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

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P3  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 T h e s i s Submitted i n P a r t i a l F u l f i l m e n t of the Requirements f o r the Degree o f Master of Science i n A g r i c u l t u r e in The Department of Agronomy  We accept t h i s t h e s i s as conforming t o the standard r e q u i r e d f o r candidates f o r the degree o f MASTER OF SCIENCE IN AGRICULTURE  Members^ of %\ie Department o f Agronomy THE UNIVERSITY OF BRITISH COLUMBIA April,  1951.  ABSTRJ-3T In use of  i n  This of  the  the  grown  the  the  study  viable  sterile  seed  of  pollen  to  the  male-sterility  w i l l It  for  to  only  as  has  i n  not  common  the  plant  Over  the  past  of  -  the  B.C.  1942-44.  was  The  low r a i n f a l l  although  sacs  f u l l  hybrid  2  of  i n  malevery  for  1:7.3,  male1:6.8,  suggest  of  that  i n  inheritance  and- b a c k c r o s s a breakdown amorphous pollen  prove  alfalfa  the  factors  mode  an  the  absence  open-pollinated  ratios  ]?2  of  of  an  produced  2  from  caused by  isolated  of  i n  material  grains.  great  seed.  alfalfa,  data.  economic  M a l e - s t e r i l i t y  although  i t  is  kingd.om. 10  years into  f i r s t  disease areas  sporadic.  is  are  showed  1:7.9,  the  by  alfalfa  conducted.  cytoplasinaic  However,  study  of  segregation  These  eventually  of  ¥±  follows:  development  been  developed  this  the  interpreted  should  lines  and  grown  count.  s t e r i l i t y  production  the  plants  as  genes.  arrested  i n  selfing  controlled by  pollen  previously  has  - i n  the  the of  a l f a l f a  is  1000  environments.  on  for  a microscopic  consistently  lines,  incomplete  male-sterility  value  In  were  f a c t o r i a l l y  that  suggestive The  an  recessive  be  appears  meiosis,  seed.  male-fertile  1:8.3,  that  seed  4 male-sterile  and  addition  no  strains  B r i t i s h - C o l u m b i a was  different  produced of  of  a l f a l f a  alfalfa,  approximately  lines  under  quantities  sterile  4  male-sterile  hybrid  by  University  revealed  the  of  produced  lines  of  finding  production  pollen at  small  hope  of  the  Witches'  serious  pointed is  known  the  Broom virus  proportions  i n  the  out  by  quadrat  to  be  distributed  province  where  i t  disease  Interior  results  is  of  obtained  throughout widespread  The in  Washington  causes of  disease  reduction crowns  i n  and  i n  size  a  of  are  of  the  Up  to  symptomless  die  period  3 months  were  shown  into  the  to  U.B.C.  plants  showed  degeneration mechanical of  with  found no  was  "be  of  not  the  the  starch was  present  i n healthy  disease  was  Out  of  142  achieved  i n  31  Seed  transmission  have  given  to  to  attempted  and  i n be  3-4  florets  produced.  Crowns  the  disease  and  gradually  plants  when  "brought  the  leaves,  the  of  diseased  a  edge  of  the  and and  outer  crowns  stage  i n  diseased  a "breakdown  i n  severely present  2  are  Valley.  affected  the  disease  plants  However,  i n  commonly  to  Affected  layer  to  are  of  are  healthy  starch  and  leaves  of  Nicola  the  decimation  plants disease,  crowns.  storage  i n  diseased  transmitted  by  crown  crowns.  found  cases  negative  of  according  found  is  xylem vessels, of  a  shoots  seed  the  A l f a l f a  "by p r o l i f e r a t i o n  stages  palisade  Storage  depleted  sucrose  but  The grafts.  to  of  of  occurs  internodes  from the the  chloropasts  leaves.  storage  However, crowns  the  "breakdown  diseased  was  of  late  from  and  reduced  2 years.  comparison  gummosis  are  r o t t i n g .  to  that  Typically,  any,  recover  greenhouse  A h i s t o l o g i c a l  i f  severe  apparently  of  spindly  crown.  u n t i l  show  plants  accompanied thin  l i t t l e ,  disease  W i t c h e s ' Broom of  inflorescences  very  of  is  diseased'  when t h e y  a  same  affected  3000  reached, in  the  shortening  leaves  single  and,  be  Drastic  chlorotic;  raceme;  to  Australia.  dwarfing  nod.es.  marginally  and r o o t s  shown  stands.  p r o d u c e d "by  per  and  severe  a l f a l f a  is  with  be  easily  grafts, positive  inoculations by  results  to  date.  union  of  scion  transmission expressed  and i n  crown  27  stock cases.  juice  Quadrats to  further  up  to  study  study  Medica^o. ready  for  were  at  plot,  Results  symptoms  of  of  time.  the  from  both  of  the  was  a l f a l f a  of  of  the  and  the  of  these  studies  w i l l  2 more at  after  be  plants  were  the  set  genus  not  and  a  be  replicate  p l a n t i n g the  badly  B.C.  years.  U.B.C.  were  at  of  experiments  members  plants to  interior  the  least  One y e a r  disease,  None  of  established  B.C. the  at  i n  Twinning  resistance  plot  Kamloops,  i n 1950  disease.  publication for  several  months  the  natural  A nursery plot  plotted  found  producing  diseased  U.B.C.  Kamloops  showed  i n any  15 signs  disease.  From diseased reduction  a  potted  plants  plant  were  i n yields  y i e l d  shown t o as  t r i a l have  compared  to  a  conducted  at  s t a t i s t i c a l l y  healthy  plants.  U.B.C.  the  significant  AG_-T0Vyj_3D GMEHTS It i s with r e a l pleasure that I take this opportunity i n thanking a l l those persons who helped i n any way with my research and i n the preparation of t h i s 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 d i r e c t i o n the research was conducted and t h i s thesis prepared. To him, I am indebted for much of the data included i n t h i s paper. . His ready and helpful advice, h i s sympathetic understanding of the other person, and h i s many kindnesses to my wife, my son and me, have contributed greatly to making the two years spent i n 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 D r . 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 a l f a l f a . An expression of sincere appreciation i s also extended to D r . H.S.Wright and D r . JTitzpatrick of the Dominion Plant Pathology Laboratory, University of B r i t i s h C o l u m b i a , t o the former for h i s help i n grafting studies, f i e l d observations and helpful advice on virus research; and to the l a t t e r for generously making available books, periodicals and laboratory equipment. I would also l i k e to thank the members of the Dominion Experiment Range Station at Kamioops, especially Mr. _ . W i l l i s and Mr. A. McLean, for providing the space for a nursery,, and for their observations and maintenance of t h i s p l o t . My thanks also go to Dr. J.D.Menzies i n Washington for providing a l f a l f a stocks containing some measure of r e s i s t ance to Witches' Broom disease for inclusion i n our f i e l d t r i a l s ; to Mr. G-. S e t t e r f i e l d of the Department of Botany, University o f - B . C . for r e s u l t s of a micro chemical test oh diseased a l f a l f a ; to D r . B.T.Dickson and associates of the D i v i s i o n of Botany, Australian Dep't of Agriculture for information on the Australian Witches' Broom disease; to Mr. A. Richman, Dep't. of H o r t i c u l t u r e , U . B . C . for his advice on, and help i n , greenhouse p r a c t i c e s . Last, but by no means l e a s t , I would l i k e to tender my sincere thanks to my wife for her w i l l i n g help i n 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) D e f i n i n g s t e r i l i t y and i n c o m p a t i b i l i t y  7  (b)  9  Causes o f s t e r i l i t y  12  (c) I n c o m p a t i b i l i t y  17  B - M a l e - s t e r i l i t y i n the h i g h e r p l a n t s  17  (a) Some general remarks (b) M a l e - s t e r i l i t y governed by one or a few r e c e s s i v e Mendelian f a c t o r s  18  (c) M a l e - s t e r i l i t y governed by a s i n g l e dominant gene  24  (d) M a l e - s t e r i l i t y governed by cytoplasmic f a c t o r s i n a d d i t i o n t o Mendelian f a c t o r s  24  (•) M a l e - s t e r i l i t y r e p o r t e d , but mode o f i n h e r i t a n c e unknown at present  29  C - Observations r e l a t i v e t o p o s s i b l e malesterility in alfalfa  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) I s t r i p p i n g necessary f o r a l f a l f a production?  32  STUDIES IN FIELD AND  seed  LABORATORY  A - M a t e r i a l s and Methods (a) P o l l e n  36  studies  (b) S e l f - p o l l i n a t i o n  studies  39  (c) O p e n - p o l l i n a t i o n s t u d i e s  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 - H i s t o r y and D i s t r i b u t i o n  65  B - Nature of the l o s s e s due t o Witches' Broom Disease o f A l f a l f a C - Other a l f a l f a v i r u s e s and v i r u s d i s e a s e s  70 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 (a)  studies  L i t e r a t u r e review  (b) Experiments  83 85  B - Mechanical I n o c u l a t i o n S t u d i e s (a) L i t e r a t u r e  89  (b) Experiments  90  C - Seed t r a n s m i s s i o n o f Witches' Broom o f 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 c u t t i n g s E - Insect transmission studies ( l i t e r a t u r e  92 only)93  II  - Yield  Trial  (a) L i t e r a t u r e  96  (b) Experiment  96  III-» F i e l d  S t u d i e s of the Witches' Broom disease o f a l f a l f a  A - Quadrat s t u d i e s 1. I n t r o d u c t i o n  lO0t  2. Study o f quadrat data taken by Dr. V. C. Brink i n 1942-44. 101 3. Quadrat s t u d i e s i n i t i a t e d the i n t e r i o r of B.C.  i n 1950 i n 102*  B - D i s t r i b u t i o n o f 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 ange S t a t i o n , Kamloops, B.C. on Sept. 17, 1951.  106  n  IV - H i s t o l o g i c a l s t u d i e s of Witches' Broom diseased a l f a l f a (a) L i t e r a t u r e review (b) Experimental  108  studies  1. Comparison o f t h e c e l l u l a r s t r u c t u r e of h e a l t h y a l f a l f a with a l f a l f a a f f e c t e d Witches' Broom d i s e a s e a) M a t e r i a l s and Mebhods b) Observations  110 111  2, Comparison of food reserves o f h e a l t h y and d i s e a s e d p l a n t s .  112  DISCUSSION  114  SUMMARY  1*7 120  LITERATURE CITED  1A#  ^  A - THE SELECTION OF ILALE-STERILE L U E S III ALFALFA. B - THE WITCHES  1  BROOM VIRUS DISEASE OF ALFALFA IN  BRITISH COLUMBIA.  INTR QDUC TION An a l f a l f a b r e e d i n g program has been c a r r i e d on at the U n i v e r s i t y of B r i t i s h Columbia s i n c e 1918 and has c o n s t i t u t e d a major a c t i v i t y of the Department of Agronomy. Work had progressed  t o such a p o i n t 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. s a t i v a ) was  r e g i s t e r e d i n Canada. An  a l f a l f a b r e e d i n g program should be c o n t i n u i n g .  r a t e d "by H o r r i s o n  ( 6 4 ) as the " i d e a l " hay crop,  Although  an a l f a l f a  s u i t a b l e f o r a l l agronomic purposes has y e t to be developed. New d i s e a s e s  and problems t h a t can be c o n t r o l l e d only through  b r e e d i n g methods are c o n t i n u a l l y p r e s e n t i n g i s a l s o i n a l f a l f a much vigour  themselves. There  a t t r i b u t a b l e to h y b r i d i t y  ( h e t e r o s i s ) y e t t o be tapped. One  aspect  i n b r e e d i n g a l f a l f a which s e e u s worthy of  s p e c i a l a t t e n t i o n 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 h e t e r o s i s or h y b r i d v i g o u r .  Harked h e t e r o s i s i s e x h i b i t e d by  c e r t a i n p l a n t s 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 f a c t o r i n u t i l i z i n g the h e t e r o s i s i n a p r a c t i c a l way by agronomists i n v o l v e s c e r t a i n p o l l i n a t i o n c o n t r o l s . a l f a l f a , which possesses p e r f e c t f l o w e r s ,  containing both  male and female p a r t s , t h i 3 n e c e s s i t a t e s the r e n d e r i n g  1  In  of the  -2-  male p a r t s of the female parent  p l a n t n o n f u n c t i o n a l . However,  hand-emasculation of the male p a r t s of the very s m a l l f l o w e r s , a v e r y slow, t e d i o u s  and expensive process,  for large scale operations.  One  way  can "be overcome i s through the use f e r t i l e plants. and u t i l i z e d ,  i s not p r a c t i c a b l e  i n which, t h i s  obstacle  of m a l e - s t e r i l e , female-  In the hope t h a t such p l a n t s c o u l d be  an i n v e s t i g a t i o n was  t h e i r presence i n our B.C.  found  i n i t i a t e d t o determine  a l f a l f a stocks.  The  first  section  of t h i s d i s s e r t a t i o n then i s a r e p o r t upon our search f o r , and study of, m a l e - s t e r i l e l i n e s .  (Since two  have been a v a i l a b l e f o r t h i s study, set has been obtained  as a r e s u l t ,  and,  growing seasons only s i n c e only one  seed  a r e p o r t on the i n h e r i t a n c e  of the p u t a t i v e 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 y e t  be  made.) While s i m i l a r i n v e s t i g a t i o n s are b e i n g taken by interested institutions  other  ( i n Nebraska, Saskatchewan, Sweden,  among o t h e r s ) , a l f a l f a i s such an important  crop i n B r i t i s h  Columbia t h a t an attempt to produce h y b r i d a l f a l f a would be worthwhile here. not e x t e n s i v e , important province  Although p r o d u c t i o n  a l f a l f a i s nevertheless  forage  i n B r i t i s h Columbia i s t h i s p r o v i n c e ' s most  crop. This crop i s grown i n a l l p a r t s of the  and i s the p r i n c i p a l hay  acreage f i g u r e s , although  species.  While  total  a v a i l a b l e are i n a c c u r a t e , an e s t -  imate of i t s importance to the p r o v i n c i a l economy can determined by the f o l l o w i n g f a c t s . source has  of winter hay  be  A l f a l f a i s the prime  f o r the range l i v e s t o c k i n d u s t r y which  an annual p r o d u c t i o n value  of $19,000,000.  One  and  one-  -3'  h a l f tons of hay are r e q u i r e d f o r each animal u n i t on the range each w i n t e r . important  I n a d d i t i o n , seed p r o d u c t i o n i s an  i n d u s t r y i n the p r o v i n c e , p r i n c i p a l l y i n the l e a c e  R i v e r area and, t o a minor extent, i n p a r t s of the Okanagon and Upper E r a s e r V a l l e y s .  There was an estimated  seed  acreage  i n 1950 i n B.C. of 8,985 acres w i t h a p r o d u c t i o n of 363,000 l b s . c l e a n 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 s e c t i o n of t h i s d i s s e r t a t i o n concerns a d i s e a s e , 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 B r i n k (17) i n 1942, which i s now becoming r a t h e r d e s t r u c t i v e to a l f a l f a i n p a r t s of the p r o v i n c e .  The d i s e a s e , 'Witches' Broom of A l f a l f a , i s  A t t r i b u t e d by Kenzies  (63) t o a v i r u s pathogen. Very few  a d d i t i o n a l f a c t s , however, r e s p e c t i n g the d i s e a s e are known. I t s s e r i o u s n e s s , g e o g r a p h i c a l d i s t r i b u t i o n , v e c t o r s , or r a t e of spread  are unknown.  I t i s not known, furthermore, whether  any n a t u r a l r e s i s t a n c e occurs i n a l f a l f a s  (Me d i e ago  spp.).  To o b t a i n much i n f o r m a t i o n i n r e s p e c t t o these f e a t u r e s w i l l require several years  of r e s e a r c h .  Some hope t h a t t h i s  dis-  ease might be c o n t r o l l e d by p l a n t b r e e d i n g methods i s o f f e r e d by the r e c e n t p r o d u c t i o n another  (49) of an a l f a l f a r e s i s t a n t t o  v i r u s d i s e a s e ( " A l f a l f a Dwarf") i n C a l i f o r n i a . 14 years  of r e s e a r c h , w i t h ample support, were r e q u i r e d t o produce t h i s variety.  A c c o r d i n g l y , our s t u d i e s i n t o A l f a l f a Witches'  Broom disease i n B.C. must be of a p r e l i m i n a r y n a t u r e .  -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 b r e e d i n g by development of h y b r i d s t r a i n s .  Inbred l i n e s , developed  c o n s i d e r a b l e c o s t , were l a t e r combined i n s u i t a b l e to produce h y b r i d s s u p e r i o r i n y i e l d , d i s e a s e and  the at  outcrosses insect  r e s i s t a n c e , and u n i f o r m i t y . Advances were so s t r i k i n g t h a t i n the U.S.A. alone 90^  of the corn acreage  i s now  of h y b r i d  s o r t s . Acreage has been r e t i r e d f o r soybeans and other crops, and y e t n a t i o n a l corn y i e l d s have i n c r e a s e d 25$. i s so marked i n h y b r i d corn t h a t mechanical techniques  are now  Uniformity  harvesting  eminently s u c c e s s f u l .  A l f a l f a breeders,  i t i s not s u r p r i s i n g , t h e r e f o r e  viewing the progress i n h y b r i d corn b r e e d i n g have wondered i f s i m i l a r successes might n o t / \ r e a l i z e d i n a l f a l f a b r e e d i n g . (1) L i k e corn, a l f a l f a i s heterozygous  and o p e n - p o l l i n a t e d ,  and marked h e t e r o s i s i s demonstrated i n h y b r i d s . (2) In a d d i t i o n , a l f a l f a , propagated  a p e r e n n i a l , can r e a d i l y be  v e g e t a t i v e l y , and as such the maintenance  r e t e n t i o n of a given genotype i s e a s i l y  and  accomplished.  (3) U n l i k e corn, which i s monecious, a l f a l f a f l o w e r s are small, hermaphroditic,  and gathered  (4) U n l i k e corn, a l f a l f a i s i n s e c t  i n t i g h t racemes. pollinated.  (5) U n l i k e corn, i n a l f a l f a only a few  seeds are produced  per  pollination.  (6)  A l f a l f a i s p o l y p l o i d y i n nature which  complicates  inheritance, and melees transfer  of characters  In addition, there i s probably cytoplasmic The fact  difficult.  influences.  that only a few seeds are produced from a  single p o l l i n a t i o n rules out the production of hybrid by physical means.  alfalfa  As i n other plants of similar nature  where the advantage of a hybrid program i s being investigated (flax, tomatoes, onions, e t c . ) ,  considerable interest  has  been drawn to the p o s s i b i l i t y of avoiding the morphological d i f f i c u l t i e s inherent i n the flower structure by the selection of m a l e - s t e r i l e , but f e m a l e - f e r t i l e ,  l i n e s which are  of setting seed but not of producing p o l l e n .  capable  Such plants,  i f of agronomic value, would serve as female parents for seed produced by natural crossing i n an i s o l a t i o n bloclc. The obvious necessity of simple inheritance of males t e r i l i t y becomes apparent i f we want to transmit this character from one l i n e to another. Hope for the existance of such l i n e s has been indicated by Tysdal and Kiesselbach who i n 1944 stated " m a l e - s t e r i l i t y , identified in alfalfa,  (91),  as such, has not been  but i t s possible  occurrence and use  should not be ignored", and by Armstrong and White (3) i n 1935, Tome (89) (27)  i n 1947 i n Argentina, and Clarice and Pryer  among other workers.  In our work, attention was directed  to both male and female s t e r i l e l i n e s but the emphasis was directed to m a l e - s t e r i l i t y . Kany d i f f i c u l t i e s  are inherent i n a l f a l f a i n working  out the inheritance of m a l e - s t e r i l i t y ,  or of any character.  The common a l f a l f a i s 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 r a r e l y siiaple. As Brink, Jones and Albrecht (16) pointed out i n discussing B a c t e r i a l Wilt disease of a l f a l f a ,  a factorial  of the inheritance of disease resistance i s at impossible.  interpretation present  Duplicate genes are often involved i n inheritance  which w i l l obviously add to the d i f f i c u l t i e s .  Atwood (5)  wrote that a l f a l f a i s highly heterozygous and as such i s using hybrid vigor n a t u r a l l y to a considerable extent. i s a very costly procedure i n a l f a l f a ,  Inbreeding  r e q u i r i n g a great  amount of tedious and slow hand labour, and the plants soon become weak and s t e r i l e ,  and as such outcrossing may r e s u l t  i n very l i t t l e , i f any, seed set. For this reason, foregoing ones, alfalfa.  and the  a program cannot be translated from corn to  However, as Tysdal and Kiesselbach (91) pointed out  in 1944 i n a crop such as a l f a l f a , easily propagated by clones,  which i s perennial and  a given genotype can be maint-  ained i n d e f i n i t e l y , thus eliminating the need of i l i z a t i o n for i t s  self-fert-  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 a b i l i t y . This i s evidenced by the general theme of such papers as Tome's (89) discussion of a l f a l f a breeding i n Argentina, Bolton's (10),  and Hayes'  (48).  Due to the foregoing reasons, and e s p e c i a l l y due to the difficulties  inherent i n polyploidy, breeders on this  7continent have l a r g e l y given up the idea of the production of hybrid a l f a l f a .  The lack of l i n e s 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 r e a l i s a t i o n that the application of the work may b e  some time away. It i s f e l t that there i s a pos-  sible use of hybrid vigor even though there are many d i f f i c ulties.  In some respects a l f a l f a lends i t s e l f  to a "hybrid"  program: i t propagates r e a d i l y by vegetative means. It  is  f e l t that i n a few years a large proportion of a l f a l f a seed producing f i e l d s w i l l D e propagated by cuttings,  and as such  the interplanting of rows of male-sterile plants with malef e r 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 i n higher plants. (a) Defining s t e r i l i t y and i n c o m p a t i b i l i t y . S t e r i l i t y i s not e a s i l y defined as i t may take several forms.  East  (38)  i n w r i t i n g of s t e r i l i t y seems to  incliide any phenomenon that r e s u l t s i n reduced or no seed set,  and considers m a l e - s t e r i l i t y to be a special case of  "self-sterility".  Armstrong and White (3) write of pollen  sterility in alfalfa  as "a very definite factor i n seed-  production - both i n determining the pod-setting and i n 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 i n c o m p a t i b i l i t y and sterility, sterile'  c o n f i n i n g t h e terms  self-sterile'  and cross1  to s t e r i l i t y which a r i s e s from d e f e c t i v e  ovules and other a b e r r a t i o n s . 'incompatibility' the  1  p o l l e n and  They would r e s t r i c t  the term  t o the f a i l u r e of the p o l l e n t o grow down  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 t o some p h y s i o l o g i c a l  hindrance.  They p o i n t  i s frequently ibility  out that  although s e l f - i n c o m p a t i b i l i t y  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-  as c r o s s - s t e r i l i t y the terms are misnomers as i n  i n c o m p a t i b i l i t y the p o l l e n on c e r t a i n p l a n t s .  and ovules are f u n c t i o n a l  at l e a s t  B e a d l e (7) d i v i d e s s t e r i l i t y i n t o two  types: 'impotence* - due t o m o r p h o l o g i c a l or p h y s i o l o g i c a l derangement o f the s e x u a l organs r e s u l t i n g i n an i n a b i l i t y t o produce v i a b l e germ c e l l s ;  and, ' i n c o m p a t i b i l i t y '  wherein  normal gametes are formed, but f e r t i l i z a t i o n may be i m p o s s i b l e i n c e r t a i n matings w h i l e i n other matings are normally productive  of o f f s p r i n g .  Riley  (78) i n 1932 admits t h a t the  t e r m i n o l o g y employed by v a r i o u s i n v e s t i g a t o r s has  n o t "been c o n s i s t e n t .  He w r i t e s t h a t  i n this f i e l d  the term  'self-sterility'  a much o l d e r term than ' s e l f - i n c o m p a t i b i l i t y ' or ' s e l f - p a r a s t e r i l i t y ' , i s a p p l i e d t o the i n a b i l i t y o f an hermaphrodite w i t h f u n c t i o n a l gametes t o 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 t o prevent s e l f - p o l l i n a t i o n . He  thinks  t h a t t h e r e should be no c o n f u s i o n between the use of ' s e l f sterility' the  and true  'sterility'  as the p r e f i x ' s e l f  qualifies  e x p r e s s i o n 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 '  t o denote those cases where p o l l e n (or  female gametophyte) fertilized)  i s incapable of f e r t i l i z i n g (or being  i n any case; and the term " i n c o m p a t i b i l i t y " to  denote those cases where f e r t i l i z a t i o n i s prevented by genetical differences between normal functional pollen and normal functional ovules and s t y l e s , (b) Causes of s t e r i l i t y S t e r i l i t y i n plants,  as has previously been noted,  may stem from a variety of causes and i t i s not surprising that many c l a s s i f i c a t i o n s  have been proposed to aid i n i t s  description. S t e r i l i t y i s c l a s s i f i e d by Crane and Lawrence (33)  into  a) "Generational s t e r i l i t y , due to the f a i l u r e of any of the processes concerned with the normal alternation of  generations,  namely, development of p o l l e n , embryo-sac, embryo, and endosperm, and the r e l a t i o n s 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 i n the i n d i v i d u a l genes of the organism, and not due to d i s s i m i l a r i t i e s  i n the gross structure of the  chromosomes; and, "chromosomal s t e r i l i t y " due to structural dissimilarities  of the chromosome b i v a l e n t s .  Genie s t e r i l i t y  in h i s sense r a r e l y involves any interference'with the chromosomal mechanism as shown by the fact that meiotic chromosome p a i r i n g 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 logous  may,  chromosomes  Chromosomal its  base,  gross of  s t e r i l i t y  into  former (or  the  latter has  Whatever many  gametic  refers for  i l i z a t i o n  Fe&erly  phenomena  to  a l l to  the may  be  resulting  flowers, Failure  such of  (88) ( 9 0 ) ;  (It  dioecious, in  which  the  as a  of  abort  entirely  pollen  this  through  to  sticky  pollen  method  (ibid), divides  (diplontic).  female  gameto-  u n f e r t i l i z e d  zygote  to  of  causes  the  for  the  male  grow  after  may  be  double to  occur  replaced (very  develop  or  many  example  many  Labiate,  type).  Anthers  (82) ( 8 3 ) . failures  genes  (42,  of may  Anthers of  at by  the  egg) fert-  plants  43),  failures  flower,  any  stage  petals  thus  occur  show  but  gynospecies  male-sterand  then  produce  no  (4)(7)(9)(40)  i n meiotic genes  (73)  as  effect  p a r t i a l l y  develop  asynaptic  of  i n h o r t i c u l t u r a l  i n  reduction-division or  an  a  may  individuals  may  of  recorded  are  develop  s t e r i l i t y ,  chrysanthemum).  whatsoever  that  population  of  common  geranium,  of  s t e r i l i t y  parts  probable  for  other  Iluntzing),  or  the  s t e r i l i t y may  (42)(43)(44)(45)(72)(73); due  the  male-sterile  the  certain  i l i t y  i n  anthers is  the  with  Anthers  plant as  of  and  at  Another  zygotic  purposes,  responsible  f i r s t  history. the  of  c l a s s i f i c a t i o n  breakdowns  rendering  s t e r i l i t y  and  has  accomplished.  Dealing  l i f e  (haplontic)  to  corn).  usage,  Dobzhansky  end  homo-  i n  deletions,  to  of  mutation  chromosomes.  and iienner  failure  been  the  according  practical  organism.  the  i n  pairing  (ibid)  inversions,  changes  of  gene  i n Dobbzansky* s  s t e r i l i t y , to  failure  "synaptic"  translocations,  (attributable  and  (ref.  morphological  phyte  involve  s t e r i l i t y ,  classifying  The  however,  a c t i v i t y  (73),  chromo-  -11somal rearrangements  (43). Pollen grains may Toe affected  "before the thickening of the w a l l , thickening, or after  or at the beginning of  thickening but before the f i r s t d i v i s i o n .  P a r t i a l or complete s t e r i l i t y may be caused by translocations, inversions, deletions,  duplications e t c .  The production of  sex c e l l s carrying abnormal gene complements i n individuals heterozygous for various chromosomal aberrations leads to a certain degree of s t e r i l i t y . may lead to p a r t i a l  ITon-dis junction of chromosomes  sterility.  Another i r r e g u l a r i t y i n meiosis may arise from allopolyploidy: mechanical d i f f i c u l t i e s may be set up to p a i r i n g 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 c h a r a c t e r i s t i c (37)  i t w i l l not germinate  i n 1932 i n an extensive  (59).  (Dwyer  test i i i A u s t r a l i a found that only  80/(> of normal pollen germinated).  Pollen may not germinate  on stigmas i f ^ p r o p e r moisture conditions are not met  (62).  Pollen may be f u l l y viable but not be able to germinate due to the stigmatic membrane being too well developed (3) unruptured (60).  The stigmatic membrane may be ruptured but  the pollen-tube may f a i l  to grow down the style  (38,  i f grown down the style f a i l to reach the ovary (38, 15, 31, 50, 60 e t c ) ; fertilize  or  or bypass the ovary (31),  any ovules (31)  or f a i l  39) j or, 39, 3,  or f a i l  to  to f e r t i l i z e the basal  ovules of the ovary (31, 13) r e s u l t i n g i n p a r t i a l  sterility.  Hie ovules themselves may be nnn-f unction al and even i f the  pollen-tube  reaches  the  ovule,  f e r t i l i z a t i o n  cannot  be  effected. Ilany  of  gametophyte  the may  gametophyte  i r r e g u l a r i t i e s also  affect,  of  but  meiosis  not  may  remain  are  present  and  the  generative  the  egg  of  the  ovule  axe  i n  effected  the  ovules  c e l l  f e r t i l i z a t i o n  following the  .'ill  is  ovules,  sac  of  In  polyploids  the  may b e  due  to  number  cannot  recent  has  been  female  nucleus  the  of  may  the  the  conjugation  (36).  abort.  somatic  pollen-tubes  p o l l e n tube  s t e r i l i t y ,  may  occur  i n  t r i p l o i d s ,  pentaploids  picture  somewnat  divide  though  and  Even  i f  Collapse  of  may  tissues  occur  adjacent  to  14).  is  unbalance  There  has  years  on  (50)  case  of  many  instances  i n  the or  more  chromosome  ordinary  other  diploid  unbalanced  complicated.  complements  S t e r i l i t y  as  an  odd  evenly.  been  a  that  under  credits  have about  families 1934  state  great  amount  incompatibility i n the  heading some  of  1764  of  of the  notable  i n V e r b as c u m . Jones  100  different  species  incompatibility. sexual  which  l i t e r a t u r e .  Since  (ibid)  i n  and  incompatibility is  f i r s t that  time  1928  scattered  Crane  published  The  r e i ^ o r t i n g the  recorded,  that  much of  s t e r i l i t y .  been  show  literature  plants,  Kolreuter with  incompatibility i n  estimated  i n  13,  of  review highlights  Jones  (34)  even  somatoplastic  foregoing  discussed  following  plant  the  male-  Incompatibility.  in  50  i n f e r t i l e  growth  (12,  the  211 p l a n t s .  (c)  or  abnormal  embryo  necessarily,  the  development.  Ovules  f e r t i l i z e d  affecting  among  Lawrence wide-spread  -13-  throughout the plant kingdom; a view substantiated by many authors. The  best known, and most satisfactory explanation of  incompatibility was presented by East ional-factor hypothesis.  (39) with his  opposit-  By t h i s postulation, incompatibility  i s determined by genes usually designated by the l e t t e r 3 and  commonly forming a multiple allelomorphic s e r i e s : S i , S2,  . . . . . . . . S i 5 , any 2 of which may be c a r r i e d by a given plant. Pollen cannot function, or shows very l i t t l e growth i n the style of a plant carrying the same incompatibility factors as the p o l l e n ; and normal pollen tube growth occurs i n s t y l a r tissue carrying a different genetic factor for ibility.  There was also discovered by East  f e r t i l i t y factor, and  3f,  self-incompat-  (ibid) a s e l f -  functional with any of the above series  dominant i n crosses. R i l e y (78)  i n 1932, working with the genus Capsella at  Princeton University found that the inheritance of selfs t e r i l i t y i n C. g r a n d i f l o r a could not be explained by the theory of oppositional f a c t o r s . self-sterile,  This species i s completely  and had remained so through 18 generations. The  3?^ from a cross between 3 other s e l f - f e r t i l e self-sterile  G. grandiflora were a l l f e r t i l e ;  into 3 s e l f - f e r t i l e s  to one s e l f - s t e r i l e .  f e r t i l e s breed true to s e l f - f e r t i l i t y ; s p l i t into a 3:1 r a t i o of f e r t i l e s self-sterile  species and the the j?2 s p l i t  1/3 of the s e l f -  while the other 2/3  to s t e r i l e s . A l l the  segregates of any ^2 family are c r o s s - s t e r i l e  among themselves. R i l e y (ibid) i n 1932 attributed self-  -14s 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) i n 1934 and 1936, he explained this self-sterility parent plants  on the basis of the sporophytic nature of the and on the interaction of two pairs of genes,  a dominant gene T which i s e p i s t a t i c to S  and their recessive  c  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 i s due to a positive chemical reaction between the pollen and the s t y l e , and not merely due to a lack of some growth substance. Selfp o l l i n a t i o n s of flowers l a t e i n the season of growth and also of flowers i n the bud stage i n certain incompatible forms of H i c o t i a n i a has r e s u l t e d i n some seed set.  Such seeds give  r i s e to plants either of the same genetic constitution as the parent or homozygous plants. with the parent.  The l a t t e r  The former f a i l r e c i p r o c a l l y are effective  when used as females,  but f a i l when used as males i n p o l l i n a t i o n s with the parent. This phenomenon i s known as " p s e u d o - f e r t i l i t y "  or"end-season"  fertility. 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  interaction grain. is  3_S2  two  i f  one  Brink 13,  14,  that  is  i n  a  allele and  31)  plastic  different  pollen  incompatible  down  that  unrelated  i n  i t .  ovule  to  things  reach  the  over  and  basal  happen: ovules,  pollen  tubes  i n  ovules  are  common  of  the  proportion  and  to  The  higher  reduce  the  of  plant  to  or  the  ovule  pollen  at  within  tendency  enter  micropyles  appears  to  be  no  becoming  frequency  of  Cooper ovary  of  for  the  pollen  and  f e r t i l e  of  which  the  that  was  normal  10-12  from  of  even f e r t i l e to  to  raise  65;  34,.j  to  7£».  extensive  grovth  ovary  an  reach  and  the  ovules  of  and  same of  arranged  to  There  percentage  the  be  to  of  15/£  embryology  ovules  one  found  from  the  may  f a i l  abortion  s e l f - f e r t i l i s a t i o n i n  the  independently  f e r t i l i z a t i o n .  betv/een  the  of  i n f e r t i l e  ovules  collapsing  contains  are  and  s t e r i l i t y  f r o m more  tubes  after  described  a l f a l f a  f e r t i l e  report  p a r t i a l  found  Crossing  accomplish  ovules  the  (12,  somato-  They  vary  remain  cavity  break  papers  frequently  f e r t i l e  pollen  may  and  They  they  and  results  correlation  f e r t i l e (30)  ovules  of  only  and may  becoming  f e r t i l i t y  tubes  4  plants.  is  tubes  ogSg  this  least  plant.  occurrence.  abortion  increased the  pollen  pollen  other.  somatoplastic  present,  ovules  3_3_and  the  a l f a l f a  many  are  same  However  other  self-incompatibility  may  the  self-incompatibility  abortion  though  ovules  style.  published  a l f a l f a  from  i n  while  Following s e l f - f e r t i l i z a t i o n  three  An  have  with  considerably  alleles  dominant  Cooper  s t e r i l i t y  opinion  of  31S1S2S2  is  dealing  3  compatible,  self-incompatibility  varies  of  of  the the  individual. alfalfa. alternately  -16along  the  condition of  the  egg  number is  ventral  suture  for  development  within  of  seed  caused by  become  the  (32)  following gradient  top  position  the  frequency  this,  of  studied,  i n  In  selfed  at  the  2  of  ovules  series  apex  of  the  to  f e r t i l i t y  although  the  proportion  ovules  a  l i t t l e  i n  position  1/3  of  than  1  to  down  the  is  that  Cooper  and  declining the  base  ovules  i n  f e r t i l e base.  somewhat becoming  after  ovules  a  to  became  ovules  higher  apex  the  the  p i s t i l s  found  of  towards  gradient  of  a l f a l f a and  i n  cross-fertilization  matings.  the  ovary  zero  the  is  from  about  series  the  of  f e r t i l i z a t i o n  disparity  proportion  types  necessary  that  and  h i s t o l o g i c a l l y ,  declining  position  self  the  is  A large  cross-pollination  i n f e r t i l i t y  ovary.  crossed  after  The  seed  the  develop  difference  and  of  order.  occurs.  f o l l o w i n g the  self-  the  serial  ovule  the  which  f e r t i l e  Albrecht  each  the  in  the  with  In  the  equal  to  f e r t i l e  selfing  approximately  of  85%  -  33;£  i n  at  the  base. Brink s t e r i l i t y develop during after of  i n t o mature early  (ibid)  a high  stages  somatic  tissue  f e r t i l i z a t i o n  zygote.  as  The  manner  well  of  c r i t i c a l  i n which  the  ovules  of  somatoplastic  f e r t i l e  collapse  collapse  adjacent  i n  described  to  c e l l the  factor  the  d i v i s i o n  endosperm for  translocated  is  food  is  i n  abnormal  sac.  to  c e l l  this  shared  growth  Shortly  i n i t i a t e d  mother  survival  f a i l  particularly  follows embryo  ovules  frequently  p o s t - f e r t i l i z a t i o n  This  active as  also  percentage  seeds:  s e l f - p o l l i n a t i o n .  integuments  the  Cooper  i n which  the  the  after  and  i n  and  stage  between  the the is the  -17-  endosperm and the integument and t h i s p a r t i t i o n depends on the rate of growth or c e l l d i v i s i o n 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 i n competition and the balance shifts  i n favor of the integuments.  Growth may continue for several hours, using breakdown of the endosperm c e l l s  as n u t r i e n t s , before collapse.  The chalazal  portion of the endosperm i s ruptured, the c e l l s of the embryo become starved,  and i s followed by a collapse, with  the resultant abortion of the remaining endosperm and embryo. B. M a l e - s t e r i l i t y i n the higher plants. (a) Some general remarks. A litex-ature search of rdcent work on m a l e - s t e r i l i t y i n plants brings to l i g h t 3 main modes of inheritance of t h i s character.  M a l e - s t e r i l i t y may be governed by a) one or a  few recessive llJendelian factors, and c) by cytoplasmic factors  or b) by dominant factors,  i n addition to Hendelian factors.  In some cases, the phenomenon i s reported but the mode of inheritance i s unknown at present.  Cytoplasmic m a l e - s t e r i l i t y  was reported, apparently erroneously, by Rick (75) i n 1948, and by Lewis (61)  i n 1941, i n l i g h t of present day findings,  to be much less common i n c u l t i v a t e d plants than genie malesterility. Lev/is (ibid) w r i t i n g i n 1941 on m a l e - s t e r i l i t y i n natural populations of hermaphrodite plants, due to a recessive  stated that male s t e r i l i t y  gene i s common as a mutant i n moreu plants.  Single examples are reported by Lewis (ibid) i n ;\ntirrhinura. Capsella spp., Lathvrus odoratus. Lyeonersicum esculentum, Oryza s a t i v a .  and Rubus idaeus.  More than 15 types of male-  s t e r i l i t y were known i n Zea mays at that time, a l l being due to the segregation of a single recessive  gene. He reported  two other cases of m a l e - s t e r i l i t y i n maize due to a single dominant gene, but which since have been shown to be i n r e a l i t y due to an i n t e r a c t i o n of cytoplasmic factors and Mendelian factors.  In 1931, m a l e - s t e r i l i t y had not been  reported i n wild populations of these plants, naturally polymorphic for m a l e - s t e r i l i t y . dioecious species f u l l y investigated,  or i n species  In a l l the gyno-  according to Lev/is (ibid)  m a l e - s t e r i l i t y i s i n h e r i t e d through the cytoplasm. (b) M a l e - s t e r i l i t y governed by one or a few recessive Mendelian f a c t o r s .  (If m a l e - s t e r i l i t y i s i n h e r i t e d  as a simple recessive,  inbreeding would favor the  production of the phenomenon.) Bohn and Uhitaker (9) i n 1949 w r i t i n g on the males t e r i l i t y i n andromonecious muskmellon report that  staminate  flowers from male-sterile plants are normal i n size and shape of a l l parts except the anther*.  These are small, f a i l  dehisce and contain empty microspore w a l l s .  to  In the male-  s t e r i l e plants meiosis i s 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. together.  The units of the tetrad remain cemented  They conclude that i n muskmellon m a l e - s t e r i l i t y  -19i s d i p l o n t i c and i s governed by a single recessive gene. Crane (33)  i n 1915 i s credited with, the f i r s t report of  m a l e - s t e r i l i t y i n the tomato.  He found that m a l e - s t e r i l i t y  "behaved as a recessive character and "believed that i t depended on a mutant gene. Rick (74) male-sterile  i n 1948, reported on a search conducted for  tomato mutants, p o t e n t i a l l y useful i n producing  _*X hybrid seed and i n cross-breeding.  Among 150 u n f r u i t f u l  plants, 12 were found to be genetically m a l e - s t e r i l e .  Breeding  r e s u l t s indicate that the m a l e - s t e r i l i t y of each mutant i s determined by a single recessive gene. One gene ms5 was recovered 4 times and 8 other n o n - a l l e l i c genes mss to msi3 were demonstrated.  Only one mutant produced any pollen that  was functional, and by using this pollen i n s e l f - f e r t i l i z a t i o n pure breeding male-sterile populations for this gene was obtained. Rick i n a previous paper (73)  i n 1945, reported on a  survey of cytogenetic causes of unfruitfillness i n the tomato. Gametic s t e r i l i t y i s l a r g e l y responsible for the u n f r u i t f u l ness of 66 plants discovered i n approximately 55,000 f i e l d plants of 3 tomato v a r i e t i e s .  F a i l u r e of normal gamete  formation was resolved to a cytogenetic basis i n every case investigated. Rick (ibid) found i n the 14 diploids discovered i n this population that 3 plants were aberrant i n gross morphology.  S t e r i l i t y of these plants i s probably conditioned  either by a p l e i o t r o p i c effect  of the gene determining the  deformity, or by the deficiency of a chromosomal segment  w h i c h i n c l u d e s a gene a f f e c t i n g t h e m o r p h o l o g i c a l and another a f f e c t i n g s t e r i l i t y .  character  R i c k ( i b i d ) f o u n d i n 3 other  p l a n t s t h a t the m a l e - s t e r i l i t y i s d e t e r m i n e d 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 o v u l e  s t e r i l i t y r e s u l t i n g i n one case f r o m a s y n a p t i c  meiosis.  Roever (81) i n 1948, w r i t i n g on a p r o m i s i n g 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  type of  production,  r e p o r t s a mutant i n w h i c h n a t u r a l s e l f i n g was p r e v e n t e d . because t h e anthers f a i l e d t o d e h i s c e .  The mutant can be  s e l f e d by hand b u t does n o t s e l f i n the f i e l d .  He c o n c l u d e s  t h a t t h e c h a r a c t e r appears t o behave as a simple L e s l e y and L e s l e y  recessive.  (59) w o r k i n g a t 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. that m a l e - s t e r i l i t y i s completely  They f o u n d  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, m s i and ms2» b o t h o f w h i c h 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 b o t h of these r e c e s s i v e c h a r a c t e r s .and produced non-viable  pollen.  Segregation  of s t e r i l e and v i a b l e p o l l e n -  was a c c o m p l i s h e d by means of g e r m i n a t i o n  t e s t s i n van Tiegliem  c e l l s u s i n g 1 5 ^ cane sugar m e d i a p l u s , ground up S c o t t and R i n e r  (82) i n 1946, r e p o r t on t h e 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 w i n t e r produce no p o l l e n .  stigmas.  squash i n w h i c h t h e male f l o w e r s  Bo d i f f e r e n c e s were o b s e r v e d i n t h e s i z e  or shape o f the m a l e - s t e r i l e v e r s u s  the normal f l o w e r , b u t  t h e r e i s a marked d i f f e r e n c e i n the appearance of the androecium of t h e m a l e - s t e r i l e and normal f l o w e r s .  I n the  m a l e - s t e r i l e f l o w e r s the androecium a b o r t s i n the bud stage  -21before The  the  stamina/be  factor  In  the  for  segregation f e r t i l e , is  open  m a l e - s t e r i l i t y  backeross  male-fertile  flowers  progenies  flowers  was  occurred.  producing  a  so  is 1  no  pollen  i n h e r i t e d as  :  1  a  segregation  observed.  In  Male-sterile  abundant  that  the  Fg  plants  seed  and  1932  on  produced.  simple for  recessive.  male-sterile:  progenies  were  f r u i t  is  f u l l y  a  3:1  female-  when f o r e i g n  pollen  added. Beadle  (7) r e p o r t e d  pollen  s t e r i l i t y  spores  and  in  female  microsporocytes degeneration from  maize.  was  or  of  varied  The ms In  a  recessive  genes  a l l  study  cases  was..:  the  meiosis  recessive  paper  are  genes  for mega-  Cytologic ally,  degeneration The  time  male-steriles almost  of  to  the  of ranging  pollen  matur-  and n o n - a l l e l o m o r p h i c .  i n 1921,  Mendelian genes  15  d e v e l o p m e n t , of  c e l l s .  different  s t a g e s -of  of  normal.  microspore  with  were  previous  a  characterized by the  the^'synizetie'  i t y .  In  gametophytes  maleriSterility  the  i n  Eryster  the  cause  (41)  of  states  that  m a l e - s t e r i l i t y  i n  maize. Emerson,  Beadle  and F r a s e r  had been  described  of  gene-steriles  these  each of and  capable  the  pollen  date  for  are  or  i n  were  the  inflorescence.  grains  are  reported  either  formed but  complete  genes A l l  recessives, or  Meiosis  before  20  i n maize.  independent  about  male  1935  m a l e - s t e r i l i t y  bringing  s t e r i l i t y i n  grains  a high occurs  anthesis  degree normally  these  abort.  Burnham of  of  to  (40)  pollen  abortion  (20)  reporting  abortion (pa)  i n  an  i n maize  on  cytogenetic  i n 1941  established  found  studies a  inbred l i n e .  gene  of  for  Plants  a  case  pollen hetero-  -22-  zygous f o r t h i s gene had serai-sterile  pollen but normal ears.  The gene was l e t h a l , or nearly so, to pollen carrying i t j but had no l e t h a l effect  on the ovules.  As a r e s u l t pa i s  trans-  mitted mainly, i f not e n t i r e l y , through the ovules, and i s located on the f i r s t linkage group. Shifriss cucurbits,  (83) w r i t i n g i n 1945 on m a l e - s t e r i l i t i e s i n  found 3 l i n e s among 200 one-generation inbreds of  Cucurbit a pepo L . which segregated for male-sterile 3?^, ^2  plants.  ^ao^cross populations showed that the m a l e - s t e r i l i t y  i 3 i n h e r i t e d as a recessive to a single gene.  character whose expression i s due  This morphological m a l e - s t e r i l i t y i s ex-  pressed i n complete abortion of the androecium at the bud stage before the staminate flowers open. Gregory (45) reported i n 1905 on the abortive development of the pollen i n certain sweet peas whose anthers were contaescent. Bateson.  These peas were produced by s e l f - f e r t i l i z a t i o n by S t e r i l i t y was found to be correlated with a somatic  character, the s t e r i l e plants possessing a green l e a f while f e r t i l e plants had red a x i l s .  axil,  A cytologic al invest-  igation revealed tha,t the whole nucleus showed i r r e g u l a r i t i e s . Reduction-division followed the normal course, pollen grains were formed.  In the contabescent  and young anther's,  growth ceases at t h i s point, the p o l l e n grains remain small and enclosed within the walls of the pollen mother-cells. Development of spindle fibres i n the cytoplasm was weak or absent.  Gregory (ibid) believed the m a l e - s t e r i l i t y character  was inherited as a simple  recessive.  Punnett showed  (70)(71),  that  sterile pollen  genetically  sweet was  sterile  pea  1937,  Institution  was  genetical  study  The  recessive  gene  color.  following (1)  Only  was  i n  second  the  Shull crosses  the  John of  Innes  a  detailed  gene  the  for  cytological  m a l e - s t e r i l i t y  was  he  of  Horticultural  for  be  linked  found  plate  chromosomes  c e l l  c e l l .  84)  odoratus. with  that  l i g h t  the  of  normal  i n meiosis  aspect  remained  i n  walls also  off  were  extending found  with  seen. only  that  some  Of  2/3  often  chambers  those of  the  more  than  being  left  whatsoever. by  a  early  change as  i n  the  diakenasis  colloidal or  c y t o p l a s m b e corner., g r a n u l a r  1927  reported  species  i n  be  recessive (88)  as  meiosis  as  la,te  very  as  rapidly  a c t i v i t y .  between the  of  He  arrested  The  meiotic  Suneson  the  divisions  cytoplasm  (  a metaphase  material,  s t e r i l i t y simply  recessive.  results  partitioned  anaphase.  arresting  in  were  the  any n u c l e a r  of  as  simple  the  i n Lathyrus  many h a d  M e i o s i s may  state  that  a  male-  s t e r i l i t y  demonstrated  cytological basis  second  across  chambers  (3)  the  cells.'  occurred,  without  that  m a l e - s t e r i l i t y  generally  few  distance  at  and  of  of  occassionally  Very  that  the  gametogenesis  plants,  occurred!  scattered  4  On a  formed;  (2)  inherited  reported  Gregory's'(ibid)  normal,  also  (42)  with  female  was  He  Paberge  and  axil  the  plants  complete.  anthers  In  working  of  interspecific to  on  finding  shepherd's Gapsella  p o l l e n - s t e r i l i t y  purse.  The  hybrid  was  4 year  test  male-  found  to  that  he  m a l e - f e r t i l i t y .  reported  i n  1940  on  a  -24p e r f ormed and _ , 2  on  a male-sterile  showed  that  male-sterility remained  the  was  mutant  mutant  inherited  shrunken,  was as  rudimentary  of  barley.  Results  male-sterile;  a. s i m p l e while  and  3?^  that  recessive.  p i s t i l l a t e  of  the  Anthers  parts  were  normal. (fe*)  M a l e - s t e r i l i t y governed Rife  variations  (76)  in  Goleus,  same  chromosome  deep  versus  f e r t i l e  studying  flowers;  the  found four  locus  shallow  by  which  lobed  both  single  dominant  of  genes  approximately  at  varying  cerain  common the  combinations  and m a l e - s t e r i l e  lobes  gene.  genetics  produce  leaves  deep  a  of  versus  and m a l e - s t e r i l i t y  male-  being  dominant. In (p)  a later  and  deep  (d)  paper,  lobes  Rife  (L)  are  (77)  points  closely  M a l e - s t e r i l i t y governed addition  to  observed  data  both  out  linked  by  cytoplasmic  male-sterility  and b o t h  cytoplasmic  Mendelian factors. on  that  To of  dominant,  factors  explain  i n  the  certain  types  m a l e - s t e r i l i t y  factors  and M e n d e l i a n f a c t o r s  are  required. Owen by  Mendelian,  both  or  influence  populations linked the  (67)  genie  the  hand,  inheritance,,  the  is  i f  a  inheritance  character  the of  from reciprocal  should.be to  male  be  of  the  from  crosses  male the  by  should  offspring; should  not  is  and  female  If. a  no  parents and  (excepting  sex-  character,  cytoplasmic,  have  to^explained  offspring,  crosses  identical.  explained  parent  characteristics reciprocal  that  characteristics  obtained  characters)  other  states  or  influence  and  populations  be  i d e n t i c a l .  on  maternal on  the  obtained If  cyto-  plasmic are  more  exert of  and  crosses  offspring, should not (50)  "chromogenes"  also of  to  Pollen  s t e r i l i t y details  have  of  the  be  i d e n t i c a l .  1950,  to  both  no  of  i n  brought which  effect  plants.  each  new the  the  i n  the  from  by  that  i n  i n  and  chromogenes  together  the  same  are  and  the  the  cytoand control  condition pollen  Plasmagenes  and  term,  their  growth  other  the  chromogenes  the  i n  case,  characteristics  chromosomes,  variable  upon  this  reciprocal  determiners  are  i n  results  terminology  about  Plasmagenes  other  on  parent  as  the  should,  obtained  genes  is  involved,  parent  proposes  designate  abortion  "both  male  populations  when w o r k i n g  effect  the  and  production.  s t e r i l i t y , no  than  are  female  designate  plasmagenes,  pollen  have  i n  "plasmagenes"  plasm.  The  influence  Jones  term  inheritance  complicated.  more  the  genie  and  structural for  plants,  independent  i n  pollen  apparently their  action. Rhoades in  Z e a mavs  i n  1931  concluded  through  the  the  cytoplasm  egg  maternal  male-sterility. through  the  before  pronounced microspores lines.  is the  difference i n normal  (72)(73)  pollen  cytoplasm.  There of  1933  that  plays  pollen  microsporogenesis occurring  and  the was  the  the  and  of  those  of  the  inherited reported  the  of  division.  cytoplasmic  m a l e - s t e r i l i t y  that  expression  plants.  degeneration  vegetative  between  i n  sterile  normal with  races  role  is  (ibid)  transmission  p a r t i a l l y  f i r s t  s t e r i l i t y  Rhoades  chief no  studying  of  character Cytologically, pollen There  elements  usually is of  male-sterile  a  -26Josephson s t e r i l i t y  i n  seriously  low  corn,  corn  the  resulted from  be  by  a minimum of point  out  that  cytoplasm, plants  plasmic  and  same  by  of  white  cause  plants.  had  from  hybrid of  the  shedding'  High  cytoplasmic also  found  to  the  male  parent,  required.  The  authors  s t e r i l i t y  is  subject  male-sterility  i n  sugar-beets  have  white  to  plants  develop  anthers. is  plants  effect  Complete  may  with  when  i n f l - . sterile  carried  male-sterility Owen  produced  reported  i n  into He  white,  by  f u l l y  and  are  on  cause  observed  empty  microspores  1947  the  cytoplasmic ally  bear  normal  no  by  factors  concluded combined  i n  by was  that  i n  cyto-  inheritance.  plants.  is  Mendelian  carried  cytoplasm.  (4)  (.ibid)  of  when  investigation  sugar-beets  to  being  for  pollen  S t e r i l i t y was  pairs  factors  empty  genie  in  f a i l  during  hybrids  contributions  male-sterility  Owen's  and  the  of  primary  male-sterile  expression  normal  in  sterile  of  tassels  apparently  several  Artschwager cytological  the  strain.  development  characterized sugar-beets,  be  working with  but  with  found the  male-  explanation fields  to  on  influence.  that  pollen  1948  of  of  certain  an  i n  a number  when  one  the  (67)  found  uence  was  only  genes  2  environmental Owen  i n  reported  Seeking  presence  contributions  cells  set  Examination  influenced  (53)  hybrids.  seed  revealed  s t e r i l i t y  1945  Jenkins  male-sterility  d i f f i c u l t y . time  and  the of  that  results pollen  when  inherited,  produced,  but the  .  abortion  completely  Normal  by  a  male-sterility  anthers.  disintegrate  of  the time  pollen  malemother  microspores the  flowers  -27open.  In  pollen  grains  Owen  (ibid)  produced ally  the  same  are  had  by  white  @e&i-male-sterile  observed  anthers  the  that  small,  anthers  sometimes  of  the  and y e l l o w  do  non-viable  not  viable  dehisce. pollen  inflorescence,  ones  are  borne  and  is occasion-  within  the  flower.  different meiotic  anther  abortion,  chromosome  divisions;  observed  a  tapetum,  important  of  sugar-beets  is  associated  tapetum  a  role.  delays  pollen  of  are  detriment  of  the  i n  hybrid  seed.  pollen  but  the  set  The  a  occur  The but  which  pollen  i n  a  is  the  while of  (52)  or  restricted  i n  c e l l u l a r  presence  to  but  not  somewhat  remains  c e l l u l a r  tetrads.  of  plays  anthers  cluster  i n  the  male-sterility a  flower  cause  (ibid)  i n which  abortion  -  abnormal  Plasmodium,  tapetum  the  of  Artschwager  a  s t i l l  with  the  He  development  sugar-beets  Cytology  of  a c t i v i t y  the  to  the  microspores. studied the  male-sterile readily  result  inherited  hypermetabolic  onion with  seed  of  Plasmodium's where  plants  degeneration  male-sterility.  developing  the  periplasmodium  may  suggests  Jone3 a n d C l a r k e s t e r i l i t y  that  explanation  cytoplasmic  Plasmodium  pollen  destroyed  periplasmodium  exhibiting young  which  is  of  however,  cytoplasmic ally  either  an  usually  development  found  abortion;  offer  of  the  flower.  microspores  the  He  with  i n hybrids  sugar-beets, type  with  single  could,not  i n  through  both  within  especially numbers,  different  an  of  hut  some b r a n c h e s  Pollen  some  formed  types,  the  hope onions  inheritance  of  the  of  male-  production  produced  no  when h y b r i d i z e d w i t h  of  viable pollen  -28from male-fertile plants.  They found 3 types of inheritance  which they explained by assuming that the m a l e - 3 t e r i l e condition r e s u l t s from an i n t e r a c t i o n between a recessive gene and  a non-nuclear or cytoplasmic f a c t o r , .  A l l plants with  normal cytoplasm (IT) produce viable seed. A l l male-sterile plants possess the s t e r i l e type of cytoplasm (s).  Ho l i g h t  was shed on the nature of the cytoplasmic factor which d i f f e r s in the 2 types.  A gene for m a l e - s t e r i l i t y (ms) also influences  pollen development when c a r r i e d by plants with S cytoplasm but has no effect when c a r r i e d by plants with N cytoplasm. Clarke and P o l l a r d (28) found i n 1949 that m a l e - s t e r i l i t y 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 s e l f ing  for a l l l i n e s tested was 4.1%.  Pineman (43) found complete pollen s t e r i l i t y i n the potato to be a r e s u l t of the f a i l u r e of normal microspore formation during meiosis.  This view i s supported by numerous  meiotic  i r r e g u l a r i t i e s she observed i n s t e r i l e - p o l l e n plants. The most common meiotic i r r e g u l a r i t i e s she observed were: (l) f i l u r e of the chromosomes to p a i r ,  (2) lagging of the  chromosomes on the spindle and (3) f a i l u r e to complete the normal reduction-division process.  P a r t i a l s t e r i l i t y of the  f e r t i l e plants i s conditioned, after what appears to be a normal meiosis, by microspore abortion, which frequently occurs after size.  the spores have reached normal pollen grain  Pineman (ibid) concluded that female parents  more pollen s t e r i l i t y than male parents.  transmit  -29Bateson and  a  and  procumbent a  Gairdner  (6)  flax  found normal  3:1  and  ividuals  i n  obtained  only  when  parent.  They  attributed  Chittenden pretation  of  interpreted specific another  plasm  the  paper  flfcootiania  pollen  i n  the  i n  of  of  P2»  race  as  due  cytoplasm  to  to  of  not  (38)  to  found  produced  the  that  Grimball  being  petals  unfolded.  had  plants  carrot  i n  shrivelled  that  Hybrid were  (90) 1947.  S  classified  cyto-  but  presence  produced  that  viable  Male-sterile the  ratio  266:232.  mode  of  inheritance  a  male-sterile  color  consisted  before  on m a l e - s t e r i l e male-sterile  and  any plant 15  normal. Frankel  (44)  working  gene  from  the  M a l e - s t e r i l i t y  37  In  of  factors i n  discovered  produced  a  single  type  cytoplasm.  and brown i n  seed  They  (erect).  a l l . .-He f o u n d  and  that  one  at  Welch  anthers  a  pollen  present.  female-fertile  wrote  sanderae  at  inter- •  race.  H.  unknown  female  "anisogeny".  of  but  a  procumbent  langsdorfii  M a l e - s t e r i l i t y reported,  as  suggested  certain  was  ind-  were  i n t e r a c t i o n of  another  male-steriles  e  a  male-sterility  f r o m IT.  presence no  with  plants used  (ibid)  the  t a l l  "anisogeny".  male-sterility with  but  derived  was  offered  Gairdner's  common  and m a l e - s t e r i l e  Sterile  results  (26)  crossed  C h i t t e n d e n i n 1927  East  self-fertiles  - (J&)  but  the  (25)  plants, produced  the  s t e r i l i t y  sanderae  cytoplasm  the  procumbent  and  determines  1932,  i n  and P e l l e w  (procumbent) In  of  the  Bateson  gene  apparently  ratio  i n 1921  i n Hew Z e a l a n d ,  observed  male-  -30'  s t e r i l i t y genus Hebe  with  Hehe, only  study  of  that  to  year  to  2  and of  loops  and  not  this  (2)  Tetrad  Ho  grains  Pollen a)  having  normal.  the  concurrent  mother h)  to  this  meiosis  i n  the  amorphous  empty  the  to  c e l l  anther  is  showed either  mass  of  co-  diakenesis  which  walls  i n :  normal.  threads  size  cavity.  regular  transplanting.  pollen  pachytene  (ibid)  causes  is  after  the  the  is  rapidly  collapse, Tapetum  cells  -  a l l  meiotic are  stages  up  formed but  to  second  collapse  rapidly.  seen.  cells  undergone  disintegrate. with  the  phenomenon  of  to  genetic  regularity,  Tetrads  degeneration  Tetrad  structure,  state  degeneration are  (3)  the  to  As  Frankel  with  an  the  simultaneously.  telophase pollen  due  "The  of  unsuited  and  up  into  remaining  is  environment  formed.  Subsequently  degenerate  is  approximately  collapse  forms  However,  the" b r e a k d o w n  dissolved". i n  i t  an* n e w  -  8  species-hybrid.  as  rapidly  individually,  bivalents,  3 years  i n  causes  prophase are  1  inheritance.  i n  occurred of  or  and  m a l e - s t e r i l i t y  study  Pachytene  Diplotene  the  year  degeneration  agulate  every  i n nature  species  environmental  A cytological  (1)  5  m a l e - s t e r i l i t y  not  from  comprising  flowers  concludes and  f e m a l e - f e r t i l i t y  a  round  change  pollen The  off, either  grains  process  and  is  degeneration  i n  about  a  day  diameter  later,  or  i n  not  wall  shrivel,  collapse,,  and  gradually  regular  and r a p i d  and  occurs  of  tapetal  walls  of  the  pollen  cells. Pollen  grains  formed,  increase  i n  size  and  c e l l  walls  -31  thickened; leading  immediately  rapidly  c). P o l l e n without  any  flowers.  G.  to  further  However,  Several  (92)  the  of  this  work.  seed  producing  by  a l f a l f a  has  and K i e s s e l b a c h Nebraska  Saskatchewan in  Argentina,  New  opening  the  to  a l f a l f a  published  Atwood  f i e l d  very  of  (5),  a  may  also The  studied report  from  reported  a  in  except  amount by  of  of  that  89%  and H a d f i e l d  and  the a  a l f a l f a study  i n  alfalfa.  where  the  (18)  Galder  not  of  crop,  although  l i m i t e d  by  c r o s s - p o l l i n at i o n  investigators.  of  Burkart  and  is  paper.  natural  several  It  and years.  Tysdal  this  c r o s s - f e r t i l i z a t i o n ; 94.2%,  and  c r o s s - f e r t i l i z e d  occur  alfalfa.  in-recent  complicate  b r i e f l y  i n  summary-repeating  peculiarities may  the  breeding  completely.  attempt  which  remain  dehisce.  G r o s s - f e r t i l i z a t i o n  seed  (46)  produced  Knowles  found  Tysdal  (56)  84.5%  reported  i n i n  crossing  44%  i n  Zealand. Cross-pollination  of  the  to  been  {92),  normally  (91)  resulted  have  discussed  been  to  and  on  self-incompatibility. in  collapse  tapetum.  articles  several  is  s e l f - f e r t i l i z a t i o n  f a i l  and  review  versus  A l f a l f a  grains  s h r i v e l l i n g  m a l e - s t e r i l i t y  thesis  are  up  of  possible  mechanism,  Self-  pollen  i n  to  cover  However,  setting  p a r t i a l l y  anthers  White  this  male-sterility, (a)  the  d i f f i c u l t i e s  Kiesselbach purpose  formed,  relative  papers  of  degeneration  notable  inherent  Extensive  collapse  grains  Observations  its  a  followed by  seeds  fact,  per  Cooper  flower  produces  pollinated  and B r i n k  (31)  a much h i g h e r than  report  average  number  s e l f - f e r t i l i z a t i o n ; a net  f e r t i l i t y  6  i n  times  -32-  as  high  Tysdal  per  flower  pollinated  and K i e s s e l b a c h  f e r t i l i t y  relationships  and  plant  breeders  Mot  only  axe  more  not  Hadfield  associates 32)  among  only results  also  to  i n  a lesser  Calder  florets  (ibid)  tripped  hand-tripped per of  100  experiments  uction on  and  50  then (b)  and  i n  a marked i n  of  possible,  of  paramount  i n  that  plants  seeds  i n  a measure seeds  and  43.0  seed  of  seed  home and  out white  Zealand, and  production,  formed  when  that  (69)  than (If  Hadfield 100  florets  were  total  more  per  the  seeds  but  whereas  heterosis.  self-pollination.  alfalfa  is  (46),  associates  p o d wa,s  s e l f - f e r t i l -  S e l f - f e r t i l i z a t i o n  were and  self-  plants.  Cooper  vigour  observed per  (22),  U.S.A.  covered  Piper  This  i n Hew  Carlson  65  than  of  importance,  Armstrong  (46)  open-pollinated,  over  pod.  decrease  selfing.  self-sterile  Sweden,  the  after  formed i n  seed  a  series  prod-  doubled 100^  p r o d u c t i o n w o u l d be  crossing incx-eased  100#. Is  tripping; necessary  complicates  "tripping"  effected.  per  vegetative  results  A morphological further  of  highly  Calder  in  1912-1914  number  i n  others  tripped.  open-pollination  were by  florets  the  (60)  92),  found  and  and  select  (91,  degree  cross-fertilization and  is  formed  L.esins  (31,  alfalfa  as  consideration  are  workers:  Brink  i n  a  following cross-  "by m a n y  and  that  set  seeds  Tysdal  state  pods  "but m o r e  i n Canada,  crossing  (91)  should  ization  (3)  after  of  the  the  a l f a l f a  peculiarity  picture  flower  According to  for  of  before  Coffman  of  seed  alfalfa  s t e r i l i t y  is  production? flowers the  f e r t i l i z a t i o n can  (29),  DeCandolie  (35)  that  necessity be in  , 1832  -33-  gave the f i r s t  explanation of the t r i p p i n g process i n which  he stated that the explosion of the flower takes place when a certain stage of i t s maturity i s reached.  Hildebrand (again  quoting Gof f man (ibid)) believed as early as 1866 that i l i z a t i o n may take place in untripped flowers. Cooper (15) flowers  fert-  Brink and  observed pollen germinating within untripped  and pollen tubes entering the styles even i n the late  bud stage.  Carlson (22)  found 27% of the flowers producing  seed without t r i p p i n g i n Utah i n 1930. that 5.9% of untripped flowers  set seed.  Hay (47)  i n 1925 found  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 i n d that 0.6% of the untripped flowers showed pollen germination.  They observed a thin membrane covering the  stigmatic surface which i s s u f f i c i e n t l y  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 s t y l e . scarification,  Stigma  and rupture of this membrane, with the release  of the stigmatic content which i n i t i a t e s  pollen germination,  i s normally achieved when the stamina! column i s released from the keel or "tripped" s t r i k i n g -the standard with, considerable 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 i n an untripped flower. Hadfield and Calder (46) write that t r i p p i n g i s a prerequisite  to seed production. B l i n n (8) i n Colorado  reporting on the r e s u l t s of an experiment on " t r i p p i n g " wrote  -34-  in 1920 that "there wa,s no clear evidence that b e e 3 or otherinsects were essential  to alfalfa, seed production. F e r t i l i z a t i o n  can and does take place without insects", i s not necessary for seed production.  and that t r i p p i n g  Brand and v/estgate (11)  i n 1909 stated that p o l l i n a t i o n i s not effective are tripped.  Burkill  until  flowers  (19) wrote that a l f a l f a pollen was shed,  in the bud stages, to which Coffman (ibid)  agrees and adds  that t r i p p i n g i s not necessary for seed production. The question has aroused many papers and i s s t i l l unanswered.  It appears to this writer that the presence or  absence of a stigmatic membrane may r e s t upon a genetic or upon a p h y s i o l o g i c a l response.  factor  The presence or absence  of t h i s membrane appears to be the determining factor i n whether or not pollen can f e r t i l i z e the ovules or grow i n the styles whether tripped or not tripped. The tripping c-f flowers i « ' normally performed by bees, and Peck and Bolton (68)  i n 1946 showed that the native bee  population was often the l i m i t i n g factor i n a l f a l f a seed production i n Northern Saskatchewan and Alberta. This view although contrary to that of B l i n n ( i b i d ) , i s generally agreed upon by the s c i e n t i s t s  working i n this f i e l d .  An idea  of the importance attached to the role of insects i n r e l a t i o n to t r i p p i n g and deed s e t t i n g . i n a l f a l f a ,  can be gleaned by  the report of the Twefth A l f a l f a Improvement Conference  (l)  wherein 9 out of the t o t a l of 23 papers read dealt with t h i s phase of seed production. Dwyer (37)  i n 1932 reported instantaneous  t r i p p i n g of  -35a l l flowers at 1 0 4 ° P under most diverse moisture conditions providing the flowers are i n a fresh turgid condition. Armstrong and White (3) state that the c o n t r o l l i n g 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 i n pollination.  However, this view i s c e r t a i n l y not agreed to  by the majority of workers. Kirk (55) reported on the finding of an autogamous a l f a l f a which, i s s e l f - t r i p p i n g , gamous alfalfas  Automatic t r i p p i n g of auto-  occur when "the dynamic force present -in the  staminal column i s s u f f i c i e n t l y greater than the s t a t i c force present i n the keel to cause the t r i p p i n g mechanism to explode simultaneously" to quote Armstrong and White Southworth (85)  (3).  attempted to r a i s e a high seed producing  l i n e by h y b r i d i z i n g M. l u p u l i n a (self-tripping)  with M. s a t i v a  (not s e l f - t r i p p i n g ) . He hoped to produce a s e l f - t r i p p i n g high seed setting alfalfa,,  and achieved a considerable measure  of success i n experiments running over 20 years. of highly s e l f - f e r t i l e ,  However, use  s e l f - t r i p p i n g l i n e s is undesirable i n  developing synthetics, because the seed y i e l d s from open progenies from such l i n e s i s similar to the seed y i e l d s from their selfed progenies  (87).  In the past few years the u t i l i z a t i o n of hybrid vigour to obtain maximum productivity of the concerned plants has assumed importance to many s c i e n t i s t s .  As Bonn and Whitaker  (9) point out, m a l e - s t e r i l i t y enables seedsmen to produce  -36hybrid seed comparatively e a s i l y and cheaply. Clarke and Fryer (27) w r i t i n g -on seed setting of  alfalfa  in 1930. found that many plants produced high percentages of sterile  (empty) pollen grains.  The percent varied among plants  but remained constant for p a r t i c u l a r plants even when the pollen was produced under different conditions.  In 1929  counts were -obtained from a number of a l f a l f a plants which, were setting p r a c t i c a l l y 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 t e r i l e p o l l e n . Armstrong and White (abid) add to t h i s i n 1935 that  plants  having a high percent of s t e r i l e pollen were deficient i n amount of pollen as w e l l ;  a high proportion of shrunken pollen  grains seems "associated with faulty dehiscence of the anthers". Tysdal and Kiesselbach (ibid) (91)  stated i n 1944 that  male s t e r i l i t y had not been i d e n t i f i e d i n a l f a l f a ,  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 i n e s of a l f a l f a  at  the University of B r i t i s h Columbia farm. A microscopic examination of the p o l l e n , produced by the 910 clonal l i n e s of Rhizoma a l f a l f a maintained by the Dep't. of Agronomy, was c a r r i e d out.  Several racemes  representative  of these l i n e s were clipped while i n a fresh turgid state and c a r r i e d , with t h e i r excised ends i n fresh water to the labor-  (  -37  atory.  Here  sample  and  mounted were  3  fresh  tripped  on  i n mineral  examined  florets  and  the  cover  125x  for  Several  observed  the  quality  were  d i s t r i b u t i o n  Following- the pollen  was  a  quantity  and  received  a numerical  was a  assessed  numerical  size  and  a  proper  shrivelled  pollen  that  44 so  could  ination, lines that  was  not 44  were was  slides  and  clonal  uction  One of  were  produced  rated  made  was up  5 both the  pollen,  so  was  slides  pollen  either  grains.  ends  of  time,  In  of  On t h e  quantity  so-called given  the  or  of  pollen  of  quality,  uniform large  shape  of  1  is  micro-pollen. during  the  summer  of  pollen;  "sterile" or  pollen  as  f i r s t  exam-  and  a l l  the  Hew  pollen  lines  rated  and  quality  lines,  showed  5  pollen  amorphic.  and  44  a higher  or -  represented"  quality  sterile  s  rounded,  the  the  grains  of  of  lines,  of  pollen  case  the  for  basis  as  20 o f  few  proper  shrivelled 49  5 both  micro-pollen  flowers  for  to  i n 1944,  quantities  A value  no  5  1  pollen  shrivelled  these  was  each  research  of  at  very  5.  absence  designated  for  for  of  pitted.)  rated  of  large  (Normal  either  This  of  an  produced  tentatively  were  pollen.  with  categorized.  lines  l j  rating  shrunken,  be  of  pollen.  lines  lines  of  represented  f i n e l y  recategorized. again  5  shape  dried-up  semi-hyaline  rating  of  quality  value  A production  numerical  rating  The  o u t l i n e d b y B u r t o n (21)  numerical  quality.  pollen  a Reichert  and the  The  from  curve.  method  assessed  of  random  affixed.  of  quantity  at  slide.  slips  power  "bin o c u l a r - m i c r o scope samples  selected  a microscopic  o i l  under  were  was  5,  a  classification.  were of prodThe  -38-  remaining 4 " s t e r i l e " l i n e s showed on a l l observations,  in  the'cases of any pollen production at a l l , only shrunken, s h r i v e l l e d pollen.  Table I shows the categorization at t h i s  date, 2 clonal cuttings of each of the 20 l i n e s designated "high pollen producing l i n e s " (See Tables I and I I I ) ,  as  and  5 cuttings of each of the 4 l i n e s designated " s t e r i l e "  (See  Tables I and II) were made, and planted i n the greenhouse November 4 and October 31, 1949 r e s p e c t i v e l y . The cuttings grew well i n the greenhouse and through the use of a r t i f i c i a l l i g h t were "forced" into flower i n A p r i l , 1950.  Pollen from the different l i n e s was. again  examined under, 125x with r e s u l t s comparable to the previous summer's observations.  (See Table I ) .  Hone of the l i n e s  designated as "male-sterile" produced .viable p o l l e n , . The plants were transplanted from the greenhouse to the A l f a l f a Hursery plots i n May 1950.  Pollen was examined on  the 18th of September, 1950, under 125x, with r e s u l t s  as  shown i n Table I. A pollen examination of the following v a r i e t i e s of alfalfa,  contained i n the Alf a l f a Hursery plots at U . B . C . ,  was also undertaken i n 1949 and 1950 for the presence of male-sterility.  In 1949 Viking, Ranger, Grimm, Ladak, Per ax,  Buffalo, Don (creeping M .  f ale at a),  and M. l u p u l i n a pollen  was examined along with .pollen from 4 strains  of Rhizoma:  H71P27, H7, H68, H190, which were not included i n the previous pollen examination.  -  In September 1950 an examination  -39-  of  the  pollen  following  from  lines:  the  II,  Hemaston,  the  lucerne,  cases (b)  666,  Kansas  M. ruthenica,  oogie  M.  and  examined  Common,  Australian  A counted  any  Hunter creeping  Vidarshov,  451,  erect  Oregon  River  the  M.  Creeper  lucerne,  lucerne.  In  the  following  and  the  f i r s t  the  latter  of  studies  by  during  and  observed,  self-pollinated  edure  I  none  each  were  f a l -  Boobor-  of  II)  of  part  of  July  of  the  selected proc-  part  of  1950.  Racemes and  a l l  cut  away  with per  adhering water.  dry,  the  standard the the After was  pollen the  with and  of  secured  might  effect  found  that  a  over  and  of  the  of  10 to  by  selected  flowers  remove  any i n  a r t i f i c i a l l y tripped of  opening  scissors  the  released  clean by  (sterilized  a wash)  between  scissors. from  were  untripped  dipping  followed by  be  were  opened  washed  pair  alcohol  to  and  standards  were  some  florets  was  closed  column  florets  a maximum  the  keel,  column w i t h the  on  ethyl  staminal  growth,  raceme  florets  the  untripped  leaving  The  points  lines  fresh  terminal  scissors  foreign  inserting  caused  buds,  raceme.  When  between  containing  leaves,  florets  bag  Grimm  plus  florets  (Table  stike  S274,  male-sterility  number  lines  the  conducted  A t l a n t i c ,  glutinosa,  was  was  Grimm Saskatchewan  Self-pollination  August,  plants  G r i m m Summer l a n d  Grimm Saskatchewan cata,  above  the  This  keel  and  force. a  raceme  raceme  cross-pollination.  cross-pollination  to  were  tripped,  exclude  (Stevenson  occurred  i n  any  a  cellulose  insects  and B o l t o n  a l f a l f a  i f  that (87)  foreign  -#o-  p o l l e n , was  applied  one  where  flowers  had  found  that  foreign  in  i f  addition  agent  to  its  mildewing  racemes  had been  identified.  the  Table  II) The  were  seeds  were  planted  and  use  i n  plant  threshed  so  by  untreated w o u l d be  a weeks  seed  formed  that  (94)  stigmas the  they  i n  the  hand  and  time to  harvested  ripen  was  to  future  the  line 25,  carried  from  at  intervals  of  October  seed of  1950,  producing  a  the 1  9I-10B. 1951,  i n  the  these  active  to  ripen.  could  The  be  periodically laboratory.  counted.  (See  greenhouse  i n  to  other  flats  containers.  Nursery  They  i n May 1951  for  studies.  5  deeds  plant Only  and  i n  i n December  A l f a l f a  and  obtained  from  the  the  latter  a, p o l l e n  1  by  selfing  seed  plant  obtained was  examination  of  i n these  out.  O p e n - p o l l i nat i o n All  flats  genetical  144-11A,  April  i n  overwintered  resulted  line  by  (c)  i n  were  to  transplanted  and  "sterile"  pollen  allowed  transplanted  flowers  any  Garl  time,)  removed  pods  to  and  tripping  .  "sterile"  flower  the  after  Tysdal  pollen  previously  was  f l a t ) ,  1  and  of  allow  seed  (40  possible  to  the  1950,  be  and  longer  added  foreign  were  developed  ripe  October per  bags  possibly  emasculated.  pollen  tagged  The  nearly  When-ripe,  from  been  own,  cellulose  prevent  w i l l  not  or  i n f e r t i l i z a t i o n 70-98^ The  when  hour  pods, few  studies when n e a r l y  days  from both l i n e s .  from the  The  ripe,  August  collected  to  the  f i r s t  "male-sterile"  and  high  seeds,  1st  were  when r i p e ,  were  part  threshed  -41and counted with r e s u l t s  as shown i n Table I I I .  The open-  p o l l i n a t e d seed from the "male-sterile" plants was planted i n f l a t s i n the greenhouse in November 1950,  and 100 plants of  each l i n e were transplanted to 5" pots i n l a t e December. The plants began to flower on A p r i l 11, and microscopic slides were prepared d a i l y of the pollen from the flowers soon as they unfolded from the bud.  as  By A p r i l 25, 71 plants  of the 20-DRC l i n e had flowered, 12 plants of 142-1033, 41 plants of 91-10B, and 44 plants of the l i n e 144-11A had come in£o flower.  Microscopic slides  of the pollen from these  plants was examined under 125x on A p r i l 26, with results  as  shown i n Table 6. B - Observations Pollen showed a great amount of d i v e r s i t y both i n shape and s i z e .  Pollen varied from normal, rounded,semi-hyaline,  and f i n e l y p i t t e d ; through e l l i p t i c a l , s t r i a t e d , dense cytoplasm; to i r r e g u l a r ,  and with  angular and of large s i z e .  Also observed was very small pollen of normal shape and density (designated  ' m i c r o - p o l l e n ' ) , and dark, amorphous,  very small granular bodies (designated 'aborted' p o l l e n ) . On the basis of pollen examination 20 l i n e s were selected being superior pollen producers,  as  and 4 l i n e s were selected  as possessing " m a l e - s t e r i l i t y " . The high pollen producing l i n e s a l l produced a large quantity of pollen (with a few exceptions)  of uniform  excellent quality except 3 l i n e s which produced,,the second year, some micro-pollen and shrunken p o l l e n .  The results  -42/ are tabulated i n Table I . The examination of the v a r i e t i e s  other than Rhizoma was  carried out as a check: most produced very good pollen and none showed any m a l e - s t e r i l i t y .  However, we had only 6 plants of  each variety i n the nursery plot to make observations  on, so  the examination would hardly be s i g n i f i c a n t . The l i n e s designated as "male-sterile" were observed i n the main production f i e l d i n August and September 1949 with the following observations recorded.  The o r i g i n a l 20-DRC l i n e  was complete and showed superior vegetative growth.  Only 2  plants were l i v i n g i n the o r i g i n a l clonal row of 91-10B: both being healthy, vigorous plants. one poor unvigorous plant l e f t  Clonal row 144-11A had only i n the row.  Clonal row 142-10B  had only one plant, - although vigorous and healthy, remaining. The o r i g i n a l observations  on the pollen taken i n 1949 of a l l  the selected "male-sterile"  and " high-pollen" l i n e s are  contained i n Table I. In a c r i t i c a l examination of the pollen of the "males t e r i l e " l i n e s on 18 Sept. 1950 the following observations were made. 20-DRC: No loose p o l l e n . The pollen sacs appear f u l l  of a  dark amorphous substance. 142-10B:  Very l i t t l e good p o l l e n , mush aborted.  Pollen sacs  f u l l of granular dark substance. 144-11A: misshapen,  Approximately equal amounts of good pollen with irregular,  very much of e i t h e r .  s h r i v e l l e d p o l l e n ; however there was not  -43-  91-10B: Very l i t t l e pollen was produced, Tout i t seemed normal pollen although very small i n size On s e l f - f e r t i l i z a t i o n  (micro).  20-DRC and 142-10B produced no  seeds; whereas 144-11A produced .0427 seeds per f l o r e t •and 91-1OB produced .008 seeds per f l o r e t selfed.  selfed,  These  amounts of seed setting agree very well with the r e s u l t s ag, oh served on the amount of pollen produced "by these l i n e s . (See Table I) Of the normal or high pollen producing l i n e s 0.654 seeds .were set per f l o r e t selfed,  with a range from 0.009 to  seeds produced per f l o r e t selfed. 0.44 seeds set per f l o r e t Cooper and Brink (31) 1  v/ere set per f l o r e t  1.877  (Clarice and Fryer (27) found  selfed i n Saskatchewan  in 1930.  found that an average of 0.80 seeds  selfed with a range of 0.16 to  1.76.  This agrees f a i r l y weifel with my findiggs.) S h r i v e l l e d , small,  and discolored seed constituted 34.84$  of the seed produced on s e l f p o l l i n a t i o n ,  and 16.19$ of the  seed produced on open-pollination. (See Tables II and III) The amount of seed produced on s e l f - p o l l i n a t i o n i s show in Table II, Table  and on open-pollination of the same l i n e s i n  III. An examination of the pollen from the 71,>]?i plants of  the 20-DRC l i n e that had. flowered at time of reporting showed 2 plants that produced no pollen at a l l , and 7 plants produced only very s h r i v e l l e d , misshapen, sterile,  pollen.  amorphic,  that  apparently  The 2 plants producing no pollen at a l l  had empty pollen sacs i n abundance, but no signs of p o l l e n , excepting a very fine dust l i k e debris which may be the remains  -44of early aborted'pollen. An examination of the pollen from the 44 3?]_ Plants of the 144-11A l i n e showed 4 plants that produced empty pollen sacs without a vestige of free p o l l e n , and 2 plants  that  produced the s h r i v e l l e d , amorphic, apparently s t e r i l e pollen as'observed in the 20-DRC segregates. The pollen from the 41  plants of 91-10B showed 1 plant  that f a i l e d to produce p o l l e n , and 5 plants that produced only misshapen ' s t e r i l e ' p o l l e n . Line 142-10B had only 12 3?^. segregates i n flower by A p r i l 26, 1951, and of these,  only 1 plant appeared, to produce  pollen that could be designated  'sterile'.  Table VT shows the frequency d i s t r i b u t i o n of the pollen of  the 3?]_ segregates of the 4 male-sterile l i n e s as they  were observed on A p r i l 26, 1951.  As previously outlined,  a value of 5 indicates pollen of uniform, semi-hyaline, rounded, shape. probably s t e r i l e  1 indicates pollen that i s misshapen and or incapable of germination.  0 indicates an absence of p o l l e n .  A value of  The intermediate grades  indicates pollen of intermediate q u a l i t y .  That i s ,  of 3 would indicate the presence of rectangular, pollen.  a value  striated  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 Qnan. Qual.  OBS. 18-24 APRIL 1950 Quantity Quality  -  20-DRC 91-10B 142-10B 144-11A  0 20 2-  0 0 0 1-  1 0 1  106-DEC 57-10B 68-10B 83-10B 102-10B 103-10B 128-10B 144-10B 15-11A JO-llA 34-11A 35-11A 59-11A 71-llA 73-11A 81-11A 94-11A 95-HA 90-11B 115-11B  55  5 5+ 5-  4+ 4+  Note:  #  4  5 5-  5-  5 5555555555555* 55*»  4+  55+ 5555555-  4+  55 5+ 5+  i n d i c a t e s presence  of  5  ©  1 0 1  5+  4+  micro - p o l l e n o r aborted  4+ 4+ 4-  4+  55-  4+  5 3  3  55  5-  5-  0 1 0 2  4+  4 4 4+ 4+  4  0 1+ 0  3-  5 5 5 5 5 5 5 5 5 3 5 5  4+  OBS. 18. SEPT. 1950. Quantity Quality  4+  5* 5+  55 5 5 5  55 5 5+  44+  4+  4+  5  5*  4+ 4-  5-  5 5 55  53 3 5  4+  35 3-  44+ 4+ 4* 4+ 4+  55 5  pollen. I I  TABLE I I Line  F u l l seeds  Male-sterile 20-DBC 142-10B 144-11A 91-10B  lines 0 0 5 1  SEEDS PRODUCED UPON SELF-POLLINATION  Small brown seeds No. % total 0 0 0 0  High p o l l e n producing l i n e s 20 106-DRG 118 0 1 37-10B 10 68-10B 8 172 27 83-10B 102-10B 10 41 103-10B 24 14 128-10B 44 15 12 125 144-10B 15-11A 27 31 11 30-11A 25 34-11A 85 8 60 33 59-11A 106 20 1 71-11A 5 13 73-11A 25 12 0 81-11A 94-11A 71 39 70 95-11A 16 90-11B 12 5 115-11B 33 5 Average  -  T o t a l seeds  0 0 5 1  No. f l o r e t s s e l f e d  Seeds/floret  217 142 117 118  0 0 0.0427 0.0085  106 106 105 106 106 107 99 100 108 103 103 110 105 103 106 100 109 103 48 107  1.302 0.009 0.171 1.877 0.481 0.355 0.596 1.370 0.537 0.350 0.903 0.845 1.200 0.058 0.358 0.120 1.009 0.835 0.354 0.355  •  14.49 100. 55.56 13.57 80.39 36.84 25.42 8.76 " 53.44 30.56 8.60 35.48 15.87 16.6? ' 34.21 18.60 70.59 13.16 34.84^  138 18 199 51 38 59 137 58 36 93 93 126 6 38 12 110 86 17 38  Average 0.654 seeds per f l o r e t s e l f e d .  TABLE I I I Line Male-sterile 20-DRC 142-10B 144-11A 91-10B  Pull  seeds  lines 491 258 528 909  SEEDS PRODUCED UPON OPEN-POLLINATION  S m a l l brown seeds No, % total 89 139 111 60  High p o l l e n produeing l i n e s . 106-DRC 44 295 37-10B 54 697 68-10B 20 74 83-lOB 1314 376 102-10B 654 58 103-10B 773 31 128-10B 41 54 144-10B 78 354 15-11A 400 54 30-11A 86 . 46 34-11A 622 36 108 569 35-llA . 470 59-11A 69 71-11A 106 6 118 7 3-HA 1165 81-11A 148 565 94-11A 368 53 95-HA 309 52 90-llB 550 61 Average  15.34 35.01 17.37 6.19 12.98 7.19 21.28 22.25 8.15 3.86 56.84 18.06 11.89 34.85 5.47 15.95 12.80 5.36 9.20 20.76 12.59 14.40 9.99 16.19^  T o t a l seeds  580 397 639 969 339 751 94 1690 712 804 95 432 454 132 658 677 539 112 1283 713 421 361 611  No. of p l a n t s  Seeds/Plant  5 5 5 5  116 79.4 127.8 193.8  2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2  169.5 375.5 47.0 845.0 356.0 402.0 47.5 216.0 227.5 66.0 329.0 338.5 269.5 56.0 641.5 356.5 210.5 180.5 305.5 Average  2 8 7 . 8 seeds per p l a n t .  -48-  TABLB IV CORRELATION OF SEED SET UNDER OPEN & SELF-POLLINATION. Line  68-10B 128-10B 71-11A 30-11A 106-DRG  95-HA  94-11A 144-10B 15-11A 59-11A 90-11B 113-1IB 34-11A 33-11A 102-lOB 81-11A .37-1GB 1I03-10B 7 3 - 11A 83-lOB  Seed s e t Open-pollinated  Self-pollinatea  #  # seeds.  seeds  Rank  47 47.5  1 2  66 169.5 180.5 210.5 216 227.5 269.5 305.5 316 329 338.5 356 356.5 375.5  4 5  56  0.171 0.596 0.058 0.350 1.302 0.835 1.009 1.370 0.537 1.200 0.354 0.355 0.903 0.845 0.461 0.120 0.00? 0.355 0.358 1.877  3  6 7 8  9 10 11 12  13 14  15 16  17  402  18 19 20  641.5 845  Rank 4 12 2  5 18 13 16  19 11  17 6 7.5 15 14 10  3  1 7.5 9 20  Using r a n k i n g c o r r e l a t i o n '.£"*'  -  210  ir  2581.0  i&  (ix) -  44,100  2  S.S.  X  £  =  y  210 2  -- 2581.5  (*.y> 2  376  S  « «y S  -  44,100 376.5  txj - 2223 r : t At  « .209  n-r2 ( l 8 ) degrees o f freedom,  pr .05.  +.0478  t a b l e d t equals 2 . 1 0 1 at  C a l c u l a t e d t i s s m a l l e r than t a b l e d t so no s i g n i f -  icance can be a s s e r t e d t o the c o r r e l a t i o n i s , t h e r e i s no c o r r e l a t i o n .  c o e f f i c i e n t . That  TABLE V RANK CORRELATION OJ? SEED SET UNDER OPEN & SELF-POLLINATION USING WILCOXON'S  .  (98) APPROXIMATE STATISTICAL PROCEDURE.  (Ranks as a s s i g n e d i n Table IV) Open-noil.  Self-poll.  1 2 3 4 5 6 7 8 9 10 11  gftflk, t o t a l s  4 12 2 5 18 13 16 19 11  5  13  6 7.5 15  15 16 17 18 19 20  10 3 1 7.5 9 20  14  25 196 25 81 529 361 529 729  14 5 9 23 19 23 2? 20 27 17 19.5 28 28 25 19 18 25.5 28  17  12  (Rftfik tQtftlg)  14 -  400  729 289 380 784 784 625 361 324 650 784 1600  40  10, 185 r 0 1  r  x r - 1. P-l _ 12 " np (p+l)  Sum (rank t o t a l s )  a a 20 X2r * = r  ~  p = 20  10. 185 70  - 3n(p+l) = 10, 185  - 126  145.5 - 126 - 1  (rank t o t a l s )  2  =  =19.5  1.03 - 1  =  +.03  To t e s t 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 e r r o r 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 t h e r e f o r e t h e c o r r e l a t i o n cannot be considered s i g n i f i c a n t as i t s value (+.03) i s l e s s than one q u a r t e r of i t s standard e r r o r (.227).  TABLE VI -  FREQUENCY DISTRIBUTION OF THE POLLEN CLASSES OF THE E l SEGREGATES 4  Line  'MALE-STERILE  No. o f p l a n t s examined  5  1  OF THE  ALFALFA LINES ON APRIL 2 6 . 1951 Category f o r q u a l i t y o f p o l l e n 4 1 2 3  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  168  2  15  39  81  15  7  Totals  Note Q u a l i t y category 1 i n d i c a t e s t h a t no v i a b l e appearing p o l l e n i s present Q u a l i t y category 0 i n d i c a t e s t h a t no p o l l e n i s produced.  i  O  I  -51-  G - Discussion.  „  L i n e s 20-DRG and 142-10B show c o n s i s t e n t l a c k o f 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 b e i n g e x t r e m e l y v i g o r o u s 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., a l t h o u g h showing m a l e - s t e r i l i t y , i s weakly  vegetative  and l o w 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 t h e s e r e a s o n s 142-1033 does n o t p o s s e s s t h e same v a l u e agronomic a l l y as the 20-DRG l i n e ; b u t 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 h a t i t i s a l s o worthy o f f u r t h e r s t u d y . I t was n o t e d t h a t the F x s e g r e g a t e s o f 20-DRG were much f u r t h e r advanced than those o f 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 s t a g e .  This i s f u r t h e r evidenced by the f a c t  t h a t 71 p l a n t s o f the 20-DRG F-j_ were i n f l o w e r b y t h e t i m e 12 142-1033 F i were a t t h e same s t a g e . L i n e s 144-11A and 91-1033 appear t o p o s s e s s p a r t i a l malesterility.  91-1033-, i n s p i t e o f t h e r e b e i n g o n l y 2 p l a n t s  i n t h e main c l o n a l row, and t h e f a c t t h a t i t produced  left  seed  on s e l f i n g , i s worthy o f f u r t h e r s t u d y due t o i t s e x c e l l e n t seed s e t on o p e n - p o l l i n at i o n and i t s v e r y l o w percentage of s m a l l , brown d i s c o l o r e d seed.  A l t h o u g h t h e same o b j e c t i o n s  can be a p p l i e d t o l i n e 144-11A, i t i s f e l t t h a t i t i s especi a l l y worthy of f u r t h e r s t u d y as 9.1% of the F  x  segregates  showed a complete absence o f p o l l e n when examined on A p r i l , 26, 1951. L i n e 57-1033 appears t o p o s s e s s a s e l f - i n c o m p a t i b i l i t y factor.  T h i s l i n e appeared w e l l above t h e average i n seed  -52^Z set  upon  open-pollination,-  per  floret  s e l f e d on  while  only  production  was  greatly  increased  by  open-pollination  (16.19$).  (Carlson  in  1931  27$  that  for  a 4  shrivelled due  to  full was  insufficient  Whatever,  results  'male-sterile' conform  to  may of  be the  lines  as  in  the  the  3?]_ i n  1:7.3.  i n 142-10B as  simply  ster i i i ty  should  However, be  show t h a t  whatever  of  the  the  ratios. of  the  1  to  season  t h e ]?]_.  the  anode o f  to  produce seed  open-pollinated on  this  basis.  difference. delayed  VI  for  do  observed.  the  not  4  appear  to  to the  7.9 to  male- -  male-fertiles  ratio  was  inheritance, the  observed m a l e - s t e r i l i t y i s  no  was  malt-  which  ~F]_  in  1:688;  male-sterility  polyploidy,  It  soma.to-  difference  male-fertility  in  by  the  to in  produces  observed If  7.7$  20-DRC  male-steriles  1:8.3..  complicated the  as  i n Table  a ratio  recessive  appear  the  over  reported  self-pollinated  explained  cause  91-10B  appeared  inherited  does  In  (24)  increase  in  significant  reported  a ratio  undoubtably  be a  late  wrinkled  from  l i m i t e d IT-^ s e g r e g a t i o n  144-11A produced  and  is  an  i n c o m p a t i b i l i t y or  fertiles. of  the  periods  cannot  ranged  that  and  f r e e z i n g weather,  case,  common M e n d e l i a n  male-steriles  set  (34.84$)  Stewart  seed  expected  same  there  and  found  before  partial  sterility  The  the  reason  that  They  our  difference  the  possible  plastic  to  in  discolored  self-pollination  dicolored  time,  However,  the  brown,  s e e d was  collected during so  small,  average.  dicolored  seed.  seed,  is  of  shrivelled, year  seeds were  self-pollination.  The seed  0.009  will  segregation  inherited  and  -53-  and i s not wholly However, b e f o r e  a t t r i b u t a b l e t o environmental m o d i f i c a t i o n .  an e x p l a n a t i o n  of the mode of i n h e r i t a n c e can  be r e s o l v e d on a g e n e t i c a l b a s i s , a more complete count of the  T?l should be obtained,  along w i t h 3? and- 'backeross 2  segregation  data. To determine the u n d e r l y i n g f e a t u r e s of the m a l e - s t e r i l i t y a histological  (and p o s s i b l y a. c y t o l o g i c a l ) examination of the  stamens w i l l have t o be performed. However, from the o b s e r v a t i o n s made, an estimate  of the cause can be made.  a t i o n and o b s e r v a t i o n s estimate  are by no means complete or f i n a l ,  i s drawn from the o b s e r v a t i o n s  "male-sterile"  As the j?x segregthis  on the p a r e n t a l 4  lines.  In the l i n e 20-DRG, no normal p o l l e n was produced, aand the p o l l e n - s a c s appeared f u l l substance.  of a dark, amorphous granular  These g r a n u l a r b o d i e s  c o u l d probably be u n i t s of  t e t r a d s cemented together w i t h d i v i s i o n a r r e s t e d , f o l l o w e d by s h r i v e l l i n g and a r e s u l t a n t i n t e n s i f y i n g of the cytoplasm to give the dark c o l o r a t i o n .  Ho d i f f e r e n c e s were observed i n  the gross morphology o f the m a l e - s t e r i l e and the male- f e r t i l e f l o w e r s ; nor i n the stamens or i n the e x t e r n a l f e a t u r e s of the p o l l e n sacs.  It i s felt  t h a t a h i s t o l o g i c a l study  of the  p o l l e n - s a c s would be w e l l worth w h i l e . In l i n e 142-10B t h e p o l l e n sacs i n most i n s t a n c e s full  appeared  of the same dark amorphous substance as i n the p r e v i o u s l y  d i s c u s s e d l i n e , but at the same time some normal, p o l l e n 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 f a c t t h a t some normal p o l l e n was produced at times,  allows  the t e n e t a t i v e c o n c l u s i o n t h a t e i t h e r the m a l e - s t e r i l i t y i s  - 5 4 -  incomplete very l a t e cells  or t h a t  the degeneration  i n microsporogenesis  are n o t e q u a l l y In the l i n e  few  w i t h misshapen,  a l l pollen-mother  equal  s a c s were o b s e r v e d  of  a single  that  no p o l l e n  examination).  the m a l e - s t e r i l i t y  penetrance.  which appeared by t h i s  basis  factor,  of the m a l e - s t e r i l i t y  early,  or at the onset,  no r e m a i n s  l i n e . However, t h e  selfing, pollen  final  division  pollen out.  grains,  in this  line,  s a c s c o u l d be e x p l a i n e d on t h e  factor  c a u s i n g a breakdown v e r y i n which  case,  observed.  91-10B, 0.8%  and a l t h o u g h t h e p o l l e n  sacs  s e e d was p r o d u c e d appeared  a s m a l l amount o f n o r m a l ,  p o l l e n was p r o d u c e d .  of course,  although present, i s of low  o f t h e p o l l e n w o u l d be  examination,  o f 44 p l a n t s  appear t h a t  of m e i o t i c d i v i s i o n ;  In the f o u r t h l i n e ,  although minute, after the  of meiosis, the t e t r a d r e s o l v e s i t s e l f  I t appears  that  s h a p e , who  i n this  line,  an answer t o t h i s  factor  affected  into  f e e d and grow and r o u n d  the p o l l e n  f o r m e d , b u t d i d n o t grow: i n w h i c h c a s e , the m a l e - s t e r i l i t y  on  empty on a  I n normal microsporogenesis,  of irregular  a  devoid of contents.  at a l l (on t h e b a s i s ,  I t would  The empty p o l l e n  shrivelled  A t t h e same t i m e  s e g r e g a t i o n g i v e s 4 p l a n t s out o f a t o t a l t o produce  amounts o f g o o d  irregular,  of e i t h e r ) .  On s e l f i n g 4.27%' s e e d was p r o d u c e d  observed  i s effected  affected.  (although very l i t t l e  pollen  and t h a t  144-11A a p p r o x i m a t e l y  p o l l e n were p r o d u c e d pollen  of the p o l l e n  g r a i n s were  i t i s possible  the t a p e t a l l a y e r .  can o n l y be o b t a i n e d b y f m r t h e r  that  However,  study.  Seed p r o d u c t i o n o f 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  self-pollination correlated.  and  o p e n - p o l l i n a t i o n was  However,  i t  was  observed  found  that  producing  line  under  s e l f - p o l l i n a t i o n v/as  producing  line  under  open-pollination.  of  the  had.  seed  set,  certain  seed-set ated  floret  seed-set  seed-set  the  l i m i t a t i o n s .  per  comparable  under  i f  per  different  selfed  of  the  an  correlation  floret  plant;  and  whereas,  selfed  had  and  the i t  been  the  seed  highest  of  made  between  total  the  open-pollin-  attempted per  of  pollinations,  would have  seed-set  not  comparison  types was  "be  highest  also  The  A comparison  selfed  the  to  been  between  floret  open-  pollinated. It as  is  f e l t  accomplished  have  very  hybrid the  vigor,  In of  plants  several These  i t  used  provide is  on  the  as  the  results  could  future  the  means  manifested  work  by  key of  to  plants  are  can  U.B.C.  resulting  exception,  is  old  their  s t a r t l i n g .  When  male-sterility,  considered whole  are  from  so  breeding  the  i n  one  can  are  door  some  the  to  of  majority  several  ro/ws  and b e s i d e  them  s e l f - p o l l i n a t i o n .  and  the  cross-pollinated  much l a r g e r ,  selfed  that,  fields  potentialities  open  alfalfa,  there  only,  are  than  be  a l f a l f a  from hybridization.  l-§- y e a r s  contrast  U.B.C.,  unleashing  cross-pollination  of  at of  from  rows  the  effects  such  resulting  vigorous  seed,  two y e a r s  male-sterility,  at  without  i t  past  of  plot  and more  when  isolation  nursery  plants  plants,  the  that  when  the  the  c o u l d be and  heterosis cases,  i n  important  Male-sterility  of  that  more  counterparts  thinks through a l l be  unlimited.  of the  what  spread,  that this  use  of  planted  to  the means,  use  of  hybrid  -56-  However, the  a  production  s t e r i l i t y scale. fully the  can  lot  of  of  testing  hybrid  become  seed  agronomic  desired  strongly i a l i t y  too,  i n  that  l i n e s .  the  have  and  "be  done  "Before  employment on  to  an  be  of  lines  checked  i t  have  male-  economic care-  combining a b i l i t y  Nevertheless,  male-sterile  vigor  to  feasible  w i l l  s u i t a b i l i t y ,  the  a hybrid  or  lines  male-parent  have  through  a r e a l i t y  The s a l e - s t e r i l e for  w i l l  is  with  f e l t ,  great  and  potent-  program.  SUMM/RY A pollen grown most  at of  the the  exhibited  study  common  f a l l  1949,  to  the  of  florets  seed  on  as  plots  were  was  3?i  follows:  from  No  observed. and  2  2  1:7.9,  modes  male-sterility Literature  is  the  clonally  and  the  for  1:7.3, seed of  of  4  the  and  and  very  i n  high  i n  the  transplanted number seed  open-pollinated  lines  produced  male the  f e r t i l e s plants  causes  of  were  grown  lines. the  observed  discussed.  on m a l e - s t e r i l i t y  No r e f e r e n c e s  were  and  found  incompatibility  c i t i n g  no  quantities.  male-sterile  inheritance  that  A counted  self-  male-sterile: 1:8.3  lines  open-pollinated  small  1:6.8,  4  and  male-sterile very  embracing  propagated  greenhouse,  line;  a l f a l f a  produced  i n May 1950.  each  of  tshat  of  and  revealed  lines  were  produced  segregation  Possible  alfalfa.  20  lines  Columbia,  c o r r e l a t i o n between  open-pollinated  reviewed.  and  U.B.C. of  1000  varieties,  lines  at  selfed  selfing,  The  a l f a l f a  These  collected. set  B r i t i s h  overwintered i n  Nursery  seed  of  m a l e - s t e r i l i t y ,  pollen.  was  approximately  University  quality of  of  is  m a l e - s t e r i l i t y  i n  57  LITERATURE  CITED  (1)  A l f a l f a Improvement July 31-August  (2)  Anonymous. 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M a l e - s t e r i l i t i e s and albinos i n cucurbits, Jour. Hered. 36: 47-51. 1945. (84.) S h u l l , G.H. Inherited pollen s t e r i l i t i e s i n Shepherd's Purse. Memoir Hort. Soc. N.Y. 3: 353-368. 1927.. (85) Sinnott, Edmund ¥ . and L.G.Dunn. P r i n c i p l e s of Genetics. 3rd E d . McGraw-Hill Book Company, New York. 1939. (86) Southworth, V . Influences which tend to affect seed production i n a l f a l f a and an attempt to raise a high seed-producing s t r a i n by h y b r i d i z a t i o n . S c i . Agr. 9: 1-29. 1928. (87) Stevenson, T.M. and J . L . B o l t o n . An evaluation of the s e l f - t r i p p i n g character i n breeding for improved seed y i e l d in a l f a l f a . Empire Jour. Exp. Agr. 15: 82-88. 1947. (88) Suneson, C . A . A male-sterile character i n barley. Hered. 31: 213-214. 1940.  Jour.  (89) Tome, Gino A. (Spanish t i t l e ) (The improvement of Rev. Argentina Agron. 14: 279-313. 1947.  alfalfa.)  -63-  (90) Tysdal, H.M. and T.A.Kiesselbach. Alf a l f a Nursery . . technique. Jour, Amer. Soc. Agr on. 31: 83-98.  1939.  (91)  . Hybrid a l f a l f a . Amer. Soc. Agron. 36: 649-667. 1944.  Jour.  (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 B l i s s M. Crandall. The polycross progeny performance as an index of the combining a b i l i t y of a l f a l f a clones. Jour. Amer. Soc. Agron. 40: 293-306. 1948. (94) ^Tysdal, H.M. and J. Russell Garl. A new method of a l f a l f a emasculation. Jour. Amer. Soc. Agron. 32: 405-407. 1940. (95) Tysdal, H.M. and H.L.Westover. Farmers B u l l . 1722. 33 p.  Growing a l f a l f a . 1949.  TJ.S.D.A.  (96) Welch, J . E . and E . L . G r i m b a l l . M a l e - s t e r i l i t y i n the carrot. Science 106: 594. 1947. (97) White, William J. Alfalfa, Improvement. Agronomy 2: 205-240. 1949.  Advances i n  (98) Wilcoxon, Frank. Some rapid approximate s t a t i s t i c a 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 f a l f a 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 A l f a l f a was f i r s t reported i n B.C.  only recently (1932 - Foster  (17)), the disease has "been  recognized i n A u s t r a l i a for 40 to 55 years. However recent i t s dicovery i n t h i s province, the disease i s developing into serious proportions i n parts of the I n t e r i o r .  The ravages of  the disease had assumed enough importance by 1948 that the Department of Agronomy at U . B . C . f e l t  the need for some invest-  igation into the nature and extent of the disease.  Since  discovery i n North America, Witches' Broom of A l f a l f a has  its as  yet received comparatively l i t t l e attention due probably to its  apparent sporadic occurrence.  In Australia,, on the other  hand, the disease has been s u f f i c i e n t l y serious to command continuing f i n a n c i a l support of f a i r l y extensive  investigations.  Investigations performed i n A u s t r a l i a and in the United States has shown the disease to be caused by a virus and to be d i f f i c u l t to transmit mechanically. probably disseminated by insects.  The pathogen  Symptomology i s well desc-  ribed by a number of workers: Edwards (12), Smith (42).  is  Menzies (34),  and  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 i n i t i a t e d at U . B . C . i n 1949,  data  regarding spread, d i s t r i b u t i o n , seriousness are to be acquired along with information regarding any resistance inherent in  65members of the genus Medieago. uction of strains or v a r i e t i e s disease.  The f i n a l goal i s the prodcapable of r e s i s t i n g the  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 l a t i o n of data of other workers relevant to the investigation. LITERATURE REVIEW A. - History and, d i s t r i b u t i o n . Witches' Broom of A l f a l f a was f i r s t recognized as a disease i n A u s t r a l i a . McCleery (36) reported that i n 1924 the disease was prevalent throughout the d r i e r parts of New South Wales.  Noble and associates (39) reported i n 1947 that,  the 417,000 acres under lucerne (alfalfa)  of  i n New South Wales,  235,000 acres occur i n areas where the Witches' Broom disease i s known to be very prevalent.  In these areas, the disease  i s considered to be the main factor l i m i t i n g 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 i s not  produced normally. Edwards (12) reported i n 1936 on investigations  into the  disease which began i n 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 i s grown for gracing purposes.  F i e l d s that were 4 to  5 years old showed 20 to 25% i n f e c t i o n while stands more than 7 to 8 years old commonly showed 70% i n f e c t i o n .  However, he '  reported that the Witches' Broom disease i s not known to occur in crops less than 15 to 18 months o l d .  Edwards (ibid)  also  reported the disease to be present, although less severe than in Hew South Wales, i n Queensland, V i c t o r i a ,  and South  Australia. The f i r s t Horth American record of Witches' Broom of A l f a l f a was made by Haskell (20) the disease as affecting Idaho.  i n 1925 with observations  of  some plants i n Salt Lake County of  In the same year, Richards (41) reported the appear-  ance of the disease i n Utah.  In 1932, Foster  (17)  reported  the disease as occurring i n 2 widely divergent parts of B . C . , but with the cause unknown.  Interest i n the disease i n Horth  America was c r y s t a l l i z e d by the work of Menzies (34) i n Washington published i n 1946.  At that time the disease dist-  r i b u t i o n was l i m i t e d 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 A l f a l f a , and a chronological l i s t i n g of the points out c l e a r l y the trend of the disease.  citations Some references  67<  to  a  (possibly  other  legumes  1922-1931;  related) is  Foster  with  a witches'  on  from  (17)  at  the  Ho  1934:  Anonymous.  other was  with  fields  older  1957:  Jones  Cariboo  laboratory disease  as  affected  from being  Smither found  and M c G i l l i v r a y ' s  fields, Broom.  which The  county  contained  disease  and  Flats  a  was  single  there  were  several also  plants  noted  diseased  i n  plant  Summerland. IT-  20%  county,  (57)  of  the  B.C.  Jones  (38)  affected i n  1940':  Cormack  and  plants  were  the  the  'Dwarf  other  reported  at  irrigated  i n  the  infected,  irrigated  being  worst  i n  parts  a  few  Agassiz  i n t e r i o r  to  be  of  the  plants  Station, of  the  prevalent  i n  the  interior  of  B.C.  of  the  B.C.  Ladak  It  province,  was  variety widely  p r i n c i p a l l y  in  areas.  mention.  Edmonton, many  reported  d i s t r i c t s  Ho  and  the  Cariboo  from  1959:  at  the the  plants  mention.  distributed the  reported  a l f a l f a  fields. Ho  were  to  old  :  places.  Witches'  Cariboo  1956:  1958:  He  2 year sent  At L y t t o n  Anonymous.  section,  were  same  i n  in  recorded  1955:  some  mention.  patches  affected  i n  •  reported  broom  1933:  certain  condition  cited.  Saanichton.  clover  broom  Ho m e n t i o n .  1932:  and  also  witches'  (5)  reported  Alberta,  others  were  were  about  1%  of  moderately  beginning  to  show  the to  plants  severely  the  i n  a  plot  affected  symptoms.  1941; Cormack (4) reported a few plants to be moderately affected i n 3 f i e l d s  at C h e r h i l l , Alt a.  5$ of the plants  were dead or severely infected i n the plot at Edmonton. 1942: Brink (2) reported the disease to be present i n one or two f i e l d s i n the N i c o l a Valley of B r i t i s h Columbia, where alfalfa, stands l a s t for only 3 or 4 years as a result disease.  of the  Brink (ibid) reported the disease to be i d e n t i c a l  with that described i n Washington. 1942; Heald and Menzies (22) report the disease to be present in 4 counties i n the state of Washington, and to be serious i n the Methow v a l l e y area of Ok an agon county.  They stated  that the disease was i d e n t i c a l with that described by Edwards in A u s t r a l i a (12). 1942: Cormack (5) reported the damage from Witches' Broom to be increased i n the plots under observation at Edmonton, Alt a.. 1945: Woolliams (4$) reported the disease as affecting  at  least 5% of the plants i n a f i e l d at Armstrong, B . C . 1943: Cormack (6) reported the disease had advanced somewhat in the f i e l d s  at C h e r h i l l , A l t a . and also i n the plot at  Edmonton, A l t a . 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 f a l f a were severely infected i n a f i e l d at Bremner, A l t a . and i n an old plot at Lacombe, A l t a . 1945: Cormack (8) reported s l i g h t damage i n the U n i v e r s i t y plots at Edmonton, A l t a .  1946:  Cormack  (9)  Saskatchewan, d i s t r i c t  Wright  (10  15%)  -  stands 1947: year and  where  and  1946:  i n  one  of  1947:  (51))  i n  observed  the  occurred  that the  at  Cariboo  15%.  reported examined  a  same  year,  he  reported  a few  clover  were  severely  affected  at  i n  the  was  affected  1948:  Mead  affected 1948:  i n  Munro  resembling 1949: in  2  central  Anonymous.  f i e l d  at  were  eight  affected, of  infected Alberta. red  A l t a. as  being  i n B . C . ,  found  a  trace  of  the  disease  quite  and  Shuswap-,'B , C . i n  the  an  odd  old  f i e l d  Sask.  stands  1949:  an  A l t a-swede  at  and Lebeau  A l t a.  of  f i e l d  (31)  Fox  alfalfa,  northern  a  in  reported central  reported  Witches  Lebeau  i n  were  i n  (35)  old  percentage  and  disease  plant  Thomson  plants  Edmonton,  and L i l l o o e t  1948:  White  proportion  plants  plants  around L y t t o n  Tisdale,  from  stands.  central  prevalent  of  the  year  d i s t r i c t  few  the  (4^) r e p o r t e d  the  seeded  In  east  5-6  A much lower  i n recently  (18)  stands  (33)  i n  time  d i s t r i c t .  i n  20% o f  four  Woolliams  2 fields  f i r s t  a r e l a t i v e l y high  in  1948:  of  that  the  Okanagon.  f i e l d  Gilpatrick  for  Loon Lake  plants  estimated  was  the  reported  a l f a l f a  infection  i n  disease  affecting  reported  Horth  was  the  was  diseased  the  damage  i t  f i e l d  (SO)  Wright old  reported  1  and Peace A few  a few  plant  A l f a l f a  i n  i n f e c t i o n was River  affected  Junction,  plants  were  found  Alberta.  clover  Broom, of  reported  Hudson Bay  a  (43)  showing the  symptoms  Cariboo.  5-tr  3-sl/240  fields  d i s t r i c t . plants  Sask.  were  seen  in  an  old  -70-  1949: in  Wright  a, 1 / 1 0  George,  (51)  a,cre  B.C.;  affected  25%  lupin  of  about  growing  of  25  found  to  were  of  Birdsfoot at  the  indet.),  George.  the  were  affecting  sp.  found  at  plants  rows  be  Broom for  clover  the  foot  (Lupinus  wild  of  plants  2  Prince  Witches'  alsike  15%  the  i n  also  plants  land  plot about  corniculatus) broom was  observed.  Only  affected  Sta,.  least  plants  at  Quesnell  i n  some  also  "Witches'  on  of  It  (Lotus  10%  grow  year  Prince  affected.  station.  at  second  Expt.  Trefoil  which  3-4  the  of  wild  uncultivated  red i n  the  clover  the  Cariboo  d i s t r i c t " . 1949:  Payette  (40)  clover  at  the  f a i l e d  to  flower,  giving  the  observed  1949:  station,  a l l  but  MacLeod  (37)  witches'  B  of  -  Nature  the  Witches' y i e l d  of  source  of  of  plant.  the  loss  Ladino  of  5  red i n  due  the  a to  clover f i e l d  of  reduction  Infected  plants  the  one  plants  trouble  year  was  old  of  of  definite  Alfalfa,. decrease  early  of  on  Broom  N.B.  the  succumb  their  Co.,  symptoms  stands  "myriads" reported  a  showing  the  demands  (ibid)  same  white  developed  but  increased  Edwards  P.Q.,  dwarfing,  the  of  to  York  Witches'  about  production  over  i n  due  the  Ladino  leaves  . The  plants  undoubtably  in  l i t t l e  clover  A l f a l f a •c a u s e s  a result  greatly  of  Pocatiere,  broom.  k i l l i n g , by  plots  county.  losses  as is  of  l a  numerous  witches'  broom  Broom  forage  of  found  resembling  Anne de  instead  stands  i n L ' I s l e t  that  Ste.  appearance  i n  inspected  reported  by  very  weakened on  readily  chief  to  condition stored  food  proliferations.  a y i e l d  experiment  i n  i n  death winter  brought reserves  -711\ Australia  conducted  determine  the  amount  fodder  of  conditions. was of  He  less  a  of  the  effect  produced found  i n progress 37.4$  over  the  period  3 years  "Witches' Broom  "by d i s e a s e d  that  during  diseased  green  of  weight  the  plants  of  disease under  period  the  given  than  the  to  on  plants  have  fodder  (1932-34)  the  f i e l d  experiment  a mean  y i e l d  unaffected  plants. Seed plants. "but  production The m a j o r i t y  occasionally  smaller  and  according  to  Smith  are  flowered  racemes  from  the  grown  total G  -  usually of  diseased  for  may  i n the  t o t a l l y  diseased "be  colour  inhibited plants  produced  than  The  the  which  normal  occasional  i n  either  (ibid)  production  adds the  are  considerably  instead  of  l i t t l e  North that  flower,  that  inflorescence. very  3  to  flowers  normal  flowers  or  diseased  inflorescence  of  report  2  i n  f a i l  groups  (ibid)  Edwards seed  the  (42).  and Edwards  respectively. is  i n  almost  of  "blooms  paler  produced  (ibid)  is  i n  affected  Both seed  is  America or areas plants  are  the  multi-  Menzies produced Australia  where  lucerne  represent  a  loss.  Other  a l f a l f a  Kenneth  viruses  Smith  (42)  and  l i s t s  virus 4  diseases.  viruses  as  affecting  alfalfa,  namely: Weimer  l)  Medicago  virus 1  2)  Medicago  virus 2 Pierce,  -  causing Zaumeyer  Common A l f a l f a a n d Wade  -  Mosaic  causing  A l f a l f a  Mosaic, 3)  Medieago  v i r u s 3 Weimer  4)  Medicago  v i r u s 4 Edwards  causing  -  causing  A l f a l f a  LVarf  Witches'  Disease,  Broom of  Alfalfa.  -721.  Common Alf a l f a Mosaic . Previous to 1931, records- of f i e l d  only were made of this disease: the existance  observations of a transmiss-  able v i r o s i s of the mosaic type affecting a l f a l f a had not been proven experimentally.  Weimer (45) transmitted i t success-  f u l l y i n 1931 with the aphid I l l i n o i a p i s i . It might be noted however that i n 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 i n C a l i f o r n i a found no severe losses from A l f a l f a Mosaic, and stated that the disease damage was l i m i t e d to a very s l i g h t dwarfing of most of the seriously infected plants. The f i r s t evidence of the disease i n a leaf i s the appearance of one or more small, more or l e s s c i r c u l a r , greenish-yellow spots.  These areas frequently consist of a  yellowish band of tissue  to 1 mm. i n width, surrounding an  island of apparently normal color -§- to 2 mm. i n diameter. There may be one or more concentric rings of green surrounding narrow bands of c h l o r o t i c t i s s u e .  The chlorosis spreads u n t i l  the rings are more or less obscured and the leaves sometimes completely c h l o r o t i c .  In some severe cases the leaves are  reduced to 3/3 their normal s i z e , less deformed.  are crinkled and more or  Although there may be some dwarfing of dis-  eased stems i n severe cases, normally there i s no reduction in s i z e .  Necrotic lesions have not been observed on the  stems, and the disease does not cause premature d e f o l i a t i o n .  -73Weimer (46)  attempted several mechanical methods of trans-  mitting the common mosaic disease."but with negative r e s u l t s . He did achieve, as noted e a r l i e r ,  successful  with the pea aphid ( I l l i n o i a p i s i ) .  transmission  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 f a l f a mosaic, frequently recorded from C a l i f ornia, is probably exist ant i n other states..  The disease i s  favored by moderately, cool damp periods i n f a l l  and spring.  Interestingly enough, i n the l a s t report of the A l f a l f a Improvement Conference (l)  the disease i s not recorded.  2. A l f a l f a mosaic. Weimer (46)  considered the virus causing this disease  to be a s t r a i n of Me die ago virus 1,. as the disease that i t causes somewhat ressembles Common Mosaic. agent d i f f e r s virus,  Inasmuch the causal  i n several important properties from the former  this pathogen has been considered .to be a separate  entity and c l a s s i f i e d  accordingly by Smith ( i b i d ) . The virus  is sap-transmis sable, whereas the former was not. I l l i n o i a p i s i , i s common to both viruses. virus caused a mosaic only of a l f a l f a ,  The vector,  Whereas the former  this virus has a wide  host range: Hyacinth bean, adzuki bean, mung bean, r i c e bean, common and Turkestan a l f a l f a ,  white sweet clover,, crimson  clover, red clover, garden pea3, spring vetch and soybeans are a l l infected. Its  affect on a l f a l f a i s more severe than the Medieago  virus 1. i n that the affected plants are decidedly dwarfed  and the l e a v e s are d i s t i n c t l y m o t t l e d  and  crinkled.  Mosaic has "been r e p o r t e d "by MacLeod (37a) of Grimm a l f a l f a at P r e d e r i c t o n , H.B., a l f a l f a near Oromocto, H.B.  Hurst  in 2 fields  and i n a f i e l d  of  (26a) r e p o r t e d i n  1945  that mosaic a f f e c t e d an o c c a s i o n a l p l a n t i n P r i n c e Edward I s l a n d f i e l d s . However, i n the U n i t e d S t a t e s , p r e v i o u s t o 1949,  the disease was In 1949,  r e c o r d e d o n l y from  Wisconsin.  McWhorter (38) r e p o r t e d a n e c r o t i c 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 e a s t e r n Washington and Oregon, and which was u s u a l l y accompanied by y e l l o w bean mosaic. o f a l f a l f a mosaic, but  the thermal  The h o s t range was  typical  i n a c t i v a t i o n p o i n t i s 10°  to 15° lower than those p r e v i o u s l y r e c o r d e d f o r t h a t v i r u s (62-64°C f o r 10  minutes).  3. A l f a l f a Dwarf D i s e a s e . •When and where t h i s disease o r i g i n a t e d i s unknown. However, i n 1919  - 1921  farmers  i n C a l i f o r n i a south o f the  Tehachapi mountains c o u l d m a i n t a i n a l f a l f a f o r 8-10  s a t i s f a c t o r y stands  y e a r s , whereas i n 1931  of  stands were seldom  worth m a i n t a i n i n g over 3 y e a r s . There has been no evidence the d i s e a s e o u t s i d e of southern C a l i f o r n i a .  of  Dwarf d i s e a s e i s  mainly r e s p o n s i b l e 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. i n 1931,  The  symptoms were f i r s t d e s c r i b e d by Weimer  and a s h o r t time l a t e r the same i n v e s t i g a t o r (47)  showed the v i r u s nature The  (44)  o f the d i s e a s e .  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 v e r y w e l l e s t a b l i s h e d i n the r o o t b e f o r e i t i s e v i d e n t i n the top.  The  first  sign i n  75-  the tops i s a shortening of the stems and a s l i g h t reduction in the size of leaves. ited.  Blossoming i s often retarded or inhib-  Progressive reduction i n size of stems and leaves  follows each cutting and i s accompanied l>y a reduction i n the number of "buds developed each time, which results reduction i n the number of stems.  i n a gradual  No chlorosis or other color  change i s evident u n t i l the l a s tflewstems die.  In the f i n a 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 disease, that can he detected, the wood just beneath the bark.  the e a r l i e s t  stage of  i s a s l i g h t yellowing of  This yellowing, a result of  gum formation-in the vessels, spreads u n t i l the entire part of the xylem i s  active  involved.  In 1941, Hewitt and Houston (24),  struck by the geograph-  i c a l association of P i e r c e ' s Disease of Grapevines and A l f a l f a Dwarf i n C a l i f o r n i a , started investigations diseases, the r e s u l t s  into the two  of which were published i n 1946  (25).  They noted that both diseases were spread by an insect vector and that they appeared i n adjacent  areas.  Both diseases appear  more frequently and more severely i n wet portions of f i e l d s or along ditches or ponds where there i s 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 P i e r c e ' s disease of grapevines  also  transmitted the A l f a l f a Dwarf disease; and that these  insects  -76effect  GOUIQ  an intertransmissability of the two diseases.  Root pieces from diseased grapevines were inserted into the • roots of healthy a l f a l f a plants and transmitted the virus i n 12 out of 35 t r i a l s ; hut root pieces from diseased  alfalfa,  s i m i l a r i l y inserted i n the stems of rooted grape cuttings, f a i l e d to transmit the disease due to f a i l u r e of union of the scions and stocks. In 1949, Houston (26) reported the release for commercial production of a dwarf r e s i s t a n t s t r a i n of a l f a l f a developed through selection in C a l i f o r n i a common a l f a l f a .  Observations  in f i e l d s of C a l i f o r n i a common a l f a l f a showed that,  after  p r a c t i c a l l y 100% of the plants were either dead or badly diseased, about 1 plant i n each 2000 square feet was  still  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 v i r u l i f e r o u s 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 C a l i f o r n i a common check plants.  These results  indicated the p o s s i b i l i t y of selecting a Dwarf resistant • s t r a i n from C a l i f o r n i a common, without changing i n any way the other desirable q u a l i t i e s of this v a r i e t y . . With the foregoing determined, 800 plants showing natural f i e l d resistance were selected and planted i n an i s o l a t i o n  -77block.  Here they were allowed to i n t e r p o l l i n a t e naturally  and set seed the following year.  Seed was harvested separately  from each of the remaining plants  (425^(the rest had died i n  transplanting,  showed virus effects,  or f a i l e d to set  seed).  This seed was planted i n 3 row blocks r e p l i c a t e d 3 times in each of 3 counties of Southern C a l i f o r n i a incommercial f i e l d s where dwarf had been very prevalent the past years. The f i n a l r e s u l t s from these plats i n the spring of 1949 showed that the progeny from 60 to 70 of the o r i g i n a l  selections  were quite tolerant to the dwarf virus and held a normal growth producing a good stand. Simultaneously, to further test the Dwarf resistant q u a l i t i e s of the o r i g i n a l selections,  cuttings from 320 of  thebetter plants were rooted i n the greenhouse, inoculated by a vector,  artificially  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 l a t t e r were dead, whereas about 65 of the former showed no effect  of the virus with the exception of root  sympt oms. Based on these r e s u l t s ,  and those of the f i e l d p l o t s ,  the more resistant selections were saved to  cross-pollinate  and set seed, which was released to the growers as C a l i f n r n i a Common '49 l a s t year.  78.  D - Other a l f a l f a diseases resembling Witches' Broom. There are two other diseases of a l f a l f a  that  superficially  resemble Witches'. Broom, and may he confused with this disease. They are the b a c t e r i a l w i l t disease and the dwarf disease, and the distinguishing features (1) B a c t e r i a l w i l t disease.  are: In common with Witches' Broom,  the B a c t e r i a l Wilt disease causes a marked dwarfing of the plant, leaf  owing to shortening of the internodes  size.  and reduction i n  The great p r o l i f e r a t i o n c h a r a c t e r i s t i c  of Witches'  Broom i s not normally a symptom of B a c t e r i a l W i l t , but i t not the distinguishing c r i t e r i o n . characteristic the vascular  is  In B a c t e r i a l Wilt the  yellowish or brownish-yellow discolouration of  cylinder of the tap root serves as a positive  d i s t i n c t i o n since the roots of Witches' Broom are normal i n colour i n the primary stages of disease. (2) Alf alf a Dwarf disease.  In common with Witches' Broom t h i s  disease causes a dwarfing of the a l f a l f a plant both i n reduction in t o t a l size and i n the length of internodes  and size of leaves.  However, i n Dwarf disease the root s are discolored, r e t a i n their normal colour or may be i n t e n s i f i e d ,  leaves  and there  is  a progressive reduction i n the number of shoots produced. In Witches' Broom the rodsts remain normal for some time, leaves are d e f i n i t e l y c h l o r o t i c , and there i s a, progressive* increase in the number of shoots'; produced.  Also of note i s the fact  that i n the former disease the roots are very badly before any symptoms appear above ground.  infected  -79*  VITGHES' Diseased in  the  their the  f i e l d pale  f i e l d  several been  plants by  due  while  is  In  the  or  the  dense  size the  the  the  afar  (hypoplasea)  patchy  number  and  appearance  of  plants.  the  highway  plants  crown of  and upper the buds  fraction  fact,  leaves  size,  that the  i t  \^as  three leaflet  with  i n  that  on  the  of  On '  disease  has  basis  of  is  spinose  of  inch  after  can  there from  a  the  period  observed. typically  crown  (Photo the  as  accom-  l ) .  In  number  severe  root,  with  the  consequent  Internodes  are  greatly  are  shoots  when  (Plate  r o t t i n g  of  the  severely Photo  1,  so.greatly a  towards  or the  to  dwarfed  2).  In  reduced  lOx  differentiated. oblong  of  n o r m a l l y !§•  examination with,  denticulations  the  however,  occasion  narrow,  is  plant  plants  long.  c o u l d be  rather  symptoms  to  the  an  leaflets  be  due  diseased  only  of  whole  of  However, over  thw  i n  on  plant.  slowly modified  portion.  observed  Witches' Broom  produced  on h e a l t h y  of  the  disease,  portions  while  were  of  the  of  infection,  shoots  reduced  (Internodes  are  a  of  is  of  dwarfing  greatly  long,  but  of  of  range  stage  stages  2-g- i n c h e s  a l f a l f a  a wide  severe  may be  symptom  infected  advanced  a  shortened.  late  i n  along  dwarfing  years,  advanced  shoots  that  and  i d e n t i f i e d from  in.Washington,  marked  p r o l i f e r a t i o n  panied by  death  and  driving  and  reduced  reduction  characteristic  of  months  the  observed  greatly  i n B.C.  SYMPTOMOLOGf.  symptoms.  appearance  very  be  coloration,  the  occasions  A l f a l f a  a  their  to  The most  of  can  yellowish  spotted  these  B R O O M OF A L F A L F A  i n  hand-lens  The  normal  elliptic-oblanceoapex.  Diseased  -80leaflets  are smaller,  ulations  and are frequently wrinkled or puckered.  The f i r s t  rounder, sometimes lack apical dentic(Plate 1 ) .  symptom of the disease to he seen i s a s l i g h t  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, "foe general a l l over the plant.  and the symptoms seem to  At t h i s time, there may he  l i t t l e or no dwarfing of the plants, hut the diseased plants can he recognized e a s i l y "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 l e a f l e t s much reduced in s i z e . P r o l i f e r a t i o n - and stunting is progressive with each successive cutting.  Both Edwards (ibid)  and Smith (ibid) report a colour  change i n infected plants when p r o l i f e r a t i o n has occurred: the foliage i s often very dark i n colour and may have a purplish tinge.  Menzies ( i b i d ) ,  on-the other hand, does not mention  this phenomenon as occurring i n Washington.  Nevertheless,  has been observed,  that the foliage  i n a few cases at U . B . C ,  it  becomes very dark, hardly p u r p l i s h , before the leaves become chlorotic,  and before crown p r o l i f e r a t i o n is pronounced. This  darkening of the foliage has occurred i n conjunction with a rosetting or c l u s t e r i n g 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 i n e ,  densely  Severely dwarfed Witches' Broom diseased plant on l e f t ; normal a l f a l f a plant on r i g h t .  -81-  matted stems.  This figure i s i n agreement with some of the  plants observed, but, i n most of the plants observed,  the  r o t t i n g of the crown had occurred to such an extent, by this p e r i o d , i n the progress of the disease that the number of shoots i s much reduced from t h i s number (3000). Immediately prece/ding death of the plant the foliage wilts severely and becomes prostrate. ination of cross-sections  At t h i s time an exam-  of these crowns has shown almost  complete disintegration of c e l l u l a r structure;  also blocking  of the vascular system has cut off the normal water and nutr i e n t supply to the f o l i a g e ,  a feature which r e s u l t s  i n wilt  and eventual death. Menzies (ibid) made no observations on the r o t t i n g of the crown, although i t was observed to precede the death of the plant i n a l l cases at U . B . C .  Edwards (ibid)  stated that i n the l a t e r stages of the disea.se, the crown and upper root tissues are often severely rotted and the plants gradually die.  He made i s o l a t i o n s from the decaying tissues  '•which y i e l d e d an u n i d e n t i f i e d s t e r i l e white fungus, Rhizoctonia bataticola,  and various types of u n i d e n t i f i e d Eusaria. The  most apparent fungus observed at U . B . C . was an unidentified Basidiomycetes. Edwards (ibid) observed p a r t i a l and complete recovery of diseased plants when transplanted to the greenhouse and maintained under conditions favourable for growth.  In many instances,  the recovery was only temporary v/ith renewal of symptoms at a l a t e r date, but i n some cases he reported complete recovery. Menzies (ibid) did not observe this phenomenon i n Washington. However, two'plants i n the greenhouse at U . B . C . ,  that exhibited  A l f a l f a plant showing apparent recovery from 'witches1 Broom disea.se.  witches' Broom diseased a l f a l f a plant showing p r o l i f e r a t i o n and dwarfing. N i c o l a Valley, B.C.  82-  positive disease symptoms i n the f a l l of 1950 (they were clones of diseased plants obtained i n June 1950 from the N i c o l a Valley) had shown apparent complete recovery from the diseased  condition i n A p r i l , 1951.  of these recovered plants. noted on Apr i l 11, 1951,  Photograph 6 shows one  Several diseased plants were to be producing normal shoots mixed  among the diseased stems, while the f a l l befox-e, shoots were produced.  only diseased  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 produced normal growth at the periphery, with reduced or dwarfed foliage i n the centre of the plant. Hot only was crown p r o l i f e r a t i o n observed, but also an abnormally large number of f i n e , 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 f a i l e d to flower,  a rather  s t r i k i n g symptom when seen during the normal flowering time in the f i e l d .  The few flowers formed on diseased plants  appear smaller i n s i z e ,  and paler i n colour.  l a t t e r point i s hard to determine, (M. media)  However,,the  as normal a l f a l f a  flowers  show a tremendous colour d i v e r s i t y , ranging from  pale blue-white, l i g h t 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 f a l f a shoot on l e f t , and v/itches' Broom diseased shoot on the r i g h t . Note c h l o r o s i s , shortened i n t e r nodes, reduced l e a f l e t size and rounder shape, and small pale inflorescence.  Normal a l f a l f a inflorescence on l e f t ; the two on the r i g h t are Witches' Broom diseased a l f a l f a inflorescences. Note reduced number of f l o r e t s and paler c o l o r .  -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 s i n g l y , or in groups of 2 to 6 with an average of 2.7 (average of 20 clusters examined at random); t h i s contrasts  sharply with the multi-flowered  raceme hearing approximately 15 flowers?  produced by healthy  plants. As i s to be expected i n l i g h t of the reduced number of flowers, very l i t t l e seed i s produced.  The small amount of  seed that was c o l l e c t e d seemed normal (perhaps a l i t t l e  small)  and germinated w e l l . INYE S TIG AT I ON AL WORK I - TRANSMISSION EXPERIMENTS A. Grafting studies (a)  L i t e r a t u r e review. Edwards (11, 12, 13), Heald and Wellman  (23),  and Menzies (34) carried out extensive graft  transmission tests  in investigating Witches' Broom of A l f a l f a .  Edwards (ibid)  achieved the f i r s t  success i n obtaining positive  transmission  of the disease by grafting. his Edwards (ibid) used, i n 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 i n an attempted c l e f t or wedge graft  into the  crown and"into the upper portion £ f the tap r o o t . He encountered  -84considerable d i f f i c u l t y i n establishing a, successful whenever a diseased plant was used as a stock.  graft  The grafts  were performed i n 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 t h i s 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 l a t t e r  11 unions, only 2 persisted after transplanting to the f i e l d and positive transmission of the disease occurred i n both instances.  Out of the former 57 unions, only 28 scions per-  sisted for 2 weeks after  transfer  to the f i e l d s ,  and a month  l a t e r 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 t h i s 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 In a l l , Edwards (ibid)  transferring  to the f i e l d .  achieved transmission i n 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 i n a l i m i t e d number of cases. Menzies (ibid)  achieved very l i t t l e success i n 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,  -85in a glassed-in humidity chamber for 10 days after  grafting.-  He reports 75$ successful grafts using t h i s method, with disease transference occurring i n 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 i n 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 A l f a 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 c u l t i e s arose from the fact  several of the legumes were annuals,  that  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 l u p u l i n a L . ) , andto Caligornaa BurClover (Medicago h i s p i d a Gaert.). (b) Experiments. !•  Several diseased plants were dug i n the Nicola,  valley of B r i t i s h Columbia, brought to the University and planted i n a greenhouse deep bench i n June 1950.  142 c l e f t  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. with r a f f i a , t h i c k l y coated v/ith p a r a f f i n ,  Grafts were bound and planted i n a  -86-  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 t h i s might give.  Although i n 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 o r i g i n .  The plants were dug up for observation on January  10, 1951, at which time there were 124 plants remaining, a l i v e . Whether the 18 plants that died, did so as a r e s u l t of the disease or from c u l t u r a l methods was not determined. ( A l l grafted plants were transplanted to pots i n November 1950.) The r e s u l t s obtained are given below; Plants healthy on January 10, 1951. Union of sciog. and stock, but scion dead. No union, scion never established or disappeared No union, both scion and stock l i v i n g separately  7 79 4  Plants diseased on January 10, 1951. Union, but scion dead Union, scion l i v i n g i n firm union with stock Union, scion l i v i n g i n very feeble, i f any, union No union, no evidence of establishment of scion  5 14 2 3  •Plants doubtful or suspicious on Jan. 10, 1951.  10 124  The 10 plants belonging to the l a t t e r category were replanted i n the greenhouse.  On the 5th of February, 1951,?3 plants  showed positive symptoms of the disease, and an additional one was suspicious i n showing a large number of small, clustered leaves;  and when observed on the 16th of February  this plant was showing p r o l i f e r a t i o n and marginal chlorosis -  -87typioal disease symptoms.  F i n a l examination of these plants  was carried out A p r i l 16 1951, when besides the 4 diseased P I A ^ - V  Mo- *S  plants, 2 appeared suspicious. One piant—(55) c l u s t e r i n g effect  showed a  of the leaves around the stem and a much  darkened color; and the otiie^—-pi-ant—(-39-) showed the c l u s t e r i n g effect  and had a c h l o r o t i c tinge to the whole plant.  Adding in the above plants to the above t o t a l s r e s u l t s 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 Union, scion l i v i n g i n firm union with stock  6 14  Union, scion l i v i n g i n 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 i n  union and both l i v i n g at time of digging, showed a disease transference. ^JOs).  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 i n removing scions every five  "  d a y s , the union must have l a s t e d l e s s than 2 weeks before t h e scion died.  In 3 cases, scion or remains of any part of the  scion couH. n o t be found although there was a positive disease transference.  In these 3 cases i t i s probable that a union d i d  exist for a period long enough for a virus transfer before a r o t t i n g of the scionoccurred.  Evidence that a union must exist  i s shown by the f i v e 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 i m i t of  time required for the appearance of symptoms - February 16th or 8 months.If the 2 doubtful or suspicious plants subsequently develop d i s e a s e symptoms, the period of incubation w i l l of course be again lengthened. 2. In May 1950, 1 2 stem c l e f t 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 wi^-h r a f f i a and kept  moistened by a string-wick  extending from a jar of water,  However, union was not observed, and t h e scions died i n about 2 weeks time.  Menzies' modifL cation of using a humidity  chamber was n o t attempted. 3. At the same time, another method of transferring the disease was attempted. sections  This consisted of cutting longitudinal  (approximately 2 inches long) out of the healthy and  diseased shoots growing i n adjoining pots, the freshly cut areas together with r a f f i a .  and firmly IpindingHowever, both the  diseased and healthy shoots, above the cut, d i e d i n a l l cases i n about 10 days time.  Mo disease transference was observed  in  any  case  during  tlae  summer,  f a l l  and winter'  i n the  greenhouse. B.  E e c h a n 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 Edwards expressed  and u n d i l u t e d  severalmethods methods, 1.  (ibid),  employing  sap of d i s e a s e d  of m e c h a n i c a l l y  t h a t he  employed,  Individual leaves  mixture  and c r u s h e d  drop  Stems were  of inoculum 3.  Stems  needle  scratched  lightly  inoculum  infected  pricked witha allowed  The  with  a  small a  tissue. needle  and  a  t h e v/ound.  longitudinally, and  plants.  as f o l l o w s .  hypodermic  into  freshly  attempted  and c o n t a i n i n g  leaf  fine  to flow  i n several places,  the  inoculating healthy  were b r u s h e d  saturated with  2.  foliage,  are o u t l i n e d b r i e f l y  cheesecloth bag of sand  as i n o c u l u m  inoculum  with  a fine  allowed  hypodermic  to flow  into  the  wound. 4. pins  Multiple p i n inoculations (using  bound  growing  shoots,  5.  vascular  leaves  entomological  of v i g o r o u s l y  stems.  woolplugs just  soaked below  i n inoculum  the crown  were  i n contact  with  tissue.  inoculated plants  2-|- t o 3-g- y e a r s , Menzies  diseased  terminal  and  i n the t a p r o o t  The from  into  leaves  Smallcotton  inserted the  together)  2o-3o  with  (ibid)  tissue  on  powder  on l e a v e s  secure  disease  observed  symptoms  i n o c u l a t e d 250  a swab  t o be  no  were  transmission  i n any  developing plants  as i n o c u l u m  inoculated.  f o r periods  using  and d u s t i n g  However, case.  i n any  ranging case.  macerated carborundum  Menzies  failed  to  -90-  (b) 1.  Crovms  ground of  of Witches*  v/ith meat  cheesecloth.  into  crowns  February cc. s i z e same  grinder,  o f 3© h e a l t h y  a  v/as w r a p p e d  o l dRhizoma  juice  fitted  with  inoculated  16 n e e d l e .  roots  A l l plants  were  and  on  and Burpe  from which  the broken  23 c o n t r o l s  thicknesses  seedlings  Fisher  a B-D  v/ith  crown m a t e r i a l  around  was  finelXy  v/as  8  o l d Rhizoma  "London" L u e r  seedlings.  together  alfalfa  through  excised  15-monilb  syringe  the macerated  greenhouse,  1951.  t h e j u i c e was  o f 12 h e a l t h y potted  observed  and  condition  rather intense  color  and  In the  from  time  field 2.  vigorously.  i n May  were  containing soil.  the other These  for further  On F e b r u a r y  seedlings  crovms  whereas,  plnted  in  observations  i n close made  One of  of  were  transplanted  to the  observations.  a  14-inch  were  contact  April  this  no f l o w e r s  i n the series  v / i l l be  size  f o l i a g e of diseased  The s e e d l i n g s  deep  However,  produced  plants  plants  28th,  the i n t e n s i f i c a t i o n  1 4 t i , 1951, 8 h e a l t h y  shredded  were  and  April  of the c o n t r o l p l a n t s .  showed  habit,  6n  shov/ed v e r y  proliferation.  series  proliferation  w e r e made  series  o n one  v/as o b s e r v e d  of the macerated  buds;  the plants  of t h e i n o c u l a t e d  plant  flowering  on  observations  coloration  flower  15  . ,  2 plants  same  5§  At the  time. Final  the  and s t r a i n e d  The r e s u l t a n t  hypodermic  time,  month  Broom d i s e a s e d  16th, 1951,using  extracted  to  Experiments  28th,  planted  6 montt*- o l d  Grimm  earthen-ware pot plants  v/elljaiixed  so that  the roots  t o the diseased  material.  1 9 5 1 shov/ed  a l l plants  v/ith and Final  t o be  -91-  normal. G  -  Seed  Transmission  Edwards with of  a  (ibid)  limited  the  young  number  plants  of  normally  e v i d e n c e , was with  considerably  obtained  to  several  symptoms  of  the  Menzies infected Broom  f a l l ,  marked  reduced,  However,  plants  to  Final  these  the  that  f i e l d , any  of  observations,  plants,  but  clustered  which  was  flowered definite  them were  plants  foliage  were  and no  taken  no  Several  infected  2 years showed  later, positive  disease.  (ibid)  plants,  infection  f i r s t  experiment  plants.  effect,  show  suspicious  the  Alfalfa.  a rosette-like  transplanting  Witches' Broom.  showed  infected  leaves,  infection.  after  from  of  transmission,  by  producing  of  seed  Broom  showed,  along  suggestive  a  seed  The  stems  Witches'  conducted  abnormalities. the  of  grew  but i n  488  f a i l e d  plants to  a year's  from  secure  seed  any  collected  evidence  of  from  Witches'  time.  Experiment Seed the  Basque  hand, to  was  was  the  as  no  162  40  and to  seed  was  the a  from diseased i n  August  germinated  to  plants  seedlings  d i s t r i c t s  B.C.  scarified,  December,  weak  ranch,  greenhouse  normal.  flats,  collected  flats were  i n  f l a t . were  (Savona,  i n  observed  pitre  than at  Ranch)  of  Savona  seed,  and  td>  numbers  pots  from which  the  was  i n  to  of  the  by  transplanted  late  other  small,  transplanting; i n  at  threshed  transplanted  plants  and  Germination  from flats  normal  time  at  plates,  1950.  were  from healthy  Basque  This  October,  plants  Larger  collected  1950.  transferred  remaining  plants  however,  same  seed  from  -92-  diseased p3a rits was gathered made.  a count of these plants v/as not  Observations made on A p r 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 r o s e t t i n g of nodal f o l i a g e . D. Plant m u l t i p l i c a t i o n through cuttings &ne  of the d i f f i c u l t , time consuming, and expensive  procedures i n the study of this virus disease w i l l be the securing of diseased plants i n s u f f i c i e n t numbers for uae i n the -Warious required r e p l i c a t e d f i e l d tests.  The diseased  plants wil^have to be laboriously dug out of normally tough sod i n f i e l d s i n the I n t e r i o r , and then c a r r i e d without delayto 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  i n the past.  Accordingly, on the 2nd of October, 1950, 3 sets of 25 stem cuttings of diseased a l f a l f a 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, c o n t r o l , dipped Into distilJe d v/ater.  as  The cuttings were  planted In clean sand i n glazed pot? kept watered, and l e f t untilDecember 4th when they v/ere dug up, observed for root formation, ani planted i n f l a t s .  7 of the cuttings  treated  with IB A none of the cuttings treated v/ith 1TAA, and 8 of the cuttings treated v/ith d i s t i l l e d v/ater were alive and showed root formation.  The cuttings' treated with IBA, although less  i,  -93-  numerous thai the controls, shwed considerably greater root formation both i n 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 ( A p r i l 28th, 1951),  a l l showing advanced  symptoms of the disease. The experiment did point out, besides being able to get the p r o l i f e r a t i o n effect  i n the absence of a crown, t h a t i t  possible to root cuttings of Witches' Broom diseased  is  alfalfa  and thus increase the number of diseased pianos for use in f i e l d experiments.  If a diseased plant produces 500 stems,  t h i s 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 t h i s melbhod should prove to be very usefulto the agronomist and plant pathologist i n m u l t i p l i c a t i o n 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 r e a d i l y for use i n resistance  and breeding studies.  E . Insect transmission studies Edwards (ibid) attempted severalexperiments i n an attempt t.o transmit the Witches.' Broom of A l f a l f a using insects as agents of inoculation (or vectors). A b r i e f resume of his work is given below. 1. 2 to 4 Jassids, f i e l d c o l l e c t e d , were allowed to feed  -'394on 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 c o l l e c t e d , 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 i n this manner, lib  transmission was observed i n 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 c o l l e c t e d on diseased plants i n the f i e l d were enclosed i n 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 l i b e r a t e d 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 c o l l e c t e d i n highly diseased f i e l d s were added p e r i o d i c a l l y .  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 i n the f i r s t 2 cages, that showed definite symptoms of Witches' Broom disease.  However none of the other  95-  plants  i n  angr  period  of  3 years,  Menzies of  insects,  feeding groups  of  caged  and  v/as  diseased plants ered  In  plants  was  12  another  containing  one  collections cages  time  retained  plants  i n  the  surviving test  5 months  Broom,  as  v/hereas  insects  and  for  none  of  after  used  added of  and b y  definitely  the  check  of  of  of  7  acutus  days to  f i r s t  on  healthy consid-  inoculation.  to  large  cages  plants.  F i e l d  some  the  months,  symptoms  inoculation,  species  transferred  couple  F i r s t  were  feed  a  short  Platymoideus  6 healthy  v/ere  a  course  one . c a s e  (ibid) to  for  species  into  symptoms.were  4  a  for  the  acquisition  acutus  plants  fin  leafhopper  2 months  checks.  after  segregated  insects  Menzies  time  plants  plants,  Broom  plant  to  a l f a l f a  were  then  experiment,  were  Witches'  suspected  approximately  cages  29  of  an  Witches'  Platymoideus  from  collections  the  allowed,  infected  of  infection.  sucking  case,  carefully  of  they  where  observed  evidence  healthy  v/ith  this  days. i n  any  infected  to  occurred  definite  test  on  which  tests  used.  for  In  them  after  hundred  although  made mass  transferred  transmission Say.  plots,  developed  (ibid)  period,  several  the  of  while  other  appeared  on  2  8-g- m o n t h s ,  1,8  of  affected  plants  with  became  infected. ( i M d ) However,  Menzies  acutus  i n  2  valley  of  Washington  rapidly. although the  He  sweeps  made at  concluded  a vector,  disease.  could not  is  i n the from  June  find  this  and  August  time  the  this,  probably  not  leafhopper of  disease  that the  1944 v/as  i n  the  Methew  spreading  PIatyrnoideus principal  x-latymoideus  acutus,  vector  of  It  was  necessity, somewhat  outside  "  YIELD  i n  after  plants the  (ibid)  f i e l d ,  the  Department  to  be  by  pathologists, of  Broom r e s i s t a n t attempted  is  Agronomy. varieties  without  of  valuable  vectors.  d i d not  over  a  the  account  higher  of  Witches'  was  cut  to  establish  healthy  the  three less hardly  y i e l d  mortality  the  recorded  of  is  plants,  foliage  37.4%  (This  1/7  35%  weights  of  an  to  The  period  y i e l d  enclosed  whenever  stage.  plants. into  25  cut  the  and  30%  plot  and  were  and  a mean  the  Menzies  selected  whole  f lower ing  take  from  reduction  The  cutting  gave  lessening  Broom  plant  regarding  diseased  Averaged  not  result  but  25  unaffected  does  have  containing from  the  the  ounce.  and  Witches'  conducted  plants.  conditions.  ely  of  of  involving  TRIAL  f i e l d  reached  province  a vector,  Literature  diseased  random  for  entomologists  the  Edwards a l f a l f a  search  regrettably,  being  (a)  a  i n breeding  may,  research II  that  trained  Some w o r k a l f a l f a  felt  of  uniform  selected  was to  the  green  immediat-  nearest  the  fodder  figure,  diseased  -g-  diseased,  weight  true  at  plants  weighed  reduction which the  Broom  unaffected  years,  a  acre  as  than i t  would  plants  thus  stand.) (lipid)  report  reported on  i n yields  (b)  Experiment.  Ten  comparable  diseased  any  due  to  sized  plants  a  serious  tests the  to  determine  the  i n  yields  amount  of  disease.  clonal  secured  reduction  cuttings  from  the  of  each  Interior,  of and  Witches' Grimm  TABLE Y I I -  YIELD TEST  WEIGHT IN GRAMS OF AIR-DRY HEALTHY AND WITCHES• BROOM DISEASED ALFALFA FODDER. A - WITCHES' BROOM DISEASED PLANTS PI. No.  1st  cutting  10.68 5.51 4.53 7.66 3.81 3.12 4.18 3.38 3.31 46.18  1 2  3  4  3 6 7  8 9  10  Totals  4.62 gms.  Averages  2nd c u t t i n g  1.91 0.89 0-|88 0.07  3.41  0.68  0.92  2.16 0.30 0.17 11.39  3rd  cutting  3.31 2.13  1  2.44  0.57 4.78 0.89 2.78 1.72 1.13 1.01 20.76 2.08 gms  1.14 gms.  B - HEALTHY PLANTS PI. No. 11  12 13 14  15 16 17 18 19 20 Totals Averages  1st  cutting 6.42  8.55 4.83 6.36  3.42  6.52 5.49 3.97 4.70 4.79  2nd c u t t i n g  3.78  2.04 1.29  3.63 1.02 2.80 1.85  1.72 2.92  2.35  3rd  cutting  4.77 3.35 2.95 4.20 1.59 4.65 2.88 2.06  3.42  2.68  55.05 g.  23.40 g.  32.55 g.  5.51 g.  2.34- g.  3.26 g.  -98-  Table 7 I I I -  C o r r e l a t i o n o f a i r - d r y weights o f fodder of  h e a l t h y and d i s e a s e d a l f a l f a on f i r s t Witches' Broom p l a n t s  Ix x n (fx) £x S.S. S.D.  2  2  X  X  S.B.z  s 46.18 : 4 . 6 2 g. = 10 = 2152.5924 , 287.7224 74.4632 t 2i-874 -  *  .9089  cutting.  Healthy plants £ y - 55.05 y 5.51 g. n - 10 (^y) -3030.5025 *y 323.2593 S.S.y 20.2090 ' *y t 1.4916 2  2  s  S  1  D  ' «y E  "  i .4-717  S.E. of d i f f . between the 2 m e a n e s t 1.024 g. D i f f . between the 2 means = 0.89 g t -  * = .86914 1.024 Tabled t at p • . 0 5 i s 2.101 at 18 D.F. Tabled t g r e a t e r than o a l e . t - so no s i g n i f i c a n t d i f f e r e n c e between the fodder produced by Witches' Broom and H e a l t h y A l f a l f a p l a n t s at beginning of the experiment. 8 9  Table IX  - C o r r e l a t i o n o f a i r - d r y weights of fodder of h e a l t h y and d i s e a s e d a l f a l f a p l a n t s on second  Witches' Broom p l a n t s  tx  Healthy p l a n t s  z 11.39 £y x 1.14 y n = 10 n -S.D.x = 1 . 0 5 2 S.D.y S.E. .3327 S.E.y S.E. of d i f f . between the 2 means= ,445T D i f f . between the 2 means= 1.20 g t ° . = 2.69 Tabled t = 2.101 .445 X  T  1  ,  2  cutting.  23.40 2.34 10 = .9333 - .2952  at 18 D.F.  C a l c . t i s g r e a t e r than t a b l e d t - t h e r e f o r e t h e r e i a a s i g n i f i c a n t d i f f e r e n c e i n y i e l d between the fodder produced by Witches* Broom and Healthy a l f a l f a p l a n t s 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 P l a n t s £x  ,  i  s  n  =  S.D. S.E. S.E.  X  X  -  Healthy p l a n t s  20.76  i y  2.08  y  10  ^  S.D,  .4124  3.26  -  n  1.304  32.55  = y  S.E.y  10 = =  1.052 .3327  d i f f e r e n c e between t h e 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  C a l c . t i s g r e a t e r than t a b l e d t . Therefore t h e r e i s a s i g n i f i c a n t dry weights third  d i f f e r e n c e between t h e a i r -  o f h e a l t h y and diseased p l a n t s ' fodder at the  cutting.  -100-  a l f a l f a  obtained  agated Sept. ized  i n 10 10,  to  pairs  1950.  avoid  reached  from of  position  Tops  allowed  to  were  return  rough  balance.  and. a  third  weighings It  are  was  were  February dried for  to  on  given  found  April  i n .Table  diseased  plants  the  ning  of  the  cuttings  the  2nd  but  48.8%  3rd  cutting, of  decreases  the  a  the  greenhouse  healthy a l l  an  oven  at  were  w e i g h i n g on  results  had har-  100°C,  time  on  random-  plants  giants  second The  prop-  periodically  and a  on M a r c h of  16,  these  V I I . f i r s t  the  y i e l d  healthy were  cutting of  plants.  comparable  there  the  (Table i n  was  no  Witches'  at  sig-  Broom  VIII).  size  that  Witches' Broom diseased  plants  upon  analyzed  cutting,  was  63.4%  of  a l f a l f a  s t a t i s t i c a l l y on  i n  ' were  This  the  begin-  experiment.  The y i e l d healthy  and  1951,  hour  i n  the  1951.  on t h e  between  the  1  16,  difference  that  16,  U.B.C.  pots  When  harvested  nificant  shows  at  air-dry-weight before  that  and  plot  watered*  effect.  They were  time  nursery  8-inch glazed  Plants  l / l 2 bloom,  vested.  the  of the  i n  the  that  2nd  Tables  fodder  produced were  IX  the  found  and X .  the  healthy to  the  by  healthy  produced by by  and  produced  produced by  fodder  i n y i e l d  the  be  plants  3rd It the  cutting was  diseased  diseased  On plants  Both  s t a t i s t i c a l l y  the are  found  plants.  plants.  and  of  that plants  the was these  significant.  -101-  Itt  -  EIELD  A.  STUDIES  A true can  given  environment. has a  many  very  such  the  injury,  an  f i e l d ) ,  due  experiment  conducted  under  t r i a l ,  previously  on  use plant  dies  and  account.  In  taken  most of  into  valuable  tools  of  a-disease,  o r . of  any  We h a d the 1944  available  Interior by  Dr.  of  B.C.  the  of  Feeling  the  thesis.  data  2 years  elapsed, between  quadrats)  additional  quadrats  were  observable.  and  accurate account)  measured  area  followed.  It  a  plant  of is  becomes  factors  can  the  progress  1942  and  this  range  plotted "in  l a i d  reading the  f a l l  the  i n  i n  data of  of  pasture.  observed  quadrat  and  or  quadrats  inadequacy  laying  winter  studying  covering  results  maintained  one  sheets  The  a  are  quadrat  spring  as  reports  affecting  the  and  i n  not  Management  i n  with  into  when  factor  i n this  are  (a  hitory  quadrat  of  plants  are  taken  observe  agronomist  reproduced (over  its  reported,  scientist  a reliable  quadrats  plant  overgrazing*  plants  disappears. short,  of  factors  to  disease  normal  the  plants  other  i n  V.C.Brink.  study,  a  Nevertheless,  effects  ( a l l  to  diseased  obtaining  and  when  be  of  recorded  sick,  and  weakened  In  quadrat  i t  The  quadrats.  through  afford  and h e a l t h y  i n y i e l d  is  as  much m e r i t .  method  of  does  healthy  diseasd  possible, and  the  diseased  decline  every  of  i t  conditions.  drought  the  y i e l d  contains  growth  the  through  i n yields  However,  only factual  on  ALFALFA.  decline  A potted-plant  experiment  and  The  is  the  comparison  ideal  report  by  prodedure  greenhouse under  of  limitations.  good  OF  Introduction picture  only he  DISEASE  s t u d i e3  Q,u a d r a t 1.  BROOM  PIT T H E W I T C H E S '  are  this the of  1950.  -102-  2. Dr. many  Study  V.C.Brink  fields  of  grasses  of  a nev; d i s e a s e  disease,  Nicola  a  the  quadrats,  f i n a l  i n the  and  A l f a l f a  were  following i n  These  of year  the  Clark  B.C.  a  due  to  order  N i c o l a i n  i n September  7  was  no  plants  to  study  square  Stock  Farms,  June,1942  and  1944.  -  1942  Plants  healthy  -  1944  33  Plants  diseased  "  27  Plants  dead  plants  diseased  Plants  dead  or  -  140  disappeared  or  1944  80  1942  93  disappeared  i n 1944  charted  i n  remaining symptoms  by of  contributed  140  to  the  may h a v e  succumbed  contributed to i n  summer,  of  and  the  of  had  plants  Witches'  Broom, but  dry  and  of  any  a l l  healthy  1944, the  of  1942:  Witches'  have  feature  1944  the  93  60  to  this  their  or  strongly  the  loss  overgrazing  were i n  27  there  Broom  only  diseased  that  management  were  advanced may  plants  factors  winter.  60  factors  healthy  Other  the  that  showed  Other  suggested  disease.  233  disappeared.  plants  of  is  i n 1942,  Broom disea.se.  is  data  93 W i t c h e s '  plotted  disappearance i t  above  died  these  the  meter  healthy  significant  the  ravages  plants  vestige  Of  replanted  a l f a l f a  233 The  great  the  dwarfing of  In  plotted  that  and b e i n g  plotted  f i e l d ,  (V.C . B r i n k ) .  i n 1941  serious  Brink  were  taken  of  stands.  Dr.  quadrats were  a  the  1942-1944  ploughed up  valley  produced  decimation  from  noticed  being  Nicola  which  observations A l f a l f a  data  (unpublished.)  r i c h  largely  B.C.  quadrat  a l f a l f a  to  plants  of  many  besides of  which  factors,  have  them may  too  -103-  3. - To A l f a l f a fields  study  disease,  Dr.  i n  v i c i n i t y  the  near  1950;  and  the  and  of  frame  of  l a i d  6  at,  or  near  Wallachin,  five  without  the  l a i d  random  long, into  -  ground  he  i n  were  metal  at  four  so  that  future  records  c o u l d be  quadrat  data  sheets  for  6  Basque  July  18,  11,  1950.  i n  the  carried  l i f e  square  the  the  plotted  plant  of  a l f a l f a  on  choice  the  Broom  three  September  frames  andt  one-inch  the  was  Interior.  following  and. one  particular  meter-quadrat  at  Foot  were  i n  the  Bridge,  quadrat  the  Witches'  near,  one  f i r s t  i n  quadrats  Spence's  The  1950  the  near  l a i d  sight  B.C.  i n  one  thrown  f e l l .  out  quadrat  were  progress  of  remaining  position:  i t  driven  recorded  pins  were  corners  made  into  of  of  the  the  area, The  osed  the  i n i t i a t e d  V.G.Brink  Ashcroft.  f i e l d  where  same  Savona,,  quadrats to  the  i n t e r i o r  points:  of  Ranch  f i e l d  studies  further  geographic  The  Quadrat  as  Appendix I  of  this  the  essay,  are  quadrats b r i e f l y  plotted,  summarized  enclas  follows. 1.  July  2.  Sept.  11,  18,  1950.  1950.  Spence's  Savona?  Bridge,  B.C.  B.C.  19 0  healthy Witches'  8 3  healthy diseased  3.  II  II  11 10  healthy diseased  4.  •II  it  7 4  he e l t h y diseased  5.  II  Wallachin,  7 5  healthy diseased  6.  II  Basque Ranch,  B.C.  Ashcroft  19 9  a l f a l f a Broom  healthy diseased.  -104-  B - DISTRIBUTION OF ALFALFA WITCHES' BROOM IN During  the summer of 1949  and 1950,  B.C.  a l f a l f a f i e l d s were  examined at many p o i n t s throughout the province of B r i t i s h Columbia (and p a r t s of the s t a t e of Washington) f o r the presence of the A l f a l f a Witches' Broom d i s e a s e * A knowledge of the disease d i s t r i b u t i o n would p o i n t out s e v e r a l s a l i e n t f e a t u r e s of the d i s e a s e .  I t would show us the e c o l o g i c a l  h a b i t a t and the c l i m a t i c c o n d i t i o n s under which the condition thrives.  diseased  (In A u s t r a l i a , Edwards (12) p o i n t s  out  t h a t the disease i s r e s t r i c t e d to areas t h a t have an annual r a i n f a l l of 17 to 21 inches.)  I f continuing, a disease  r i b u t i o n would a l s o g i v e us p e r t i n e n t i n f o r m a t i o n on of spread  which may,  i b l e vector  or may  dist-  direction  not, be c o r r e l a t e d with the  poss-  spread.  F i e l d s were examined during the two  years  i n the  as i n d i c a t e d i n the f o l l o w i n g t a b l e s XI & X I I and accompanying d i s t r i b u t i o n map.  A l l fields  (except  i n d i c a t e d by jf) were examined by Dr. V.C.Brink i n some cases by the a u t h o r ) .  on  districts the  where  (accompanied  A l f a l f a f i e l d s i n 35  districts  were found t o c o n t a i n at l e a s t 5^ of Witches' Broom d i s e a s e d plants.  The  disease was  not observed i n f i e l d s i n 31  d i s t r i c t s , but the c o n c l u s i o n i s not to be drawn t h a t disease i s not  present  i n these  d i s t r i c t s , but  just  other the  not  observed. A survey  of the North Thompson v a l l e y i s planned f o r  l a t e r on i n the i n v e s t i g a t i o n .  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 c o n t a i n i n g at l e a s t 5% A l f a l f a Witches' Broom p l a n t s .  '% - A l f a l f a f i e l d s t h a t d i d not c o n t a i n A l f a l f a Witches' Broom p l a n t s .  x  Table X l -  Districts  -105'  alfalfa i n w h i c h / f i e l d s were examined that d i d  \  not  oontain A l f a l f a Witches' Broom P l a n t s . Rosk Creek Grand Forks Castlegar Balfonr Creston Moyie Cranbrook Newgate Canal f l a t s Edgewater Shftmway Lake Kamloops Pavillion Stump Lake L y t t o n (south o f )  Saanich Peniaaula U.B.C. Niooman I s l a n d Chilliwack Rosedale Princeton Hedly Keremeos Okanagon P a l l s Summerland West Bank Kelowna Vernon Falkland Lumby Bridesville Table X I I - 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  t h a t contained  at l e a s t 5% A l f a l f a Witches' Broom  Plants. Peace R i v e r # Lake Cathlyn (near Smithers) Smithers Fort F r a s e r Vanderhoof Quesnel Williams Lake Riske Creek Spring House A l k a l i Lake Canoe Creek Clinton Pavillion Hat Creek Lillooet Lytton McGillvray Ashcroft  Wallishin Savona Tranquille Kamloops Vinsula Nicola Nutsford Shumway Lake Merritt Dot Aspen Grove Monte Creek Falkland Armstrong Salmon Arm Methew V a l l e y  (Washington)  (Ag*SS«Z?)#  jf i n d i c a t e s f i e l d s i n which Withhes' Broom of A l f a l f a has been reported by other i n v e s t i g a t o r s that those o f the Department o f Agronomy, U.B.C.  -106-  C - Notes on A l f a l f a grown at the Dominion Range S t a t i o n , Kamloops, B.C. At  on September 17»  1951>  the same time that the Nursery A l f a l f a P l o t  p l a n t e d at the U n i v e r s i t y o f B r i t i s h Columbia, p l o t was  was  a replicated  p l a n t e d at the Dominion Range S t a t i o n , Kamloops,  B.C.  A l f a l f a p l a n t s , grown from seed at the U.B.C. greenhouse, t r a n s p l a n t e d to the f i e l d  at Kamloops on June 1,  Seed f o r the Rhizoma s t r a i n s was 1948  were  1949.  harvested i n the f a l l  of  from o p e n - p o l l i n a t e d c l o n a l rows i n the main p r o d u c t i o n  field  at U.B.C.  and Ranger was u l t u r e , Ottawa.  Seed of V i k i n g , Ladak, Grimm, Ferax, B u f f a l o  r e c e i v e d from the Dominion Department of A g r i c Grimm Vidarshov seed was  Grimm Summerland S274 from Dom.  obtained from Sweden)  Exp't. S t a . , Summerland,  and Grimm Sask. 666 and 451 were both obtained from the Forage Lab., Saskatoon,  and 1950.  furrow i r r i g a t e d both i n the  Observations i n the f a l l  showed a l l p l a n t s t o be v i g o r o u s and h e a l t h y . s p r i n g of 1950  some of the p l a n t s appeared  of  1949  However, i n the  very p o o r l y and  d u r i n g the summer began t o develop d i s e a s e symptoms. 17, 1950  Dom.  Sask.  The Kamloops n u r s e r y was summer of 1949  B.C.,  On Sept.  s e v e r a l p l a n t s were observed t o be showing v a r i o u s  stages of the Witches' Broom d i s e a s e : t h i s i s shown i n the accompanying t a b l e . the  A l f a l f a Nursery at U.B.C. f a i l e d t o d i s c l o s e any  showing at  Observations of the r e p l i c a t e d p l a n t s i n  any symptoms of the Witches' Broom d i s e a s e .  U.B.C. had r e c e i v e d comparable  plants The n u r s e r y  c u l t u r a l treatment t o t h e  Kamloops p l o t , with the e x c e p t i o n t h a t s p r i n k l e r  irrigation  was used at U.B.C. and furrow i r r i g a t i o n at Kamloops.  TABLE X I I I - OBSERVATIONS OH ALFALFA GROWN AT THE DOM. RANGE STA.. KAMLOOPS. B.C. No. p l a n t s  Row  6 6 6 6 6 5 6 6 6 6 6 6  1 2 3  4 5 6 7 8  9 10 11 12 13  (6  4 orig) 6  14 15 16  4 (6 o r i g ) 6 6 6 6 6 6 6 6  17 18 19 20 21 22 23  24 25  6 6  Variety  No. of  No. of p l a n t s showing symptoms of Witshes* Br6om of A i f a l f a ~ d i s e a s e  Rhizoma (30-11A) » (24-DRC) « (29-11B) (45-11A) » (H71P27) " ( 4-11A) « (39-DRC) « (66-10A) (51-DRB) • (28-11B) " (H156P18) Ranger Buffalo  0 0 0 0 0 4 0 0 0 0 0 0 0  Vftgtr  17/9/50  Seed s e t (graded 1-5)  0 0-1 0-1 d r y 0-1 0-2 dry 0-1-2 d r y 0-2 d r y 0-3 0-3 0-1  Ferax  0  Excellent Excellent Fair Fair Fair Poor F a i r except jf6 Good Good Good t o poor Good Good t o poor Fair( 2 p i . missing) F a i r (3 weak)  Grimm  ft  Good (2 miss.)  3  Ladak Viking Grimm Sask. 451  6  0 4  666 n  0 0  S274  5  M  "  tt  it  Summerland ti  n  Grimm V i d a r s h o v »  n  (primary)  3  0 0 0  (advanced)  3 5 5 on one  5 on othe: 5 onone 2-3 other  Fair 5+ V. good (2 poor) 4+ V. good 5+ r e s t p Good 0-1 d r y Fair 0-1 Very good 2-3 d r y Good 0 dry Good 0-1 a r y Good 0-2 d r y Good hutone 5+  H O <! I  -108-  Iv  " » H i s t o l o g i c a l 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 alfalfa.  However there exists several fine review a r t i c l e s  on the h i s o l o g i c a l aspects of plant virus diseases i n 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 i n the xylem of  various plants under normal conditions, but i s frequently more often observed i n the tissues of plants affected by various pathogens  and physiological disturbances.  She reports  that most workers think that the gum i s derived from the decomposition of starch, hydrates;  and to a lesser extent,  other carbo-  and that the products of decomposition migrate from  the l i v i n g c e l l s into the tracheary elements.  Gummosis i s a  very frequent phenomenon of virus i n f e c t i o n , but i s not specific to this type of disease.  Jones and McCulloch (27) report the  presence of gum i n the vascular system of a l f a l f a  affected with  the B a c t e r i a l Wilt disease. Wynd (53) reviews the metabolic phenomena associated with virus infection i n plants.  He found that the two major phys-  i o l o g i c a l effects of virus infection i n plants ares (l) Respiratory a c t i v i t i e s  are markedly increased very early  during the course of i n f e c t i o n which corresponds to the period of a r r i v a l of virus p a r t i c l e s from the point of i n f e c t i o n . The  109-  plant recovers  from t h i s  initial  of v i r u s p r o t e i n c o n t i n u e s (2)  The  permeability  altered the  i n respect  accumulation  to  of c y t o p l a s m o r  to  soluble  i t s membranes i s g r e a t l y  substances.  of c a r b o h y d r a t e s  i n the  This  accounts f o r  leaves,  i n the r o o t s  (ibid)  i n d i s c u s s i n g the  in chlorotic  leaves,  ution  of t h e  c h l o r o p h y l l i s due,  decrease per  total  c h l o r o p l a s t i d , but  of c h l o r o p l a s t i d s . may  be  points  cause  phyll  and  a t the  the  expense  c a u s e d by  He  the  believes  v i r u s , not  a deranged metabolism.  out  of d i m i n i s h e d  that part not  only  of  that  the m o t t l i n g but  However, o t h e r  i s a f a c t o r i n the  green  The  Jones  (21);  (28).  food reserves have  increase  h i s t o l o g y of normal  by Hayward  a greater  increased  and  the  Graber of  and  a l f a l f a plants.  proportion  fibrous  t i s s u e ; and  reserve  foods,  the  root  associates  of  carotene the  fully  describe  light leaves.  described described the  normal  R o o t s of h i g h r e s e r v e  plants  of p a r e n c h y m a i n r e l a t i o n  starch,  which  destruction  t i s s u e s are  (19)  parenchyma c e l l s  principally  accord-  over normal c o l o r e d  a l f a l f a i s very  crown  and  o f 96%  leaves  acidity  (mosaic) i s enhanced; i n f a c t ,  a r e a s showed an  of  through products  Although c h l o r o p h y l l i s decreased,  of leaves  number  investigators,  accompanies v i r u s d i s e a s e s  content  the  dimin-  actual  lessened  that  chlorophyll.  to  to the  directly,  chloro-  of t h e  also  i n g t o Wynd ( i b i d ) b e l i e v e  by  quantity  tops. Vynd  of  a l t h o u g h the  increase.  accumulation of n u t r i e n t i o n s the  shock,  1  are p a c k e d  to  the  with  -110-  b)  Experimental 1.  C o m p a r i s o n "of with  Materials (1)  and h e a l t h y  at  blue. (2)  15  tone  at  Stained  to  (3)  40-50  several  previous  of  transversely Leaves  (grwwn  i n  sections  (Nov.  to  with  Fixed  Imbedded  the  rotary  and  blue,  with  K i l l e d  with i n  micro-  methylene  s l i d i n g  with  B.C.  (Found b e t t e r  micro-  F i x . staining  ammonium m o l y b d a t e ,  experiment of  plants  that  the  mod.  with  and m e t h .  not  i n  Navashin. mu w i t h Haup#ts  become at  transplanted  90%  i n  Jan.  used)  at  As  40-50  from  U.B.C.  for  mu.  alcohol  rotary  B.C.  balsam.  the  Cariboo  were  i n Feb. to  sectioned  1951  remove  i n parawax,  microtone.  adhesive,  were  (3)  plants  Imbedded  1951,  diseased  Mounted i n Canada  and h e a l t h y  blue.  had  greenhouse  dipped  10-14  completed  cutting  blue.  plants,  to  was  s l i d i n g microtone.  slides  solution.  sectioned  17/50).  and methylene  at  1950.  balsam.  greenhouse),  safranine  and r o o t  methylene  Rand.  June  and  balsam.  from diseased  verse  Navashin's  s l i d i n g microtone  on  i n  and m e t h y l e n e b l u e .  summer  with  Ashcroft  safranine  diseased  Fixed  secured  buds  flowers  with  crowns  Safranine  the  (5)  for  using  Crowns  a l f a l f a  stems,  Stained  microns  the  (4)  healthy  disease.'  cut  transmission  sectioned  of  the'-Witches' Broom  were  crown  i n Canada  of  with  sections  mu.  safranine  After  Fix.  20  Grimm  when mordant Mounted  -  m o d i f i c a t i o n of  Mounted i n Canada  Normal  structure  rootlets,  from Clinton  paraffin,, transverse tone  c e l l u l a r  Methods  obtained  Randolph's  the  a l f a l f a, a f f e c t e d  and  Diseased  were  studies  stained  Mounted i n Canada  air, trans-  Sections with  balsam.  -Ill-  Observations CrossSections for  differences  main,  i f  not  diseased of  In  i n  stem  has  without  tissues  a  a  tendency  tissue  supporting  corky  elements  which  stained  in  (see  the  noted  size.  The  The  smaller  the In  than  the  r i n g  of  with  whole  the  those  are  being  more the  and  formed.  a r e l a t i v e l y  simple  network  undifferentiated structure  well  plants,  cross-section  a radiating  young,  bundles  diseased  bundle has  i n  bundles  vascular  i n  photo)  that  vascular  square  the  infrequently,  In  the  observed. diseased  the  small  leaf Quite  leaf,  i n  by  not  tissue  or  the  outer  areas, a  of  appear  found  yellowish  a member  crown  surrounded  does  frequently  either  numbers  throughout  crown  were  w h i c h was  great  v/ere  remain  consisting  cells;  the  ring.  interspaced  diseased  Xylem  of  tissues.  i n  much  compared  being  of  storage  surrounded  by  epidermis.  The  one  was  very  was  stems  vascular  crown  vascular  a  to  continuous  structure,  i t  stem,  older  continuous  normal  were  were  diseased  observed  Material,  i n  cellular  and  cells  and  "buds,  (pygmismic).  whereas  a  The  difference  healthy  form  nodes,  healthy  cross-section,  separated; less  stems,  between  plants  the  sqarish  roots,  only,  plant  healthy  or  of  several  a breakdown  of  i n  v/ith  the  somewhat healthy of  outer  palisade  (photo)  organisms,  class  appear  Interspersed  plants  points  the  gum  Secondary  tissue.  diseases  appearing  frequently,  altered.  Basidiomycetes  differentiated  tissues,  f i l l e d  red.  rotted  of  greatly  v/ere  found,  gummotic,  cambial contrast  portion layer  of was  that  area. were the noted.  The  palisade  rounded noted at  cells  lost  and r e s e m b l e d  that  a l l ,  these  of  spongy  c e l l s ,  possessed  leaves  their  but  diseased  i f  a  t y p i c a l l y mesophyll  they  very  plants,  few.  have  number  vascular  of  bundles  of  the  The  to  palisade  leaves.  layer  would  allow  occur.  leaf-comparison,  calcium  of  of  strength-giving  eased  macrosymptoms  edges  the  the  chloroplasts  chlorotic  of  i n  also  and  breakdown  noted  any  was  a puckering  marginally  Also  It  became  shown  the  condition  The  shape,  often  of  puckering  cells.  contained  wrinkling  this  elongate  oxalate  diseased  was  crystals  leaves  the  greatly  surrounding  over  those  of  incr-  the  the  -  healthy  leaves. 2.  Comparison  of  food  reserves  of  healthy  and  diseased  plants. Material  and  The showing the  Methods.  diseased varying  a l f a l f a  stages  N i c o l a valley,  material from  was  2 year A  the  obtained  series  of  to  for  plants.  Cross-sections  starch  a razor blade same  a l l  color  for  starch  6 months  series,  was  and  I>^1.  i n  the  crown  with  and  IgKI.  with  them,  i n  root  70%  and  plants  originated  i n  plant  seedlings,  and  greenhouse.  a graduation conditions  from were  representative were  made  Leaves  ethyl  then  from  healthy  Grimm  diseased  boiled  from  The  showing  along  stained  were  removed  using  of  and w h i c h  old  grown  badly  reserves,  obtained  summer.  plants  very  was  disease  previous  diseased  examined  the  the  old Rhizoma plants  diseased  of  of  from  slightly  with  material  they  freehand  from  alcohol were  healthy  plants  u n t i l tested  At of  the  sive  the  suggestion  Department  of  of  the  Botany  micro-chemical tests  Broom  diseased  diseased)  plants  a microchemistry tests  for  commonly  14  of  found  carried  on  root  stages  (2  and h e a l t h y  a l f a l f a  problem for the  i n  author,  organic  Mr.  out  a  crowns -  Botany  stems  534.  was  He  more of  diseased  This  materials  Setterfield  somewhat  and  mildly  plants.  George  Witches*  and  severely  performed  carried  and f o o d  exten-  as  out  storage  forms  plants.  Observations The  crown  series  amount  of  crowns  contained  a  starch  graduation  in  the  plant  s t i l l  stored a  down  poorest  there the  storage was  healthy He  stage  of  Healthy a l l  more  starch  plant  i n  the  the  that  not  of  stored  support starch  being  present  i n  Although  starch crowns,  with  the  i n  the  the  the  the  was  crown  my f i n d i n g s . present  i n  the  disease. starch,  series.  the  the  storage  significant  disease  i n  the  showed  crown;  starch  the  the  diseased  sucrose in  and  crowns  Mildly  showed no  quantity  results  a, - v e r y  plant  quantities  i n  of  to  of  considerable as  correlation  Healthy  with crowns  poorest  leaves,  a  observed. tissues  and  However,  he  diseased  stems  their  found  that  than  in  stems.  found  cases.  quantity  no  decline,  3tage  the  to  Setterfield's starch  a, d e f i n i t e  with  large  showed  progressive  showed  storage negative plants  while  storage  stored  stems  i n  c o r r e l a t i o n between  i n  of  tests  stored  severely i n the  either  sucrose  the  for  diseased  i n  crowns. i n  moderate  plants,  showed  tissue.  healthyoor  the  sucrose  sucrose  crowns,  crown  i n  the  a  large  Sucrose  diseased  was  plants.  -114-  PISCUSSION Considerable in  B r i t i s h  has  been  additional  Columbia of  gained  already  of  distribution i n  of  Kamloops  (and  Cascade the  and  radially)  i t  is  exception  the  disease a l l  the  is  observed  of  as  a  i n  the  climate  to  this  recovery  of  studies  at  explain  the  management  Australia,  disease  inch  the  This where  annual  same the  with  masking  diseased U.B.C.,  plants,  and  observed  of  also  the  the  w o u l d be  masked.  have  is  With of  r a i n f a l l  only  restricted  pattern  affects  i t  a  is  dry possible,  d i s t r i b u t i o n .  the  from  i t  is  where  been  need  and  i n  the  disease  observed)  symptoms,  observed  of  areas.  observed  r a i n f a l l ,  the  d i s t r i b u t i o n  distribution picture.  sufficient  report  heavy  vectors,  reported  the  regions.  disease  disease  v i c i n i t y  northward  of  since  r a i n f a l l  virus  the  Saskatchewan,  disease  a, m a x i m u m p o p u l a t i o n ;  of  symptoms  of  i n  centre  would extend  low r a i n f a l l (not  i n  oral  presence  1937  disease  The  between  a l f a l f a  Agassiz  notorious  correlated  and  at  areas.  17-21  phenomenon  i n  be  extends  A l b e r t a and  the  occurrences  reach is  i n  report  low r a i n f a l l i n  of  to  regions  The  A l f a l f a  completed.  and  on  d i s t r i b u t i o n  of  An u n p u b l i s h e d  north.  occurring  Leafhoppers,  The  far  the  Broom  appears  Valley,  Alaska  from parts  noted  to  that  province  i n  on  studies  low r a i n f a l l  appearing  the  plants  the  quite  reported  area,  the  H ic o l a  occurring  distribution  again,  i n  by  this  the  Witches'  and R o c k y m o u n t a i n s .  disease  also  the  knowledge  and  the  greenhouse  Australia, Under possible,  may  good that  the  -115-  Besides plants other  i n  some  salient  disea.se one  the  has  year  cases, points  been  after  Kamloops  observation  cases.  that  the  i n  study,  to  6 months plot,  recovery  disease  shown  nursery  some  the  of  so  far,  young  seedlings  of  the  primary  disease  has  diseased  brought  symptoms  is  of  very  plants  planted  were  out  Witches'  a l f a l f a were  A continuing observation  onset  the  symptomology.  affect  old  of  -  in  the  appearing  these  rapid,  i n  plants  as  by  Broom  shov/ed  the v/ere  end  of  4  noted. at had  months  shown  of  2 weeks  of  necrotic  that  with  the  detailed and  later,  leaf  uently  of  than i n  the i n  the  diseased  the  in  crowns  older was  progress  of  the  the  of  sucrose  It  is  form  crowns.  plants. the  which  to  be  and was is and  not  another very  This time,  month. l i t t l e  and h e a l t h y  further  Food  tissues,  exceptions,  observed  crown  v/as  of  A l l to  found  of  diminished v/ith  stored  indeed  by  food  i n healthy  probable,  be  diseased  storage  replaced  grafting  primary  months  Gummosis  w«,a  suspicious  marked  be  were  portions.  disease;  possible,  to  by  abnormalities.  plants  found  disease  chlorosis.  i n yet  the  of  as  another  diseased and  xylem elements  i n  i n  palisade  thenormal  especially healthy  size,  the  marginal  appears  between  of  cells,  smaller  there  the  transference  developed and  of  recorded  appearing  exception of  disease  dwarfing  symptoms  differnce  symptoms  plants  time  severe  Histologically, mechanical  the  p r o l i f e r a t i o n  f o l l o w e d by  v/ith  on  have  inside  symptoms was  advanced  Observations  U.B.C. ;  very  that  tissues much freqplants  starch the storage  i n  a l f a l f a the  virus  -116-  may so  upset, that  also and  or  modify,  this  noted the  food  that  rot  storage  a l l  was  the  not  any  and  change  brought  about.  i n  crowns by  The  were  (caused  presence  sections  of  the  of  conjecture:  storage  of  sucrose,  implementing  (sucrose  t,o h a s t e n crown by In  a  is  a  the  the  by  favored  death  of  comparison  observed.  leaves  In  of  the  a mechanical  observed.to  orientation,  (which  would  of  leaf).  lower  the  give The  -  diseased  leaves  The  this  of  The  chlorotic  plant  is  the  more  and  the  of  rotted,  fungus  was crowns.  production  growth or  the  breakdown  of  of  of  the  nutrient),  r o t t i n g  the  healthy  very of  of  cells  chlorotic  palisade rounded  of  up  to  palisade  this layer  would  of  the  leaves  i n  manifested  by  the  common  symptom  produce  distances.  of  f i e l d  infected  symptoms  accompanied  In  fact,  their  outer of  puckering.  the  symptoms  and  area  strength  appearance  layer  chloroplasts  coloration  a marginal  diseased  differences  marginally  devoid  the  and  marked  normal  palisade  chlorotic  pronounced  to  portion  appeared  appearance,  combined  considerable  The  showing  chlorotic  one  by  chlorosis)  breakdown  structural  portions  the  was  undetermined  the  medium  plant  It  healthy  Does  cross-sections  outer  occur.  lacked  the  the  crown  fungus?  leaves (exhibiting marginal were  fungous  an  this  normal  interesting  the  severely  by?)  an  help  was  is  raises  fungus  the  of  accompanied fungus.  This  balance  diseased  Basidiomycetous observed  enzymatic  by  that many  of  plants the  dwarfing were  fields  disease. of  visible were  produced  the at observed  -117-  from  the  highway  confirmed  135  province at  correlated  not  the  a l f a l f a  was  anything but  always.  i n i t i a t e d  hay  last  f i e l d  diseased  diseased  the  that  fate  of  plotted  These  w i l l  and more To 4?  observations  the 50  to  Trnaquille  farm,  point  of out  of  the  these  fields  incidence  were  results  should  of  60%  practices  plants  The  Interior  Disease  be  the  the  the  of  10  the  healthy  comparable  healthy  a  only 27  and  plants  10  of  33  of  140  of  the  was fields,  observed  i n  and  this  f i e l d  quadrat  study  a  many  features  gain  intervals  be  charted.  size  i n  started  s t a t i s t i c a l l y  to  of  140  for  the the  the  plants flower.  significant  were It  and  any  next  and  i n  the plants  80  quadrats d i s t r i c t .  few  years  was  carried  winter. pots,  harvested shown  differnce  93  information  t r i a l  was  of  healthy  past  propagated  1942,  Savona  a y i e l d  data  plants  diseased,  in  during  were  diseased  the  i n  f i e l d j 6 more  reports,  U.B.C.  plotted  additional  largely  at  quadrat  remained  of  the  of  healthy  them were  plants, in  at  study When  out  To  quadrat  greenhouse  the  was  w i l l  a  vestige  Interior,  observed  augment  no  altogether. a l f a l f a  i n  total  1944, and  from  1942-44.  a  state,  quadrats  Cuttings  there  In  plants;  i n  i n  contained  disappeared  were  out  the  emerged  Brink  plants.  remained  on  diseased  (1950)  fact  by Dr.  quadrats  had  and  plants.  mismanaged.  f a l l  i n  management  at  -  respect.  collected  93  with  plants  fields.  summers,  Several  A s t a r t l i n g  the  two  the  examined  infected  5%  but  to  were  the  least  diseased  v i s i t s  fields  during  generally  this  contain  by.actual  Over  showed  to  i n  and whenever that y i e l d  -118of  the  healthy  cutting, to  the  the  same  However, the "by  3rd the  of  2nd  diseased  data  on  the  host  closely related  designed.  randomized block  past  planted ations  and  i n  made  to  the  i n the  from D r .  J.D .Menzies  contain  inherent  Disease to  healthy  carried  out 142  i n  the  of  and  healthy Rhizoma  end  of  in  31  U.B.C.  the  are  from  diseased of  summer  of  was  1950.  crown  grafts,  stocks,  124  Successful and  a  v/ill  sources be  and  observ-  occurs. received  supposed  to  resitance.  b y means  cases,  plants are  i n  w i l l  transference  test  varieties  other  plants  compiling experiment  these  healthy plants  disease  stocks  of  planted To  from  diseased  plants  attempted  7 months.  to  under  transference  a l f a l f a  from  i f  v/ere  received  fodder  plants.  twinning 40  and  host  hope  1950,  belong  51.2^  on t h e  the  1st  concerned.  i n native  a  of  a l f a l f a at  of  was  healthy  light  i n Washington that  disease  Out  achieved  plants  varieties  the  In  summer  of  proximity  Included  cast  genera.  nursery  determine  to  on r e s i s t a n c e  the  Cuttings  fodder  i n p r o d u c t i o n of  on r e s i s t a n c e ,  species  spring,  close  and  During  winter.  i n  also  of  On t h e  shown to  a reduction  36.6^  possibly  related  range  and  this  when  were  weight  showed,  may  closely  this  as  compared  been  added,  far  plants  has  be  as  plants.  plants  of  virus,  or  diseased  a reduction  reports  the  the  diseased  cutting  cutting  a l f a l f a  and  population  the  of  over  healthy  Quadrat range  plants  graft  plants of  of  at  diseased v/ere  scion  a l f a l f a  experiments  observed  using  union  disease  plants  U.B.C. scions  l i v i n g and  tranference  at  stock  was  • the  was  observed  -119-  in  27  cases.  union  and  A l l cases  both  l i v i n g  A grafting of  showing  a  purposes, to  the  that  In  without  with  several  the  the  insect  only  and  known  the  vector  of  Platymoideus  attempts  being  be  the  chief  employed essential  to  F i e l d merely  the  presence can  of  readily  healthy  the  of  plant  the be  plants,  that  the  disease. determined as  i n  the  strains  tested  plants.  disea.se  is  axsutus,  has  indeed,  disease. i t  not  i n  The by  not  can  an  resisted  is  f i e l d  immunity,  grow  successfully f i e l d  interplanting diseased experiment  not  resistance.  true  of  to  easily  advantageous,  possession  twinning  the  considered  determining  imply  plants  Alfalfa,  Anyway,  w o u l d be  available,  performance.  successfully is  When  and  simple  Broom of  diseases,  matter.  known  Witches'  indeed, diseases,  virus  simple  a. r e l a t i v e l y  and  virus  transmitted  of  and,  breeder.  the  plant  plant  numbers  to  than  a  large  type  •  recognition  on  The  diseases,  a  i n  outside  pathologist  mosaic  such  were  disease  i n nature.  testing  not  breeder may  for  places  fungous  virus  the  although  resistance fact  is  reared,  vector  vector,  and  stock  performed,  plant  modifying the  t h e n become  leafhppper, at  for  d i f f i c u l t l y  of  was  the  available  testing  tests  to  and  transference.  sequence  transmitted  the  vector  resistance The  i n  as  receives  bacterial  are  scion  disease  graft  techniques  easily  However, transmission  interest  ever)  procedures  a  symptom  seriously  with  the  such  A c r w n  (if  requires  dealing  even  more  seldom  breeder plants  of  good  agronomist.  i t  showed  experiment,  very  is  wherein  but i n  the  resistance and  previously  -120-  discussed,  preferably  naturally  and  With  i n mind,  be  this  i n i t i a t e d  disease  is  where  this  i n  the  an  area where  natural  agents  experiments year  at  the  of  points  of  this  i n  disease  inoculation  type  the  occurs  are  Interior  reside.  planned where  to the  endemic.  SUM1ARY The in  distribution  B r i t i s h  low  Columbia  r a i n f a l l .  basis  of  f i e l d  shown  y i e l d  and  and  to  have  compared  diseased  disease  mechanical starch food  is  is  stored  crown  i n  sap  Experiments disease  are  of  be  plants  Seed  the  the  A l f a l f a  areas  discussed  on  of the  Diseased  is  same  plants  reduction conditions.  enlarged.  Recovery' study  vessels  crowns,  of  ceils  in  the  of  i n  the the  crowns,  of  leaves. and  the and Storage  instead  sucrose. of  the  transmission,  negative  discussed.  of  A histological  depleted  designed  to  significant under  palisade  of  is  included.  disease  i n  confined  disease  are  transmission  gave  be  Broom disease  observations.  noted.  form  grafts.  expressed  the  is  breakdown  Successful by  of  to  to  s t a t i s t i c a l l y  gummosis  shown  Witches*  the  data  healthy  plants  shows  of  quadrat  a  to  the  shown  y i e l d  Symptomology of  is  Severity  Quadrat are  of  results  for  the  disease and  was  achieved  inoculations  at  present.  future  study  of  the  with  121• LITERATURE CITED . (1  Anonymous. A l f a l f a Improvement Conference, Twelfth Annual Report. July 31 - August 2, 1950. Lethhridge, A l t a .  (2  Brink, V . C . ' Witches' Broom on a l f a l f a . Rept. C an. PI. D i s . Sur. 1942.  (3)  Cormack, M.W. Witches' Broom of A l f a l f a . In 20th Ann. 1940. Rept. Can. P I . D i s . Sur.  (4) (5,) 6; (e') (7) t'  (8) (9) (10 (11  Rept. ""Can. PI. D i s . Sur. 1941. Rept. C an. P I . D i s . Sur. 1942. Rept. C an. P I . D i s . Sur. 1943. Rept. Can. P I . D i s . Sur. 1944. Rept. Can. PI. D i s . Sur. 1945. Rept. Can. P I . D i s . Sur. 1946. Dickson, B . T . MacDonald Edwards, E . T . lucerne. 1935.  In 22nd. Ann.  • In 21st Ann. • In 22nd Ann. *  In 23rd Ann.  • In 24 th Ann.  • In 25 th Ann. • In 26th Ann.  Studies concerning mosaic diseases. College, Tech, B u l l . 2. 125 p. 1922. Witches' Broom, a new virus disease of Jour Australian Inst. Agr. S c i . 1: 31-32.  (12  The Witches' Broom disease of lucerne. Dept. Agr..Sew South Wales S c i . B u l l . 52. 1936.  (13  . Witches' Broom of lucerne. Agric. Gaz. of New South Wales 47; 424-426. 1936.  (14  Esau, Katherine. Some anatomical aspects of plant virus disease problems. I I . Bot. Rev. 14: 413449. 1948.  (15)  Esau, Katherine. 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Witches' broom of Ladino white c l o v e r . 29th Ann. Rept. Can. P I . D i s . Sur. 1949.  (41)  Richards, B . L . Dept. Agr.  (42)  Smith, Kenneth. Textbook of Churchill Press. 1939.  (43)  Thomson, In  (44)  Weimer, J . L . A l f a l f a dwarf, a hitherto Phytopath. 21: 71. 1931.  (45)  Frank P. A l f a l f a An a b s t r a c t .  Witches' P I . D i s .  on r e d c l o v e r . Sur. 1949. virus  N.  In  Can.  Phytopath  29th  39:  broom, cause unknown. In Rept. 71: 309-310. 1929. plant  virus  861.  In  U.S.  diseases.  J.E.J, and J . B . L e b e a u . W i t c h e s ' b r o o m on alfalfa. 28th Ann. Rept. Can. P I . D i s . Sur. 1948.  A l f a l f a mosaic. (abstract). 1931.  unreported  Phytopath.  21:  disease.  122-123.  Weimer, J . L . Studies on a l f a l f a mosaic. 24: 239-247. 1934.  Phytopath.  " A l f a l f a dvfarf, a v i r u s disease transmiasable "by g r a f t i n g . Jour. Agr. Res. 53: 333-347. 1936. Woolliams, Witches' broom on a l f a l f a . Ann. Rept. Can. P I . D i s . Sur. 1943. . Ann. Rept. Can. P I . D i s . Sur.  In 28th 1948.  Wright, U . S . Witches' broom on a l f a l f a . Ann. Rept. Can. P I . D i s . Sur. 1946. Ann. Rept. Can. P I . D i s . Sur.  In 23rd  In  . In 1947.  . Witches' broom i n B . C . Rept. Can. P I . D i s . Sur. 1949.  26th 27th  In 49th Ann.  Wynd, P . L . Metabolic phenomena associated with v i r u s i n f e c t i o n i n p l a n t s . B o t . Rev. 9: 395-465. 1943.  Quadrat No  Date Charted.  i.r.JC.9..  /?  it . r^ ......J  (  «  H  •  )\  0  \  '////  >. j £ )  ////;/  /  (  * t K  /  S J J ,  &IQ *r & g & to/sire  ' ' ' , / Jt  « ;  i  <  1  <  1 7T~. if /S  *T  ••  1  G v4  )/  * / **  V •1  <3  i  i  ASQ.reacm.in  •  •D O<JV *y  SA/IO  ^  ' T  x *.  O  0 Species  ///  0  )  ;• *  N .msof co ul  it gh LeaveAsverage HeS et ms  Seek  Prod.  REMARKS  Vigour  i • •(  fy t a  mfrt 0/1*** F A  <  -  —  .  : ~~  -  -  QUADRAT No. L:..5° Date Charted fit-sT  u - e .  A L F A L F A  l&iA&CTeP  :  ^1  HtfrH  W « y  Character of Site. (b) Altitude  j  Sfeuce  f i t . i T  CMLt>»£C  F L A T  O P  F i t L b  ttt««-»t  j  U C O » T  C  o f  & i L e u e )  Roc I t *  ow  • * • • £ £ n B o u e  B E M C H  M A » « I ,  (a) Exposure and slope *0°'.....  Plant Type.  (,  Location  <W  ^ O A O S I O S  & - V J •  " O I T W  ".  s i n e  H«»Y  ~  u m u  C o M f l t  J O S T  —  (c) Soil..  -  (a) FormaUon^}^.^^..-.^ :*:^^  O F  i»J  -  -  ~,  (b) Association.  1  (c) Principal species  S U M M A R Y OF QUADRAT D A T A (a) Grasses and Sedges.  Average height '  Totals  i e a v e s  l  m  Area J s q - c occupied OL  s  | |  m  (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  S ' * H T ,  6*  Quadrat No..  s*  Date Charted,. 1  / « v > — v  ;  Oif -  •  Z  i —  O  O  ;  < y  /V" )  M l '  /  H" t  '1  * ^^^^  O" A " <  \ CD  •  w 6 *  IX » ft  4'  CD,  \  1®.-  j  -1 .  .  . t  d  < £ J .  CP  )  V .  1  fir.  ^  /A  © No.  \  Species  Asr n. qe .acim  St.*' *' * ?y/9 £ A/9*/=M ' 9  J  No. of culms  '"S.  L.  1  i z  it ge hm ts LeaveAsverage HeS  Seed Prod.  Vigour  REMARKS  QUADRAT No.  6-50 Location....A h.crpft..Manor.^ s  Date Charted-  near main highway at the foot o f the Oregon Jack Creek Road; l i n e up outhouse and conspicuous r e d s o I T outcrop; % paces "from deep with Clematis l i g r e s t i o i f o l l a . Character of Site. (b) Altitude Plant Type.  N  (a) Exposure and slope...  ,  }?. 0.?:.  (c) Soil (a) Forniation..^^ .?.?.^....-....^ .^.?. J!.^. . .?.*. 1  0  0 11  .-.  .  I„„„„L  l*8ht . * r ^ ^ (b) Association...J  [  (c) Principal species..  SUMMARY OF QUADRAT DATA (a) Grasses and Sedges.  Totals  Average J i e d v e ! > height 1 dims  Area J S q ' C occupied OL  |  (b) Non-Grasses.  :  veg. height j flower  Area 1 occupied ] or q -  •  Relative Forage Value of Quadrat  ,  % Carry-over.  Distance of Quadrat from nearest watering place Other Factors affecting grazing...... Remarks  ....  . , ; Totals  Mileage No. 139.2  V.C.Brink  QUADRAT No.......2."....5.0. , . Date Charted  Sept. 18, 1950  4  , Location  ;  S o f main highway - Kamloops - Savonaj near telephone side" Is)' s i d e 6 ? Bi'ghway; quad .with..2 above^.mention  pole 11/34 KA 112 with c r o s s support  on opposite  (a) Exposure and slope..;.-  Character of Site. (b) Altitude  .?..?..9.°„!  Plant Type.  (a) Formation  (c) Principal species  1  (c) Soil....M.8.k*...!?.^ (b) Association....;.  .....AA?*^.?.*..."...?.^.?.?.!?..  (a) Grasses and Sedges. Symbol  SAVONA  SUMMARY OF QUADRAT DATA  Totals  ...........  No. of SpecimensAverage height  leaves.. culms...  No. of culms Seed production.... Area occupied  sq. cm.  % Totals  (b) Non-Grasses. -  veg. Average growth height "j flower  •  i Area J L occupied i o/  j  1  n  Relative Forage Value of Quadrat Distance of Quadrat from nearest watering place. Other Factors affecting grazing......... Remarks  % Carry-over..  No.  Species  Asr qe .acimn.  N .msof co ul  it ge ht LeaveAsverage HeS ms  Seed Prod.  S "  ff  /o  i  Vigour  REMARKS  V.C.Brink  QUADRAT No A..= J?° Date Charted.. Near  ..®?..V.. ...V... ... S  t  l8  Location  19 5 0  (W ojf) Qua  •No H u n t i n g ' s i g n ; quadrat l o c a t e d 21 paces S of fence Character of Site.  (a) Exposure and slope  (b) Altitude  -  (a) Formation  Plant Type.  ^B...t?.*...Qt~%~&9. -  (c) Soil  A°.  (b) Association..  A°.  (c) Principal species  ;  SUMMARY OF QUADRAT DATA (a) Grasses and Sedges.  Average J  Totals  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  - --  -—  -  -  V.CBrink  mileage No. 142.4  QUADRAT No.. .....S..ePl.V...l8......l.?.5.?  Date Charted  „ j , .  West o f Q 2-5.0. and 0^. J telephone p o l e s 9/7 KA 5 9 and 9/6; Character of Site.  (a) Exposure and slope...  (b) Altitude (a) Formation... A ^ .  1  L o c a  i  i o n  ™™t.  8  ..?.?....^.^.S)?L........!l*^.?.?. about 2 paces 'inside" "^'S )"" f e"ne e VWe si" "'of gate, w a y  quadrat l o c a t e d  v  b  w  :  v  : (c) Soil..  Plant Type.  *..-J.9...  l i g h t brown, sandy  . ^  (c) Principal species  SUMMARY OF QUADRAT D A T A  (a) Grasses and Sedges.  Totals  Symbol No. of Specimens.. Average { height 1  ___  l e a v e s  culms  No. of culms Seed production... Area occupied  sq. cm-  Totals  (b) Non-Orasses. •  veg. height "j flower  Area J q " occupied i OL  "  ..,  Relative Forage Value of Quadrat Distance of Quadrat from nearest watering placeOther Factors affecting grazing.... Remarks...  ............  % Carry-over..  No.  Asrqe .aciinn.  Species  Seed, Prod. • +n  A  7  —  it ge ht LeaveAsverage HeS ms  < 3.  3+*  ...  N .msof co ul  Vigour  REMARKS  Mileage No. 165.65  QUADRAT No Date Charted .'...A?_L..-i Near "Hoodoo C l i f f s " W a l l a c h i n ;  L:J1  Location, Hay stacker and lone deciduous t r e e make a l i n e  , on which quadrat  i s l o c a t e d i n the f i e l d , S o f highway t o Cache Creek, 9 paces from fence and i r r i g a t i o n ; " ( f f a y W r a ' s l Character of Site. (a) Exposure and slope  I :*™'  (b) Altitude  <c) Soil  3  (a) Formation  Plant Type.  A l f a l f a . „-„K ....M.Uftgras.S...  ditch.  ... (b) Association...... '.  (c) Principal species.  SUMMARY OF QUADRAT DATA  (a) Grasses and Sedges.  Totals  Symbol No. of Specimens.. Average { height |  " ...  l e a v e s  c u l m s  No. of culms  ...  Seed production.... Area occupied  sq. cm.. Totals  (b) Non-Grasses. SymbolNo. of specimens.. veg. Average growthheight • flower stalks.... No. flower stalks,. Seed production.... Area 1 s ^ c )ied jj o/ occupied  m  '  Q  % Carry-over..  Relative Forage Value of Quadrat Distance of Quadrat from nearest watering placeOther Factors affecting grazing. Remarks  ........  

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