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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 submit ted i n p a r t i a l f u l f i l m e n t o f the requirements f o r the Degree o f MASTER OF SCIENCE I N AGRICULTURE i n t h e Department o f AGRONOMY The U n i v e r s i t y o f B r i t i s h Columbia A p r i l 1948 ABSTRACT Rhizoma, a new al f a l f a variety possessing a dis-tinctive spreading ha<bit, was developed from six 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 its unusual origin, i t differs from other al f a l f a varieties grown in 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 six hybrids were triploids (S s 24) and three were tetraploids (S = 32), the latter having received sixteen chromosomes from the female parent, possibly through the formation of unreduced gametes. It was concluded that the new variety Rhizoma is a permanent tetraploid hybrid (S s 32) containing an equal complement of chromosomes from each parent. Pollination 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 aid 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 line as a male and female parent must be determined for a true criterion of combining a b i l i t y . In addition, i t was found that, in contrast to other varieties, the seed set after controlled cross-pollination did not greatly exceed the seed set after self-pollination. ACKNOWLEDGMENTS The w r i t e r wishes t o acknowledge w i t h deep a p p r e c i a t i o n the keen i n t e r e s t and in 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 of the Department of Agronomy, U n i v e r s i t y o f B r i t i s h Columbia; the val 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 connection w i t h 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 rendered by Mr. M. F. Clarke, S e n i o r A s s i s t a n t , Dominion Experimental Farm, Agass i z ; and the i n t e r e s t and c y t o l o g i c a l data s u p p l i e d by Dr. A. Hi Hutchinson, Department of 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 are a l s o extended t o Dr. D. C. Cooper, Department of Genetics, U n i v e r s i t y of Wisconsin, and to Mr. 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 regarding the c y t o l o g i c a l s t u d i e s , and to Dr. P. T r u s s e l and Mr. Guy Palmer of the B r i t i s h Columbia Research C o u n c i l f o r t h e i r v a l u a b l e a s s i s t a n c e i n connection w i t h the photomicrographs. The w r i t e r e s p e c i a l l y wishes t o express h i s s i n c e r e thanks to Dr. 7. C. Br i n k , Department of Agronomy, U n i v e r s i t y of 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 which have been given so f r e e l y throughout these s t u d i e s . TABLE OF CONTENTS INTRODUCTION 1 A. CYTOLOGICAL STUDIES OF MEDICAGO SATIVA AND MEDICAGO FALCATA, AND OF THE SIX 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 - photo-micrographs. 9 B. SELF-POLLINATION, OPEN^POLLINATION, 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 16 S e l f - p o l l i n a t i o n s t u d i e s 21 O p e n - p o l l i n a t i o n studies 21 S e l f - and - o p e n - p o l l i n a t i o n compared 27 I I EASE OF FLORET TRIPPING AND OPEN-POLLINATION 33 I I I COMBINING ABILITY Test of e f f e c t i v e n e s s of a l c o h o l emasculation method. 39 Combining a b i l i t y r e s u l t s 41 IV SELF-AND CROSS-FERTILITY COMPARED 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 spreading habit,has been released t h i s year, 1948, by the Department of Agronomy, U n i v e r s i t y of B r i t i s h Columbia. The o r i g i n of 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 cross between two s p e c i e s , Medioago f a l o a t a (var. Don) and Medicago s a t i v a (var. Grimm or Ontario V a r i e g a t e d ) . For the h i s t o r y of the cross the reader i s r e f e r r e d to the w r i t i n g s of Moe (1928) and Rogers (1941). I t i s r e l e v a n t , however, to note t h a t rows of Grimm or Ontario Variegated (the p o l l e n parents) or both were grown beside the Don (the seed p a r e n t ) . From the l a t t e r , two hundred and f i f t y seeds were c o l l e c t e d and grown. Only 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 the s i x h y b r i d s a breeding program, founded mainly on mass s e l e c t i o n , was i n i t i a t e d . Rhizoma represents s e l e c t i o n over s i x generations. Rhizoma d i f f e r s i n growth h a b i t s and breeding behavior from other commercial v a r i e t i e s of a l f a l f a . T h i s anomaly i n phenotypic 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 unusual o r i g i n and, more d i r e c t l y the e f f e c t of chro-mosomal i r r e g u l a r i t i e s . To f i n d the reason f o r such irregufarADenavioi*, the most l o g i c a l course i s , t h e r e f o r e , t o study the 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 the U n i v e r s i t y o f B r i t i s h Columbia on the parents and the F 1 hybrids from which Rhizoma was developed. Another approach to s o l v i n g the i r r e g u l a r i t i e s of breeding behavior of Rhizoma was afforded at the Dominion Experimental Farm, Agassiz, B r i t i s h Columbia, where 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 were conducted on s e l e c t e d c l o n a l l i n e s of the F 4 generation. 3. PART A: CYTOLOGICAL STUDIES OF MEDICAGO SATIVA AND  MEDICAGO FALCATA, AND OF THE SIX F x HYBRIDS, PARENT STOCK OF RHIZOMA ALFALFA. Although Rhizoma a l f a l f a has been under develop-ment f o r a number of years, i t s o y t o l o g i c a l behavior has not been f u l l y i n v e s t i g a t e d . As p r e v i o u s l y s t a t e d , i t i s a h y b r i d of M. f a l o a t a x M. s a t i v a possessing c h a r a c t e r -i s t i c s of both s p e c i e s . To what extent each species has c o n t r i b u t e d t o i t s genetic and hence phenotypic c h a r a c t e r i s -t i c s , however, has been a mystery. Since i t i s a h y b r i d from a cross between two species i t s c y t o l o g i c a l behavior might w e l l be q u i t e i r r e g u l a r . An i n d i c a t i o n that 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 gathered from the p e c u l i a r i t i e s which e x i s t i n the phenotypic behavior of t h i s a l f a l f a . For i n s t a n c e , Moe (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 i n t e r c r o s s - f e r t i l i t y i n the F^ h y b r i d s . Subsequent gener-a t i o n s , however, were q u i t e s e l f - f e r t i l e . Throughout the breeding program simple Mendelian segregations have not been observed f o r any morphological f a c t o r s . Odland and Lepper (1939) studying the 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 hot been,able t o p l a c e the segregations on a simple Mendelian b a s i s . I n a d d i t i o n , Moe (1928) has noted dwarf and odd type p l a n t s segregating i n the F 2 generation. I t has been viewed f o r some time t h a t the 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 a l f a l f a 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 explore 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. cytology of the p l a n t s of the two species i n v o l v e d i n the o r i g i n a l cross and, of the s i x h y b r i d s were conducted at 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. Phenotypie anomalies such as those reported i n Rhizoma stock have been observed 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 other genera. Through i n v e s t i g a t i o n s of the parent species and t h e i r hybrids i t has been found that 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 the species had d i f f e r e n t chromo-some numbers, examples of which are reported i n N i c o t i a n a by Clausen (1928b), Goodspeed and Clausen (1927a and b) and East (1928), and i n T r i t i c u m by Watkins (1924). Hybrids from crosses between the species M. f a l c a t a and M. s a t i v a have been produced and s t u d i e d w i t h a view to agronomic usefulness by O l i v e r (1913), Hagen (1919), Hansen (1909), Waldron (1920) and Witte (1921). More r e c e n t l y , Ledingham (1939) has examined the c y t o l o g y of hybrids a r i s i n g from crosses between a d i p l o i d M. f a l c a t a (S e 16) and a t e t r a p l o i d M. s a t i v a (S * 32). When the f a l c a t a was the female parent he obtained seventeen some-what s i m i l a r hybrids whose chromosome numbers were 32. The f e r t i l i t y of these p l a n t s was low. To e x p l a i n the t e t r a p l o i d h y b r i d s he suggested that e i g h t e x t r a chromosomes had come from the d i p l o i d parent through the formation of unreduced gametes. 5. When t h e c a l oat a was the male parent, Ledingham obtained o n l y two h y b r i d s t h a t were u n l i k e eaoh other and whose chromosome numbers were 24. On s e l f i n g and c r o s s i n g these h y b r i d s , no seeds were obtained. N i l s s o n and Andersson (1941) and J u l e n (1943) have.studied " d i p l o i d " (S s 32)* " 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 treatments of s a t i v a and Ultuna a l f a l f a d e r i v e d from the cross M. s a t i v a x M. f a l c a t a . 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 " Ultuna were crossed. The " t r i p l o i d s " were more vigorous than the " d i p l o i d s " or " 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 of the " 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 the workers eould f i n d no c o r r e l a t i o n between m e i o t i c disturbances and p o l l e n f e r t i l i t y . 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 i n t o the c y t o l o g y of the s a t i v a and f a l c a t a parents and the s i x hybrids from which 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 (unpublished d a t a ) . Although, no doubt, c o n s c i e n t i o u s l y d e r i v e d at a time when the cytology of a l f a l f a was very 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 . Hutchison and F a r l e y found the gametic production o f the hybrids to 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 to c o n t a i n from ei g h t to s i x t e e n chromosomes. *The b a s i c chromosome number of a l f a l f a i s con-s i d e r e d by the Scandinavian workers t o be 16. Workers on t h i s continent consider 8 t o be the b a s i c chromosome number w i t h which the w r i t e r i s i n accord. 6. The p l a n t s o f the two s p e c i e s o f Medicago t h a t were c rossed at the 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 c o n s i d e r a b l y i n h a b i t o f g r o w t h . The f a l c a t a ( v a r . Don) was a v e r y low growing p l a n t , c h a r a c t e r i z e d by s h o r t s e m i -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 or t h r e e i n c h e s below the s u r f a c e o f t h e ground, and b r e a k i n g through the s u r f a c e at v a r y i n g d i s t a n c e s from the crown. The p l a n t s produced y e l l o w f l o w e r s and were cons idered to be q u i t e s e l f - s t e r i l e . I t might be noted 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 s t r a i n s t o e x i s t i n N o r t h A m e r i c a . I n s t r a i n s I and I I the 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 .and I I I were p r o s t r a t e and bore f a l c a t e -shaped pods, 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 but had f a l c a t e pods. 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 the chromosome number o f M . s a t i v a i s 32; Reeves (1930) and F r y e r (1930) . I t i s o f importance to note h e r e , t h a t v e r y o f t e n Grimm and O n t a r i o V a r i e g a t e d are p l a c e d i n the s p e c i e s "Medicago m e d i a " . There i s l i t t l e doubt t h a t ' l l , media" arose 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 an 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 the 7. s e p a r a t i o n of the species "media" and s a t i v a does not e x i s t , the d e s i g n a t i o n M. s a t i v a i s used i n these s t u d i e s . The s i x hybrids produced from the i n t e r s p e c i f i c cross have been s p e c i f i e d as H - 7, H-56, H-68, H-71, H-156, H-190. Although complete d e s c r i p t i o n s o f them are not a v a i l a b l e they 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. For instance, they a l l produced rhizomes an expression o f the f a l c a t a parent. Apart from a s l i g h t procumbent h a b i t of the stems, however, the top growth of the hybrids resembled the s a t i v a parent. The f l o w e r c o l o u r was predominantly v a r i e g a t e d , 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 o r introduced by the s a t i v a 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 respect to seed s e t and rhizome production. On repeated s e l f i n g s , the 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 they i n t e r c r o s s e d f r e e l y . H ybrid 7 was the highest seed s e t t e r f o l l o w e d by H-190 and H-156. Hybrid 71, however, was o u t s t a n d i n g l y low i n t h i s respect (Eek - 1943). Concerning rhizome production, H-156 and H-190 produced abundant rhizomes and hence were e x c e l l e n t spreaders. On the other hand, H-7 and i t s progeny produced so few rhizomes t h a t they were e n t i r e l y discarded from the fihizoma breeding program. 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 respect to stem c o l o u r , pubescence and shape of s t i p u l e . 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 parent, of a Grimm p l a n t , and o f the s i x 8 . h y b r i d s were grown i n the greenhouse from 1946 to 1948. Bud m a t e r i a l , f o r the s tudy of the 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) f o r two h o u r s , then washed and s t o r e d i n 80% a l c o h o l . Root t i p s f o r the somatic chromos-ome counts were taken from c u t t i n g s o f the c lones which 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 hours , 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 the same purpose as and t r e a t e d s i m i l a r l y to the r o o t t i p s . 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 the aceto-oarmine 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 to the 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 R e i c h a r t m i c r o s c o p e . Camera l u c i d a drawings were made at X 1620 and X 3550. Photomicrographs 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 X980. S ince the 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 , the de termina-t i o n o f the m e i o t i c behaviour and chromosome numbers was v e r y 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 to the d i f f i c u l t i e s , the s t a i n i n g o f the margdinsoaboutbfche C h r o -mosomes of the h y b r i d s d u r i n g m e i o s i s took on a f u z z y appearance which d i d not 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 the s t a i n i n g might have been an i n d i c a t i o n that chromatin was d i f f u s i n g t o o r from the' chromosomes of the hybrids during m e i o s i s , a c h a r a c t e r i s t i c which might have been the e f f e c t s o f h y b r i d i t y . I t was impossible to d i s t i n g u i s h the f a l c a t a and s a t i v a ohromos-omes i n the h y b r i d s . A summary of the behaviour during microsporogenesis and the chromosome counts f o r the parents and the s i x hybrids i s as f o l l o w s : M« s a t i v a (Grimm) - Although not c e r t a i n t h a t t h i s i s the male parent, there should be l i t t l e d i f f e r -ence between the c y t o l o g i c a l behaviour of i t and Ontario Variegated. I t s me&tio behaviour i s f a i r l y r e g u l a r w i t h the formation of 16 l o o s e l y assoc-i a t e d b i v a l e n t s at l a t e d iakenesis (Figure 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 could be found at l a t e diaken-e s i s ( f i g u r e 12). M. f a l c a t a (Don) - Thisvas 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 photo 1 ) . E i g h t p a i r s could 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 the chromosome p a i r s could 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 , the meios i s at F.M.O. was q u i t e r e g u l a r . HyBrlrd 7 - I t s meio s i s was i r r e g u l a r w i t h an un-equal r a t e of d i v i s i o n i n the second anaphase ( f i g u r e 7 ) . This 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 are oamera l u e i d a drawings o f m l o r o -sporogenes i s and somatic chromosomes o f Don (Medlcago  f a l c a t a ) Grimm (Medlcago s a t i v a ) and o f the s i x F i h y b r i d s f rom aeeto 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 r e m a i n i n g f i g u r e s . F i g u r e 1 . Don (M. f a l o a t a ) , somatic chromosomes, S - 16. F i g u r e 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. F i g u r e 3 . H - 6 8 , somatic chromosomes, S - 24. F i g u r e 4 . H-190, m e i o s i s , second anaphase, 16 and 16. F i g u r e 5 . H-190, somatic chromosomes, S s 32. F i g u r e 6 . H-156, somatic chromosomes, S r 32. F i g u r e 7 . H - 7 , m e i o s i s , second anaphase, unequal 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 . -F i g u r e 8 . H-56 , m e i o s i s , l a t e second anaphase, a f t e r unequal 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. F i g u r e 9 . H - 7 1 , meiosi3, f i r s t anaphase, 16 and 16 . PLATE II PLATE I I F i g u r e 10• H-68 , m e i o s i s , l a t e f i r s t metaphase, n u c l e o l u s p e r s i s t e n t . F i g u r e 1 1 . H-68 , m e i o s i s , l a t e anaphase, d i v i d i n g b i v a l e n t a t one p o l e . F i g u r e 12 . Grimm, m e i o s i s , l a t e d i a k e n e s i s , 14 b i v a -l e n t s . F i g u r e 1 3 . H - 7 , m e i o s i s , f i r s t metaphase, b r i d g e s and u n i v a l e n t s . F i g u r e 14. H-56 , 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 . F i g u r e 15 . H - 7 , m e i o s i s , f i r s t metaphase, ch iasmata , t r i v a l e n t F i g u r e 16. H - 7 , m e i o s i s , e a r l y prophase , prochromo-somes. F i g u r e 17 . H-190, m e i o s i s , l a t e d i a k e n e s i s , chiasmata F i g u r e 18 . H - 7 , m e i o s i s , e a r l y prophase, 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 the c e l l and o f a s m a l l e r number o f chromosomes go ing t o t h e p o l e s i n t h e o t h e r h a l f o f the c e l l . L a g g i n g chromo-somes were p r e s e n t . 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 the 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 unequal numbers o f chromosomes, u s u a l l y around twelve ( 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 1 4 ) . H y b r i d 68 - The somatic chromosome number was found 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 1 1 ) . 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 ( f i g u r e 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 ) . M e i o s i s was r e g u l a r w i t h equal d i s t r i b u t i o n o f chromosomes i n the 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 f o r m a t i o n o f normal chiasmata ( f i g u r e 1 7 ) , and an e q u a l d i s t r i b u t i o n o f ohromosomes i n the second d i v i s i o n ( f i g u r e 4 ) . PLATE I I I PLATE I I I Photos 1-5 Photomicrographs o f miorosporogenes is 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 ) , somatic chromosomes, S = 16. Photo 2 . Heo Grimm, m e i o s i s , d i a k e n e s i s , 16 b i v a l e n t s . Photo 3 . H-190 somatio chromosomes, S r 32. Photo 4 . H-156, somat ic chromosomes, S . s 32 . Photo 5 . H - 7 , m e i o s i s , e a r l y prophase , t h r e e n u c l e o l i . 1 1 . An o u t s t a n d i n g f e a t u r e o f t h i s s t u d y i s the f a c t t h a t one o f the p a r e n t s , namely the female f a l c a t a , had o n l y o n e - h a l f the chromosome complement o f the s a t i v a p a r e n t . The parent s tock o f Rhizoma, t h e n , not o n l y arose from an i n t e r s p e c i f i c c ross 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 unusual on t h i s c o n t i n e n t s i n c e most o t h e r commercial v a r i e t i e s have stemmed from former 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 should be ment ioned, 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 to Rhizoma, can boast o f t h i s unique o r i g i n . The s tock from which i t was produced i s b e l i e v e d to have been developed from the s i x U.B .C. h y b r i d s . Erom a cross 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 expec ted . Ledingham (1940) o b t a i n e d t r i p l o i d s o n l y when the d i p l o i d parent was the male , but o b t a i n e d t e t r a p l o i d s when the 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 ^ i n d i c a t e t h a t H - 7 , H - 5 6 , ; and H-68 were t r i p l o i d s . T h e i r m e i o t i c behaviour 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 the b a s i s o f the 8 chromosomes o f 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 the 16 chromo-somes o f s a t i v a at 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 to t r a v e l fio one p o l e o r 12 . to the other? 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 case , the d i v i s i o n i n second anaphase would be unequal w i t h the h a l f c e l l c o n t a i n i n g the 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 produced, two o f which would be normal and two t h a t would be s m a l l s i n c e the l a t t e r c o n t a i n e d Only 8 chromosomes. I t i s i n t e r e s t i n g to note t h a t 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 out by W i l s o n Stewart (unpubl i shed 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 the 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 71 , 156, and 190 were t e t r a p l o i d s i s a n o t h e r noteworthy r e s u l t o f t h i s s t u d y . These h y b r i d s , t h e n , must c o n t a i n 16 chromosomes from the f a l c a t a p a r e n t . 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 the i n c r e a s e d number o f chromosomes coming from the d i p l o i d parent i s on the assumption t h a t i t formed unreduced gametes. That t h e i r m i i o t i o behaviour 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 , s i n c e i t was then 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 of the 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 chromat in 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 prophase . Reeves (1930), i n h i s s tudy 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 one. S i n c e the number o f n u c l e o l i i s I S . u s u a l l y an i n d i c a t i o n o f the number o f chromosome genomes, the .appearance o f more than 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 they c o n t a i n two o r p o s s i b l y three genomes. S ince the h y b r i d s d i f f e r c o n s i d e r a b l y 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 phenotypic 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 should more c l o s e l y resemble the Grimm p a r e n t . 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 hand, should 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 the t r i p l o i d s s i n c e the 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 the 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 - 1928) . Because o f t h i s f a i l i n g , these progeny were d i s c a r d e d . On the o t h e r hand, H-156 and H-190 produced progeny t h a t gave an abundanoe o f r h i z o m e s . From the c y t o l o g i o a l s t u d i e s , these d i f f e r e n c e s i n rhizome p r o d u c t i o n on the 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 of the h y b r i d s i s a l s o o f c o n s i d e r a b l e i n t e r e s t . On s e l f i n g , they 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 they set seed p r o f u s e l y . I n t h i s r e s -p e c t , 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 behaviour would be expected to be l e s s f e r t i l e than the 14. t e t r a p l o i d s . 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 not behave e x a c t l y i n a c c o r -dance w i t h the 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 the 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 the most s t e r i l e . N e v e r t h e l e s s , the r e s t o f the h y b r i d s behaved n o r n a l l y as the 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 f e r t i l i t y . 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 Andersson (1943) and J u l e n (1944) were more f e r t i l e than the " d i p l o i d s " (S s 32) and as f e r t i l e as the " t e t r a p l o i d s " (S e 6 4 ) . They concluded 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 f e r t i l i t y . 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 such a h i g h chromosome number. U . B . O S i m i l a r l y , i t might be conc luded , t h a t s i n c e the/ h y b r i d s are 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 unequal d i s t r i b u t i o n o f chromosomes t o the v a r i o u s c e l l s , e s p e c i a l l y i n the t r i p l o i d s , may not e f f e c t too g r e a t l y t h e i r f e r t i l i t y . Another p o i n t t h a t must be remembered when account-i n g f o r the anomalies i n the h y b r i d s i s the f a c t t h a t one o f the p a r e n t s , namely the 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 cross between s a t i v a and f a l c a t a and p r o b a b l y capable o f p r o d u c i n g gametes o f v a r y i n g chromosome numbers. F r y e r (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 , which was 1 5 . des ignated as Medieago media , 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 m e i o s i s . H u t c h i n s o n and F a r l e y (unpubl i shed 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 the 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 c e r t a i n 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 the t r i p l o i d s were present i n the h y b r i d s and p r o b a b l y p e r s i s t e d d u r i n g the 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 through 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 t h e c h a r a c t e r i s t i c s shown by the t e t r a p l o i d s , t h e r e i s v e r y l i t t l e t r i p l o i d y present i n the Rhizoma s tock o f l a t e r g e n e r a t i o n s . I n f a o t , Armstrong (persona l correspondence) has found the chromosome number o f c e r t a i n p l a n t s of the F4. g e n e r a t i o n t o be 32. M e i o s i s was r e g u l a r w i t h the f o r m a t i o n o f 16 b i v a l e n t s . From the evidence presented i t i s p o s s i b l e to conc lude , t h a t the 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 : 1 c o n t a i n i n g s i x t e e n chromosomes from the M . f a l c a t a parent 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. Experiments r e l a t i n g t o the b r e e d i n g behaviour o f c e r t a i n promis ing l i n e s o f a l f a l f a , s e l e c t e d from the parent s tocks 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 Farm, 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 p l a n t s had been sent to 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 to 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 they were e s t a b l i s h e d as l i n e s o f twenty- f ive* c l o n e s . A number o f the most p r o m i s i n g l i n e s were s e l e c t e d and t h e i r b r e e d i n g behaviour determined by experiments 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 combining a b i l i t y , and ease of f l o r e t t r i p p i n g . These experiments and t h e i r r e s u l t s a re presented i n t h i s r e p o r t . I SELF-POLLINATION AND OPEN-POLLINATIOH. I t has been assumed f o r many years 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 always takes place i n seed f o r m a t i o n . That the assumption i s j u s t i f i e d seems to be borne out i n recent s t u d i e s . B u r k h a r t (1937) , f o r example, i n A r g e n t i n a , found t h a t , i n d i f f e r e n t l i n e s , 67% to 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 -a t i o n . In 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 westover (1942) 17. o b t a i n e d from three t e s t a o f seed produced under open-p o l l i n a t i o n an 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) found averages o f 94.2$ and over 90$ c r o s s i n g r e s p e c t i v e l y . 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 , many p l a n t s a r e q u i t e s e l f - f e r t i l e and under c e r t a i n c i rcumstances s e l f - p o l l i n a t i o n may o c c u r to an a p p r e c i a b l e degree . Knowles (1943) i n a eoramon commercial 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 par f l o r e t , i n o t h e r s o n l y 0.56 seeds . Bo l ton^and F r y e r (1937) r e p o r t e d 3 .8$ and 54.7$ pods per f l o r e t s t r i p p e d when s e l f i n g a group o f s t e r i l e p l a n t s and f e r t i l e p l a n t s r e s p e c t i v e l y . l a t e l y , 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 from 0.02 to 5 .22 w i t h an average o f 1.58 seeds per f l o r e t , i n a d d i t i o n he has 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 s e l f and c r o s s - f e r t i l i t y . Many workers have r e p o r t e d t h a t i n s e c t s , e s p e c i a l l y b e e s , are the Indispensable agents i n the c r o s s - p o l l i n a t i o n o f a l f a l f a . 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 process i n which anthers and s t y l e s are l i b e r a t e d from confinement i n the k e e l and s t r i k e a g a i n s t the s tandard p e t a l . In 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 , which may c a r r y i t to the s t igmas o f o t h e r f l o r e t s . Armstrong and White (19354 r e p o r t t h a t p o l l e n germinat ion 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 rom 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 . E e c e n t l y the same author (1946) r e p o r t s t h a t l e s s than 5$ o f the f l o r e t s se 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 out t h a t , i n some a r e a s , a l f a l f a can s e t seed -.-Without t r i p p i n g and hence w i t h o u t the agency o f i n s e c t s . B r i n k and cooper (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 but o n l y 12$ o f the 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 not 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 to the genera Megachi le ( the L e a f - e u t t e r B e e s ) , Bombus ( the Bumble Bees)and Nomia ( the A l k a l i B e e s ) . B n g e l b e r t (1932) , T y s d a l (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 over the e f f e c t i v e n e s s o f honey bees as t r i p p i n g agents . C a r l s o n (1935) r e p o r t s l i t t l e t r i p p i n g b y them i n U t a h . Knowles (1943) c la ims t h a t they p l a y a minor r o l e i n Saskatchewan. T y s d a l (1940) found honey bees to be i n e f f e c t i v e as they o n l y t r i p p e d 1 to 2$ of the f l o r e t s , on the o t h e r h a n d , Le Jeune and Olson (1940) , 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 t h a t honey bees are e f f e c t i v e t r i p p e r s . The l a t t e r r e p o r t t h a t the honey bees which 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 are the v a l u a b l e t r i p p e r s . That the f a c t o r s o f weather are important i n t r i p p i n g and seed s e t t i n g has been r e p o r t e d by 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 found wind to be unimpor tant . 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 i n c r e a s e 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 , they 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 has been noted 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 se t seed abundant ly w h i l e o t h e r s s e t seed s p a r i n g -l y . The h i g h seed s e t on some p l a n t s i s v e r y unusual 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 iew o f the work done elsewhere i t I s necessary to 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 s e t s . m 1946, s i x t e e n l i n e s and i n 1947 twenty-seven l i n e s were s e l e c t e d f o r s t u d y , s e l e c t i o n was m a i n l y based on h a b i t o f growth and v i g o r . 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 by 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 the f l o r e t s under vegetab le cookery parchment bags . The bags were n o t removed u n t i l the pods were mature . 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 too many pods were knocked o f f . i n a d d i t i o n , i t appeared t h a t the seed s e t was net i m p a i r e d by e n c l o s i n g the d e v e l o p i n g pods i n l i g h t bags, i n p a s s i n g i t might be noted t h a t K i r k (1927) and Stewart (1934) found 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 of seeds were counted f o r each l i n e s e l f e d . From t h i s data the percent f l o w e r s f o r m i n g pods , seeds per pod and seeds per f l o r e t t r i p p e d were c a l c u l a t e d . The o p e n - p o l l i n a t e d seed s e t was determined by an 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 to 10) o f the pod number per l i n e i n 1946 and 194? and a l s o by the average weight o f seed per c lone o f each l i n e i n 1946. 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 c o r r e l a t i o n c o e f f i c i e n t Wilooxon (1945) was u s e d . T h i s s t a t i s t i c i s an 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 . I t i s r e a l l y 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 t reatments 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 not 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 h e r e . The procedure i s as f o l l o w s : The l i n e s are ranked a c c o r d i n g to t h e i r performance i n the t rea tments . The formula 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 i s then a p p l i e d . a P ( P + l ) Surafrank t o t a l s ) • 3n(p+I) where n number o f t reatments p number o f l i n e s 12 and 13 are constants <v,2 The J£ r and the rank d i f f e r e n c e c o r r e l a t i o n c o e f f i c i e n t (r^"J are r e l a t e d , i n f a c t 1 ~2 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 zero 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 - 1 R a t i o » r 1 ^ S e l f - p o l l i n a t i o n s t u d i e s . Tables 1 and 2 present the s e l f - p o l l i n a t e d data o f the s e l e c t e d l i n e s f o r 1946 and 1947. Based on seeds per f l o r e t , 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 years was h i g h l y v a r i a b l e . Noteworthy 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 e . S e l f - f e r t i l i t y data f o r b o t h y e a r s , 1946 and 1947, was a v a i l a b l e f o r o n l y twelve) l i n e s . T h a t , however, the s e l f -f e r t i l i t y o f a g i v e n p l a n t i s a g e n e t i c c h a r a c t e r i s t i c , o r i n o t h e r words , t h a t 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 constant year a f t e r y e a r , i s to be concluded from the v e r 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 o b t a i n e d (Table 1 3) from the a v a i l a b l e d a t a . O p e n - p o l l i n a t i o n S t u d i e s . Tables 4 and 5 present the o p e n - p o l l i n a t e d data 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. T A B L E 1 SELF-POLLINA.TION Or SELECTED CLONAL LINES BASED OH PERCENT FLORETS FORMING PODS, SEED PER POD, AND SEED PER FLORET - 1946 LINE FLORETS PODS SEED % FLORETS FORMING PODS SEEDS PER POD SEEDS PER FI 45-3 386 147 415 37.8 2.8 1.4 -10 963 216 449 22.4 2.0 0.4 -12 289 74 102 25.6 1.3 0.3 - 29 509 130 176 25.5 1.3 0.3 - 32 320 95 144 29.0 1.5 0.4 - 42 374 103 120 25.6 1.1 0.3 - 45 362 38 59 3.0.4 1.6 0.1 - 46 612 357 628 58*3 1.7 1.0 - 61 445 44 74 9.8 1.6 0.1 - 64 489 131 263 26.7 2.0 0.5 - 84 656 70 85 10.6 1.2 0.1 -112 560 32 34 5.7 1.0 0.06 -121 261 148 305 56.7 2.0 1.0 -134 692 147 259 21.2 1.7 0.5 -150 230 77 114 33.4 1.4 0.4 -151 195 36 63 18.6 1.7 0.3 23. T A B L E 2 SELF-POLLIMTION OF SELECTED CLONAL LINES BASED ON PERCENT FLORETS FORMING PODS, SEED PER POD, AND SEED PER FLORET - 1947 LINE FLORETS PODS SEEDS % FLORETS FORMING PODS S BED PER POD SEED PER FL< 45-6 200 76 85 38.0 1.1 0.4 ' 200 77 131 38.5 1.6 0.6 ) - 8 200 117 191 58.5 1.6 0.9 - 12 200 53 70 26.5 1.3 0.3 - 15 200 64 136 32.0 2.0 0.6 - 22 200 31 34 15.5 1.0 0.1 - 29 200 37 84 18.5 2.2 .04 200 46 44 23.0 0.9 0.2 - 33 . 100 67 121 33.5 1.8 1.2 100 65 132 32.5 2.0 1.3 - 39 200 61 78 30.5 1.2 0.3 - 42 200 66 84 33.0 1.2 0.4 - 45 200 0 0 0.0 0.0 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 1.2 0.4 - 61 i 200 40 49 20.0 1.2 0.2 - 64 200 67 128 38.5 1.9 0.6 - 73 200 1 1 0.5 1.0 0.005 - 84 200 15 10 7.5 0.6 0.05 - 85 200 13 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 0.4 - 103 200 90 223 45.0 2.4 1.0 - 112 200 0 0 0.0 0.0 0.0 - 134 200 94 184 47.0 1.9 0.9 - 150 200 68 61 34.0 0.8 0.3 - 151 200 53 70 26.5 •1.3 0.3 .5 • Lines "selfed" at different dates during the svnnmero f 1947. 24. T A B L S 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 -112 -134 -150 -151 1946 6.5 6.5 6.5 9.0 1.0 1G.0 2.5 11.0 12.0 2.5 4.0 6.5 1947 6.5 6.5 4.0 11.0 1.5 9.0 3.0 10.0 12.0 1.5 6.5 6.5 Total 13.0 13.0 10.5 20.0 2.5 19.0 5.5 21.0 24.0 4.0 10.5 13.0 n . 2 p • 12 r 1 • .852 S.B. m .301 Ratio - .652 m 2.49 .301 Sinoe the r 1 value is over twice its standard error the correlation is highly significant. 25. f T A B L E 4 OPEN-POLLINATION OF SELECTED CLONAL LINES BASED ON POD NUMBER VALUE (0 to 10) AND SEED YIELD - 1946. POD NUMBER VALUE P (0-10) > - 3 24.4 9 - 10 7.3 6 - 12 3.7 6 - 29 6.6 8 - 32 14.6 8 - 42 3.1 8 -45 3.3 7 - 46 9.0 9 - 53 15.0 10 - 61 8.5 6 - 64 3.1 7 - 84 2.1 3 -112 3.9 3 -121 16.2 8 -134 14.6 8 -150 8.1 7 -151 5.1 6 SEED YIELD LINE ° (9 ma. Per Clone) 26. T A B L E 5 OPEN-POLLINATION OF SELECTED CLONAL LINES BASED ON POD NUMBER VALUE (0 to 10) - 1947 POD NUMBER VALUE LINE (0 - 10) 45-6 10 - 8 9 - 12 3 - 15 5 - 22 4 - 29 2 - 33 10 - 39 7 - 42 5 -45 5 - 46 8 - 51 3 - 60 8 - 61 4 - 64 7 - 65 1 - 73 3 - 84 5 - 85 2 - 90 9 - 91 1 - 92 8 - 105 9 - 112 2 - 115 2 - 134 4 - 150 3 - 151 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 determined b y w e i g h i n g . S i n c e 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 o c u l a r T" method was checked by comparing the data 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 data o b t a i n e d by the w e i g h i n g method i n the same year f o r the same l i n e s . The methods were compared by the rank c o r r e l a t i o n c o e f f i c i e n t . 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 p o i n t o f i n t e r e s t to the a l f a l f a breeder i s whether p l a n t s which se 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 s e t 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 e x t e n s i v e l y , whether c e r t a i n p l a n t s are good seed producers r e g a r d l e s s o f the p o l l i n a t i o n regime. A comparison o f the " s e l f e d " and "open" seed s e t s o f 1946 and 1947 p o i n t s to an answer i n the a f f i r m a t i v e . (Tables 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 i n d i c a t e t h a t , c l o n a l l i n e s , which 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 f a c t o r s , under these c o n d i t i o n s i t might be concluded t h a t 28.* T A B L E 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 - 2.81 .250 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. 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 Value Self Fert- 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 i l i t y 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 & - 2 p - 16 r 1 - .688 S.E. z .258 Ratio e .688 s 2.66 .258 T A B L E 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 - 1947 LINE 45-6 -€ -12 -15 -22 -29 -33 -39 -42 -45 -46 -51 -60 -61 -64 -73 -84 -85 -90 -91 -92 Pod No. 1.5 4.0 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 Self Ferti-l i t y 8.0 4.0 16.0 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 g . ' • . • • • • H I . . . . . i i [ i • II • w • • i i i i II n i i i i • Total 9.5 8.0 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 LINE 45-103 -112 -134 -150 «L51 Pod No. 4.0 24.5 17.0 20.5 24.5 n m P -2 26 r 1 . S.E. -Ratio — .646 •200 .646 .200 Self Ferti-l i t y 2.0 26.0 4.0 16.0 16.0 • i Total 6.0 50.5 21.0 36.5 40.5 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 " seed . I t has been noted 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 the 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 e f f e c t i v e t r i p p i n g i n s e c t s has been v e r y l o w . There have been, however, l a r g e numbers o f honey bees present d u r i n g the l a s t two summers a t A g a s s i z but from repeated 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 by them. The w r i t e r has y e t to 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 . I n s t e a d , the honey bees procure the n e c t a r by 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 noted t h i s behaviour i n t h e i r " n e c t a r c o l l e c t i n g " honey bee . T h i s bee " i n s e r t e d i t s probiseus between the o v e r l a p p i n g wing and the s tandard 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 bees were the e f f e c t i v e t r i p p e r s but there were v e r y few o f them. Leaf c u t t e r bees were ex t remely s c a r c e . I n these years 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 observed there 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 ad jacent l i n e s bore v e r y few. A t no time d i d there appear to be enough bumble bees to aeoount f o r the h i g h seed s e t o f the 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 i n connec t ion 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 examples , the o p e n - p o l l i n a t e d data 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 , 45 -32 , 45 -53 , 45-121 and 45-134 were e x c e p t i o n a l l y h i g h seed s e t t e r s . These l i n e s were l aden w i t h pods . L i n e 45-3 w i t h i t s 24*4 32. . grams o f seed per c lone had ad jacent to i t l i n e s 45-10 w i t h 7,3 grams per c lone and 45-12 w i t h 3 , 7 grams par 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 16 .2 grams o f seed per c lone had l i n e s 45-84 and 45-112 n e x t to 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 r e s p e c t i v e l y . 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 seeds per 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 gave o n l y 14 and •3 seeds per f l o r e t r e s p e c t i v e l y . S i m i l a r l y , l i n e 45-121 gave 1.0 seeds per 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 . S i m i l a r 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 data o f 1947. (Table 2 and 5) I t i s v e r y l i k e l y then t h a t a f a i r l y h i g h percent o f 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 accomplished w i t h o u t the a i d o f bees . - B r i n k and cooper (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. No doubt such a phenomenon must l e a d to s e l f - p o l l i n a t i o n . I t can f i n a l l y be c o n c l u d e d , t h e r e f o r e , that under such a c o n d i t i o n the h i g h e r the self** 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 s e t . 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 se t determined 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 t r i p p i n g . 33. I I 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 o p e n - p o l l i n a t e d 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 by 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 than i n o t h e r s . F u r t h e r , 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 might be due to 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 by the bees . I n t h i s c o n n e c t i o n , i t has 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 process 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 o t h e r s . i n 1947^ an experiment was conducted t o determine the ease of 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 . In 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 se t had f l o r e t s that 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 the 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 as suggested by T y s d a l (1946) . I n h i s s t u d i e s he has found » 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 * to t r i p the f l o w e r s " . F o r t h i s experiment t en a l c o h o l s o l u t i o n s o f s t r e n g t h s rang ing from 50$-55$*60$ —-95$ were u s e d . F i f t y drops o f each s o l u t i o n were p l a c e d by 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 . The number o f f l o r e t s 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 presents 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 - 1947 TRIPPING TRIPPING LINE 1 2 3 4 5 6 7 8 9 10 TOTALS VALUE 50£ 55# > 60$ 65$ 70# 75# 85# 90# 95$ 45-6 1 6 4 12 24 16 26 41 44 42 216 7 - 8 2 6 5 22 26 40 38 40 35 45 259 5 - 12 2 16 25 49 49 49 50 49 49 50 388 3 - 22 0 3 15 18 29 45 46 50 50 50 306 5 - 29 0 4 19 36 43 44 46 45 43 47 327 4 - 39 0 4 5 19 28 39 40 48 47 49 279 5 - 45 0 5 13 28 38 45 50 50 50 50 329 4 - 46 0 3 12 22 38 48 48 49 50 50 320 5 - 60 0 19 27 30 47 41 43 49 47 50 348 3 - 90 0 1 2 2 3 3 8 24 26 18 87 9 - 91 0 6 8 16 17 33 60 46 45 43 274 6 - 92 0 7 10 24 29 35 50 50 50 50 305 5 -103 0 7 26 21 30 38 44 48 46 48 308 3 -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 . By a d d i t i o n the t o t a l number o f f l o r e t s 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 to a s s i g n to each l i n e a t r i p p i n g v a l u e . Each s o l u t i o n was g i v e n a value, i . e . 50$ 1 , 95$ 10. The v a l u e o f the weakest s o l u t i o n which 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 then the t r i p p i n g v a l u e . 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 to 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 are presented i n Table 9 . By use o f the rank 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 expec ted , t h a t the t r i p p i n g v a l u e s and the t o t a l 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 1 0 ) . 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 (Table 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 f i f t e e n l i n e s were c o r r e l a t e d by 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 1 1 ) . 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 l i n e s 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 not g ive 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 low seed s e t t e r . On the o t h e r hand the most d i f f i c u l t l i n e to t r i p , was a h i g h seed s e t t e r , l i n e 45-90. These r e s u l t s tend to support the p r e v i o u s c o n c l u s i o n that f o r many l i n e s the h i g h seed se t i s not n e c e s s a r i l y a s s o c i a t e d w i t h i n s e c t v i s i t a t i o n s . 36. T A B L E 10 TRIPPLIHG VALUE AND TRIPPING TOTAL FOR FIFTEEN CLONAL LINES COMPARED BY THE RANK FORRELATION COEFFICIENT - 1947 LINE 45-6 -6 -12 -22 -29 -39 -45 -46 -60 -90 -91 -92 -103 -134 -150 Tripp-ing Valua 13 9 2 9 5 9 5 9 2 1 5 12 9 2 5 14 Tripp-ing Total 1 3 1 2 1 8 4 10 3 6 2 15 1 1 9 7 5 1 4 Total 26 21 3 17 9 19 8 15 4 30 23 18 9 10 28 n - 2 p - 15 r l - .857 S.E. - .267 Ratio - .857 - 3.20 .267 37. T A B L E 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 Trip-ping 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 a - 2 P - 15 r 1 - .448 S.E. - .267 Ratio - ^  .448 - -1.67 .267 38, I I I COMBINING ABILITY. Experiments were conducted i n 1946 and 1947 a t A g a s s i z to determine the combining a b i l i t y o f Rhizoma a l f a l f a stock: , i n p a r t i c u l a r , the purpose o f the experiments was to l e a r n i f d i f f e r e n c e s i n combining 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 to f i n d out i f d i f f e r e n c e s i n the male and female b e h a v i o u r o f each l ine3were p r e s e n t . The combining a b i l i t y based on seed s e t was determined b y d i a l l e l c r o s s i n g ( c r o s s i n g i n a l l p o s s i b l e c o m b i n a t i o n s ) . 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 1946, and t h i r t e e n l i n e s d i a l l e l c rossed i n 1947. L i n e 45-65 was used as a male parent o n l y i n 1947. S i x o f the twenty-one l i n e s were employed b o t h y e a r s . 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 -f e r t i l i t y * the a l c o h o l method o f e m a s c u l a t i o n as used by T y s d a l and G a r l (1940) Was employed. T h i s method was v e r y r a p i d and q u i t e e f f e c t i v e . About a dozen f l o r e t s on two a d j a c e n t racemes were handled t o g e t h e r . The s tandard 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 igma and stamens r e l e a s e d from 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 , they were d ipped i n 57$ e t h y l a l c o h o l f o r 10-12 seconds and then Immediately r i n s e d i n tap w a t e r . The f l o r e t s were then d r i e d and the f o r e i g n p o l l e n a p p l i e d . 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 by a camel h a i r brush o r t o o t h p i c k t o the prepared s t i g m a . T y s d a l and westover (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. At Agassiz, however, the selected l i n e s were quite f a r apart and to avoid the tedious process of c o l l e c t i n g pollen i n v i a l s a method, not apparently used by other investigators, was devised. i n this method, instead of c o l l e c t i n g pollen from the pollen parent, racemes of f r e s h l y matured f l o r e t s were quickly gathered and taken to the female parent l i n e . There a f l o r e t was removed and the base of 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 anthers were projected onto the standard petal. The standard would then be f a i r l y covered with pollen and, thus, by touching the standard petal to the stigmas of 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 with-very l i t t l e waste of pollen. A f t e r the twelve treated f l o r e t s on the two racemes had been pollinated, Cookery parchment bags were placed on f o r i s o l a t i o n . F i f t y f l o r e t s were pollinated f o r each cross between two l i n e s . The bags were l e f t on u n t i l the pods had matured. Then both bags and pods were harvested. The pod3 from each cross 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 calculated. Test of Effectiveness of Alcohol Emasculation Method. To test the effectiveness of the alcohol method of emasculation, checks were conducted on rather highly s e l f -f e r t i l e l i n e s . The f l o r e t s were treated as outlined i n the 40 T A B L E 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 1947 - 29 44 1 0.02 - 64 48 1 0.02 - 64 135 9 0.06 -121 156 6 0.03 - 4 1 . method but they were i s o l a t e d under bags wi thout the a d d i t i o n o f f o r e i g n p o l l e n , on m a t u r i t y the pods were g a t h e r e d , threshed and the seeds from them counted . (Table 1 2 ) * From those t e s t s i t appeals t h a t by u s i n g the a l c o h o l method 100% emascula t ion 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% emascula t ion from t h e i r method i n the greenhouse. A c c o r d i n g to them," 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 igma i n a d d i t i o n to i t s own i t i s the e f f e c t i v e agent i n f e r t i l i z a t i o n from 70%-80% of the t i m e , (unpubl ished 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 (1940) , 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 crosses i s h y b r i d seed . Combining A b i l i t y R e s u l t s . Tables 13 and 14 r e c o r d the counts and c a l c u l a t i o n s from the d i a l l e l c rosses conducted i n 1946 and 194$ r e s p e c t -i v e l y . The e f f e c t i v e n e s s of a c l o n a l l i n e as female parent and as a male parent was used as the c r i t e r i o n o f the combining a b i l i t y o r c r o s s - c o m p a t i b i l i t y o f t h a t 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 year the f o l l o w i n g procedure was employed. The f l o w e r s , pods , and seeds from every 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 data the seed per 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 c r i t e r i o n o f the e f f e c t i v e n e s s o f tha t l i n e as a female p a r e n t . The 42. T A B L E 13 RESULTS FROM DIALLEL CROSSING NINE SELECTED CLONAL LINES - . 1946 Cross Florets Pods Seed : % Floret Foradng Pods Seeds per Pod Seeds per 45-3 x 45-12 69 23 75 33.3 3.2 1.0 -3 x -29 91 27 80 29.6 2.9 0.8 -3x -45 62 29 106 46.7 3.6 1.7 -3 X -46 69 20 66 28.9 3.3 0.9 -3 X -64 65 33-, 159 57.6 4.8 2.4 -3 X -84 67 25 78 37.3 3.1 1.1 -3 X -134 59 12 23 20.3 1.9 0.3 -3 x -151 70 13 29 18.5 2.2 0.3 45-12 x 45-3 66 37 72 56.0 1.9 1.0 -12 x -29 69 N 30 50 43.6 1.6 0.7 -12 x -45 68 12 19 17.6 1.5 0.2 -12 x -46 73 8 13 10.9 1.6 0.1 -12 X -64 59 32 80 54.2 2.5 1.3 -12 x -94 70 17 25 24.2 1*4. 0.3 -12 x -134 71 44 84 61.9 1.8 1.1 -12 x -151 68 24 41 35.2 1.7 0.6 45-29 x 45-3 60 33 121 55.0 3.6 2.0 -29 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 2.1 -29 x -64 77 34 115 44.1 3.3 1.4 -29 x -84 70 25 77 35.6 3.0 1.1 -29 x -134 57 31 110 54.3 3.5 1.9 -29 x -151 74 23 47 31.0 2.2 0.6 45-45 x 45-3 75 47 209 62.6 4.4 2.7 -45 x -12 65 23 24 35.3 1.0 0.3 -45 x -29 71 35 45 35.2 1.8 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 73 46 134 63.0 2.9 1.8 -45 x -84 76 29 66 38.3 2.2 0.8 -45 * -134 69 45 174 65.2 3.8 2.5 -45 x -151 71 37 125 52.1 3.4 1.7 45-46 x 45-3 71 34 112 47.8 3.2 1.7 046 x -12 59 29 64 49.1 2.2 1.0 -46 x -29 117 55 134 47.0 2.4 1.1 -46 x -45 71 41 171 57.7 4.1 2.4 -46 x -64 71 52 188 73.2 3.6 2.6 -46 x -84 1 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 69 36 174 52.1 4.8 2.5 -64-x -12 26 - 14 - 0.5 -64 x -29 92 28 111 30.0 3.9 , 1.2 -64 x -45 80 29 110 36.2 3.7 1.3 -64 X -46 66 23 65 34.8 2.8 0.9 -64 x -84 58 15 31 25.8 2.0 0,5 -64 X -134 68 - 76 - - 1.1 -64 x -151 - - - - - -45-84 X 45-3 55 - 89 - - 1.6 -84 X -12 19 - 33 - - 1.7 -84 X -29 - 140 37 85 26.4 2.3 0.6 684 X -45 65 23 64 35.3 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 -134 mm - - - - --84 x -151 - - - - - -45-134x 45- 3 70 43 176 61.4 4.0 2.5 -134 x -12 71 44 198 61.9 4,5 2.8 -134 x -29 141 91 401 64.5 4.4 2,8 Table 13 0On»t 4 4 . 3 Cross Florets Pods Seeds % Florets Fonolng Pods Seeds Per Pod Seeds I 45-134 X 45-45 72 42 196 58.3 4.6 2.7 -134 x -46 72 34 134 47.2 3.9 1.8 -134 x -64 70 40 202 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 65 36 106 55.3 2.9 1.6 -151 x -12 58 20 44 34.4 2.2 0.7 -151 x -29 48 24 42 50.0 1.7 0.8 -151 x -45 67 24 81 37.3 3.3 1.2 -151 x -46 70 26 89 37.1 3.4 1.2 -151 x - 64 63 31 117 49.2 3.7 1.8 -151 x -84 70 21 40 30.0 1.9 0.5 -151 x -134 68 26 64 38.2 2.4 0.9 4 5 . I A B L B 14 RESULTS FROM DIALLEL CROSSING THIRTEEN SELECTED CLONAL LINES - 1947 Cross Florets Pods Seeds % Florets Forming Poda Seeds per Pod Seeds per Floret 45-12 x 45-22 50 13 13 26.0 1.0 0.2 -12 x -29 45 8 11 17.7 1.3 0.2 -12 x -30 50 19 33 38.0 1.7 0.6 -12 x -45 50 25 37 50.0 0.7 -12 x -46 50 16 29 32.0 1.8 0.5 -12 x -60 50 20 37 40.0 1.8 0.7 -12 x -65 46 14 22 30.4 1.5 0.4 -12 x -91 50 23 55 46.0 2.3 1.1 -12 x - 92 50 22 32 44.0 1.4 0.6 -12 x -103 45 12 18 26.6 1.5 0.4 -12 x -134 50 8 9 16.0 1.1 0.1 -12 x -151 50 16 32 32.0 2.0 0.6 45-22 x 45-12 50 10 21 , 20.0 2.1 0.4 -22 x -29 50 14 16 28.0 1.2 0.3 -22 x -30 50 16 21 32.0 1.3 0.4 -22 x -45 50 25 44 50.0 1.7 0.8 -22 x -46 50 11 18 22.0 1.6 0.3 -22 x -60 50 25 90 50.0 3.6 1.8 -22 x -65 50 14 45 28.0 3.2 0.9 4 -22 x -91 50 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 50 9 12 18.0 1.3 0.2 -22 x -131 50 13 11 26.0 0.8 , 0.2 45-29 x 45-12 50 19 61 38.0 3.2 1.2 -29 X -22 50 12 23 24.0 1.9 0.4 -29 x -39 50 25 67 50.0 2.6 1.3 -29 x -45 50 25 84 50.0 3.3 1.6 -29 x -46 52 2 0 0.0 0.0 0.0 -29 x 660 50 26 81 52.0 3.1 1.6 46. 2 Table 14 Con»t Cress Florets Pods Seeds % Florets Forming Pods Seeds per Pod Seeds 45-29 x 45-65 50 25 108 50.0 4.3 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 50 23 74 46.0 3.2 1.4 -29 x -151 46 2 5 4.3 2.5 0.1 45-39 x 45-12 50 30 67 60.0 2.2 1.3 -39 X -22 50 26 44 52.0 1.6 0.8 -39 x -29 50 26 85 52.0 3.2 1.7 -39 x -45 50 21 64 42.0 3.0 1.2 -39 x -46 50 31 106 62.0 3.4 2.1 -39 x -60 50 20 69 40.0 3.4 1.3 -39 x -65 50 35 100 70.0 2.8 2.0 -39 x -91 50 30 166 60.0 3.5 2.1 -39 x -92 50 39 134 78.0 3.4 2.6 -39 x -103 50 40 156 80.0 3.9 3.1 -39 x -134 50 20 53 40.0 2.6 1.0 -39 x -151 50 22 66 44.0 3.0 1.3 45-45 x 45-12 50 18 27 36.0 1.5 0.5 -45 x -22 45 26 78 57.0 3.0 1.7 -45 x -29 42 3 0 0.0 0.0 0.0 -45 x -39 50 21 57 42.0 2.7 1.1 -45 x -46 45 12 28 21.6 2.3 0.6 -45 x -60 50 23 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 -46 x -22 50 35 153 70.0 4.2 3.0 -46 x -29 50 31 149 62.0 4.8 2.9 47. 3 Table 14 Con»t Cross Florets Pods Seeds % Florets Fonning Pods Seeds per Pod Seeds per Floret 45-46 x 45-39 50 33 116 66.0 3.5 2.3 -46 x -45 50 37 145 74.0 3.9 2.9 -46 x -60 41 21 58 51.1 2.7 1.4 -46 x -65 50 26 82 52.0 3.3 1.6 -46 x -91 39 28 109 71.7 3.8 2.7 -46 x -©2 42 20 75 47.6 3.7 1.7 -46 x -103 40 32 120 80.0 3.7 3.0 -46 x -134 5o 39 158 78.0 4.0 3.1 -46 x -151 50 32 97 64.0 3.0 1.9 45-60 x 45-12 50 14 20 28.0 1.4 0.4 -60 x -22 50 12 28 24.0 ' 2.3 0.5 -60 x -29 50 19 54 38.0 2.8 1.0 -60 x -39 50 22 65 44.0 2.9 1.3 -60 x -45 50 31 133 62.0 4.3 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 50 25 87 50.0 3.4 1.7 -60 x -92 50 25 80 50.0 3.2 1.6 -60 x -103 50 25 71 50.0 2.4 1.4 -60 x -134 50 28 131 56.0 4.6 2.6 -60 x -151 50 33 191 66.0 5.8 3.8 45-91 x 45-12 50 18 29 36.0 1.6 0.5 -91 x -22 26 13 33 46.4 2.5 1.1 -91 x -29 50 33 151 66.0 4.5 3.0 -91 x -39 50 24 88 48.0 3.6 1.7 -91 x -45 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 31 108 62.0 3.4 2.1 -91 x -92 40 14 55 35.0 3.9 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 50 11 28 22.0 2.5 0.5 -92 x -22 50 25 80 50.0 3.2 1.6 -92 x -29 50 23 56 46.0 2.4 1.1 -92 x -39 50 25 60 50.0 2.4 1.2 -02 x -45 50 29 141 58.0 4.8 2.8 -92 x -46 50 31 93 62.0 3.0 1.8 -92 x -60 50 3 0 6.0 0.0 0.0 -92 x -65 48 27 98 56.2 3.6 2.0 -92 x «91 50 23 56 46.0 1.1 -92 x -103 50 26 84 52.0 3.6 1.4 -92 x -134 50 13 36 26.0 2.7 0.7 -92 x -151 50 21 62 42.0 2.9 1*2 45-403 x 45-12 50 16 56 32.0 3.5 1.1 -103 x -22 50 21 46 42.0 2.1 0.9 -103 x -29 50 29 116 58.0 4.0 2.3 -103 x -39 50 22 74 44.0 3.3 1.4 -103 x -45 50 23 99 46.0 4.3 1.9 -103 x -46 50 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 2.1 -103 x -134 50 29 81 58.0 2.7 1.6 -103 x -451 50 24 95 48.0 3.9 1.9 45-134 x 45-12 50 19 32 38.0 1.6 0.6 -134 x -15 -134 x -22 50 34 183 68.0 5.3 3.6 -134 x -29 50 36 201 72.0 5.6 4.0 -134 x -39 50 32 108 64.0 3.3 2.1 -134 X -45 50 38 201 76.0 5.2 4.0 -134 x -46 50 19 52 38.0 2.7 1.0 -134 x -60 50 32 130 64.0 4.0 2.6 -134 X -65 50 0 0 0.0 0.0 0.0 49. 5 Table 14 Con«t Cross Florets Pods Seeds % Florets Forming Pods Seeds per Pod Seeds 45-134 x 45-91 50 16 40 32.0 2.5 0.8 -134 x -92 50 28 97 56.0 3.4 1.9 -134 x -1Q3 50 26 110 52.0 4.2 2.2 -134 X -151 50 18 45 36.0 2.5 0.9 45-151 x 45-12 50 27 60 54.0 2.2 1.2 151 x -22 50 24 61 48.0 2.5 1.2 -151 x -29 50 29 91 58.0 3.1 1.8 -151 x -39 50 27 65 54.0 2.3 1.3 -151 x -45 50 34 117 68.0 3.4 2.3 -151 x -46 50 32 105 64.0 3.2 2.1 -151 x -60 50 35 119 70.0 3.2 2.3 -151 x -65 50 22 49 44.0 2.2 0.9 -151 x -91 50 36 132 72.0 3.6 2:6 -151 x -92 46 16 47 32.0 2.9 1.0 -151 x -103 50 28 77 56.0 2.9 1.5 -151 x -134 50 14 14 28.0 1.0 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 parent was s i m i l a r l y found by employing the data where the l i n e appeared as a male parent i n the c rosses (Tables 13 and 14 ) . I n t a b l e 15 i s recorded ths data 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 parents i n 1946 and 1947, i n o r d e r o f d e c r e a s i n g seed per 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 t a b l e . Some l i n e s show a h i g h combining a b i l i t y s ince they are h i g h l y e f f e c t i v e as both male and female p a r e n t s , on the o t h e r hand, 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 both female and male p a r e n t s , a n d , hence,, poor combiners . S t i l l o t h e r l i n e s appear to be e i t h e r good female parents but poor male p a r e n t s , o r , good male parents but poor female p a r e n t s . A p o s s i b i l i t y o f f i n d i n g out whether any one o f the p r e v i o u s t rends i n b r e e d i n g behaviour 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 by use o f the rank c o r r e l a t i o n c o e f f i c i e n t s t a t i s t i c , m t a b l e 16 the c o n s i s t -ency o f the r a n k i n g o f the l i n e s as to t h e i r e f f e c t i v e n e s s as female o r as male parents i s compared (1946 and 1947) . S ince the c o r r e l a t i o n s are d e f i n i t e l y not s i g n i f i c a n t , there appear to be no c o n s i s t e n t t rends o f b r e e d i n g behaviour o f the l i n e s . A h i g h l y s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n would have i n d i c a t e d that l i n e s whieh are good o r poor female parents are a l s o good o r poor male p a r e n t s , on the o t h e r hand, a h i g h l y s i g n i f i c a n t negat ive c o r r e l a t i o n would have 5 1 . T A B L E IS FEMALE AND MALE EFFECTIVENESS OF SELECTED CLONAL LINES, BASED ON SEED PER FLORET} in ORDER OF DECREASING EFFECTIVENESS - 1946 and 1947 F E M A L E MALE SEED PER SEED PER LINE FLORET PODS SEED FLORET LINE FLORET PODS SEED FLORET 1946 45-134 625 360 1506 2.40 45- 3 531 - 1059 1.99 45-46 601 343 1087 1.80 45-64 549 298 1060 1.93 45-29 1013 524 1682 1.66 45-45 556 250 967 1.73 45-45 575 290 860 1.49 45-134 463 720 1.55 45-64 459 581 1.26 45-151 424 188 563 1.32 45-151 509 208 583 1.14 45-12 427 0 542 1.26 45-3 552 182 616 1.11 45-29 769 327 948 1.23 45-84 420 - 400 1.05 45-46 543 197 616 1.13 45-12 544 204 384 0.70 45-84 540 185 424 0.78 1947 45-46 562 368 1387 2.48 45-45 550 320 1268 2.30 45-91 539 309 1271 2.31 45-29 537 251 932 1.73 45-134 600 298 1199 1.99 45-103 541 250 848 1.56 45-39 600 340 1050 1.75 45-60 544 248 835 1.53 45-103 600 291 1029 1.71 45-91 542 242 820 1.51 45-151 593 346 979 1.65 45-151 477 207 711 1.49 45-60 600 282 985 1.64 45-46 547 257 794 1.45 45-92 598 257 794 1.32 45-22 523 241 742 141 45-29 611 209 675 1.10 45-134 547 243 767 1.40 45-45 600 175 318 0.53 45-39 550 266 754 1.37 45-12 586 196 328 0.55 45-92 532 222 689 1.29 45-22 600 175 318 0.53 45-12 550 249 526 0.95 52 . i n d i c a t e d t h a t l i n e s which are good female parents a re poor male parents 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 rends o f b e h a v i o u r were d i s c o v e r e d might be expected s i n c e i n d i v i d u a l l i n e s appear to 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 to t h e i r e f f e c t i v e n e s s as female o r male p a r e n t s . F o r example, on the b a s i s o f seed per f l o r e t (Table 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 as both male and female parents w h i l e , on the o t h e r hand, l i n e s 45-84 and 45-12 were f a i r l y i n e f f e c t i v e n a s e i t h e r p a r e n t . L i n e s 45-46 and 45-29 appeared to be v e r y e f f e c t i v e as female parents but i n e f f e c t i v e as male parents w h i l e 45-3 was d e f i n i t e l y a good male parent but a r e l a t i v e l y poor female p a r e n t , i n 1947, l i n e s were found which 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 parents 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 . L i n e 45-46 was d e f i n i t e l y a good female parent but a poor male parent w h i l e l i n e s 45-29 and 45-45 were good male parents 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 rosses i n 1946 and 1947, was c o n s i s t e n t l y low as a male and as a female p a r e n t . S ince s i x l i n e s had been used i n both years 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 of t h e i r female and male e f f e c t i v e n e s s from one year to the next by use o f the rank 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 not h i g h i n d i c a t e s t h a t , from one year to the 53, I A BL B 16 FEMALE AND MALE EFFECTIVENESS OF SELECTED CLONAL LINES COMPARED BY THE RANK CORRELATION COEFFICIENT - 1946 and 1947 1946 LINE 45-3 -12 -29 -45 -46 -64 -84 -134 -151 Female 7 9 3 4 2 5 8 1 6 Male 1 6 7 3 8 2 9 4 5 Total 8 15 10 7 10 7 17 5 11 n — 2 P 9 r l - .016 S.E. — ,354 Ratio - .016 - 0.04 .354 1947 LINE 45-12 -22 -29 -39 -45 -46 -60 -91 -92 -103 -134 -151 Female 11 12 9 4 10 1 7 2 8 5 3 6 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 n - 2 p - 12 r l - 0 S.E. - .301 Ratio - 0 — 0.0 .301 54. T A B L E 17 THE CONSISTENCY OF FEMALE AND MALE EFFECTIVENESS OF SIX CLONAL LINES FOR 1946 and 1947 TESTED BY THE RANK CORRELATION COEFFICIENT. FEMALE EFFECTIVENESS LINE 45-12 -29 -45 -46 -134 -151 1946 6 3 4 2 1 5 1947 6 4 5 1 2 " 3 Total 12 7 9 3 3 8 n — 2 P 6 r l - .770 S.E. .448 Ratio - .770 - 1.711 .448 MALE EFFECTIVENESS LINE 45-12 -29 -45 -46 -134 -151 1946 4 5 1 6 2 3 1947 6 2 1 4 5 3 Total 10 7 2 10 7 6 n - 2 p - 6 r 1 - .256 S.I. - .448 Ratio - .256 - .571 .448 55 . n e x t , the male and female behaviour o f each l i n e was not very c o n s i s t e n t . Too much importance must not 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 data o f Table 17 shows t h a t some l i n e s behave q u i t e c o n s i s t e n t l y . F o r i n s t a n c e , i n both years 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 parents w h i l e l i n e 45-12 was v e r y poor , on the o t h e r hand, l i n e 45-45 was a c o n s i s t e n t l y good male parent 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 presented 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 tudy can d i f f e r from one l i n e to the next s i n c e the e f f e c t i v e n e s s o f those l i n e s as female and male parents d i f f e r . The d i f f e r e n c e s may be due to 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 to the d i f f e r e n c e s i n g e n e t i c a l c h a r a c t e r i s t i c s o f the v a r i o u s c l o n a l l i n e s , whether the d i f f e r e n c e s are due to the former o r the l a t t e r cannot , however, be c l e a r l y determined u n t i l d i a l l e l c r o s s i n g data f o r one o r two more years i s o b t a i n e d from the l i n e s . I n c o n c l u s i o n i t must be remembered that the combining a b i l i t y o f the l i n e s was determined from c o n t r o l l e d crosses and does not 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 s e l f -p o l l i n a t i o n appears to p l a y a s i g n i f i c a n t p a r t i n the open-p o l l i n a t e d seed set o b t a i n e d , must be taken 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 tock can be 56 . e v a l u a t e d as to t h e i r combining a b i l i t y o r c r o a s - e o m p a t i l i t i t y under n a t u r a l c o n d i t i o n s . IV SELF- AND CROSS-FERTILITY COMPARED. A l f a l f a b e i n g a n a t u r a l l y c r o s s - p o l l i n a t e d c r o p , one would expect the *» c rossed *» seed s e t to exceed the » s e l f a d " seed s e t . Many workers have found t h i s to be t r u e . F o r 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 , found 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 se 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 se t a f t e r c o n t r o l l e d c r o s s -i n g than a f t e r s e l f i n g . They have e x p l a i n e d t h a t the d i f f e r -ences are due to 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 to the 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 per p o d , w h i l e on s e l f i n g o n l y 1.36 seeds per p o d . B o l t o n (1948) r e p o r t s tha t s e l f - f e r t i l i t y ranged from .02 to 5 . 2 2 seeds per f l o w e r s e l f e d w i t h an average o f 1 . 58 , and the c r o s s - f e r t i l i t y v a r i e d from 2.67 to 8.26 seeds per pod w i t h an average of 5 . 54 . 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 data a t A g a s s i z i t was p o s s i b l e to 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 of o t h e r w o r k e r s . 57 • The average seeds per f l o r e t and seeds per pod from the s e l f - f e r t i l i t y data (Table 1 and 2 J and the d i a l l e l c r o s s i n g data (Table 13 and 14) were found f o r 1946 and 1947. 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 presented 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 to 0.06 - 1 . 6 1 seeds per pod Range from 2 .8 to 1.0 G r o s s - f e r t i l i t y — 1 . 3 ? seeds per f l o r e t Range from 2.8 to 0 . 1 - 2 . 9 4 seeds per pod Range from 4 . 8 to 1.0 1947 S e l f - f e r t i l i t y — .46 seeds per f l o r e t Range from 1.3 to 0.03 - 1.39 seeds per pod Range from 2.41 to 1.0 C r o s s - f e r t i l i t y - 1.52 seeds per f l o r e t Range from 4 .0 to 1.0 - 2.98 seeds per pod Range from 6.3 to 0.6 From the data 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 per f l o r e t and per pod than 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 t h i s a l f a l f a do n o t , however, seem to be as g r e a t as the d i f f e r e n c e s found f o r o t h e r a l f a l f a s , i n f a c t , the c r o s s - p o l l i n a t e d seed se t i s o n l y three t imes the s e l f - p o l l i n a t e d seed s e t . I t might 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 , t h i s a l f a l f a i s more h i g h l y s e l f - f e r t i l e than o t h e r a l f a l f a s . I t i s i n t e r e s t i n g o t note t h a t both the n c rossed *» and 58. » s e l f e d " seed se t are c o n s i s t e n t l y below tfte seed se t ob ta ined by o t h e r w o r k e r s . The reason f o r t h i s lower 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 , to 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 to the i n h e r e n t , hence g e n e t i c a l , nature o f t h i s a l f a l f a . 39. SUMMARY AND CONCLUSIONS 1. Rhizoma a l f a l f a , a new v a r i e t y possessing a spreading h a b i t , o r i g i n a t e d from s i x h y b r i d s of a c r o s s between the <&4&£,e&&s* species Medic ago f a l c a t a var. Don (the seed parent) and M. s a t i v a var. Grimm or Ontario Variegated ( p o l l e n p a r e n t ) . I t represents s e l e c t i o n over s i x generations. 2. Because of the 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 of growth h a b i t and breeding behaviour that do not appear i n other commercial v a r i e t i e s of a l f a l f a . 3. To explore the reason f o r i t s unusual behaviour, 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 i n t o the p l a n t s of the parent species and of the s i x F^ ^ hybrids from which Rhizoma was developed were conducted a t the 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 of the parent p l a n t s revealed 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 the s a t i v a parent 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 parent, o f t e n c a l l e d M. media, might be i r r e g u l a r because of i t s h y b r i d (M. s a t i v a x M. f a l c a t a ) origin., was recognized. 3. Chromosome s t u d i e s of the s i x h y b r i d s i n d i c a t e d t h a t three were t r i p l o i d s (S = 24) and three were t e t r a p l o i d s (s = 32). 6. The m e i o t i c behaviour and r e s u l t i n g gametic p r o d u c t i o n 6 0 . of the t r i p l o i d s was i r r e g u l a r while that o f the 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 th a t the t e t r a p l o i d s r e c e i v e d a double complement o f f a l c a t a chromosomes from the Don parent through the 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 the more d e s i r a b l e t e t r a p l o i d types, and 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 of c e r t a i n p l a n t s of the F^ generations, i t was supposed t h a t t r i p l o i d s do not e x i s t i n the new v a r i e t y . 9. The c y t o l o g i c a l s t u d i e s have exp l a i n e d phenotypie pecu-l i a r i t i e s of Rhizoma which h i t h e r t o f o r e were unexplain-able 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 , presumably, equal complements of chromosomes from each parent s p e c i e s . 10. To f i n d the reason f o r anomalies i n the breeding behaviour of Rhizoma stock, s e l f - p o l l i n a t i o n , open-p 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 , c r o s s - f e r t i l i t y , and combining a b i l i t y s t u d i e s have been conducted on selected c l o n a l l i n e s of the F^ generation at the Dominion Experimental Farm a t Agassiz. 11. 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 set very l i t t l e seed. The e f f e c t i v e bee population was very low and could not account for the high seed s e t . 12. A 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 f o r 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 t h a t " s e l f e d " seed was c o n t r i b u t i n g to the op e n - p o l l i n a t e d seed s e t , and that, the.greater the s e l f - f e r t i l i t y o f a l i n e , the greater would be i t s seed set 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 of f l o r e t t r i p p i n g and op e n - p o l l i n a t e d seed set i n d i c a t e d t h a t probably much of the seed was being set without the a i d o f i n s e c t v i s i t a t i o n and hence supported 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 deter-mined by c o n t r o l l e d d i a l l e l crosses was found t o vary . considerably. I n a d d i t i o n i t was found t h a t the e f f e c t i v e n e s s of a l i n e as a female and as 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 of emasculation was checked and found t o be s a t i s f a c t o r y . A l s o , 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 they are not cl o s e to each o t h e r , one not used by other ..workers, was r e p o r t e d . 1 6 . The value o f determining both male and female e f f e c -t i v e n e s s o f a l i n e as a c r i t e r i o n of i t s combining a b i l i t y was emphasized. 1 7 . The cross-and s e l f - f e r t i l i t y v alues of a number o f c l o n a l l i n e s was compared. I n 1946 and 1 Q47 the average s e l f - f e r t i l i t y was 0.44 and 0.46 seeds per f l o r e t , w i t h a range from 0.6 to 1.4 and 0.3 to 1.3 seeds per 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 both years was 1.37 and 1 . 3 2 seeds per 6 2 . f l o r e t , w i t h a range from 0 . 1 t o 2 . 8 and 1 . 0 t o 4.0 r e s p e c t i v e l y . From the 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 concluded that c r o s s - p o l l i n a t i o n "by bees was not as frequent 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 that the seed set a f t e r c o n t r o l l e d c r o s s - p o l l i n a t i o n does not g r e a t l y exceed the seed set a f t e r s e l f -p o l l i n a t i o n . Since the 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 the Fj_ generation o f Rhizoma s t o c k , no attempt was made to e x p l a i n the anomalies of breeding behaviour found i n the F A g e n e r a t i o n of Rhizoma stock 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. Sci. 25:161-179. Atwood, S. S., 1947. Cytogenetics and breeding of forage crops. Advances in 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 in relation to certain methods of selection. Sci. Agr. 28:97-126. Bolton, J. L., and J. R. Fryer, 1937. Inter-plant variations in certain seed-setting processes in a l f a l f a . Sci. Agr. 18:148-160. Brink, R. A., and D. C. Cooper, 1936. The mechanism of pollination in a l f a l f a ( Medicago sativa). Amer. Jour. Bot. 23:678-683. Burkhart, A, 1937. Frequency of cross-fertilization in 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 in Utah. Utah Agr. Exp. Stat. Bull. 258, 47 pp. Clausen, R. E., 1928 (b). Interspecific hybridization in I&jootiana 711 The cytology of hybrids of the synthetic species, digluta, with i t s parents, glutinosa and Tabacum. Univ. Calif. Pub. Bot. 11:177-211. Cooper, D. C, and R. A. Brink, 1940. Partial self-incompat-i b i l i t y and the collapse of f e r t i l e ovules as factors affecting seed formation in a l f a l f a . Jour. Agr. Res. 60:453-472. Darlington, C. D., and La Cour, L. F., 1942. The Handling of Chromosomes. Macmillan Co. East, E. M., 1928. The genetics of the Genus Nicotiana. Bibliographia Genetica, 4:243-320. Eek, C J., 1943. Association of Economic Characters in Rhizoma A l f a l f a . M. S. A. thesis University of British Columbia (unpublished). Englebert, 7. A., 1932. A study of various factors influencing seed production in a l f a l f a (Medicago sativa). Sci. Agr. 12:593-603. 64. Fryer, J. R., 1930. Cytological studies in MEdicago, Melilotus and Trigonella. Can. Jour. Res. 3:3-50. Goodspeed, T. H., and Clausen, R. E., 1927 (b). Interspecific hybridization in Nicotiana V. Cytological features of two F-i hybrids made with Nicotiana Bigelovii as a parent. Univ. of Calif. Pub. Bot. 11:117-125. Gray, H. E., 1925. Observations on tripping of al 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. falcata. Genetica 2:535-536. Hansen, N. E., 1909. The wild alfalfas and clovers of Siberia, with a perspective view of the alfalfas of the world. U. S. D. A. Bull. 150. Hanson, Angus A., 1944. A digest of reports relating $ro "Rhizoma" a l f a l f a . Undergraduate Essay University of British 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 cytological studies of al f a l f a (Medioago). Thesis for 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 diploid, t r i p l o i d and tetraploid 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 relation to forage crop improvement. Sci. Agr. 8:1-40. Knowles, R. P. 1943. The---role of insects, weather conditions, and plant character in seed setting of a l f a l f a . Sci. Agr. 24:29-50. Ledingham, G. F., 1940. Cytological and developmental studies of hybrids between Medioago sativa and a diploid form of M. falcata. Genetics 25:1-15. Lejeune, A. J., and P. J. Olson, 1940. Seed setting i n a l f a l f a . Sci. Agr. 20:570-573. Lepper, R.,Jr., and T. E. Odland, 1939. Inheritance of flower : .... -color in 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 certain 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 Hereditas, 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 pastures. U. S. D. A. Bureau of P l a n t Industry, B u l l . , 258. Peek, 0., and J . L. Bo l t o n , 1946. A l f a l f a seed production i n northern Saskatchewan as a f f e c t e d by bees, w i t h a report on means of i n c r e a s i n g the p o p u l a t i o n of n a t i v e bees. S c i . Agr. 26:388-418. Reeves, R. G., 1930. Nuclear and cytoplasmic d i v i s i o n i n the microsporogenesis of a l f a l f a . Am. J . Botany Vol.17, 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 reference to a l f a l f a (Medicago sp.) M. S. A. Thesis, U n i v e r s i t y of B r i t i s h Columbia, (unpublished). Stewart, G., 1934. E f f e c t s of inbreeding on v a r i a b i l i t y i n a l f a l f a . Jour. Agr. Research, 49:669-694. Tysdal, H. M., 1940. I s t r i p p i n g necessary f o r seed s e t t i n g i n a l f a l f a , Jour. Amer. Soc. Agron. 32:570-585. Tysdal, 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. Jo. 32:405-407. Tysdal, H. M., 1946. Inf l u e n c e of t r i p p i n g , s o i l moisture, p l a n t spacing and l o d g i n g on a l f a l f a seed production. Jour. Amer. Soc. Agron. 38:515-535. Tys 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. Univ. of Neb. Exp. S t a t . Res. B u l l . 124 46pp. Tysdal, H. M., and Westover, H. L., 1937. A l f a l f a improvement. Yearbook of 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 . Jour. Amer. Soc. Agron. 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 . Am. Soc. Agron. Jo. 11: 259-266. Watkins, A. E., 1924. Genetic 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 of Genetics 14: 129-171. Wilcoxon, Frank, 1946. I n d i v i d u a l comparisons of grouped data by ranking method. Jour. Ec.i Ent. 39: 269. 

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