . However, i n our c a s e , t h e s e l f - p o l l i n a t e d s e e d was c o l l e c t e d d u r i n g t h e same p e r i o d s as t h e o p e n - p o l l i n a t e d s e e d , so t h e d i f f e r e n c e c a n n o t be e x p l a i n e d on t h i s b a s i s . W hatever, t h e r e a s o n t h e r e i s a s i g n i f i c a n t d i f f e r e n c e . I t i s p o s s i b l e t h a t p a r t i a l i n c o m p a t i b i l i t y or d e l a y e d soma.to- p l a s t i c s t e r i l i t y may be t h e c a u s e o f t h e d i f f e r e n c e o b s e r v e d . The r e s u l t s o f t h e l i m i t e d IT-̂  s e g r e g a t i o n f o r t h e 4 ' m a l e - s t e r i l e ' l i n e s as r e p o r t e d i n T a b l e V I do n o t a p p e a r t o c o n f o r m t o common M e n d e l i a n r a t i o s . 20-DRC p r o d u c e s m a l e - s t e r i l e s i n t h e 3?]_ i n a r a t i o o f 1 t o 7.9 t o t h e m a l e - - f e r t i l e s . 144-11A p r o d u c e d m a l e - s t e r i l e s t o m a l e - f e r t i l e s i n a r a t i o o f 1:7.3. I n 91-10B t h e o b s e r v e d r a t i o was 1:688; and i n 142-10B a p p e a r e d 1:8.3.. I f t h e m a l e - s t e r i l i t y was i n h e r i t e d as s i m p l y r e c e s s i v e t o m a l e - f e r t i l i t y no m a l t - s t e r i i i t y s h o u l d appear i n t h e ]?]_. However, w h a t e v e r t h e anode o f i n h e r i t a n c e , w h i c h w i l l u n d o u b t a b l y be c o m p l i c a t e d by p o l y p l o i d y , t h e ~F]_ s e g r e g a t i o n does show t h a t t h e o b s e r v e d m a l e - s t e r i l i t y i s i n h e r i t e d and - 5 3 - and i s not wholly attributable to environmental modification. However, before an explanation of the mode of inheritance can be resolved on a genetical basis, a more complete count of the T?l should be obtained, along with 3?2 and- 'backeross segregation data. To determine the underlying features of the m a l e - s t e r i l i t y a h i s t o l o g i c a l (and possibly a. cytological) examination of the stamens w i l l have to be performed. However, from the observations made, an estimate of the cause can be made. As the j?x segreg- ation and observations are by no means complete or f i n a l , t h i s estimate i s drawn from the observations on the parental 4 "male-sterile" l i n e s . In the l i n e 20-DRG, no normal pollen was produced, aand the pollen-sacs appeared f u l l of a dark, amorphous granular substance. These granular bodies could probably be units of tetrads cemented together with d i v i s i o n arrested, followed by s h r i v e l l i n g and a resultant i n t e n s i f y i n g of the cytoplasm to give the dark coloration. Ho differences were observed i n the gross morphology of the male-sterile and the male- f e r t i l e flowers; nor i n the stamens or i n the external features of the pollen sacs. It i s f e l t that a h i s t o l o g i c a l study of the pollen-sacs would be well worth while. In l i n e 142-10B the pollen sacs i n most instances appeared f u l l of the same dark amorphous substance as i n the previously discussed l i n e , but at the same time some normal, pollen was produced. Ho seed was produced on s e l f - p o l l i n a t i o n . Hov/ever, the fact that some normal pollen was produced at times, allows the tenetative conclusion that either the m a l e - s t e r i l i t y i s - 5 4 - incomplete or t h a t the degeneration of the p o l l e n i s e f f e c t e d v ery l a t e i n microsporogenesis and t h a t a l l pollen-mother c e l l s are not e q u a l l y a f f e c t e d . In the l i n e 144-11A approximately equal amounts of good p o l l e n were produced w i t h misshapen, i r r e g u l a r , s h r i v e l l e d p o l l e n (although very l i t t l e of e i t h e r ) . At the same time a few p o l l e n sacs were observed which appeared d e v o i d of con t e n t s . On s e l f i n g 4.27%' seed was produced by t h i s l i n e . However, the se g r e g a t i o n g i v e s 4 p l a n t s out of a t o t a l of 44 p l a n t s observed t o produce no p o l l e n at a l l (on the b a s i s , of course, of a s i n g l e examination). I t would appear t h a t i n t h i s l i n e , t h a t the m a l e - s t e r i l i t y f a c t o r , although present, i s of low penetrance. The empty p o l l e n sacs c o u l d be e x p l a i n e d on the b a s i s of the m a l e - s t e r i l i t y f a c t o r c a u s i n g a breakdown v e r y e a r l y , or at the onset, of m e i o t i c d i v i s i o n ; i n which case, no remains of the p o l l e n would be observed. In the f o u r t h l i n e , 91-10B, 0.8% seed was produced on s e l f i n g , and although the p o l l e n sacs appeared empty on a p o l l e n examination, a sma l l amount of normal, although minute, p o l l e n was produced. In normal micro s p o r o g e n e s i s , a f t e r the f i n a l d i v i s i o n of m e i o s i s , the t e t r a d r e s o l v e s i t s e l f i n t o p o l l e n g r a i n s , of i r r e g u l a r shape, who f e e d and grow and round out. I t appears t h a t i n t h i s l i n e , the p o l l e n g r a i n s were formed, but d i d not grow: i n which case, i t i s p o s s i b l e t h a t the m a l e - s t e r i l i t y f a c t o r a f f e c t e d the t a p e t a l l a y e r . However, an answer to t h i s can only be o b t a i n e d by fmrther study. Seed p r o d u c t i o n of the h i g h p o l l e n p r o d u c i n g l i n e s under s e l f - p o l l i n a t i o n a n d o p e n - p o l l i n a t i o n w a s f o u n d t o "be n o t c o r r e l a t e d . H o w e v e r , i t w a s o b s e r v e d t h a t t h e h i g h e s t s e e d p r o d u c i n g l i n e u n d e r s e l f - p o l l i n a t i o n v /as a l s o t h e h i g h e s t p r o d u c i n g l i n e u n d e r o p e n - p o l l i n a t i o n . T h e c o m p a r i s o n o f o f t h e s e e d s e t , u n d e r t h e d i f f e r e n t t y p e s o f p o l l i n a t i o n s , h a d . c e r t a i n l i m i t a t i o n s . A c o m p a r i s o n w a s m a d e b e t w e e n t h e s e e d - s e t p e r f l o r e t s e l f e d s e l f e d a n d t h e t o t a l o p e n - p o l l i n - a t e d s e e d - s e t o f t h e p l a n t ; w h e r e a s , i t w o u l d h a v e b e e n c o m p a r a b l e i f a n c o r r e l a t i o n h a d b e e n a t t e m p t e d b e t w e e n s e e d - s e t p e r f l o r e t s e l f e d a n d s e e d - s e t p e r f l o r e t o p e n - p o l l i n a t e d . I t i s f e l t t h a t t h e i s o l a t i o n o f m a l e - s t e r i l i t y , s u c h a s a c c o m p l i s h e d i n t h e p a s t t w o y e a r s w o r k a t U . B . C . , c o u l d h a v e v e r y i m p o r t a n t e f f e c t s o n t h e f u t u r e o f a l f a l f a b r e e d i n g M a l e - s t e r i l i t y c o u l d b e u s e d a s t h e k e y t o o p e n t h e d o o r t o h y b r i d v i g o r , a n d p r o v i d e t h e m e a n s o f u n l e a s h i n g s o m e o f t h e h e t e r o s i s t h a t i s m a n i f e s t e d b y a l f a l f a , i n t h e m a j o r i t y o f c a s e s , w h e n i t r e s u l t s f r o m h y b r i d i z a t i o n . I n t h e n u r s e r y p l o t a t U . B . C . t h e r e a r e s e v e r a l r o / w s o f p l a n t s r e s u l t i n g f r o m c r o s s - p o l l i n a t i o n a n d b e s i d e t h e m s e v e r a l r o w s o f p l a n t s r e s u l t i n g f r o m s e l f - p o l l i n a t i o n . T h e s e p l a n t s a r e l-§- y e a r s o l d o n l y , a n d t h e c r o s s - p o l l i n a t e d p l a n t s , w i t h o u t e x c e p t i o n , a r e s o m u c h l a r g e r , m o r e s p r e a d , a n d m o r e v i g o r o u s t h a n t h e i r s e l f e d c o u n t e r p a r t s t h a t t h e c o n t r a s t i s s t a r t l i n g . W h e n o n e t h i n k s o f w h a t t h i s m e a n s , w h e n i t c a n b e c o n s i d e r e d t h a t , t h r o u g h t h e u s e o f u s e o f m a l e - s t e r i l i t y , w h o l e f i e l d s c a n a l l b e p l a n t e d t o h y b r i d s e e d , t h e p o t e n t i a l i t i e s a r e u n l i m i t e d . - 5 6 - H o w e v e r , a l o t o f t e s t i n g w i l l h a v e t o "be d o n e " B e f o r e t h e p r o d u c t i o n o f h y b r i d s e e d t h r o u g h t h e e m p l o y m e n t o f m a l e - s t e r i l i t y c a n b e c o m e a r e a l i t y o r f e a s i b l e o n a n e c o n o m i c s c a l e . T h e s a l e - s t e r i l e l i n e s w i l l h a v e t o b e c h e c k e d c a r e - f u l l y f o r a g r o n o m i c s u i t a b i l i t y , a n d c o m b i n i n g a b i l i t y w i t h t h e d e s i r e d m a l e - p a r e n t l i n e s . N e v e r t h e l e s s , i t i s f e l t , a n d s t r o n g l y t o o , t h a t t h e m a l e - s t e r i l e l i n e s h a v e g r e a t p o t e n t - i a l i t y i n a h y b r i d v i g o r p r o g r a m . SUMM/RY A p o l l e n s t u d y o f a p p r o x i m a t e l y 1 0 0 0 l i n e s o f a l f a l f a g r o w n a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , a n d e m b r a c i n g m o s t o f t h e c o m m o n a l f a l f a v a r i e t i e s , r e v e a l e d 4 l i n e s t h a t e x h i b i t e d m a l e - s t e r i l i t y , a n d 2 0 l i n e s t s h a t p r o d u c e d v e r y h i g h q u a l i t y p o l l e n . T h e s e l i n e s w e r e c l o n a l l y p r o p a g a t e d i n t h e f a l l o f 1 9 4 9 , o v e r w i n t e r e d i n t h e g r e e n h o u s e , a n d t r a n s p l a n t e d t o t h e N u r s e r y p l o t s a t U . B . C . i n M a y 1 9 5 0 . A c o u n t e d n u m b e r o f f l o r e t s w e r e s e l f e d o f e a c h l i n e ; a n d o p e n - p o l l i n a t e d s e e d w a s c o l l e c t e d . N o c o r r e l a t i o n b e t w e e n s e l f - a n d o p e n - p o l l i n a t e d s e e d s e t w a s o b s e r v e d . 2 o f t h e m a l e - s t e r i l e l i n e s p r o d u c e d n o s e e d o n s e l f i n g , a n d 2 p r o d u c e d v e r y s m a l l q u a n t i t i e s . T h e 3 ? i s e g r e g a t i o n f o r m a l e - s t e r i l e : m a l e f e r t i l e s w e r e a s f o l l o w s : 1 : 7 . 9 , 1 : 7 . 3 , 1 : 6 . 8 , 1 : 8 . 3 i n t h e p l a n t s g r o w n f r o m o p e n - p o l l i n a t e d s e e d o f t h e 4 m a l e - s t e r i l e l i n e s . P o s s i b l e m o d e s o f i n h e r i t a n c e a n d c a u s e s o f t h e o b s e r v e d m a l e - s t e r i l i t y i s d i s c u s s e d . L i t e r a t u r e o n m a l e - s t e r i l i t y a n d i n c o m p a t i b i l i t y i s r e v i e w e d . N o r e f e r e n c e s w e r e f o u n d c i t i n g m a l e - s t e r i l i t y i n a l f a l f a . 5 7 L I T E R A T U R E C I T E D ( 1 ) A l f a l f a I m p r o v e m e n t C o n f e r e n c e , T w e l f t h A n n u a l R e p o r t . J u l y 3 1 - A u g u s t 2 , 1 9 5 0 . L e t h b r i d g e , A l t a . ( 2 ) A n o n y m o u s . G e n e r a l s e e d c r o p r e p o r t . P r o d u c t i o n S e r v i c e , P l a n t P r o d u c t s D i v i s i o n , C a n . D e p ' t . A g r i c . 5 p . m i m e o . N o v . 1 5 , 1 9 5 0 . ( 3 ) A r m s t r o n g , J . M . a n d V . J . W h i t e . F a c t o r s i n f l u e n c i n g --' s e e d - s e t t i n g i n a l f a l f a . J o u r . A g r . S c i . 2 5 : 1 6 1 - 1 7 9 . 1 9 3 5 . ( 4 ) A r t s c h w a g e r , E r n s t . P o l l e n d e g e n e r a t i o n i n m a l e - s t e r i l e s u g a r - b e e t s , w i t h s p e c i a l r e f e r e n c e t o t h e t a p e t a l P l a s m o d i u m . J o u r . A g . R e s . 7 5 : 1 9 1 - 1 9 7 . 1 9 4 7 . ( 5 ) A t w o o d , S a n f o r d E . C y t o g e n e t i c s a n d b r e e d i n g o f f o r a g e c r o p s . R e c e n t A d v . I n G e n e t i c s 1 : 1 - 6 7 . 1 9 4 7 . ( 6 ) B a t e s o n , W . a n d A . E . G a i r d n e r . M a l e - s t e r i l i t y i n f l a x , s u b j e c t t o 2 t y p e s o f s e g r e g a t i o n . J o u r . G e n e t . 1 1 : 2 6 9 - 2 7 5 . 1 9 2 1 . ( 7 ) B e a d l e , G . W . G e n e s i n m a i z e f o r p o l l e n s t e r i l i t y . G e n e t i c s 1 7 : 4 1 3 - 4 3 1 . 1 9 3 2 . ( 8 ) B l i n n , P h i l o K . F a c t o r s t h a t a f f e c t a l f a l f a s e e d y i e l d s . C o l o . A g r i c . C o l l . E x p t . S t a . B u l l . 2 5 7 . 1 9 2 0 . ( 9 ) B o n n , G . W . a n d T h o m a s W . W h i t a k e r . A g e n e f o r m a l e - s t e r i l i t y i n t h e m u s k m e l l o n . P r o c . A m e r . S o c . H o r t , S c i . 5 3 : 3 0 9 - 3 1 4 . 1 9 4 9 . ( 1 0 ) B o l t o n , J . L . A s t u d y o f c o m b i n i n g a b i l i t y i n a l f a l f a ' i n r e l a t i o n t o c e r t a i n m e t h o d s o f s e l e c t i o n . S c i . A g r i c . 2 9 : 9 7 - 1 2 6 . 1 9 4 8 . ( 1 1 ) B r a n d , C . J . a n d J . M . W e s t g a f e e . A l f a l f a i n c u l t i v a t e d r o w s . f o r s e e d p r o d u c t i o n i n s e m i - a r i d r e g i o n s . U . S . D . A . D e p . G i r o . 2 4 . 2 3 p . 1 9 0 9 . ( 1 2 ) B r i n k , R . A . a n d D . C . C o o p e r . S o m a t o p l a s t i c s t e r i l i t y i n M e d i c a g o s a t i v a . S c i e n c e 9 0 : 5 4 5 - 5 4 6 . 1 9 3 9 . ( 1 3 ) , m I n c o m p l e t e s e e d f a i l u r e - a s a r e s u l t o f s o m a t o p l a s t i c . s t e r i l i t y . G e n e t i c s 2 6 : 4 8 7 - 5 0 5 . 1 9 4 1 . ( 1 4 ) . S o m a t o p l a s t i c s t e r i l i t y a s a f u n c t i o n o f t h e e n d o s p e r m g e n o t y p e . G e n e t i c s 2 7 ; 1 3 4 . 1 9 4 2 . A n a b s t r a c t . -58- (15) Brink, R.A. and D.C .Cooper. The mechanism of poll ination in a l fa l fa (Medicago sativa). Amer. Jour. Bot. 23: 678-683. 1936. (16) Brink, R . A . , F.R.Jones and H.R.Albrecht. Genetics of "" resistance to bacterial wilt in a l fa l fa . Jour. Ag. Res. 49: 635-642. 1934. (17) Brink, V .C . Witches' Broom on Al fa l fa . In 22nd Ann. Rept. Can. PI. Dis . Sur. 1942. (18) Burkart, A. (Spanish t i t l e ) (Alfa l fa production in Argentina.) Herbage Abs. 7: 296-297. 1937. (19) B u r k i l l , T .H. On the f e r t i l i z a t i o n of some of the species of Medic ago L. in England. Proc. Camb. P h i l . Soc. 8: 142-143. 1894. (20) Burnham, C.R. Cytogenetic studies of a case of pollen . _ abortion in maize. Genetics 26: 460-468. 1941. (21) Burton, G.W. Estimating individual forage plant yields . - .. Jour. Amer. Soc. Agr on. 36: 709-712. 1944. (22) Carlson, J.W. A r t i f i c i a l tripping of flowers in a l fa l fa in relat ion to seed production. Jour. Amer. Soc . Agron. 22: 780-786. 1930. (23) Al fa l f a seed investigations in Utah. Utah . Agr. Exp. Sta. B u l l . 258: 1935. (24) and G. Stewart. Al fa l f a seed production. Utah Agr. Exp. Sta. B u l l . 226,. 1931. (25) Chittenden, R . J . Cytoplasmic inheritance in flax. Jour. Hered. 18: 336-343. 1927. (26) Chittenden, R . J . and Caroline Pellew. A suggested inter- petation of certain cases of anisogeny. Nautute 119: 10-12. 1927. (27) Clarke, A .E . and J.R.Fryer. Seed-setting in a l fa l fa . Sc i . Agric. 11: 38-43. 1930. (28) Clarke, A . E . and L.H.Pol lard. The amount_of se l f -pol l in- ation in male-sterile onion l ines . Proc. Amer. Soc. Hort. Sc i . 53: 299-301. 1949. (29) Coffman, F .A . Pol l inat ion in a l fa l fa . Bot. Gaz. 74: 197-203. 1922. (30) Cooper, D.C. Macrosporogenesis and embryology of Medicago. Jour. Agr. Res. 471-477. 1933. ( 3 1 ) C o o p e r , D . C . a n d R . A . B r i n k . P a r t i a l s e l f - i n c o m p a t i b i l i t y a n d . t h e c o l l a p s e , o f f e r t i l e o v u l e s a s f a c t o r s a f f e c t i n g s e e d f o r m a t i o n " i n a l f a l f a . J o u r . A g . R e s . 6 0 : 4 5 3 - 4 7 2 . 1 9 4 0 . ( 3 2 ) a n d H . R . A l b r e c h t . E m b r y o m o r t a l i t y i n r e l a t i o n t o s e e d f o r m a t i o n i n a l f a l f a , ( M e d i c a g o s a t i v a ) . A m e r . J o u r . B o t . 2 4 : 2 0 3 - 3 1 3 . 1 9 3 7 . ( 3 3 ) C r a n e , M . B . H e r e d i t y o f t y p e s o f i n f l o r e s c e n c e a n d f r u i t s i n t o m a t o . J o u r . G e n e t i c s . 5 : 1 - 1 1 . 1 9 1 5 . ( 3 4 ) C r a n e , M B . a n d W . J . C . L a w r e n c e . T h e G e n e t i c s o f G a r d e n P l a n t s . M a c m i l l a n a n d C o . , L o n d o n . 2 3 0 p . 1 9 3 4 . ( 3 5 ) L e C a n d o l l e , A . P . P h y s i o l o g i e V e g e t a l e . P a r i s . 5 4 8 . - p . 1 8 3 2 . ( 3 6 ) D o b z h a n s k y , T . G e n e t i c s a n d t h e o r i g i n o f s p e c i e s . . . C o l u m b i a U n i v e r s i t y P r e s s . 2 n d E d . 4 4 6 p . 1 9 4 7 . ( 3 7 ) D w y e r , R . E . F u r t h e r o b s e r v a t i o n s o n p o l l i n a t i o n a n d s e e d - s e t t i n g i n l u c e r n e . A g r i c . G a z . H e w S o u t h W a l e s . ^ 4 3 : 1 4 1 - 1 4 6 . 1 9 3 2 . ( 3 8 ) E a s t , E . M . S t u d i e s o n s e l f - s t e r i l i t y . I X . T h e b e h a v i o u r o f c r o s s e s b e t w e e n s e l f - s t e r i l e a n d s e l f - f e r t i l e p l a n t s . G e n e t i c s 1 7 : 1 7 5 - 2 0 2 . 1 9 3 2 . ( 3 9 ) a n d S . H . Y a r n e l l . S t u d i e s o n s e l f - s t e r i l i t y . V I I I . S e l f - s t e r i l i t y a l l e l o m o r p h s . G e n e t i c s 1 4 : 4 5 5 - ^ 4 8 7 . 1 9 2 9 . ( 4 0 ) E m e r s o n , R . A . , G . W . B e a d l e a n d A . C . E r a s e r . A s u m m a r y o f l i n k a g e s t u d i e s i n m a i z e . C o r n e l l U n i v . A g r i c . E x p t . " S t a . M e m o i r 1 8 0 : 8 3 p . 1 9 3 5 . ( 4 1 ) E r y s t e r , L . A . H e r i t a b l e c h a r a c t e r s o f m a i z e . V I I . M a l e - s t e r i l e . J o u r . H e r e d . 1 2 : 1 3 8 - 1 4 1 . 1 9 2 1 . . . - ( 4 2 ) F a b e r g e , A . C . T h e c y t o l o g y f o t h e m a l e - s t e r i l e L a t h y r u s - o d o r a t u s . . G e n e t i c a 1 9 : 4 2 3 - 4 3 0 . 1 9 3 7 . ( 4 3 ) F i n e m a n , Z o l a M . E l i m i n a t i o n a n d r e t e n t i o n o f p o l l e n s t e r i l i t y i n p o t a t o i m p r o v e m e n t . J o u r . A g . R e s . 7 5 : 1 3 5 - 1 4 5 . 1 9 4 7 . ( 4 4 ) F r a n k e l , O . H . S t u d i e s i n H e b e I I . T h e s i g n i f i c a n c e o f m a l e - s t e r i l i t y i n t h e g e n e t i c s y s t e m s . J o u r . H e r e d . 4 0 : 1 7 1 - 1 8 4 . 1 9 4 0 . ( 4 4 a ) F r y e r , J . R . C y t o l o g i c a l s t u d i e s i n M e d i c a g o . M e l i l o t u s a n d T r i g o n e l l a . C a n . J o u r . R e s . 3 : 3 - 5 0 . 1 9 3 0 . ( 4 5 ) G r e g o r y , R . P . T h e a b o r t i v e d e v e l o p m e n t o f t h e p o l l e n i n c e r t a i n s w e e t p e a s . P r o c . C a m b . P h i l . S o c . 1 3 : 1 4 8 - 1 5 7 . 1 9 0 5 . ( 4 6 ) H a d f i e l d , J . W . a n d R . A . G a l d e r . L u c e r n e ( M e d i c a g o s a t i v a ) i n v e s t i g a t i o n s r e l a t i v e t o p o l l i n a t i o n a n d s e e d p r o d u c t i o n i n H e w Z e a l a n d . H . Z . J o u r . S c i . a n d T e c h . 1 7 : 5 7 7 - 5 9 4 . 1 9 3 6 . ( 4 7 ) H a y , W . D . D o e s a r t i f i c i a l t r i p p i n g o f a l f a l f a b l o s s o m s " i n c r e a s e s e e d s e t t i n g ? S c i . A g r . 5 : 2 8 9 - 2 9 0 . 1 9 2 5 . ( 4 8 ) H a y e s , H . K . Y i e l d g e n e s , h e t e r o s i s a n d c o m b i n i n g a b i l i t y . A m e r . H a t . 8 0 : 4 3 0 - 4 4 5 . 1 9 4 6 . ( 4 9 ) H o u s t o n , B y r o n R . D w a r f r e s i s t a n t a l f a l f a . S e e d o f n e w C a l . C o m m o n 4 9 t o b e r e l e a s e d n e x t s e a s o n f o r c o m m e r c i a l h a y p r o d u c t i o n i n c e r t a i n a r e a s . G a l . A g r i c . 3 : 3 - . 1 9 4 9 . ( 5 0 ) J o n e s , D . P . S e l e c t i v e f e r t i l i z a t i o n . T h e U n i v e r s i t y o f C h i c a g o S c i e n c e S e r i e s . 1 6 3 p . 1 9 2 8 . ( 5 1 ) J o n e s , D o n a l d P . T h e i n t e r r e l a t i o n o f p l a s m a g e n e s a n d c h r o m o g e n e s i n p o l l e n p r o d u c t i o n i n m a i z e . G e n e t i c s 3 5 : 5 0 7 - 5 1 2 . 1 9 5 0 . ( 5 2 ) J o n e s , H . A . a n d A . E . C l a r i c e . I n h e r i t a n c e o f m a l e - s t e r i l i t y i n t h e o n i o n a n d t h e p r o d u c t i o n o f h y b r i d s e e d . P r o c . A m e r . S o c . H o r t . S c i . 4 3 : 1 8 9 - 1 9 4 . 1 9 4 3 . ( 5 3 ) J o s e p h s o n , L . M . a n d M e r l e T . J e n k i n s . M a l e - s t e r i l i t y i n c o r n h y b r i d s . J o u r . A m e r . S o c . . A g r o n . 4 0 : 2 6 7 - 2 7 4 i 1 9 4 8 . ( 5 4 ) J u l e n , G . I n v e s t i g a t i o n s o n d i p l o i d , t r i p l o i d , a n d t e t r a p l o i d l u c e r n e . H e r e d i t a s 3 0 : 5 6 7 - 5 8 2 . 1 9 4 4 . ( 5 5 ) K i r k , L . E . a n d W . J . W h i t e . A u t o g a m o u s a l f a l f a . S c i . A g r . . . 1 3 : 5 9 1 - 5 9 3 . . 1 9 3 3 . ( 5 6 ) K n o w l e s , R . P . T h e r o l e o f i n s e c t s , w e a t h e r c o n d i t i o n s ' a n d p l a n t c h a r a c t e r o n s e e d p r o d u c t i o n i n a l f a l f a . S c i . A g r . 2 4 : 2 9 - 5 0 . 1 9 4 3 . ( 5 7 ) L e d i n g h a m , G . F . C y t o l o g i c a l a n d d e v e l o p m e n t a l s t u d i e s o f h y b r i d s b e t w e e n M e d i c a g ; o s a t i v a a n d a d i p l o i d f o r m o f M . f a l e a t a . G e n e t i c s 2 5 : 1 - 1 5 . 1 9 4 0 . ( 5 8 ) L e o n a r d , W a r r e n H . a n d A n d r e w G . C l a r k e . F i e l d P l o t T e c h n i q u e . B u r g e s s P u b . C o . 1 9 5 0 . ( 5 9 ) L e s l e y , J . W . a n d M a r g a r e t L e s l e y . U n f r u i t f u l n e s s i n ' t h e t o m a t o c a u s e d "by m a l e - s t e r i l i t y . J o u r . A g . R e s . 5 8 : 6 2 1 - - 6 3 0 . 1 9 3 9 . ( 6 0 ) L e s i n s , . E a r l i s . I n v e s t i g a t i o n s i n t o s e e d - s e t t i n g o f l u c e r n e a t U l t a n a , S w e d e n , 1 9 4 5 - 1 9 4 9 . A n n a l s o f t h e R o y a l A g r i c u l t u r a l C o l l e g e o f S w e d e n . 1 7 : 4 4 2 - 4 8 3 . 1 9 5 0 . ( 6 1 ) L e v / i s , D. M a l e - s t e r i l i t y i n n a t u r a l p o p u l a t i o n s o f h e r m a p h r o d i t e p l a n t s . N e w P h y t o t . 4 0 : . 5 6 - 6 3 . 1 9 4 1 . ( 6 2 ) M a r t i n , J . N . R e l a t i o n o f m o i s t u r e t o s e e d p r o d u c t i o n i n a l f a l f a . I o w a A g r i c . J U x p . S t a . B u l l . 2 3 : 3 0 3 - 3 2 4 . 1 9 1 5 . ( 6 3 ) M e n z i e s , J . D . W i t c h e s ' b r o o m o f a l f a l f a i n N o r t h Miexic a P h y t o p a t h . 3 6 : 7 6 2 - 7 7 4 . 1 9 4 6 . ( 6 4 ) M o r r i s o n , P r a n k B . F e e d s a n d f e e d i n g . 2 1 s t E d . T h e M o r r i s o n P u b . C o . 1 9 4 9 . ( 6 5 ) N i l a n , R . A . R h i z o m a a l f a l f a : C h r o m o s o m e s t u d i e s o f t h e p a r e n t s t o c k s . S c i . A g r . 3 1 : 1 2 3 - 1 2 6 . 1 9 5 1 . ( 6 6 ) N i l s s o n , P . a n d E . A n d e r s s o n . P o l y p l o i d y i n t h e g e n u s M e d i c a g o . H e r e d i t a s 2 9 : 1 9 7 - 1 9 8 . 1 9 4 3 . ( 6 7 ) O w e n , F . V . C y t o p l a s m i c a l l y i n h e r i t e d m a l e - s t e r i l i t y i n s u g a r - b e e t s . J o u r . A g . R e s . 7 1 : 4 2 3 - 4 4 0 . 1 9 4 5 . ( 6 8 ) P e c k , 0 . a n d J . L . B ' . o l t o n . A l f a l f a s e e d p r o d u c t i o n i n ' n o r t h e r n . S a s k a t c h e w a n a s a f f e c t e d b y b e e s . S c i . A g r . 2 6 : 3 8 8 - 4 1 8 . 1 9 4 6 . ( 6 9 ) P i p e r , C . V . , M . W . E v a n s , R. M c K e e , a n d W . J . M o r s e . A l f a l f a s e e d p r o d u c t i o n : p o l l i n a t i o n s t u d i e s . U . S . D . A . B u l l . 7 5 : 1 9 1 4 . ( 7 0 ) P u n n e t t , R . C . L i n k a g e g r o u p s a n d c h r o m o s o m e n u m b e r ' i n L a t h y r u s . P r o c . R o y . S o c . 1 0 2 : 2 3 6 - 2 3 8 . 1 9 2 7 . ( 7 1 ) F u r t h e r s t u d i e s o f l i n k a g e i n t h e s w e e t p e a . J o u r . G e n e t i c s 2 6 : 9 7 - 1 1 2 . 1 9 3 2 . ( 7 2 ) R h o a d e s , M . C y t o p l a s m i c i n h e r i t a n c e o f m a l e - s t e r i l i t y i n Z e a m a v s . S c i e n c e 7 3 : 3 4 0 - 3 4 1 . 1 9 3 1 . ( 7 3 ) C y t o p l a s m i c i n h e r i t a n c e o f m a l e - s t e r i l i t y i n Z e a m a y s . J o u r . G e n e t i c s 2 7 : 7 1 - 9 3 . 1 9 3 3 . ( 7 4 ) R i c k , C M . A s u r v e y o f c y t o g e n e t i c c a u s e s o f u n f r u i t - f u l n e s s i n t h e t & m a t o . G e n e t i c s 3 0 : 3 4 7 - 3 6 2 . 1 9 4 5 . -62- (75) Rick, C.M. Genetics and development of 9 male-sterile tomato mutants. Hi&gardia 18: 599-633. 1948. (76) Rife , David G. The genetics of certain common variations in Goleus. Ohio Jour. Sc i . 44: 18-24. 1944. (77) . Simply inherited variations in Coleus. "~ Jour, of Hered. 39: 85-91. 1948. (78) Ri ley, Herbert Parkes. Se l f - s t e r i l i ty in Shepherd's Purse. Genetics 17: 231-295. 1932. (79) ; . A further test showing the dominance of s e l f - f e r t i l i t y to s e l f - s t e r i l i t y i n ' Shepherd's Purse. -Amer. Naturalist 68: 60-64. 1934. (80) - .- The genetics and physiology of s e l f - s t e r i l i t y in the genus Gapsella. Genetics 21: 24-39. 1936. (81) Roever, V.E. A promising type of male-steri l i ty for use in hybrid tomato seed production. Science 107: 506. 1948. (82) Scott, D.H. and M.E.Riner. Inheritance of male-steri l i ty in winter squash. ' Proc. Amer. Soc. Hort. Sc i , 47: 375-377. 1946. (83) Shifr iss , Oved. Male-ster i l i t ies and albinos in cucurbits, Jour. Hered. 36: 47-51. 1945. (84.) Shull , G.H. Inherited pollen s t e r i l i t i e s in Shepherd's Purse. Memoir Hort. Soc. N.Y. 3: 353-368. 1927.. (85) Sinnott, Edmund ¥ . and L.G.Dunn. Principles of Genetics. 3rd Ed. McGraw-Hill Book Company, New York. 1939. (86) Southworth, V. Influences which tend to affect seed production in a l fa l fa and an attempt to raise a high seed-producing strain by hybridization. Sc i . Agr. 9: 1-29. 1928. (87) Stevenson, T.M. and J .L.Bolton. An evaluation of the self-tr ipping character in breeding for improved seed y i e l d in a l fa l fa . Empire Jour. Exp. Agr. 15: 82-88. 1947. (88) Suneson, C.A. A male-sterile character in barley. Jour. Hered. 31: 213-214. 1940. (89) Tome, Gino A. (Spanish t i t l e ) (The improvement of alfalfa.) Rev. Argentina Agron. 14: 279-313. 1947. -63- (90) Tysdal, H.M. and T.A.Kiesselbach. Alf alf a Nursery . . technique. Jour, Amer. Soc. Agr on. 31: 83-98. 1939. (91) . Hybrid a l fa l fa . Jour. Amer. Soc. Agron. 36: 649-667. 1944. (92) , and H.L.Westover. Alf alf a breeding. Nebr. Agr. Expt...Sta. nea. B u l l . 124. 46 p. 1942. (93) Tysdal, H.M. and Bl i s s M. Crandall. The polycross progeny performance as an index of the combining abi l i ty of a l fa l fa clones. Jour. Amer. Soc. Agron. 40: 293-306. 1948. (94) ^Tysdal, H.M. and J. Russell Garl. A new method of a l fa l fa emasculation. Jour. Amer. Soc. Agron. 32: 405-407. 1940. (95) Tysdal, H.M. and H.L.Westover. Growing a l fa l fa . TJ.S.D.A. Farmers B u l l . 1722. 33 p. 1949. (96) Welch, J .E . and E.L.Grimball . Male-s ter i l i ty in the carrot. Science 106: 594. 1947. (97) White, William J. Alfalfa, Improvement. Advances in Agronomy 2: 205-240. 1949. (98) Wilcoxon, Frank. Some rapid approximate s ta t i s t i ca l •procedures. Annals of t he New York Acad, of Sciences. 52: 808-814. 1950. (99) Wilse, C.P. and John Skory. S e l f - f e r t i l i t y of erect and pasture type a l fa l fa clones as related to the vigour and f e r t i l i t y of their inbred and outcrossed progenies. Jour. Amer. Soc. Agron. 40: 786-794. 1948. B - THE WITCHES' BROOM VIRUS DISEASE OF ALFALFA IN B.C. INTRODUCTION Although Witches' Broom of Al fa l fa was f i r s t reported in B.C. only recently (1932 - Foster (17)), the disease has "been recognized in Austral ia for 40 to 55 years. However recent i t s dicovery in this province, the disease is developing into serious proportions in parts of the Interior. The ravages of the disease had assumed enough importance by 1948 that the Department of Agronomy at U.B.C. fe l t the need for some invest- igation into the nature and extent of the disease. Since i t s discovery in North America, Witches' Broom of Al fa l fa has as yet received comparatively l i t t l e attention due probably to i t s apparent sporadic occurrence. In Australia,, on the other hand, the disease has been suff iciently serious to command continuing f inancial support of f a i r l y extensive investigations. Investigations performed in Australia and in the United States has shown the disease to be caused by a virus and to be d i f f i cu l t to transmit mechanically. The pathogen is probably disseminated by insects. Symptomology is well desc- ribed by a number of workers: Edwards (12), Menzies (34), and Smith (42). However, very l i t t l e basic information has been acquired so far on the true nature and fundamentals of the disease. Cut of the studies in i t ia ted at U.B.C. in 1949, data regarding spread, distr ibution, seriousness are to be acquired along with information regarding any resistance inherent in 65- members of the genus Medieago. The f ina l goal is the prod- uction of strains or varieties capable of res is t ing the disease. It i s hoped that information on methods of trans- mission, insect vectors of the disease, and the host range of the virus w i l l "be obtained by plant pathologists and entomologists. A program, such as outlined, w i l l require many years to consummate. Therefore, my part of the investigation w i l l consist of setting up foundation experiments, and the comp- i la t ion of data of other workers relevant to the investigation. LITERATURE REVIEW A. - History and, dis tr ibution. Witches' Broom of Al fa l fa was f i r s t recognized as a disease in Australia. McCleery (36) reported that in 1924 the disease was prevalent throughout the drier parts of New South Wales. Noble and associates (39) reported in 1947 that, of the 417,000 acres under lucerne (alfalfa) in New South Wales, 235,000 acres occur in areas where the Witches' Broom disease is known to be very prevalent. In these areas, the disease is considered to be the main factor l imi t ing the profitable l i f e of lucerne stands. Economic stands could only be main- tained 4 to 5 years before ploughing up and re sowing was necessitated. In areas where lucerne was grown for seed, the diseased plants represent a total loss, as seed is not produced normally. Edwards (12) reported in 1936 on investigations into the disease which began in 1931 (with preliminary work dating back to 1929). At that time he reported the disease to be very widespread throughout the inland areas of Hew South Vales where, under an average annual r a i n f a l l of 17 to 21 inches, lucerne is grown for gracing purposes. Fields that were 4 to 5 years old showed 20 to 25% infection while stands more than 7 to 8 years old commonly showed 70% infection. However, he ' reported that the Witches' Broom disease is not known to occur in crops less than 15 to 18 months old. Edwards (ibid) also reported the disease to be present, although less severe than in Hew South Wales, in Queensland, Vic tor ia , and South Australia. The f i r s t Horth American record of Witches' Broom of Al fa l fa was made by Haskell (20) in 1925 with observations of the disease as affecting some plants in Salt Lake County of Idaho. In the same year, Richards (41) reported the appear- ance of the disease in Utah. In 1932, Foster (17) reported the disease as occurring in 2 widely divergent parts of B . C . , but with the cause unknown. Interest in the disease in Horth America was crystal l ized by the work of Menzies (34) in Washington published in 1946. At that time the disease dist- ribution was l imited to the area between the Cascade and Rocky mountains excepting an eastern extension of the disease into Alberta, and a single report from Vancouver Island. The Canadian Plant Disease Survey Annual Reports for the years 1922 to 1949 contains 27 mentions of the Witches' Broom of Alfa l fa , and a chronological l i s t i n g of the citations points out clearly the trend of the disease. Some references 67< t o a ( p o s s i b l y r e l a t e d ) w i t c h e s ' b r o o m c o n d i t i o n i n s o m e o t h e r l e g u m e s i s a l s o c i t e d . 1 9 2 2 - 1 9 3 1 ; H o m e n t i o n . • : 1 9 3 2 : F o s t e r ( 1 7 ) r e p o r t e d 2 y e a r o l d a l f a l f a p l a n t s a f f e c t e d w i t h a w i t c h e s ' b r o o m w e r e s e n t t o t h e l a b o r a t o r y f r o m S m i t h e r a n d f r o m S a a n i c h t o n . H e r e p o r t e d t h e d i s e a s e a s b e i n g f o u n d o n c l o v e r a t t h e s a m e p l a c e s . 1 9 3 3 : H o m e n t i o n . 1 9 3 4 : A n o n y m o u s . A t L y t t o n a n d M c G i l l i v r a y ' s F l a t s t h e r e w e r e c e r t a i n p a t c h e s i n t h e f i e l d s , w h i c h c o n t a i n e d s e v e r a l p l a n t s a f f e c t e d w i t h W i t c h e s ' B r o o m . T h e d i s e a s e w a s a l s o n o t e d i n o t h e r f i e l d s i n C a r i b o o c o u n t y a n d a s i n g l e d i s e a s e d p l a n t w a s r e c o r d e d f r o m S u m m e r l a n d . 1 9 5 5 : A n o n y m o u s . I T - 20% o f t h e p l a n t s i n t h e i r r i g a t e d s e c t i o n , C a r i b o o c o u n t y , B . C . w e r e i n f e c t e d , b e i n g w o r s t i n o l d e r f i e l d s . 1 9 5 6 : H o m e n t i o n . 1 9 5 7 : J o n e s ( 5 7 ) r e p o r t e d ' D w a r f t o b e p r e v a l e n t i n t h e C a r i b o o d i s t r i c t s a n d o t h e r p a r t s o f t h e i n t e r i o r o f B . C . 1 9 5 8 : J o n e s ( 3 8 ) r e p o r t e d a f e w p l a n t s o f t h e L a d a k v a r i e t y w e r e a f f e c t e d a t t h e A g a s s i z S t a t i o n , B . C . I t w a s w i d e l y d i s t r i b u t e d i n t h e i n t e r i o r o f t h e p r o v i n c e , p r i n c i p a l l y i n t h e i r r i g a t e d a r e a s . 1 9 5 9 : H o m e n t i o n . 1 9 4 0 ' : C o r m a c k ( 5 ) r e p o r t e d a b o u t 1% o f t h e p l a n t s i n a p l o t a t E d m o n t o n , A l b e r t a , w e r e m o d e r a t e l y t o s e v e r e l y a f f e c t e d a n d m a n y o t h e r s w e r e b e g i n n i n g t o s h o w t h e s y m p t o m s . 1941; Cormack (4) reported a few plants to be moderately affected in 3 f ields at Cherhi l l , Alt a. 5$ of the plants were dead or severely infected in the plot at Edmonton. 1942: Brink (2) reported the disease to be present in one or two f ie lds in the Nicola Valley of B r i t i s h Columbia, where alfalfa, stands last for only 3 or 4 years as a result of the disease. Brink (ibid) reported the disease to be identical with that described in Washington. 1942; Heald and Menzies (22) report the disease to be present in 4 counties in the state of Washington, and to be serious in the Methow valley area of Ok an agon county. They stated that the disease was identical with that described by Edwards in Australia (12). 1942: Cormack (5) reported the damage from Witches' Broom to be increased in the plots under observation at Edmonton, Alt a.. 1945: Woolliams (4$) reported the disease as affecting at least 5% of the plants in a f i e l d at Armstrong, B .C. 1943: Cormack (6) reported the disease had advanced somewhat in the f ields at Cherh i l l , Alta . and also in the plot at Edmonton, Alta. 1944: Cormack (7) reported that the f i e l d under observation at Edmonton was so thinned out by the disease that i t was ploughed up. In the same year, he reported occasional plants of a l fa l fa were severely infected in a f i e l d at Bremner, Alta . and in an old plot at Lacombe, Alta . 1945: Cormack (8) reported slight damage in the University plots at Edmonton, Alta . 1 9 4 6 : C o r m a c k ( 9 ) r e p o r t e d t h e d i s e a s e f o r t h e f i r s t t i m e f r o m S a s k a t c h e w a n , w h e r e i t w a s a f f e c t i n g 2 f i e l d s i n t h e W h i t e F o x d i s t r i c t a n d o n e f i e l d i n t h e L o o n L a k e d i s t r i c t . 1 9 4 6 : W r i g h t (SO) r e p o r t e d t h a t a r e l a t i v e l y h i g h p r o p o r t i o n ( 1 0 - 15%) o f d i s e a s e d p l a n t s o c c u r r e d i n 5 - 6 y e a r o l d a l f a l f a , s t a n d s i n t h e H o r t h O k a n a g o n . 1 9 4 7 : W r i g h t (51)) r e p o r t e d t h a t 20% o f t h e p l a n t s i n a n e i g h t y e a r o l d a l f a l f a f i e l d i n t h e C a r i b o o d i s t r i c t w e r e a f f e c t e d , a n d d a m a g e w a s e s t i m a t e d a t 1 5 % . A m u c h l o w e r p e r c e n t a g e o f i n f e c t i o n w a s o b s e r v e d i n r e c e n t l y s e e d e d s t a n d s . 1 9 4 7 : G i l p a t r i c k ( 1 8 ) r e p o r t e d a f e w p l a n t s w e r e i n f e c t e d i n f o u r o f t h e s t a n d s e x a m i n e d i n c e n t r a l a n d n o r t h e r n A l b e r t a . I n t h e s a m e y e a r , h e r e p o r t e d a f e w p l a n t s o f A l t a - s w e d e r e d c l o v e r w e r e s e v e r e l y a f f e c t e d a t E d m o n t o n , A l t a . 1 9 4 8 : W o o l l i a m s ( 4 ^ ) r e p o r t e d t h e d i s e a s e a s b e i n g q u i t e p r e v a l e n t a r o u n d L y t t o n a n d L i l l o o e t i n B . C . , a n d t h e o d d p l a n t w a s a f f e c t e d i n a f i e l d a t S h u s w a p - , ' B , C . 1 9 4 8 : M e a d ( 3 3 ) f o u n d a t r a c e o f t h e d i s e a s e i n a n o l d f i e l d e a s t o f T i s d a l e , S a s k . 1 9 4 8 : T h o m s o n a n d L e b e a u r e p o r t e d ( 4 3 ) a f e w p l a n t s w e r e f o u n d a f f e c t e d i n 2 s t a n d s i n c e n t r a l A l b e r t a . 1 9 4 8 : M u n r o ( 3 5 ) r e p o r t e d a c l o v e r p l a n t s h o w i n g s y m p t o m s r e s e m b l i n g W i t c h e s 1 B r o o m , o f A l f a l f a i n t h e C a r i b o o . 1 9 4 9 : L e b e a u ( 3 1 ) r e p o r t e d i n f e c t i o n w a s 5 - t r 3 - s l / 2 4 0 f i e l d s i n c e n t r a l A l t a . a n d P e a c e R i v e r d i s t r i c t . 1 9 4 9 : A n o n y m o u s . A f e w a f f e c t e d p l a n t s w e r e s e e n i n a n o l d f i e l d a t H u d s o n B a y J u n c t i o n , S a s k . - 7 0 - 1 9 4 9 : W r i g h t ( 5 1 ) o b s e r v e d . W i t c h e s ' B r o o m f o r t h e s e c o n d y e a r i n a, 1 / 1 0 a , c r e p l o t o f a l s i k e c l o v e r a t t h e E x p t . S t a , . P r i n c e G e o r g e , B . C . ; a b o u t 1 5 % o f t h e p l a n t s w e r e a f f e c t e d . I t a l s o a f f e c t e d 25% o f t h e p l a n t s o f B i r d s f o o t T r e f o i l ( L o t u s c o r n i c u l a t u s ) i n 2 2 5 f o o t r o w s a t t h e s t a t i o n . " W i t c h e s ' b r o o m w a s a l s o f o u n d t o b e a f f e c t i n g a t l e a s t 1 0 % o f t h e w i l d l u p i n p l a n t s ( L u p i n u s s p . i n d e t . ) , w h i c h g r o w o n u n c u l t i v a t e d l a n d a b o u t P r i n c e G e o r g e . O n l y 3 - 4 p l a n t s o f r e d c l o v e r g r o w i n g w i l d w e r e f o u n d a f f e c t e d a t Q u e s n e l l i n t h e C a r i b o o d i s t r i c t " . 1 9 4 9 : P a y e t t e ( 4 0 ) r e p o r t e d t h a t i n s o m e p l o t s o f L a d i n o w h i t e c l o v e r a t t h e s t a t i o n , S t e . A n n e d e l a P o c a t i e r e , P . Q . , p l a n t s f a i l e d t o f l o w e r , b u t i n s t e a d n u m e r o u s l i t t l e l e a v e s d e v e l o p e d g i v i n g t h e a p p e a r a n c e o f w i t c h e s ' b r o o m . . T h e s a m e t r o u b l e w a s o b s e r v e d i n a l l s t a n d s o f L a d i n o c l o v e r o v e r o n e y e a r o l d i n s p e c t e d i n L ' I s l e t c o u n t y . 1 9 4 9 : M a c L e o d ( 3 7 ) f o u n d 5 r e d c l o v e r p l a n t s s h o w i n g s y m p t o m s r e s e m b l i n g w i t c h e s ' b r o o m i n a f i e l d i n Y o r k C o . , N . B . B - N a t u r e o f t h e l o s s e s d u e t o W i t c h e s ' B r o o m o f A l f a l f a , . W i t c h e s ' B r o o m o f A l f a l f a • c a u s e s a d e f i n i t e d e c r e a s e i n y i e l d o f f o r a g e a s a r e s u l t o f t h e d w a r f i n g , b u t t h e c h i e f s o u r c e o f l o s s i s t h e r e d u c t i o n o f s t a n d s b y t h e e a r l y d e a t h o f t h e p l a n t . I n f e c t e d p l a n t s s u c c u m b v e r y r e a d i l y t o w i n t e r k i l l i n g , u n d o u b t a b l y d u e t o t h e i r w e a k e n e d c o n d i t i o n b r o u g h t a b o u t b y t h e g r e a t l y i n c r e a s e d d e m a n d s o n s t o r e d f o o d r e s e r v e s i n t h e p r o d u c t i o n o f " m y r i a d s " o f p r o l i f e r a t i o n s . E d w a r d s ( i b i d ) r e p o r t e d o n a y i e l d e x p e r i m e n t i n - 7 1 - 1\ A u s t r a l i a c o n d u c t e d o v e r a p e r i o d o f 3 y e a r s ( 1 9 3 2 - 3 4 ) t o d e t e r m i n e t h e e f f e c t o f t h e " W i t c h e s ' B r o o m d i s e a s e o n t h e a m o u n t o f f o d d e r p r o d u c e d "by d i s e a s e d p l a n t s u n d e r f i e l d c o n d i t i o n s . H e f o u n d t h a t d u r i n g t h e p e r i o d t h e e x p e r i m e n t w a s i n p r o g r e s s t h e d i s e a s e d p l a n t s h a v e g i v e n a m e a n y i e l d o f 3 7 . 4 $ l e s s g r e e n w e i g h t o f f o d d e r t h a n t h e u n a f f e c t e d p l a n t s . S e e d p r o d u c t i o n i s a l m o s t t o t a l l y i n h i b i t e d i n d i s e a s e d p l a n t s . T h e m a j o r i t y o f t h e d i s e a s e d p l a n t s f a i l t o f l o w e r , " b u t o c c a s i o n a l l y " b l o o m s m a y "be p r o d u c e d w h i c h a r e c o n s i d e r a b l y s m a l l e r a n d p a l e r i n c o l o u r t h a n t h e n o r m a l i n f l o r e s c e n c e a c c o r d i n g t o S m i t h ( 4 2 ) . T h e o c c a s i o n a l f l o w e r s t h a t a r e p r o d u c e d a r e u s u a l l y i n g r o u p s o f 2 o r 3 i n s t e a d o f t h e m u l t i - f l o w e r e d r a c e m e s o f t h e n o r m a l i n f l o r e s c e n c e . B o t h M e n z i e s ( i b i d ) a n d E d w a r d s ( i b i d ) r e p o r t v e r y l i t t l e s e e d i s p r o d u c e d f r o m t h e d i s e a s e d f l o w e r s i n e i t h e r N o r t h A m e r i c a o r A u s t r a l i a r e s p e c t i v e l y . E d w a r d s ( i b i d ) a d d s t h a t i n a r e a s w h e r e l u c e r n e i s g r o w n f o r s e e d p r o d u c t i o n t h e a f f e c t e d p l a n t s r e p r e s e n t a t o t a l l o s s . G - O t h e r a l f a l f a v i r u s e s a n d v i r u s d i s e a s e s . K e n n e t h S m i t h ( 4 2 ) l i s t s 4 v i r u s e s a s a f f e c t i n g a l f a l f a , n a m e l y : l ) M e d i c a g o v i r u s 1 W e i m e r - c a u s i n g C o m m o n A l f a l f a M o s a i c 2 ) M e d i c a g o v i r u s 2 P i e r c e , Z a u m e y e r a n d W a d e - c a u s i n g A l f a l f a M o s a i c , 3 ) M e d i e a g o v i r u s 3 W e i m e r - c a u s i n g A l f a l f a L V a r f D i s e a s e , 4 ) M e d i c a g o v i r u s 4 E d w a r d s - c a u s i n g W i t c h e s ' B r o o m o f A l f a l f a . -72- 1. Common Alf alf a Mosaic . Previous to 1931, records- of f i e l d observations only were made of this disease: the existance of a transmiss- able viros is of the mosaic type affecting a l fa l fa had not been proven experimentally. Weimer (45) transmitted i t success- ful ly in 1931 with the aphid I l l i n o i a p i s i . It might be noted however that in 1922, Dickson (10) working at MacDonald College transmitted a mosaic disease of clover to Medica^o sativa using the same vector. Veimer (46) reporting on further studies in Cal i fornia found no severe losses from Al fa l f a Mosaic, and stated that the disease damage was l imited to a very slight dwarfing of most of the seriously infected plants. The f i r s t evidence of the disease in a leaf is the appearance of one or more small, more or less c ircular , greenish-yellow spots. These areas frequently consist of a yellowish band of tissue to 1 mm. in width, surrounding an island of apparently normal color -§- to 2 mm. in diameter. There may be one or more concentric rings of green surrounding narrow bands of chlorotic tissue. The chlorosis spreads unt i l the rings are more or less obscured and the leaves sometimes completely chlorotic . In some severe cases the leaves are reduced to 3/3 their normal size, are crinkled and more or less deformed. Although there may be some dwarfing of dis- eased stems in severe cases, normally there is no reduction in size. Necrotic lesions have not been observed on the stems, and the disease does not cause premature defoliation. -73- Weimer (46) attempted several mechanical methods of trans- mitting the common mosaic disease."but with negative results . He did achieve, as noted ear l ier , successful transmission with the pea aphid ( I l l i n o i a p i s i ) . The period of incubation was found to be 7 to 14 days from time of inoculation to the appearance of primary symptoms. Common a l fa l fa mosaic, frequently recorded from Cal i f - ornia, is probably exist ant in other states.. The disease is favored by moderately, cool damp periods in f a l l and spring. Interestingly enough, in the last report of the Al fa l fa Improvement Conference (l) the disease is not recorded. 2. Al fa l fa mosaic. Weimer (46) considered the virus causing this disease to be a strain of Me die ago virus 1,. as the disease that i t causes somewhat ressembles Common Mosaic. Inasmuch the causal agent differs in several important properties from the former virus, this pathogen has been considered .to be a separate entity and class i f ied accordingly by Smith ( ibid) . The virus is sap-transmis sable, whereas the former was not. The vector, I l l i n o i a p i s i , is common to both viruses. Whereas the former virus caused a mosaic only of a l fal fa , this virus has a wide host range: Hyacinth bean, adzuki bean, mung bean, r ice bean, common and Turkestan al fa l fa , white sweet clover,, crimson clover, red clover, garden pea3, spring vetch and soybeans are a l l infected. Its affect on a l fa l fa is more severe than the Medieago virus 1. in that the affected plants are decidedly dwarfed and the leaves are d i s t i n c t l y mottled and c r i n k l e d . Mosaic has "been reported "by MacLeod (37a) i n 2 f i e l d s of Grimm a l f a l f a at Predericton, H.B., and i n a f i e l d of a l f a l f a near Oromocto, H.B. Hurst (26a) reported i n 1945 that mosaic affected an occasional plant i n Prince Edward Island f i e l d s . However, i n the United States, previous to 1949, the disease was recorded only from Wisconsin. In 1949, McWhorter (38) reported a necrotic s t r a i n of a l f a l f a mosaic v i r u s which he i s o l a t e d from a l f a l f a and peas i n eastern Washington and Oregon, and which was usually accompanied by yellow bean mosaic. The host range was t y p i c a l of a l f a l f a mosaic, but the thermal i n a c t i v a t i o n point i s 10° to 15° lower than those previously recorded f o r that v i r u s (62-64°C f o r 10 minutes). 3. A l f a l f a Dwarf Disease. •When and where t h i s disease originated i s unknown. However, i n 1919 - 1921 farmers i n C a l i f o r n i a south of the Tehachapi mountains could maintain s a t i s f a c t o r y stands of a l f a l f a f o r 8-10 years, whereas i n 1931 stands were seldom worth maintaining over 3 years. There has been no evidence of the disease outside of southern C a l i f o r n i a . Dwarf disease i s mainly responsible f o r the s h o r t - l i v e d stands of a l f a l f a i n t h i s area. The symptoms were f i r s t described by Weimer (44) i n 1931, and a short time l a t e r the same investigator (47) showed the virus nature of the disease. The a a r l i e s t symptoms of the disease cannot be detected above ground, and A l f a l f a Dwarf i s very well established i n the root before i t i s evident i n the top. The f i r s t sign i n 7 5 - the tops is a shortening of the stems and a slight reduction in the size of leaves. Blossoming is often retarded or inhib- i ted. Progressive reduction in size of stems and leaves follows each cutting and is accompanied l>y a reduction in the number of "buds developed each time, which results in a gradual reduction in the number of stems. No chlorosis or other color change is evident u n t i l the last flew stems die. In the f ina l stage of the disease only one stem, or at the most a few stems, are produced and are only a few inches high. Not infrequently, the leaves of the affected plants are darker green than healthy plants. In roots, the earliest stage of the disease, that can he detected, is a slight yellowing of the wood just beneath the bark. This yellowing, a result of gum formation-in the vessels, spreads u n t i l the entire active part of the xylem is involved. In 1941, Hewitt and Houston (24), struck by the geograph- ica l association of Pierce's Disease of Grapevines and Al fa l fa Dwarf in Cal i fornia , started investigations into the two diseases, the results of which were published in 1946 (25). They noted that both diseases were spread by an insect vector and that they appeared in adjacent areas. Both diseases appear more frequently and more severely in wet portions of f ie lds or along ditches or ponds where there is a luxuriant growth of grasses heavily infested by leafhoppers. Through a series of experiments they proved that the same 4 species of leafhoppers that transmitted the Pierce's disease of grapevines also transmitted the Al fa l f a Dwarf disease; and that these insects -76- GOUIQ effect an intertransmissability of the two diseases. Root pieces from diseased grapevines were inserted into the • roots of healthy a l fa l fa plants and transmitted the virus in 12 out of 35 t r i a l s ; hut root pieces from diseased al fa l fa , s imi lar i ly inserted in the stems of rooted grape cuttings, - fa i led to transmit the disease due to fai lure of union of the scions and stocks. In 1949, Houston (26) reported the release for commercial production of a dwarf resistant strain of a l fa l fa developed through selection in Cal i fornia common al fa l fa . Observations in f ields of Cal i fornia common al fa l fa showed that, after pract ical ly 100% of the plants were either dead or badly diseased, about 1 plant in each 2000 square feet was s t i l l making a normal top growth, even.though root symptoms showed that the plant had been diseased for a period of one to Wo years. A number of such plants were selected, transplanted and, selfed to produce progeny. The progeny were a r t i f i c i a l l y inoculated using viruliferous vectors and observed over a. period of 2 years. A portion of the progeny appeared quite tolerant to the virus and maintained normal growth 2 to 3 times that of Ca l i fornia common check plants. These results indicated the poss ib i l i ty of selecting a Dwarf resistant • strain from Cal i fornia common, without changing in any way the other desirable qualities of this variety. . With the foregoing determined, 800 plants showing natural f i e l d resistance were selected and planted in an isolat ion -77- block. Here they were allowed to interpollinate naturally and set seed the following year. Seed was harvested separately from each of the remaining plants (425^(the rest had died in transplanting, showed virus effects, or fa i led to set seed). This seed was planted in 3 row blocks replicated 3 times in each of 3 counties of Southern Cal i fornia incommercial f ields where dwarf had been very prevalent the past years. The f ina l results from these plats in the spring of 1949 showed that the progeny from 60 to 70 of the original selections were quite tolerant to the dwarf virus and held a normal growth producing a good stand. Simultaneously, to further test the Dwarf resistant qualities of the original selections, cuttings from 320 of thebetter plants were rooted in the greenhouse, a r t i f i c i a l l y inoculated by a vector, and transplanted to an isolated block in a f i e l d along with a number of common plants inoculated at the smae time. At the end of the second &ea,son of growth,, a l l the latter were dead, whereas about 65 of the former showed no effect of the virus with the exception of root sympt oms. Based on these results , and those of the f i e l d plots, the more resistant selections were saved to cross-pollinate and set seed, which was released to the growers as Cal i fnrnia Common '49 last year. 78. D - Other a l fa l fa diseases resembling Witches' Broom. There are two other diseases of a l fa l fa that superficial ly resemble Witches'. Broom, and may he confused with this disease. They are the bacterial wilt disease and the dwarf disease, and the distinguishing features are: (1) Bacterial wil t disease. In common with Witches' Broom, the Bacterial Wilt disease causes a marked dwarfing of the plant, owing to shortening of the internodes and reduction in leaf size. The great proli feration characteristic of Witches' Broom is not normally a symptom of Bacterial Wil t , but i t is not the distinguishing cr i ter ion. In Bacterial Wilt the characteristic yellowish or brownish-yellow discolouration of the vascular cylinder of the tap root serves as a positive dist inction since the roots of Witches' Broom are normal in colour in the primary stages of disease. (2) Alf alf a Dwarf disease. In common with Witches' Broom this disease causes a dwarfing of the a l fa l fa plant both in reduction in total size and in the length of internodes and size of leaves. However, in Dwarf disease the root s are discolored, leaves retain their normal colour or may be intensif ied, and there is a progressive reduction in the number of shoots produced. In Witches' Broom the rodsts remain normal for some time, leaves are definitely chlorotic , and there is a, progressive* increase in the number of shoots'; produced. Also of note i s the fact that in the former disease the roots are very badly infected before any symptoms appear above ground. - 7 9 * V I T G H E S ' B R O O M OF A L F A L F A S Y M P T O M O L O G f . D i s e a s e d p l a n t s c a n b e o b s e r v e d a n d i d e n t i f i e d f r o m a f a r i n t h e f i e l d b y t h e i r g r e a t l y r e d u c e d s i z e ( h y p o p l a s e a ) a n d t h e i r p a l e y e l l o w i s h c o l o r a t i o n , a n d t h e p a t c h y a p p e a r a n c e o f t h e f i e l d d u e t o t h e r e d u c t i o n i n t h e n u m b e r o f p l a n t s . O n ' s e v e r a l o c c a s i o n s i n B . C . a n d i n . W a s h i n g t o n , t h e d i s e a s e h a s b e e n s p o t t e d w h i l e d r i v i n g a l o n g t h e h i g h w a y o n t h e b a s i s o f t h e s e s y m p t o m s . T h e m o s t c h a r a c t e r i s t i c s y m p t o m o f W i t c h e s ' B r o o m o f A l f a l f a i s t h e m a r k e d d w a r f i n g o f t h e p l a n t . H o w e v e r , a s t h e a p p e a r a n c e o f i n f e c t e d p l a n t s i s s l o w l y m o d i f i e d o v e r a p e r i o d o f m o n t h s o r y e a r s , a w i d e r a n g e o f s y m p t o m s c a n b e o b s e r v e d . I n t h e a d v a n c e d s t a g e o f i n f e c t i o n , t h e r e i s t y p i c a l l y a d e n s e p r o l i f e r a t i o n o f s h o o t s p r o d u c e d f r o m t h e c r o w n a c c o m - p a n i e d b y a s e v e r e d w a r f i n g o f t h w whole p l a n t ( P h o t o l ) . I n v e r y a d v a n c e d s t a g e s o f t h e d i s e a s e , h o w e v e r , t h e n u m b e r o f s h o o t s m a y b e g r e a t l y r e d u c e d d u e t o t h e s e v e r e r o t t i n g o f t h e c r o w n a n d u p p e r p o r t i o n s o f t h e r o o t , w i t h t h e c o n s e q u e n t d e a t h o f t h e b u d s i n t h a t p o r t i o n . I n t e r n o d e s a r e g r e a t l y s h o r t e n e d . ( I n t e r n o d e s o n h e a l t h y p l a n t s a r e n o r m a l l y ! § • t o 2-g- i n c h e s l o n g , w h i l e i n d i s e a s e d s h o o t s w h e n s e v e r e l y d w a r f e d a r e b u t a f r a c t i o n o f a n i n c h l o n g . ( P l a t e 1 , P h o t o 2 ) . I n f a c t , l e a v e s w e r e o b s e r v e d o n o c c a s i o n s o . g r e a t l y r e d u c e d i n s i z e , t h a t i t \^as o n l y a f t e r e x a m i n a t i o n w i t h , a l O x h a n d - l e n s t h a t t h e t h r e e l e a f l e t s c o u l d b e d i f f e r e n t i a t e d . T h e n o r m a l a l f a l f a l e a f l e t i s r a t h e r n a r r o w , o b l o n g o r e l l i p t i c - o b l a n c e o - l a t e w i t h s p i n o s e d e n t i c u l a t i o n s t o w a r d s t h e a p e x . D i s e a s e d  -80- leaflets are smaller, rounder, sometimes lack apical dentic- ulations and are frequently wrinkled or puckered. (Plate 1 ) . The f i r s t symptom of the disease to he seen is a slight marginal, chlorosis of the leaves giving the plants a yellowish cast or tinge. Primary symptoms of infection usually are seen when new growth starts after cutting, and the symptoms seem to "foe general a l l over the plant. At this time, there may he l i t t l e or no dwarfing of the plants, hut the diseased plants can he recognized easily "by the greatly increased number of stems, the yellowish cast, and a tendency for the growth to "be very erect. (Photos 3, 4, 5) After the next cutting, the new growth w i l l usually he decidedly dwarfed and the leaflets much reduced in size. Prol i ferat ion - and stunting is progressive with each successive cutting. Both Edwards (ibid) and Smith (ibid) report a colour change in infected plants when proli feration has occurred: the foliage is often very dark in colour and may have a purplish tinge. Menzies ( ibid) , on-the other hand, does not mention this phenomenon as occurring in Washington. Nevertheless, i t has been observed, in a few cases at U . B . C , that the foliage becomes very dark, hardly purplish, before the leaves become chlorotic, and before crown prol i ferat ion is pronounced. This darkening of the foliage has occurred in conjunction with a rosetting or clustering of the nodal foliage about the nodes. Several hundred spindly stems are common on infected plants and Menzies (ibid) reports that severely infected crowns have been found with as many as 3000 very f ine, densely Severely dwarfed Witches' Broom diseased plant on l e f t ; normal a l fa l fa plant on r ight . -81- matted stems. This figure is in agreement with some of the plants observed, but, in most of the plants observed, the rott ing of the crown had occurred to such an extent, by this period,in the progress of the disease that the number of shoots is much reduced from this number (3000). Immediately prece/ding death of the plant the foliage wilts severely and becomes prostrate. At this time an exam- ination of cross-sections of these crowns has shown almost complete disintegration of cel lular structure; also blocking of the vascular system has cut off the normal water and nut- rient supply to the foliage, a feature which results in wilt and eventual death. Menzies (ibid) made no observations on the rotting of the crown, although i t was observed to precede the death of the plant in a l l cases at U.B.C. Edwards (ibid) stated that in the later stages of the disea.se, the crown and upper root tissues are often severely rotted and the plants gradually die. He made isolations from the decaying tissues '•which yielded an unidentified ster i le white fungus, Rhizoctonia bataticola, and various types of unidentified Eusaria. The most apparent fungus observed at U.B.C. was an unidentified Basidiomycetes. Edwards (ibid) observed part ia l and complete recovery of diseased plants when transplanted to the greenhouse and maint- ained under conditions favourable for growth. In many instances, the recovery was only temporary v/ith renewal of symptoms at a la ter date, but in some cases he reported complete recovery. Menzies (ibid) did not observe this phenomenon in Washington. However, two'plants in the greenhouse at U . B . C . , that exhibited A l f a l f a p l a n t s h o w i n g a p p a r e n t r e c o v e r y f r o m ' w i t c h e s 1 B r o o m d i s e a . s e . w i t c h e s ' B r o o m d i s e a s e d a l f a l f a p l a n t s h o w i n g p r o l i f e r a t i o n a n d d w a r f i n g . N i c o l a V a l l e y , B . C . 82- positive disease symptoms in the f a l l of 1950 (they were clones of diseased plants obtained in June 1950 from the Nicola Valley) had shown apparent complete recovery from the diseased condition in A p r i l , 1951. Photograph 6 shows one of these recovered plants. Several diseased plants were noted on Apr i l 11, 1951, to be producing normal shoots mixed among the diseased stems, while the f a l l befox-e, only diseased shoots were produced. These normal shoots bore flowers by A p r i l , while very few of the diseased stems bore flowers or even buds at this date. It was commonly observed that newly diseased plants pro- duced normal growth at the periphery, with reduced or dwarfed foliage in the centre of the plant. Hot only was crown proli feration observed, but also an abnormally large number of f ine, elongated shoots have been observed arising from the nodes of the stems, and then more shoots arising from the nodes of these elongated shoots, with the resultant production of a thick, abnormal, bushy type of gr owth. The majority of diseased plants fa i led to flower, a rather str iking symptom when seen during the normal flowering time in the f i e l d . The few flowers formed on diseased plants appear smaller in size, and paler in colour. However,,the latter point is hard to determine, as normal a l fa l fa flowers (M. media) show a tremendous colour diversity, ranging from pale blue-white, l ight greens, several shades of purple, to the odd yellowish flower. Nevertheless, in the few cases where healthy periphereal shoots were producing flowers at the Hormal a l fa l fa shoot on l e f t , and v/itches' Broom diseased shoot on the r ight . Note chlorosis, shortened inter- nodes, reduced leaflet size and rounder shape, and small pale inflorescence. Normal a l fa l fa inflorescence on l e f t ; the two on the right are Witches' Broom diseased a l fa l fa inflorescences. Note reduced number of f lorets and paler color. -83- same time as the central diseased shoots were producing flowers, the central flowers appeared paler in colour. The flowers "borne on diseased shoots were "borne singly, or in groups of 2 to 6 with an average of 2.7 (average of 20 clusters examined at random); this contrasts sharply with the multi-flowered raceme hearing approximately 15 flowers? produced by healthy plants. As is to be expected in l ight of the reduced number of flowers, very l i t t l e seed is produced. The small amount of seed that was collected seemed normal (perhaps a l i t t l e small) and germinated well . INYE S TIG AT I ON AL WORK I - TRANSMISSION EXPERIMENTS A. Grafting studies (a) Literature review. Edwards (11, 12, 13), Heald and Wellman (23), and Menzies (34) carried out extensive graft transmission tests in investigating Witches' Broom of Al fa l fa . Edwards (ibid) achieved the f i r s t success in obtaining positive transmission of the disease by grafting. his Edwards (ibid) used, in a majority of/cases, scions from diseased plants introduced by means of a side or veneer graft into the upper portion of the tap root of healthy plants just below the crown. He also used healthy scions and dis- eased stocks in an attempted cleft or wedge graft into the crown and"into the upper portion £ f the tap root. He encountered -84- considerable d i f f i cu l ty in establishing a, successful graft whenever a diseased plant was used as a stock. The grafts were performed in a greenhouse, bound with r a f f i a and then coated with wax. Two months after the grafts were attempted the plants were transplanted to the f i e l d ; at this time he had achieved 57 unions out of 100 attempted using diseased scions and healthy stocks; and 11 unions out of 58 attempted using healthy scions and diseased stocks. Out of the lat ter 11 unions, only 2 persisted after transplanting to the f i e l d and positive transmission of the disease occurred in both instances. Out of the former 57 unions, only 28 scions per- sisted for 2 weeks after transfer to the f ie lds , and a month later there were only 15 of these scions s t i l l surviving. Out of the instances where the scions had died out prior to transplanting, 4 showed positive disease transmission when examined 2-g- months after transplanting. At this time 13 of 15 plants showing scion survival also showed positive disease transmission; and 9 of the plants were diseased whose'scions only persisted for 2 weeks after transferring to the f i e l d . In a l l , Edwards (ibid) achieved transmission in 28 plants out of 158 attempted grafts, or out of 68 achieved grafts. He aid and "Wellman (ibid) reported that inoculations by grafting from diseased to healthy plants had reproduced the symptoms in a l imited number of cases. Menzies (ibid) achieved very l i t t l e success in root- grafting tests, so he used instead a modification of the shoot-grafting technique. This modification consisted of keeping the delicate grafts, firmly bound with fine thread, -85- in a glassed-in humidity chamber for 10 days after grafting.- He reports 75$ successful grafts using this method, with disease transference occurring in a l l cases where the diseased scions became united v/ith the healthy stocks. In a scion removal experiment, he found that virus transfer commenced between the 10th and 12th day following grafting and was completed by 25 days. Using shoot grafts, disease symptoms were apparent on the average in 61 days; while v/ith root-approach grafts, symptoms took on the average 118 days to be manifested. It thus appears in the former case, that the actual latent period of the virus i s from 40 to 50 days using shoot-grafts. Menzies (ibid) also attempted a study of the hpsvt/'itange of Witches' Broom of Al fa l f a by means of cross-grafting. He obtained 87 apparently successful grafts on 12 species of legumes. Serious d i f f i cu l t i e s arose from the fact that several of the legumes were annuals, and did not remain alive long enough for the symptoms, i f any, to be recognized. However, he was successful in transferring the disease to Black Medic (Me die ago lupulina L . ) , andto Caligornaa Bur- Clover (Medicago hispida Gaert.). (b) Experiments. ! • Several diseased plants were dug in the Nicola, valley of B r i t i s h Columbia, brought to the University and planted in a greenhouse deep bench in June 1950. 142 cleft crown grafts, using healthy 8 months old Rhizoma plants as stocks and Witches' Broom scions from the above plants, were attempted from June 13 to 16, 1950. Grafts were bound with ra f f i a , thickly coated v/ith paraffin, and planted in a - 8 6 - deep greenhouse "bench. The healthy foliage was cut "back at this time, and several times during the summer and f a l l the healthy foliage was cut hack to allow the diseased scions any advantage that this might give. Although in several cases an apparent disease transfer was suspected, the nature of a crown graft rendered i t impossible to observe whether the disease symptoms appearing were from disease transfer to the healthy stocks, or i f the diseased foliage appearing was scional only in origin. The plants were dug up for observation on January 10, 1951, at which time there were 124 plants remaining, alive. Whether the 18 plants that died, did so as a result of the disease or from cultural methods was not determined. (A l l grafted plants were transplanted to pots in November 1950.) The results obtained are given below; Plants healthy on January 10, 1951. Union of sciog. and stock, but scion dead. 7 No union, scion never established or disappeared 79 No union, both scion and stock l i v i n g separately 4 Plants diseased on January 10, 1951. Union, but scion dead 5 Union, scion l i v i n g in firm union with stock 14 Union, scion l i v i n g in very feeble, i f any, union 2 No union, no evidence of establishment of scion 3 •Plants doubtful or suspicious on Jan. 10, 1951. 10 124 The 10 plants belonging to the latter category were replanted in the greenhouse. On the 5th of February, 1951,?3 plants showed positive symptoms of the disease, and an additional one was suspicious in showing a large number of small, clustered leaves; and when observed on the 16th of February this plant was showing prol i ferat ion and marginal chlorosis - -87- typioal disease symptoms. F ina l examination of these plants was carried out Apr i l 16 1951, when besides the 4 diseased P I A ^ - V Mo- *S plants, 2 appeared suspicious. One piant—(55) showed a clustering effect of the leaves around the stem and a much darkened color; and the otiiê —-pi-ant—(-39-) showed the clustering effect and had a chlorotic tinge to the whole plant. Adding in the above plants to the above totals results in figures given below. Plants healthy Union of scion and stock, but scion dead. 7 No' union, scion never established or disappeared 83 No union, both scion and stock l i v i n g separately 5 Plants diseased Union, but scion dead 6 Union, scion l i v i n g in firm union with stock 14 Union, scion l i v i n g in feeble union v/ith stock 4 No union, no evidence of establishment of scion 3 Plants doubtful ot suspicious 2 l£4~~ Out of 142 attempted crown grafts, 124 plants were l i v i n g after 7. months, successful union of scion and stock was achieved in 31 cases, and disease transference was observed in 27 cases. All^cases wherein the scion and stock were in union and both l i v i n g at time of digging, showed a disease transference. ĴOs). 7 cases were observed, where the scion and stock" had united, but the scion was dead by January 10. How long the union lasted is not known,, but judging from the " result s of Menzies experiment in removing scions every five days, the union must have lasted l e s s than 2 weeks before the scion died. In 3 cases, scion or remains of any part of the scion couH. not be found although there was a positive disease transference. In these 3 cases i t is probable that a union d i d exist for a period long enough for a virus transfer before a rott ing of the scionoccurred. Evidence that a union must exist i s shown by the five ca.ses where both scions- and stocks were l i v i n g in very close proximity to one another, but with no disease transmission. A rough measure of the upper l imit of time required for the appearance of symptoms - February 16th or 8 months.If the 2 doubtful or suspicious plants subsequently develop d isease symptoms, the period of incubation w i l l of course be again lengthened. 2. In May 1950, 1 2 stem cleft grafts, using diseased scions and healthy plants were attempted in- the greenhouse in co-operation w i t h If. S. Wright of the Dominion Plant Pathology Laboratory. The grafts were bound wî -h ra f f i a and kept moistened by a string-wick extending from a jar of water, However, union was not observed, and the scions died in about 2 weeks time. Menzies' modifL cation of using a humidity chamber was not attempted. 3. At the same time, another method of transferring the disease was attempted. This consisted of cutting longitudinal sections (approximately 2 inches long) out of the healthy and diseased shoots growing in adjoining pots, and firmly Ipinding- the freshly cut areas together with ra f f i a . However, both the diseased and healthy shoots, above the cut, d i e d in a l l cases i n about 10 days time. Mo disease transference was observed i n any c a s e d u r i n g tlae summer, f a l l and winter' i n t h e g r e e n h o u s e . B. Ee chan i c a l I n o o u l a t i on S t u d i e s ' (a) L i t e r a t u r e E d w a r d s ( i b i d ) , e m p l o y i n g as i n o c u l u m the f r e s h l y e x p r e s s e d and u n d i l u t e d sap of d i s e a s e d f o l i a g e , a t t e m p t e d s e v e r a l m e t h o d s o f m e c h a n i c a l l y i n o c u l a t i n g h e a l t h y p l a n t s . The met h o d s , t h a t he employed, a r e o u t l i n e d b r i e f l y as f o l l o w s . 1. I n d i v i d u a l l e a v e s were b r u s h e d l i g h t l y w i t h a s m a l l c h e e s e c l o t h b a g s a t u r a t e d w i t h i n o c u l u m and c o n t a i n i n g a m i x t u r e of s a n d and c r u s h e d i n f e c t e d l e a f t i s s u e . 2 . Stems were p r i c k e d w i t h a f i n e h y p o d e r m i c n e e d l e and a d r o p o f i n o c u l u m a l l o w e d t o f l o w i n t o t h e v/ound. 3 . Stems s c r a t c h e d l o n g i t u d i n a l l y , w i t h a f i n e h y p o d e r m i c n e e d l e i n s e v e r a l p l a c e s , and i n o c u l u m a l l o w e d t o f l o w i n t o the wound. 4 . M u l t i p l e p i n i n o c u l a t i o n s ( u s i n g 2 o - 3 o e n t o m o l o g i c a l p i n s b o u n d t o g e t h e r ) i n t o t e r m i n a l l e a v e s of v i g o r o u s l y g r o w i n g s h o o t s , l e a v e s and stems. 5 . S m a l l c o t t o n w o o l p l u g s s o a k e d i n i n o c u l u m were i n s e r t e d i n t h e t a p r o o t j u s t b e l o w t h e crown i n c o n t a c t w i t h t h e v a s c u l a r t i s s u e . The i n o c u l a t e d p l a n t s were o b s e r v e d f o r p e r i o d s r a n g i n g f r o m 2-|- t o 3-g- y e a r s , w i t h no symptoms d e v e l o p i n g i n any c a s e . M e n z i e s ( i b i d ) i n o c u l a t e d 250 p l a n t s u s i n g m a c e r a t e d d i s e a s e d t i s s u e on a swab as i n o c u l u m and d u s t i n g c a r b o r u n d u m powder on l e a v e s t o be i n o c u l a t e d . However, M e n z i e s f a i l e d t o s e c u r e d i s e a s e t r a n s m i s s i o n i n any c a s e . - 9 0 - (b) E x p e r i m e n t s 1. Crovms o f W i t c h e s * Broom d i s e a s e d a l f a l f a v/as f i n e l X y g r o u n d v / i t h meat g r i n d e r , and s t r a i n e d t h r o u g h 8 t h i c k n e s s e s o f c h e e s e c l o t h . The r e s u l t a n t e x c i s e d j u i c e was i n o c u l a t e d i n t o crowns o f 3© h e a l t h y 15-monilb o l d R h i z o m a s e e d l i n g s on F e b r u a r y 1 6 t h , 1 9 5 1 , u s i n g a "London" L u e r F i s h e r and B u r p e 5§ cc. s i z e h y p o d e r m i c s y r i n g e f i t t e d v / i t h a B-D 16 n e e d l e . At t h e same t i m e , t h e m a c e r a t e d crown m a t e r i a l f r o m which t h e j u i c e was e x t r a c t e d v/as wrapped a r o u n d the b r o k e n r o o t s o f 12 h e a l t h y 15 month o l d R h i z o m a s e e d l i n g s . A l l p l a n t s were p o t t e d I n t h e g r e e n h o u s e , t o g e t h e r w i t h 23 c o n t r o l s and o b s e r v e d f r o m t i m e t o t i m e . . , F i n a l o b s e r v a t i o n s on t h e p l a n t s were made 6n A p r i l 2 8 t h , 1951. 2 p l a n t s of t h e i n o c u l a t e d s e r i e s shov/ed v e r y deep c o l o r a t i o n and r a t h e r i n t e n s e p r o l i f e r a t i o n . However, t h i s same c o n d i t i o n v/as o b s e r v e d on one o f t h e c o n t r o l p l a n t s . One p l a n t o f the m a c e r a t e d s e r i e s showed t h e i n t e n s i f i c a t i o n o f c o l o r and p r o l i f e r a t i o n h a b i t , and p r o d u c e d no f l o w e r s of f l o w e r b u d s ; w h e r e a s , t h e o t h e r p l a n t s i n t h e s e r i e s were f l o w e r i n g v i g o r o u s l y . T h e s e p l a n t s v / i l l be t r a n s p l a n t e d t o the f i e l d i n May f o r f u r t h e r o b s e r v a t i o n s . 2. On F e b r u a r y 1 4 t i , 1951, 8 h e a l t h y 6 montt*- o l d Grimm s e e d l i n g s were p l n t e d in a 1 4 - i n c h s i z e e a r t h e n - w a r e p o t c o n t a i n i n g s h r e d d e d f o l i a g e of diseased p l a n t s v/elljaiixed v / i t h t h e s o i l . The s e e d l i n g s were p l a n t e d so t h a t t h e r o o t s and crovms were i n c l o s e c o n t a c t t o t h e d i s e a s e d m a t e r i a l . F i n a l o b s e r v a t i o n s made A p r i l 2 8 t h , 1951 shov/ed a l l p l a n t s t o be - 9 1 - n o r m a l . G - S e e d T r a n s m i s s i o n o f W i t c h e s ' B r o o m o f A l f a l f a . E d w a r d s ( i b i d ) c o n d u c t e d a s e e d t r a n s m i s s i o n , e x p e r i m e n t w i t h a l i m i t e d n u m b e r o f s e e d f r o m i n f e c t e d p l a n t s . S e v e r a l o f t h e y o u n g p l a n t s s h o w e d , b y t h e f i r s t f a l l , m a r k e d f o l i a g e a b n o r m a l i t i e s . T h e l e a v e s , c o n s i d e r a b l y r e d u c e d , w e r e c l u s t e r e d a l o n g t h e s t e m s p r o d u c i n g a r o s e t t e - l i k e e f f e c t , w h i c h w a s s u g g e s t i v e o f i n f e c t i o n . H o w e v e r , t h e s e p l a n t s f l o w e r e d n o r m a l l y a f t e r t r a n s p l a n t i n g t o t h e f i e l d , a n d n o d e f i n i t e e v i d e n c e , w a s o b t a i n e d t o s h o w t h a t a n y o f t h e m w e r e i n f e c t e d w i t h W i t c h e s ' B r o o m . F i n a l o b s e r v a t i o n s , t a k e n 2 y e a r s l a t e r , s h o w e d s e v e r a l s u s p i c i o u s p l a n t s , b u t n o p l a n t s s h o w e d p o s i t i v e s y m p t o m s o f t h e d i s e a s e . M e n z i e s ( i b i d ) g r e w 4 8 8 p l a n t s f r o m s e e d c o l l e c t e d f r o m i n f e c t e d p l a n t s , b u t f a i l e d t o s e c u r e a n y e v i d e n c e o f W i t c h e s ' B r o o m i n f e c t i o n i n a y e a r ' s t i m e . E x p e r i m e n t S e e d w a s c o l l e c t e d f r o m d i s e a s e d p l a n t s a t S a v o n a a n d a t t h e B a s q u e r a n c h , B . C . i n A u g u s t 1 9 5 0 . T h i s s e e d , t h r e s h e d b y h a n d , w a s s c a r i f i e d , g e r m i n a t e d i n p i t r e p l a t e s , a n d t r a n s p l a n t e d t o t h e g r e e n h o u s e t o f l a t s i n O c t o b e r , 1 9 5 0 . G e r m i n a t i o n w a s n o r m a l . 1 6 2 p l a n t s w e r e t r a n s f e r r e d f r o m f l a t s td> p o t s i n l a t e D e c e m b e r , a n d t h e r e m a i n i n g p l a n t s w e r e t r a n s p l a n t e d t o o t h e r f l a t s , 4 0 t o a f l a t . L a r g e r t h a n n o r m a l n u m b e r s o f s m a l l , w e a k s e e d l i n g s w e r e o b s e r v e d a t t i m e o f t r a n s p l a n t i n g ; h o w e v e r , a s n o s e e d w a s c o l l e c t e d f r o m h e a l t h y p l a n t s i n t h e s a m e d i s t r i c t s ( S a v o n a , B a s q u e R a n c h ) f r o m w h i c h t h e s e e d f r o m -92- diseased p3a rits was gathered a count of these plants v/as not i, made. Observations made on Apr i l 28th, 1951, showed in at r although ndplants shov/ed Witches' Broom symptoms, several plants were very small and the odd plant chlora/tic and exfcklMted rosetting of nodal foliage. D. Plant multipl icat ion through cuttings &ne of the d i f f icul t , time consuming, and expensive procedures in the study of this virus disease w i l l be the securing of diseased plants in sufficient numbers for uae in the -Warious required replicated f i e l d tests. The diseased plants wil^have to be laboriously dug out of normally tough sod in f ields in the Interior, and then carried without delay- to the coast. Here the plants v/ii-^be divided and planted as quickly as possible. However, very high mortality has been observed in a l l such transfers in the past. Accordingly, on the 2nd of October, 1950, 3 sets of 25 stem cuttings of diseased a l fa l fa were treated with the following procedure. The basal ends of sets of cuttings were dipped for 36 seconds into 4000- ppm solutions of indole butyric acid (IBA) alpha-napthalene acetic acid (BAA), with, the 3rd set, as control, dipped Into distilJe d v/ater. The cuttings were planted In clean sand in glazed pot? kept watered, and le f t untilDecember 4th when they v/ere dug up, observed for root formation, ani planted in f la t s . 7 of the cuttings treated with IB A none of the cuttings treated v / i th 1TAA, and 8 of the cuttings treated v/ith distilled v/ater were alive and showed root formation. The cuttings' treated with IBA, although less -93- numerous thai the controls, shwed considerably greater root formation both in size of roots and extent of rooting area.. 2 of the cuttings subsequently died after transplanting, but the remaining 13 have grown, into f a i r l y large plants by the time of reporting (April 28th, 1951), a l l showing advanced symptoms of the disease. The experiment did point out, besides being able to get the proliferation effect in the absence of a crown, thatit is possible to root cuttings of Witches' Broom diseased a l fa l fa and thus increase the number of diseased pianos for use in f i e l d experiments. If a diseased plant produces 500 stems, this means that the one plant can produce, using my results of 26% rooting, 130 diseased plants? and this could be done about once a month.. It isfeflt, however, that with improvement in techniques much higher percentage of rooting of cuttings couB. be achieved, and., thai this melbhod should prove to be very usefulto the agronomist and plant pathologist in multiplication of diseased stocks. It has the advantage to the plant breeder inth at a great number of plants, a l l with the same genotype, and at the same s^age of the disease, can be secured readily for use in resistance and breeding studies. E . Insect transmission studies Edwards (ibid) attempted severalexperiments in an attempt t.o transmit the Witches.' Broom of Alfa l fa using insects as agents of inoculation (or vectors). A brief resume of his work is given below. 1. 2 to 4 Jassids, f i e l d collected, were allowed to feed -'394- on a diseased plant for 7 to 9 days and then transferred to healtny plants for 5 - 10 days. Twenty eiglxt 5 - 6 month old plants were treated this: way. 12 - 24 thripsi, also f i e l d collected, were allowed a 5 - 7 day acquisition feed on a diseased plant and then transferred to a healthy plant for 5 - 7 days. 16 healthy plants were treated in this manner, lib transmission was observed in any case. 2. Two 24 plant plots were established for preliminary work, with possible insect vectors. Plots were enclosed with, wooden frames covered wit h fine white madopolam. Mixed populations o£ insects collected on diseased plants in the f i e l d were enclosed in eaci frame. After 3 months, the experiment was modified by replacing the central 4 plants by a laarge diseased plant to produce a continuous source of inoculum. At the same time , massed populations of f i e l d collected insects were liberated inside the cages. 86% of these insects belonged to the Thysanoptera and the Hemiptera. The reiaining 20% consisted of species belongingoto the Diptera, Coleoptera, and Lepidopter a. In addition, in the following year 6 more plots were added to the experiment, 2 of which were kept sprayed and. served as controls. The plots were cut as requir ed, and massed populations of insects collected in highly diseased fields were added periodical ly . 9 such additions were made to the latter 4 transmission cages, and 11 to the former 2. 15 months after establishment of the f i r s t part of "theexperiment, one plant was found in the f i r s t 2 cages, that showed definite symptoms of Witches' Broom disease. However none of the other 9 5 - p l a n t s i n angr o f t h e p l o t s , a l t h o u g h o b s e r v e d c a r e f u l l y f o r a p e r i o d o f 3 y e a r s , d e v e l o p e d a n y e v i d e n c e o f i n f e c t i o n . M e n z i e s ( i b i d ) m a d e m a s s c o l l e c t i o n s o f s u s p e c t e d s p e c i e s o f i n s e c t s , c a g e d t h e m o n i n f e c t e d a l f a l f a p l a n t s f o r a s h o r t f e e d i n g p e r i o d , a f t e r w h i c h t h e y w e r e s e g r e g a t e d i n t o s p e c i e s g r o u p s a n d t r a n s f e r r e d t o h e a l t h y p l a n t s , fin t h e c o u r s e o f s e v e r a l h u n d r e d t e s t s v / i t h s u c k i n g i n s e c t s o n e . c a s e o f t r a n s m i s s i o n o c c u r r e d w h e r e t h e l e a f h o p p e r P l a t y m o i d e u s a c u t u s S a y . v /as u s e d . I n t h i s c a s e , a n a c q u i s i t i o n f e e d o f 7 d a y s o n d i s e a s e d p l a n t s w a s a l l o w e d , t h e n i n s e c t s t r a n s f e r r e d t o h e a l t h y p l a n t s f o r 1 2 d a y s . W i t c h e s ' B r o o m s y m p t o m s . w e r e f i r s t c o n s i d - e r e d d e f i n i t e i n a p p r o x i m a t e l y 2 m o n t h s a f t e r i n o c u l a t i o n . I n a n o t h e r e x p e r i m e n t , M e n z i e s ( i b i d ) u s e d l a r g e c a g e s c o n t a i n i n g o n e i n f e c t e d p l a n t a n d 4 t o 6 h e a l t h y p l a n t s . F i e l d c o l l e c t i o n s o f P l a t y m o i d e u s a c u t u s v / e r e a d d e d t o s o m e o f t h e t e s t c a g e s f r o m t i m e t o t i m e f o r a c o u p l e o f m o n t h s , w h i l e o t h e r c a g e s w e r e r e t a i n e d a s c h e c k s . F i r s t s y m p t o m s a p p e a r e d o n 2 p l a n t s i n 5 m o n t h s a f t e r i n o c u l a t i o n , a n d b y 8-g- m o n t h s , 1,8 o f t h e 2 9 s u r v i v i n g t e s t p l a n t s w e r e d e f i n i t e l y a f f e c t e d w i t h W i t c h e s ' B r o o m , v / h e r e a s n o n e o f t h e c h e c k p l a n t s b e c a m e i n f e c t e d . ( i M d ) H o w e v e r , M e n z i e s c o u l d n o t f i n d t h i s l e a f h o p p e r x - l a t y m o i d e u s a c u t u s i n 2 s w e e p s m a d e i n J u n e a n d A u g u s t o f 1 9 4 4 i n t h e M e t h e w v a l l e y o f W a s h i n g t o n a t t h e t i m e t h e d i s e a s e v/as s p r e a d i n g r a p i d l y . H e c o n c l u d e d f r o m t h i s , t h a t P I a t y r n o i d e u s a c u t u s , a l t h o u g h a v e c t o r , i s p r o b a b l y n o t t h e p r i n c i p a l v e c t o r o f t h e d i s e a s e . I t w a s f e l t t h a t a s e a r c h f o r a v e c t o r , i n v o l v i n g b y n e c e s s i t y , t r a i n e d e n t o m o l o g i s t s a n d p l a n t p a t h o l o g i s t s , i s s o m e w h a t o u t s i d e t h e p r o v i n c e o f t h e D e p a r t m e n t o f A g r o n o m y . Some w o r k i n b r e e d i n g o f W i t c h e s ' B r o o m r e s i s t a n t v a r i e t i e s o f a l f a l f a m a y , r e g r e t t a b l y , h a v e t o b e a t t e m p t e d w i t h o u t v a l u a b l e r e s e a r c h b e i n g c o n d u c t e d r e g a r d i n g v e c t o r s . I I " Y I E L D T R I A L ( a ) L i t e r a t u r e E d w a r d s ( i b i d ) s e l e c t e d a n d e n c l o s e d a n 1 / 7 a c r e a l f a l f a f i e l d c o n t a i n i n g f r o m 30% t o 35% o f W i t c h e s ' B r o o m d i s e a s e d p l a n t s . T h e w h o l e p l o t w a s c u t t o e s t a b l i s h u n i f o r m c o n d i t i o n s . 2 5 d i s e a s e d a n d 2 5 h e a l t h y p l a n t s , s e l e c t e d a t r a n d o m i n t h e f i e l d , w e r e c u t w h e n e v e r t h e u n a f f e c t e d p l a n t s r e a c h e d t h e f l o w e r i n g s t a g e . T h e f o l i a g e w a s w e i g h e d i m m e d i a t - e l y a f t e r c u t t i n g a n d t h e w e i g h t s r e c o r d e d t o t h e n e a r e s t -g- o u n c e . A v e r a g e d o v e r a p e r i o d o f t h r e e y e a r s , t h e d i s e a s e d , p l a n t s g a v e a m e a n y i e l d o f 3 7 . 4 % l e s s g r e e n w e i g h t f o d d e r t h a n t h e u n a f f e c t e d p l a n t s . ( T h i s i s h a r d l y a t r u e f i g u r e , a s i t d o e s n o t t a k e i n t o a c c o u n t t h e y i e l d r e d u c t i o n w h i c h w o u l d r e s u l t f r o m t h e h i g h e r m o r t a l i t y o f t h e d i s e a s e d p l a n t s t h u s l e s s e n i n g t h e s t a n d . ) M e n z i e s ( l i p i d ) r e p o r t e d a s e r i o u s r e d u c t i o n i n y i e l d s b u t d i d n o t r e p o r t o n a n y t e s t s t o d e t e r m i n e t h e a m o u n t o f r e d u c t i o n i n y i e l d s d u e t o t h e d i s e a s e . ( b ) E x p e r i m e n t . T e n c o m p a r a b l e s i z e d c l o n a l c u t t i n g s o f e a c h o f W i t c h e s ' B r o o m d i s e a s e d p l a n t s s e c u r e d f r o m t h e I n t e r i o r , a n d G r i m m TABLE YII - YIELD TEST WEIGHT IN GRAMS OF AIR-DRY HEALTHY AND WITCHES• BROOM DISEASED ALFALFA FODDER. A - WITCHES' BROOM DISEASED PLANTS PI. No. 1st cutting 2nd cutting 3rd cutting 1 2 3 4 3 6 7 8 9 10 10.68 5.51 4.53 7.66 3.81 3.12 4.18 3.38 3.31 1.91 0.89 0-|88 0.07 3.41 0.68 0.92 2.16 0.30 0.17 3.31 2.13 1 2.44 0.57 4.78 0.89 2.78 1.72 1.13 1.01 Totals 46.18 11.39 20 76 Averages 4.62 gms. 1.14 gms. 2.08 gms B - HEALTHY PLANTS PI. No. 1st cutting 2nd cutting 3rd cutting 11 12 13 14 15 16 17 18 19 20 6.42 8.55 4.83 6.36 3.42 6.52 5.49 3.97 4.70 4.79 3.78 2.04 1.29 3.63 1.02 2.80 1.85 1.72 2.92 2.35 4.77 3.35 2.95 4.20 1.59 4.65 2.88 2.06 3.42 2.68 Totals 55.05 g. 23.40 g. 32.55 g. Averages 5.51 g. 2.34- g. 3.26 g. -98- Table 7III - Correlation of air-dry weights of fodder of healthy and diseased a l f a l f a on f i r s t cutting. Witches' Broom plants Healthy plants Ix s 46.18 £y - 55.05 x : 4 . 6 2 g. y - 5.51 g. n = 10 n - 10 ( f x ) 2 = 2152.5924 ( ^ y ) 2 - - 3 0 3 0 . 5 0 2 5 £ x 2 , 287.7224 *y 2 1 3 2 3 . 2 5 9 3 S.S. X 74 .4632 S.S.y - 20 .2090 S.D.X t 2i-874 s ' D * y - t 1.4916 S.B.z - * .9089 S ' E « y " i .4-717 S.E. of d i f f . between the 2 meanest 1.024 g. D i f f . between the 2 means = 0.89 g t - * 8 9 = .86914 1.024 Tabled t at p • . 0 5 i s 2.101 at 18 D.F. Tabled t greater than oale. t - so no s i g n i f i c a n t difference between the fodder produced by Witches' Broom and Healthy A l f a l f a plants at beginning of the experiment. Table IX - Correlation of air-dry weights of fodder of healthy and diseased a l f a l f a plants on second cutting. Witches' Broom plants Healthy plants tx z 11 .39 £y - 2 3 . 4 0 x - 1.14 y 2.34 n = 10 n -- 10 S.D.x = 1 . 0 5 2 S.D.y = .9333 S.E. X .3327 S.E.y - .2952 S.E. of d i f f . between the 2 means= ,445T D i f f . between the 2 means= 1.20 g T t 1 , 2 ° . = 2 .69 Tabled t = 2.101 at 18 D.F. .445 Calc. t i s greater than tabled t - therefore there i a a s i g n i f - icant difference i n y i e l d between the fodder produced by Witches* Broom and Healthy a l f a l f a plants on the second cutting. -99- Table X - CORRELATION OF AIR-DRY WEIGHTS OF FODDER OF HEALTHY AND DISEASED ALFALFA PLANTS ON THIRD CUTTING. Witches' Broom Plants Healthy plants £x , 20.76 i y ^ 32.55 i s 2.08 y - 3.26 n = 10 n = 10 S.D.X - 1.304 S.D,y = 1.052 S.E. X - .4124 S.E.y = .3327 S.E. difference between the 2 means- .5298 D l f f . between means - 1.18 gms. t = 2.2 27 Tabled t = i s 2.101 at 18 D.F. and p a .05 Calc. t i s greater than tabled t . Therefore there i s a s i g n i f i c a n t difference between the a i r - dry weights of healthy and diseased plants' fodder at the t h i r d cutting. - 1 0 0 - a l f a l f a o b t a i n e d f r o m t h e n u r s e r y p l o t a t U . B . C . ' w e r e p r o p - a g a t e d i n 1 0 p a i r s o f 8 - i n c h g l a z e d p o t s i n t h e g r e e n h o u s e o n S e p t . 1 0 , 1 9 5 0 . P l a n t s w e r e w a t e r e d * a n d p e r i o d i c a l l y r a n d o m - i z e d t o a v o i d p o s i t i o n e f f e c t . W h e n t h e h e a l t h y p l a n t s h a d r e a c h e d l / l 2 b l o o m , F e b r u a r y 1 6 , 1 9 5 1 , a l l g i a n t s w e r e h a r - v e s t e d . T o p s w e r e d r i e d f o r 1 h o u r i n a n o v e n a t 1 0 0 ° C , a n d a l l o w e d t o r e t u r n t o a i r - d r y - w e i g h t b e f o r e w e i g h i n g o n a r o u g h b a l a n c e . T h e y w e r e h a r v e s t e d a s e c o n d t i m e o n M a r c h 1 6 , a n d . a t h i r d t i m e o n A p r i l 1 6 , 1 9 5 1 . T h e r e s u l t s o f t h e s e w e i g h i n g s a r e g i v e n i n . T a b l e V I I . I t w a s f o u n d t h a t o n t h e f i r s t c u t t i n g t h e r e w a s n o s i g - n i f i c a n t d i f f e r e n c e b e t w e e n t h e y i e l d o f t h e W i t c h e s ' B r o o m d i s e a s e d p l a n t s a n d t h e h e a l t h y p l a n t s . ( T a b l e V I I I ) . T h i s s h o w s t h a t t h e c u t t i n g s w e r e c o m p a r a b l e i n s i z e a t t h e b e g i n - n i n g o f t h e e x p e r i m e n t . T h e y i e l d o f t h e W i t c h e s ' B r o o m d i s e a s e d p l a n t s a n d t h e h e a l t h y a l f a l f a p l a n t s u p o n t h e 2 n d a n d t h e 3 r d c u t t i n g a r e s t a t i s t i c a l l y a n a l y z e d i n T a b l e s I X a n d X . I t w a s f o u n d t h a t o n t h e 2 n d c u t t i n g , t h e f o d d e r p r o d u c e d b y t h e d i s e a s e d p l a n t s w a s b u t 4 8 . 8 % o f t h a t p r o d u c e d b y h e a l t h y p l a n t s . O n t h e 3 r d c u t t i n g , t h e f o d d e r p r o d u c e d b y t h e d i s e a s e d p l a n t s w a s 6 3 . 4 % o f t h a t p r o d u c e d b y t h e h e a l t h y p l a n t s . B o t h o f t h e s e d e c r e a s e s i n y i e l d w e r e f o u n d t o b e s t a t i s t i c a l l y s i g n i f i c a n t . -101- Itt - E I E L D S T U D I E S PIT T H E W I T C H E S ' BROOM D I S E A S E OF A L F A L F A . A . Q,u a d r a t s t u d i e 3 1 . I n t r o d u c t i o n A t r u e p i c t u r e o f t h e d e c l i n e i n y i e l d s d u e t o a d i s e a s e c a n o n l y h e g i v e n b y a n e x p e r i m e n t c o n d u c t e d u n d e r n o r m a l p l a n t e n v i r o n m e n t . A p o t t e d - p l a n t y i e l d t r i a l , a s p r e v i o u s l y r e p o r t e d , h a s m a n y l i m i t a t i o n s . H o w e v e r , i t d o e s a f f o r d t h e s c i e n t i s t w i t h a v e r y g o o d c o m p a r i s o n o f t h e h e a l t h y a n d d i s e a s e d p l a n t s a n d a s s u c h t h e p r o d e d u r e c o n t a i n s m u c h m e r i t . N e v e r t h e l e s s , i n a g r e e n h o u s e e x p e r i m e n t d i s e a s e d a n d h e a l t h y p l a n t s a r e m a i n t a i n e d u n d e r i d e a l g r o w t h c o n d i t i o n s . T h e e f f e c t s o f o v e r g r a z i n g * w i n t e r i n j u r y , a n d d r o u g h t o n d i s e a s d w e a k e n e d p l a n t s a r e n o t o b s e r v a b l e . T h e o n l y f a c t u a l m e t h o d o f o b t a i n i n g a r e l i a b l e a n d a c c u r a t e r e p o r t o n t h e d e c l i n e i n y i e l d ( a l l f a c t o r s t a k e n i n t o a c c o u n t ) i s t h r o u g h t h e u s e o f q u a d r a t s . I n q u a d r a t s ( a m e a s u r e d a r e a o f f i e l d ) , e v e r y p l a n t i s r e c o r d e d a n d i t s h i t o r y f o l l o w e d . I t i s p o s s i b l e , t h r o u g h q u a d r a t s t u d y , t o o b s e r v e w h e n a p l a n t b e c o m e s s i c k , a n d w h e n i t d i e s a n d d i s a p p e a r s . M a n a g e m e n t f a c t o r s c a n b e t a k e n i n t o a c c o u n t . I n s h o r t , q u a d r a t r e p o r t s a r e o n e o f t h e m o s t v a l u a b l e t o o l s o f t h e a g r o n o m i s t i n s t u d y i n g t h e p r o g r e s s o f a - d i s e a s e , o r . o f a n y o t h e r f a c t o r a f f e c t i n g r a n g e o r p a s t u r e . We h a d a v a i l a b l e q u a d r a t s h e e t s c o v e r i n g q u a d r a t s l a i d i n t h e I n t e r i o r o f B . C . i n t h e s p r i n g o f 1 9 4 2 a n d o b s e r v e d i n 1 9 4 4 b y D r . V . C . B r i n k . T h e r e s u l t s o f t h i s q u a d r a t d a t a a r e r e p r o d u c e d i n t h i s t h e s i s . F e e l i n g t h e i n a d e q u a c y o f t h i s d a t a ( o v e r 2 y e a r s e l a p s e d , b e t w e e n l a y i n g a n d r e a d i n g t h e q u a d r a t s ) a d d i t i o n a l q u a d r a t s w e r e p l o t t e d " i n t h e f a l l o f 1 9 5 0 . - 1 0 2 - 2. S t u d y o f q u a d r a t d a t a f r o m 1 9 4 2 - 1 9 4 4 ( V . C . B r i n k ) . D r . V . C . B r i n k ( u n p u b l i s h e d . ) n o t i c e d i n 1 9 4 1 t h a t a g r e a t m a n y f i e l d s o f a l f a l f a w e r e b e i n g p l o u g h e d u p a n d b e i n g r e p l a n t e d t o g r a s s e s i n t h e r i c h N i c o l a v a l l e y o f B . C . d u e t o t h e r a v a g e s o f a n e v ; d i s e a s e w h i c h p r o d u c e d a s e r i o u s d w a r f i n g o f a l f a l f a p l a n t s a n d a d e c i m a t i o n o f t h e s t a n d s . I n o r d e r t o s t u d y t h e d i s e a s e , t h e f o l l o w i n g y e a r D r . B r i n k p l o t t e d 7 s q u a r e m e t e r q u a d r a t s , l a r g e l y i n t h e C l a r k f i e l d , N i c o l a S t o c k F a r m s , N i c o l a B . C . T h e s e q u a d r a t s w e r e p l o t t e d i n J u n e , 1 9 4 2 a n d f i n a l o b s e r v a t i o n s w e r e t a k e n i n S e p t e m b e r 1 9 4 4 . A l f a l f a p l a n t s h e a l t h y - 1 9 4 2 1 4 0 P l a n t s h e a l t h y - 1 9 4 4 3 3 P l a n t s d i s e a s e d - " 2 7 P l a n t s d e a d o r d i s a p p e a r e d 1 9 4 4 8 0 A l f a l f a p l a n t s d i s e a s e d 1 9 4 2 9 3 P l a n t s d e a d o r d i s a p p e a r e d 1 9 4 4 9 3 2 3 3 2 3 3 T h e s i g n i f i c a n t f e a t u r e o f t h e a b o v e d a t a i s t h a t t h e r e w a s n o v e s t i g e i n 1 9 4 4 o f a n y o f t h e 9 3 W i t c h e s ' B r o o m d i s e a s e d p l a n t s c h a r t e d i n 1 9 4 2 : a l l h a d d i e d o r d i s a p p e a r e d . O f t h e 1 4 0 h e a l t h y p l a n t s p l o t t e d i n 1 9 4 2 , o n l y 6 0 w e r e r e m a i n i n g b y 1 9 4 4 , a n d o f t h e s e 6 0 p l a n t s 2 7 s h o w e d a d v a n c e d s y m p t o m s o f t h e W i t c h e s ' B r o o m d i s e a . s e . O t h e r f a c t o r s m a y h a v e c o n t r i b u t e d t o t h e d i s a p p e a r a n c e o f t h e h e a l t h y p l a n t s b e s i d e s W i t c h e s ' B r o o m , b u t i t i s s t r o n g l y s u g g e s t e d t h a t m a n y o f t h e m m a y h a v e s u c c u m b e d t o t h i s d i s e a s e . O t h e r f a c t o r s w h i c h m a y h a v e c o n t r i b u t e d t o t h e i r l o s s w e r e m a n a g e m e n t f a c t o r s , t o o d r y i n s u m m e r , a n d o v e r g r a z i n g i n t h e w i n t e r . - 1 0 3 - 3 . Q u a d r a t s t u d i e s i n i t i a t e d i n 1 9 5 0 i n t h e I n t e r i o r . - T o f u r t h e r s t u d y t h e p r o g r e s s o f t h e W i t c h e s ' B r o o m o f A l f a l f a d i s e a s e , D r . V . G . B r i n k l a i d 6 q u a d r a t s i n a l f a l f a f i e l d s i n t h e i n t e r i o r o f B . C . a t , o r n e a r , t h e f o l l o w i n g g e o g r a p h i c p o i n t s : o n e n e a r S p e n c e ' s B r i d g e , t h r e e i n t h e v i c i n i t y o f S a v o n a , , o n e n e a r W a l l a c h i n , a n d . o n e o n t h e B a s q u e R a n c h n e a r A s h c r o f t . T h e f i r s t q u a d r a t w a s p l o t t e d J u l y 1 1 , 1 9 5 0 ; a n d t h e r e m a i n i n g f i v e w e r e l a i d S e p t e m b e r 1 8 , 1 9 5 0 . T h e q u a d r a t s w e r e l a i d w i t h o u t p a r t i c u l a r c h o i c e i n t h e f i e l d t o p o s i t i o n : t h e m e t e r - q u a d r a t f r a m e s w e r e c a r r i e d i n t o t h e f i e l d a n d t h r o w n a t r a n d o m - a n d t h e p l a n t l i f e r e c o r d e d w h e r e i t f e l l . F o o t l o n g , o n e - i n c h s q u a r e m e t a l p i n s w e r e d r i v e n o u t o f s i g h t i n t o t h e g r o u n d a t t h e f o u r c o r n e r s o f t h e q u a d r a t f r a m e s o t h a t f u t u r e r e c o r d s c o u l d b e m a d e o f t h e s a m e a r e a , T h e q u a d r a t d a t a s h e e t s f o r t h e 6 q u a d r a t s p l o t t e d , e n c l - o s e d a s A p p e n d i x I o f t h i s e s s a y , a r e b r i e f l y s u m m a r i z e d a s f o l l o w s . 1 . J u l y 1 1 , 1 9 5 0 . S p e n c e ' s B r i d g e , B . C . 1 9 h e a l t h y a l f a l f a 0 W i t c h e s ' B r o o m 2. S e p t . 1 8 , 1 9 5 0 . S a v o n a ? B . C . 8 h e a l t h y 3 d i s e a s e d 3 . II II 1 1 h e a l t h y 1 0 d i s e a s e d 4. •II it 7 h e e l t h y 4 d i s e a s e d 5 . II W a l l a c h i n , B . C . 7 h e a l t h y 5 d i s e a s e d 6 . II B a s q u e R a n c h , A s h c r o f t 1 9 h e a l t h y 9 d i s e a s e d . -104- B - DISTRIBUTION OF ALFALFA WITCHES' BROOM IN B.C. During the summer of 1949 and 1950, a l f a l f a f i e l d s were examined at many points throughout the province of B r i t i s h Columbia (and parts of the state of Washington) for the presence of the A l f a l f a Witches' Broom disease* A knowledge of the disease d i s t r i b u t i o n would point out several sa l i e n t features of the disease. It would show us the e c o l o g i c a l habitat and the climatic conditions under which the diseased condition t h r i v e s . (In A u s t r a l i a , Edwards (12) points out that the disease i s r e s t r i c t e d to areas that have an annual r a i n f a l l of 17 to 21 inches.) I f continuing, a disease d i s t - r i b u t i o n would also give us pertinent information on d i r e c t i o n of spread which may, or may not, be correlated with the poss- i b l e vector spread. Fields were examined during the two years in the d i s t r i c t s as indicated i n the following tables XI & XII and on the accompanying d i s t r i b u t i o n map. A l l f i e l d s (except where indicated by jf) were examined by Dr. V.C.Brink (accompanied in some cases by the author). A l f a l f a f i e l d s i n 35 d i s t r i c t s were found to contain at least 5^ of Witches' Broom diseased plants. The disease was not observed i n f i e l d s i n 31 other d i s t r i c t s , but the conclusion i s not to be drawn that the disease i s not present i n these d i s t r i c t s , but just not observed. A survey of the North Thompson v a l l e y i s planned for l a t e r on i n the investigation. THE DISTRIBUTION OF WITCHES' BROOM OF ALFALFA IN BRITISH COLUMBIA On A p r i l 3 0 , 1 9 5 1 . M - A l f a l f a f i e l d s containing at least 5% A l f a l f a Witches' Broom plants. '% - A l f a l f a f i e l d s that did not contain A l f a l f a Witches' Broom plants.  x -105' a l f a l f a Table X l - D i s t r i c t s i n which/fields were examined that did - \ not oontain A l f a l f a Witches' Broom Plants. Saanich Peniaaula U.B.C. Niooman Island Chilliwack Rosedale Princeton Hedly Keremeos Okanagon P a l l s Summerland West Bank Kelowna Vernon Falkland Lumby B r i d e s v i l l e Rosk Creek Grand Forks Castlegar Balfonr Creston Moyie Cranbrook Newgate Canal f l a t s Edgewater Shftmway Lake Kamloops P a v i l l i o n Stump Lake Lytton (south of) Table XII - D i s t r i c t s i n which a l f a l f a f i e l d s were examined that contained at least 5% A l f a l f a Witches' Broom Plants. Peace River # Wallishin Lake Cathlyn (near Smithers) Savona Smithers Tranquille Fort Fraser Kamloops Vanderhoof Vinsula Quesnel Nicola Williams Lake Nutsford Riske Creek Shumway Lake Spring House Merritt A l k a l i Lake Dot Canoe Creek Aspen Grove Clinton Monte Creek P a v i l l i o n Falkland Hat Creek Armstrong L i l l o o e t Salmon Arm Lytton Methew Vall e y (Washington) McGillvray (Ag*SS«Z?)# Ashcroft jf indicates f i e l d s i n which Withhes' Broom of A l f a l f a has been reported by other investigators that those of the Department of Agronomy, U.B.C. - 1 0 6 - C - Notes on A l f a l f a grown at the Dominion Range Station, Kamloops, B.C. on September 17» 1951> At the same time that the Nursery A l f a l f a Plot was planted at the University of B r i t i s h Columbia, a replicated plot was planted at the Dominion Range Station, Kamloops, B.C. A l f a l f a plants, grown from seed at the U.B.C. greenhouse, were transplanted to the f i e l d at Kamloops on June 1, 1949. Seed for the Rhizoma strains was harvested i n the f a l l of 1948 from open-pollinated clonal rows in the main production f i e l d at U.B.C. Seed of Viking, Ladak, Grimm, Ferax, Buffalo and Ranger was received from the Dominion Department of Agric- ulture, Ottawa. Grimm Vidarshov seed was obtained from Sweden) Grimm Summerland S274 from Dom. Exp't. Sta., Summerland, B.C., and Grimm Sask. 666 and 451 were both obtained from the Dom. Forage Lab., Saskatoon, Sask. The Kamloops nursery was furrow i r r i g a t e d both in the summer of 1949 and 1950. Observations i n the f a l l of 1949 showed a l l plants to be vigorous and healthy. However, in the spring of 1950 some of the plants appeared very poorly and during the summer began to develop disease symptoms. On Sept. 17, 1950 several plants were observed to be showing various stages of the Witches' Broom disease: th i s i s shown i n the accompanying table. Observations of the replicated plants i n the A l f a l f a Nursery at U.B.C. f a i l e d to disclose any plants showing any symptoms of the Witches' Broom disease. The nursery at U.B.C. had received comparable c u l t u r a l treatment to the Kamloops plot, with the exception that sprinkler i r r i g a t i o n was used at U.B.C. and furrow i r r i g a t i o n at Kamloops. TABLE XIII - OBSERVATIONS OH ALFALFA GROWN AT THE DOM. RANGE STA.. KAMLOOPS. B.C. 17/9/50 Row No. plants Variety No. of No. of plants  showing symptoms of Witshes* Br6om of Aifalfa~disease Vftgtr Seed set (graded 1 - 5 ) 1 6 Rhizoma (30-11A) 0 Excellent 0 2 6 » (24-DRC) 0 Excellent 0 - 1 3 6 « (29-11B) 0 Fair 0-1 dry 4 6 M (45-11A) 0 Fa i r 0-1 5 6 » (H71P27) 0 Fair 0-2 dry 6 5 " ( 4-11A) 4 Poor 0-1-2 dry 7 6 « (39-DRC) 0 Fa i r except jf6 0-2 dry 8 6 « (66-10A) 0 Good 0-3 9 6 (51-DRB) 0 Good 0-3 10 6 • (28-11B) 0 Good to poor 0-1 11 6 " (H156P18) 0 Good 3 12 6 Ranger 0 Good to poor 5 13 (6 4 orig) Buffalo 0 Fair( 2 p i . missing) 5 on one 5 on othe: 14 6 Ferax 0 Fair (3 weak) 5 onone 2-3 other 15 4 Grimm ft Good (2 miss.) 3 (6 orig) (primary) 16 6 Ladak 6 F a i r 5+ 17 6 Viking 0 V. good (2 poor) 4+ 18 6 Grimm Sask. 451 4 V. good 5+ rest p 19 6 3 Good 0-1 dry 20 6 " 666 0 Fa i r 0-1 21 6 it tt n 0 (advanced) Very good 2-3 dry 22 6 Summerland S274 5 Good 0 dry 23 6 ti n 0 Good 0-1 ary 24 6 Grimm Vidarshov 0 Good 0-2 dry 25 6 » n 0 Good hutone 5+ H O <! I -108- I v "» Histological studies of Witches' Broom diseased alfalfa , a) Literature review. Neither Edi^ards (12) or Menzies (26) reported on any attempt at studying the tissues of Witches' Broom affected al fa l fa . However there exists several fine review articles on the hisological aspects of plant virus diseases in ganeral. The anatomical aspects of plant virus disease problems are reviewed very completely by Miss Esau (14, 15, 16). She reports that gum deposition and tylose development in the tracheary elements of the xylem are found to be frequently occurring phenomenon. Guramosis may occur in the xylem of various plants under normal conditions, but is frequently more often observed in the tissues of plants affected by various pathogens and physiological disturbances. She reports that most workers think that the gum is derived from the decomposition of starch, and to a lesser extent, other carbo- hydrates; and that the products of decomposition migrate from the l i v i n g cel ls into the tracheary elements. Gummosis i s a very frequent phenomenon of virus infection, but is not specific to this type of disease. Jones and McCulloch (27) report the presence of gum in the vascular system of a l fa l fa affected with the Bacterial Wilt disease. Wynd (53) reviews the metabolic phenomena associated with virus infection in plants. He found that the two major phys- iological effects of virus infection in plants ares (l) Respiratory act iv i t ies are markedly increased very early during the course of infection which corresponds to the period of arr ival of virus particles from the point of infection. The 109- p l a n t r e c o v e r s from t h i s i n i t i a l shock, although the q u a n t i t y of v i r u s p r o t e i n c o n t i n u e s to i n c r e a s e . ( 2 ) The p e r m e a b i l i t y of cytoplasm or i t s membranes i s g r e a t l y a l t e r e d i n r e s p e c t to s o l u b l e substances. This accounts f o r the accumulation of carbohydrates i n the l e a v e s , and the accumulation of n u t r i e n t ions i n the r o o t s at the expense of the tops. Vynd ( i b i d ) i n d i s c u s s i n g the cause of d i m i n i s h e d c h l o r o - p h y l l i n c h l o r o t i c l e a v e s , p o i n t s out t h a t p a r t of the dimin- u t i o n of the t o t a l c h l o r o p h y l l i s due, not only t o a c t u a l decrease per c h l o r o p l a s t i d , but a l s o to the l e s s e n e d number of c h l o r o p l a s t i d s . He b e l i e v e s that the m o t t l i n g of l e a v e s may be caused by the v i r u s , not d i r e c t l y , but through products of a deranged metabolism. However, other i n v e s t i g a t o r s , accord- i n g to Wynd ( i b i d ) b e l i e v e that the i n c r e a s e d a c i d i t y which accompanies v i r u s d i s e a s e s i s a f a c t o r i n the d e s t r u c t i o n of c h l o r o p h y l l . Although c h l o r o p h y l l i s decreased, carotene content of leaves (mosaic) i s enhanced; i n f a c t , the l i g h t green areas showed an i n c r e a s e of 96% over normal c o l o r e d l e a v e s . The h i s t o l o g y of normal a l f a l f a i s very f u l l y d e s c r i b e d by Hayward (21); and the crown and r o o t t i s s u e s are d e s c r i b e d by Jones (28). Graber and a s s o c i a t e s (19) d e s c r i b e the normal food r e s e r v e s of a l f a l f a p l a n t s . Roots of h i g h r e s e r v e p l a n t s have a g r e a t e r p r o p o r t i o n of parenchyma i n r e l a t i o n to the f i b r o u s t i s s u e ; and the parenchyma c e l l s are packed w i t h r e s e r v e foods, p r i n c i p a l l y starch, 1 -110- b ) E x p e r i m e n t a l s t u d i e s 1 . C o m p a r i s o n " o f t h e c e l l u l a r s t r u c t u r e o f h e a l t h y a l f a l f a w i t h a l f a l f a, a f f e c t e d w i t h t h e ' - W i t c h e s ' B r o o m d i s e a s e . ' M a t e r i a l s a n d M e t h o d s ( 1 ) D i s e a s e d a n d h e a l t h y r o o t l e t s , s t e m s , f l o w e r s a n d b u d s w e r e o b t a i n e d f r o m C l i n t o n - A s h c r o f t i n J u n e 1 9 5 0 . F i x e d w i t h R a n d o l p h ' s m o d i f i c a t i o n o f N a v a s h i n ' s s o l u t i o n . I m b e d d e d i n p a r a f f i n , , t r a n s v e r s e s e c t i o n s w e r e c u t w i t h t h e r o t a r y m i c r o - t o n e a t 1 5 t o 2 0 m u . S t a i n e d w i t h s a f r a n i n e a n d m e t h y l e n e b l u e . M o u n t e d i n C a n a d a b a l s a m . ( 2 ) N o r m a l G r i m m c r o w n a n d r o o t s e c t i o n e d w i t h s l i d i n g m i c r o - t o n e a t 4 0 - 5 0 m i c r o n s ( N o v . 1 7 / 5 0 ) . K i l l e d w i t h B . C . F i x . S t a i n e d s a f r a n i n e a n d m e t h y l e n e b l u e . ( F o u n d b e t t e r s t a i n i n g w h e n m o r d a n t f o r m e t h y l e n e b l u e , a m m o n i u m m o l y b d a t e , n o t u s e d ) M o u n t e d i n C a n a d a b a l s a m . ( 3 ) A f t e r t r a n s m i s s i o n e x p e r i m e n t w a s c o m p l e t e d i n J a n . 1 9 5 1 , s e v e r a l o f t h e c r o w n s o f p l a n t s t h a t h a d b e c o m e d i s e a s e d w e r e s e c t i o n e d u s i n g s l i d i n g m i c r o t o n e c u t t i n g a t 4 0 - 5 0 m u . B . C . F i x . S a f r a n i n e a n d m e t h y l e n e b l u e . M o u n t e d i n C a n a d a b a l s a m . ( 4 ) C r o w n s o f d i s e a s e d p l a n t s , t r a n s p l a n t e d f r o m t h e C a r i b o o t h e p r e v i o u s s u m m e r t o t h e g r e e n h o u s e a t U . B . C . w e r e s e c t i o n e d t r a n s v e r s e l y o n s l i d i n g m i c r o t o n e . A s f o r ( 3 ) ( 5 ) L e a v e s f r o m d i s e a s e d a n d h e a l t h y p l a n t s i n F e b . 1 9 5 1 ( g r w w n i n g r e e n h o u s e ) , d i p p e d i n 90% a l c o h o l t o r e m o v e a i r , F i x e d w i t h R a n d . m o d . N a v a s h i n . I m b e d d e d i n p a r a w a x , t r a n s - v e r s e s e c t i o n s a t 1 0 - 1 4 mu w i t h r o t a r y m i c r o t o n e . S e c t i o n s s e c u r e d t o s l i d e s w i t h H a u p # t s a d h e s i v e , s t a i n e d w i t h s a f r a n i n e a n d m e t h . b l u e . M o u n t e d i n C a n a d a b a l s a m . - I l l - O b s e r v a t i o n s C r o s s S e c t i o n s o f r o o t s , s t e m s , n o d e s , " b u d s , w e r e c o m p a r e d f o r d i f f e r e n c e s b e t w e e n h e a l t h y a n d d i s e a s e d t i s s u e s . T h e m a i n , i f n o t o n l y , d i f f e r e n c e o b s e r v e d w a s i n s i z e . T h e d i s e a s e d p l a n t t i s s u e s c e l l s w e r e v e r y m u c h s m a l l e r t h a n t h o s e o f h e a l t h y p l a n t s ( p y g m i s m i c ) . I n t h e h e a l t h y Material, i t w a s n o t e d t h a t i n t h e y o u n g , s q a r i s h i n c r o s s - s e c t i o n , s t e m , t h e v a s c u l a r b u n d l e s a r e w e l l s e p a r a t e d ; w h e r e a s i n o l d e r s t e m s t h e v a s c u l a r b u n d l e s m o r e o r l e s s f o r m a c o n t i n u o u s r i n g . I n t h e d i s e a s e d p l a n t s , t h e s t e m h a s a t e n d e n c y t o r e m a i n s q u a r e i n c r o s s - s e c t i o n a n d w i t h o u t a c o n t i n u o u s v a s c u l a r b u n d l e r i n g b e i n g f o r m e d . T h e n o r m a l c r o w n ( s e e p h o t o ) h a s a r e l a t i v e l y s i m p l e c e l l u l a r s t r u c t u r e , c o n s i s t i n g o f a r a d i a t i n g n e t w o r k o f v a s c u l a r t i s s u e i n t e r s p a c e d w i t h u n d i f f e r e n t i a t e d s t o r a g e a n d s u p p o r t i n g c e l l s ; t h e w h o l e s t r u c t u r e b e i n g s u r r o u n d e d b y a c o r k y e p i d e r m i s . T h e d i s e a s e d c r o w n d o e s n o t a p p e a r g r e a t l y a l t e r e d . X y l e m e l e m e n t s w e r e f r e q u e n t l y f o u n d f i l l e d v / i t h g u m ( p h o t o ) w h i c h s t a i n e d e i t h e r y e l l o w i s h o r r e d . S e c o n d a r y o r g a n i s m s , o n e o f w h i c h w a s a m e m b e r o f t h e B a s i d i o m y c e t e s c l a s s a p p e a r i n g r e a t n u m b e r s i n t h e o u t e r r o t t e d t i s s u e . I n t e r s p e r s e d t h r o u g h o u t t h e c r o w n t i s s u e o f d i s e a s e s p l a n t s v / e r e f o u n d , i n f r e q u e n t l y , s m a l l a r e a s , a p p e a r i n g s o m e w h a t g u m m o t i c , t h a t v / e r e s u r r o u n d e d b y a d i f f e r e n t i a t e d h e a l t h y c a m b i a l a r e a . I n t h e l e a f t i s s u e s , s e v e r a l p o i n t s o f c o n t r a s t w e r e o b s e r v e d . Q u i t e f r e q u e n t l y , i n t h e o u t e r p o r t i o n o f t h e d i s e a s e d l e a f , a b r e a k d o w n o f t h e p a l i s a d e l a y e r w a s n o t e d . T h e p a l i s a d e c e l l s l o s t t h e i r t y p i c a l l y e l o n g a t e s h a p e , b e c a m e r o u n d e d a n d r e s e m b l e d s p o n g y m e s o p h y l l c e l l s . I t w a s a l s o n o t e d t h a t t h e s e c e l l s , i f t h e y c o n t a i n e d a n y c h l o r o p l a s t s a t a l l , p o s s e s s e d b u t a v e r y f e w . T h e m a c r o s y m p t o m s o f t h e l e a v e s o f d i s e a s e d p l a n t s , h a v e o f t e n s h o w n a p u c k e r i n g a n d w r i n k l i n g o f t h e m a r g i n a l l y c h l o r o t i c e d g e s o f l e a v e s . T h e b r e a k d o w n o f t h e s t r e n g t h - g i v i n g p a l i s a d e l a y e r w o u l d a l l o w t h i s p u c k e r i n g c o n d i t i o n t o o c c u r . A l s o n o t e d i n t h e l e a f - c o m p a r i s o n , w a s t h e g r e a t l y i n c r - e a s e d n u m b e r o f c a l c i u m o x a l a t e c r y s t a l s s u r r o u n d i n g t h e - v a s c u l a r b u n d l e s o f d i s e a s e d l e a v e s o v e r t h o s e o f t h e h e a l t h y l e a v e s . 2 . C o m p a r i s o n o f f o o d r e s e r v e s o f h e a l t h y a n d d i s e a s e d p l a n t s . M a t e r i a l a n d M e t h o d s . T h e d i s e a s e d a l f a l f a m a t e r i a l w a s o b t a i n e d f r o m p l a n t s s h o w i n g v a r y i n g s t a g e s o f t h e d i s e a s e a n d w h i c h o r i g i n a t e d i n t h e N i c o l a v a l l e y , t h e p r e v i o u s s u m m e r . T h e h e a l t h y p l a n t m a t e r i a l w a s o b t a i n e d f r o m 6 m o n t h s o l d G r i m m s e e d l i n g s , a n d f r o m 2 y e a r o l d R h i z o m a p l a n t s g r o w n i n t h e g r e e n h o u s e . A s e r i e s o f d i s e a s e d p l a n t s s h o w i n g a g r a d u a t i o n f r o m s l i g h t l y d i s e a s e d t o v e r y b a d l y d i s e a s e d c o n d i t i o n s w e r e e x a m i n e d f o r s t a r c h r e s e r v e s , a l o n g w i t h r e p r e s e n t a t i v e h e a l t h y p l a n t s . C r o s s - s e c t i o n s o f c r o w n a n d r o o t w e r e m a d e f r e e h a n d w i t h a r a z o r b l a d e a n d s t a i n e d w i t h IgKI. L e a v e s f r o m p l a n t s o f t h e s a m e s e r i e s , w e r e b o i l e d i n 70% e t h y l a l c o h o l u n t i l a l l c o l o r w a s r e m o v e d f r o m t h e m , a n d t h e n t h e y w e r e t e s t e d f o r s t a r c h u s i n g I>^1. A t t h e s u g g e s t i o n o f t h e a u t h o r , M r . G e o r g e S e t t e r f i e l d o f t h e D e p a r t m e n t o f B o t a n y c a r r i e d o u t a s o m e w h a t m o r e e x t e n - s i v e m i c r o - c h e m i c a l t e s t s o n r o o t c r o w n s a n d s t e m s o f W i t c h e s * B r o o m d i s e a s e d p l a n t s (2 s t a g e s - m i l d l y d i s e a s e d a n d s e v e r e l y d i s e a s e d ) a n d h e a l t h y a l f a l f a p l a n t s . T h i s w a s p e r f o r m e d a s a m i c r o c h e m i s t r y p r o b l e m f o r B o t a n y 5 3 4 . H e c a r r i e d o u t t e s t s f o r 1 4 o f t h e o r g a n i c m a t e r i a l s a n d f o o d s t o r a g e f o r m s c o m m o n l y f o u n d i n p l a n t s . O b s e r v a t i o n s T h e c r o w n s e r i e s s h o w e d a, d e f i n i t e c o r r e l a t i o n t o t h e a m o u n t o f s t a r c h s t o r e d w i t h t h e 3tage o f t h e d i s e a s e . H e a l t h y c r o w n s c o n t a i n e d a l a r g e q u a n t i t y o f s t o r a g e s t a r c h , w i t h a g r a d u a t i o n d o w n t o n o s t a r c h b e i n g p r e s e n t i n t h e c r o w n s i n t h e p o o r e s t p l a n t i n t h e s e r i e s . A l t h o u g h t h e p o o r e s t p l a n t s t i l l s h o w e d c o n s i d e r a b l e s t a r c h i n t h e l e a v e s , a p r o g r e s s i v e d e c l i n e , a s i n t h e c r o w n s , w a s o b s e r v e d . S e t t e r f i e l d ' s r e s u l t s w i t h t h e c r o w n t i s s u e s a n d t h e i r s t a r c h s t o r a g e s u p p o r t m y f i n d i n g s . H o w e v e r , h e f o u n d t h a t t h e r e w a s m o r e s t a r c h p r e s e n t i n t h e d i s e a s e d s t e m s t h a n i n t h e h e a l t h y s t e m s . H e f o u n d a, - v e r y s i g n i f i c a n t c o r r e l a t i o n b e t w e e n t h e s t a g e o f t h e d i s e a s e a n d t h e s t o r a g e o f s u c r o s e i n t h e c r o w n s . H e a l t h y p l a n t c r o w n s s h o w e d n e g a t i v e t e s t s f o r s u c r o s e i n a l l c a s e s . M i l d l y d i s e a s e d p l a n t s s t o r e d s u c r o s e i n m o d e r a t e q u a n t i t i e s i n t h e c r o w n ; w h i l e s e v e r e l y d i s e a s e d p l a n t s , t h a t s h o w e d n o s t a r c h s t o r a g e i n t h e c r o w n s , s h o w e d a l a r g e q u a n t i t y o f s u c r o s e s t o r e d i n t h e c r o w n t i s s u e . S u c r o s e w a s n o t s t o r e d i n t h e s t e m s i n e i t h e r h e a l t h y o o r d i s e a s e d p l a n t s . - 1 1 4 - P I S C U S S I O N C o n s i d e r a b l e a d d i t i o n a l k n o w l e d g e o n t h e d i s t r i b u t i o n i n B r i t i s h C o l u m b i a o f t h e W i t c h e s ' B r o o m o f A l f a l f a d i s e a s e h a s b e e n g a i n e d a l r e a d y b y t h e s t u d i e s c o m p l e t e d . T h e c e n t r e o f d i s t r i b u t i o n i n t h i s p r o v i n c e a p p e a r s t o b e i n t h e v i c i n i t y o f K a m l o o p s a n d t h e H i c o l a V a l l e y , a n d e x t e n d s n o r t h w a r d ( a n d r a d i a l l y ) i n t h e l o w r a i n f a l l r e g i o n s b e t w e e n t h e C a s c a d e a n d R o c k y m o u n t a i n s . A n u n p u b l i s h e d o r a l r e p o r t o f t h e d i s e a s e o c c u r r i n g i n A l a s k a o n a l f a l f a w o u l d e x t e n d t h e d i s t r i b u t i o n q u i t e f a r n o r t h . T h e p r e s e n c e o f t h e d i s e a s e i s a l s o r e p o r t e d f r o m p a r t s o f A l b e r t a a n d S a s k a t c h e w a n , w h e r e a g a i n , i t i s a p p e a r i n g i n t h e l o w r a i n f a l l r e g i o n s . W i t h t h e e x c e p t i o n o f a r e p o r t i n 1 9 3 7 ( n o t s i n c e o b s e r v e d ) o f t h e d i s e a s e a s o c c u r r i n g a t A g a s s i z i n t h e h e a v y r a i n f a l l a r e a , a l l n o t e d o c c u r r e n c e s o f t h e d i s e a s e h a v e b e e n r e s t r i c t e d t o t h e l o w r a i n f a l l a r e a s . T h i s s a m e d i s t r i b u t i o n p a t t e r n i s o b s e r v e d i n A u s t r a l i a , w h e r e t h e d i s e a s e o n l y a f f e c t s p l a n t s i n t h e 1 7 - 2 1 i n c h a n n u a l r a i n f a l l a r e a s . L e a f h o p p e r s , n o t o r i o u s v i r u s v e c t o r s , n e e d a d r y c l i m a t e t o r e a c h a, m a x i m u m p o p u l a t i o n ; a n d i t i s p o s s i b l e , t h a t t h i s i s c o r r e l a t e d w i t h t h e o b s e r v e d d i s t r i b u t i o n . T h e p h e n o m e n o n o f m a s k i n g o f d i s e a s e s y m p t o m s , a n d t h e r e c o v e r y o f d i s e a s e d p l a n t s , o b s e r v e d i n t h e g r e e n h o u s e s t u d i e s a t U . B . C . , a n d a l s o r e p o r t e d f r o m A u s t r a l i a , m a y e x p l a i n t h e o b s e r v e d d i s t r i b u t i o n p i c t u r e . U n d e r g o o d m a n a g e m e n t a n d s u f f i c i e n t r a i n f a l l , i t i s p o s s i b l e , t h a t t h e d i s e a s e s y m p t o m s w o u l d b e m a s k e d . - 1 1 5 - B e s i d e s t h e o b s e r v a t i o n o f r e c o v e r y o f t h e d i s e a s e d p l a n t s i n s o m e c a s e s , t h e s t u d y , s o f a r , h a s b r o u g h t o u t o t h e r s a l i e n t p o i n t s i n d i s e a s e s y m p t o m o l o g y . W i t c h e s ' B r o o m d i s e a . s e h a s b e e n s h o w n t o a f f e c t y o u n g a l f a l f a p l a n t s - o n e y e a r a f t e r 6 m o n t h s o l d s e e d l i n g s w e r e p l a n t e d i n t h e K a m l o o p s n u r s e r y p l o t , p r i m a r y s y m p t o m s w e r e a p p e a r i n g i n s o m e c a s e s . A c o n t i n u i n g o b s e r v a t i o n o f t h e s e p l a n t s s h o v / e d t h a t t h e o n s e t o f t h e d i s e a s e i s v e r y r a p i d , a s b y t h e v / e r e e n d o f 4 m o n t h s v e r y a d v a n c e d s y m p t o m s o f t h e d i s e a s e w«,a n o t e d . O b s e r v a t i o n s o n t h e d i s e a s e t r a n s f e r e n c e b y g r a f t i n g a t U . B . C . h a v e s h o w n t h a t p l a n t s r e c o r d e d a s s u s p i c i o u s h a d ; i n s i d e o f 2 w e e k s t i m e d e v e l o p e d t h e m a r k e d p r i m a r y s y m p t o m s o f p r o l i f e r a t i o n a n d m a r g i n a l c h l o r o s i s . T h i s w a s f o l l o w e d b y s e v e r e d w a r f i n g i n a n o t h e r m o n t h s t i m e , v / i t h n e c r o t i c s y m p t o m s a p p e a r i n g i n y e t a n o t h e r m o n t h . H i s t o l o g i c a l l y , t h e r e a p p e a r s t o b e v e r y l i t t l e m e c h a n i c a l d i f f e r n c e b e t w e e n d i s e a s e d a n d h e a l t h y t i s s u e s , w i t h t h e e x c e p t i o n o f s i z e , a n d t h e f u r t h e r e x c e p t i o n s , d e t a i l e d l a t e r , o f l e a f p a l i s a d e a b n o r m a l i t i e s . A l l t i s s u e s a n d c e l l s , o f t h e d i s e a s e d p l a n t s w e r e o b s e r v e d t o b e m u c h s m a l l e r t h a n i n t h e n o r m a l p l a n t s . G u m m o s i s v /as f o u n d f r e q - u e n t l y i n t h e x y l e m e l e m e n t s o f t h e c r o w n o f d i s e a s e d p l a n t s e s p e c i a l l y i n t h e o l d e r p o r t i o n s . F o o d s t o r a g e o f s t a r c h i n h e a l t h y c r o w n s w a s f o u n d t o b e d i m i n i s h e d v / i t h t h e p r o g r e s s o f t h e d i s e a s e ; a n d w a s r e p l a c e d b y f o o d s t o r a g e i n t h e f o r m o f s u c r o s e w h i c h i s n o t s t o r e d i n h e a l t h y a l f a l f a c r o w n s . I t i s p o s s i b l e , a n d i n d e e d p r o b a b l e , t h a t t h e v i r u s -116- m a y u p s e t , o r m o d i f y , t h e e n z y m a t i c b a l a n c e o f t h e c r o w n s o t h a t t h i s f o o d s t o r a g e c h a n g e i s b r o u g h t a b o u t . I t w a s a l s o n o t e d t h a t a l l d i s e a s e d c r o w n s w e r e s e v e r e l y r o t t e d , a n d t h e r o t w a s a c c o m p a n i e d b y ( c a u s e d b y ? ) a n u n d e t e r m i n e d B a s i d i o m y c e t o u s f u n g u s . T h e p r e s e n c e o f t h i s f u n g u s w a s n o t o b s e r v e d i n a n y s e c t i o n s o f t h e n o r m a l h e a l t h y c r o w n s . T h i s r a i s e s a n i n t e r e s t i n g c o n j e c t u r e : D o e s t h e p r o d u c t i o n a n d s t o r a g e o f s u c r o s e , b y i m p l e m e n t i n g t h e g r o w t h o f t h e f u n g u s ( s u c r o s e i s a f a v o r e d f u n g o u s m e d i u m o r n u t r i e n t ) , h e l p t ,o h a s t e n t h e d e a t h o f t h e p l a n t b y t h e r o t t i n g o f t h e c r o w n b y t h e f u n g u s ? I n a c o m p a r i s o n o f c r o s s - s e c t i o n s o f h e a l t h y a n d d i s e a s e d l e a v e s ( e x h i b i t i n g m a r g i n a l c h l o r o s i s ) v e r y m a r k e d d i f f e r e n c e s w e r e o b s e r v e d . I n t h e o u t e r p o r t i o n o f m a r g i n a l l y c h l o r o t i c l e a v e s a m e c h a n i c a l b r e a k d o w n o f t h e n o r m a l p a l i s a d e l a y e r w a s o b s e r v e d . t o o c c u r . T h e p a l i s a d e c e l l s r o u n d e d u p a n d l a c k e d o r i e n t a t i o n , a n d a p p e a r e d d e v o i d o f c h l o r o p l a s t s ( w h i c h w o u l d g i v e t h e c h l o r o t i c c o l o r a t i o n t o t h i s a r e a o f t h e l e a f ) . T h e b r e a k d o w n o f t h e p a l i s a d e l a y e r w o u l d l o w e r t h e s t r u c t u r a l s t r e n g t h o f t h e l e a v e s i n t h e i r o u t e r p o r t i o n s - t h i s i s m a n i f e s t e d b y t h e c o m m o n s y m p t o m o f d i s e a s e d l e a v e s s h o w i n g a m a r g i n a l p u c k e r i n g . T h e c h l o r o t i c a p p e a r a n c e o f t h e i n f e c t e d p l a n t s p r o d u c e d o n e o f t h e m o r e p r o n o u n c e d f i e l d s y m p t o m s o f t h e d i s e a s e . T h e c h l o r o t i c a p p e a r a n c e , a c c o m p a n i e d b y d w a r f i n g o f t h e p l a n t c o m b i n e d t o p r o d u c e s y m p t o m s t h a t w e r e v i s i b l e a t c o n s i d e r a b l e d i s t a n c e s . I n f a c t , m a n y f i e l d s w e r e o b s e r v e d - 1 1 7 - f r o m t h e h i g h w a y t o c o n t a i n d i s e a s e d p l a n t s - o b s e r v a t i o n s c o n f i r m e d b y . a c t u a l v i s i t s t o t h e f i e l d s . O v e r 1 3 5 f i e l d s w e r e e x a m i n e d i n t h e I n t e r i o r o f t h e p r o v i n c e d u r i n g t h e t w o s u m m e r s , a n d 5 0 t o 60% o f t h e s e f i e l d s s h o w e d a t l e a s t 5% i n f e c t e d p l a n t s . D i s e a s e i n c i d e n c e w a s g e n e r a l l y c o r r e l a t e d w i t h m a n a g e m e n t p r a c t i c e s o f t h e f i e l d s , b u t n o t a l w a y s . S e v e r a l d i s e a s e d p l a n t s w e r e o b s e r v e d i n t h e a l f a l f a h a y f i e l d a t t h e T r n a q u i l l e f a r m , a n d t h i s f i e l d w a s a n y t h i n g b u t m i s m a n a g e d . T h e r e s u l t s o f a q u a d r a t s t u d y i n i t i a t e d l a s t f a l l ( 1 9 5 0 ) s h o u l d p o i n t o u t m a n y f e a t u r e s i n t h i s r e s p e c t . A s t a r t l i n g f a c t e m e r g e d f r o m a s t u d y o f q u a d r a t d a t a c o l l e c t e d b y D r . B r i n k i n 1 9 4 2 - 4 4 . W h e n p l o t t e d i n 1 9 4 2 , t h e q u a d r a t s c o n t a i n e d a t o t a l o f 1 4 0 h e a l t h y p l a n t s a n d 9 3 d i s e a s e d p l a n t s . I n 1 9 4 4 , n o v e s t i g e r e m a i n e d o f a n y o f t h e 9 3 d i s e a s e d p l a n t s ; a n d o n l y 3 3 o u t o f t h e 1 4 0 h e a l t h y p l a n t s r e m a i n e d i n t h a t s t a t e , 2 7 o f t h e m w e r e d i s e a s e d , a n d 8 0 h a d d i s a p p e a r e d a l t o g e t h e r . T o g a i n a d d i t i o n a l i n f o r m a t i o n o n t h e f a t e o f a l f a l f a p l a n t s , i n t h e f i e l d j 6 m o r e q u a d r a t s w e r e p l o t t e d i n t h e I n t e r i o r , l a r g e l y i n t h e S a v o n a d i s t r i c t . T h e s e w i l l b e o b s e r v e d a t i n t e r v a l s f o r t h e n e x t f e w y e a r s a n d m o r e q u a d r a t s w i l l b e c h a r t e d . T o a u g m e n t t h e q u a d r a t r e p o r t s , a y i e l d t r i a l w a s c a r r i e d o u t 4? t h e g r e e n h o u s e a t U . B . C . d u r i n g t h e p a s t w i n t e r . C u t t i n g s o f c o m p a r a b l e s i z e w e r e p r o p a g a t e d i n p o t s , a n d t h e 1 0 h e a l t h y a n d 1 0 d i s e a s e d p l a n t s w e r e h a r v e s t e d w h e n e v e r t h e h e a l t h y p l a n t s s t a r t e d t o f l o w e r . I t w a s s h o w n t h a t t h e r e w a s a s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r n c e i n y i e l d - 1 1 8 - o f t h e h e a l t h y p l a n t s o v e r t h e d i s e a s e d p l a n t s . O n t h e 1 s t c u t t i n g , t h e h e a l t h y a n d d i s e a s e d p l a n t s w e r e s h o w n t o b e l o n g t o t h e s a m e p o p u l a t i o n a s f a r a s w e i g h t o f f o d d e r w a s c o n c e r n e d . H o w e v e r , t h e 2 n d c u t t i n g showed, a r e d u c t i o n o f 5 1 . 2 ^ a n d t h e 3 r d c u t t i n g a r e d u c t i o n o f 3 6 . 6 ^ i n p r o d u c t i o n o f f o d d e r "by t h e d i s e a s e d p l a n t s w h e n c o m p a r e d t o t h e h e a l t h y p l a n t s . Q u a d r a t r e p o r t s m a y p o s s i b l y c a s t l i g h t o n t h e h o s t r a n g e o f t h e v i r u s , a n d a l s o o n r e s i s t a n c e i n n a t i v e s t o c k s o f a l f a l f a o r c l o s e l y r e l a t e d g e n e r a . I n t h e h o p e o f c o m p i l i n g d a t a o n t h e h o s t r a n g e a n d o n r e s i s t a n c e , a t w i n n i n g e x p e r i m e n t h a s b e e n d e s i g n e d . D u r i n g t h e s u m m e r o f 1 9 5 0 , 4 0 v a r i e t i e s a n d c l o s e l y r e l a t e d s p e c i e s o f a l f a l f a v / e r e p l a n t e d i n a r a n d o m i z e d b l o c k i n t h e n u r s e r y a t U . B . C . T o t h e s e v / i l l b e a d d e d , t h i s s p r i n g , p l a n t s r e c e i v e d f r o m o t h e r s o u r c e s t h i s p a s t w i n t e r . C u t t i n g s f r o m d i s e a s e d p l a n t s w i l l b e p l a n t e d i n c l o s e p r o x i m i t y t o t h e h e a l t h y p l a n t s a n d o b s e r v - a t i o n s m a d e t o d e t e r m i n e i f d i s e a s e t r a n s f e r e n c e o c c u r s . I n c l u d e d i n t h e v a r i e t i e s u n d e r t e s t a r e p l a n t s r e c e i v e d f r o m D r . J . D . M e n z i e s i n W a s h i n g t o n t h a t a r e s u p p o s e d t o c o n t a i n i n h e r e n t d i s e a s e r e s i t a n c e . D i s e a s e t r a n s f e r e n c e f r o m d i s e a s e d p l a n t s o f a l f a l f a t o h e a l t h y a l f a l f a p l a n t s b y m e a n s o f g r a f t e x p e r i m e n t s c a r r i e d o u t i n t h e s u m m e r o f 1 9 5 0 . w a s o b s e r v e d a t U . B . C . O u t o f 1 4 2 a t t e m p t e d c r o w n g r a f t s , u s i n g d i s e a s e d s c i o n s • a n d h e a l t h y R h i z o m a s t o c k s , 1 2 4 p l a n t s v / e r e l i v i n g a t t h e e n d o f 7 m o n t h s . S u c c e s s f u l u n i o n o f s c i o n a n d s t o c k w a s a c h i e v e d i n 3 1 c a s e s , a n d d i s e a s e t r a n f e r e n c e w a s o b s e r v e d - 1 1 9 - i n 2 7 c a s e s . A l l c a s e s w h e r e i n t h e s c i o n a n d s t o c k w e r e i n u n i o n a n d b o t h l i v i n g s h o w e d a d i s e a s e t r a n s f e r e n c e . A g r a f t i n g e x p e r i m e n t , s u c h a s w a s p e r f o r m e d , o u t s i d e • o f s h o w i n g a v e r y g o o d s y m p t o m s e q u e n c e f o r d i s e a s e r e c o g n i t i o n p u r p o s e s , i s o f m o r e i n t e r e s t t o t h e p l a n t p a t h o l o g i s t t h a n t o t h e a g r o n o m i s t . A c r w n g r a f t p l a c e s s t r a i n s o n a p l a n t t h a t i t s e l d o m ( i f e v e r ) r e c e i v e s i n n a t u r e . T h e p l a n t b r e e d e r r e q u i r e s t e c h n i q u e s f o r t e s t i n g l a r g e n u m b e r s o f p l a n t s w i t h o u t s e r i o u s l y m o d i f y i n g t h e t e s t e d p l a n t s . I n d e a l i n g w i t h b a c t e r i a l a n d f u n g o u s d i s e a s e s , a n d , i n d e e d , e v e n w i t h t h e e a s i l y t r a n s m i t t e d m o s a i c t y p e v i r u s d i s e a s e s , s e v e r a l p r o c e d u r e s a r e a v a i l a b l e t o t h e b r e e d e r . H o w e v e r , i n t h e d i f f i c u l t l y t r a n s m i t t e d v i r u s d i s e a s e s , t r a n s m i s s i o n a n d t e s t i n g i s n o t s u c h a s i m p l e m a t t e r . W h e n t h e i n s e c t v e c t o r o f t h e v i r u s d i s e a . s e i s k n o w n a n d a v a i l a b l e , r e s i s t a n c e t e s t s t h e n b e c o m e a. r e l a t i v e l y s i m p l e p e r f o r m a n c e . T h e o n l y k n o w n v e c t o r o f W i t c h e s ' B r o o m o f A l f a l f a , t h e l e a f h p p p e r , P l a t y m o i d e u s a x s u t u s , h a s s u c c e s s f u l l y r e s i s t e d a t t e m p t s a t b e i n g r e a r e d , a n d i n d e e d , i s n o t c o n s i d e r e d t o b e t h e c h i e f v e c t o r o f t h e d i s e a s e . A n y w a y , a n e a s i l y e m p l o y e d v e c t o r , a l t h o u g h i t w o u l d b e a d v a n t a g e o u s , i s n o t e s s e n t i a l t o t h e p l a n t b r e e d e r i n d e t e r m i n i n g f i e l d r e s i s t a n c e . F i e l d r e s i s t a n c e m a y n o t i m p l y t r u e i m m u n i t y , b u t m e r e l y t h e f a c t t h a t t h e p l a n t s c a n g r o w s u c c e s s f u l l y i n t h e p r e s e n c e o f t h e d i s e a s e . T h e p o s s e s s i o n o f f i e l d r e s i s t a n c e c a n r e a d i l y b e d e t e r m i n e d b y i n t e r p l a n t i n g d i s e a s e d a n d h e a l t h y p l a n t s , a s i n t h e t w i n n i n g e x p e r i m e n t p r e v i o u s l y -120- d i s c u s s e d , p r e f e r a b l y i n a n a r e a w h e r e t h e d i s e a s e o c c u r s n a t u r a l l y a n d w h e r e t h e n a t u r a l a g e n t s o f i n o c u l a t i o n r e s i d e . W i t h t h i s i n m i n d , e x p e r i m e n t s o f t h i s t y p e a r e p l a n n e d t o b e i n i t i a t e d t h i s y e a r a t p o i n t s i n t h e I n t e r i o r w h e r e t h e d i s e a s e i s e n d e m i c . SUM1ARY T h e d i s t r i b u t i o n o f t h e W i t c h e s * B r o o m d i s e a s e o f A l f a l f a i n B r i t i s h C o l u m b i a i s s h o w n t o b e c o n f i n e d t o t h e a r e a s o f l o w r a i n f a l l . S e v e r i t y o f t h e d i s e a s e i s d i s c u s s e d o n t h e b a s i s o f f i e l d a n d q u a d r a t o b s e r v a t i o n s . Q u a d r a t a n d y i e l d d a t a a r e i n c l u d e d . D i s e a s e d p l a n t s a r e s h o w n t o h a v e a s t a t i s t i c a l l y s i g n i f i c a n t r e d u c t i o n i n y i e l d c o m p a r e d t o h e a l t h y p l a n t s u n d e r s a m e c o n d i t i o n s . S y m p t o m o l o g y o f t h e d i s e a s e i s e n l a r g e d . R e c o v e r y ' o f d i s e a s e d p l a n t s i s n o t e d . A h i s t o l o g i c a l s t u d y o f t h e d i s e a s e s h o w s g u m m o s i s i n t h e v e s s e l s o f t h e c r o w n s , a n d m e c h a n i c a l b r e a k d o w n o f p a l i s a d e c e i l s o f t h e l e a v e s . S t o r a g e s t a r c h i s s h o w n t o b e d e p l e t e d i n t h e c r o w n s , a n d i n s t e a d f o o d i s s t o r e d i n f o r m o f s u c r o s e . S u c c e s s f u l t r a n s m i s s i o n o f t h e d i s e a s e w a s a c h i e v e d b y c r o w n g r a f t s . S e e d t r a n s m i s s i o n , a n d i n o c u l a t i o n s w i t h e x p r e s s e d s a p g a v e n e g a t i v e r e s u l t s a t p r e s e n t . E x p e r i m e n t s d e s i g n e d f o r t h e f u t u r e s t u d y o f t h e d i s e a s e a r e d i s c u s s e d . 121- (1 (2 (10 (11 (12 (13 (14 • LITERATURE CITED . Anonymous. Al fa l f a Improvement Conference, Twelfth Annual Report. July 31 - August 2, 1950. Lethhridge, Alta . Brink, V . C . ' Witches' Broom on al fa l fa . In 22nd. Ann. Rept. C an. PI. Dis . Sur. 1942. (3) Cormack, M.W. Rept. Can. Witches' PI. Dis . Broom Sur. of Al fa l fa . 1940. In 20th Ann. (4) • In 21st Ann. (5,) Rept. ""Can. PI. Dis . Sur. 1941. • In 22nd Ann. 6 ; Rept. C an. PI. Dis . Sur. 1942. (e') * In 23rd Ann. Rept. C an. PI. Dis . Sur. 1943. (7) • In 24 th Ann. t' Rept. Can. PI. Dis . Sur. 1944. (8) • In 25 th Ann. Rept. Can. PI. Dis . Sur. 1945. (9) • In 26th Ann. Rept. Can. PI. Dis . Sur. 1946. Dickson, B .T . Studies concerning mosaic diseases. MacDonald College, Tech, B u l l . 2. 125 p. 1922. Edwards, E . T . Witches' Broom, a new virus disease of lucerne. Jour Australian Inst. Agr. Sc i . 1: 31-32. 1935. The Witches' Broom disease of lucerne. Dept. Agr..Sew South Wales Sc i . B u l l . 52. 1936. . Witches' Broom of lucerne. Agric. Gaz. of New South Wales 47; 424-426. 1936. Esau, Katherine. Some anatomical aspects of plant virus disease problems. II. Bot. Rev. 14: 413- 449. 1948. (15) Esau, Katherine. Anatomic effects of the viruses of Pierce's disease and-the phony peach. Hilgardia 18: • 423 - 464. 16 plates. 1948. (16) Phloem structure in the grapevine, and i t s seasonal changes. Hilgardia 18: 217-296. 1948. - 1 2 2 - ( 1 7 ) P o s t e r , W . R . A l f a l f a W i t c h e s ' B r o o m , c a u s e u n d e t e r i n i r i e d . I n A n n R e p t . C a n , P I . D i s . S u r . 1 2 : 2 5 . 1 9 3 2 ( 1 8 ) G i l p a t r i c k , J . D . W i t c h e s ' B r o o m o f A l f a l f a . W i t c h e s ' B r o o m o f A l t a s w e d e r e d c l o v e r . I n 2 7 t h A n n R e p t C a n P I D i s S u r . 1 9 4 7 . ( 1 9 ) G r a h e r , N e l s o n , N . T . , L e u k e l , W . A . , a n d W . B . A l b e r t . O r g a n i c f o o d r e s e r v e s i n r e l a t i o n t o t h e g r o w t h o f a l f a l f a a n d o t h e r p e r e n n i a l h e r b a c i o u s p l a n t s . W i s e . A g r . E x p . S t a . R e s . B u l l . 8 0 ; 1 9 2 7 ( 2 0 ) H a s k e l l , R . J . D i s e a s e s o f c e r e a l c r o p s a n d f o r a g e c r o p s i n t h e U n i t e d S t a t e s i n 1 9 2 5 . I n U . S . D e p t . A g r . P I . D i s . R e p t . S u p p . 4 8 : 3 6 7 . 1 9 2 6 . ( 2 1 ) H a y w a r d , H e r m a n E . M e d i c a g o s a t i v a . I n T h e S t r u c t u r e o f E c o n o m i c P l a n t s . T h e M a c m i l l a n C o m p a n y . 1 9 3 8 . ( 2 2 ) H e a l d , E . D . a n d J . D . M e n z i e s . W i t c h e s ' B r o o m o n a l f a l f a . 2 2 n d . A n n . R e p t . C a n . P I D i s . S u r . 1 9 4 2 . ( 2 3 ) H e a l d , E . D . a n d R . H . W e l l m a n . E t i o l o g y a n d p r e v e n t i o n o f a l f a l f a f a i l u r e s . I n 4 9 t h A n n . R e p t . W a s h . A g r . E x p . S t a . B u i . 3 8 4 : 5 7 . 1 9 3 9 . ( 2 4 ) H e w i t t , W m . B, a n d B y r o n H o u s t o n . A s s o c i a t i o n o f P i e r c e ' s d i s e a s e , o f g r a p e v i n e s a n d a l f a l f a d w a r f d i s e a s e - i n C a l i f o r n i a . U . S . D . A . P I . D i s . R e p t . 2 5 : 4 7 5 - 4 7 6 . 1 9 4 1 . ( 2 5 ) H e w i t t , Wm. B . , B y r o n H o u s t o n , H . W . E r a z i e r a n d J . E r i e t a g . L e a f h o p p e r t r a n s m i s s i o n o f t h e . v i r u s c a u s i n g P i e r c e ' s d i s e a s e o f g r a p e s a n d d w a r f o f a l f a l f a . P h y t o p a t h . . 3 6 : 1 1 7 - 1 2 8 . ^ 1 9 4 6 . ( 2 6 ) H o u s t o n , B y r o n R . D w a r f r e s i s t a n t a l f a l f a . S e e d o f n e w C a l . c o m m o n 4 9 t o b e r e l e a s e d n e x t s e a s o n f o r c o m m e r c i a l h a y p r o d u c t i o n i n c e r t a i n a r e a s . C a l . A g r i c . 3 : 1 0 - I l l u s . 1 9 4 9 . ( 2 § a ) H u r s t , R . R . M o s a i c i n P . E . I . I n 2 5 t h A n n . R e p t . C a n . P I . D i s . S u r . 1 9 4 5 . ( 2 7 ) J o n e s , E . R . , a n d L . M c C u l l o e h . . A r o o t r o t a n d b a c t e r i a l w i l t o f a l f a l f a c a u s e d b y . . A p l a n o b a c t e r i n s i d i o s u m L . M c C . J o u r . A g r . R e s . 3 3 : 4 9 3 - 5 2 1 . 1 9 2 6 . ( 2 8 ) J o n e s , E . R . a n d J . L . W e i m e r . W i n t e r i n j u r y o f a l f a l f a . J o u r . A g . R e s . 3 7 * . 1'8'9 - . ' " 2 1 1 . 1 9 2 8 . ( 2 9 ) Jone3_, W a l t e r . W i t c h e s ' B r o o m o f A l f a l f a . I n 1 7 t h A n n . K e p t . C a n . P I . D i s . S u r . 1 9 3 7 . - 1 2 3 - ( 3 0 ) J o n e s . W a l t e r . W i t c h e s ' / b r o o m , o f a l f a l f a . I n 1 8 t h A n n . R e p t . C a n . P I . D i s . S u r . 1 9 3 8 ( 3 1 ) L e b e a u , J . B . V / i t c h e s * b r o o m o n a l f a l f a . I n 2 9 t h A n n . R e p t . C a n . P I . D i s . S u r . 1 9 4 9 ( 3 2 ) L e o n a r d a n d C l a r k . F i e l d P l o t T e c h n i q u e . B u r g e s s P u b . C o . 1 9 3 9 . ( L e o n a r d , W a r r e n H. & A n d r e w G . C l a r k ) ( 3 3 ) H e a d , H . W . W i t c h e s ' b r o o m o f a l f a l f a . I n 2 8 t h A n n . R e p t . C a n . P I . D i s . S u r . 1 9 4 8 . ( 3 4 ) M e n z i e s , J . D . W i t c h e s ' b r o o m o f a l f a l f a i n F o r t h A m e r i c a . P h y t o p a t h . 3 6 : 7 6 2 - 7 7 4 . 1 9 4 6 . ( 3 5 ) M u n r o , J . B . W i t c h e s ' b r o o m o n r e d c l o v e r . I n 2 8 t h A n n . R e p t . C a n . P I . D i s . S u r . 1 9 4 8 . ( 3 6 ) M c C l e e r y , F . C . L u c e r n e W i t c h e s ' B r o o m . I n P l a n t D i s e a s e s R e c o r d e d i n H e w S o u t h W a l e s . D e p t . A g r . N . S . W . S c i . B u i . 4 6 . . 1 9 3 4 . ( 3 7 a ) M a c L e o d , D . J . A l f a l f a M o s a i c . I n 2 5 t h A n n . R e p t . C a n . P I . D i s . S u r . 1 9 4 5 . ( 3 7 ) M a c L e o d . D . J . W i t c h e s ' b r o o m o n r e d c l o v e r . I n 2 9 t h A n n . R e p t . C a n . P I . D i s . S u r . 1 9 4 9 . ( 3 8 ) M c W h o r t e r , F r a n k P . A l f a l f a v i r u s N . P h y t o p a t h 3 9 : 8 6 1 . 1 9 4 9 . A n a b s t r a c t . ( 3 9 ) N o b l e , R . J . e t a l . T h e o c c u r r e n c e o f p l a n t d i s e a s e s I n New S o u t h W a l e s . D e p t . A g r i c . N . S . W . S c i . B u i 5 7 : 1 7 . 1 9 2 7 . ( 4 0 ) P a y e t t e , A . W i t c h e s ' b r o o m o f L a d i n o w h i t e c l o v e r . I n 2 9 t h A n n . R e p t . C a n . P I . D i s . S u r . 1 9 4 9 . ( 4 1 ) R i c h a r d s , B . L . W i t c h e s ' b r o o m , c a u s e u n k n o w n . I n U . S . D e p t . A g r . P I . D i s . R e p t . 7 1 : 3 0 9 - 3 1 0 . 1 9 2 9 . ( 4 2 ) S m i t h , K e n n e t h . T e x t b o o k o f p l a n t v i r u s d i s e a s e s . C h u r c h i l l P r e s s . 1 9 3 9 . ( 4 3 ) T h o m s o n , J . E . J , a n d J . B . L e b e a u . W i t c h e s ' b r o o m o n a l f a l f a . I n 2 8 t h A n n . R e p t . C a n . P I . D i s . S u r . 1 9 4 8 . ( 4 4 ) W e i m e r , J . L . A l f a l f a d w a r f , a h i t h e r t o u n r e p o r t e d d i s e a s e . P h y t o p a t h . 2 1 : 7 1 . 1 9 3 1 . (45) A l f a l f a m o s a i c . P h y t o p a t h . 2 1 : 1 2 2 - 1 2 3 . ( a b s t r a c t ) . 1 9 3 1 . Weimer, J .L . Studies on a l fa l fa mosaic. Phytopath. 24: 239-247. 1934. " A l f a l f a dvfarf, a virus disease transmias- able "by grafting. Jour. Agr. Res. 53: 333-347. 1936. Woolliams, Witches' broom on a l fa l fa . In 23rd Ann. Rept. Can. PI . Di s . Sur. 1943. . In 28th Ann. Rept. Can. PI. Di s . Sur. 1948. Wright, U.S. Witches' broom on a l fa l fa . In 26th Ann. Rept. Can. PI. Di s . Sur. 1946. . In 27th Ann. Rept. Can. PI . D i s . Sur. 1947. . Witches' broom in B .C . In 49th Ann. Rept. Can. PI . Di s . Sur. 1949. Wynd, P . L . Metabolic phenomena associated with virus infection in plants. Bot. Rev. 9: 395-465. 1943. Quadrat No i.r.JC.9.. Date Charted. it . r ^ H . . . . . . J « ( >.  j £ ) /? )\ • 0 ( ' / / / / / / / / ; / / / \ * t K S J J , Jt ' ' ' , / « ; i ;• * ) 0 < < O v4 x *. G ^ ) * / * * V 1 0 / •1 i i <3 &IQ *r & g & to/sire ' T / / / Species Area in SQ. cm. No. of culms Average Height Leaves Stems Seek Prod. i Vigour REMARKS 1 7T~. * if T • •  1 • /S •D O<JV *y fyat • •( SA/IO mfrt 0/1*** F A < - — . : ~~ - - QUADRAT No. L:..5° Date Charted ^1 : Location • * • • £ £ f i t - s T A L F A L F A u - e . S f e u c e F L A T (, ttt««-»t U C O » T & i L e u e ) o w B E M C H n B o u e & - V J • s i n e O F u m u l&iA&CTeP H t f r H W « y j f i t . i T CMLt>»£C O P F i t L b j C o f Roc I t * M A » « I , <W ^ O A O S I O S " O I T W ". H « » Y C o M f l t J O S T i»J S ' * H T , Character of Site. (a) Exposure and slope ~ — - (b) Altitude * 0 ° ' . . . . . (c) Soil.. - - ~ , Plant Type. (a) FormaUon^}^.^^..-.^1:*:^^ (b) Association. (c) Principal species SUMMARY OF QUADRAT D A T A (a) Grasses and Sedges. Totals Average i e a v e s height ' l m s | Area J s q - c m occupied OL | (b) Non-Grasses. Totals veg. 1 height 1 flower < Area J s q - - occupied ] OL Relative Forage Value of Quadrat. % Carry-over. Distance of Quadrat from nearest watering place.. Other Factors affecting grazing Remarks i Quadrat No.. 6* s* Date Charted,. v ; ; * ^ ^ ^ ^ / « v > — i — 1 OifZ- O M l ' O Ht" /V" ) / ' 1 • < y O" A" \ CD < • w 6 * IX » ft 4' \ CD, 1 ® . - j . . d -1 . t < £ J . ) CP V . fir. ^ 1 '"S. L. /A © \ i z 1 No. Species St.*'9*' * ?y /9 £ A/9*/=MJ' Area in sq. cm. No. of culms Average Height Leaves Stems Seed Prod. Vigour REMARKS QUADRAT No. 6 - 5 0 Location....Ash.crpft..Manor.^ Date Charted- near main highway at the foot of the Oregon Jack Creek Road; l i n e up outhouse and conspicuous red soIT outcrop; % paces "from deep with Clematis l i g r e s t i o i f o l l a . N Character of Site. (a) Exposure and slope... , .-. . (b) Altitude }?.0.?:. (c) Soil l*8ht . * r ^ ^ I„„„„L Plant Type. (a) Forniation..^^1.?.?.^....-....^0.^.?. J!.̂ .0.11.?.*. (b) Association...J [ (c) Principal species.. SUMMARY OF QUADRAT DATA (a) Grasses and Sedges. Totals Average J iedve!> height 1 dims | Area J S q ' C occupied OL (b) Non-Grasses. : . , ; Totals veg. height j flower Area 1 q - occupied ] or • Relative Forage Value of Quadrat , % Carry-over. Distance of Quadrat from nearest watering place Other Factors affecting grazing...... .... Remarks  Mileage No. 139.2 V.C.Brink QUADRAT No.......2."....5.0. , 4 . Sept. 18, 1950 , SAVONA Date Charted ; Location S of main highway - Kamloops - Savonaj near telephone pole 11/34 KA 112 with cross support on opposite side" Is)' s i d e 6 ? Bi'ghway; quad .with..2 above^.mention Character of Site. (a) Exposure and slope..;.- (b) Altitude .?..?..9.°„! 1 (c) Soil....M.8.k*...!?.^ Plant Type. (a) Formation (b) Association....;. (c) Principal species .....AA?*^.?.*..."...?.^.?.?.!?.. (a) Grasses and Sedges. SUMMARY OF QUADRAT DATA Totals Symbol ........... No. of Specimens- leaves.. culms... No. of culms Seed production.... Average height Area occupied sq. cm. % (b) Non-Grasses. Totals - veg. Average growth - height "j flower • i j 1 Area J L occupied i o/n Relative Forage Value of Quadrat Distance of Quadrat from nearest watering place. Other Factors affecting grazing......... Remarks % Carry-over.. No. Species Area in sq. cm. No. of culms Average Height Leaves Stems Seed Prod. Vigour REMARKS S " ff /o i V.C.Brink QUADRAT No A..= J?° Date Charted.. .S.®?t..V..l8...V...19.5.0. Location Near (W ojf) Qua •No Hunting' sign; quadrat located 21 paces S of fence Character of Site. (a) Exposure and slope ^B...t?.*...Qt~%~&9. (b) Altitude - - (c) Soil A°. Plant Type. (a) Formation A°. (b) Association.. (c) Principal species ; SUMMARY OF QUADRAT DATA (a) Grasses and Sedges. Totals Average J i e a v e s > height 1 l m s | | Area . s q - " occupied OL | (b) Non-Grasses. Totals veg. height 1 flower Area J q > c occupied j OL - Relative Forage Value of Quadrat < % Carry-over. Distance of Quadrat from nearest watering place Other Factors affecting grazing : Remarks... - - -- -— - - i i  mileage No. 142.4 V.CBrink QUADRAT No.. *..-J.9... Date Charted .....S..ePl.V...l8......l.?.5.? „ j , . L o c a i i o n  8™™t. West of Q 2-5.0. and 0̂. J ..?.?....̂ .̂ .S)?Lw.a.y....b..!l*w .̂?.:?. telephone poles 9/7 KA 5 9 and 9/6; quadrat located about 2 paces 'inside" "̂ 'S )""vf e"ne e V vWe si" "'of gate, Character of Site. (a) Exposure and slope... : l i g h t brown, sandy (b) Altitude (c) Soil.. Plant Type. (a) Formation... A ^ . 1 . ^ (c) Principal species (a) Grasses and Sedges. SUMMARY OF QUADRAT D A T A Totals Symbol No. of Specimens.. Average { l e a v e s - height 1 c u l m s ___ No. of culms Seed production... sq. cm- Area occupied (b) Non-Orasses. Totals • veg. height "j flower Area J q " " occupied i OL Relative Forage Value of Quadrat Distance of Quadrat from nearest watering place- Other Factors affecting grazing.... Remarks... ............ .., % Carry-over.. No. Species Area in sq. cin. No. of culms Average Height Leaves Stems Seed, Prod. Vigour REMARKS 3 + * < 3. A • +n 7 ... — Mileage No. 165.65 QUADRAT No L:J1 Date Charted .'...A?_L..-i Location, , Near "Hoodoo C l i f f s " Wallachin; Hay stacker and lone deciduous tree make a l i n e on which quadrat is located in the f i e l d , S of highway to Cache Creek, 9 paces from fence and i r r i g a t i o n d i t c h . ; " ( f f a y W r a ' s l Character of Site. (a) Exposure and slope (b) Altitude I3:*™' <c) Soil ... Plant Type. (a) Formation A l f alfa. „-„K ....M.Uftgras.S... (b) Association...... '. (c) Principal species. (a) Grasses and Sedges. SUMMARY OF QUADRAT DATA Totals Symbol No. of Specimens.. Average { l e a v e s " height | c u l m s . . . No. of culms ... Seed production.... Area occupied sq. cm.. (b) Non-Grasses. Totals Symbol- No. of specimens.. veg. Average height • growth- flower stalks.... No. flower stalks,. Seed production.... Area 1 s ^ c m ' )ied j occup j o/Q Relative Forage Value of Quadrat Distance of Quadrat from nearest watering place- Other Factors affecting grazing. Remarks ........ % Carry-over..

Cite

Citation Scheme:

    

Usage Statistics

Country Views Downloads
United States 13 0
China 8 0
France 1 0
Egypt 1 0
Russia 1 0
City Views Downloads
Beijing 8 0
Unknown 6 5
Clarks Summit 4 0
Wilkes Barre 1 0
Mountain View 1 0
Redmond 1 0
Madinat an Nasr 1 0
Sunnyvale 1 0
Ashburn 1 0

{[{ mDataHeader[type] }]} {[{ month[type] }]} {[{ tData[type] }]}

Share

Share to:

Comment

Related Items