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UBC Theses and Dissertations

Breeding behavior in interspecific hybrids (Medicago falcata x M. sativa), parent stock of Rhizoma alfalfa Nilan, Robert A. 1948

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££3  JB 7  Cap. /  BREEDING BEHAVIOR I N INTERSPECIFIC HYBRIDS (Medioago f a l o a t a x M . s a t i v a ) , PARENT STOCK OF RHTZOMA ALFALFA by Robert A . N i l a n  A t h e s i s submitted  in partial  fulfilment  o f t h e r e q u i r e m e n t s f o r the Degree o f MASTER OF SCIENCE I N AGRICULTURE i n t h e Department of AGRONOMY  The U n i v e r s i t y o f B r i t i s h Columbia April  1948  ABSTRACT Rhizoma, a new a l f a l f a variety possessing a d i s t i n c t i v e spreading ha<bit, was developed from s i x hybrids of a cross between the species Kedicago falcata var. Don (seed parent) and Medicago sativa var, Grimm or Ontario Variegated (pollen parent).  Because of i t s unusual o r i g i n , i t d i f f e r s  from other a l f a l f a v a r i e t i e s grown i n North America, Cytological investigations showed the falcata parent to be a low chromosome a l f a l f a (S  =  16) and the  sativa parent to be high chromosome a l f a l f a (S - 32).  Three  of the s i x hybrids were t r i p l o i d s (S s 24) and three were tetraploids (S  =  32), the l a t t e r having received sixteen  chromosomes from the female parent, possibly through the formation of unreduced gametes.  It was concluded that the  new variety Rhizoma i s a permanent t e t r a p l o i d hybrid (S  s  32)  containing an equal complement of chromosomes from each parent. P o l l i n a t i o n and f e r t i l i t y studies on selected clonal lines of the  generation revealed that a consider-  able amount of seed set under open-pollination was selfed seed and that much of the seed was being set without the a i d of bees.  The combining a b i l i t y of the lines varied con-  siderably and i t was concluded that the effectiveness of each l i n e as a male and female parent must be determined for a true c r i t e r i o n of combining a b i l i t y .  In addition, i t was  found that, i n contrast to other v a r i e t i e s , the seed set after controlled cross-pollination did not greatly exceed the seed set after s e l f - p o l l i n a t i o n .  ACKNOWLEDGMENTS  The w r i t e r w i s h e s t o acknowledge w i t h deep a p p r e c i a t i o n t h e keen i n t e r e s t and i n f o r m a t i o n  regarding  Rhizoma a l f a l f a s u p p l i e d by Dr. G. G. Moe, Eead o f t h e Department o f Agronomy, U n i v e r s i t y o f B r i t i s h Columbia; t h e v a l u a b l e a s s i s t a n c e and c r i t i c i s m s i n c o n n e c t i o n  with the  f e r t i l i t y and p o l l i n a t i o n s t u d i e s r e n d e r e d by Mr. M. F. C l a r k e , S e n i o r A s s i s t a n t , Dominion E x p e r i m e n t a l Farm, A g a s s i z ; and t h e i n t e r e s t and c y t o l o g i c a l d a t a s u p p l i e d by Dr. A. H i H u t c h i n s o n , Department o f B i o l o g y and Botany, U n i v e r s i t y o f B r i t i s h Columbia.  Thanks a r e a l s o extended t o  Dr. D. C. Cooper, Department o f G e n e t i c s , W i s c o n s i n , and t o Mr.  University of  J . Armstrong, C e n t r a l Experimental  Farm, Ottawa, f o r t h e i r i n f o r m a t i o n r e g a r d i n g t h e c y t o l o g i c a l s t u d i e s , and t o Dr. P. T r u s s e l and Mr.  Guy Palmer o f t h e  B r i t i s h Columbia Research C o u n c i l f o r t h e i r assistance i n connection  valuable  w i t h the photomicrographs.  The w r i t e r e s p e c i a l l y w i s h e s t o e x p r e s s h i s s i n c e r e thanks t o D r . 7. C. B r i n k , Department o f Agronomy, U n i v e r s i t y o f B r i t i s h Columbia, f o r h i s i n s p i r i n g c r i t i c i s m s and d i r e c t i o n , and i n v a l u a b l e a i d w h i c h have been g i v e n so f r e e l y throughout t h e s e s t u d i e s .  TABLE OF CONTENTS INTRODUCTION  1  A. CYTOLOGICAL STUDIES OF MEDICAGO SATIVA AND MEDICAGO FALCATA, AND OF THE S I X F  x  HYBRIDS,  PARENT STOCK OF RHIZOMA ALFALFA  3  C y t o l o g i c a l r e s u l t s - drawings - photomicrographs. B. SELF-POLLINATION, OPEN^POLLINATION,  9  CROSS-FERTILITY  AND COMBINING ABILITY, AND EASE OF FLORET TRIPPING, IN PROMISING LINES OF RHIZOMA ALFALFA PARENT STOCK 16 I  SELF-POLLINATION AND OPEN-POLLINATION S e l f - p o l l i n a t i o n studies Open-pollination studies S e l f - and - o p e n - p o l l i n a t i o n compared  16 21 21 27  I I EASE OF FLORET TRIPPING AND OPENPOLLINATION  33  I I I COMBINING ABILITY Test o f e f f e c t i v e n e s s o f a l c o h o l e m a s c u l a t i o n method. Combining a b i l i t y r e s u l t s I V SELF-AND CROSS-FERTILITY COMPARED  39 41 56  SUMMARY AND CONCLUSIONS  59  LITERATURE CITED  63  1. INTRODUCTION  Rhizoma, a new  a l f a l f a v a r i e t y possessing a d i s -  t i n c t i v e s p r e a d i n g h a b i t , h a s been r e l e a s e d t h i s y e a r ,  1948,  by t h e Department o f Agronomy, U n i v e r s i t y o f B r i t i s h Columbia.  The  o r i g i n o f t h i s a l f a l f a i s unique i n a l f a l f a  improvement, s i n c e i t was  developed  from a n a t u r a l c r o s s  between two s p e c i e s , Medioago f a l o a t a ( v a r . Don)  and  Medicago s a t i v a ( v a r . Grimm o r O n t a r i o V a r i e g a t e d ) .  For  t h e h i s t o r y o f the c r o s s t h e r e a d e r i s r e f e r r e d t o the w r i t i n g s o f Moe  (1928) and Rogers (1941).  I t i s relevant,  however, t o note t h a t rows o f Grimm o r O n t a r i o V a r i e g a t e d (the p o l l e n p a r e n t s ) o r b o t h were grown b e s i d e the (the seed p a r e n t ) .  Don  From t h e l a t t e r , two hundred and  seeds were c o l l e c t e d and grown.  fifty  O n l y s i x p l a n t s were  s e l e c t e d t h a t showed d e f i n i t e h y b r i d i t y . From t h e s i x h y b r i d s a b r e e d i n g program, founded m a i n l y on mass s e l e c t i o n , was  initiated.  Rhizoma r e p r e s e n t s  s e l e c t i o n over s i x g e n e r a t i o n s . Rhizoma d i f f e r s i n growth h a b i t s and  breeding  b e h a v i o r from o t h e r commercial v a r i e t i e s o f a l f a l f a . anomaly i n p h e n o t y p i c  This  e x p r e s s i o n i s ; p o s s i b l y the e f f e c t of  i t s u n u s u a l o r i g i n and, more d i r e c t l y t h e e f f e c t o f c h r o mosomal i r r e g u l a r i t i e s .  To f i n d t h e r e a s o n f o r such  irregufarADenavioi*, t h e most l o g i c a l c o u r s e i s , t h e r e f o r e , t o s t u d y t h e chromosomes concerned.  To t h i s  end,  e y t o l o g i c a l i n v e s t i g a t i o n s were conducted a t t h e U n i v e r s i t y  o f B r i t i s h Columbia on t h e p a r e n t s and t h e F  1  h y b r i d s from  w h i c h Rhizoma was d e v e l o p e d . Another approach t o s o l v i n g t h e i r r e g u l a r i t i e s b r e e d i n g b e h a v i o r o f Rhizoma was a f f o r d e d at t h e Dominion E x p e r i m e n t a l Farm, A g a s s i z , B r i t i s h Columbia, where pollination  and f e r t i l i t y s t u d i e s were conducted on  selected c l o n a l l i n e s of the F  4  generation.  of  3. PART A:  CYTOLOGICAL STUDIES OF MEDICAGO SATIVA MEDICAGO FALCATA, AND  OF THE  SIX F  x  AND  HYBRIDS,  PARENT STOCK OF RHIZOMA ALFALFA.  A l t h o u g h Rhizoma a l f a l f a has been under d e v e l o p ment f o r a number o f y e a r s , i t s o y t o l o g i c a l b e h a v i o r not been f u l l y i n v e s t i g a t e d . a hybrid of  M.  f a l o a t a x M.  i s t i c s of both species.  has  As p r e v i o u s l y s t a t e d , i t i s sativa  possessing  character-  To what e x t e n t each s p e c i e s  has  c o n t r i b u t e d t o i t s g e n e t i c and hence p h e n o t y p i c c h a r a c t e r i s t i c s , however, has been a m y s t e r y . S i n c e i t i s a h y b r i d from a c r o s s between two s p e c i e s i t s c y t o l o g i c a l b e h a v i o r m i g h t w e l l be irregular.  quite  An i n d i c a t i o n t h a t t h i s s u p p o s i t i o n i s c o r r e c t  i s g a t h e r e d from t h e p e c u l i a r i t i e s w h i c h e x i s t i n t h e phenotypic behavior of t h i s a l f a l f a . Moe  For  instance,  (19S8) r e p o r t s v e r y low s e l f - f e r t i l i t y but a h i g h  inter cross-fertility  i n t h e F^ h y b r i d s .  a t i o n s , however, were q u i t e s e l f - f e r t i l e .  Subsequent generThroughout t h e  b r e e d i n g program s i m p l e M e n d e l i a n s e g r e g a t i o n s been o b s e r v e d f o r any m o r p h o l o g i c a l  factors.  have not Odland  and  L e p p e r (1939) s t u d y i n g t h e i n h e r i t a n c e o f f l o w e r c o l o u r i n t h i s a l f a l f a have h o t been,able t o p l a c e t h e on a s i m p l e M e n d e l i a n b a s i s .  segregations  I n a d d i t i o n , Moe  (1928) has  n o t e d dwarf and odd t y p e p l a n t s s e g r e g a t i n g i n t h e generation. I t has been viewed f o r some time t h a t  the  F  2  4. p e c u l i a r i t i e s i n the phenotypie behaviour o f t h i s  alfalfa  might be r e s u l t i n g from c y t o l o g i e s ! i r r e g u l a r i t i e s .  To  e x p l o r e t h i s p o s s i b i l i t y i n v e s t i g a t i o n s i n t o the. c y t o l o g y o f t h e p l a n t s o f t h e two s p e c i e s i n v o l v e d i n t h e o r i g i n a l c r o s s and, o f t h e s i x h y b r i d s were conducted a t thw U n i v e r s i t y o f B r i t i s h Columbia from 1946 t o 1948. P h e n o t y p i e anomalies such as t h o s e r e p o r t e d i n Rhizoma s t o c k have been o b s e r v e d i n i n t e r s p e c i f i c h y b r i d s w i t h i n o t h e r genera.  Through i n v e s t i g a t i o n s o f t h e p a r e n t  s p e c i e s and t h e i r h y b r i d s i t has been found t h a t o f t e n such p e c u l i a r i t i e s a r i s e when t h e s p e c i e s had d i f f e r e n t chromosome numbers, examples o f w h i c h a r e r e p o r t e d i n N i c o t i a n a by C l a u s e n (1928b), Goodspeed and C l a u s e n (1927a and b) and E a s t (1928), and i n T r i t i c u m by Watkins (1924). H y b r i d s from c r o s s e s between t h e s p e c i e s M.  falcata  and M. s a t i v a have been produced and s t u d i e d w i t h a v i e w t o agronomic u s e f u l n e s s by O l i v e r (1913), Hagen (1919), Hansen (1909), Waldron (1920) and W i t t e (1921).  More  r e c e n t l y , Ledingham (1939) has examined t h e c y t o l o g y o f h y b r i d s a r i s i n g from c r o s s e s between a d i p l o i d M.  falcata  (S e 16) and a t e t r a p l o i d M. s a t i v a (S * 3 2 ) . When t h e f a l c a t a was t h e female p a r e n t he o b t a i n e d seventeen somewhat s i m i l a r h y b r i d s whose chromosome numbers were 32. The f e r t i l i t y o f t h e s e p l a n t s was l o w .  To e x p l a i n t h e  t e t r a p l o i d h y b r i d s he suggested t h a t e i g h t e x t r a  chromosomes  had come from t h e d i p l o i d p a r e n t t h r o u g h t h e f o r m a t i o n o f unreduced gametes.  5.  When t h e c a l oat a was t h e male p a r e n t , Ledingham o b t a i n e d o n l y two h y b r i d s t h a t were u n l i k e eaoh o t h e r and whose chromosome numbers were 24.  On s e l f i n g and  c r o s s i n g t h e s e h y b r i d s , no seeds were o b t a i n e d . N i l s s o n and A n d e r s s o n (1941) and J u l e n (1943) h a v e . s t u d i e d " d i p l o i d " (S  s  3 2 ) * " t e t r a p l o i d " (S = 64) and  " t r i p l o i d " (S = 48) a l f a l f a p l a n t s .  The " t e t r a p l o i d s "  were produced from c o l c h i c i n e t r e a t m e n t s o f s a t i v a and U l t u n a a l f a l f a d e r i v e d from t h e c r o s s M. s a t i v a x M. falcata.  The " t r i p l o i d s " o r i g i n a t e d when " t e t r a p l o i d "  s a t i v a and " d i p l o i d " U l t u n a were c r o s s e d .  The  "triploids"  were more v i g o r o u s t h a n t h e " d i p l o i d s " o r " t e t r a p l o i d s " . The p o l l e n f e r t i l i t y o f t h e " t r i p l o i d s " and " t e t r a p l o i d s " was s a t i s f a c t o r y and t h e w o r k e r s e o u l d f i n d no c o r r e l a t i o n between m e i o t i c d i s t u r b a n c e s and p o l l e n f e r t i l i t y . Preliminary investigations into the cytology of t h e s a t i v a and f a l c a t a p a r e n t s and t h e s i x h y b r i d s from w h i c h Rhizoma has been developed  have been conducted  by  R o l l i d a y (1932),and H u t c h i s o n and F a r l e y ( u n p u b l i s h e d d a t a ) . A l t h o u g h , no doubt, c o n s c i e n t i o u s l y d e r i v e d a t a t i m e when t h e c y t o l o g y o f a l f a l f a was v e r y new, H o l l i d a y * s r e s u l t s n  appeared t o be q u i t e u n r e l i a b l e .  H u t c h i s o n and F a r l e y  found t h e gametic p r o d u c t i o n o f t h e h y b r i d s t o be q u i t e i r r e g u l a r s i n c e gametes were b e l i e v e d t o c o n t a i n from e i g h t t o s i x t e e n chromosomes. *The s i d e r e d by t h e t h i s continent w i t h which the  b a s i c chromosome number o f a l f a l f a i s conS c a n d i n a v i a n workers t o be 16. Workers on c o n s i d e r 8 t o be t h e b a s i c chromosome number w r i t e r i s i n accord.  6. The p l a n t s o f t h e two s p e c i e s o f Medicago t h a t were c r o s s e d a t t h e U n i v e r s i t y o f B r i t i s h Columbia d i f f e m a considerably i n habit of growth. was a v e r y l o w g r o w i n g p l a n t ,  The f a l c a t a  characterized  ( v a r . Don)  by s h o r t  semi-  e r e c t stems i n a t h i c k matted growth w i t h rhizomes coming o f f about two o r t h r e e i n c h e s below t h e s u r f a c e o f t h e g r o u n d , and b r e a k i n g t h r o u g h t h e s u r f a c e a t v a r y i n g distances  from t h e c r o w n .  The p l a n t s produced y e l l o w  f l o w e r s and were c o n s i d e r e d t o be q u i t e  self-sterile.  I t might be n o t e d i n p a s s i n g , t h a t F r y e r  (1930),  i n h i s s t u d i e s on Medicago f a l c a t a has found t h r e e  strains  to e x i s t i n North America.  I n s t r a i n s I and I I t h e  somatio chromosome number was 3E, w h i l e i n s t r a i n I I I i t was 16.  S t r a i n s 1 . a n d I I I were p r o s t r a t e  and bore f a l c a t e -  shaped p o d s , w h i l e s t r a i n I I was e r e c t i n growth h a b i t b u t had f a l c a t e p o d s . The s a t i v a p a r e n t ,  a l t h o u g h not d e f i n i t e l y known  whether i t was Grimm o r O n t a r i o V a r i e g a t e d , had e r e c t , f a i r l y l o n g stems and produced no r h i z o m e s .  The f l o w e r  c o l o u r c o u l d have been e i t h e r p u r p l e o r v a r i e g a t e d . S e v e r a l workers have r e p o r t e d t h a t t h e chromosome number o f M . s a t i v a i s 32; Reeves (1930) and F r y e r I t i s o f importance t o n o t e h e r e ,  (1930).  t h a t v e r y o f t e n Grimm and  Ontario Variegated are placed i n the s p e c i e s "Medicago media".  There i s l i t t l e doubt t h a t ' l l , m e d i a " a r o s e by  n a t u r a l h y b r i d i z a t i o n between M . s a t i v a and M . f a l c a t a , and hence i s a n a l l o p o l y p l o i d .  S i n c e a sound b a s i s f o r t h e  7. s e p a r a t i o n o f t h e s p e c i e s "media" and s a t i v a does not the d e s i g n a t i o n M. The  s a t i v a i s used i n t h e s e s t u d i e s .  s i x h y b r i d s produced from t h e  c r o s s have been s p e c i f i e d as H - 7, H-56, H-190.  Although  exist,  interspecific  H-68,  H-71,  complete d e s c r i p t i o n s o f them a r e  H-156,  not  a v a i l a b l e t h e y d i d e x h i b i t a c h a r a c t e r i s t i c s o f both  parents.  F o r i n s t a n c e , t h e y a l l produced r h i z o m e s an e x p r e s s i o n o f the f a l c a t a parent.  A p a r t from a s l i g h t procumbent h a b i t o f  t h e stems, however, the t o p growth o f the h y b r i d s r e s e m b l e d the s a t i v a p a r e n t .  The  f l o w e r c o l o u r was  predominantly  variegated, a c h a r a c t e r i s t i c of h y b r i d i t y or introduced the s a t i v a  by  parent. The h y b r i d s d i f f e r e d q u i t e d e f i n i t e l y i n r e s p e c t  t o seed s e t and rhizome p r o d u c t i o n .  On r e p e a t e d  selfings,  t h e p l a n t s vere d e f i n i t e l y s t e r i l e , but when i s o l a t e d intercrossed freely. f o l l o w e d by H-190  H y b r i d 7 was  and H-156.  they  t h e h i g h e s t seed s e t t e r  H y b r i d 71, however,  was  o u t s t a n d i n g l y low i n t h i s r e s p e c t (Eek - 1943). Concerning  rhizome p r o d u c t i o n , H-156  and  H-190  produced abundant rhizomes and hence were e x c e l l e n t s p r e a d e r s . On the o t h e r hand, H-7  and i t s progeny produced so  rhizomes t h a t t h e y were e n t i r e l y d i s c a r d e d from the b r e e d i n g program.  few fihizoma  I n a d d i t i o n , a l l h y b r i d s showed s l i g h t  d i f f e r e n c e s i n r e s p e c t t o stem c o l o u r , pubescence and shape of stipule. For the c y t o l o g i c a l i n v e s t i g a t i o n s , clones of the o r i g i n a l f a l c a t a p a r e n t , o f a Grimm p l a n t , and o f t h e s i x  8. h y b r i d s were grown i n the greenhouse from 1946 t o  1948.  Bud m a t e r i a l , f o r the s t u d y o f t h e chromosomes d u r i n g m e i o s i s i n p o l l e n mother c e l l s was c o l l e c t e d and f i x e d - i n C a r n o y ' s medium (6 : 2:2) s t o r e d i n 80% a l c o h o l .  f o r two h o u r s , t h e n washed and  Root t i p s f o r t h e somatic chromos-  ome counts were t a k e n from c u t t i n g s o f the c l o n e s w h i c h had been s e t out i n sand t r a y s .  They were f i x e d i n Carney*s  medium ( 6 : 3 : 1 ) f o r t w e n t y - f o u r h o u r s , and washed and s t o r e d i n 95% a l c o h o l .  I n a d d i t i o n , rhizome t i p s were c o l l e c t e d  f o r t h e same purpose as and t r e a t e d s i m i l a r l y t o t h e r o o t tips.  The most s a t i s f a c t o r y p r e p a r a t i o n s were o b t a i n e d by  u s i n g t h e a c e t o - o a r m i n e smear t e c h n i q u e .  Many p a r a f f i n  s e o t i o n s o f t i p s and buds were s t a i n e d w i t h G e n t i a n V i o l e t , but due t o t h e d i f f i c u l t i e s encountered t h i s method was soon abandoned. The p r e p a r a t i o n s were examined under a b i n o c u l a r Reichart microscope. X 1620 and X 3550.  Camera l u c i d a drawings were made a t P h o t o m i c r o g r a p h s were t a k e n , u s i n g a  Bausch and Lomb m i c r o s c o p e , a t X 9 8 0 . S i n c e t h e chromosomes o f t h e a l f a l f a were so s m a l l and l a c k i n g i n i n d i v i d u a l i t y d u r i n g m e i o s i s , t h e d e t e r m i n a t i o n o f the m e i o t i c b e h a v i o u r and chromosome numbers was very d i f f i c u l t .  O f t e n many p r e p a r a t i o n s were s t u d i e d b e f o r e  a s u i t a b l e chromosome f i g u r e c o u l d be f o u n d .  To add t o t h e  d i f f i c u l t i e s , t h e s t a i n i n g o f t h e margdinsoaboutbfche C h r o mosomes o f t h e h y b r i d s d u r i n g m e i o s i s t o o k on a f u z z y appearance w h i c h d i d n o t appear i n s i m i l a r m a t e r i a l o f  the  9  parents.  The f u z z i n e s s o f t h e s t a i n i n g m i g h t have been an  i n d i c a t i o n t h a t c h r o m a t i n was d i f f u s i n g t o o r from t h e ' chromosomes o f t h e h y b r i d s d u r i n g m e i o s i s , a c h a r a c t e r i s t i c which m i g h t have been t h e e f f e c t s o f h y b r i d i t y . I t was i m p o s s i b l e t o d i s t i n g u i s h t h e f a l c a t a and s a t i v a ohromosomes i n t h e h y b r i d s . A summary o f t h e b e h a v i o u r  during  and t h e chromosome counts f o r t h e p a r e n t s  microsporogenesis  and the s i x h y b r i d s  i s as f o l l o w s : M« s a t i v a (Grimm) - A l t h o u g h  not c e r t a i n t h a t t h i s  i s t h e male p a r e n t , t h e r e s h o u l d be l i t t l e ence between t h e c y t o l o g i c a l b e h a v i o u r Ontario Variegated.  differ-  o f i t and  I t s me&tio b e h a v i o u r  is fairly  r e g u l a r w i t h t h e f o r m a t i o n o f 16 l o o s e l y a s s o c i a t e d b i v a l e n t s a t l a t e d i a k e n e s i s ( F i g u r e 2 and photo 2 ) . Some c e l l s , however, were observed were o n l y 14 b i v a l e n t s c o u l d be found a t l a t e  diaken-  esis (figure 12). M. f a l c a t a (Don) - T h i s v a s Sound t o be a d i p l o i d a l f a l f a c o n t a i n i n g 16 chromosomes  ( f i g u r e 1 and  p h o t o 1 ) . E i g h t p a i r s c o u l d e a s i l y be d i s t i n g u i s h e d and i n d i v i d u a l i t y i n t h e chromosome p a i r s c o u l d be recognized.  From p r e l i m i n a r y i n v e s t i g a t i o n s , t h e  m e i o s i s a t F.M.O. was q u i t e r e g u l a r . HyBrlrd 7 - I t s m e i o s i s was i r r e g u l a r w i t h an une q u a l r a t e o f d i v i s i o n i n t h e second anaphase ( f i g u r e 7 ) . T h i s r e s u l t e d i n 16 chromosomes  going  PLATE  I  PLATE I  F i g u r e s 1-18 a r e oamera l u e i d a drawings o f m l o r o s p o r o g e n e s i s and s o m a t i c chromosomes o f Don (Medlcago f a l c a t a ) Grimm (Medlcago s a t i v a ) and o f t h e s i x F i h y b r i d s f r o m a e e t o carmine smears" M a g n i f i c a t i o n reduced \ to- X 8I.D f o r f i g u r e s 3, 1 £ , and 16 and t o X 17 7 5 f o r the remaining figures.  Figure 1.  Don (M. f a l o a t a ) ,  s o m a t i c chromosomes, S -  16.  Figure 2.  Grimm ( M . s a t i v a ) * m e i o s i s . l a t e second anaphase, 16 and 16.  Figure 3.  H - 6 8 , s o m a t i c chromosomes, S -  Figure 4.  H - 1 9 0 , m e i o s i s , second anaphase, 16 and 1 6 .  Figure 5.  H-190, s o m a t i c chromosomes, S s  32.  F i g u r e 6.  H-156, s o m a t i c chromosomes, S r  32.  F i g u r e 7.  H - 7 , m e i o s i s , second anaphase, u n e q u a l r a t e o f d i v i s i o n , l a g g i n g o f chromosomes, p o s s i b l y 16 and 8 . -  Figure 8.  H - 5 6 , m e i o s i s , l a t e second anaphase, a f t e r u n e q u a l d i s t r i b u t i o n o f chromosomes, l a g g i n g o f chromosomes.  Figure 9.  H - 7 1 , meiosi3, f i r s t anaphase, 16 and 1 6 .  24.  PLATE  II  PLATE I I  F i g u r e 10•  H - 6 8 , m e i o s i s , l a t e f i r s t metaphase, nucleolus persistent.  Figure 11.  H - 6 8 , m e i o s i s , l a t e anaphase, b i v a l e n t a t one p o l e .  Figure 12.  Grimm, m e i o s i s , lent s.  Figure 13.  H-7, meiosis, univalents.  F i g u r e 14.  H - 5 6 , m e i o s i s l a t e f i r s t anaphase, l a g g i n g chromosomes and m i c r o n u c l e i .  Figure 15.  H-7, meiosis, trivalent  f i r s t metaphase,  F i g u r e 16.  H-7, meiosis, somes.  e a r l y prophase,  F i g u r e 17.  H-190, m e i o s i s ,  F i g u r e 18.  H-7, meiosis,  dividing  late diakenesis,  14 b i v a -  f i r s t metaphase, b r i d g e s and  chiasmata, prochromo-  late diakenesis,  e a r l y prophase,  chiasmata  two n u c l e o l i .  10. t o each p o l e o f o n e - h a l f o f t h e c e l l and o f a s m a l l e r number o f chromosomes g o i n g t o t h e p o l e s i n the other h a l f of the c e l l . somes were p r e s e n t .  L a g g i n g chromo-  Chiasmata were u s u a l l y r e g u l a r  but o f t e n t h e r e was an i n d i c a t i o n o f t r i v a l e n t s and b r i d g e s ( f i g u r e s 13 and 1 5 ) .  O f t e n , as i n t h e  o t h e r h y b r i d s , two ( f i g u r e 18) o r t h r e e (photo 5) n u c l e o l i and prochromosomes ( f i g u r e 16) were found i n the e a r l y p r o p h a s e . H y b r i d 56 ~ I t s m e i o s i s was i r r e g u l a r w i t h the f o r m a t i o n o f gametes c o n t a i n i n g u n e q u a l numbers o f chromosomes, u s u a l l y around t w e l v e ( f i g u r e  8),  l a g g i n g chromosomes and m i c r o - n u c l e i ( f i g u r e  14).  H y b r i d 68 - The s o m a t i c chromosome number was f o u n d t o be 2 * - ( f i g u r e 3 ) .  M e i o s i s was i r r e g u l a r  w i t h p e r s i s t i n g n u c l e o l i ( f i g u r e 10) and u n d i v i d e d b i v a l e n t s g o i n g t o one p o l e ( f i g u r e  11).  H y b r i d 71 - M e i o s i s was r e g u l a r w i t h 16 b i v a l e n t s d i v i d i n g a t f i r s t metaphase  (figure  9).  H y b r i d 156 - The somatic chromosome number was found t o be 32 ( f i g u r e 6) and (photo 4 ) .  Meiosis  was r e g u l a r w i t h e q u a l d i s t r i b u t i o n o f chromosomes i n t h e second d i v i s i o n . H y b r i d 190 - The somatio chromosome number was 32 ( f i g u r e 5 and photo 3 ) .  M e i o s i s was r e g u l a r w i t h  the formation of normal chiasmata ( f i g u r e 17),  and  an e q u a l d i s t r i b u t i o n o f ohromosomes i n t h e second division (figure  4).  PLATE  III  PLATE  III  Photos  1-5  P h o t o m i c r o g r a p h s o f m i o r o s p o r o g e n e s i s and somatio chromosomes o f Don (M. f a l c a t a ) , Grimm (M. s a t i v a and o f h y b r i d s 7, 156 and 190.  Photo 1 .  Don (M. f a l c a t a ) ,  s o m a t i c chromosomes,  Photo 2 .  Heo Grimm, m e i o s i s , d i a k e n e s i s ,  Photo 3 .  H-190 somatio chromosomes,  Photo 4 .  H - 1 5 6 , s o m a t i c chromosomes, S . s  Photo 5 .  H - 7 , m e i o s i s , e a r l y prophase, three n u c l e o l i .  S r  16  S  =  16.  bivalents.  32. 32.  11. An o u t s t a n d i n g f e a t u r e t h a t one o f t h e p a r e n t s ,  of t h i s study i s the  namely the female f a l c a t a ,  o n e - h a l f the chromosome complement o f the s a t i v a  fact  had o n l y  parent.  The p a r e n t s t o c k o f Rhizoma, t h e n , not o n l y a r o s e from an i n t e r s p e c i f i c c r o s s hut a l s o from a c r o s s where a s p e c i e s had d i f f e r e n t chromosome numbers.  Such an o r i g i n f o r an  a l f a l f a v a r i e t y i s q u i t e u n u s u a l on t h i s c o n t i n e n t  since  most o t h e r commercial v a r i e t i e s have stemmed from f o r m e r v a r i e t i e s and s t r a i n s .  I n p a s s i n g i t s h o u l d be m e n t i o n e d ,  t h a t a new v a r i e t y , N a r a g a n s e t t ,  d i s t r i b u t e d by the  Rhode I s l a n d E x p e r i m e n t a l S t a t i o n ,  and r a t h e r s i m i l a r t o  Rhizoma, can b o a s t o f t h i s unique o r i g i n .  The s t o c k from  w h i c h i t was produced i s b e l i e v e d t o have been developed from the s i x U.B.C. h y b r i d s . Erom a c r o s s between a d i p l o i d and a t e t r a p l o i d , t r i p l o i d h y b r i d s would n o r m a l l y be e x p e c t e d .  Ledingham  (1940) o b t a i n e d t r i p l o i d s o n l y when t h e d i p l o i d p a r e n t was t h e m a l e , but o b t a i n e d t e t r a p l o i d s when t h e d i p l o i d was t h e female. The s t u d i e s o f the s i x ^ and H-68 were t r i p l o i d s .  indicate that H-7, H-56,  ;  T h e i r m e i o t i c b e h a v i o u r was q u i t e  I r r e g u l a r w i t h l a g g i n g chromosomes and f o r m a t i o n o f gametes o f unequal numbers o f chromosomes.  These i r r e g u l a r i t i e s  can be e x p l a i n e d on t h e b a s i s o f t h e 8 chromosomes  of  f a l c a t a forming u n i v a l e n t s o r t r i v a l e n t s w i t h t h e 16 chromosomes o f s a t i v a a t f i r s t metaphase.  I t was p o s s i b l e f o r any  number o f the f a l c a t a chromosomes t o t r a v e l fio one p o l e o r  12. t o the o t h e r ?  O f t e n , as supposed i n H - 7 , the 8 f a l c a t a  chromosomes would go t o one p o l e r e s u l t i n g i n o n e - h a l f o f the c e l l h a v i n g 16 chromosomes w h i l e t h e o t h e r h a l f had o n l y 8 s a t i v a chromosomes.  I n t h i s c a s e , t h e d i v i s i o n i n second  anaphase would be u n e q u a l w i t h t h e h a l f c e l l c o n t a i n i n g t h e fewer chromosomes d i v i d i n g f i r s t .  F i n a l l y , f o u r c e l l s , would  be p r o d u c e d , two o f w h i c h would be n o r m a l and two t h a t would be s m a l l s i n c e t h e l a t t e r c o n t a i n e d O n l y 8 chromosomes. I t i s i n t e r e s t i n g to note that p r e l i m i n a r y p o l l e n s t u d i e s c a r r i e d o u t by W i l s o n S t e w a r t  (unpublished data)  showed some h y b r i d s t o c o n t a i n a few p o l l e n c e l l s w h i c h were o n l y h a l f t h e s i z e o f the normal p o l l e n g r a i n s .  These  reduced g r a i n s were not shrunken and appeared q u i t e f e r t i l e . That h y b r i d s 7 1 , 156,  and 190 were t e t r a p l o i d s  i s another noteworthy r e s u l t of t h i s study.  These h y b r i d s ,  t h e n , must c o n t a i n 16 chromosomes from t h e f a l c a t a  parent.  The o n l y s u i t a b l e e x p l a n a t i o n f o r t h e i n c r e a s e d number o f chromosomes coming from t h e d i p l o i d p a r e n t i s on t h e assumption t h a t i t formed unreduced gametes.  That t h e i r  m i i o t i o b e h a v i o u r was q u i t e r e g u l a r was v e r y f e a s i b l e ,  since  i t was t h e n p o s s i b l e f o r a u t o s y n d e t i c p a i r i n g o f the 16 f a l c a t a and o f t h e 16 s a t i v a chromosomes. H y b r i d i t y i n the'F-^ p l a n t s was a l s o demonstrated by the f o r m a t i o n o f prochromosomes o r c h r o m a t i n masses, and o f two o r t h r e e n u c l e o l i i n e a r l y p r o p h a s e .  Reeves ( 1 9 3 0 ) ,  i n h i s s t u d y o f Grimm, found o c c a s i o n a l l y two o r more n u c l e o l i , but u s u a l l y o n e .  S i n c e the number o f n u c l e o l i i s  IS. u s u a l l y an i n d i c a t i o n o f t h e number o f chromosome genomes, the .appearance o f more t h a n one n u c l e o l i i n the U . B . C . h y b r i d s i s j u s t i f i e d as t h e y c o n t a i n two o r p o s s i b l y t h r e e genomes. Since the hybrids d i f f e r considerably i n t h e i r chromosome numbers, marked d i f f e r e n c e s i n t h e i r p h e n o t y p i c e x p r e s s i o n would be e x p e c t e d .  The t r i p l o i d s w i t h t h e i r  preponderance o f s a t i v a chromosomes s h o u l d more resemble t h e Grimm p a r e n t .  closely  The t e t r a p l o i d h y b r i d s , on t h e  o t h e r h a n d , s h o u l d show more f a l c a t a c h a r a c t e r i s t i c s  than  t h e t r i p l o i d s s i n c e t h e chromosome c o n t r i b u t i o n from each parent i s s i m i l a r . A l t h o u g h t h e s u p p o s i t i o n s are not g e n e r a l l y t h e r u l e i n these h y b r i d s , one p o s i t i v e example can be c i t e d . I t was found t h a t H-7 produced progeny o f which o n l y 25% developed rhizomes (Moe - 1 9 2 8 ) . t h e s e progeny were d i s c a r d e d .  Because o f t h i s  failing,  On t h e o t h e r h a n d , H-156  and H-190 produced progeny t h a t gave an abundanoe o f rhizomes.  From t h e c y t o l o g i o a l s t u d i e s , t h e s e  differences  i n rhizome p r o d u c t i o n on t h e p a r t o f the h y b r i d s and t h e i r progeny would be expected s i n c e H-7 i s a t r i p l o i d , and H-156 and H-190 are t e t r a p l o i d s . The f e r t i l i t y o f t h e h y b r i d s i s a l s o o f interest.  On s e l f i n g , t h e y were s t e r i l e ,  but on i n t e r c r o s s -  i n g under i s o l a t i o n t h e y s e t seed p r o f u s e l y . pect,  considerable  In this  res-  the t r i p l o i d s w i t h t h e i r i r r e g u l a r m e i o t i c  b e h a v i o u r would be e x p e c t e d t o be l e s s f e r t i l e t h a n t h e  14. tetraploids.  I n f a c t , Ledingham (1940) found t h i s t o be  t r u e f o r h i s t r i p l o i d and t e t r a p l o i d h y b r i d s .  The s i x  U . B . O . h y b r i d s , h o w e v e r , d i d n o t behave e x a c t l y i n a c c o r dance w i t h t h e e x p e c t a t i o n .  H y b r i d 7, a t r i p l o i d ,  e x h i b i t e d t h e h i g h e s t f e r t i l i t y , whereas H - 7 1 , a t e t r a p l o i d , was t h e most s t e r i l e .  Nevertheless,  the r e s t o f the hybrids  behaved n o r n a l l y as t h e t e t r a p l o i d s H-156 and H-190 were q u i t e f e r t i l e , w h i l e t r i p l o i d s H-56 and H-68 showed reduced fertility. The " t r i p l o i d s " (S  s  48) s t u d i e d by N i l s s o n and  A n d e r s s o n (1943) and J u l e n (1944) were more f e r t i l e t h a n t h e " d i p l o i d s " (S s 32) and as f e r t i l e as t h e " t e t r a p l o i d s " (S e 6 4 ) .  They c o n c l u d e d from t h e i r s t u d i e s t h a t no c o r r -  e l a t i o n e x i s t e d between m e i o t i c i r r e g u l a r i t y and p o l l e n fertility.  Such a c o n e l u s i o n i s no doubt f e a s i b l e  i n conn-  e c t i o n w i t h p o l y p l o i d s o f s u c h a h i g h chromosome number. U.B.O S i m i l a r l y , i t m i g h t be c o n c l u d e d , t h a t s i n c e the/ h y b r i d s a r e p o l y p l o i d s , the m e i o t i c i r r e g u l a r i t i e s w i t h the r e s u l t i n g u n e q u a l d i s t r i b u t i o n o f chromosomes t o t h e v a r i o u s  cells,  e s p e c i a l l y i n t h e t r i p l o i d s , may not e f f e c t t o o g r e a t l y  their  fertility. A n o t h e r p o i n t t h a t must be remembered when a c c o u n t i n g f o r t h e anomalies i n t h e h y b r i d s i s t h e f a c t t h a t one o f the parents,  namely t h e v a r i e g a t e d s a t i v a i s a h y b r i d o f a  c r o s s between s a t i v a and f a l c a t a and p r o b a b l y c a p a b l e o f p r o d u c i n g gametes o f v a r y i n g chromosome numbers.  Fryer  (1930) found i n h i s s t u d i e s o f a G r i m m - l i k e p l a n t , w h i c h was  15. d e s i g n a t e d as Medieago m e d i a , and w h i c h produced gametes w i t h d i f f e r e n t complements o f chromosomes because o f i r r e g u l a r i t i e s i n i t s meiosis.  H u t c h i n s o n and F a r l e y ( u n p u b l i s h e d  data), i n t h e i r s t u d i e s o f Grimm, found i r r e g u l a r i t i e s Bft a t m e i o s i s and an unequal d i s t r i b u t i o n o f chromosomes t o gametes.  The w r i t e r has p r e v i o u s l y i n d i c a t e d  the  certain  m e i o t i c p e c u l i a r i t i e s i n the Grimm p l a n t s t u d y . A l t h o u g h t h e t r i p l o i d s were p r e s e n t  i n the hybrids  and p r o b a b l y p e r s i s t e d d u r i n g t h e second and t h i r d g e n e r a t i o n , i t i s l i k e l y t h a t t h r o u g h t h e i r ^ i n s t a b i l i t y and s e l e c t i o n f o r the c h a r a c t e r i s t i c s  shown by t h e t e t r a p l o i d s , t h e r e  v e r y l i t t l e t r i p l o i d y present generations.  In faot,  i n the Rhizoma s t o c k o f  Armstrong ( p e r s o n a l  is  later  correspondence)  has found the chromosome number o f c e r t a i n p l a n t s o f t h e F4. g e n e r a t i o n t o be 32. o f 16  M e i o s i s was r e g u l a r w i t h t h e f o r m a t i o n  bivalents. From the e v i d e n c e p r e s e n t e d i t i s p o s s i b l e  to  c o n c l u d e , t h a t t h e new v a r i e t y Rhizoma i s a permanent t e t r a p l o i d h y b r i d : c o n t a i n i n g s i x t e e n chromosomes from the 1  M . f a l c a t a p a r e n t and s i x t e e n chromosomes from the M . s a t i v a parent•  16. PART B ;  S2LF-POLLINATION, OPEN-POLLINATION  t  C ROSS-FE UTILITY  AND COMBINING A B I L I T Y , AND EASE OF FLORET TRIPPING I N PROMISING LINES OF RHIZOMA ALFALFA PARENT STOCK.  E x p e r i m e n t s r e l a t i n g t o the b r e e d i n g b e h a v i o u r o f c e r t a i n promising l i n e s of a l f a l f a , selected  from t h e p a r e n t  s t o c k s o f the Rhizoma v a r i e t y , were conduoted a t the Dominion E x p e r i m e n t a l F a r m , A g a s s l z , B r i t i s h Columbia d u r i n g the summers o f 1946 and 1947.  P r e v i o u s l y , i n 1945, F  4  plants  had been s e n t t o A g a s s i z from the Department o f Agronomy, U n i v e r s i t y o f B r i t i s h Columbia t o form the b a s i s o f a c o o p e r a t i v e b r e e d i n g program f o r t h i s a l f a l f a ,  on a r r i v a l  t h e y were e s t a b l i s h e d a s l i n e s o f t w e n t y - f i v e * c l o n e s . A number o f t h e most p r o m i s i n g l i n e s were  selected  and t h e i r b r e e d i n g b e h a v i o u r determined b y e x p e r i m e n t s on s e l f - p o l l i n a t i o n , o p e n - p o l l i n a t i o n , c r o s s - f e r t i l i t y and c o m b i n i n g a b i l i t y , and ease o f f l o r e t t r i p p i n g .  These  e x p e r i m e n t s and t h e i r r e s u l t s a r e p r e s e n t e d i n t h i s I  report.  SELF-POLLINATION AND OPEN-POLLINATIOH.  I t has been assumed f o r many y e a r s t h a t i n a l f a l f a c r o s s o r o p e n - p o l l i n a t i o n n e a r l y a l w a y s t a k e s p l a c e i n seed formation.  That the a s s u m p t i o n i s j u s t i f i e d seems to be  borne o u t i n r e c e n t s t u d i e s . i n A r g e n t i n a , found t h a t ,  B u r k h a r t ( 1 9 3 7 ) , f o r example,  i n d i f f e r e n t l i n e s , 67% t o 98%  (average 84.5%) o f the seed was the r e s u l t o f c r o a s - p o l l i n ation.  I n N e b r a s k a , T y s d a l , K i e s s e l b a e h and w e s t o v e r  (1942)  17. o b t a i n e d from t h r e e t e s t a o f seed produced under o p e n p o l l i n a t i o n a n average o f 8 9 . 1 $ .  A g a i n , i n Saskatchewan,  u s i n g t e s t p l a n t s t h a t were t r u e b r e e d i n g f o r w h i t e f l o w e r , Knowlea (1943) a n d , more r e c e n t l y , B o l t o n (1948) f o u n d averages o f 9 4 . 2 $ and o v e r 90$ c r o s s i n g  respectively.  While i t appears, to be g e n e r a l l y t r u e t h a t a l f a l f a i s n o r m a l l y c r o s s - p o l l i n a t e d and c r o a s - f e r t i l e , are quite s e l f - f e r t i l e  and under c e r t a i n  many p l a n t s  circumstances  s e l f - p o l l i n a t i o n may o c c u r t o an a p p r e c i a b l e d e g r e e . Knowles (1943) i n a eoramon c o m m e r c i a l v a r i e t y found i n some p l a n t s , a r t i f i c i a l l y s e l f - p o l l i n a t e d , a s e t o f 1.65 seeds p a r f l o r e t , i n o t h e r s o n l y 0.56 s e e d s .  B o l t o n ^ a n d F r y e r (1937) r e p o r t e d  3 . 8 $ and 54.7$ pods p e r f l o r e t s t r i p p e d when s e l f i n g a group of s t e r i l e  p l a n t s and f e r t i l e  plants respectively.  lately,  B o l t o n (1948) r e p o r t s a rang© o f s e l f - f e r t i l i t y f r o m 0 . 0 2 t o 5 . 2 2 w i t h an average o f 1.58 seeds p e r f l o r e t ,  i n addition  he h a s found a s l i g h t s i g n i f i c a n t c o r r e l a t i o n between  self  and c r o s s - f e r t i l i t y . Many w o r k e r s have r e p o r t e d t h a t i n s e c t s , b e e s , are the I n d i s p e n s a b l e of a l f a l f a .  especially  a g e n t s i n the c r o s s - p o l l i n a t i o n  Through an i n s e c t v i s i t the f l o r e t may be t r i p p e d ,  a p r o c e s s i n w h i c h a n t h e r s and s t y l e s a r e l i b e r a t e d f r o m confinement i n the k e e l and s t r i k e a g a i n s t petal.  the s t a n d a r d  I n s t r i k i n g the s t a n d a r d , p o l l e n i s showered o u t , much  o f i t on the v i s i t i n g i n s e c t , stigmas o f other f l o r e t s .  w h i c h may c a r r y i t t o the  A r m s t r o n g and White (19354 r e p o r t  t h a t p o l l e n g e r m i n a t i o n i n t r i p p e d f l o w e r s was 84$ w h i l e i n  18. u n t r l p p e d f l o w e r s i t was o n l y 1 $ .  T y s d a l (1940) c l a i m e d ,  f r o m s t u d i e s i n N e b r a s k a , t h a t f o r the p r o d u c t i o n o f commercial s e e d , a l f a l f a f l o r e t s must be t r i p p e d .  Eecently  the same a u t h o r (1946) r e p o r t s t h a t l e s s t h a n 5$ o f the f l o r e t s s e t seed w i t h o u t t r i p p i n g ,  on the o t h e r h a n d , i t  has been p o i n t e d o u t t h a t , i n some a r e a s , a l f a l f a can s e t seed -.W i t h o u t t r i p p i n g and hence w i t h o u t t h e agency o f  insects.  B r i n k and c o o p e r (1936) r e p o r t t h a t , d u r i n g a p e r i o d o f h o t , d r y weather i n W i s c o n s i n , 34$ f l o r e t s s e t seed b u t o n l y 12$ o f t h e f l o r e t s had bean t r i p p e d ,  i n U t a h , C a r l s o n (1935)  c l a i m s t h a t t r i p p i n g i s n o t e s s e n t i a l i n seed s e t t i n g . The bees most e f f e c t i v e  i n p o l l i n a t i o n b e l o n g t o the  genera M e g a c h i l e ( the L e a f - e u t t e r B e e s ) , Bombus ( t h e Bumble Bees)and Nomia ( t h e A l k a l i B e e s ) . B n g e l b e r t  (1932), Tysdal  (1940) peck and B o l t o n (1941) and Knowles (1943)  There I s ,  however, c o n s i d e r a b l e c o n t r o v e r s y o v e r the e f f e c t i v e n e s s honey bees a s t r i p p i n g a g e n t s . t r i p p i n g b y them i n U t a h .  of  C a r l s o n (1935) r e p o r t s l i t t l e  Knowles (1943) c l a i m s t h a t  p l a y a m i n o r r o l e i n Saskatchewan.  they  T y s d a l (1940) found honey  b e e s t o be i n e f f e c t i v e as t h e y o n l y t r i p p e d 1 t o 2$ o f t h e florets,  on the o t h e r h a n d , Le Jeune and Olson ( 1 9 4 0 ) , Hare  and T a n s e l l (1946) and V a n s e l l and Todd (1946) b e l i e v e honey bees a r e e f f e c t i v e  trippers.  The l a t t e r r e p o r t  that that  the honey bees w h i c h c o l l e c t p o l l e n i n s t e a d o f n e c t a r a r e the valuable t r i p p e r s . That the f a c t o r s o f weather a r e i m p o r t a n t i n t r i p p i n g and seed s e t t i n g h a s been r e p o r t e d b y s e v e r a l w o r k e r s .  Gray  19. (1925), Knowles (1943) and T y s d a l (1946) have f o u n d wind t o be u n i m p o r t a n t .  The two l a t t e r a u t h o r s have f o u n d , however,  r a i n to increase  t r i p p i n g , b u t , s i n c e i t prevented c r o s s -  p o l l i n a t i o n , i t c o u l d n o t , t h e y c o n c l u d e d , be a f a c t o r i n i n c r e a s i n g seed s e t t i n g . i n a l f a l f a p l a n t s o f Rhizoma s t o c k , i t h a s been n o t e d a t A g a s s i z and a t the u n i v e r s i t y o f B r i t i s h Columbia (M©e 1928) some p l a n t s s e t seed a b u n d a n t l y w h i l e o t h e r s s e t seed s p a r i n g ly.  The h i g h seed s e t on some p l a n t s i s v e r y u n u s u a l i n t h a t  the e f f e c t i v e _ p o p u I a t I o n o f bees i s l o w .  I n v i e w o f the work  done elsewhere i t I s n e c e s s a r y t o account f o r the p e c u l i a r i t i e s o f seed s e t t i n g i n s t o c k s o f the I&Izoma v a r i e t y . Of some i n t e r e s t  i n t h i s c o n n e c t i o n would be the r e l a t -  i o n s h i p o f s e l f - p o l l i n a t e d and o p e n - p o l l i n a t e d seed  sets.  m 1946, s i x t e e n l i n e s and i n 1947 t w e n t y - s e v e n l i n e s were s e l e c t e d f o r s t u d y , o f growth and v i g o r .  s e l e c t i o n was m a i n l y based on h a b i t  The seed s e t t i n g a b i l i t y o f the l i n e s  appeared q u i t e v a r i a b l e . The s e l f - p o l l i n a t i o n b e h a v i o u r o f each l i n e was d e t e r mined b y t r i p p i n g two hundred o r more f r e s h f l o r e t s w i t h a t o o t h p i c k and i s o l a t i n g t h e f l o r e t s under v e g e t a b l e parchment b a g s . were m a t u r e .  cookery  The bags were n o t removed u n t i l t h e pods  I t was d i s c o v e r e d t h a t i f the bags were removed  e a r l i e r t o o many pods were knocked o f f .  i n addition, i t  appeared t h a t the seed s e t was n e t i m p a i r e d b y e n c l o s i n g t h e d e v e l o p i n g pods i n l i g h t b a g s ,  i n p a s s i n g i t m i g h t be n o t e d  t h a t K i r k (1927) and S t e w a r t (1934) f o u n d r e a s o n a b l y f a v o r a b l e  so. seed s e t under bags i n i n b r e e d i n g programs. The number o f pods and the number o f seeds were counted f o r each l i n e s e l f e d .  From t h i s d a t a the p e r c e n t  flowers  f o r m i n g p o d s , seeds p e r pod and seeds p e r f l o r e t t r i p p e d were calculated. The o p e n - p o l l i n a t e d seed s e t was d e t e r m i n e d b y a n o c u l a r o b s e r v a t i o n o r r a t i n g ( from 0 t o 10) o f the pod number p e r l i n e i n 1946 and 194? and a l s o b y the average w e i g h t o f seed p e r c l o n e o f each l i n e i n 1 9 4 6 . To c o r r e l a t e  the s e l f and o p e n - p o l l i n a t i o n data the rank  correlation coefficient statistic  W i l o o x o n (1945) was u s e d .  This  i s a n approximate measure and i s i d e a l l y s u i t e d  f o r o c u l a r and o t h e r s e m i - q u a n t i t a t i v e d a t a .  It is really a  t e s t o f the c o n s i s t e n c y o f the r a n k i n g o f l i n e s i n v a r i o u s treatments  i . e . s e l f and o p e n - p o l l i n a t i o n seed s e t .  S i n c e the method i s n o t r e a d i l y a v a i l a b l e i t i s g i v e n here.  The procedure i s a s f o l l o w s :  a c c o r d i n g to t h e i r performance  The l i n e s a r e ranked  i n the t r e a t m e n t s .  f o r m u l a f o r the rank c o r r e l a t i o n c o e f f i c i e n t  a  where n p  P(P+l)  The  i s then a p p l i e d .  Surafrank t o t a l s ) • 3n(p+I)  number o f t r e a t m e n t s number o f l i n e s  12 and 13 a r e c o n s t a n t s <v,2 The J£ r and the rank d i f f e r e n c e (r^"J a r e r e l a1 t e~2 d,in fact  correlation  coefficient  21. T© t e s t whether the r * v a l u e d i f f e r s s i g n i f i c a n t l y front z e r o i t i s compared w i t h the s t a n d a r d e r r o r S. E . .  1  *0 Ratio »  -1  r ^ 1  Self-pollination  studies.  T a b l e s 1 and 2 p r e s e n t the s e l f - p o l l i n a t e d d a t a o f selected  l i n e s f o r 1946 and 1947.  Based on seeds per  the  floret,  s e l f - f e r t i l i t y o f the c l o n a l l i n e s f o r b o t h y e a r s was h i g h l y variable.  N o t e w o r t h y s e l f - f e r t i l i t y o c c u r r e d i n l i n e s 45-3,  45-46 and 45-121 i n 1946, and i n l i n e s 45^-33, 45-46, 45-1G3 and 45-134 i n 1947.  I n 1946 l i n e s 45-45, 45-61 and 45-^84  were c o n s p i c u o u s l y s e l f - f e r t i l e ;  i n 1947 l i n e s 45-22, 45-45,  45-51, 45-73, 45^84 and 45-85 were h i g h l y  S e l f - f e r t i l i t y data f o r both y e a r s , a v a i l a b l e f o r o n l y twelve) l i n e s .  self-fertile.  1946 and 1947, was  T h a t , however, the  f e r t i l i t y of a given plant i s a genetic  self-  characteristic,  i n other words, that i t s r e l a t i v e s e l f - f e r t i l i t y i s a  or constant  y e a r a f t e r y e a r , i s t o be c o n c l u d e d from the v e r y s i g n i f i c a n t r a n k c o r r e l a t i o n c o e f f i c i e n t o b t a i n e d (Table available  1  3) from the  data.  Open-pollination Studies. Tables 4 and 5 p r e s e n t the o p e n - p o l l i n a t e d d a t a f o r the c l o n a l l i n e s f o r 1946 and 1947.  i n 1946 l i n e s 45-3, 45-32,  45-121 and 45-134 s e t abundant seed under o p e n - p o l l i n a t e d c  22.  TA BLE  1  SELF-POLLINA.TION Or SELECTED CLONAL LINES BASED OH PERCENT FLORETS FORMING PODS, SEED PER POD, AND SEED PER FLORET - 1946 LINE FLORETS 45-3 386  PODS 147  SEED 415  % FLORETS FORMING PODS SEEDS PER POD SEEDS PER FI 2.8 1.4 37.8 0.4 2.0 22.4  -10  963  216  -12 - 29  74 130  - 32 - 42  289 509 320 374  449 102 176  95 103  144 120  29.0 25.6  - 45  362  38  59  - 46  612  357  - 61  445  - 64  25.6 25.5  1.3 1.3 1.5  0.3 0.3  1.1  0.4 0.3  3.0.4  1.6  0.1  628  58*3  1.0  44  74  9.8  1.7 1.6  489  131  263  26.7  2.0  0.5  - 84  656  70  85  10.6  0.1  -112 -121  560 261  32 148  34 305  5.7 56.7  1.2 1.0  -134  692  147  259  -150  230  77  -151  195  36  0.1  2.0  0.06 1.0  21.2  1.7  0.5  114  33.4  1.4  0.4  63  18.6  1.7  0.3  23. TABLE  2  SELF-POLLIMTION OF SELECTED CLONAL LINES BASED ON PERCENT FLORETS FORMING PODS, SEED PER POD, AND SEED PER FLORET LINE FLORETS 45-6 200 -8 - 12  200 200  - 15  200 200  - 22  200  - 29  200  PODS 76 77 117 53 64  - 1947  SEEDS % FLORETS FORMING PODS SBED PER POD SEED PER FL< 85 38.0 0.4 ' 1.1 .5 0.6 ) 38.5 1.6 131 58.5 1.6 0.9 191 26.5 0.3 70 1.3 0.6 136 32.0 2.0 34  15.5  1.0  0.1  37  84  18.5  2.2  .04  200  46  44  23.0  0.9  0.2  . 100 100  67  121  33.5  1.8  65  132  32.5  2.0  1.2 1.3  - 39  200  61  78  30.5  1.2  0.3  - 42 - 45  200 200  66  84  33.0  0.4  0  0  0.0  1.2 0.0  - 46  200  90  182  45.0  2.0  0.9  - 51  200  9  9  4.5  1.0  0.04  - 60  200  67  88  38.5  - 61 i 200  40  49  20.0  1.2 1.2  0.4 0.2  - 64  200  67  128  38.5  1.9  0.6  - 73  200  1  0.5  1.0  0.005  - 84 - 85  200  1 15  7.5  0.6  0.05  200  13  10 7  6.5  0.5  0.03  - 90  200  66  83  33.0  1.1  0.4  - 91  200  63  91  31.5  1.4  0.4  - 92  200  65  99  32.5  1.5  - 103  200  90  223  45.0  2.4  0.4 1.0  - 112  200  0  0  0.0  0.0  0.0  - 134  200  47.0 34.0  0.9  200  184 61  1.9  - 150  94 68  0.8  0.3  - 151  200  53  70  26.5  •1.3  0.3  - 33  31  • Lines "selfed" at different dates during the svnnmero f 1947.  0.0  24.  TA BLS  3  SELF-POLLINATION OF SELECTED CLONAL LINES FOR 1946 and 19*7 COMPARED BY THE RANK CORRELATION COEFFICIENT.  LINE 45-12 -29 -42 -45 -46 -61 -64 -84 1946 6.5 6.5 6.5 9.0 1.0 1G.0 2.5 11.0 1947  6.5 6.5  4.0 11.0  Total 13.0 13.0 10.5 20.0  n .  1.5  9.0 3.0 10.0  2.5 19.0 5.5  -112 12.0  -134 -150 -151 2.5 4.0 6.5  12.0  1.5  21.0 24.0 4.0  6.5  6.5  10.5 13.0  2  p • 12 r  1  • .852  S.B.  m  .301  Ratio -  .652 .301  m  2.49  Sinoe the r value is over twice its standard error the correlation is highly 1  significant.  f  25.  TABLE  4  OPEN-POLLINATION OF SELECTED CLONAL LINES BASED ON POD NUMBER VALUE (0 to 10) AND SEED YIELD - 1946.  LINE > -3  °  SEED YIELD (9 ma. Per Clone)  POD NUMBER VALUE P (0-10)  24.4 7.3 3.7  9 6 6  6.6  8  14.6  8 8  -45  3.1 3.3  - 46  9.0  9  - 53  15.0 8.5  10 6 7  - 84  3.1 2.1  -112  3.9  3  -121 -134  16.2  8  14.6  8  -150  8.1  7  -151  5.1  6  - 10 - 12 - 29 - 32 - 42  - 61 - 64  7  3  26. TABLE  5  OPEN-POLLINATION OF SELECTED CLONAL LINES BASED ON POD NUMBER VALUE (0 to 10) - 1947  LINE 45-6 -8 - 12 - 15  POD NUMBER VALUE (0 - 10) 10 9 3 5  - 22 - 29  4  - 33  10  - 39  7 5  - 42 -45  2  5  - 46  8  - 51  3  - 60  8  - 61  4  - 64 - 65  7  - 73 - 84 - 85  3 5  - 90  9  - 91  1  - 92  8  - 105  9  - 112  2  - 115  2  - 134  4  - 150  3  - 151  2  1  2  27. c o n d i t i o n s w h i l e l i n e s 45-64 and 45-84 s a t v e r y l i t t l e  seed,  i n 1947 l i n e s 45-6 and 45-33 were h i g h seed s e t t e r s w h i l e l i n e s 45-65 and 45-91 were l o w . The pod number, a s p r e v i o u s l y r e p o r t e d , was determined u s i n g an o c u l a r s c a l e w i t h u n i t s a r b i t r a r i l y chosen from 0 t o 10;  the seed y i e l d was d e t e r m i n e d b y w e i g h i n g .  Since  the pod number was used i n c o r r e l a t i n g the o p e n - p o l l i n a t e d and s e l f - p o l l i n a t e d seed s e t the v a l i d i t y o f the  ocular  T"  method was checked b y comparing the d a t a f o r twelve? l i n e s o b t a i n e d b y t h a t method i n 1946 w i t h the d a t a o b t a i n e d by the w e i g h i n g method i n the same y e a r f o r the same l i n e s . The methods were compared b y the rank c o r r e l a t i o n  coefficient.  The v a l i d i t y o f the o c u l a r method i s demonstrated by a h i g h l y s i g n i f i c a n t c o r r e l a t i o n v a l u e (Table 6 ) . S e l f - and O p e n - p o l l i n a t i o n S t u d i e s compared. A point of i n t e r e s t  t o the a l f a l f a b r e e d e r i s whether  p l a n t s w h i c h s e t seed r e a d i l y when s e l f - p o l l i n a t e d a l s o seed r e a d i l y when o p e n - p o l l i n a t e d , o r , more  set  extensively,  whether c e r t a i n p l a n t s a r e good seed p r o d u c e r s r e g a r d l e s s the p o l l i n a t i o n regime.  of  A comparison o f the " s e l f e d " and  " o p e n " seed s e t s o f 1946 and 1947 p o i n t s t o an answer i n the affirmative.  ( T a b l e s 7 and 8 ) .  H i g h l y s i g n i f i c a n t rank c o r r e l a t i o n c o e f f i c i e n t s that,  indicate  c l o n a l l i n e s , w h i c h were h i g h l y s e l f - f e r t i l e were a l s o  heavy seed s e t t e r s under o p e n - p o l l i n a t i o n .  B o l t o n (1946)  found a s l i g h t r e l a t i o n i n h i s s t u d i e s between these two factors,  u n d e r these c o n d i t i o n s i t m i g h t be c o n c l u d e d t h a t  28.*  TABLE  6  THE TWO METHODS OF DETERMINING OPEN-POLLINATED SEED SET, POD NUMBER (from 0 to 10) AND SEED YIELD (WEIGHED), COMPARED BY THE RASK CORRELATION COEFFICIENT - 1946  LINE  45-3 -10 -12 -29 -32 -42 -45 -46 -S3 -61 -64 -84 -112 -1ZL—134 -150 -151  Pod No.  2.5 13.5 6.0 6.0 6.0 10.5 2.5 2.5 1.0 13.5 9.0 16.5 16.5 6.0 6.0 10.5 13.5  Seed Yield 1.0  9.0 13.0 10.0 4.0 15.5 14.0 6.0 3.0 7.0 15.5 17.0 12.0 2.0 5.0 8.0 11.0  Total 3.5 24.5 21.5 16.0 10.0 2L5 24.5 8.5 4.0 20.5 24.5 33.5 28.5 8.0 11.0 18.5 24.5  N • 2 P »17 „  .703  JS.E. -  .250  Ratio  -  .703 .250  -  2.81  29. T A BL B  7  SELF-POLLINATION AND OPEN-POLLINATION OF SELECTED CLONAL LINES BASED ON SELF-FERTILITY' AND POD NUMBER VALUE, RESPECTIVELY, CORRELATED BY THE RANK CORRELATION COEFFICIENT 1946.  LINE  45-3 -10 -12 -29 -32 -42 -45 -46 -*61 -64 -84 -112 -121 -134 -150 -151  Pod Ho. Value  1.5 12.5 12.5 5.0 5.0 5.0 9.5 1.5 12.5 8.0 15.5 15.5 5.0  5.0  9.5 12.5  Self Fertility  2.0 7.0 10.5 10.5 7.0 10.5 14.0 2.0 14.0 4.5 14.0 16.0 2.0  4.5  7.0 10.5  Total  3.5 19.5 23.0 15.5 12. 15.5 23.5 3.5 26.5 12.5 29.5 31.5 7  9.5 16.5 23.0  & p  - 2 - 16 r  -  .688  S.E. z  .258  Ratio  .688  1  e  .258  s  2.66  TABLE  8  SELF—POLLINATION AND OPEU-POLLINATION OF SELECTED CLONAL LINES BASED ON SELJ-FERTILITr AND POD NUMBER VALUE, RESPECTIVELY, CORRELATED BY THE RANK CORRELATION COEFFICIENT LINE  45-6 -€  Pod No.  1.5 4.0  .  LINE Pod No.  45-103  20.5 13.5 17.0 24.5 1.5 10.0 13.5 13.5 7.0 20.5 7.0 17.0 10.0 20.5 13.5 24.5 4.0 10.0 7.0  '  6.5 20.0 16.0 1.0 16.0 11.0 25.0 4.0 22.0 11.0 19.0 6.5 24.0 23.0 21.0 11.0 11.0 11.0  •  .  • • • • H I  .  .  .  .  .  i  i  [  i  II • w  •  ••  i  i  i  i II n  i  i  i  i  36.5 20.0 37.0 40.5 2.5 26.0 24.5 38.5 11.0 42.5 18.0 36.0 16.5 44.5 36.5 45.5 15.0 21.0 18.0  -112 -134 -150 «L51 n  4.0  1947  -12 -15 -22 -29 -33 -39 -42 -45 -46 -51 -60 -61 -64 -73 -84 -85 -90 -91 -92  Self Fertil i t y 8.0 4.0 16.0 Total 9.5 8.0  -  24.5  17.0  20.5  24.5  m  2  P - 26 r . .646 •200 S.E. .646 Ratio — 1  Self Fertility  2.0  26.0  Total  6.0  50.5 21.0 36.5 40.5  4.0  16.0  16.0  .200  •i  g •  31. much o f the seed produced by h i g h l y s e l f - f e r t i l e p l a n t s under o p e n - p o l l i n a t i o n was r e a l l y " s e l f e d " s e e d . I t has been n o t e d p r e v i o u s l y t h a t a t A g a s s i z and e t u n i v e r s i t y o f B r i t i s h Columbia the p o p u l a t i o n o f t r i p p i n g i n s e c t s has been v e r y l o w .  the  effective  There have b e e n , however,  l a r g e numbers o f honey bees p r e s e n t d u r i n g the l a s t two summers a t A g a s s i z b u t from r e p e a t e d o b s e r v a t i o n s v e r y l i t t l e t r i p p i n g was done b y them.  The w r i t e r has y e t t o w i t n e s s a  honey bee t r i p p i n g an a l f a l f a f l o r e t .  Instead,  the honey  bees p r o c u r e the n e c t a r b y p e n e t r a t i n g the n e c t a r y a t the base o f the f l o r e t .  V a n a e l l and Todd (1946) have n o t e d t h i s  b e h a v i o u r i n t h e i r " n e c t a r c o l l e c t i n g " honey b e e .  T h i s bee  " i n s e r t e d i t s p r o b i s e u s between the o v e r l a p p i n g w i n g and the s t a n d a r d p e t a l w i t h o u t c o n t a c t i n g the t r i p p i n g mechanism". A t A g a s s i z the bumble b e e s were the e f f e c t i v e there were v e r y few o f them.  t r i p p e r s but  L e a f c u t t e r b e e s were e x t r e m e l y  scarce. I n t h e s e y e a r s when r e l a t i v e l y l i t t l e t r i p p i n g was o b s e r v e d t h e r e would be l i n e s , however, t h a t were l a d e n w i t h pods w h i l e a d j a c e n t l i n e s bore v e r y f e w .  A t no time d i d there appear t o  be enough bumble bees to aeoount f o r the h i g h seed s e t o f former l i n e s .  Moe (1928) has made s i m i l a r o b s e r v a t i o n s  the  in  c o n n e c t i o n w i t h Bhiaoma stock: a t the U n i v e r s i t y o f B r i t i s h Columbia. To c i t e some e x a m p l e s , the o p e n - p o l l i n a t e d d a t a o b t a i n e d a t A g a s s i z i n 1946  ( Table 4) r e v e a l s t h a t l i n e s 4 5 - 3 ,  4 5 - 5 3 , 45-121 and 45-134 were e x c e p t i o n a l l y h i g h seed These l i n e s were l a d e n w i t h p o d s .  45-32, setters.  L i n e 45-3 w i t h i t s 24*4  32.  .  grams o f seed p e r c l o n e had a d j a c e n t to i t l i n e s 45-10  with  7,3 grams per c l o n e and 45-12 w i t h 3 , 7 grams p a r c l o n e . S i m i l a r l y , l i n e 45-121 g i v i n g 1 6 . 2 grams o f seed p e r c l o n e had l i n e s 45-84 and 45-112 n e x t t o i t w i t h o p e n - p o l l i n a t e d seed s e t a o f 2 . 1 grams and 3 . 9  grams p e r c l o n e  respectively.  The s e l f - f e r t i l i t y o f these l i n e s f o l l o w s a s i m i l a r p a t t e r n (Table 1 ) .  i n 1946 l i n e s 45-3 gave 1.4  f l o r e t s e l f e d w h i l e l i n e s 45-10  and 45-12  •3 seeds p e r f l o r e t r e s p e c t i v e l y .  seeds p e r  gave o n l y 14 and  S i m i l a r l y , l i n e 45-121  gave 1.0 seeds p e r f l o r e t » s e l f e d » w h i l e l i n e s 45-112 and 45-84 gave o n l y 0.06 and 0 . 1 seeds r e s p e c t i v e l y .  Similar  examples o f h i g h l y s e l f - f e r t i l e l i n e s s e t t i n g abundant seed under o p e n - p o l l i n a t i o n c o u l d be c i t e d from the d a t a o f  1947.  (Table 2 and 5) It  i s v e r y l i k e l y then that a f a i r l y high percent of  the  Rhizoma o p e n - p o l l i n a t e d seed s e t a t A g a s s i z and a t the u n i v e r s i t y o f B r i t i s h Columbia i s b e i n g a c c o m p l i s h e d w i t h o u t the a i d o f b e e s . - B r i n k and c o o p e r (1936) and C a r l s o n (1935) have r e p o r t e d on a s i m i l a r phenomenon. must l e a d t o s e l f - p o l l i n a t i o n . therefore,  No doubt such a phenomenon  I t c a n f i n a l l y be c o n c l u d e d ,  t h a t under such a c o n d i t i o n the h i g h e r t h e s e l f * *  f e r t i l i t y o f a p l a n t , the g r e a t e r w i l l be i t s o p e n - p o l l i n a t e d seed  set. To f u r t h e r i n v e s t i g a t e t h i s phenomenon i t i s proposed  t h a t a number o f h i g h l y s e l f - f e r t i l e  p l a n t s w i l l be caged and  t h e i r seed s e t d e t e r m i n e d w i t h o u t the a i d o f i n s e c t o r a r t i f i c i a l tripping.  33.  II  BASK OF, FLORET TRIPPING AND OPEN-POLLINATION.  TO account f o r the marked d i f f e r e n c e s  i n open-pollinated  seed s e t o f the c l o n a l l i n e s o f Rhizoma s t o c k a t A g a s s i z i t was reasoned t h a t p o s s i b l y the f l o r e t s were b e i n g t r i p p e d b y bees more f r e q u e n t l y i n c e r t a i n l i n e s t h a n i n o t h e r s .  Further,  t h i s i n c r e a s e d t r i p p i n g i n some l i n e s m i g h t be due t o the f a c t t h a t the f l o r e t s o f those l i n e s were more e a s i l y t r i p p e d b y the b e e s .  I n t h i s c o n n e c t i o n , i t h a s been n o t e d , from h a n d l i n g  the f l o r e t s o f v a r i o u s l i n e s i n the p r o c e s s o f s e l f i n g and c r o s s i n g , t h a t the f l o r e t s o f some l i n e s t r i p p e d much more r e a d i l y than the f l o r e t s o f  others.  i n 1947^ an experiment was conducted t o determine the ease o f t r i p p i n g o f f i f t e e n l i n e s w i t h the purpose o f oomparing the t r i p p i n g data w i t h the o p e n - p o l l i n a t e d seed s e t o f those l i n e s . I n p a r t i c u l a r , the purpose was t o l e a r n i f l i n e s t h a t gave a h i g h seed s e t had f l o r e t s  t h a t were e a s i l y t r i p p e d .  The method o f d e t e r m i n i n g t h e ease o f f l o r e t t r i p p i n g was a m o d i f i c a t i o n o f the a l c o h o l method a s suggested b y Tysdal (1946).  I n h i s s t u d i e s he has f o u n d » a good c o r r e l -  a t i o n between t h i s method and the p h y s i c a l f o r c e r e q u i r e * t o t r i p the f l o w e r s " . F o r t h i s experiment t e n a l c o h o l s o l u t i o n s o f r a n g i n g f r o m 50$-55$*60$  — - 9 5 $ were u s e d .  strengths  F i f t y drops o f  each s o l u t i o n were p l a c e d b y means o f a t o o t h p i c k i n t o the » t h r o a t a » o f f i f t y f r e s h l y matured f l o r e t s . florets  The number o f  t r i p p e d by each s o l u t i o n was r e c o r d e d .  Table 9 p r e s e n t s  the number o f f l o r e t s  t r i p p e d per l i n e  34.  T A B LB 9 BASE OF FLORET TRIPPING DATA OF FIFTEEN SELECTED CLONAL LINES LINE 45-6 -8 - 12  1 2 3 4 5 6 7 8 9 50£ 55#> 60$ 65$ 70# 75# 85# 90# 1 6 4 12 24 16 26 41 44 2 6 5 22 26 40 38 40 35 2 16 25 49 49 49 50 49 49  10 95$ 42 45  -  1947 TRIPPING TRIPPING TOTALS VALUE 216 259  7 5  50  388  3  18 29 45 46 50 50 50 36 43 44 46 45 43 47 19 28 39 40 48 47 49 28 38 45 50 50 50 50  306  5  327 279  4 5  329  4  320 348  5 3  87  9  274 305  6 5  308  3  - 22  0  3 15  - 29 - 39  0 0  4 19 4 5  - 45  0  5 13  - 46 - 60  0 3 12 0 19 27  - 90  0  1  2  22 38 48 48 49 50 50 30 47 41 43 49 47 50 2 3 3 8 24 26 18  - 91  0  6  8  16 17 33 60 46 45 43  - 92  0  7 10  -103  0  7 26  24 29 35 50 50 50 50 21 30 38 44 48 46 48  -134  0  4  8  39 40 38 47 47 50 50  323  4  -150  0  0  0  1 15 18 24 32 34 40  164  8  35. b y each s o l u t i o n .  B y a d d i t i o n the t o t a l number o f  florets  t r i p p e d f o r each l i n e by the t a n s o l u t i o n s was f o u n d . I t was a l s o p o s s i b l e t o a s s i g n to each l i n e a t r i p p i n g value.  Each s o l u t i o n was g i v e n a v a l u e , i . e . 50$ 1 , 95$  10.  The v a l u e o f the weakest s o l u t i o n w h i c h t r i p p e d t w e n t y - f i v e f l o r e t s o r more out o f the f i f t y f l o r e t s t r e a t e d was t h e n the tripping value.  F o r example, the t r i p p i n g v a l u e f o r l i n e  45-8 would be 5 s i n c e 70$ was the weakest a l c o h o l s o l u t i o n t o t r i p t w e n t y - f i v e o r more f l o r e t s .  The t r i p p i n g v a l u e s f o r  the f i f t e e n l i n e s a r e p r e s e n t e d i n Table  9.  By use o f the r a n k c o r r e l a t i o n c o e f f i c i e n t i t was f o u n d , as would be e x p e c t e d , t h a t the t r i p p i n g v a l u e s and the f l o r e t s t r i p p e d c o i n c i d e d v e r y s i g n i f i c a n t l y (Table  total  10).  To l e a r n i f the l i n e s t h a t were h i g h seed s e t t e r s h a d , i n t u r n , f l o r e t s t h a t were e a s i l y t r i p p e d , the o p e n - p o l l i n a t e d seed s e t  ( T a b l e 5) and the t o t a l f l o r e t s t r i p p e d o f the  fifteen  l i n e s were c o r r e l a t e d b y the rank c o r r e l a t i o n c o e f f i c i e n t (Table  (  11).  The f a i r l y h i g h n e g a t i v e c o r r e l a t i o n i n d i c a t e s t h a t  lines  t h a t had f l o r e t s which were e a s i l y t r i p p e d d i d n o t g i v e the h i g h e s t o p e n - p o l l i n a t e d seed s e t .  F o r example, l i n e  45-1S  was the e a s i e s t l i n e to t r i p but was a c o n s i s t e n t l y l o w seed setter.  On the o t h e r hand the most d i f f i c u l t l i n e to  trip,  was a h i g h seed s e t t e r , l i n e 4 5 - 9 0 . These r e s u l t s t e n d to s u p p o r t the p r e v i o u s c o n c l u s i o n t h a t f o r many l i n e s the h i g h seed s e t i s n o t associated with insect v i s i t a t i o n s .  necessarily  36. TABLE  10  TRIPPLIHG VALUE AND TRIPPING TOTAL FOR FIFTEEN CLONAL LINES COMPARED BY THE RANK FORRELATION COEFFICIENT  -  LINE  1947  45-6  -6  -12  -22  -29  Tripping Valua  13  9  2  9  5  Tripping Total  1312  1  8  Total  26  21  3  17  n  -  2  p  -  15 -  .857 .267 .857  rl S.E. Ratio  .267  4 9  -  -39 -45 -46 -60 -90 -91 -92 -103 -134 -150 9 5  9  10  3  6  19  8  15  3.20  2 1 5  2  12  15 4  30  9 2  5  14  1 1 9 7 5 1 4 23  18  9  10  28  37.  TABLE  11  EASE OF FLORET TRIPPING AND OPEN-POLLINATION OF FIFTEEN CLONAL LINES, BASED ON TRIPPING TOTAL AND POD NUMBER VALUE, RESPECTIVELY, CORRELATED BY THE RANK CORRELATION COEFFICIENT.  LINE  45-6 -8 -12 -22 -29- -39 -45 -46 -60 -90 -91 -92 -103 -134 -150  Pod No.  1.0 3.0 13.5 11.5 15.0 8.5 10.0 6.0 6.0 3.0 8.5 6.0 3.0  11.5 13.5  Total 13.0 12.0 1.0 8.0 4.0 10.0 3.0 6.0 2.0 15.0 11.0 9.0 7.0  5.0 14.0  Total 14.0 15.0 14.5 19.5 19.0 18.5 13.0 12.0 8.0 18.0 19.5 15.0 10.0  16.5 27.5  Tripping  a P  - 2 - 15 r  1  S.E. Ratio  -  .448  -  .267  - ^ .448 .267  -  -1.67  38, I I I COMBINING A B I L I T Y . E x p e r i m e n t s were conducted i n 1946 and 1947 a t A g a s s i z t o determine the c o m b i n i n g a b i l i t y o f Rhizoma a l f a l f a stock:,  in  p a r t i c u l a r , the purpose o f the e x p e r i m e n t s was to l e a r n i f differences  i n c o m b i n i n g a b i l i t y e x i s t e d between c l o n a l l i n e s ,  and t o f i n d o u t i f d i f f e r e n c e s o f each l i n e 3 w e r e  i n the male and female  behaviour  present.  The combining a b i l i t y based on seed s e t was d e t e r m i n e d by d i a l l e l crossing (crossing i n a l l possible  combinations).  E i g h t s e l e c t e d c l o n a l l i n e s were d i a l l e l c r o s s e d i n 1 9 4 6 , and t h i r t e e n l i n e s d i a l l e l c r o s s e d i n 1947. used a s a male p a r e n t o n l y i n 1947. l i n e s were employed b o t h  L i n e 45-65 was  S i x o f the twenty-one  years.  S i n c e some o f the l i n e s showed a h i g h degree o f s e l f fertility*  the a l c o h o l method o f e m a s c u l a t i o n a s used b y  T y s d a l and G a r l (1940) Was employed. r a p i d and q u i t e  T h i s method was v e r y  effective.  About a dozen f l o r e t s on two a d j a c e n t racemes were handled together.  The s t a n d a r d p e t a l o f a f r e s h f l o r e t was  c l i p p e d o f f and the s t i g m a and stamens r e l e a s e d f r o m the k e e l . A f t e r the twelve f l o r e t s had been p r e p a r e d , t h e y were d i p p e d i n 5 7 $ e t h y l a l c o h o l f o r 10-12 seconds and then r i n s e d i n tap water.  Immediately  The f l o r e t s were t h e n d r i e d and the  foreign pollen applied. when a r t i f i c i a l l y p o l l i n a t H j t j a l f a l f a , the p o l l e n i s u s u a l l y c o l l e c t e d i n v i a l s and t r a n s f e r r e d b y a camel h a i r b r u s h o r t o o t h p i c k t o the p r e p a r e d s t i g m a . T y s d a l and w e s t o v e r (1937)  L a t e l y e r o s s i n a b l o c k s have been used to speed up  39. the p o l l i n a t i n g process. A t A g a s s i z , however, the  selected  l i n e s were q u i t e f a r a p a r t and to a v o i d the t e d i o u s process o f c o l l e c t i n g p o l l e n i n v i a l s a method, not a p p a r e n t l y used by o t h e r i n v e s t i g a t o r s , was  devised.  i n t h i s method, i n s t e a d o f c o l l e c t i n g p o l l e n from  the  p o l l e n parent, racemes o f f r e s h l y matured f l o r e t s were q u i c k l y gathered and was  taken to the female parent l i n e .  There a f l o r e t  removed and the base o f i t squeezed between the thumb and  the f i r s t f i n g e r u n t i l the stigma and a n t h e r s were p r o j e c t e d onto the standard p e t a l .  The standard would then be  fairly  covered with p o l l e n and, thus, by t o u c h i n g the standard p e t a l to the stigmas o f the prepared f l o r e t s , p o l l i n a t i o n was  quickly  and e f f e c t i v e l y c a r r i e d out, and w i t h - v e r y l i t t l e waste o f pollen. A f t e r the twelve t r e a t e d f l o r e t s on the two  racemes had  been p o l l i n a t e d , Cookery parchment bags were p l a c e d on f o r isolation.  F i f t y f l o r e t s were p o l l i n a t e d f o r each c r o s s  between two  lines.  The bags were l e f t on u n t i l the pods had matured. both bags and pods were h a r v e s t e d .  The  Then  pod3 from each c r o s s  were counted, hand threshed, and the seeds from them  counted.  From that data the percent f l o r e t s forming pods, seeds per pod and seeds per f l o r e t were c a l c u l a t e d . Test o f E f f e c t i v e n e s s o f A l c o h o l Emasculation Method. To t e s t the e f f e c t i v e n e s s o f the a l c o h o l method o f emasculation, checks were conducted fertile lines.  on r a t h e r h i g h l y s e l f -  The f l o r e t s were t r e a t e d as o u t l i n e d i n the  40 TABLE  12  EFFECTIVENESS OF THE ALCOHOL METHOD OF EMASCULATION AS SHOWN BY" SEEDS PER FLORET SET SELECTED ON CLONAL LINES 1945 and 1947  1946  LINE  FLORETS  SEED  SEED PER FLORET  45-45  69  0  0.0  -46  55  4  0.07  -46  55  3  0.05  -134  79  2  0.02  - 29  44  1  0.02  - 64  48  1  0.02  - 64  135  9  0.06  -121  156  6  0.03  1947  -  41.  method but they were i s o l a t e d under bags w i t h o u t the a d d i t i o n of foreign pollen,  on m a t u r i t y the pods were  t h r e s h e d and the seeds from them c o u n t e d .  gathered,  (Table  12)*  From those t e s t s i t a p p e a l s t h a t by u s i n g the a l c o h o l method 100% e m a s c u l a t i o n cannot be o b t a i n e d .  T y s d a l and  G a r l (1940) o b t a i n e d , however, 100% e m a s c u l a t i o n from t h e i r method i n the greenhouse.  A c c o r d i n g t o t h e m , " when the  f o r e i g n p o l l e n i s a p p l i e d to a s t i g m a i n a d d i t i o n t o i t s own i t i s the e f f e c t i v e the t i m e ,  agent i n f e r t i l i z a t i o n from 70%-80% o f  (unpublished d a t a ) * .  Because o f t h i s phenomenon  which has a l s o been r e p o r t e d by B r i n k and Cooper ( 1 9 4 0 ) , and because a f a i r l y h i g h c o n t r o l o f s e l f - p o l l i n a t i o n was o b t a i n e d , i t i s reasonable  toSassume t h a t 100% o f the  seed  o b t a i n e d from the A g a s s i z c r o s s e s i s h y b r i d s e e d . Combining A b i l i t y  Results.  T a b l e s 13 and 14 r e c o r d the c o u n t s and c a l c u l a t i o n s from the d i a l l e l c r o s s e s conducted i n 1946 and 194$ r e s p e c t ively. The e f f e c t i v e n e s s o f a c l o n a l l i n e as female p a r e n t and as a male parent was used as the c r i t e r i o n o f the c o m b i n i n g a b i l i t y or c r o s s - c o m p a t i b i l i t y of that l i n e .  To determine  the  female and male e f f e c t i v e n e s s o f each l i n e f o r each y e a r the f o l l o w i n g procedure was employed.  The f l o w e r s , p o d s , and  seeds from e v e r y c r o s s (Table  13 and 14) where a l i n e was a  female parent were t o t a l l e d .  From the summed d a t a the  p e r f l o r e t p o l l i n a t e d was c a l c u l a t e d , t h i s b e i n g the o f the e f f e c t i v e n e s s o f t h a t l i n e as a female p a r e n t .  seed  criterion The  42. TABLE  13  RESULTS FROM DIALLEL CROSSING NINE SELECTED CLONAL LINES  Cross  - . 1946  Florets Pods Seed :% Floret Foradng Pods Seeds per Pod Seeds per  45-3 x 45-12  1.0 0.8  23 27  75  33.3  3.2  80  29  106  29.6 46.7  2.9 3.6  20  66  28.9  3.3  0.9  67  33-, 159 25 78  57.6 37.3  4.8 3.1  2.4 1.1  -134  59  12  23  20.3  1.9  -3 x -151  70  13  29  18.5  2.2  0.3 0.3  45-12 x 45-3  66  37  72  56.0  1.9  1.0  30  50  43.6  1.6  0.7  1.5 1.6 2.5  0.2  -3 x  -29  -3x  -45  -3  X  -46  -3 X -3 X  -64 -84  -3  X  69 91 62 69 65  1.7  -12 x  -29  69  -12 x  -45  68  12  19  17.6  -12 x  -46  73  -64  59  13 80  10.9  -12  8 32  -12 x -94 -12 x -134  70  17  25  54.2 24.2  71  84  61.9  -12 x -151  68  44 24  1*4. 1.8  41  35.2  1.7  1.1 0.6  60  33  121  55.0  3.6  2.0  X  45-29 x 45-3 -29  N  0.1 1.3 0.3  X  -12  60  33  90  55.0  2.7  1.5  -20 x  -45  71  50  220  70.4  4.4  3.0  -»z  -46  48  25  102  52.8  4.0  -29 x  -64  77  34  115  44.1  3.3  2.1 1.4  -29 x  -84  70  25  77  35.6  3.0  -29 x -134 -29 x -151  57  31 23  110  54.3  3.5  1.1 1.9  47  31.0  2.2  0.6  47  209  62.6  4.4  2.7  23  24  35.3  1.0  45-45 x 45-3  74  -45 x  -12  75 65  -45 x  -29  71  35  45  35.2  1.8  0.3 0.6  -45 x  -46  75  28  83  37,3  2.9  1.1  43*  2  Table 13 Con't. CROSS  Florets Pods Seeds % Florets Foaming Pods Seeds per Pod Seeda per Floret  45-45 X 45-64 -45 x -84 -45 * -45 x  -134 -151  45-46 x 45-3 046 x -12  73 76  46 29  134 66  63.0 38.3  69 71  45 37  174 125  65.2 52.1  71 59  34 29  112 64  47.8 49.1  2.9 2.2 3.8 3.4  1.8 0.8 2.5 1.7  -46 x  -29  117  55  134  47.0  3.2 2.2 2.4  -46 x  -45  71  41  171  57.7  4.1  1.1 2.4  -46 x  -64  71  52  188  73.2  3.6  2.6  -46 x  -84  70  30  62  42.8  2.0  0.8  -46 x - 134  71  54  189  76.0  3.5  2.6  -46 x -151  71  48  167  67.6  3.4  2.3  45-64 x 45-3 -64-x -12  69  36  52.1  4.8  2.5  26  -  174  -64 x  -29  92  28  111  30.0  3.9  , 1.2  -64 x  -45  36.2  3.7  1.3  X  -46  29 23  110  -64  80 66  65  34.8  2.8  0.9  -64 x  -84  58  15  25.8  2.0  0,5  X  -134 -64 x -151  68  -  31 76  45-84 X 45-3  55  -  89  -64  -84  X  -84  X  684  X  1  -  -12 19 -29 - 140 -45 65  14  33  -  37 23  85  26.4  64  -  -  -  -  1.7 1.0  0.5  1.1  -  1.6 1.7 0.6  35.3  2.3 2.7  0.9  -84 x -46  70  33  64  49.1  1.9  0.9  -84 x -64  71  30  65  42.2  2.1  0.9  -84 x -151  -  -  45-134x 45- 3  70  43  176  -84 x -134  mm  -  -  -  -  -  61.4  4.0  2.5 2.8 2,8  -134 x  -12  71  44  198  61.9  4,5  -134 x  -29  141  91  401  64.5  4.4  44.  3  Table 13 0 n»t O  Cross  Florets Pods Seeds % Florets Fonolng Pods Seeds Per Pod Seeds I 72 45-134 X 45-45 196 42 4.6 58.3 2.7 -134 x -46 72 47.2 34 134 3.9 1.8 -134 x -64 70 202 40 57.1 5.0 2.8 -134 x -84 59 23 45 38.9 1.5 0.7 -134 x -151 70 43 154 61.4 3.5 2.2 45-151 x 45 -3 -151 x -12 -151 x -29 -151 x -45  65 58  36 20  48 67 70  24 24  106 44 42 81  55.3 34.4 50.0 37.3  89 117  2.9 2.2  1.6 0.7  1.7 3.3  0.8 1.2  37.1 49.2  3.4 3.7  1.2 1.8  -151 x -46 -151 x - 64  63  26 31  -151 x  -84  70  21  40  30.0  1.9  0.5  -151 x -134  68  26  64  38.2  2.4  0.9  45. IABL B  14  RESULTS FROM DIALLEL CROSSING THIRTEEN SELECTED CLONAL LINES Cross  -  1947  Florets Pods Seeds % Florets Forming Poda Seeds per Pod Seeds per Floret  45-12 x 45-22 -12 x -29  50 45  13 8  13 11  26.0 17.7  1.0 1.3  -30  50  19  33  38.0  1.7  0.2 0.2 0.6  -12 x -45 -12 x -46 -12 x -60 -12 x -65  50 50 50 46  25  50.0 32.0 40.0 30.4  1.8  0.7 0.5  20 14  37 29 37 22  1.8 1.5  0.7 0.4  -12 x -91  50  23  55  46.0  2.3  -12 x - 92  22  32  -12 x -103  50 45  12  18  44.0 26.6  1.4 1.5  1.1 0.6  -12 x -134  50  8  9  16.0  -12 x -151  50  16  32  32.0  1.1 2.0  0.1 0.6  45-22 x 45-12  50 50  10  21 16 21  , 20.0  2.1  0.4  28.0  1.2  0.3  32.0 50.0  1.3 1.7  0.4 0.8  22.0  1.6  0.3  -12 x  -22 x -29  16  0.4  -22 x -30 -22 x -45  50  14 16  50  25  -22 x -46  50  11  44 18  -22 x  -60  50  25  90  50.0  3.6  1.8  -22 x -65 4 -22 x -91  50  45  28.0  3.2  0.9  50  14 15  10  30.0  0.6  0.2  -22 x -103  50  13  12  26.0  0.9  0.2  -22 x -92  50  10  16  20.0  1.6  0.3  -22 x - 134 -22 x -131  50  9  12  18.0  1.3  0.2  50  13  11  26.0  0.8  , 0.2  45-29 x 45-12  50  19  61  38.0  3.2  1.2  12 25 25  23  24.0  0.4  67  50.0  1.9 2.6  84  50.0  3.3  2 26  0  0.0  0.0  81  52.0  3.1  -29 X  -22  50  -29 x -29 x  -39 -45  50 50  -29 x  -46  52  -29 x  660  50  1.3 1.6 0.0 1.6  2  46. Table 14 Con»t Cress Florets Pods Seeds % Florets Forming Pods Seeds per Pod Seeds 45-29 x 45-65 25 50 108 4.3 50.0 2.1 -29 x -91 53 16 56 30.1 3.5 1.0 -29 x -92 54 12 44 22.2 3.6 0.8 -29 x -103 56 22 72 39.2 3.2 1.2 -20 x -134 -29 x -151  50 46  23  45-39 x 45-12  46.0  2  74 5  50  30  67  60.0  4.3  3.2 2.5  1.4 0.1 1.3  1.2 2.1  -39 X -39 x  -22 -29  50 50  26 26  44 85  52.0 52.0  2.2 1.6 3.2  -39 x  -45  21  64  42.0  3.0  -39 x  -46  50 50  31  106  62.0  3.4  -39 x  -60  50  20  69  40.0  3.4  -39 x  -65  50  35  100  70.0  2.8  -39 x  -91  50  30  166  60.0  3.5  -39 x  -92  50  39  134  78.0  3.4  2.1 2.6  -39 x -103  50  40  156  80.0  3.9  3.1  -39 x -134  50  20  53  2.6  1.0  -39 x -151  50  22  66  40.0 44.0  3.0  1.3  45-45 x 45-12  50 45  18 26  27  36.0 57.0  1.5  0.5 1.7  42  3  0.0  3.0 0.0  42.0  2.7  1.3 2.0  -45 x -45 x  -22 -29  -45 x  -39  50  21  -45 x -45 x  -46 -60  45  28  21.6  2.3  50  12 23  1.1 0.6  76  46.0  3.3  1.5  -45 x  -65  50  24  83  48.0  3.4  1.6  -45 x  -91  50  0  0  0.0  0.0  0.0  -45 x  -92  50  4  0  8.0  0.0  0.0  -45 x -103  50  2  0  4.0  0.0  0.0  -45 x *134  50  30  120  60.0  4.0  2.4  45-46 x 45-12  50  34  125  68.0  3.4  2.5  50  35  153  4.2  3.0  50  31  149  70.0 62.0  4.8  2.9  -46 x -46 x  -22 -29  78 0  0.8 1.7  57  0.0  47.  3  Table 14 Con»t Cross Florets Pods Seeds % Florets Fonning Pods Seeds per Pod Seeds per Floret 45-46 x 45-39 -46 x -45  50 50  33 37  116 145  66.0 74.0  3.5 3.9  2.3 2.9  -46 x  -60  41  21  58  51.1  2.7  1.4  -46 x  -65  50  26  52.0  3.3  -46 x -91 -46 x -©2 -46 x -103  39 42 40  28  82 109 75  71.7 47.6  3.8 3.7 3.7  1.6 2.7 1.7  -46 x -134 -46 x -151  5o 50  39 32  45-60 x 45-12  50  -60 x  -22  -60 x  20 32  3.0  158  80.0 78.0  4.0  97  64.0  3.0  14  20  28.0  1.4  50  12  28  24.0  ' 2.3  0.4 0.5  -29  50  19  54  38.0  2.8  1.0  -60 x -60 x  -39 -45  50 50  22 31  65  44.0  2.9  133  62.0  4.3  1.3 2.6  -60 x  -46  50  36  97  72.0  2.7  1.9  -60 x  -65  50  12  28  24.0  2.3  0.5  -60 x  -91 -92  50  25  87  50.0  3.4  50  25  80  50.0  3.2  1.7 1.6  -60 x -103  50  25  71  50.0  2.4  1.4  -60 x -134 -60 x -151  50 50  28  131  4.6  2.6  33  191  56.0 66.0  5.8  3.8  45-91 x 45-12  50  18  29  36.0  1.6  0.5  33  46.4  2.5  151  66.0  1.1 3.0 1.7  -60 x  120  3.1 1.9  -91 x  -22  26  -91 x  -29  50  13 33  -91 x  50  24  88  48.0  -91 x  -39 -45  4.5 3.6  50  32  203  64.0  6.3  4.0  -91 x  -46  50  35  165  70.0  4.7  3.3  -91 x  -60  53  29  125  54.7  4.3  2.3  -91 x  -65  50  31  62.0  3.4  -91 x  -92  40  14  108 55  35.0  3.9  2.1 1.3  -91 x  -103  50  24  128  48.0  5.3  2.5  -91 x  -134  47  30  79  63.7  2.6  1.6  -91 X  -151  31  26  107  83.9  4.1  3.4  48.  4  Table 14 Con't Cross Florets Pods Seeds gPlorets Forming Pods Seeds per Pod Seeds per Floret 45-92 x 45-12 -92 x -22  50 50  11 25  3.2  23  80 56  22.0 50.0  x  -29  50  46.0  2.4  -92 x  -39  50  25  60  50.0  2.4  -02 x  -45 -46  50 50  29 31  141 93  58.0 62.0  4.8 3.0  2.8  -60 -65  50  0  6.0  0.0  0.0  48  27  56.2  3.6  2.0  «91 -103  50 50  23 26  98 56  3.6  -134 -151  50 50  13  84 36  21  45-403 x 45-12 -103 x -22  50  -92  -92 x -92 x -92 x -92 x -92 x -92 x -92 x  3  28  46.0 52.0  2.5  0.5 1.6 1.1 1.2 1.8  2.7  62  26.0 42.0  1.1 1.4 0.7  2.9  1*2  16  56  32.0  3.5  1.1  50  21  46  42.0  2.1 4.0  0.9 1.4  -103 x  -29  50  29  116  58.0  -103 x  -39  50  22  74  44.0  -103 x  -45  23  99  46.0  -103 x  -46  50 50  3.3 4.3  32  101  64.0  3.1  2.0  -103 x  -60  50  14  50  28.0  3.5  1.0  -103 x  -65  50  19  33  38.0  1.7  0.6  -103 x  -91  50  30  169  60.0  5.6  3.3  -103 x  -92  50  32  109  64.0  3.4  -103 x  -134 -451  50  29  81  58.0  2.7  2.1 1.6  50  24  95  48.0  3.9  1.9  50  19  32  38.0  1.6  0.6  183  68.0  3.6  201  72.0  5.3 5.6  -103 x  45-134 x 45-12 -134 x  -15  -134 x  -22  50  -134 x -134 x  -29  50  34 36  50  32  108  64.0  X  -39 -45  50  38  201  76.0  -134 x  -46  50  19  52  38.0  -134 x  -60  50  32  130  -134  -65  50  0  0  -134  X  3.3 5.2  2.3 1.9  4.0 2.1 4.0 1.0  64.0  2.7 4.0  0.0  0.0  0.0  2.6  49.  5  Table 14 C «t on  Cross Florets Pods Seeds % Florets Forming Pods Seeds per Pod Seeds 45-134 x 45-91 16 50 40 32.0 2.5 0.8 -134 x -92 28 50 97 56.0 3.4 1.9 -134 x -1Q3 26 50 110 52.0 4.2 2.2 -134 X -151 50 18 45 36.0 2.5 0.9 45-151 x 45-12 151 x -22 -151 x -29  50 50  27  50  29  24  60 61 91  54.0 48.0 58.0  65 117 105  54.0 68.0 64.0  119  70.0  3.2  2.3 0.9 2:6  2.2 2.5 3.1 2.3  1.2 1.2 1.8  -151 x -151 x -151 x  -39 -45  50 50  27 34  -46  50  -151 x  -60  50  32 35  -151 x  -65  50  22  49  44.0  -151 x -151 x  -91 -92  50 46  36 16  132 47  72.0  2.2 3.6  32.0  2.9  1.0  -151 x -103  50  28  77  56.0  1.5  -151 x -134  50  14  14  28.0  2.9 1.0  3.4 3.2  1.3 2.3 2.1  0.2  50. e f f e c t i v e n e s s o f the l i n e as a male p a r e n t was s i m i l a r l y found by e m p l o y i n g the data where the l i n e appeared as a male parent i n the c r o s s e s ( T a b l e s 13 and 1 4 ) .  In table  15  i s r e c o r d e d ths d a t a c o n c e r n i n g the e f f e c t i v e n e s s o f the l i n e s as female and male p a r e n t s i n 1946 and 1947,  i n order  o f d e c r e a s i n g seed p e r f l o r e t v a l u e . That the male and female e f f e c t i v e n e s s was q u i t e v a r i a b l e w i t h i n l i n e s and between l i n e s i s e v i d e n t from the  table.  Some l i n e s show a h i g h c o m b i n i n g a b i l i t y s i n c e t h e y a r e h i g h l y e f f e c t i v e as b o t h male and female p a r e n t s ,  on the  o t h e r h a n d , o t h e r l i n e s were q u i t e i n e f f e c t i v e as b o t h female and male p a r e n t s , a n d , hence,, poor c o m b i n e r s .  Still  o t h e r l i n e s appear t o be e i t h e r good female p a r e n t s but poor male p a r e n t s , o r , good male p a r e n t s but poor female parents. A p o s s i b i l i t y o f f i n d i n g o u t whether any one o f  the  p r e v i o u s t r e n d s i n b r e e d i n g b e h a v i o u r was c o n s i s t e n t o r p r e dominant throughout the l i n e s i 3 a f f o r d e d b y use o f the rank correlation coefficient s t a t i s t i c ,  m t a b l e 16 the  consist-  ency o f the r a n k i n g o f the l i n e s a s t o t h e i r e f f e c t i v e n e s s as female o r as male p a r e n t s i s compared (1946 and 1 9 4 7 ) . S i n c e the c o r r e l a t i o n s are d e f i n i t e l y n o t s i g n i f i c a n t , t h e r e appear to be no c o n s i s t e n t t r e n d s o f b r e e d i n g b e h a v i o u r o f the l i n e s .  A highly significant positive correlation  would have i n d i c a t e d t h a t l i n e s whieh a r e good o r poor female p a r e n t s are a l s o good o r poor male p a r e n t s ,  on the  other  h a n d , a h i g h l y s i g n i f i c a n t n e g a t i v e c o r r e l a t i o n would have  51. TABLE  IS  FEMALE AND MALE EFFECTIVENESS OF SELECTED CLONAL LINES, BASED ON SEED PER FLORET} in ORDER OF DECREASING EFFECTIVENESS  -  1946 and 1947  FEMALE LINE 1946  FLORET PODS SEED  MALE SEED PER FLORET  LINE  FLORET PODS SEED  -  1059  1.99  298 250  1060 967  1.93 1.73  720  1.55  188  563  0  542  1.32 1.26  769  327  948  1.23  45-46  543  197  616  1.13  0.70  45-84  540  185  424  0.78  1387  2.48  45-45  550  320  1268  2.30  932  1.73 1.56  360  1506  2.40  45- 3 531  343 524  1087 1682  1.80 1.66  45-64 45-45  575 459  290  860  1.49 1.26  509  208  1.14  45-134 463 45-151 424 45-12 427  45-3  552  182  1.11  45-29  45-84 45-12  420  -  616 400  1.05  544  204  384  562  368  45-134 625 601 45-46 45-29 1013 45-45 45-64 45-151  1947 45-46  SEED PER FLORET  581 583  549 556  45-91 45-134  539  309  1271  2.31  45-29  537  251  600  298  1199  1.99  45-103 541  250  45-39  600  340  1050  1.75  45-60  544  248  835  1.53  45-103  600  1029  542  242  820  1.51  593  979  1.71 1.65  45-91  45-151  291 346  207  711  1.49  45-60  600  282  985  1.64  45-151 477 45-46 547  257  794  1.45  45-92  598  257  794  1.32  45-22  523  241  141  45-29  611  675  1.10  45-134 547  243  45-45  600  209 175  742 767  318  0.53  550  266  754  1.37  45-12  586  196  328  0.55  45-39 45-92  532  222  689  1.29  45-22  600  175  318  0.53  45-12  550  249  526  0.95  848  1.40  52. i n d i c a t e d t h a t l i n e s which a r e good female p a r e n t s a r e poor male p a r e n t s o r v i s a v e r s a . The f a c t t h a t no d e f i n i t e t r e n d s o f b e h a v i o u r were d i s c o v e r e d m i g h t be expected s i n c e i n d i v i d u a l l i n e s appear t o d i f f e r q u i t e d e f i n i t e l y from each o t h e r as t o t h e i r e f f e c t i v e n e s s a s female o r male p a r e n t s .  F o r e x a m p l e , on  the b a s i s o f seed p e r f l o r e t ( T a b l e 1 5 ) , i n 1946 l i n e s 45-45 and 45-134 were e f f e c t i v e a s b o t h male and female p a r e n t s w h i l e , on the o t h e r h a n d , l i n e s 45-84 and 45-12 were f a i r l y ineffectivenas e i t h e r parent.  L i n e s 45-46 and 45-29 appeared  t o be v e r y e f f e c t i v e as female p a r e n t s b u t i n e f f e c t i v e as male p a r e n t s w h i l e 45-3 was d e f i n i t e l y a good male p a r e n t b u t a r e l a t i v e l y p o o r female p a r e n t ,  i n 1947, l i n e s were found  w h i c h behaved i n a s i m i l a r ' m a n n e r .  F o r i n s t a n c e , l i n e s 45-91  and 45-103 were good female and male p a r e n t s w h i l e l i n e s 45-12 and 45-22 were d e f i n i t e l y i n e f f e c t i v e as e i t h e r p a r e n t .  Line  45-46 was d e f i n i t e l y a good female parent b u t a poor male p a r e n t w h i l e l i n e s 45-29 and 45-45 were good male p a r e n t s but d e f i n i t e l y poor female p a r e n t s ,  i n c i d e n t a l l y , l i n e 45-12,  one o f the s i x l i n e s employed i n the d i a l l e l c r o s s e s i n 1946 and 1947, was c o n s i s t e n t l y l o w as a male and a s a female parent. S i n c e s i x l i n e s had been used i n b o t h y e a r s i t was p o s s i b l e £ o check the c o n s i s t e n c y o f t h e i r female and male effectiveness  from one y e a r t o the n e x t b y use o f the r a n k  c o r r e l a t i o n method. (Table 17)  The f a c t t h a t the c o r r e l a t i o n  c o e f f i c i e n t s were n o t h i g h i n d i c a t e s t h a t , from one y e a r t o the  53, I A B L B 16 FEMALE AND MALE EFFECTIVENESS OF SELECTED CLONAL LINES COMPARED BY THE RANK CORRELATION COEFFICIENT  -  1946  and 1947  1946 LINE Female  45-3 7  -12 9  -29 3  Male  1  6  7  Total  8  15  10  -  0.04  n P rl  —  -45 4  -46 2  -64 5  8  2  7  10  7  3  -84 8  -134 1  -151 6  9  4  5  17  5  11  2 9  -  .016  S.E. — ,354 Ratio - .016 .354  1947 -91 -92 -103 -134 -151 6 5 3 2 8  45-12  -22  -29  -39  -45  -46  -60  11  12  9  4  10  1  7  Male  12  8  2  10  1  7  4  5 11  3  9  6  Total  23  20  11  14  11  8  11  7 19  8  12  12  LINE Female  n  -  p  - 12  rl  -  S.E. Ratio -  2 0 .301 0 .301  —  0.0  54. TABLE  17  THE CONSISTENCY OF FEMALE AND MALE EFFECTIVENESS OF SIX CLONAL LINES FOR 1946 and 1947 TESTED BY THE RANK CORRELATION COEFFICIENT. FEMALE EFFECTIVENESS -45 -46 45-12 -29 6 3 4 2  LINE 1946 1947  6  4  Total  12  7  9  n  2  -  1.711  —  P rl  5  -134  -151  1  5  1  2 "  3  3  3  8  6 -  .770  .448 S.E. Ratio - .770 .448  MALE EFFECTIVENESS -29  -45  -46  -134  -151  4  5  1  6  2  3  1947  6  2  1  4  5  3  Total  10  7  2  10  7  6  -  .571  45-12  LINE 1946  n p r  1  -  2  -  6  -  .256  S.I. -  .448  Ratio - .256 .448  55. next,  the male and female b e h a v i o u r o f each l i n e was n o t v e r y  consistent.  Too much importance must n o t be put on t h i s  c o n c l u s i o n , however, s i n c e the b e h a v i o u r o f o n l y s i x l i n e s was compared. The ranked d a t a o f Table 17 shows t h a t some l i n e s behave quite consistently.  For instance,  i n b o t h y e a r s l i n e s 45-46  and 45-134 were h i g h l y e f f e c t i v e female p a r e n t s w h i l e l i n e 45-12 was v e r y p o o r ,  on the o t h e r h a n d , l i n e 45-45 was a  c o n s i s t e n t l y good male p a r e n t w h i l e l i n e s 45-12 and 45-46 gave i n d i c a t i o n s o f b e i n g q u i t e p o o r . The r e s u l t s t h a t have been p r e s e n t e d i n d i c a t e t h a t the combining a b i l i t y o f the c l o n a l l i n e s under s t u d y c a n d i f f e r from one l i n e t o the n e x t s i n c e the e f f e c t i v e n e s s l i n e s as female and male p a r e n t s d i f f e r .  of those  The d i f f e r e n c e s  may be due t o e n v i r o n m e n t a l f a c t o r s o r may be i n h e r e n t and hence due t o the d i f f e r e n c e s the v a r i o u s c l o n a l l i n e s ,  i n genetical characteristics  of  whether the d i f f e r e n c e s a r e due  t o the f o r m e r o r the l a t t e r c a n n o t , however, be c l e a r l y d e t e r m i n e d u n t i l d i a l l e l c r o s s i n g d a t a f o r one o r two more y e a r s i s o b t a i n e d f r o m the l i n e s . I n c o n c l u s i o n i t must be remembered t h a t the c o m b i n i n g a b i l i t y o f the l i n e s was d e t e r m i n e d from c o n t r o l l e d c r o s s e s and does n o t n e c e s s a r i l y i n d i c a t e the combining a b i l i t y o f the l i n e s under o p e n - p o l l i n a t i o n .  The f a c t t h a t  self-  p o l l i n a t i o n appears t o p l a y a s i g n i f i c a n t p a r t i n the openp o l l i n a t e d seed s e t o b t a i n e d , must be t a k e n i n t o c o n s i d e r a t i o n before  the c l o n a l l i n e s o f Ehizoma s t o c k c a n be  56. e v a l u a t e d a s t o t h e i r combining a b i l i t y o r under n a t u r a l  IV  croas-eompatilitity  conditions.  S E L F - AND CROSS-FERTILITY COMPARED.  A l f a l f a being a n a t u r a l l y cross-pollinated would e x p e c t the *» c r o s s e d seed s e t .  c r o p , one  *» seed s e t t o exceed t h e » s e l f a d "  Many workers have f o u n d t h i s t o be t r u e .  For  example, T y s d a l (1940) i n N e b r a s k a , and C a r l s o n (1935) i n U t a h , f o u n d s e l f - f e r t i l i z a t i o n r e s u l t e d i n much l e s s seed s e t per f l o w e r .  Cooper and B r i n k (1940) r e p o r t ,  from W i s c o n s i n ,  f i v e and one h a l f -times more seed s e t a f t e r c o n t r o l l e d i n g than a f t e r s e l f i n g .  cross-  They have e x p l a i n e d t h a t the d i f f e r -  ences a r e due t o p a r t i a l s e l f - i n c o m p a t i b i l i t y and t o t h e a b o r t i o n o f many s e l f - f e r t i l i z e d embryos.  Hembold ( c i t e d i n  T y s d a l and Westover-1937) found t h a t 24.86% f l o r e t s gave pods on c r o s s i n g w i t h f o r e i g n p o l l e n w h i l e o n l y 17.54% gave pods on s e l f i n g .  I n a d d i t i o n , he found on c r o s s i n g an average o f  2.34 seeds p e r p o d , w h i l e on s e l f i n g o n l y 1.36 seeds p e r p o d . B o l t o n (1948) r e p o r t s  t h a t s e l f - f e r t i l i t y ranged from . 0 2 t o  5 . 2 2 seeds p e r f l o w e r s e l f e d w i t h a n average o f 1 . 5 8 , and the c r o s s - f e r t i l i t y v a r i e d f r o m 2.67 t o 8.26 seeds p e r pod w i t h an average o f 5 . 5 4 . From the c o n t r o l l e d c r o s s i n g and s e l f - p o l l i n a t i o n d a t a a t A g a s s i z i t was p o s s i b l e  t o compare the s e l f - and c r o s s - f e r t i l i t y  o f Rhizoma s t o c k and check the r e s u l t s w i t h r e s u l t s workers.  of other  57 • The average seeds p e r f l o r e t and seeds p e r pod from the s e l f - f e r t i l i t y d a t a (Table 1 and 2 J and the d i a l l e l d a t a (Table 13 and 14) were f o u n d f o r 1946 and 1947.  crossing These  average v a l u e s a l o n g w i t h the h i g h e s t and the l o w e s t v a l u e s are p r e s e n t e d as f o l l o w s : 1946  S e l f - f e r t i l i t y — - . 4 4 s e e d s per f l o r e t Range from 1.4 t o 0.06 - 1 . 6 1 seeds p e r pod Range from 2 . 8 t o 1.0 G r o s s - f e r t i l i t y — 1 . 3 ? seeds p e r f l o r e t Range from 2 . 8 t o 0 . 1 - 2 . 9 4 seeds p e r pod Range from 4 . 8 to 1.0  1947  S e l f - f e r t i l i t y — .46 seeds p e r f l o r e t Range from 1.3 t o 0.03 - 1.39 seeds p e r pod Range from 2 . 4 1 t o 1.0 C r o s s - f e r t i l i t y - 1.52 seeds p e r f l o r e t Range from 4 . 0 t o 1.0 - 2.98 seeds p e r pod Range from 6.3 t o 0.6  From the d a t a i t i s q u i t e c l e a r t h a t f o r Rhizoma a l f a l f a c r o s s - p o l l i n a t i o n g i v e s a h i g h e r seed s e t p e r f l o r e t and p e r pod t h a n does s e l f - p o l l i n a t i o n .  The d i f f e r e n c e s f o r  a l f a l f a do n o t , however, seem to be as g r e a t as the found f o r o t h e r a l f a l f a s ,  in fact,  differences  the c r o s s - p o l l i n a t e d seed  s e t i s o n l y t h r e e times the s e l f - p o l l i n a t e d seed s e t . m i g h t be reasones t h e n , t h a t on a r e l a t i v e b a s i s , i s more h i g h l y s e l f - f e r t i l e  this  than other  It  this a l f a l f a  alfalfas.  I t i s i n t e r e s t i n g o t note t h a t b o t h the  n  c r o s s e d *» and  58. » s e l f e d " seed s e t are c o n s i s t e n t l y below tfte seed o b t a i n e d by o t h e r w o r k e r s .  set  The r e a s o n f o r t h i s l o w e r seed  s e t might be a t t r i b u t e d to the c l i m a t e a t A g a s s i z , t o  the  presence o f the bags d u r i n g pod and seed f o r m a t i o n o r t o the i n h e r e n t , hence g e n e t i c a l , n a t u r e o f t h i s  alfalfa.  39.  SUMMARY AND CONCLUSIONS 1.  Rhizoma a l f a l f a , a new v a r i e t y p o s s e s s i n g a s p r e a d i n g h a b i t , o r i g i n a t e d f r o m s i x h y b r i d s o f a c r o s s between the  <&4&£e&&s* s p e c i e s Medic ago ,  falcata  v a r . Don ( t h e seed p a r e n t ) and M. s a t i v a v a r . Grimm o r Ontario Variegated (pollen p a r e n t ) .  I t represents  s e l e c t i o n over s i x g e n e r a t i o n s . 2.  Because o f t h e unusual o r i g i n o f t h i s v a r i e t y , i t e x h i b i t s c h a r a c t e r i s t i c s o f growth h a b i t and b r e e d i n g b e h a v i o u r t h a t do n o t appear i n o t h e r commercial v a r i e t i e s of a l f a l f a .  3.  To e x p l o r e t h e r e a s o n f o r i t s unusual b e h a v i o u r ,  cyto-  l o g i c a l i n v e s t i g a t i o n s i n t o t h e p l a n t s o f t h e parent s p e c i e s and o f t h e s i x F^^ h y b r i d s f r o m which Rhizoma was developed were conducted a t t h e U n i v e r s i t y o f B r i t i s h Columbia. 4.  Chromosome s t u d i e s o f t h e p a r e n t p l a n t s r e v e a l e d t h a t the f a l c a t a parent was a d i p l o i d (S z 16) and t h e s a t i v a p a r e n t was a t e t r a p l o i d (S = 3 2 ) .  The f a c t , t h a t  the s a t i v a p a r e n t , o f t e n c a l l e d M. media, might b e i r r e g u l a r because o f i t s h y b r i d (M. s a t i v a x M.  falcata)  o r i g i n . , was r e c o g n i z e d . 3.  Chromosome s t u d i e s o f t h e s i x h y b r i d s i n d i c a t e d  that  t h r e e were t r i p l o i d s (S = 24) and t h r e e were t e t r a p l o i d s (s = 6.  32).  The m e i o t i c b e h a v i o u r and r e s u l t i n g gametic p r o d u c t i o n  60.  of t h e t r i p l o i d s was i r r e g u l a r w h i l e t h a t o f t h e t e t r a p l o i d s was q u i t e r e g u l a r . 7»  I t was assumed t h a t the t e t r a p l o i d s  received a double  complement o f f a l c a t a chromosomes f r o m the Don p a r e n t through t h e f o r m a t i o n o f unreduced gametes. 8.  Because o f t h e i r i n s t a b i l i t y , s e l e c t i o n o f t h e more d e s i r a b l e t e t r a p l o i d t y p e s , and c y t o l o g i c a l  investiga-  t i o n s o f c e r t a i n p l a n t s of t h e F^ g e n e r a t i o n s ,  i t was  supposed t h a t t r i p l o i d s do n o t e x i s t i n t h e new 9.  The c y t o l o g i c a l  variety.  s t u d i e s have e x p l a i n e d p h e n o t y p i e pecu-  l i a r i t i e s o f Rhizoma w h i c h h i t h e r t o f o r e  were  unexplain-  a b l e and have shown t h a t t h i s v a r i e t y i s a permanent t e t r a p l o i d h y b r i d (S - 32) w i t h , p r e s u m a b l y ,  equal  complements o f chromosomes f r o m each p a r e n t s p e c i e s . 10.  To f i n d t h e r e a s o n f o r a n o m a l i e s i n t h e b r e e d i n g b e h a v i o u r o f Rhizoma s t o c k , s e l f - p o l l i n a t i o n , openp o l l i n a t i o n , ease o f f l o r e t t r i p p i n g ,  cross-fertility,  and combining a b i l i t y s t u d i e s have b e e n conducted on s e l e c t e d c l o n a l l i n e s o f t h e F^ g e n e r a t i o n a t t h e Dominion E x p e r i m e n t a l 11.  Farm a t A g a s s i z .  Under open p o l l i n a t i o n , some c l o n a l l i n e s s e t abundant seed w h i l e o t h e r l i n e s s e t v e r y l i t t l e s e e d .  The  e f f e c t i v e bee p o p u l a t i o n was very l o w a n d c o u l d not account f o r the h i g h seed s e t . 12.  A highly s i g n i f i c a n t rank correlation  coefficient for  s e l f - p o l l i n a t e d seed s e t and o p e n - p o l l i n a t e d seed s e t of a number-of l i n e s , i n d i c a t e d  that "selfed"  seed was  c o n t r i b u t i n g t o t h e o p e n - p o l l i n a t e d seed s e t , and that, t h e . g r e a t e r the s e l f - f e r t i l i t y o f a l i n e , t h e g r e a t e r would be i t s seed s e t under o p e n - p o l l i n a t i o n . 13.  A f a i r l y s i g n i f i c a n t negative rank c o r r e l a t i o n c o e f f i c i e n t f o r ease o f f l o r e t t r i p p i n g and o p e n - p o l l i n a t e d seed s e t i n d i c a t e d t h a t p r o b a b l y much o f t h e seed was b e i n g s e t w i t h o u t t h e a i d o f i n s e c t v i s i t a t i o n and hence s u p p o r t e d c o n c l u s i o n 12.  14.  The combining a b i l i t y o f a number o f l i n e s was d e t e r mined by c o n t r o l l e d d i a l l e l considerably.  c r o s s e s was found t o v a r y .  I n a d d i t i o n i t was found t h a t t h e  e f f e c t i v e n e s s of a l i n e a s a female and a s a male parent c o u l d a l s o d i f f e r . 15.  The e f f i c i e n c y o f t h e a l c o h o l method o f e m a s c u l a t i o n was checked and found t o be s a t i s f a c t o r y .  Also, a  method o f c r o s s - p o l l i n a t i n g l i n e s when t h e y a r e n o t c l o s e t o each o t h e r , one n o t used by o t h e r ..workers, was r e p o r t e d . 16.  The v a l u e o f d e t e r m i n i n g both male and female e f f e c t i v e n e s s o f a l i n e a s a c r i t e r i o n o f i t s combining a b i l i t y was  17.  emphasized.  The c r o s s - a n d s e l f - f e r t i l i t y v a l u e s o f a number o f c l o n a l l i n e s was compared.  I n 1946 and 1 4 7 t h e Q  average s e l f - f e r t i l i t y was 0.44 and 0.46 seeds p e r f l o r e t , w i t h a range f r o m 0.6 to 1.4 and 0.3 t o 1.3 seeds p e r f l o r e t r e s p e c t i v e l y .  The c o n t r o l l e d c r o s s -  f e r t i l i t y f o r b o t h y e a r s was 1.37 and 1 . 3 2 seeds p e r  62. f l o r e t , w i t h a range f r o m 0 . 1 t o 2 . 8 and 1 . 0 t o 4.0 respectively. From t h e p o l l i n a t i o n and f e r t i l i t y s t u d i e s i t was c o n c l u d e d t h a t c r o s s - p o l l i n a t i o n "by b e e s was n o t as f r e q u e n t i n Rhizoma as i n o t h e r a l f a l f a v a r i e t i e s , and t h a t t h e seed s e t a f t e r c o n t r o l l e d  cross-pollination  does not g r e a t l y exceed t h e seed s e t a f t e r s e l f pollination. S i n c e t h e c y t o l o g i c a l s t u d i e s were l i m i t e d t o t h e Fj_ g e n e r a t i o n o f Rhizoma s t o c k , no attempt was made t o e x p l a i n the anomalies o f breeding behaviour found i n the F  A  g e n e r a t i o n o f Rhizoma s t o c k on a chromosomal o r  genetic b a s i s .  63  LITERATURE CITED Armstrong, J . M., and W. J . White, 1935. Factors influencing seed-setting i n a l f a l f a . Jour. Agr. S c i . 25:161-179. Atwood, S. S., 1947. Cytogenetics and breeding of forage crops. Advances i n Genetics 1:3-67. Bolton, J . L., 1948. A study of combining a b i l i t y of a l f a l f a i n r e l a t i o n to certain methods of selection. S c i . Agr. 28:97-126. Bolton, J . L., and J . R. Fryer, 1937. Inter-plant variations i n certain seed-setting processes i n a l f a l f a . S c i . Agr. 18:148-160. Brink, R. A., and D. C. Cooper, 1936. The mechanism of p o l l i n a t i o n i n a l f a l f a ( Medicago s a t i v a ) . Amer. Jour. Bot. 23:678-683. Burkhart, A, 1937. Frequency of c r o s s - f e r t i l i z a t i o n i n lucerne based on experiments with recessive white-flowering plants, and considerations on the improvement of this forage plant. Herb. Abst. 7:296-297. Carlson, J . W., 1935. Alfalfa-seed investigations i n Utah. Utah Agr. Exp. Stat. B u l l . 258, 47 pp. Clausen, R. E., 1928 (b). I n t e r s p e c i f i c hybridization i n I&jootiana 711 The cytology of hybrids of the synthetic species, digluta, with i t s parents, glutinosa and Tabacum. Univ. C a l i f . Pub. Bot. 11:177-211. Cooper, D. C , and R. A. Brink, 1940. P a r t i a l self-incompati b i l i t y and the collapse of f e r t i l e ovules as factors a f f e c t i n g seed formation i n a l f a l f a . Jour. Agr. Res. 60:453-472. Darlington, C. D., and La Cour, L. F., 1942. Chromosomes. Macmillan Co.  The Handling of  East, E. M., 1928. The genetics of the Genus Nicotiana. Bibliographia Genetica, 4:243-320. Eek, C J . , 1943. Association of Economic Characters i n Rhizoma A l f a l f a . M. S. A. thesis University of B r i t i s h Columbia (unpublished). Englebert, 7. A., 1932. A study of various factors influencing seed production i n a l f a l f a (Medicago s a t i v a ) . S c i . Agr. 12:593-603.  64.  Fryer, J . R., 1930. Cytological studies i n MEdicago, Melilotus and Trigonella . Can. Jour. Res. 3:3-50. Goodspeed, T. H., and Clausen, R. E . , 1927 (b). I n t e r s p e c i f i c hybridization i n Nicotiana V. Cytological features of two F-i hybrids made with Nicotiana B i g e l o v i i as a parent. Univ. of C a l i f . Pub. Bot. 11:117-125. Gray, H. E., 1925. Observations on t r i p p i n g of a l f a l f a blossoms. Can. Ent. 57:235-237. Hagen, Oscar, 1920. Some F and F_ generations of the hybrid Medioago sativa x M. f a l c a t a . Genetica 2:535-536. ?  Hansen, N. E., 1909. The wild a l f a l f a s and clovers of Siberia, with a perspective view of the a l f a l f a s of the world. U. S. D. A. B u l l . 150. Hanson, Angus A., 1944. A digest of reports r e l a t i n g $ro "Rhizoma" a l f a l f a . Undergraduate Essay University of B r i t i s h Columbia ( unpublished). Hayes, H. K., and F. R. Immer, 1942. Methods of plant breeding. MeGraw-Hill Book Co. Inc., New York and London. 432pp. Holliday, E. M., 1932. Morphological, physiological and c y t o l o g i c a l studies of a l f a l f a (Medioago). Thesis f o r the degree of Master of Arts i n the Dept. of Botany, U. B. C. ( unpublished). Johansen, D. A., 1940. Plant Microtechnique  McGraw-Hill.  Julen, Gosta, 1944. Investigations on d i p l o i d , t r i p l o i d and t e t r a p l o i d lucerne. Hereditas, Lund 30:567-582. Kirk, L. E., 1927. S e l f - f e r t i l i z a t i o n i n r e l a t i o n to forage crop improvement. S c i . Agr. 8:1-40. Knowles, R. P. 1943. The---role of insects, weather conditions, and plant character i n seed setting of a l f a l f a . S c i . Agr. 24:29-50. Ledingham, G. F., 1940. Cytological and developmental studies of hybrids between Medioago sativa and a d i p l o i d form of M. f a l c a t a . Genetics 25:1-15. Lejeune, A. J . , and P. J . Olson, 1940. Seed setting i n a l f a l f a . S c i . Agr. 20:570-573. Lepper, R.,Jr., and T. E. Odland, 1939. Inheritance of flower : .... -color i n a l f a l f a . Jour. Amer. Soc. Agron. 31:209-216. Moe,  G. G., 1928. A l f a l f a studies: A preliminary study of the inheritance of c e r t a i n morphological characters. Doctorate thesis (unpublished).  65. N i l s s o n , F., and Andersson, E., 1943. P o l y p l o i d y i n genus Medioago H e r e d i t a s , Lund 29, 197-198. O l i v e r , G. W., 1913. Some new a l f a l f a v a r i e t i e s f o r p a s t u r e s . U. S. D. A. Bureau o f P l a n t I n d u s t r y , B u l l . , 258. Peek, 0., and J . L. B o l t o n , 1946. A l f a l f a seed p r o d u c t i o n i n n o r t h e r n Saskatchewan as a f f e c t e d b y b e e s , w i t h a r e p o r t on means o f i n c r e a s i n g t h e p o p u l a t i o n o f n a t i v e bees. S c i . A g r . 26:388-418. Reeves, R. G., 1930. N u c l e a r and c y t o p l a s m i c d i v i s i o n i n t h e m i c r o s p o r o g e n e s i s o f a l f a l f a . Am. J . Botany V o l . 1 7 , 1:29-40. Rogers, C. B. W., 1941. Rhizome development i n p l a n t s w i t h s p e c i a l r e f e r e n c e t o a l f a l f a (Medicago sp.) M. S. A. T h e s i s , U n i v e r s i t y o f B r i t i s h Columbia, ( u n p u b l i s h e d ) . S t e w a r t , G., 1934. E f f e c t s o f i n b r e e d i n g on v a r i a b i l i t y i n alfalfa. J o u r . A g r . Research, 49:669-694. T y s d a l , H. M., 1940. I s t r i p p i n g n e c e s s a r y f o r seed s e t t i n g i n a l f a l f a , J o u r . Amer. Soc. A g r o n . 32:570-585. T y s d a l , H. M., and G a r l , J". R u s s e l l , 1940, A new method f o r a l f a l f a emasculation. Am. Soc. Agron. J o . 32:405-407. T y s d a l , H. M., 1946. I n f l u e n c e o f t r i p p i n g , s o i l m o i s t u r e , p l a n t s p a c i n g and l o d g i n g on a l f a l f a seed p r o d u c t i o n . J o u r . Amer. Soc. A g r o n . 38:515-535. T y s d a l , H. M., T. A. K i e s s e l b a c h , and H. L, Westover, 1942. A l f a l f a breeding. U n i v . o f Neb. E x p . S t a t . Res. B u l l . 124 46pp. T y s d a l , H. M., and Westover, H. L., 1937. A l f a l f a improvement. Yearbook o f A g r i c u l t u r e : 1122-1153. V a n s e l l , G. H., and F. E. Todd, 1946. A l f a l f a t r i p p i n g by i n s e c t s . J o u r . Amer. Soc. A g r o n . 38: 470-488. Waldron, L. R., 1919. 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 . Soc. A g r o n . J o . 11: 259-266.  Am.  Watkins, A. E., 1924. G e n e t i c and - c y t o l o g i c a l s t u d i e s i n wheat I . J o u r n a l o f G e n e t i c s 14: 129-171. W i l c o x o n , Frank, 1946. I n d i v i d u a l comparisons o f grouped d a t a by r a n k i n g method. J o u r . Ec.i E n t . 39: 269.  

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