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Reproduction and breeding in a dioecious bluegrass, Poa confinis Vasey Hanna, Michael Ross 1955

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REPRODUCTION AND BREEDING IN A DIOECIOUS BLUEGRASS, Poa  c o n f i n i s VASEY  by  MICHAEL ROSS HANNA  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS  FOR THE DEGREE OF  MASTER OF SCIENCE IN AGRICULTURE i n the Department of Agronomy  We accept t h i s t h e s i s as conforming to the standard r e q u i r e d from candidates f o r the degree o f MASTER OF SCIENCE IN AGRICULTURE.  Members o f the Department o f Agronomy  THE UNIVERSITY OF BRITISH COLUMBIA June 1955  ABSTRACT  Dune b l u e g r a s s , Poa  c o n f i n i s Vasey, i s a d i o e -  c i o u s grass n a t i v e to the P a c i f i c Coast of North  America.  I t has a very r e s t r i c t e d h a b i t a t , being c o n f i n e d  almost  e n t i r e l y to the s e m i - s t a b i l i z e d , porous sand areas of the coast.  Herbarium s t u d i e s , and f i r s t - h a n d o b s e r v a t i o n s of  a number of P. c o n f i n i s s i t e s i n d i c a t e t h a t i t i s a v e r y uniform s p e c i e s . Under c u l t i v a t i o n on The  U n i v e r s i t y of B r i t i s h  Columbia farm P. c o n f i n i s grows v i g o r o u s l y and forms a t h i c k sod.  I t s f i n e - l e a v e d growth, rhizomatous r o o t  system and r a p i d l y - s p r e a d i n g h a b i t , together with sandy nature of i t s n a t i v e h a b i t a t , a l l suggest may  f i n d use as a t u r f s p e c i e s f o r sandy g o l f  the  that i t  courses  and s i m i l a r areas along the c o a s t . A survey of the l i t e r a t u r e on r e p r o d u c t i o n i n the bluegrasses r e v e a l s t h a t apomixis Poa,  i s widespread i n  and t h a t apospory or d i p l o s p o r y f o l l o w e d by pseudo-  gamous embryo development i s the u s u a l form of apomictic reproduction.  Breeding procedures  with apomictic  grasses muct be c o n s i d e r a b l y m o d i f i e d , but the techniques  of improvement are s t i l l  able to the b l u e g r a s s  blue-  standard  theoretically  avail-  breeder.  A c y t o l o g i c a l study of embryo sac development  i n p i s t i l l a t e p l a n t s i n d i c a t e s t h a t i t f o l l o w s the "normal scheme, and t h a t r e p r o d u c t i o n i s s e x u a l . t a i n s a s i n g l e EMC  Each ovule con-  which undergoes a r e g u l a r m e i o t i c  divi-  s i o n , g i v i n g r i s e to a t r i a d or t e t r a d of megaspores, of  which forms the h a p l o i d embryo s a c .  one  Somatic chromosome  numbers of 2n = 42 are found i n two EMCa i n d i a k i n e s i s . The presence of twin embryo sacs i s observed i n two ovules Very marked a n t i p o d a l development, with an i n c r e a s e i n s i z e and number of c e l l s , cell,  and i n the number of n u c l e i per  i s c h a r a c t e r i s t i c of the mature female  gametophyte.  M i c r o s p o r o g e n e s i s i n the staminate p l a n t s appears "normal . 11  also  S t u d i e s of anthers i n p i s t i l l a t e  floret  and ovules i n staminate f l o r e t s show t h a t t h e i r development proceeds n o r m a l l y up to a c e r t a i n p o i n t and then breaks down. Embryo and endosperm  development can be seen i n  s e c t i o n s of ovules prepared a f t e r the p o l l i n a t i o n of P. c o n f i n i s by P. p r a t e n s i s .  The embryos are b e l i e v e d t o be  the  product of t r u e h y b r i d i z a t i o n .  the  c r o s s i n g s among P. c o n f i n i s , P. macrantha, P. p r a t e n -  s i s and P. compressa u s i n g the f i r s t  Seed forms a f t e r a l l  two s p e c i e s as female  parents, but the seed i n v a r i a b l y s h r i v e l s s h o r t l y before reaching maturity. The e x c i s i o n of h y b r i d embryos from seeds formed a f t e r some of the i n t e r s p e c i f i c c r o s s e s , and the c u l t u r e  of these embryos on agar media have been s u c c e s s f u l l y c a r r i e d out.  Tissues of p u t a t i v e macrantha x c o n f i n i s  hybrids are growing, and showing some d i f f e r e n t i a t i o n . The r e s u l t s i n d i c a t e t h a t hybrids between P. c o n f i n i s and other blue grasses can probably be obtained through embryo c u l t u r e techniques.  ACKNOWLEDGEMENTS The, w r i t e r wishes te» express h i s appreci a t i o n to D r . V. C. B r i n k , D i v i s i o n of P l a n t ence, f o r h i s suggestion  Sci-  of t h e Poa c o n f i n i s .  problem and f o r h i s a s s i s t a n c e and guidance the course of the study.  during  The t r i p s to t h e Poa  c o n f i n i s h a b i t a t s i t e s on Vancouver I s l a n d and i n Washington State were o n l y made p o s s i b l e through Dr* Brink's, assistance:. Grateful, acknowledgement i s a l s o made t o Dr. T. M. C. T a y l o r , Department of B i o l o g y and Botany, f o r a r r a n g i n g e r i a l s from, other  i n s t i t u t i o n s ; t o Mr. S. F.  F l o r i a n f o r preparing and  the lioan o f herbarium mat-  the media f o r embryo) c u l t u r e ;  to Miss* M. Wilson f o r donating  her time towards  the f i n a l p r e p a r a t i o n of the manuscript* The; f i n a n c i a l support  o f t h e N a t i o n a l Research  C o u n c i l of Canada, i s acknowledged with  thanks.  TABLE OF CONTENTS Page I II  INTRODUCTION  1  THE SYSTEMATICS OF THE BLUEGRASSES, WITH PARTICULAR REFERENCE TO Poa c o n f i n i s  5  A. B. III  T r i b a l , g e n e r i c , and s p e c i f i c relationships A study of p r e s c r i b e d herbarium specimens of Poa s p e c i e s  THE DISTRIBUTION AND ECOLOGY OF Poa conf i n i s AND SOME GENERAL COMMENTS ON ITS INTRODUCTION TO CULTIVATION  12  A. D i s t r i b u t i o n B. Ecology C. I n t r o d u c t i o n to c u l t i v a t i o n IV  REPRODUCTION IN THE BLUEGRASSES A. B. C.  V  VI  A g e n e r a l statement on l i t e r a t u r e review The terminology o f the a p o m i c t i c phenomena Reproduction i n i n d i v i d u a l Poa s p e c i e s  BREEDING IN THE BLUEGRASSES A. B.  B.  53  I n t r a s p e c i f i c improvement Interspecific hybridization  STUDIES OF REPRODUCTION IN Poa c o n f i n i s A.  25  Attempts t o induce f l o w e r i n g under a r t i f i c i a l conditions 1. Introduction 2. M a t e r i a l s and methods 3. Results Cytology and h i s t o l o g y 1. Megasporogenesis i n p i s t i l l a t e flowers (a) M a t e r i a l s and methods (b) Observations 2. Embryo and endosperm f o r m a t i o n f o l l o w i n g p o l l i n a t i o n o f Poa c o n f i n i s by Poa p r a t e n s i s Ca) M a t e r i a l s and methods (b) Observations -ii-  65  TABLE OF CONTENTS (CONTINUED) Page 3.  4.  5.  C.  VII VIII IX  Anther development i n p i s t i l l a t e flowers (a) M a t e r i a l s and methods (b) Observations M i c r o s p o r o g e n e s i s i n staminate flowers (a) M a t e r i a l s and methods (b) Observations Ovule development i n staminate flowers (a) M a t e r i a l s and methods  H y b r i d i z a t i o n techniques and attendant macroscopic o b s e r v a t i o n s 1. Techniques used t o secure c r o s s e s 2. Observations f o l l o w i n g p o l l i n a t i o n s 3. Embryo c u l t u r e (a) M a t e r i a l s and methods (b) O b s e r v a t i o n s and r e s u l t s  DISCUSSION  108  SUMMARY  124  LITERATURE CITED  127 138  X. APPENDIX  -iii-  LIST OF FIGURES Figure 1  2  3  4  5  6  Page P a r k s v i l l e , V. I . The d r i e d grass on the bank above the l i t t o r a l area i s m o s t l y P. c o n f i n i s and the t a l l e r P. p r a t e n s i s •.•.  14  P a r k s v i l l e , V. I . S c a t t e r e d t u f t s o f P. c o n f i n i s can be seen i n the open sand i n the foreground, j u s t above t h e upper l i m i t of the d r i f t w o o d a r e a . The shrub i s G r i n d e l i a squarrosa  15  P a r k s v i l l e , V. I . Close-up of the P. c o n f i n i s which can be seen i n the f o r e ground i n Figure 2  15  Dune area of Twin Harbours S t a t e Park, Gray's Harbour, Wash., where P. macrantha and a s p e c i e s of Ammophila are the dominant grasses  17  Close-up of p o r t i o n of foreground i n F i g ure 4 showing l a r g e t u f t s o f P. ma era nt ha. Some P. c o n f i n i s i s a l s o found i n t h i s s t a b i l i z e d area  18  Small t u f t s o f P. c o n f i n i s growing i n a sandy vacant l o t near P o i n t C h e h a l i s , Gray's Harbour  19  7  Megasporogenesis i n Poa c o n f i n i s  73  8  Megasporogenesis i n Poa c o n f i n i s  74  9  Megasporogenesis i n Poa c o n f i n i s  76  10  One-celled embryo sacs i n Poa c o n f i n i s  .»  78  11  Embryo sac development i n Poa c o n f i n i s  ...  80  12 13  Embryo sac development i n Poa c o n f i n i s ». A, B, C, and D - Female gametophyte development i n Poa c o n f i n i s . E and F - Embryo and endosperm f o r m a t i o n f o l l o w i n g p o l l i n a t i o n of p i s t i l l a t e Poa c o n f i n i s f l o r e t s with Poa p r a t e n s i s pollen -iv-  83  84  LIST OF FIGURES (CONTINUED) Figure 14 15 16 17  Page Anther development of Poa c o n f i n i s  in pistillate  plants 90  M i c r o s p o r o g e n e s i s i n staminate p l a n t s of Poa c o n f i n i s  94  M i c r o s p o r o g e n e s i s i n staminate p l a n t s of Poa c o n f i n i s  96  P l a n t s used i n the attempted c o n f i n i s x P. p r a t e n s i s  99  -v-  cross P.  REPRODUCTION AND BREEDING IN A DIOECIOUS BLUEGRASS, Poa c o n f i n i s VASEI I  INTRODUCTION Many grasses need only t o be given study to  demonstrate t h e i r usefulness to man*  Although grass i s  one of the world's greatest n a t u r a l resources, u n t i l r e l a t i v e l y recent times very l i t t l e has bee.n done to conserve, improve, r e p l e n i s h or i n many instances even make any use of more than a small p r o p o r t i o n of the t o t a l number of grass species.  The c e r e a l s and sugar-supplying species  have been c u l t i v a t e d and improved by man f o r c e n t u r i e s , and the c u l t i v a t e d and range forage grasses are now r e c e i v i n g considerable a t t e n t i o n . ' Nonetheless, the potent i a l i t i e s of a vast number of the 4,000 or more grass species are v i r t u a l l y unknown. There are some species of Poa, the bluegrasses, among these l i t t l e - k n o w n grasses, although the members of t h i s genus are probably more widely known than those belonging to many other grass genera.  P a r t of t h e i r recog-  n i t i o n i s due t o the p a r t i c u l a r importance of a few of the i n d i v i d u a l Poa s p e c i e s , e s p e c i a l l y P. p r a t e n s i s * . Kentucky bluegrass, but many other bluegrasses are a l s o of great value.  The challenge of the d i f f i c u l t taxonomy  * F u l l b o t a n i c a l nomenclature f o r a l l species named appears.in Appendix.  -2-  of Poa has r e s u l t e d i n much study on the i n d i v i d u a l s p e c i e s , subspecies and forms i n attempts to determine and e v o l u t i o n a r y patterns w i t h i n the genus.  relationships The genus i s  of added i n t e r e s t because of i t s cosmopolitan character. Although p r i m a r i l y grasses of the temperate regions of the world, bluegrasses are found on every cont i n e n t and i n a wide v a r i e t y of h a b i t a t s . D i f f e r e n t bluegrasses t h r i v e i n a r c t i c , a n t a r c t i c , and a l p i n e h a b i t a t s , i n woodlands, meadows, marshes and a r i d p l a i n s , and on rocky seashores and c o a s t a l dunes. w i t h i n Poa i s a l s o considerable:  The v a r i e t y of form annuals and p e r e n n i a l s ,  rhizomatous, s t o l o n i f e r o u s , and bunchgrass types, some as t a l l as a man and others only a few inches i n h e i g h t — a l l are found among the bluegrasses. Among the Poa species about which very l i t t l e i s known i s Poa c o n f i n i s or dune bluegrass, a s m a l l , dioecious species n a t i v e t o the sandy meadows and dune areas of the P a c i f i c Coast of North America.  Established  i n t r i a l p l o t s on The U n i v e r s i t y of B r i t i s h Columbia farm s e v e r a l years ago, a biotype of P. c o n f i n i s o r i g i n a t i n g from Vancouver I s l a n d has shown s e v e r a l c h a r a c t e r i s t i c s which merit p a r t i c u l a r a t t e n t i o n .  I t s very f i n e - l e a v e d  growth and i t s strong, r a p i d l y - s p r e a d i n g rhizomatous  root  system, together w i t h the nature of i t s n a t i v e h a b i t a t , have a l l suggested that i t might prove of value as a t u r f  s p e c i e s f o r sandy g o l f c o u r s e s , p a r k s and along the coast.  similar locations  A f u r t h e r p o s s i b i l i t y i s t h a t some o f  the more d e s i r a b l e f e a t u r e s o f P. c o n f i n i s might be combined, through i n t e r s p e c i f i c h y b r i d i z a t i o n , w i t h those o t h e r b l u e g r a s s e s , p a r t i c u l a r l y P. p r a t e n s i s and P.  of  com-  pressa. The  f a c t t h a t dune b l u e g r a s s i s a d i o e c i o u s  s p e c i e s i s of added i n t e r e s t , as s c a n t a t t e n t i o n seems t o have been p a i d t o d i o e c i s m i n g r a s s e s .  No l e s s t h a n  eight North American bluegrasses are d i o e c i o u s , yet i s known of t h e r e p r o d u c t i v e f e a t u r e s or o t h e r  little  character-  i s t i c s of most o f them. The  d i s c o v e r y d u r i n g t h e l a s t two  the w i d e s p r e a d o c c u r r e n c e among t h e Poa  New  o f a p o m i c t i c seed  formation  s p e c i e s has a r o u s e d a g r e a t d e a l o f  among p l a n t b r e e d e r s and icance.  decades o f  interest  i s p r o v i n g t o be o f g r e a t  o p p o r t u n i t i e s f o r i n t r a - and  signif-  interspecific  h y b r i d i z a t i o n have been opened up, and t h e development o f a l m o s t l i m i t l e s s numbers o f new t i a l l y possible.  Poa forms now  seems p o t e n -  But the p r e s e n c e o f a p o m i x i s g r e a t l y  m o d i f i e s the standard p l a n t breeding  procedures;  for this  r e a s o n i t i s e s s e n t i a l t o e s t a b l i s h the method o f r e p r o d u c t i o n i n p l a n t s w i t h w h i c h b r e e d i n g work i s b e i n g templated  and which a r e r e l a t e d t o known a p o m i c t i c  conforms.  W i t h t h i s o b j e c t i n mind, a d e t a i l e d c y t o l o g i c a l s t u d y o f  r e p r o d u c t i o n i n P» c o n f i n i s was undertaken..  This study  was complemented by a t t e m p t s a t h y b r i d i z a t i o n between  - 5 -  II  THE  SYSTEMATICS OF THE  REFERENCE TO Poa A.  BLUEGRASSES, WITH PARTICULAR  confinis  T r i b a l , g e n e r i c , and s p e c i f i c  relationships  The t r i b e F e s t u c e a e , t o which t h e genus Poa b e l o n g s , r a n k s w i t h t h e t r i b e s Hordeae, Aveneae, Oryzeae and Andropogoneae as a most I m p o r t a n t economic group o f t h e Gramineae.  F e s t u c a , Bromus, D a c t y l i s , E r a g r o s t i s  and K o e l e r i a a r e o t h e r s o f importance among t h e e i g h t y t h r e e genera w i t h i n t h e t r i b e  (89).  The t r i b e i s d i s t i n g u i s h e d from t h e o t h e r t e n t r i b e s r e c o g n i z e d by H a c k e l ( 4 9 )  and Rendle ( 8 9 )  by t h e  f o l l o w i n g combined c h a r a c t e r s : Culms herbaceous, a n n u a l ; l e a f b l a d e s e s s i l e , not j o i n t e d t o t h e s h e a t h ; the s p i k e l e t s upon d i s t i n c t p e d i c e l s and a r r a n g e d i n p a n i c l e s or racemes; s p i k e l e t s c o n t a i n i n g two o r more f l o r e t s ; t h e f l o w e r i n g glumes g e n e r a l l y l o n g e r t h a n the empty glumes, and unawned o r w i t h a s t r a i g h t awn from t h e p o i n t ; t h e r a c h i l l a j o i n t e d above t h e empty glumes, which remain a f t e r t h e f r u i t i n g glumes have f a l l e n ; d i s t i n c t i n t e r n o d e s produced between t h e f l o r e t s . The genus Poa i t s e l f i s d e s c r i b e d by H i t c h c o c k (54):  " S p i k e l e t s 2 - to s e v e r a l - f l o w e r e d , the r a c h i l l a d i s a r t i c u l a t i n g above the glumes and between t h e f l o r e t s , t h e uppermost f l o r e t r e duced o r r u d i m e n t a r y ; glumes a c u t e , k e e l e d , somewhat u n e q u a l , the f i r s t u s u a l l y 1 - n e r v e d , t h e second u s u a l l y 3 - n e r v e d ; lemmas somewhat k e e l e d , acute or a c u t i s h , r a r e l y obtuse, awnless, membranaceous, o f t e n somewhat s c a r i o u s a t t h e summit, 5 - n e r v e d ( i n t e r m e d i a t e n e r v e s , t h a t i s ,  -6t h e p a i r between the k e e l and the m a r g i n a l n e r v e s , r a r e l y o b s o l e t e ) , the n e r v e s sometimes pubescent. Low or r a t h e r t a l l s l e n d e r a n n u a l s or u s u a l l y p e r e n n i a l s w i t h s p i k e l e t s i n open or c o n t r a c t e d p a n i c l e s , the r e l a t i v e l y narrow b l a d e s f l a t , f o l d e d , o r i n v o l u t e , ending i n a boat-shaped t i p . " Hitchcock  (54) d i v i d e d the 60-odd Poa  species  found i n t h e U n i t e d S t a t e s i n t o seven s e c t i o n s :  the  Annuae, P r a t e n s e s , P a l u s t r e s , A l p i n a e , E p i l e s , S c a b r e l l a e and Nevadenses.  These s e p a r a t i o n s he based p r i n c i p a l l y  on s i n g l e c h a r a c t e r s . The  s p e c i e s w i t h i n the s e c t i o n P r a t e n s e s  d e f i n e d by H i t c h c o c k i n g rhizomes,  were  (54) as p e r e n n i a l p l a n t s from  creep-  h a v i n g compressed s p i k e l e t s , w i t h the  glumes and lemmas b e i n g k e e l e d .  The  sixteen species  which he p l a c e d i n t h i s s e c t i o n i n c l u d e s i x d i o e c i o u s species:  Poa  c o n f i n i s , P. macrantha, P. a r a c h n i f e r a .  P. D o u g l a s i i , P. r h i z o m a t a  and P. a t r o p u r p u r e a .  The  re-  m a i n i n g s p e c i e s a r e P. p r a t e n s i s , P. compressa. P. c u r t a . P. n e r v o s a . P. K e l l o g g i i . P. l a x i f l o r a . P. P. a r i d a . P. g l a u c i f o l i a and P. a r c t i c a . d i d not a c t u a l l y d e s c r i b e P. r h i z o m a t a as d i o e c i o u s s p e c i e s , but Marsh (68)  over a l m o s t i t s e n t i r e range.  Hitchcock  and P.  (54)  atropurpurea  d e s c r i b e s them as  such i n a more r e c e n t taxonomic t r e a t m e n t . has a l s o found t h a t P. nervosa  cuspidata.  Grun (42,  43)  has o n l y a b o r t i v e a n t h e r s  -7P. c o n f i n i s , P. macrantha, and P. D o u g l a s i i a r e a l l P a c i f i c Coast sand dune s p e c i e s and have been g e n e r a l l y c o n s i d e r e d t o be c l o s e l y r e l a t e d by o t h e r taxonomists  (33, 1 4 ) , since Hitchcock (54) f i r s t  them t o g e t h e r w i t h i n the P r a t e n s e s . ( 5 3 ) and Clausen et a l . "rhizomes"  placed  However, Hartung  ( 2 2 ) i n d i c a t e t h a t the  so-called  o f P. macrantha and P. D o u g l a s i i a r e a c t u a l l y  l o n g a e r i a l r u n n e r s o r w h i p - l i k e stems w h i c h bend o v e r and r o o t a t the nodes.  These have the appearance o f  rhizomes when b u r i e d under sand.  A c c o r d i n g t o Hartung  ( 5 3 ) t h e s e a e r i a l r u n n e r s a r e not found i n any o t h e r  North  American Poa s p e c i e s . B e e t l e ( 1 4 ) c o n s i d e r s a l l t h r e e o f t h e s e dune s p e c i e s t o be c l o s e l y r e l a t e d t o P. eminens of c o a s t a l n o r t h e a s t e r n A s i a and a r c t i c America and P. l a b r a d o r i c a o f the c o a s t o f L a b r a d o r .  Marsh ( 6 8 ) says t h a t Poa  con-  f i n i s " s h o u l d not be confused w i t h any o t h e r s p e c i e s " and t h a t i t s c l o s e s t r e l a t i v e seems t o be P.  atropurpurea.  a s p e c i e s w i t h a v e r y r e s t r i c t e d range i n Bear V a l l e y i n t h e San Bernadino  Mountains,  Southern  California.  Marsh (68.) has proposed a d i v i s i o n D i o e c i a t o i n c l u d e a l l the c o m p l e t e l y d i o e c i o u s s p e c i e s i n the U n i t e d S t a t e s and s o u t h e r n Canada.  I n t h i s group he p l a c e s P.  c o n f i n i s , P. macrantha, P. D o u g l a s i i . P.  atropurpurea.  P. P i p e r i , , P. r h i z o m a t a . P. P r i n g l e i , and P. a r a c h n i f e r a .  The  d e s c r i p t i o n o f P. c o n f i n i s g i v e n by Marsh  (68) i s : " P e r e n n i a l ; w i t h c r e e p i n g rhizomes., o f t e n 1 m. or more l o n g . Culms 10-35 ( u s u a l l y 15-20) cm. t a l l , e r e c t , r a r e l y somewhat s p r e a d i n g , g l a b r o u s , smooth or s t r i a t e , l e a f y t o above t h e m i d d l e or n e a r l y t o t h e i n f l o r e s c e n c e i n many s m a l l p l a n t s . Sheaths 1-3 ( u s u a l l y 2) per culm, g l a b r o u s , u s u a l l y s t r i a t e ; g r e e n , tawny or p u r p l i s h i n c o l o r ; shorter than internodes; closed 1/3 - 1/2 t h e i r l e n g t h . Culm b l a d e s 0.5-2.5 cm. l o n g , 1-2 mm. wide, i n v o l u t e , g l a b r o u s on d o r s a l s u r f a c e , g l a b r o u s t o scabrous or m i n u t e l y pubescent on v e n t r a l s u r f a c e . I n n o v a t i o n b l a d e s v a r y i n g i n l e n g t h t o as much as 20 cm., o f t e n as l o n g o r l o n g e r t h a n the culms, f o l d e d o r i n v o l u t e , u s u a l l y more scabrous or pubescent on v e n t r a l s u r f a c e . L i g u l e s up t o 1.5 (2.2) mm. l o n g , obt u s e t o a c u t e , scabrous on d o r s a l s u r f a c e . Pani c l e s e r e c t , c o n t r a c t e d , 1-7 ( u s u a l l y 3-4) cm. l o n g , up t o 2 ( u s u a l l y 1) cm. w i d e . R a c h i s g l a b r o u s , i t s branches u s u a l l y s p a r s e l y s c a b r o u s . P l a n t s c o m p l e t e l y d i o e c i o u s . S p i k e l e t s 2- t o 5 - f l o w e r e d , 3-6 (8) mm. l o n g , 2-4 mm. wide. First glume 1- or 3-nerved; k e e l e d , a c u t e 1/2 - 2/3 the l e n g t h of the 1 s t f l o r e t , about 2/3 t h e l e n g t h of the 2nd glume, g l a b r o u s o r s l i g h t l y scabrous on k e e l near t i p . Second glume u s u a l l y about 3 mm. l o n g , 3-nerved, k e e l e d , b r o a d l y a c u t e t o obt u s e , w i d e r t h a n 1 s t glume, 2/3 - 3/4 t h e l e n g t h o f 2nd f l o r e t , scabrous on upper 1/2 - 1/4 o f k e e l . Lemmas 2-3*5 (4*5) mm. l o n g , a c u t e t o o b t u s e , k e e l e d , g l a b r o u s t o s p a r s e l y and m i n u t e l y scabrous a l l over the back, o c c a s i o n a l l y s c a n t l y pubescent on the back, u s u a l l y a few ( o f t e n c o p i o u s ) cobwebby h a i r s p r e s e n t a t base of l o w e r f l o r e t s . P a l e a s 7/8 as l o n g as lemmas, or i n upper f l o r e t s as l o n g as t h e lemmas, scabrous on k e e l s . R a c h i l l a s g l a b r o u s t o m i n u t e l y p u b e s c e n t . A n t h e r s 1.52 mm. l o n g . " The  only recorded  chromosome count f o r Poa  f i n i s i s t h a t made by Hartung ( 5 3 ) , who the h e x a p l o i d number f o r  Poa.  found 2n •  con42,  B.  A s t u d y o f p r e s c r i b e d h e r b a r i u m specimens o f Poa s p e c i e s An e x a m i n a t i o n was made o f a number o f h e r b a r i u m  specimens o f Poa c o n f i n i s i n o r d e r t o check s e v e r a l c h a r a c t e r i s t i c s o f t h e s p e c i e s a g a i n s t t h o s e p r e s e n t e d by Marsh (68)  ( a b o v e ) , and t o note any v a r i a t i o n s i n c h a r a c t e r s  t h a t m i g h t be r e l a t e d t o g e o g r a p h i c d i s t r i b u t i o n .  Differ-  ences were a l s o n o t e d between P. c o n f i n i s and s e v e r a l o t h e r Poa s p e c i e s , p a r t i c u l a r l y P. a b b r e v i a t a , a n a r c t i c s p e c i e s ( 1 1 , 4 1 , 104) which r e s e m b l e s P. c o n f i n i s q u i t e c l o s e l y i n outward a p p e a r a n c e . The specimens o f P. c o n f i n i s examined were b o r rowed from t h e U n i v e r s i t y o f C a l i f o r n i a , t h e N a t i o n a l Museum o f Canada, and The U n i v e r s i t y o f B r i t i s h C o l u m b i a . These specimens r e p r e s e n t e d c o l l e c t i o n s made i n B r i t i s h Columbia, Washington, Oregon and C a l i f o r n i a . The numbers o f t h e h e r b a r i u m s h e e t s were: U n i v e r s i t y o f C a l i f o r n i a Herbarium numbers:  563639,  580408,  926146,  801585, 759245, 759092, 759091,  525623,  183532,  6O8I7,  249669,  570515,  570516,  525631,  525624,  121967, 128074,  187226, 603582,  586098. N a t i o n a l Museum o f Canada Herbarium numbers:  34712,  34711, 34710, 34709, 34708, 34707,. 34706, 161661, 161769.  *10-  The U n i v e r s i t y of B r i t i s h Columbia Herbarium number:  20,211.  The f o l l o w i n g c h a r a c t e r i s t i c s were noted: Presence or absence of rhizomes; l e a f length i n r e l a t i o n to culm length; culm l e n g t h ; p a n i c l e l e n g t h ; s p i k e l e t length; number of f l o r e t s per s p i k e l e t ; length of 1st and 2nd glumes, and number of nerves; scabrousness of k e e l of 1st glume; l e n g t h of lemma of 1st f l o r e t and number of nerves; prominence of web a t base of lemma; sex of p l a n t . In n e a r l y a l l features the specimens examined were e s s e n t i a l l y the same as described by Marsh  (68),  although minor d i f f e r e n c e s were noted. A l l p l a n t s had s t r o n g l y developed rhizomes. Leaf length i n r e l a t i o n to culm l e n g t h , culm length i t s e l f , and p a n i c l e length were much as described by Marsh, although the p a n i c l e s were more o f t e n 3 cms. or l e s s i n l e n g t h , somewhat shorter than h i s f i g u r e . ences i n s e v e r a l other features were noted:  Slight differthe f i r s t  glume was never found to be 1-nerved, and was o c c a s i o n a l l y f a i n t l y 5-nerved; the second glume was a l s o o c c a s i o n a l l y 5-nerved, but u s u a l l y 3-nerved l i k e the f i r s t glume.  The  scabrousness of the k e e l of the f i r s t glume was somewhat v a r i a b l e , but was u s u a l l y q u i t e evident a t the t i p of the  -11glume.  The web  a t t h e base of t h e lemma seemed a v e r y  v a r i a b l e c h a r a c t e r , i n agreement w i t h Marsh's d e s c r i p t i o n ; i n a few cases i t was i t was  almost e n t i r e l y l a c k i n g , i n others  v e r y pronounced, but i n t e r m e d i a t e t y p e s predominated.  Of the 31 specimens examined 18 were p i s t i l l a t e p l a n t s 13 were s t a m i n a t e ; rudimentary  i n a l m o s t a l l f l o r e t s examined t h e  sex organs o f t h e o p p o s i t e sex c o u l d be  and t h e r e was  and  no i n s t a n c e o b s e r v e d o f the s t r o n g  seen,  develop-  ment o f b o t h s e t s of sex organs i n a s i n g l e f l o r e t or a single plant. On the whole, the p l a n t s examined appeared t o be r e m a r k a b l y u n i f o r m , and t h e v a r i a t i o n s t h a t d i d e x i s t i n some c h a r a c t e r s were t o t a l l y u n r e l a t e d t o the n o r t h south d i s t r i b u t i o n o f t h e s p e c i e s a l o n g the P a c i f i c The  Coast.  specimens of P. a b b r e v i a t a examined d i f -  f e r e d m a r k e d l y from P. c o n f i n i s i n s e v e r a l f e a t u r e s . were h e r m a p h r o d i t i c  and none had rhizomes*  l e n g t h of the 1 s t and mas, tive.  and  copious web  The  equal  2nd glumes, pubescence of the lemo f a l l p l a n t s s t u d i e d were d i s t i n c -  The p l a n t s were g e n e r a l l y l e s s t h a n 10 cms.  A l l the specimens o f P. a b b r e v i a t a r e p r e s e n t e d made i n a r c t i c  All  tall.  collections  regions.  No o t h e r c o a s t a l s p e c i e s examined bore any resemblance t o P.  confinis.  close  -12III  THE DISTRIBUTION AND ECOLOGY OF Poa c o n f i n i s . AND SOME GENERAL COMMENTS ON ITS INTRODUCTION TO CULTIVATION A.  Distribution Poa  confinis i s l i m i t e d e n t i r e l y t o the P a c i f i c  Coast o f N o r t h America (54). A l t h o u g h  i t i slisted i na  number o f f l o r a s a s b e i n g found a s f a r n o r t h a s A l a s k a (54,  57, 104), no specimens have been seen by t h e w r i t e r  from f a r t h e r n o r t h t h a n U c l u e l e t , on t h e West Coast o f Vancouver I s l a n d , and Comox, on t h e e a s t e r n s i d e .  Hulte'n,  i n h i s f l o r a o f A l a s k a and t h e Yukon (56) says t h a t he d i d not see any A l a s k a n specimens o f P. c o n f i n i s .  Collections  from t h e n o r t h e r n c o a s t a l r e g i o n s o f B r i t i s h Columbia a r e r a t h e r i n f r e q u e n t , and t h e s p e c i e s may e x i s t c o n s i d e r a b l y f a r t h e r n o r t h t h a n Vancouver I s l a n d .  However, a t t h e  moment t h e northernmost l i m i t o f P. c o n f i n i s remains unknown. H o w e l l (55) has found P. c o n f i n i s a s f a r south as P o i n t Reyes P e n i n s u l a , C a l i f o r n i a , j u s t n o r t h o f t h e Golden Gate, a l t h o u g h he s t a t e s t h a t on r e c e n t he has been u n a b l e t o f i n d i t a g a i n i n t h i s  occasions  location.  I t c e r t a i n l y o c c u r s f r e q u e n t l y f a r t h e r n o r t h i n Mendocino and Humboldt C o u n t i e s , C a l i f o r n i a , and many c o l l e c t i o n s have a l s o been made a l o n g t h e Oregon and Washington c o a s t s .  C o l l e c t i o n s i n B r i t i s h Columbia have been made a t a number o f p o i n t s , i n c l u d i n g t h e west and e a s t c o a s t s o f Vancouver I s l a n d , t h e G u l f I s l a n d s and Boundary B.  Bay.  Ecology The r e s t r i c t e d h a b i t a t o f Poa c o n f i n i s i s one  o f i t s most d i s t i n c t i v e f e a t u r e s , making i t an interesting  subject f o r e c o l o g i c a l study.  extremely  Throughout  i t s d i s t r i b u t i o n range i t i s c o n f i n e d a l m o s t  completely  t o sand dunes, sandy meadows, s p i t s and f l a t s a l o n g ocean c o a s t .  P r a c t i c a l l y every h e r b a r i u m  the  sheet o f dune  b l u e g r a s s examined has borne a note emphasizing  the sandy  n a t u r e o f i t s c o l l e c t i o n s i t e , and c e r t a i n l y a l l s i t e s seen a t f i r s t hand have been p r e d o m i n a n t l y The  sandy a r e a s .  e c o l o g y of c o a s t a l dune a r e a s ,  particularly  o f Great B r i t a i n , has been t h o r o u g h l y examined by S a l i s b u r y ( 9 1 ) , and the w r i t e r i s not equipped  t o extend  knowledge o f dune s p e c i e s and t h e i r environment.  the  However,  many of the s i t e s v i s i t e d m e r i t some d e s c r i p t i v e study  be-  cause o f t h e i r r a t h e r s p e c i a l i z e d n a t u r e and because the t o t a l area o f such l o c a l i t i e s i n t h e w o r l d i s p r o b a b l y not v e r y g r e a t .  The d e s c r i p t i o n s p r o v i d e d here s h o u l d  regarded as c a s u a l o b s e r v a t i o n s r a t h e r t h a n a t t e m p t s present accurate e c o l o g i c a l data.  to  be  -14Poa  c o n f i n i s was  found a t the f o l l o w i n g  during the course of the study: Whiffin Island  Spit,  Sooke H a r b o u r ;  View B e a c h ,  Parksville; located  Saanichton; Englishman's  on t h e n o r t h - w e s t Island.  Gray's Harbour  Roberts,  Boundary  At raised  beach  and  Bay.  somewhat more d e t a i l  the  spit  In the United  or f l a t  v e r y sandy h i g h water  soil,  Point  Metchosin; River  I t was  also just o f f  S t a t e s P.  confinis places  and a l s o a t  Point  others. P.  confinis  grows b o t h on t h e  " d e l t a " area of Englishman's and  "delta,"  coast at s e v e r a l  Chehalis,  at  Some o f t h e s e s i t e s were s t u d i e d i n  than  Parksville,  Island  on James I s l a n d ,  f o u n d on t h e w e s t e r n W a s h i n g t o n  around  in  W i t t y ' s Lagoon,  and F r e n c h C r e e k , Q u a l i c u m .  Sidney, Vancouver was  on V a n c o u v e r  locations  also  mark ( F i g s . 1,  i n almost pure 2 and  River,  sand j u s t  3).  F i g u r e 1: P a r k s v i l l e , V. I . The d r i e d g r a s s on t h e bank a b o v e t h e l i t t o r a l a r e a i s m o s t l y P. c o n f i n i s and t h e t a l l e r P. p r a t e n s i s .  above  -15In  the  P.  confinis  and  P.  leaved shrubby  former  area,  grows  compressa  behind  the  littoral  i n close association and  a  number  species under  a  s c a t t e r e d cover  Rosa  of  other  area with  itself, P.  grasses of  pratensis and  F i g u r e 2: P a r k s v i l l e , V. I . Scattered t u f t s o f P. c o n f i n i s c a n b e s e e n i n t h e open sand i n the f o r e g r o u n d , j u s t above the upper l i m i t of the d r i f t w o o d a r e a . The s h r u b i s G r i n d e l i a squarrosa.  be  seen  broad-  low-grovring,  gymno c a r p a .  t h e P. c o n f i n i s w h i c h c a n foreground i n Figure 2.  the  in  the  -16In t h i s area the dune bluegrass i s a common though not dominant species and does not appear to be competing s t r o n g l y with the a s s o c i a t e d p l a n t s *  On the low bank  j u s t above the l i t t o r a l area ( F i g . 1) the P. c o n f i n i s i s more evident and seems to be e f f e c t i v e l y holding the sandy s o i l along parts of the bank edge, i n conjunction w i t h the t a l l e r P. p r a t e n s i s . A l l the dune bluegrass i s characteri z e d by a strong rhizome system which connects the more or l e s s s c a t t e r e d t u f t s of grass and acts as a sandbinding agent.  On the beach i t s e l f , j u s t behind the d r i f t -  wood logs at the high t i d e l i n e ( F i g s . 2 and 3 ) , the t u f t s are somewhat more s c a t t e r e d , although s t i l l connected by ' the rhizomatous stems. At French Creek, Qualicum, only a few s c a t t e r e d p l a n t s of dune bluegrass occur i n the r a i s e d beach sand area close to a short road leading to the shore. area i s considerably d i s t u r b e d .  This  A few Canada bluegrass  p l a n t s are a l s o scattered i n the beach sand. At Witty's Lagoon P. c o n f i n i s grows on the sandy s p i t which separates the lagoon from the ocean.  It i s  found on the exposed areas of sand among the broom ( C y t i sus) bushes which dominate much of the c e n t r a l r i d g e of the spit.  The dune bluegrass i s absent from' the areas where  a number of grasses ( i n c l u d i n g P.. p r a t e n s i s and P. pressa) and broad-leaved  com-  p l a n t s have formed a more or l e s s  -17solid  cover over the sandy s o i l .  The whole area i s sub-  j e c t to heavy g r a z i n g by c a t t l e , sheep and horses. On James I s l a n d and I s l a n d View Beach S p i t P. c o n f i n i s i s a g a i n found i n open, porous sand j u s t behind the l i t t o r a l  area.  The l a r g e r P. macrantha i s  w e l l e s t a b l i s h e d on I s l a n d View Beach S p i t , along with the P. c o n f i n i s , P. p r a t e n s i s , and other p l a n t s .  A  small area of P. macrantha i s a l s o present beside the roadway on the north-west s p i t , James I s l a n d , i n open sand. On the l a r g e , d r i f t i n g at  expanse of sand dunes  Twin Harbours S t a t e Park, by Gray's Harbour, Washing-  ton,  P. macrantha and a coarse Ammophila s p e c i e s are the  dominant p l a n t s ( F i g . 4).  F i g u r e 4: Dune area of Twin Harbours State Park, Gray's Harbour, Wash., where P. macrantha and a species of Ammophila are the dominant g r a s s e s .  -18Poa  confinis  but  occurs  which this  i s not  in  shows  found  small patches  i n the  low-growing  the  portion  coarse  on  open,  more  of  cover  the of  A  sand P.  drifting  stabilized  i n F i g . 4.  bunches  broad-leaved  the  i n the  foreground  well-stabilized  shows m o s t l y  here  the  of 5),  (Fig.  macrantha,  i n between  area  closeup  dunes  sand,  with  the  bunches.  F i g u r e 5: Closeup of p o r t i o n of foreground i n F i g . 4 s h o w i n g l a r g e t u f t s o f P. m a c r a n t h a . Some P. c o n f i n i s i s a l s o found i n t h i s s t a b i l i z e d area.  Poa behind  the  confinis  actual  dune  stabilized  banks  along  in  sandy  vacant  a  these dune  flat,  locations area.  P.  are  i s also or  confinis  beach  the  more  found  in  area,  roadways  than and  P.  a  few  in  and  l o t ( F i g . 6).  several sandy i n one However,  hundred  places semiinstance none  yards  p r a t e n s i s occur  in  of  from close  the  -19-  association on one sandy bank just behind the dunes, close to the Point Chehalis lighthouse.  Figure 6: Small t u f t s of P. confinis growing i n a sandy vacant l o t near Point Chehalis, Gray's Harbour. The only s i t e v i s i t e d when P. confinis plants were i n flower was at Point Roberts, Boundary Bay.  Here,  large areas of P. confinis are found on the slopes of the sand banks at the back of the raised beach area.  On  these sloping banks the dune bluegrass receives l i t t l e competition from other plants and sometimes forms extensive patches twenty to t h i r t y feet i n diameter.  The  plants in these patches are connected by the characteri s t i c rhizome system, and each patch seems to be composed of either a l l female or a l l male plants.  -20At none of the sites v i s i t e d during the studyi s there any sign of seedling plants of dune bluegrass. As perhaps the s p e c i f i c epithet confinis implies, dune bluegrass exists i n a very circumscribed habitat. The general impression gained from the study of the v a r i ous sites i s that P. c o n f i n i s i s adapted p a r t i c u l a r l y to the semi-stabilized, porous sand areas which often l i e behind the l i t t o r a l zone along the P a c i f i c Coast.  It i s  not often found i n the more seaward l o c a t i o n s — t h e pebble or sand beaches which must often be subject to windblown salt spray—where the Lathyrus (beach.pea), and Elymus species frequently f l o u r i s h .  Glaux  Nor i s P. con-  f i n i s ever seen on the muddy f l a t s or saltmarsh areas where the S a l i c o r n i a . D i s t i c h l i s . A t r i p l e x and P u c c i n e l l i a halophytes t h r i v e .  It does sometimes occur on the more  s t a b i l i z e d sandy f l a t s where a heavier cover of other grasses and legumes has formed, but even i n these areas i t does not seem to be well-adapted.  The narrow confines  of the dune bluegrass habitat are rather unique, as almost a l l the other species of higher plants with which P. conf i n i s can be found associated possess much wider ranges of adaptation.  -21C.  Introduction to c u l t i v a t i o n The P. confinis material on which most of the  c y t o l o g i c a l and h y b r i d i z a t i o n a l studies were carried out came o r i g i n a l l y from Whiffin Spit, Sooke Harbour, V. I., where Dr. V. C. Brink made a c o l l e c t i o n of a few plants i n the summer of 1948.  These plants were grown i n a  single row on the University of B r i t i s h Columbia farm and showed great vigour and spreading capacity. Unfortunately, detailed notes were not kept on the grass at t h i s time, and i t i s not known whether any male plants were among the o r i g i n a l group.  However, a number of these plants  gave a heavy seed set following t h e i r establishment on the farm.  This seed was used to sow several metre square  plots i n a bluegrass nursery close to the o r i g i n a l row of plants. The growth and vigour of the P. confinis plants i n the metre square plots was again good.  However, no  note was taken of t h e i r composition with respect to the proportion of male and female plants, although i t i s known that a good seed set was obtained on at least some of the plants. When the bluegrass nursery had to be abandoned, the dune bluegrass was perpetuated through the e s t a b l i s h ment of a new plot by clonal propagation from the metre  -22square p l o t s .  Rhizomes were spread and  l a y e r of s o i l i n a 6 x 12 f o o t p l o t * known, the rhizomes used f o r the new  p l o t were taken from a f a i r l y  metre square p l a n t i n g s .  b u r i e d under a  As  f a r as i s  now  establishment of t h i s l a r g e p o r t i o n of  Therefore,  the  the  p l a n t s i n the  6 x 12 f o o t p l o t are presumed to have been reasonably representative seed.  of the  earlier material  established  from  However, when these p l a n t s f i r s t flowered a f t e r  the w r i t e r undertook the dune b l u e g r a s s study, i n May i t was  found t h a t the e n t i r e p l o t contained  plants.  i n May  T h i s was  female  confirmed when the p l a n t s a g a i n flowered  1955* P.  c o n f i n i s has  done e x c e p t i o n a l l y w e l l on  U n i v e r s i t y farm, both i n the roiir  plantings.  and  the p l a n t s  have shown an creeping plot  only  1954,  The  s o l i d p l a n t i n g s and  single-row spacings, p a r t i c u l a r l y ,  extremely r a p i d spread by means of The  formation.  s p r i n g u n t i l the middle of A p r i l  years during which the w r i t e r made  However, the  foot  dune b l u e g r a s s on the U n i v e r s i t y farm d i d  not begin growth i n the i n the two  the  s o l i d p l a n t i n g i n the 6 x 12  shows a v e r y t h i c k , rhizomatous sod The  i n single  f i n e - l e a v e d growth i s v e r y a t t r a c t i v e ,  i n the  rhizomes.  the  observations.  i n f l o r e s c e n c e s appeared v e r y r a p i d l y a f t e r  growth began, and  l i t t l e vegetative  growth preceded  the  -23emergence of the p a n i c l e s .  In the f i r s t year of  t i o n (1954) the p a n i c l e s emerged on May about two  2nd, and  observaflowered  weeks l a t e r ; the f o l l o w i n g year f l o w e r i n g took  p l a c e almost  two  weeks l a t e r , due to the l a t e s p r i n g .  In both years t h e r e were v e r y few other b l u e grasses f l o w e r i n g as e a r l y as P. c o n f i n i s . except b l u e g r a s s , P. annua.  In May  1955  p l a n t s flowered a t approximately  a row  o f P.  annual  alpina  the same time as P.  con-  f i n i s . but a t s e v e r a l hundred f e e t d i s t a n t from the nearest p l a n t s of the l a t t e r  species.  Kentucky b l u e g r a s s i n the  nursery, and growing i n nearby u n c u l t i v a t e d a r e a s ,  flowered  a few days a f t e r P. c o n f i n i s had f i n i s h e d f l o w e r i n g ; Canada b l u e g r a s s flowered c o n s i d e r a b l y l a t e r . The w r i t e r has not found any  seeds formed on  the  dune b l u e g r a s s p l a n t s growing on the U n i v e r s i t y farm. Bagged p l a n t s have set no  seed.  A number of seed c o l l e c t i o n s were made from P. ous  c o n f i n i s p l a n t s and from other b l u e g r a s s e s a t the  vari-  s i t e s a l o n g the coast which were v i s i t e d d u r i n g the  study.  Seed from these c o l l e c t i o n s was  sand i n the greenhouse i n December 1954. obvious  sowed i n f l a t s of There was  s i g n of any h y b r i d s among the thousands of  no seed-  l i n g s from the v a r i o u s c o l l e c t i o n s , although t h i s f e a t u r e could not be checked too c l o s e l y and a l l s e e d l i n g s c o u l d not be  saved.  In February,  s e e d l i n g s from each c o l l e c t i o n  -24were t r a n s f e r r e d to very l i g h t , greenhouse bench.  sterilized  Most of these s e e d l i n g s were p l a n t e d  i n spaced rows i n the U n i v e r s i t y n u r s e r y i n Poa represented  soil in a  May.  c o n f i n i s from the f o l l o w i n g p l a c e s i s  i n the nursery;  now  W h i f f i n S p i t ( o r i g i n a l mat-  e r i a l ) ; I s l a n d View Beach S p i t ; W i t t y ' s Lagoon; Parksville;  Twin.Harbours S t a t e Park; P o i n t  Roberts.  -25-  IV  REPRODUCTION IN THE BLUEGRASSES A.  A g e n e r a l statement on l i t e r a t u r e r e v i e w The l i t e r a t u r e on a p o m i c t i c r e p r o d u c t i o n i n  b o t h t h e lower and h i g h e r p l a n t s has reached  substantial  p r o p o r t i o n s s i n c e t h e f i r s t o b s e r v a t i o n o f seed f o r m a t i o n w i t h o u t f e r t i l i z a t i o n was made i n t h e m i d d l e o f t h e l a s t c e n t u r y (44)* A comprehensive  c o l l a t i o n of observations  was not u n d e r t a k e n u n t i l 1930, when Rosenberg  (90) pub-  l i s h e d a summary o f t h e l i t e r a t u r e on a p o m i x i s .  Stebbins  (96), f o r t h e h i g h e r p l a n t s , extended and brought up t o date t h e Rosenberg  summary, and emphasized  that the  a p o m i c t i c p r o c e s s e s were more t h a n " f r e a k " phenomena* G r a d u a l l y , t h e v e r y g e n e r a l o c c u r r e n c e and s i g n i f i c a n c e of a p o m i x i s was r e a l i z e d , and i n some r e s p e c t s an e x p r e s s i o n o f t h i s was t h e p u b l i c a t i o n o f G u s t a f s s o n ' s (44, 45, 46) monograph on a p o m i x i s i n t h e angiosperms.  Most o f  t h e more r e c e n t f i n d i n g s have been r e v i e w e d by Nygren ( 8 3 ) , A l a r g e l i t e r a t u r e on r e p r o d u c t i o n i n Poa has developed because o f t h e common o c c u r r e n c e o f a p o m i x i s i n t h i s genus, and because o f t h e wide d i s t r i b u t i o n and economic importance o f t h e Poa s p e c i e s .  Much o f t h i s  l i t e r a t u r e has been r e v i e w e d h e r e j n o n e t h e l e s s , many o f t h e papers r e a d have n o t been c i t e d , w h i l e a few papers  -26l i s t e d are c i t a t i o n s of other authors which t h e w r i t e r was unable t o r e a d . B.  The t e r m i n o l o g y o f t h e a p o m i c t i c phenomena One o f t h e d i f f i c u l t problems c o n f r o n t i n g t h e  student o f a p o m i x i s  i s that of terminology.  As G u s t a f s -  son (44) has p o i n t e d o u t , t h e t e r m i n o l o g y may appear unn e c e s s a r i l y c o m p l i c a t e d because o f t h e number o f terms t h a t have been used i n d e s c r i p t i o n s o f a p o m i c t i c phenomena by v a r i o u s a u t h o r s .  Furthermore,  t h e r e have been  d i f f e r e n c e s o f o p i n i o n over t h e c o r r e c t usage o f some o f t h e s e terms.  The c o m p l e x i t y o f t h e phenomena  themselves,  t h e i r r a d i c a l d e p a r t u r e from those encountered  i n sexually-  r e p r o d u c i n g p l a n t s , and the f a i l u r e o f e a r l y w o r k e r s t o d i s t i n g u i s h c l e a r l y between t h e s p o r o p h y t i c and  gametophytic  g e n e r a t i o n s i n some a p o m i c t i c s p e c i e s have a l s o been a d vanced as reasons f o r t h e d i f f i c u l t i e s over (44, 97)*  terminology  A c e r t a i n s t a b i l i z a t i o n o f t e r m i n o l o g y seems  t o be r e c o g n i z a b l e now i n t h e l i t e r a t u r e , a l t h o u g h f e r e n t systems and d e f i n i t i o n s o f terms s t i l l  dif-  occur.  The system o f t e r m i n o l o g y g i v e n by G u s t a f s s o n (44, 48) i s t h e one used here and t h e one now adopted by the m a j o r i t y o f w r i t e r s .  T h i s system has been based, i n  t u r n , on t h a t o f W i n k l e r (106, 108, 109) and Edman (35). W i n k l e r emphasized p a r t i c u l a r l y t h a t two o f t h e most s i g n i f i c a n t p o i n t s i n t h e normal s e x u a l c y c l e , m e i o s i s and  -27f e r t i l i z a t i o n , are completely  separated  by the gametophyte  g e n e r a t i o n , and a r e t h e r e f o r e l i k e l y i n f l u e n c e d by d i f f e r e n t environmental summarized by S t e b b i n s  entirely  and g e n e t i c f a c t o r s ( 1 0 7 ) *  As  ( 9 7 ) , "Every harmonious a p o m i c t i c  c y c l e must t h e r e f o r e p r o v i d e e i t h e r a s i n g l e s u b s t i t u t e f o r b o t h of these p r o c e s s e s  or a s e p a r a t e  substitute for  each of them, w i t h c o o r d i n a t i o n o f these two s u b s t i t u t e s . A l l p l a n t s may  f i r s t o f a l l be grouped i n t o  main c l a s s e s a c c o r d i n g t o t h e i r mode of  two  reproduction.  Those i n which s e x u a l d i f f e r e n t i a t i o n and  fertilization  have not y e t been e v o l v e d can be d e s c r i b e d as a m i c t i c . Those w h i c h have d i f f e r e n t i a t e d s e x u a l l y may m i o t i c or amphimictic.  be termed  W i t h i n the l a t t e r group t h e r e i s  a f u r t h e r subgroup o f p l a n t s whose r e p r o d u c t i o n does not i n v o l v e f e r t i l i z a t i o n and a r e termed a p o m i c t i c .  Thus,  ". . . a m i x i s i s a p r i m i t i v e s t a g e , a p o m i x i s a d e r i v e d stage i n which t h e f e r t i l i z i n g mechanism once a c q u i r e d has a g a i n been l o s t . " According  (44)•  to these d e f i n i t i o n s ,  vegetative  r e p r o d u c t i o n by means o f b u l b i l s , b u l b s , r h i z o m e s , e t c . can be c o n s i d e r e d as a p o m i c t i c r e p r o d u c t i o n when t h e sexual process  i s n o n - f u n c t i o n a l or g r e a t l y suppressed.  Thus, v i v i p a r y . i n w h i c h v e g e t a t i v e p r o p a g u l e s a r i s e i n the i n f l o r e s c e n c e s o f p l a n t s , r e p l a c i n g o r  reducing  s t a m i n a l and o v u l a r development, i s sometimes i n c l u d e d a s  1 1  -28a form of apomixis.  However, apomixis has commonly come  to have a more r e s t r i c t e d meaning, synonymous with agamospermy. or seed formation without  fertilization.  The normal sexual cycle i n higher plants includes an alternation of a sporophytic and a much reduced garnetophytic generation. gametophytic  In certain agamospermous species' the  generation i s omitted completely and the  embryo develops d i r e c t l y from somatic c e l l s i n the ovule of the parent sporophyte as integumentary outgrowths.  or nucellar  This type of agamospermy i s termed adventi-  tious embryony» and l i k e vegetative reproduction i t i s an example of a single process substituting f o r both meiosis and f e r t i l i z a t i o n i n the l i f e  cycle.  In those agamospermous species which s t i l l maintain a complete a l t e r n a t i o n of generations the gametophyte can originate i n one of two fundamental ways.  The  gametophyte can develop by diplospory from a true archesporial c e l l (macrospore mother c e l l ) , or i t can develop by apospory from a somatic c e l l of the nucellus or chalaza. Whichever of these two general schemes of development i s followed, the gametophyte receives the unreduced chromosome number.  The mature embryo sac i s usually i n a l l other r e -  spects p e r f e c t l y normal, with three antipodals, two polar n u c l e i , two synergids and an egg c e l l .  However, as happens  i n some sexual species, exceptions may occur i n the number of c e l l s present i n the mature embryo sac.  4  -29D i p l o s p o r o u s and aposporous gametophyte ment can be q u i t e e a s i l y d i f f e r e n t i a t e d  develop-  cytologically i n  s p e c i e s h a v i n g a u n i c e l l u l a r a r c h e s p o r e , as i n Poa. s p e c i e s w i t h a m u l t i c e l l u l a r a r c h e s p o r e , which may  In develop  a number o f EMCs*, i t i s o f t e n hard t o determine whether the  embryo sac i n i t i a l c e l l a c t u a l l y o r i g i n a t e s i n a r c h -  e s p o r i a l o r somatic t i s s u e .  Transitions " . . .  between  o r d i n a r y EMCs, p o t e n t i a l EMCs and somatic c e l l s . • . ." a l s o seem t o o c c u r , c o m p l i c a t i n g t h e s i t u a t i o n (44)* F a g e r l i n d ' s (38,  39)  terminology concerning  d i p l o s p o r y and apospory s h o u l d be mentioned here because i t i s s t i l l f o l l o w e d by some a u t h o r s .  T h i s scheme i s  based on the c h a r a c t e r of t h e f i r s t n u c l e a r d i v i s i o n i n the  embryo sac i n i t i a l c e l l as w e l l as on t h e o r i g i n o f  this c e l l .  The development  of t h e a p o m i c t i c gametophyte  i s s u b d i v i d e d i n t o ( a ) D i p l o s p o r y (b)  Semi-apospory  ( c ) G e n e r a t i v e apospory and (d) Somatic a p o s p o r y . d i p l o s p o r y a m e i o t i c d i v i s i o n i n t h e EMC the  In  i s f o l l o w e d by  f o r m a t i o n o f a r e s t i t u t i o n n u c l e u s ; i n semi-apospory  the EMC  d i v i s i o n i s pseudohomeotypic**;  * EMC cell.•  = embryo sac mother c e l l , PMC  i n generative = p o l l e n mother  ** I n a pseudohomeotypic d i v i s i o n , a c c o r d i n g t o Gusta f s s o n ( 4 7 ) , who proposed t h e term, t h e EMC undergoes t h e s t a r t o f a . m e i o t i c prophase but b i v a l e n t s a r e not formed. I n s t e a d , the chromosomes a r e g a t h e r e d . a s u n i v a l e n t s a t the c e n t r a l p l a t e and s p l i t i n t o c h r o m a t i d s . I n t h i s way, t h e d i v i s i o n i s completed m i t o t i c a l l y and.two i d e n t i c a l daughter c e l l s a r e formed.  apospory t h e EMC d i v i s i o n i s m i t o t i c .  The n e t r e s u l t o f  any o f t h e s e m o d i f i c a t i o n s o f normal EMC b e h a v i o u r i s t h e f o r m a t i o n o f an embryo sac w i t h t h e somatic chromosome number.  The t h r e e terms a r e thus c o l l e c t i v e l y e q u i v a l e n t  to  d i p l o s p o r y i n G u s t a f s s o n ' s ( 4 4 , 4 8 ) sense o f t h e word.  In  somatic apospory t h e embryo sac i n i t i a l c e l l i s n o t  an EMC, and i s t h e r e f o r e t h e same as G u s t a f s s o n ' s ( 4 4 , 48)  apospory. M e i o s i s h a v i n g been c i r c u m v e n t e d by d i p l o s p o r o u s  or aposporous embryo sac development, t h e subsequent a t i o n o f an embryo w i t h o u t f e r t i l i z a t i o n i n one o f two ways.  form-  can be a c h i e v e d  Such an embryo can develop from t h e  egg c e l l , a p r o c e s s termed p a r t h e n o g e n e s i s . o r from one of  t h e v e g e t a t i v e c e l l s o f t h e embryo sac by apogamety.  A l t h o u g h common i n t h e p t e r i d o p h y t e s , apogamety i s apparently rare i n higher plants ( 4 4 , 9 8 ) . In  some p l a n t s d e v e l o p i n g a p o m i c t i c embryos,  p o l l i n a t i o n i s s t i l l r e q u i r e d f o r seed development, u s u a l l y because a f e r t i l i z a t i o n o f t h e f u s i o n n u c l e u s is  n e c e s s a r y t o produce t h e endosperm, even though t h e  o t h e r h a l f o f double f e r t i l i z a t i o n  i s omitted.  In other  p l a n t s p o l l i n a t i o n p e r se may m e r e l y p r o v i d e a s t i m u l a t i o n f o r a p o m i c t i c development.  Pseudogamy i s a c o l l e c t i v e  term employed f o r any a p o m i c t i c seed f o r m a t i o n f o r which p o l l i n a t i o n i s s t i l l necessary.  I n other apomicts t h e  -31-  seed formation may be autonomous (44)• In sexually-reproducing plants an egg c e l l sometimes develops without f e r t i l i z a t i o n , forming an embryo with the reduced chromosome number ( i . e . a haploid).  This has been termed haplo-parthenogenesis  to distinguish i t from parthenogenesis or diploparthenogenesis as i t occurs i n diplosporous or aposporous apomicts.  Haploid plants produced by haplo-  parthenogenesis w i l l deviate morphologically from the parent plant and may  show a wide range of v a r i a t i o n ,  i n contrast to the uniformity of progenies a r i s i n g by adventitious embryony, diplospory or apospory. The terminology on apomictic seed formation can be adequately summarized by stating that there are three p r i n c i p a l processes concerned (44): ( 1 ) Diplospory, followed by parthenogenesis or apogamety. (2) Apospory, followed by parthenogenesis or apogamety. (3) Adventitious embryony. Stebbins (97) has suggested gametophytic apomixis as a c o l l e c t i v e term f o r the f i r s t two types of agamospermy, which emphasizes the fact that a gametophytic generation i s s t i l l maintained i n the l i f e  cycle.  -32-  C. R e p r o d u c t i o n i n i n d i v i d u a l Poa s p e c i e s Poa p r a t e n s i s (Kentucky b l u e g r a s s ) B e i n g t h e most i m p o r t a n t and widespread member of  i t s genus, Poa p r a t e n s i s has r e c e i v e d more a t t e n t i o n  than any o t h e r Poa s p e c i e s .  I t i s an e x t r e m e l y complex  and polymorphous s p e c i e s , and w i t h chromosome numbers r a n g i n g i n a n e u p l o i d s e r i e s from about 2n = 38 - 124 ( 8 3 ) . Consequently, t h e taxonomic made d i f f i c u l t .  t r e a t m e n t o f Poa p r a t e n s i s i s  Certain d i v i s i o n s w i t h i n the species are  g e n e r a l l y r e c o g n i z e d and some t a x o n o m i s t s d e f i n e t h e s e a s s u b s p e c i e s and o t h e r s (65) d e f i n e them as s e p a r a t e s p e c i e s . The d e l e g a t i o n o f t h e s e t a x a t o s u b s p e c i f i c rank, a s f o l lowed by Nygren ( 8 3 ) , i s r e c o g n i z e d here s i m p l y f o r convenience i n d i s c u s s i o n . R e p r o d u c t i o n has been s t u d i e d i n many w i l d and c u l t i v a t e d b i o t y p e s o f P. p r a t e n s i s from a l l over t h e world, mostly belonging to the subspecies eupratensis which embraces t h e m a j o r i t y o f c u l t i v a t e d forms ( 8 4 ) . T h i s 'is t h e s u b s p e c i e s g e n e r a l l y r e f e r r e d t o m e r e l y a s P. p r a t e n s i s . a c o n v e n t i o n f o l l o w e d i n t h i s paper.  Very  few i n v e s t i g a t o r s have r e f e r r e d s p e c i f i c a l l y t o t h e o t h e r t h r e e s u b s p e c i e s , a l p i g e n a . a n g u s t i f o l i a and i r r i g a t a : a few o f t h e f i n d i n g s i n t h e s e l a s t s u b s p e c i e s w i l l be mentioned separately l a t e r .  -33-  Muntzing (69) f i r s t reported the presence of agamospermy i n the genus, concluding that t h i s was  the  method of seed formation i n c e r t a i n Swedish biotypes of P. p r a t e n s i s . He gave as c r i t e r i a of apomixis: (1)  Extreme u n i f o r m i t y w i t h i n a b i o t y p e .  (2)  A constant aneuploid chromosome number f o r  the b i o t y p e . (3)  Good seed production, even i n p l a n t s e v i d -  encing great meiotic i r r e g u l a r i t i e s , Eancken (87) used these same c r i t e r i a i n establ i s h i n g the occurrence of apomictic seed production i n F i n n i s h s t r a i n s of Kentucky bluegrass.  Confirmation of  apomixis i n many biotypes of t h i s species has been made by o t h e r s , i n c l u d i n g Akerberg (3, 6, 7), who was a l s o the f i r s t to show that p o l l i n a t i o n i s necessary f o r seed development i n s e v e r a l apomictic s t r a i n s ( 7 ) . He  found  that P. a l p i n a p o l l e n was almost as e f f e c t i v e as p r a t e n s i s p o l l e n i n i n i t i a t i n g seed development i n these  apomicts  and t h a t the seed thus formed gave r i s e to p l a n t s i d e n t i c a l w i t h the maternal p r a t e n s i s parent,  P. glauca and  P. compressa p o l l e n were somewhat l e s s e f f e c t i v e , but where seed d i d form t h i s a l s o gave r i s e to matroclinous offspring.  N i l s s o n (80), Muntzing  (71). Akerberg (1) and  Engelbert (37) confirmed t h i s pseudogamous development of seed primordia i n t o seed.  Engelbert (37) studied p o l l e n  -34tube growth on stigmas of P. p r a t e n s i s and concluded t h a t merely the growth of p o l l e n tubes down the s t y l e s a c t e d as a s t i m u l a n t to seed f o r m a t i o n .  Akerberg  sexual p r a t e n s i s b i o t y p e s among those he  (7) found f o u r  investigated.  The u n i f o r m i t y of s i n g l e p l a n t progenies l e c t e d i n Maryland was  noted by Kemp (58), and i n b i o t y p e s  from E a s t e r n Canada by Armstrong (101) f i r s t used the progeny ing  col-  (12).  Tinney and Aamodt  t e s t as a means o f i d e n t i f y -  a p o m i c t i c s t r a i n s of Kentucky  bluegrass.  They grew  p o p u l a t i o n s o f p l a n t s o r i g i n a t i n g from o p e n - p o l l i n a t e d seed c o l l e c t e d from s i n g l e p l a n t s , and r a t e d the p l a n t s f o r uniformity.  Of 102 progenies from s i n g l e p l a n t c l o n e s  from widely s c a t t e r e d areas i n Europe and North o n l y two f a i l e d  America  to show extreme u n i f o r m i t y , and 48  t a i n e d no v a r i a n t s a t a l l .  con-  M o r p h o l o g i c a l constancy i n  p r a t e n s i s progenies i s held by a l l w r i t e r s to be proof of a p o m i c t i c r e p r o d u c t i o n j c o n v e r s e l y , l a c k of u n i f o r m i t y or  the presence of a b e r r a n t i n d i v i d u a l s i n otherwise u n i -  form progenies i s c o n s i d e r e d as evidence of s e x u a l r e p r o d u c t i o n i n the parents (15, 16, 17, 21, 25, 26, 80, 85, 72,  93, 94,  Andersen  27,  100). (10) f o l l o w e d the development of the  female gametophyte i n P. p r a t e n s i s but saw nothing unu s u a l and concluded t h a t r e p r o d u c t i o n was  normal, a l t h o u g h  she observed no f e r t i l i z a t i o n of the egg c e l l .  Nishimura  (82) observed the f o r m a t i o n of embryos of s p o r o p h y t i c  -35o r i g i n by a d v e n t i t i o u s embryony i n c o n j u n c t i o n with development of the normal embryo from the process. by one  fertilization  In most cases the normal embryo was  or more adventive  embryos.  supplanted  Nishimura (82)  con-  s i d e r e d t h i s development to be the r e s u l t of g a l l s by i n s e c t a t t a c k s . of any  the  There appears to be no other  caused  record  i n s t a n c e s of a d v e n t i t i o u s embryony o c c u r r i n g i n  the genus Poa,  and Nygren (84)  says t h a t Nishimure  " m i s i n t e r p r e t e d " the e m b r y o l o g i c a l  evidence.  Armstrong (12) attempted to e x p l a i n the constancy of an a n e u p l o i d  chromosome number i n p r a t e n s i s  b i o t y p e s by p o s t u l a t i n g t h a t o n l y gametes w i t h a c e r t a i n chromosome number could f u n c t i o n i n f e r t i l i z a t i o n . p o s s i b i l i t y of such a scheme had by Muntzing ( 6 9 ) . l o g i c a l evidence  been p r e v i o u s l y r e j e c t e d  Akerberg (1) suggested t h a t the presented  first  cyto-  i n Armstrong's paper d i d not  r u l e out the p o s s i b i l i t y of a p o m i c t i c The  The  reproduction.  i n v e s t i g a t o r to o u t l i n e the  cyto-  l o g i c a l b a s i s of agamospermy i n Kentucky bluegrass  was  Akerberg ( l ) .  cells  He observed the presence of somatic  of the n u c e l l u s developing producing first  d i p l o i d embryo sacs by apospory.  d e t a i l e d these  biotypes.  as embryo sac i n i t i a l s ,  developments thoroughly  He found t h a t .the EMC  Tinney in  thus (100)  Wisconsin  u s u a l l y underwent a some-  what i r r e g u l a r m e i o t i c d i v i s i o n , g i v i n g r i s e to a t r i a d  -36or  a complete t e t r a d of macrospores, a l l of which  integrated.  An aposporous  dis-  c e l l of the n u c e l l u s or c h a l a z a  took over the f u n c t i o n of a macrospore and formed a d i p l o i d embryo sac.  As a r u l e , he found o n l y one embryo sac per  ovule, although he found two  i n several instances.  he a t t r i b u t e d to independent development aposporous  cells.  These  from separate  The egg c e l l of the d i p l o i d embryo sac  f u n c t i o n e d p a r t h e n o g e n e t i c a l l y t o form a proembryo even before a n t h e s i s o c c u r r e d .  He was unable to determine  whether endosperm development  was a s s o c i a t e d w i t h p o l l i n a -  tion. A d e t a i l e d study of apospory was a l s o out  by K i e l l a n d e r (63).  U n l i k e Tinney (100)  (5) he found two or t h r e e aposporous  carried  and Akerberg  embryo sacs per  ovule to be the commonest number, w i t h f o u r or f i v e and even up to seven f a i r l y f r e q u e n t .  Through  c o m p e t i t i o n be-  tween embryo sacs u s u a l l y o n l y one or two developed' t o maturity.  The o r g a n i z a t i o n o f a l e g i t i m a t e ( s e x u a l )  embryo sac r a r e l y o c c u r r e d . 0  Akerberg (4,  5)  and N i e l s e n (78)  have a l s o shown  t h a t the embryo develops by parthenogenesis, as by Tinney (100).  N i e l s e n (78)  established  found 8- t o 16-celled  proembryos a day b e f o r e f l o w e r i n g .  Akerberg (5)  found  t h a t embryo f o r m a t i o n occurs r e g u l a r l y even i n emasculated f l o r e t s of a p o m i c t i c P. p r a t e n s i s but t h a t seed f o r m a t i o n  -37f a i l e d because no endosperm formed. was induced by p o l l i n a t i o n .  Endosperm development  In one instance, the f i r s t  ever recorded i n a Poa apomict, A k e r b e r g  (5) was able to  show that the central fusion nucleus was f e r t i l i z e d before dividing, forming pentaploid* endosperm.  He thus-con-  cluded that autonomous embryo development (parthenogenesis) with induced endosperm formation (probably through  ferti-  l i z a t i o n ) i s a general phenomenon i n apomictic types of P. pratensis. Polyembryony, the formation of two or more embryos i n a single ovule, i s of f a i r l y common occurrence in Kentucky bluegrass.  The origins of twin and t r i p l e t  seedlings have been studied by Andersen (10), Armstrong (12),  I k e r b e r g (1), Tinney (100), Ghristoff (20), Muntzing  ( 7 0 ) , Brittingham (16) and Nielsen ( 7 5 , 7 9 ) .  Armstrong  (12) noted differences between apomictic strains i n t h e i r capacities to form two or more embryos within one seed. Both Akerberg (1) and Muntzing  (70) found the frequency  of twins to be lower i n sexual tha.n i n apomictic biotypes * The terms "haploid," " d i p l o i d , " " t r i p l o i d , " "pentap l o i d i " etc. are used i n a.loose sense i n t h i s paper to refer to the chromosome complement present. Since the basic genome number i n Poa i s 7, a true d i p l o i d would have a somatic count of 2n - 14, a t r i p l o i d 2n - 21 etc. But the extreme range of chromosome numbers i n P. pratensis (and other Poa species), and the occurrence of many aneuploid numbers, prevent the e f f e c t i v e use of a s t r i c t e r terminology.  -38-  o f P. p r a t e n s i s .  T h i s t h e y assumed t o be i n agreement w i t h  t h e e m b r y o l o g i c a l data on Kentucky b l u e g r a s s , as o n l y one EMC  normally  i s found and only.one macrospore f u n c t i o n s  i n the o v u l e s o f the s e x u a l b i o t y p e s .  In apomictic b i o -  t y p e s , on the o t h e r hand, t h e r e i s a tendency f o r s e v e r a l aposporous c e l l s t o develop  i n a s i n g l e o v u l e , and  there  i s a l s o a tendency f o r b o t h s e x u a l and aposporous embryosacs t o develop  s i m u l t a n e o u s l y i n the same o v u l e .  Brit-  tingham's ( 1 6 ) a n a l y s i s o f t h e p r o g e n i e s o f 1 1 5 openp o l l i n a t e d Kentucky b l u e g r a s s p l a n t s i n d i c a t e d a  signif-  i c a n t ( a l t h o u g h o n l y b a r e l y so) n e g a t i v e c o r r e l a t i o n  be-  tween polyembryony and v a r i a b i l i t y ( v a r i a b i l i t y b e i n g a c r i t e r i o n of sexual r e p r o d u c t i o n ) . summarized h i s own  N i e l s e n ( 7 9 ) has  f i n d i n g s and those o f p r e v i o u s  authors  i n r e s p e c t o f the o r i g i n o f t w i n and t r i p l e t p l a n t s i n t h i s species.  Development o f embryos from two or more  s e x u a l embryo sacs a r i s i n g from t h e same o r  different  EMCs i s p o s s i b l e ; development from two members o f the egg a p p a r a t u s and  o f a s e x u a l or aposporous sac ( e . g . normal  s y n e r g i d embryos) i s p r o b a b l y uncommon; development  from a s e x u a l and an aposporous s a c , o r from two o r more aposporous sacs seems t o be the most common cause o f polyembryony.  Haploid-diploid, diploid-diploid,'and  diploid-triploid  r e l a t i o n s h i p s have been found i n t w i n s  -39depending upon t h e o r i g i n o f t h e embryos and whether  ferti-  l i z a t i o n was e f f e c t e d b e f o r e t h e i r development ( 7 9 ) • The f o r m a t i o n o f h a p l o i d p l a n t s by h a p l o parthenogenesis  o c c u r s o f t e n i n Poa p r a t e n s i s ( 8 ) ,  Akerberg  ( l ) , B r i t t i n g h a m ( 1 7 ) , and Tinney and  Aamodt ( 1 0 1 ) , among o t h e r s , have r e p o r t e d d i f f e r e n t degrees o f a p o m i c t i c seed f o r m a t i o n i n t h e b i o t y p e s w i t h which t h e y worked, based on t h e f r e q u e n c y o f m o r p h o l o g i c a l aberrants.  Although apomictic, semi-apomictic  and s e x u a l  types a l l occur w i t h i n the s p e c i e s , the apomictic types a r e t h e most f r e q u e n t ( 8 ) .  The f r e q u e n c y o f s e x u a l Ken-  tucky bluegrass biotypes i n nature i s quite low ( 6 ) . Most o f t h e f i n d i n g s i n t h e s u b s p e c i e s t e n s i s seem t o be a p p l i c a b l e t o t h e o t h e r t h r e e  euprasubspecies.  Apospory o c c u r s i n subsp. a l p i g e n a ( 8 4 ) , subsp. a n g u s t i folia  ( 4 ) and subsp, i r r i g a t a  (65).  A v i v i p a r o u s form o f  subsp. a l p i g e n a o c c u r s i n n o r t h e r n Scandanavia  and has  been i n v e s t i g a t e d by Nygren ( 8 4 ) .  I t producesr.good  pollen,  and aposporous c e l l s a r e developed  i n t h e o v u l e s ; however,  t h e s e c e l l s r a r e l y produce mature embryo sacs and t h e v i v i parous p r o p a g u l e s almost i n v a r i a b l y f u n c t i o n i n t h e r e p r o d u c t i o n o f t h e p l a n t s , a l t h o u g h i t may be p o s s i b l e f o r some seed t o form.  The I c e l a n d i c form o f subsp. i r r i g a t a  stud-  i e d by Love (65) a p p a r e n t l y never forms s e x u a l embryo s a c s ,  -40although h y b r i d i z a t i o n i s s t i l l p o s s i b l e through the f e r t i l i z a t i o n o f t h e d i p l o i d , aposporous Poa compressa  egg c e l l ,  (Canada b l u e g r a s s )  Poa compressa  i s found a l m o s t t h r o u g h o u t t h e  N o r t h American c o n t i n e n t , h a v i n g much t h e same range as P. p r a t e n s i s , a l t h o u g h i t i s c h a r a c t e r i s t i c a l l y found on somewhat p o o r e r s o i l s t h a n t h e l a t t e r s p e c i e s .  L i k e Ken-  t u c k y b l u e g r a s s , i t i s a r h i z o m a t o u s p e r e n n i a l used m a r i l y as a p a s t u r e s p e c i e s . ness as a lawn g r a s s .  pri-  I t a l s o f i n d s some u s e f u l -  I t i s of less.importance i n  Europe than on t h i s c o n t i n e n t ( 4 0 , 54, 9 2 ) , Canada b l u e g r a s s has n o t h i n g l i k e t h e v a r i a b i l i t y i n morphology bluegrass.  and chromosome number o f Kentucky  The range i n 2n chromosome number i s almost  a e u p l o i d s e r i e s — n u m b e r s o f 35, 42, 49, 50 and 56 b e i n g known ( 8 3 ) . Andersen development  (10) i n v e s t i g a t e d female gametophyte  i n P. compressa b u t c o n c l u d e d t h a t r e p r o d u c -  t i o n was s e x u a l .  A p o m i x i s i n t h i s s p e c i e s was f i r s t  demonstrated by B r i t t i n g h a m ( 1 5 ) , who p o l l i n a t e d h e a t emasculated f l o r e t s o f P. compressa pollen. the  w i t h P, p r a t e n s i s  He o b t a i n e d o n l y one h y b r i d among t h e progeny,  remainder b e i n g m a t e r n a l - t y p e p l a n t s developed t h r o u g h  pseudogamy.  C h r i s t o f f (19) showed t h a t  aposporous  -41development  was  involved,  chromosome c o u n t s fusion nucleus  in  as  i n P.  pratensis,  endosperm t i s s u e ,  requires  fertilization  and,  through  proved t h a t for  the  apomictic  seed  is  a form  com-  chromosome numbers r u n n i n g  from  formation, Poa a r c t i c a  (Arctic  bluegrass)  Poa a r c t i c a , plex,  w i t h a range  of  like 2n  P.  pratensis, i  about  38 t o 100  c u m b o r e a l and a l p i n e i n Europe ainous  it  is  regions It  As i t s  (83)*  distribution.  It  (54)*  some o f t h e  earlier  were c a r r i e d were d e f i n e d .  a number o f studies  out b e f o r e Flovik  was p r o b a b l y a p o m i c t i c  interspecific  the  mount-  of  the  however, arctica  species  seed  formation  f o r m s w h i c h he e x a m i n e d ,  irregularities  basis  on w e s t e r n G r e e n l a n d P.  showed t h a t  i n b o t h EMCs a n d  of  her  arctica  percentage  emasculation, germination  plants,  Engelbert  s e e d f o r m a t i o n was p s e u d o g a m o u s .  no endosperm d e v e l o p e d u n t i l  were p o l l i n a t e d ,  and  Scandanavian  on r e p r o d u c t i o n i n P.  the  On t h e  in  (83, 84);  c r o s s - p o l l i n a t i o n and p o l l e n  (37) c o n c l u d e d t h a t  florets  continent  s e e d s e t t i n g was v e r y g o o d and t h e  o f good p o l l e n h i g h .  also  subspecies  cir-  perennial.  (41) c o n c l u d e d t h a t  a s he n o t e d many m e i o t i c  studies  some o f  such d i v i s i o n s  in  has a  timberline  a rhizomatous  has b e e n d i v i d e d b y into  yet  is  it  On t h i s  found usually- above  taxonomists  PMCs,  name i m p l i e s ,  although  She  emasculated  embryo f o r m a t i o n  was  -42autonomous ( 3 6 , 3 7 ) .  Although  she made no chromosome  counts she s a i d t h a t germination o f t h e p o l l e n on the stigmas s t i m u l a t e d t h e development o f endosperm, the p o l l e n tube f u n c t i o n i n g as an a c t i v a t o r and "not i n a genetic capacity" ( 3 7 ) . Nygren (84) has found t h a t the subspecies c a e s p i t a n s . depauperata. aposporous.  elongata and microglumis a r e a l l  In c a e s p i t a n s no p o l l e n i s formed ( 7 4 , 8 4 ) ,  and i t i s c o n s i d e r e d by Nygren to be a non-pseudogamous apomict.  Some s e x u a l as w e l l as aposporous embryo sacs  a r i s e , and the f u s i o n n u c l e i o f both types a r e o c c a s i o n a l l y f e r t i l i z e d by f o r e i g n Poa p o l l e n , as evidenced by the occurrence o f both t r i p l o i d  and p e n t a p l o i d endosperm;  however, most counts i n endosperm n u c l e i r e v e a l  tetraploid  numbers, i n d i c a t i n g t h a t the m a j o r i t y of embryo sacs a r e aposporous and that no f e r t i l i z a t i o n these f o r endosperm f o r m a t i o n . a "rare and i n t e r e s t i n g  i s required i n  Nygren (84) c i t e s t h i s as  case."  In the subspecies depauperata  and elongata t h e  presence o f o n l y aposporous embryo s a c s , together with the very e a r l y autonomous d i v i s i o n o f the egg c e l l s i n these, and the low percentage of good p o l l e n produced, exclude most chances o f f e r t i l i z a t i o n .  " T h i s mechanism ex-  p l a i n s why the two a c t u a l subspecies have not been s p l i t up i n a m a n i f o l d of forms as i s the r u l e i n Poa a r c t i c a . "  -43(84)•  I n t h e s u b s p e c i e s m i c r o g l u m i s , on t h e o t h e r hand,  the aposporous egg c e l l sometimes d i v i d e s l a t e , t h e time of d i v i s i o n a p p a r e n t l y v a r y i n g a c c o r d i n g to the p o i n t when t h e p o l a r n u c l e i f u s e . percentage  This, together with a high  o f good p o l l e n f o r m a t i o n , i n d i c a t e s t h a t m i c r o -  g l u m i s can p r o b a b l y be i n v o l v e d f a i r l y r e a d i l y as e i t h e r t h e female o r male p a r e n t i n h y b r i d i z a t i o n s w i t h o t h e r Poa populations (84). The  s u b s p e c i e s s t r i c t a i s v i v i p a r o u s , and has  been i n v e s t i g a t e d by F l o v i k ( 4 1 ) , Nygren ( 8 4 ) • to  N a n n f e l d t (74) and  I t i s o b l i g a t e l y v i v i p o r o u s , and i s n o t a b l e  produce e i t h e r p o l l e n o r mature female  gametophytes.  Poa a l p i n a ( A l p i n e b l u e g r a s s ) Poa a l p i n a . a l t h o u g h n o t c u l t i v a t e d , has some v a l u e as a range s p e c i e s i n a l p i n e meadows and a r c t i c r e g i o n s o f N o r t h A m e r i c a and n o r t h e r n Europe ( 5 4 ) * S e x u a l , a p o m i c t i c (agamospermous), and v i v i p a r ous b i o t y p e s o f P. a l p i n a e x i s t .  A s e r i e s o f 2n chromo-  some numbers r a n g i n g from 14 t o 57 a r e found i n t h e s e s t r a i n s (83). A t t h e same time a s he d i s c o v e r e d a p o m i x i s i n Swedish P. p r a t e n s i s b i o t y p e s , Muntzing  (69) a l s o deter-  mined a p o m i x i s i n Swedish P. a l p i n a forms, u s i n g t h e same c r i t e r i a o f a p o m i c t i c seed f o r m a t i o n .  On t h e o t h e r hand,  he found c e r t a i n Swiss P. a l p i n a t y p e s t o be s e x u a l .  Pseu-  dogamous seed f o r m a t i o n was shown by E n g e l b e r t ( 3 7 ) , who  -44c o n c l u d e d t h a t s t i m u l a t i o n o f b o t h endosperm and development was  embryo  i n v o l v e d , w i t h o u t any f e r t i l i z a t i o n  of  e i t h e r egg o r f u s i o n n u c l e u s . M u n t z i n g (70)  established that diplospory  the b a s i s of a p o m i c t i c seed f o r m a t i o n , t h e EMC through  was  developing  successive m i t o t i c d i v i s i o n s d i r e c t l y into a  m o r p h o l o g i c a l l y n o r m a l , a l t h o u g h d i p l o i d , embryo sac ( i . e . g e n e r a t i v e apospory under F a g e r l i n d ' s t e r m i n o l o g y ) . Hakansson (51)  s t u d i e d t h e embryology o f b o t h s e x u a l  a p o m i c t i c s t r a i n s o f P. a l p i n a i n more d e t a i l and firmed Muntzing s f i n d i n g s . 1  o f the f u s i o n n u c l e u s  He a l s o showed  and  con-  fertilization  i n two a p o m i c t i c s t r a i n s ,  resulting  i n the f o r m a t i o n o f p e n t a p l o i d endosperm, i n c o n t r a d i c t i o n to E n g e l b e r t ' s (37) was  conclusions.  An example of apogamety  a l s o found i n which a d i p l o i d , s y n e r g i d c e l l i n an  a p o m i c t i c embryo sac developed  i n t o an embryo.  The  pro-  d u c t i o n o f h a p l o i d p l a n t s among s e x u a l s t r a i n s i n d i c a t e d the autonomous development o f egg c e l l s i n some s e x u a l embryo s a c s .  I n a l a t e r paper (52),  Hakansson r e p o r t e d  t h e development of b o t h h a p l o i d and d i p l o i d embryo sacs i n apomictic s t r a i n s .  I n the d i p l o i d sacs the egg, c e l l  u s u a l l y d i v i d e d autonomously, w h i l e f e r t i l i z a t i o n was a l ways n e c e s s a r y b e f o r e t h e f u s i o n n u c l e u s c o u l d d i v i d e i n both s e x u a l or a p o m i c t i c embryo s a c s ; the endosperm then b e i n g t r i p l o i d or p e n t a p l o i d , r e s p e c t i v e l y .  -45F l o v i k (41) and Nygren (84) have s t u d i e d  vivipor-  ous P . a l p i n a forms and found t h a t i n s p i t e of many m e i o t i c i r r e g u l a r i t i e s i n the PMCs a h i g h percentage of good p o l l e n i s formed.  On the female s i d e , the beginning of d i p l o s p o r -  ous embryo sac development o c c u r s , but t h e r e i s no chance f o r seed to form as the v i v i p o r o u s propagules develop a t an e a r l y stage i n the s p i k e l e t s and c a r r y on a l l reproduction  (84).  Poa p a l u s t r i s (Fowl b l u e g r a s s ) Poa p a l u s t r i s i s widespread a c r o s s the North American  c o n t i n e n t at low and medium a l t i t u d e s from New-  foundland to C a l i f o r n i a and as f a r south as New  Mexico.  I t i s o c c a s i o n a l l y c u l t i v a t e d i n meadow mixtures but seldom  important a g r i c u l t u r a l l y on t h i s c o n t i n e n t (54).  In n o r t h e r n Sweden, however, P. p a l u s t r i s i s an  important  c u l t i v a t e d hay and pasture s p e c i e s (81). K i e l l a n d e r (60) found both h a p l o i d and embryo sacs i n the m a t e r i a l he examined.  diploid  He showed t h i s  to be due to d i p l o s p o r y through a m i t o t i c d i v i s i o n o f the EMC  d i r e c t l y i n t o an embryo sac, and i n one  p l a n t he found t h a t the EMC i c a l l y as m e i o t i c a l l y ( 6 2 ) . gamy was  particular  d i v i d e d about as o f t e n m i t o t He concluded t h a t pseudo-  not v e r y probable i n t h i s s p e c i e s , as he saw  p o l l e n tubes i n the s l i d e p r e p a r a t i o n s u n t i l the and  no  egg-cells  endosperm n u c l e i had d i v i d e d s e v e r a l times ( 6 0 ) .  , -46However, A k e r b e r g  (7)  which, K i e l l a n d e r (62)  demonstrated pseudogamous development said " . . .  must be e x p l a i n e d by  the  p o l l e n tube i n d u c i n g t h e development o f the embryo a t some d i s t a n c e from the egg  cell."  No f u r t h e r e m b r y o l o g i c a l  work appears t o have been c a r r i e d on w i t h t h i s s p e c i e s . Poa  glauca Poa g l a u c a i s a t u f t e d p e r e n n i a l , c l o s e l y r e l a t e d  t o Poa a l p i n a , which i s found i n a r c t i c r e g i o n s and a l p i n e summits on t h i s c o n t i n e n t as f a r south as New s h i r e and Vermont  on Hamp-  (54).  F l o v i k (41)  found m e i o s i s t o be v e r y  irregular  i n the PMCs o f the a r c t i c m a t e r i a l he examined; however, the p o l l e n produced was  good and t h e progeny d i d not show  v a r y i n g chromosome numbers, i n d i c a t i n g t h a t a p o m i c t i c seed f o r m a t i o n was  probable.  K i e l l a n d e r ( c i t e d i n 66) made  e m b r y o l o g i c a l s t u d i e s and found i t to. be  diplosporous.  As i n t h e o t h e r d i p l o s p o r o u s b l u e g r a s s e s t h e d i p l o s p o r y took the form o f m i t o t i c d i v i s i o n s i n the EMCs.  An aneu-  p l o i d s e r i e s of chromosome numbers i s known f o r the s p e c i e s . Poa  nemoralis Poa n e m o r a l i s . a n o t h e r t u f t e d p e r e n n i a l , has been  i n t r o d u c e d onto t h i s c o n t i n e n t from Europe and i s o c c a s i o n a l i n meadows a c r o s s t h e n o r t h e r n U n i t e d S t a t e s (54).  It is  used t o some e x t e n t i n Sweden as a lawn g r a s s f o r shaded a r e a s (81).  The o n l y r e p o r t e d e m b r y o l o g i c a l s t u d y i s t h a t o f  -47K i e l l a n d e r ( c i t e d i n 66), who w i t h the EMC Poa nervosa  found  i t t o be d i p l o s p o r o u s ,  d i v i s i o n again m i t o t i c . (Wheeler b l u e g r a s s )  Poa nervosa  i s a rhizomatous  s p e c i e s , a l l i e d to  Kentucky b l u e g r a s s , which i s found a t medium a l t i t u d e s i n western  Canada and the U n i t e d S t a t e s ( 5 4 ) . Grun (43) found t h a t , " B a r r i n g r a r e e x c e p t i o n s ,  throughout  i t s range i n C a l i f o r n i a and most of i t s range  i n Oregon, Washington, Montana, Idaho, Colorado, Utah and Nevada i t i s female,  i t s flowers containing either  anthers or o n l y aborted a n t h e r s . " vosa does c o n t a i n v a r i a n t forms. t i o n has  N e v e r t h e l e s s , P.  ner-  E m b r y o l o g i c a l examina-  shown t h a t t h i s grass reproduces  by d i p l o s p o r y , the EMC  no  apomictically  d i v i s i o n being m i t o t i c ( 4 2 ) .  When  P. nervosa p l a n t s were put i n p o l l e n - p r o o f cages p r i o r t o blowing  "...  the seed set was  n e v e r t h e l e s s normal and  the germination of the seeds h i g h , " d e s p i t e the complete absence of p o l l e n ( 4 2 ) .  The  endosperm formed i s 4n, r e -  s u l t i n g from the f u s i o n of a p a i r of 2n p o l a r n u c l e i . P. nervosa and P. a r c t i c a subsp. c a e s p i t a n s are the o n l y b l u e g r a s s e s i n which t h i s completely autonomous development of both endosperm and established.  embryo has d e f i n i t e l y been  V a r i a t i o n i n P. nervosa  i s b e l i e v e d due  to  some f o r m a t i o n of sexual embryo sacs w i t h chance c r o s s i n g by p o l l e n from other Poa  species ( 4 2 ) .  -48Poa  .jemtlandica Poa  .jemtlandica, b e l i e v e d t o be the n a t u r a l h y b r i d  between P. a l p i n a and P. l a x a subsp. f l e x u o s a , i s a comp l e t e l y v i v i p a r o u s s p e c i e s having a r e s t r i c t e d  distribution  i n c e n t r a l Scandanavia and S c o t l a n d ( 7 3 , 8 3 , 84, 9 5 ) .  Ac-  cording t o Nygren (84) i t i s ". . . a b s o l u t e l y uniform i n i t s whole d i s t r i b u t i o n sphere."  I t produces l i t t l e  o r no  v i a b l e p o l l e n , and female gametophyte development always f a i l s completely,  so t h a t i t i s probably q u i t e i s o l a t e d  from other Poa p o p u l a t i o n s  (84).  Although  female gameto-  phyte f o r m a t i o n i s never completed, the EMC development i s a p p a r e n t l y q u i t e normal up u n t i l the time the ovules abort and a r e superseded by the v i v i p a r o u s  propagules  (84). Poa her.jedalica Poa - her.jedalica i s d e s c r i b e d as the n a t u r a l hyb r i d between v i v i p a r o u s P. a l p i n a and P. p r a t e n s i s subsp. a l p i g e n a . and, l i k e P. .jemtlandica. i s v i v i p a r o u s ( 7 3 , 8 3 , 84, 9 5 ) .  However, i t has a much wider d i s t r i b u t i o n i n  the Scandanavian-mountains than P. j e m t l a n d i c a , i s not found  i n S c o t l a n d , and has a wide range o f m o r p h o l o g i c a l  types forming an a n e u p l o i d  series (83, 84).  A l l strains  examined by Nygren (84) formed good p o l l e n , although i n v a r y i n g percentages,  and the s p e c i e s i s t h e r e f o r e not  i s o l a t e d from other b l u e g r a s s e s as i s P. .jemtlandica.  -49Although female gametophytes never mature, the presence of aposporous  c e l l s i n the o v u l a r t i s s u e s of s e v e r a l  P.  (84).  her.jedalica s t r a i n s has been r e p o r t e d by Nygren Poa bulbosa (Bulbous b l u e g r a s s )  Poa bulbosa i s a p e r e n n i a l s p e c i e s i n t r o d u c e d i n t o America.  In Sweden and the U n i t e d S t a t e s i t i s com-  p l e t e l y v i v i p o r o u s , producing b u l b i l s i n the f l o r e t s ; stamens or seeds are not formed  (44,  59)*  In England,  on the other hand, a sexual form of P. bulbosa i s i n d i g e nous, and the v i v i p a r o u s form i n t r o d u c e d (44)•  The s e x u a l  form has 2n » 28, w h i l e the v i v i p a r o u s forms have 2n • 42 or 2n -  56.  Poa ampla ( B i g b l u e g r a s s ) Poa ampla i s an important p e r e n n i a l which i s widespread  bunchgrass  on western rangelands of North  America  and which has been c u l t i v a t e d t o a l i m i t e d extent (40, 54).  I t has been u t i l i z e d  e x t e n s i v e l y i n the Carnegie  I n s t i t u t i o n of Washington b l u e g r a s s b r e e d i n g program, and a number of the most s u c c e s s f u l i n t e r s p e c i f i c  hybrid  s t r a i n s developed i n t h i s program i n v o l v e P. ampla as of the parents (21, development (83,  83).  85).  P. ampla has aposporous  In two  one  seed  s t r a i n s of b i g b l u e g r a s s i t  has been found t h a t the d i p l o i d egg c e l l remains u n d i v i d e d i n the mature embryo sac and  can e a s i l y be f e r t i l i z e d  by  p o l l e n g r a i n s from the same or other P. ampla p l a n t s , or  -50from other Poa s p e c i e s ( 8 5 ) .  Nygren (85) s t a t e s t h a t t h e r e  was ". . . n o d i f f i c u l t y i n o b t a i n i n g h y b r i d s when e i t h e r of these two ampla s t r a i n s was used as the female Poa  s c a b r e l l a (Pine Poa  found  parent."  bluegrass)  s c a b r e l l a i s another  i n the north-western  p e r e n n i a l bunchgrass,  S t a t e s and C a l i f o r n i a a t low  and medium a l t i t u d e s (54)* I t i s o f some s i g n i f i c a n c e as a range s p e c i e s ( 9 2 ) . I t i s r e p o r t e d by Nygren (83) t o be aposporous, although no f u r t h e r i n f o r m a t i o n has been p u b l i s h e d .  P.  s c a b r e l l a has been used s u c c e s s f u l l y i n a l a r g e number o f c r o s s e s with P. p r a t e n s i s , P. ampla. P. compressa and s e v e r a l other s p e c i e s i n the Carnegie  I n s t i t u t i o n of Wash-  i n g t o n program (24, 28, 29, 83, 102). Clausen  et a l . (22)  r e p o r t the use o f a " p a r t i a l l y s e x u a l " s t r a i n i n c r o s s e s w i t h P. a r a c h n i f e r a . Poa a r a c h n i f e r a (Texas Poa  bluegrass)  a r a c h n i f e r a i s a rhizomatous s p e c i e s , v a l u -  a b l e as a source o f p a l a t a b l e w i n t e r forage i n the southern Great P l a i n s r e g i o n o f the U n i t e d S t a t e s , where i t i s native (40, 64, 9 2 ) .  So f a r , however, i t has not been w i d e l y  c u l t i v a t e d because of a webby lemma, " . . . which makes h a r v e s t i n g , p r o c e s s i n g , or seeding o f t h i s s p e c i e s impossible w i t h o r d i n a r y machinery." ( 6 4 ) . Texas b l u e g r a s s i s completely  d i o e c i o u s , and one  of the o n l y d i o e c i o u s Poa s p e c i e s whose r e p r o d u c t i o n has been s t u d i e d .  I t i s s e x u a l , and has been used  f u l l y both as a male and female parent  success-  i n crosses i n t h e  bluegrass breeding program o f the Carnegie  I n s t i t u t i o n of  Washington (22, 102). I t i s r e f e r r e d to as v i v i p a r o u s i n the t a b l e of a p o m i c t i c p l a n t s presented although  the w r i t e r has been unable t o f i n d any other  r e c o r d o f i t being anything but completely unusual  by Nygren ( 8 3 ) ,  sexual.  An  f e a t u r e of e i g h t h y b r i d s produced i n the Carnegie  program from the c r o s s a r a c h n i f e r a x p r a t e n s i s i s t h a t a l l were m o r p h o l o g i c a l l y i n d i s t i n g u i s h a b l e from P. a r a c h n i f e r a . although  seven out of eight were shown from chromosome  counts to be d e r i v e d from the f u s i o n o f a reduced egg nucleus with a reduced of the remaining  p o l l e n nucleus  (102).  The o r i g i n  h y b r i d i s not e x p l a i n e d .  Despite the a p p a r e n t l y  sexual mode of reproduc-  t i o n , chromosome numbersof 2n • 42, 54 t o 56, 58 and 63 have been r e p o r t e d (18, 53, 2 2 ) . Other  bluegrasses A number of b l u e g r a s s e s which a r e p o s s i b l y apo-  m i c t i c , but on which no d e t a i l e d s t u d i e s have been made, have been used i n the Carnegie b l u e g r a s s breeding t h a t Poa nevadensis  program.  I n s t i t u t i o n of Washington  Clausen  et a l . (27) suggest  i s probably a p o m i c t i c .  Successful  h y b r i d s have been obtained u s i n g P. Canbyi and P. g r a c i l l i m a  -52but  i t i s not known whether these  sexual. Zealand  species are apomictic or  A t l e a s t one s t r a i n o f P. c a e s p i t o s a from New i s sexual and has been h y b r i d i z e d with P. praten-  s i s , P. ampla, P. a r a c h n i f e r a and P. compressa (22, 103). S e v e r a l other b l u e g r a s s e s u a l l y reproducing. a s m a l l extent one  a r e known t o be sex-  P. t r i v i a l i s . which i s c u l t i v a t e d t o  i n Europe, i s e n t i r e l y sexual, and i s a l s o  of the few d i p l o i d  s p e c i e s i n the genus (2n • 14)  (2, 6, 8 1 ) . P. l a x a subsp. f l e x u o s a i s o f some i n t e r e s t because i t has been found t o be sexual and almost completely s e l f - f e r t i l i z i n g .  The e a r l y development o f both  embryo sacs and p o l l e n i s a "severe  o b s t a c l e " to c r o s s -  f e r t i l i z a t i o n , and s e l f - p o l l i n a t i o n i s a l r e a d y when the f l o r e t s open ( 8 4 ) .  finished  -53-  V  BREEDING IN THE BLUEGRASSES A.  I n t r a s p e c i f i c improvement M u n t z i n g ' s i n i t i a l r e p o r t on a p o m i x i s  i n Poa  has been termed by Atwood ( 1 3 ) , • • • a t u r n i n g p o i n t i n 11  the c y t o g e n e t i c and b r e e d i n g i n v e s t i g a t i o n s w i t h b l u e grass."  This i s c e r t a i n l y true i n respect of i n t e r s p e c i f i c  h y b r i d i z a t i o n i n t h e genus, a phase o f b r e e d i n g i n w h i c h advantage has been t a k e n o f t h e a p o m i c t i c means o f r e p r o duction t o perpetuate d e s i r a b l e combinations  species hybrids without the l o s s of through  a p a r t from improvement t h r o u g h  s e g r e g a t i o n ( 2 1 ) . However, individual plant selection  (below) t h e r e appear t o have been no g r e a t s t r i d e s i n i n t r a s p e c i f i c improvement, d e s p i t e t h e f a c t t h a t t h e same methods a r e a v a i l a b l e t o t h e breeder a s have been u t i l i z e d i n i n t e r s p e c i f i c h y b r i d i z a t i o n work ( 1 7 , 8 6 ) . B r i t t i n g h a m (17) concluded  from h i s s t u d y o f r e p r o d u c t i o n i n P. p r a -  t e n s i s b i o t y p e s t h a t t h e s t a n d a r d t e c h n i q u e s o f "• . • s e l e c t i o n , s t r a i n b u i l d i n g , and i n t r a - and i n t e r s p e c i f i c h y b r i d i z a t i o n are t h e o r e t i c a l l y a p p l i c a b l e to breeding problems i n Kentucky b l u e g r a s s . " Single plant s e l e c t i o n , or the selection of groups o f i n d i v i d u a l p l a n t s , has u n d o u b t e d l y been t h e most s i g n i f i c a n t form o f improvement, even b e f o r e t h e d i s c o v e r y of a p o m i x i s  i n Poa ( 8 1 , 8 6 ) . Among a p o m i c t i c  bluegrasses,  -54which i n c l u d e s the m a j o r i t y of s p e c i e s o f a g r i c u l t u r a l importance, i n d i v i d u a l p l a n t s e l e c t i o n i s a sound  practice.  The most d e s i r a b l e p l a n t types can be perpetuated i n d e f i n i t e l y by a p o m i c t i c a l l y - f o r m e d seed.  Some o f t h e e a r l i e r  workers assumed that improvement i n Poa i s l i m i t e d t o i n d i v i d u a l plant selection. (81),  N i l s s o n - L e i s s n e r and N i l s s o n  f o r i n s t a n c e , s t a t e d t h a t because  " . . . crosses  can be made o n l y i n e x c e p t i o n a l cases . • • combination breeding i s t h e r e f o r e o f s m a l l importance." Muntzing of  (70) s u c c e s s f u l l y produced a number  h y b r i d s between predominantly s e x u a l and predominantly  a p o m i c t i c s t r a i n s of P. a l p i n a . and found that the h y b r i d s were mostly t h e r e s u l t o f the f e r t i l i z a t i o n egg c e l l by a reduced male gamete. from the f u s i o n o f an unreduced gamete.  o f a reduced  A few, however, a r o s e  egg and a reduced male  A l l the F i p l a n t s were s e x u a l , as were a l l the  F2 p r o g e n i e s , l e a d i n g Muntzing "Apomixis  (70) to conclude t h a t ,  cannot be due to a s i n g l e gene but r a t h e r to  s p e c i a l c o n s t e l l a t i o n s o f genes brought about by n a t u r a l selection."  The r e s u l t s from o t h e r s t u d i e s c o r r o b o r a t e  t h i s view, a l t h o u g h as y e t t h e r e i s r e a l l y v e r y l i t t l e known o f the i n h e r i t a n c e o f apomixis i n Poa. ( 2 9 ) . From Muntzing s 1  of the  study (70) i t appears t h a t t h e establishment  a p o m i c t i c h y b r i d s t r a i n s o f a s p e c i e s i s dependent  upon  use o f a p o m i c t i c plants, as both male and female parents  -55i n a cross.  The  t e c h n i q u e s of i n t r a s p e c i f i c h y b r i d i z a t i o n  must t h e r e f o r e be e s s e n t i a l l y l i k e t h o s e of h y b r i d i z a t i o n i n Poa, B.  described  i n the f o l l o w i n g s e c t i o n .  Interspecific hybridization The  tween Poa  f i r s t r e c o r d e d a r t i f i c i a l h y b r i d i z a t i o n be-  s p e c i e s seems t o be t h a t made by George  O l i v e r i n 1908.  W.  He c r o s s e d a female Poa . a r a c h n i f e r a  p l a n t w i t h a P. p r a t e n s i s p l a n t and a b l e F]_ progeny ( 1 0 5 ) . time.  interspecific  obtained  No f u r t h e r work was  a highly varidone a t  this  A r e p e t i t i o n of the same c r o s s by E. M a r i o n Brown  of the U n i t e d S t a t e s Department of A g r i c u l t u r e a l s o gave a v a r i a b l e F^, and  and  i n c l u d e d p l a n t s showing g r e a t e r  drought-resistance  and p r o d u c t i v i t y t h a n P.  heat-  pratensis  (105). A k e r b e r g (3) produced a progeny of 17  plants  from a s e m i - s t e r i l e P. p r a t e n s i s p l a n t p o l l i n a t e d w i t h P. a l p i n a p o l l e n .  Only one  of the p l a n t s was  a hybrid,  t h e remainder .being m a t e r n a l t y p e s produced a p o m i c t i c a l l y t h r o u g h pseudogamous development.  The  the f e r t i l i z a t i o n of a reduced egg  c e l l by a reduced male  gamete. •  I n o t h e r h y b r i d i z a t i o n s between t h e same s p e c i e s  Akerberg obtained and  hybrid arose from  h y b r i d s b o t h from the u n i o n o f r e d u c e d  of unreduced e g g - n u c l e i  w i t h reduced p o l l e n n u c l e i ,  t h e m a j o r i t y b e i n g of the former t y p e ( 1 , 4, 7 ) .  These  -56observations and  were based on  chromosome counts i n the  parents  hybrids. Hybrids between P. p r a t e n s i s and  P.  much more r a p i d germination than P. p r a t e n s i s  a l p i n a showed ". •  .a  c h a r a c t e r which i t would be  h i g h l y d e s i r a b l e to be able to  transmit  slow g e r m i n a t i o n of which i s  to the l a t t e r , the  f r e q u e n t l y a cause of poor establishment." hybrids a l s o reached the p a n i c l e - b e a r i n g quickly.  A h y b r i d from the by Akerberg (4).  one  h y b r i d i n the c r o s s P. p a l u s t r i s x P.  a p l a n t of an apomictic female parent and  stage more  K i e l l a n d e r (6l)  obtained  (15)  These  cross P. p r a t e n s i s x P.  was  Brittingham  (6).  glauca  a l s o found  compressa.  produced a s i n g l e h y b r i d between  s t r a i n of P.  compressa as  the  a P. p r a t e n s i s p l a n t as p o l l e n p a r e n t .  On the b a s i s of p o l l e n g r a i n s i z e comparisons i n parents and  h y b r i d i t was  concluded t h a t the h y b r i d arose from i  the f u s i o n of an unreduced compressa egg reduced p r a t e n s i s p o l l e n n u c l e u s .  nucleus and  (Muntzing (70)  showed the p o s i t i v e c o r r e l a t i o n between c e l l s i z e chromosome number f o r P. p r a t e n s i s ) .  The  first and  h y b r i d appeared  to have s e v e r a l d e s i r a b l e q u a l i t i e s , i n c l u d i n g good a good spreading  h a b i t and  l a r g e r and  fewer) p a n i c l e s than e i t h e r p a r e n t . The  a  heavier  vigour,  (although  Seed set was  high.  leaves of the h y b r i d were r e l a t i v e l y f r e e from l e a f  spot and  r u s t , which were severe on the parent p l a n t s .  -57I t a l s o seemed somewhat l e s s s u s c e p t i b l e to mildew. 0  Akerberg and B i n g e f o r s between sexual p r a t e n s i s and and  can be  (8) r e c o r d that, "Crosses  a l p i n a give normal  e a s i l y produced." Some of the d i f f i c u l t i e s  and  hybrids  involved  i n both i n t r a -  i n t e r s p e c i f i c h y b r i d i z a t i o n between apomictic  forms  are brought out by Nygren ( 8 6 ) , a l t h o u g h h i s comments a p p l y p a r t i c u l a r l y to Poa  pratensis.  The  best a v a i l a b l e p l a n t s  w i l l be the ones sought by the breeder as p o t e n t i a l parents i n a c r o s s , but u n f o r t u n a t e l y  these w i l l o f t e n  be  the most uniform s t r a i n s as a r e s u l t of improvement through selection.  For P. p r a t e n s i s . " E m b r y o l o g i c a l l y  t h a t they do not  only have a strong tendency t o aposporous  embryo sac f o r m a t i o n ,  but a l s o t h a t the  aposporous sacs d i v i d e s i n such an fertilization  t h i s means  egg  c e l l i n these  e a r l y stage t h a t a  i s more or l e s s excluded.  T h i s makes the  breeding work v e r y d i f f i c u l t , i f such a good s t r a i n i s wanted as mother i n the combination." ( 8 6 ) .  I f the  other  s t r a i n or s p e c i e s to be used i n the c r o s s has a tendency to a l a t e r e g g - c e l l d i v i s i o n i t can be used as the parent; however, t h i s may  a l s o be disadvantageous i f  unreduced e g g - c e l l s tend to be f e r t i l i z e d of h y b r i d s .  female  i n the  formation  Thus, i f the b e t t e r s t r a i n i s used as a male  parent, because of i t s u n i f o r m i t y ,  i t may  only  contribute  -58approximately one t h i r d of the t o t a l chromosome complement of the h y b r i d .  The disadvantage of such an  inheritance  p a t t e r n i s obvious; degeneration of the b e t t e r r a t h e r than improvement, may  result.  strain,  Nygren (86)  suggests  t h a t , "In order to estimate the chances to o b t a i n a s p e c i a l h y b r i d combination an e m b r y o l o g i c a l i n v e s t i g a t i o n ought to be made on the planned mother s t r a i n . " way  In t h i s  a study of the r e l a t i o n between the number of s e x u a l  and a p o m i c t i c embryo sacs can be made, and the time of d i v i s i o n of the egg n u c l e i i n these can be observed. importance  The  of such a study i s a l s o emphasized by G u s t a f s -  son's c o n c l u s i o n (44)  that " . . .  hybrid formation  . . .  i n c r e a s e s i n the measure t h a t reduced embryo-sacs a r i s e or t h a t the f i r s t One  d i v i s i o n of the e g g - c e l l begins  of the most ambitious and  i n g programs with the genus Poa  late."  successful  i n North America  breed-  has been  c a r r i e d on by the Carnegie I n s t i t u t i o n of Washington workers a t S t a n f o r d , C a l i f o r n i a , i n c o o p e r a t i o n w i t h the S o i l Conservation S e r v i c e of the U n i t e d S t a t e s Department of A g r i c u l t u r e .  T h i s program, s t a r t e d i n 1942-1943, arose  out of the growing  r e a l i z a t i o n of the d e p l e t i o n of the  Western rangelands and of the p o s s i b i l i t y of r e s t o r i n g to them some of t h e i r former p r o d u c t i v i t y w i t h the a i d of improved  b l u e g r a s s forms (25,  26).  new,  -59The b l u e g r a s s e s , because of the extent to which p o l y p l o i d y has a r i s e n i n them, and because of t h e i r dominantly  pre-  a p o m i c t i c means of r e p r o d u c t i o n , were seen to  have unique p o s s i b i l i t i e s . Carnegie group saw  The f a r s i g h t e d members of the  t h a t the b l u e g r a s s e s are among the  groups of p l a n t s which have reached an e v o l u t i o n a r y l e v e l i n which "• • • the genome, r a t h e r than the gene, becomes the b a s i c e v o l u t i o n a r y u n i t . " One  (25).  of the b a s i c p r i n c i p l e s behind the program  has been to combine the best s t r a i n s a v a i l a b l e of s p e c i e s belonging to d i f f e r e n t  s e c t i o n s of the genus.  These r e -  motely r e l a t e d s p e c i e s have a l s o been chosen f o r t h e i r c o n t r a s t i n g r e g i o n s of a d a p t a t i o n .  T h i s has meant c r o s s -  i n g forms from high a l t i t u d e s with those from low  alti-  tudes, c r o s s i n g c o a s t a l types w i t h i n t e r i o r t y p e s , l a t i t u d e forms w i t h low l a t i t u d e forms and The  high  so on (25,  reason f o r such a course i s t h a t , as p o i n t e d out  Clausen  et a l . ( 2 9 ) ,  "The  26). by  p r o b a b i l i t i e s of success i n  such an endeavour are enhanced by c r o s s i n g forms t h a t do not occur together i n the w i l d j i n those t h a t occur t o gether h y b r i d i z a t i o n and n a t u r a l s e l e c t i o n have presuma b l y a l r e a d y s o r t e d out the best combinations."  By com-  b i n i n g the genomes of forms from o p p o s i t e extremes of environment the Carnegie workers have found to produce h y b r i d s adaptable  i t possible  to i n t e r m e d i a t e c o n d i t i o n s .  -60-  Other p a r t i c u l a r c o n s i d e r a t i o n s have been made i n the s e l e c t i o n o f types t o be c r o s s e d .  One o f these  has been to combine the g r e a t e r drought r e s i s t a n c e o f the bunchgrass of  with the s u p e r i o r s o i l - h o l d i n g  the rhizomatous type and to ". . • u t i l i z e  capacity the b e t t e r  storage f a c i l i t i e s o f the rhizome g r a s s as an a d d i t i o n a l p r o t e c t i o n a g a i n s t o v e r g r a z i n g and f i r e . "  (26).  Other  o b j e c t i v e s have been t h e l e n g t h e n i n g o f the season o f use by combining w i n t e r - and s p r i n g - a c t i v e w i t h summer-active rhizome  bunchgrasses  g r a s s e s , and t o breed f o r more  d i s e a s e - r e s i s t a n t and winter-hardy forms.  In many o f the  h y b r i d i z a t i o n s i t has been p o s s i b l e to aim f o r s e v e r a l o b j e c t i v e s a t once. Because t h e p a r e n t a l s p e c i e s a r e predominantly a p o m i c t i c types the percentage o f h y b r i d s t o be expected i n any c r o s s i s very low.  F o r t h i s reason a mass-  p o l l i n a t i o n technique i s employed whereby the p l a n t s to be c r o s s e d a r e grown i n pots sunk s i d e by s i d e i n t h e ground. A p o l l e n - t i g h t t e n t cage, designed f o r outdoor i s p l a c e d over two p l a n t s .  pollination,  Pollen i s collected daily i n  parchment bags p l a c e d over the i n f l o r e s c e n c e s and r e c i p r o cal  p o l l i n a t i o n s made by exchanging  t h e bags and shaking  the p o l l e n over t h e r e s p e c t i v e i n f l o r e s c e n c e s . ( 2 6 ) .  The  seed from c r o s s e s i s sown i n t h e greenhouse and the seedl i n g s p r i c k e d out i n t o f l a t s  and spaced c a r e f u l l y so that  -61they can be examined i n d i v i d u a l l y .  The h y b r i d s can be  d e t e c t e d when they a r e two t o t h r e e months o l d , i n about the 8- to 1 2 - l e a f stage. inated (27).  The non-hybrids are then e l i m -  The frequency  of the h y b r i d s v a r i e s be-  tween about 0.2 and 5 per cent i n d i f f e r e n t In 1952, Clausen  combinations.  (21) s t a t e d t h a t the Carnegie  program  had r e s u l t e d i n the p r o d u c t i o n of some 700 F i h y b r i d s out of about 80,000 s e e d l i n g s i n the 70 attempted c r o s s ings up to t h a t  time.  Some combinations have been much more s u c c e s s f u l than o t h e r s , and many of these have been made s e v e r a l times, u s i n g d i f f e r e n t r a c e s .  The most s u c c e s s f u l c r o s s e s  have been those i n v o l v i n g P. ampla. P. s c a b r e l l a . P. p r a t e n s i s . P. compressa and P. a r i d a , which " . . . appear to have the gene s e t s (genomes) from which new forms can most s u c c e s s f u l l y be s y n t h e s i z e d . " ( 2 9 ) . attempts,  Despite many  no h y b r i d s have been o b t a i n e d u s i n g P. p r a t e n s i s  as the female parent,  i n f a c t , v e r y few have been obtained  u s i n g any member of the Pratenses  as the maternal  parent.  Chromosome counts o f the p a r e n t a l s t r a i n s and the h y b r i d s have r e v e a l e d t h a t most of the h y b r i d s have r e s u l t e d from the f u s i o n of a reduced  egg with a  reduced  p o l l e n n u c l e u s , while a s m a l l e r number have a r i s e n from the f u s i o n of an unreduced egg w i t h a reduced  pollen  -62nucleus  ( 3 2 , 8 3 ) . One p a r t i c u l a r P. ampla s t r a i n used a s  a female p a r e n t produced h y b r i d s from reduced eggs o n l y when p o l l i n a t e d by one s t r a i n o f P. p r a t e n s i s . but from unreduced eggs o n l y when p o l l i n a t e d by a second p r a t e n s i s s t r a i n (32). Normally,  i n c r o s s i n g w i d e l y separated n a t u r a l  p l a n t f o r m s , t h e b a l a n c e d gene systems o f t h e p a r e n t s a r e d i s t u r b e d , and t h e s e g r e g a t i o n p r o d u c t s w h i c h a r i s e i n t h e F2 and subsequent g e n e r a t i o n s a r e t o o i l l - a d a p t e d to be o f any v a l u e .  But as has been mentioned p r e v i o u s l y ,  where a p o m i c t i c r e p r o d u c t i o n p r e v a i l s i t i s p o s s i b l e t o o b t a i n non-segregating  h y b r i d s which can be p e r p e t u a t e d  i n d e f i n i t e l y w i t h o u t f u r t h e r g e n e t i c change.  The h y b r i d  v i g o u r c h a r a c t e r i s t i c o f many f i r s t g e n e r a t i o n h y b r i d s can t h e r e f o r e be m a i n t a i n e d  (25, 26, 27, 2 8 ) . I n p r a c t i c e ,  Clausen and h i s co-workers have found t h a t about two t h i r d s o f t h e i n t e r s p e c i f i c h y b r i d s between p r e d o m i n a n t l y  apomictic  s t r a i n s reproduce s e x u a l l y , and o n l y one t h i r d a p o m i c t i c a l l y ( 2 8 , 29, 3 0 ) . T h i s c h a r a c t e r i s t i c o f t h e h y b r i d s was une x p e c t e d , b u t has s i n c e proved t o be o f immense v a l u e i n the b r e e d i n g program because t h e s e x u a l l y - r e p r o d u c i n g h y b r i d s tend t o segregate  i n t o a number o f F2 l i n e s about  h a l f o f which a r e a p o m i c t i c ( 8 3 ) .  Thus, t h e s e x u a l mode  of reproduction i n the hybrids permits t h e r e l e a s e o f a  -63c e r t a i n amount o f v a r i a b i l i t y , y e t s e g r e g a t i o n stops i n those F 2 l i n e s i n which apomixis  i s reinstated. Selection  among these a p o m i c t i c F 2 l i n e s g i v e s the breeder p o r t u n i t y o f choosing  constant l i n e s t h a t s t i l l  the opretain  much of the i n i t i a l v i g o u r o f the F]_ h y b r i d and t h a t show the other d e s i r a b l e c h a r a c t e r i s t i c s being sought.  The  s e g r e g a t i o n i n t o numerous F 2 l i n e s a l s o i n c r e a s e s the chances o f f i n d i n g new, d e s i r a b l e s t r a i n s because, as a l r e a d y noted, the i n i t i a l number o f h y b r i d s formed i s so low. The Poa program i s now under completion by the Carnegie  group, and i s being c a r r i e d on by o t h e r  t i o n s and government agencies  (23, 24).  institu-  Some 45 s t a b i l -  i z e d h y b r i d l i n e s have been produced, o f which as many as 10 t o 15 may prove t o be o f immediate v a l u e i n the western S t a t e s ( 2 3 ) .  Some quadruple  ampla x ampla-pratensis) may be used ( 1 0 2 ) .  hybrids (e.g. a r i d a -  have a l s o been s y n t h e s i z e d and  The t e s t i n g of h y b r i d l i n e s i s g r e a t l y  f a c i l i t a t e d by the a p o m i c t i c r e p r o d u c t i o n , because can be used t o propagate s t r a i n s without  g e n e t i c change.  Many l i n e s have been on t e s t under v a r y i n g c l i m a t i c d i t i o n s i n d i f f e r e n t areas throughout and  seed  con-  the U n i t e d S t a t e s  i n a number of European c o u n t r i e s ; some may be u t i l i -  zable i n these areas ( 3 1 ) . As Clausen  et a l . ( 2 3 ) p o i n t out, "In Poa the  p o t e n t i a l i t i e s f o r producing a great range o f new a p o m i c t i c  -64s t r a i n s t h r o u g h i n t e r s e c t i o n a l h y b r i d i z a t i o n a r e almost unlimited."  More o f such b r e e d i n g work i n t h e b l u e g r a s s e s  w i l l u n d o u b t e d l y be c a r r i e d on.  Furthermore, the breeding  p r i n c i p l e s t h a t have been developed may a l s o be a p p l i c a b l e t o o t h e r groups o f economic p l a n t s ( 2 3 , 2 4 ) .  -65-  VI  STUDIES OF REPRODUCTION IN Poa c o n f i n i s A.  A t t e m p t s t o i n d u c e f l o w e r i n g under a r t i f i c i a l conditions 1.  Introduction  One o f t h e d i f f i c u l t i e s throughout most o f t h e s t u d y was t h e i n a b i l i t y t o i n d u c e f l o w e r i n g i n P. c o n f i n i s p l a n t s grown i n t h e greenhouse.  T h i s meant t h a t t h e r e was  a l a c k o f f r e s h m a t e r i a l f o r c y t o l o g i c a l s t u d y d u r i n g much o f t h e course o f t h e i n v e s t i g a t i o n .  Furthermore, the  h y b r i d i z a t i o n e x p e r i m e n t s were c o n s i d e r a b l y this  delayed.  For  r e a s o n , a s e r i e s o f a t t e m p t s were made t o i n d u c e  f l o w e r i n g i n P. c o n f i n i s p l a n t s , n o t a s an experiment i n the i n d u c t i o n o f f l o w e r i n g as such, b u t m e r e l y t o p r o v i d e m a t e r i a l f o r t h e c y t o l o g i c a l and h y b r i d i z a t i o n a l work. 2.  M a t e r i a l s and methods  A number o f p l a n t s o f t h e W h i f f i n S p i t  biotype  were t a k e n from t h e 6 x 12 f o o t U n i v e r s i t y farm p l o t and potted 1953  i n sandy loam s o i l i n t h e greenhouse i n September  a t the s t a r t of the study.  They were s u p p l i e d  with  complete f e r t i l i z e r and k e p t under a p p r o x i m a t e l y a 14-hour day d u r i n g t h e w i n t e r months w i t h t h e a i d o f e l e c t r i c lights.  D u r i n g t h e summer months t h e y were l e f t under t h e  p r e v a i l i n g day-length c o n d i t i o n s . t a i n e d t h r o u g h December 1 9 5 4 .  These p l a n t s were main-  -66Ih October 1954 a s e r i e s of treatments were started on P. c o n f i n i s p l a n t s brought i n a t that time from the p l o t of W h i f f i n S p i t m a t e r i a l and placed i n l a r g e p o r c e l a i n pots. treatments,  S i x plants were used f o r each of nine  and w i t h i n each treatment three p l a n t s were  potted i n pure beach sand and three p l a n t s i n f a i r l y orchard  sandy  soil. The nine treatments were as f o l l o w s :  (1)  "Drought":  the p l a n t s were given enough water merely  to keep them a l i v e . (2)  Carbohydrate:  sucrose was supplied a t .5 grams per  pot per week. (3)  Nitrogen:  ammonium n i t r a t e ( 3 3 per cent nitrogen)  was supplied a t .6 grams per pot per month. (4)  Salt:  sodium c h l o r i d e was supplied a t .5 grams per pot per month.  (5)  Complete f e r t i l i z e r :  10-20-10  was supplied a t  1  gram  per pot per month. (6)  Cold shock: at  (7)  Cold shock: at  (8)  32°  32°  p l a n t s were given three weeks' treatment F. p l a n t s were given eight weeks' treatment F.  Continuous l i g h t :  a r t i f i c i a l l i g h t was supplied by a  500-watt bulb, three f e e t above the p l a n t s . (9)  Control:  no treatment.  These treatments were continued f o r j u s t over f i v e months, .during which time no a r t i f i c i a l l i g h t  was  used (except f o r the continuous l i g h t t r e a t m e n t ) . A number of P. c o n f i n i s p l a n t s brought  from  Gray's Harbour, Washington, on November 5, 1954, were potted i n sand i n the greenhouse a f t e r t h e i r  collection*  S e v e r a l p l a n t s from t h e U n i v e r s i t y p l o t were a l s o brought i n t o the greenhouse and p o t t e d i n sand i n e a r l y December 1954.  No a r t i f i c i a l l i g h t was s u p p l i e d f o r e i t h e r o f  these two groups of p l a n t s . In t h e middle of February 1955 t h r e e p l a n t s from the  U n i v e r s i t y were p o t t e d i n sand i n the greenhouse and  put  under the l i g h t  (i.e.  c o n d i t i o n s p r e v a i l i n g i n e a r l y May  approximately a 13-hour d a y - l e n g t h ) . At two-week  i n t e r v a l s other P. c o n f i n i s p l a n t s from the farm p l o t were brought i n and p l a c e d under the same c o n d i t i o n s ; t h i s was continued u n t i l the middle of A p r i l . During a l l these v a r i o u s attempts t o induce f l o w e r i n g i n the greenhouse  t h e r e was o f t e n c o n s i d e r a b l e  f l u c t u a t i o n i n temperature; no r e c o r d s o f the temperature were kept.  A l l p l a n t s , except those under the "drought"  c o n d i t i o n s were watered r e g u l a r l y .  Aphids were p a r t i c u -  l a r l y troublesome on the dune b l u e g r a s s p l a n t s , and r e g u l a r f u m i g a t i o n was n e c e s s a r y .  -683*  Besuits  None of the p l a n t s i n the group i n i t i a l l y brought i n t o the greenhouse f l o w e r e d .  N e a r l y a l l p l a n t s showed a  f a i r l y v i g o r o u s v e g e t a t i v e growth d u r i n g the e n t i r e teen months t h a t they were kept i n the  fif-  greenhouse. 1954  A l l the nine treatments s t a r t e d i n October were u n s u c c e s s f u l i n i n d u c i n g f l o w e r i n g i n any of the plants.  One  s t r i k i n g f e a t u r e noted was  treatments the p l a n t s grown i n beach  t h a t i n a l l the  sand maintained a f a r  more h e a l t h y and v i g o r o u s v e g e t a t i v e growth than t h o s e grown i n s o i l . u n i f o r m l y poor.  Growth of the p l a n t s i n s o i l was P l a n t s i n both sand and  s p i n d l y under continuous l i g h t . c o l d temperature  almost  s o i l were v e r y  A l l p l a n t s i n the  two  treatments kept i n r e l a t i v e l y good con-  d i t i o n , although the p l a n t s were somewhat e t i o l a t e d the l o n g e r treatment.  after  N e v e r t h e l e s s , they a l l r e c o v e r e d  q u i c k l y and resumed normal v e g e t a t i v e growth when r e t u r n e d to  the greenhouse.  There was  p l a n t s kept under drought  very l i t t l e  conditions.  growth by the  There were no  vis-  i b l e d i f f e r e n c e s between the p l a n t s o f the carbohydrate, s a l t , n i t r o g e n , complete  f e r t i l i z e r and c o n t r o l treatments  — a g a i n , v e g e t a t i v e growth was  good i n sand and poor i n  soil. The p l a n t s taken i n t o the greenhouse from  Gray s 1  Harbour and from the U n i v e r s i t y p l o t i n e a r l y November and  -69e a r l y December, r e s p e c t i v e l y , d i d not growth f o r almost two  weeks.  show any  A f t e r t h i s time, however,  they began v e g e t a t i v e growth but d i d not Almost a l l the P.  signs o f  flower.  c o n f i n i s p l a n t s brought i n t o  the greenhouse during February, March and A p r i l 1955, duced i n f l o r e s c e n c e s w i t h i n two about two  weeks.  They  flowered  weeks a f t e r the f i r s t appearance of the p a n i c l e s .  On a l l these p l a n t s f l o w e r i n g was  very  sparse,  a l a r g e s e c t i o n of the rhizomatous sod o n l y one i n f l o r e s c e n c e s would develop. not flower a t , a l l .  Two  grown i n the  and or  from two  or t h r e e p l a n t s d i d  T h i s p a u c i t y of i n f l o r e s c e n c e s i s  i n d i r e c t c o n t r a s t to the profuse  f l o w e r i n g of the  plants  field.  The m a t e r i a l which flowered was  pro-  i n the greenhouse  used p a r t l y f o r c y t o l o g i c a l study and  p a r t l y f o r the  h y b r i d i z a t i o n experiments. B.  Cytology 1.  and  histology  Megasporogenesis i n p i s t i l l a t e (a)  M a t e r i a l s and  flowers  methods  Most of the m a t e r i a l used i n the c y t o l o g i c a l and P.  h i s t o l o g i c a l s t u d i e s was  obtained  from the p l o t of  c o n f i n i s on the U n i v e r s i t y farm when the p l a n t s  ered i n May  1954.  flow-  Some a d d i t i o n a l study of p a r t i c u l a r  -70s t a g e s o f development was made on t h e p l a n t s o f t h i s same W h i f f i n S p i t b i o t y p e which were i n d u c e d t o f l o w e r i n t h e greenhouse i n March 1955*  As p r e v i o u s l y r e c o r d e d , t h e  e n t i r e p l o t was composed o f p i s t i l l a t e  plants*  F i x a t i o n s were s t a r t e d a s soon a s t h e f i r s t p a n i c l e s were observed emerging from t h e sheath on May 2nd* Whole i n f l o r e s c e n c e s were t a k e n a t random from throughout t h e p l o t and p l a c e d i n R b l l i n - C a r n o y k i l l i n g  and f i x i n g  s o l u t i o n * f o r 6 t o 10 h o u r s , t h e l o n g e r t i m e s b e i n g used for  the l a t e r stages.  for  r i n s i n g o f f t h e i n f l o r e s c e n c e s a f t e r f i x a t i o n and f o r  s t o r i n g them.  Seventy p e r cent a l c o h o l was used  M a t e r i a l n o t imbedded i m m e d i a t e l y was k e p t  i n t h e 70 p e r c e n t . a l c o h o l i n t h e r e f r i g e r a t o r . C o l l e c t i o n s were made d a i l y u n t i l a n t h e s i s o c c u r r e d about f o u r t e e n days a f t e r t h e f i r s t appearance o f the p a n i c l e s .  These c o l l e c t i o n s were t a k e n i n b o t h  even-  i n g s and mornings, a l t h o u g h more f r e q u e n t l y i n t h e f o r m e r . S e v e r a l a d d i t i o n a l c o l l e c t i o n s were made subsequent t o anthesis. S p i k e l e t s from t h e f i x e d m a t e r i a l were d i s s e c t e d from t h e o u t e r glumes, w i t h o u t t h e a i d o f a l e n s o r m i c r o scope, and imbedded i n t a c t .  The s p i k e l e t s were dehydrated  * 85 c c . 70 p e r cent e t h y l a l c o h o l : a c e t i c a c i d : 5 c c . commercial f o r m a l i n .  5 cc. glacial  -71i n a l c o h o l i n t h e u s u a l manner, and a s m a l l amount o f E r y t h r o s i n B was added a t t h e 95 per cent a l c o h o l l e v e l to  l e n d a p i n k t i n g e t o them, thus i m p r o v i n g t h e i r  b i l i t y when imbedded.  visi-  They were c l e a r e d i n x y l o l and  imbedded i n t i s s u e mat o r Parowax. The s p i k e l e t s were c u t on t h e microtome a t v a r y i n g a n g l e s a t a l l s t a g e s , and a t t h i c k n e s s e s v a r y i n g from 8 t o 22 m i c r o n s , depending  on t h e i r m a t u r i t y .  a r a b i c was used a s an a d h e s i v e , and a 1 p e r cent of  Gum  solution  p o t a s s i u m d i c h r o m a t e used t o s t r a i g h t e n t h e r i b b o n s . A f t e r mordanting  i n 4 p e r cent i r o n - a l u m s o l u -  t i o n t h e s e c t i o n s were s t a i n e d i n H a r r i s  1  hematoxylin f o r  2\ t o 4 h o u r s , d e s t a i n e d i n 2 p e r cent i r o n - a l u m under the m i c r o s c o p e and c o u n t e r s t a i n e d w i t h l i g h t  green;  A l l drawings were made w i t h t h e a i d o f a camera lucida. (b)  Observations  A t t h e time o f emergence o f t h e p a n i c l e from t h e s h e a t h t h e s i n g l e o v u l e i n each o f t h e b a s a l f l o r e t s o f the s p i k e l e t s i s a l r e a d y w e l l developed. o u t e r integument,  An i n n e r and  each composed o f two l a y e r s o f c e l l s ,  surround t h e n u c e l l u s , a l t h o u g h a t t h i s stage t h e i n t e g u ments have not y e t grown up around t h e n u c e l l u s t o form t h e m i c r o p y l e ( F i g . 7, A ) .  The n u c e l l u s c o n t a i n s a s i n g l e ,  -72o f t e n somewhat wedge-shaped EMC, w i t h a l a r g e t y p i c a l l y c l o s e r t o the m i c r o p y l a r  nucleus  end o f t h e c e l l , but  sometimes c e n t r a l l y p l a c e d o r near t h e c h a l a z a l end. The  chromatin m a t e r i a l i n the nucleus  i n the  m a j o r i t y o f EMCs a t t h i s time shows a c h a r a c t e r i s t i c s y n i z e s i s - - a stage o f the m e i o t i c prophase i n which the chromatin forms a spireme and i s aggregated towards one s i d e o f the nucleus  ( F i g s . 7, A and B ) .  The t h r e a d - l i k e  o r g a n i z a t i o n o f the spireme i s u s u a l l y evident zesis. although  A single, large nucleolus i s also  i n syni-  present,  i n one EMC a second, s m a l l e r n u c l e o l u s can be  seen. The EMC appears t o undergo a normal f i r s t tic  division.  meio-  P a i r e d chromosomes at- zygotene a r e evident  i n a number o f s l i d e s ( F i g . 7, C ) . F i g . 8, A shows a pachytene or d i p l o t e n e stage.  L a t e r stages  i n diakinesis  have i n v a r i a b l y shown a l l chromosomes t o be a s s o c i a t e d i n p a i r s , as f a r as can be determined ( F i g s . 7, D and E j F i g . 8, B ) . The two c e l l s drawn i n F i g s . 7, D and E a r e t h e o n l y EMCs i n which an almost p o s i t i v e count can be obtained; both c e l l s c o n t a i n 21 p a i r s of chromosomes. the l a r g e n u c l e o l u s i s s t i l l o r g a n i z e d ,  I n F i g . 7, D  and one b i v a l e n t  i s c l o s e l y a s s o c i a t e d w i t h i t j i n F i g . 7, E t h e n u c l e o l u s has disappeared grouped.  and the 21 b i v a l e n t s a r e more c l o s e l y  S a t e l l i t e s , or t r a b a n t s , a r e observable on  - 7 3 -  Figure 7 :  Megasporogenesis  i n Poa c o n f i n i s  A.  Ovule a t time of emergence o f p a n i c l e ; s i n g l e EMC i n m e i o t i c prophase ( s y n i z e s i s ) j the i n n e r and outer integuments developing around the nucellus.  B.  S y n i z e s i s i n EMC  C.  Zygotene i n EMC  (Prophase I ) , (Prophase I ) ,  D. and E. D i a k i n e s i s i n EMC (Prophase I ) ; 2 1 b i valent s present. Note the t r a b a n t s ( s a t e l l i t e s ) on s e v e r a l p a i r s o f chromosomes i n both c e l l s . (D c o n s t r u c t e d from two a d j a c e n t s e r i a l s e c t i o n s . ) F.  Metaphase I i n EMC.  G.  Dyad c e l l s i n i n t e r k i n e s i s . (all x  The m i c r o p y l a r drawings.  1 7 6 0 )  end i s to the top o f , t h e page i n a l l  -74-  F i g u r e 8:  Megasporogenesis  A.  Diplotene  i n EMC  B.  D i a k i n e s i s i n EMC  C.  Metaphase I i n  i n Poa  confinis  (Prophase I ) . (Prophase I ) .  EMC.  ( a l l x 2640)  -75several p a i r s of chromosomes i n these and other EMCs a t diakinesis. An elongation of the EMC accompanies the o r ganization of the spindle f i g u r e and the alignment of the b i v a l e n t s a t the metaphase p l a t e ( F i g . 7, E; F i g * 8, C). Although good sections a t anaphase and telophase of the heterotypic d i v i s i o n a r e d i f f i c u l t t o f i n d , the separation of the homologous p a i r s appears normal, with no evidence of u n i v a l e n t s or other i r r e g u l a r i t i e s * The f i r s t meiotic d i v i s i o n always takes place p a r a l l e l t o the long a x i s of the ovule.  Dyad c e l l s with n u c l e i i n  i n t e r k i n e s i s are i l l u s t r a t e d i n F i g . 7, C, i n which a r e o r g a n i z a t i o n of the nucleolus i n each c e l l appears t o have taken place* The  second meiotic d i v i s i o n e v i d e n t l y f o l l o w s  very r a p i d l y upon the f i r s t .  A t y p i c a l t r i a d stage i s  shown i n F i g . 9, A, i n which the d i v i s i o n i n the mierop y l a r c e l l of the dyad has been preceded by the d i v i s i o n i n the c h a l a z a l c e l l .  The mieropylar  dyad c e l l often  d i v i d e s i n a plane a t r i g h t angles t o the long a x i s of the ovule, forming a T-shaped t e t r a d ( F i g . 9, B), but more f r e q u e n t l y i n a plane p a r a l l e l t o the long a x i s of the ovule ( F i g s . 9, C, D and E ) . of the mieropylar  Delayed second d i v i s i o n  dyad c e l l i s common, and i n some cases  the d i v i s i o n f a i l s a l t o g e t h e r ( F i g . 9, F ) .  The d i v i s i o n  -76-  Figure 9s  Megasporogenesis i n Poa c o n f i n i s  A.  Triad.  B.  Tetrad; d i v i s i o n i n the chalazal dyad c e l l has preceded that i n the mieropylar c e l l ; note the T-shaped form of the t e t r a d .  C.  Linear tetrad; delayed disintegration of the three upper non-functional spores of the tetrad; the chalazal megaspore has already divided to form a 2-celled embryo sac.  D.  Linear tetrad; the second megaspore from the chalazal end i s functioning as the embryo sac initial.  £.  "Umbrella" stage following tetrad formation; the chalazal megaspore functioning as the embryo sac i n i t i a l .  F.  "Umbrella" stage following t r i a d formation; the chalazal c e l l i s the functional megaspore. ( a l l x 1520)  The mieropylar end i s to the top of the page i n a l l drawings.  -77of  the c h a l a z a l d y a d . c e l l i s i n v a r i a b l y i n the plane of  the l o n g i t u d i n a l a x i s of the o v u l e . stages are shown i n F i g s . 9,  C and D.  Complete t e t r a d In F i g . 9,  C the  b a s a l c e l l o f the t e t r a d i s f u n c t i o n i n g as the embryo sac initial  c e l l and i s a l r e a d y d i v i d i n g before the  r a t i o n of the upper t h r e e megaspores.  disinteg-  In F i g . 9, D the  second megaspore from the c h a l a z a l end i s probably funct i o n i n g as the embryo sac i n i t i a l i n p r e f e r e n c e to the b a s a l megaspore. T y p i c a l "umbrella" stages, seen i n a v e r y l a r g e 9,  number of s l i d e p r e p a r a t i o n s , are i l l u s t r a t e d i n F i g s . E, F and 10,  A and B.  The f l a t t e n i n g of the m i e r o p y l a r  c e l l of the spore t r i a d or t e t r a d , t o g e t h e r w i t h the  dis-  i n t e g r a t i o n o f the lower, n o n - f u n c t i o n a l megaspores, g i v e s the umbrella- or mushroom-like appearance.  In F i g . 9,  the two m i e r o p y l a r spores have a l r e a d y d i s i n t e g r a t e d ,  E and  the second megaspore from the c h a l a z a l end a l s o shows s i g n s of b r e a k i n g down.  F i g . 9, F i s a s i m i l a r stage,  cept t h a t the second d i v i s i o n i n the upper dyad c e l l not been completed,  and o n l y a t r i a d has formed.  ex-  has  The  second megaspore from the c h a l a z a l end of the ovule i s seen d i s i n t e g r a t i n g above the embryo sac i n i t i a l Fig.  10,  C.  cell in  -78-  F i g u r e 10:  O n e - c e l l e d embryo  sacs i n Poa  confinis  A. and B. "Umbrella" stages; the c h a l a z a l megaspore i s the.embryo sac i n i t i a l c e l l i n each case.  ( x 1075) C.  The c h a l a z a l megaspore i s f u n c t i o n a l ; the lower member o f the l i n e o f d i s i n t e g r a t i n g megaspores i s v i s i b l e on the r i g h t .  ( x 1610) The m i c r o p y l a r end i s to the r i g h t hand s i d e i n a l l three photographs.  i  -79Complete d i s i n t e g r a t i o n o f the n o n - f u n c t i o n a l megaspores i s accompanied by the i n c r e a s e d and  growth of the f u n c t i o n a l megaspore, or u n i n u c l e a t e  embryo sac* may  va>cuolation  The f i r s t m i t o t i c d i v i s i o n i n the embryo sac  take p l a c e e i t h e r p a r a l l e l t o or a c r o s s the l o n g i t u d -  i n a l a x i s ( F i g s . 11, A and B ) . anaphase - e a r l y t e l o p h a s e telophase  stage, and F i g . 11, B a l a t e  stage i n which the n u c l e a r membranes have formed  around the chromosomes.  The remains of the spore t e t r a d  are evident a t the m i e r o p y l a r Two-celled  F i g * 11, A shows a l a t e  end of both embryo s a c s .  embryo sacs are.seen  i n F i g s . 11,  C and 12,  A.  In the former, the t h r e e n o n - f u n c t i o n a l megaspores are s t i l l d i s c e r n i b l e , although u s u a l l y by t h i s stage i t i s d i f f i c u l t to p i c k them out i n d i v i d u a l l y from the  cell  debris. In o n l y two  i n s t a n c e s can more than one  sac be seen i n an o v u l e .  One  embryo  of these i n s t a n c e s i s i l -  l u s t r a t e d i n F i g . 11, D, which i s taken from a t r a n s v e r s e s e c t i o n of a p a i r of t w o - c e l l e d embryo sacs l y i n g s i d e i n the n u c e l l a r c a v i t y .  The  s i d e by  f i r s t mitotic division  i n the embryo sac on the r i g h t has j u s t been completed. One  of the two  telophase f i g u r e s i n t h i s sac can be  w h i l e the second i s i n a n e i g h b o r i n g  serial section.  seen, In  the second example of polyembryony found a p a i r of f o u r -  -80-  F i g u r e 11:  Embryo sac development i n Poa c o n f i n i s  A.  F i r s t d i v i s i o n i n the embryo s a c — l a t e anaphase - e a r l y telophase; d i s i n t e g r a t e d megaspores a t micropylar. end*  B.  Two-celled embryo s a c — t h e d i v i s i o n j u s t comp l e t e d ; d i s i n t e g r a t e d megaspores e v i d e n t .  C.  Two-celled embryo sac; t h e three d i s i n t e g r a t e d megaspores c l e a r l y d i s t i n g u i s h a b l e .  D.  Polyembryony; two embryo sacs l y i n g side by s i d e i n the n u c e l l a r chamber ( t r a n s v e r s e s e c t i o n ) .  E. and F. 0.  F o u r - c e l l e d embryo  sacs.  E i g h t - c e l l e d embryo sac. ( c o n s t r u c t e d eral serial sections).  from sev-  ( a l l x 1370) The  m i c r o p y l a r end i s t o the top o f the page i n a l l drawings.  -81c e l l e d embryo sacs a r e p l a c e d  s i d e by s i d e i n the o v u l e .  T y p i c a l f o u r - c e l l e d embryo sacs show the vacuol a t i o n o f the center of the sac, where the cytoplasm forms a t h i n , p e r i p h e r a l l a y e r a g a i n s t the w a l l ( F i g s , 11, E and F ) .  The number of n u c l e o l i i n the c e l l s of t h e  embryo sac seems q u i t e v a r i a b l e , a l t h o u g h one i s most usual.  Two, or o c c a s i o n a l l y t h r e e , may be found, and  there i s no tendency f o r c e l l s with more than one lus  nucleo-  to be found more o f t e n a t one end o f the embryo sac  than the o t h e r . Development of an e i g h t - c e l l e d embryo sac, two to three days b e f o r e a n t h e s i s , i s accompanied by f u r t h e r v a c u o l a t i o n and i n c r e a s e i n s i z e ( F i g , 11, G),  A polar  nucleus from each group of f o u r c e l l s a t e i t h e r end o f the embryo sac migrates toward t h e center of the sac where they fuse to form a s i n g l e c e l l  ( t h e f u s i o n nucleus) ( F i g ,  13, A) or,remain c l o s e l y a s s o c i a t e d but unfused ( F i g , 13, B),  At the m i c r o p y l a r  end of t h e embryo sac the three  c e l l s remaining there become d i s t i n g u i s h a b l e as an egg and a p a i r o f synergids  ( F i g . 12, B; F i g s . 13, A and B ) ,  In F i g , 13, B the egg c e l l i s l a r g e r than the s y n e r g i d s and  contains two n u c l e o l i . Marked a n t i p o d a l c e l l development i s evident i n  all  embryo sacs, and begins j u s t p r i o r t o a n t h e s i s .  This  -82takes the form not only of an i n c r e a s e i n the number o f antipodals crease  ( F i g s . 13, A and B) but l a t e r a l s o a huge i n -  i n t h e i r s i z e and i n the number o f n u c l e i per  antipodal c e l l  ( F i g s . 12, C; F i g . 13, C ) .  Division  f i g u r e s can be seen i n two of the t h r e e o r i g i n a l podal c e l l s i n F i g . 13, A. c e l l s are present.  anti-  In F i g . 13, B f o u r a n t i p o d a l  A t l e a s t e i g h t o r t e n a n t i p o d a l s are  seen i n almost every embryo sac a t a n t h e s i s , and as many as twenty can be counted i n s e v e r a l i n s t a n c e s . teristic  c h a n n e l l i n g or furrowing  of the dense,  A  granular  cytoplasm appears t o d i v i d e o f f the m u l t i n u c l e a t e podals ( F i g . 12, C and F i g . 13, C ) .  charac-  anti-  In most embryo  sacs  the a n t i p o d a l c e l l s f i l l up as much as two t h i r d s o f t h e sac, a l t h o u g h there does not appear t o be any compression of the u n d e r l y i n g f u s i o n nucleus and egg a p p a r a t u s . F l o r e t s s e c t i o n e d f i v e days a f t e r the beginning of a n t h e s i s appear much the same as f l o r e t s a t a n t h e s i s . In none of the embryo sacs examined was any d i v i s i o n o f e i t h e r egg c e l l or f u s i o n nucleus seen.  In the s m a l l  number of f l o r e t s examined seven days a f t e r a n t h e s i s s e v e r a l o f the embryo sacs are presumably aborted,  taking  o n l y a heavy s t a i n a l l over, through which i t i s imposs i b l e t o d i s c e r n any c e l l u l a r  detail.  -83-  F i g u r e 12: A.  Embryo sac development i n Poa  Two-celled  confinis  embryo s a c . (x  B.  1610)  M i e r o p y l a r end of mature embryo sac, showing egg c e l l and two s y n e r g i d s . (x  C.  2150)  A n t i p o d a l c e l l development i n mature embryo sac; note f u r r o w i n g of the cytoplasm between the m u l t i n u c l e a t e a n t i p o d a l s . (x  1075)  «  c  -84Figure  13:  A, B, C, and D - Female gametophyte development i n Poa c o n f i n i s . E and F - Embryo and endosperm f o r m a t i o n f o l l o w i n g p o l l i n a t i o n of p i s t i l l a t e Poa conf i n i s f l o r e t w i t h Poa p r a t e n s i s p o l l e n .  A and  B. Embryo sacs a few days p r i o r to a n t h e s i s ; A shows an egg and s y n e r g i d s , f u s i o n nucleus and d i v i s i o n s i n a n t i p o d a l c e l l s ; i n B the egg i s w e l l d i f f e r e n t i a t e d from the two syne r g i d s , the p o l a r n u c l e i have not yet f u s e d , and f o u r a n t i p o d a l s are p r e s e n t . (Both c o n s t r u c t e d  C.  from s e r i a l  sections; x  Extreme a n t i p o d a l c e l l development; note the c h a n n e l l i n g between the c e l l s , and the numerous n u c l e i per c e l l . ( x 815  D.  from two  adjacent s e c t i o n s ; x  370)  Neighboring s e c t i o n through same ovule as E; p e r i p h e r a l l a y e r of endosperm forms l a t e r a l l y ; note d i v i s i o n f i g u r e s i n s e v e r a l n u c l e i . (x  The  520)  Young embryo d e v e l o p i n g a f t e r p o l l i n a t i o n w i t h p r a t e n s i s p o l l e n ; a m u l t i - c e l l e d endosperm has a l r e a d y formed and surrounds the embryo; the two dark b o d i e s on e i t h e r s i d e of the embryo are the disintegrated synergids. (x  F.  )  Mature embryo sac s e v e r a l days a f t e r the s t a r t of a n t h e s i s ; the l a r g e egg l i e s between two pear-shaped s y n e r g i d s , the p o l a r n u c l e i are s t i l l unfused, and the a n t i p o d a l development i s strong. (Constructed  E.  1610)  m i c r o p y l a r end a l l drawings.  370)  i s to the bottom of the page i n  -85-  Chromosome counts cannot even be approximated i n any of the c e l l s o f the embryo sacs seen throughout the various  stages o f  development.  Chromosome counts i n the c e l l s o f the somatic t i s s u e s surrounding the d e v e l o p i n g gametophyte are a l s o v e r y d i f f i c u l t , as the c e l l s a r e normally s m a l l  and  crowded, w i t h the chromosomes hard to d i s t i n g u i s h  even  i n good p o l a r views Of metaphase p l a t e s .  very  Several  rough counts i n n u c e l l a r c e l l s i n d i c a t e no more than t h a t the  somatic chromosome number o f 42 i s p o s s i b l e , but the  most a c c u r a t e count o b t a i n e d i s about 40 1 5* 2.  Embryo and endosperm development p o l l i n a t i o n of Poa (a)  Materials  Because no seed was material  following  c o n f i n i s by Poa  pratensis  and methods set i n the W h i f f i n  i n the U n i v e r s i t y p l o t s i n May  Spit  1954 no c y t o l o g i c a l  study beyond the stage of the mature female gametophyte could be made.  However, i n A p r i l 1955, when f l o w e r i n g  induced i n P. c o n f i n i s p l a n t s c y t o l o g i c a l study was w i t h P. p r a t e n s i s  i n the greenhouse,  was  further  made f o l l o w i n g p o l l i n a t i o n s made  pollen.  The ,P. c o n f i n i s s p i k e l e t s were f i x e d a t a s e r i e s of time i n t e r v a l s f o l l o w i n g p o l l i n a t i o n .  Due  to the  -86.sparse f l o w e r i n g o n l y t h r e e or f o u r s p i k e l e t s used f o r each of the The  (1) At the  following  pollination pollination  (4) 16 hours a f t e r  pollination  (5) 25 hours a f t e r  pollination  (6)  pollination  50 hours a f t e r hours a f t e r  pollination  f i x a t i o n , s t a i n i n g and  other features  permanent s l i d e p r e p a r a t i o n s were e x a c t l y  as f o r the  study o f megasporogenesis*  cut a t a t h i c k n e s s of 24 (b)  times:  pollination  (3) 11 hours a f t e r  The the  time of  6 hours a f t e r  (7) 122  be  treatments*  f i x a t i o n s were made a t the  (2)  could  the  of  same  A l l s e c t i o n s were  microns.  Observations  At the time of p o l l i n a t i o n w i t h p r a t e n s i s the mature female gametophyte of P*  confinis  s t r o n g l y developed a n t i p o d a l s t r u c t u r e n u c l e u s and  egg  apparatus.  p o l a r n u c l e i have not  I n F i g . 13,  pollen  shows the  and  a normal  fusion  0,  however,  the  y e t a c t u a l l y formed a s i n g l e  nucleus.  Two  somewhat pear-shaped s y n e r g i d c e l l s l i e r a t h e r below  the  l a r g e egg  cell.  S i x hours a f t e r p o l l i n a t i o n the g r a i n s can  be  seen g e r m i n a t i n g on the  pratensis  stigmas of  the  pollen  -87confinis florets. ful  In the 11- and l 6 - h o u r stages a c a r e -  search f o r p o l l e n tubes i n the s t y l a r and  t i s s u e s r e v e a l s s e v e r a l probable examples, but  nucellar their  presence i s very hard t o c o n f i r m . One  s l i d e a t the 16-hour stage shows the  ini-  t i a l d i v i s i o n of the endosperm nucleus t a k i n g p l a c e , while the egg c e l l i s u n d i v i d e d . l i n a t i o n an endosperm of about  At 25 hours a f t e r  e i g h t c e l l s can be  pol-  seen  i n one o v u l e , and i n another an endosperm of s i x c e l l s . In  both these the endosperm i s developing around  the  a n t i p o d a l mass, and the egg c e l l remains u n d i v i d e d .  A  t h i r d f l o r e t a t the 25-hour stage shows what i s almost c e r t a i n l y the t e r m i n a l end of a p o l l e n tube l y i n g to  the egg c e l l .  next  The a c t u a l stage of f e r t i l i z a t i o n i s  e i t h e r l a c k i n g or e l s e cannot be i n t e r p r e t e d a c c u r a t e l y i n any o f the s l i d e s examined. At 50 hours a f t e r p o l l i n a t i o n t h r e e o v u l e s appear to c o n t a i n t w o - c e l l e d embryos, a l t h o u g h the d i s i n t e g r a t i o n of the s y n e r g i d s and what are p o s s i b l y the r e mains of the p o l l e n tubes make i t d i f f i c u l t to i n t e r p r e t the s l i d e s a t t h i s s t a g e .  The  endosperm has about  twenty-  f o u r c e l l s i n one of these o v u l e s , and i s forming a p e r i p h e r a l l a y e r a g a i n s t the w a l l of the n u c e l l a r chamber, and  surrounding the a n t i p o d a l c e l l s , which are o n l y j u s t  -88showing s i g n s o f . d i s i n t e g r a t i o n .  The t w o - c e l l e d embryo  i n t h i s ovule i s b u r i e d i n -the endosperm a t the microp y l a r end of the n u c e l l a r  cavity*  At 122 hours a f t e r p o l l i n a t i o n , e i g h t - to s i x t e e n - c e l l e d embryos are present i n h a l f a dozen observable instances.  F i g . 13, E shows an approximately  eight-*celled embryo surrounded by endosperm t i s s u e .  The  dark bodies on e i t h e r s i d e of the b a s a l end o f the embryo are probably the two  disintegrated synergid c e l l s .  Fig.  13, F, from a n e i g h b o r i n g s e c t i o n through the same o v u l e , shows the p e r i p h e r a l endosperm.formation.  The a n t i p o d a l  c e l l s have been i n g e s t e d by the endosperm t i s s u e .  Sev-  e r a l telophase f i g u r e s a r e observable i n the endosperm i n F i g s . 13, E and F, and the endosperm s t i l l  shows f r e e  n u c l e a r development a t t h i s time. The h i s t o l o g i c a l study was  not c a r r i e d beyond  t h i s stage. 3*  Anther development i n p i s t i l l a t e f l o w e r s (a)  M a t e r i a l s and methods  Anther development i n the p i s t i l l a t e f l o w e r s of P. c o n f i n i s was  s t u d i e d c o n c u r r e n t l y with the study of  female gametophyte development, and no s l i d e s were p r e pared s p e c i f i c a l l y f o r the  purpose.  -89(b)  Observations  In the f l o r e t s of the young s p i k e l e t s there i s little  d i f f e r e n t i a t i o n of s t a m i n a l t i s s u e s a t the time  the p a n i c l e f i r s t  emerges from the  sheath.  In the upper-  most f l o r e t the f i l a m e n t i s b a r e l y e v i d e n t , and  the  epidermal l a y e r of c e l l s of the anther surrounds a homogeneous mass of c e l l s i n which even the  provascular  s t r a n d i s not d i f f e r e n t i a t e d . Successive in  of development can be  seen  the stamens from the uppermost to the lowermost f l o r e t s .  A provascular ened. in  stages  s t r a n d i s developed and  the f i l a m e n t  length-  An area of sporogenous t i s s u e becomes d i f f e r e n t i a t e d  each of the f o u r a n t h e r - l o b e s  which form.  In the  primary f l o r e t s of a l l but the youngest s p i k e l e t s ( i . e . those on the lower branches of the p a n i c l e ) the a n t h e r s show a s i n g l e epidermal l a y e r , a t a p e t a l l a y e r the  sporogenous c e l l s of each a n t h e r - l o b e ,  surrounding  and a c e n t r a l  v a s c u l a r strand surrounded by parenchyma t i s s u e ( F i g . 14, A).  The  little  t h i n - w a l l e d a r c h e s p o r i a l c e l l s a t t h i s stage are  d i f f e r e n t from the surrounding  plasm i s t h i n and and  cells.  Their  cyto-  l i g h t l y - s t a i n i n g ; t h e i r n u c l e i are  c h a r a c t e r i s t i c a l l y c o n t a i n one  These c e l l s enlarge  to t h r e e  small  nucleoli.  somewhat as development proceeds but  otherwise undergo o n l y s l i g h t change i n appearance u n t i l about the two-  or f o u r - c e l l e d stage of the embryo sac i n  -90-  F i g u r e 14:  Anther development i n p i s t i l l a t e Poa c o n f i n i s  plants of  A.  Transverse s e c t i o n o f anther i n primary f l o r e t a t time o f emergence o f p a n i c l e ; sporogenous c e l l s developing i n f o u r lobes o f t h e a n t h e r .  B.  L a t e r stage showing v a c u o l a t i o n and r e t a r d a t i o n of cytoplasmic membranes from c e l l w a l l s i n t h e p o t e n t i a l PMCs.  C.  D i s i n t e g r a t i o n o f the p o t e n t i a l PMCs and the surrounding t a p e t a l l a y e r .  D.  Complete d i s i n t e g r a t i o n o f a l l the i n n e r a t t h e time o f f l o w e r i n g .  tissue  ( a l l x 520)  -91the accompanying p i s t i l .  At t h i s time t h e a r c h e s p o r i a l  c e l l s begin t o take the hematoxylin  s t a i n more h e a v i l y .  They become v a c u o l a t e d , the n u c l e i e n l a r g e , and the cytoplasmic membrane s t a r t s t o p u l l away from the c e l l w a l l ( F i g . 14, B ) . Complete d i s i n t e g r a t i o n o f a l l the p o t e n t i a l PMCs f o l l o w s r a p i d l y , accompanied by the d i s i n t e g r a t i o n of  the t a p e t a l l a y e r , which shows p r a c t i c a l l y no d i f f e r -  e n t i a t i o n throughout 14, C ) . lar  the development o f the anther ( F i g .  By the time a n t h e s i s o c c u r s , most of the c e l l u -  contents o f both sporogenous and t a p e t a l r e g i o n s have  been d i g e s t e d by the surrounding epidermal t i s s u e ( F i g . 14, E ) . The coalescence o f the sporangia i n t o two spore chambers or p o l l e n sacs i s not completed, angia remaining In  the f o u r spor-  separated, as shown i n F i g s . 14, C and D.  no i n s t a n c e were any but m i t o t i c  figures  seen i n the a n t h e r s , and even these r a t h e r i n f r e q u e n t l y . The t i m i n g o f the f o r e g o i n g sequence o f events i n r e l a t i o n to female  gametophyte development seems t o  be somewhat v a r i a b l e .  Complete d i s i n t e g r a t i o n o f the  anther contents has been seen o c c a s i o n a l l y as e a r l y as the "umbrella" stage i n the accompanying p i s t i l , and sometimes not u n t i l a f t e r a n t h e s i s .  D i s i n t e g r a t i o n ap-  peared t o take p l a c e somewhat e a r l i e r i n the p i s t i l l a t e p l a n t s which flowered i n the greenhouse than i n those which flowered i n the U n i v e r s i t y farm  plot.  -924.  M i c r o s p o r o g e n e s i s i n staminate f l o w e r s (a)  M a t e r i a l s and methods  M i c r o s p o r o g e n e s i s was s t u d i e d i n the staminate P. c o n f i n i s p l a n t s which were c o l l e c t e d a t P o i n t Roberts on A p r i l 17th and A p r i l 23, 1955, and grown i n beach i n the greenhouse.  sand  A t t h e time o f c o l l e c t i o n the p a n i -  c l e s were a l r e a d y showing, and some f i x a t i o n s were made at  the s i t e a t P o i n t Roberts on t h e second date o f c o l -  lection.  However, most o f the stages o f development  were  found i n the f i x a t i o n s t h a t were made a f t e r the p l a n t s were t r a n s f e r r e d t o the greenhouse. Permanent s l i d e s were made i n the same way as for  the study o f female gametophyte development. A l l  s e c t i o n s were cut a t t h i c k n e s s e s o f 8 t o 14 microns. A d d i t i o n a l examination o f the e a r l i e r stages o f development  was made through t h e p r e p a r a t i o n o f a c e t o -  carmine anther squashes.  Whole s p i k e l e t s were p l a c e d i n  Carnoy"s F l u i d No. 1* f o r 1^ t o 2 hours and then washed first  i n 70 per cent a l c o h o l and then i n d i s t i l l e d  water.  The anthers were d i s s e c t e d out o f a l l t h e f l o r e t s i n a s p i k e l e t and p l a c e d i n a drop of acetocarmine on a s l i d e for  about one minute.  The a n t h e r s were then  squashed  under a cover s l i p and t h e excess s t a i n removed w i t h paper * Three p a r t s a b s o l u t e a l c o h o l : acetic acid.  one p a r t  glacial  -93-  towelling.  After the s l i d e was warmed b r i e f l y over a  flame the edges of the cover s l i p were sealed with n a i l polish. (b)  Observations  Anthers showing almost a l l stages of a normal microsporogenesis can be seen i n the permanent and temporary s l i d e s . PMCs i n synizesis f i l l the anther sacs i n the e a r l i e s t stages found ( F i g . 15, A). nucleolus i s seen i n each c e l l .  A single, large  At t h i s time the sur-  rounding c e l l s of the anther are already well compressed by the growth of the archesporial c e l l s , and i t i s d i f f i c u l t to see c l e a r l y the number of outer c e l l l a y e r s . However, a single layer of darkly-staining tapetal c e l l s i s always present, l i n i n g the sporangial cavity and surrounding the PMCs.  One or two layers of compressed c e l l s  appear to l i e between the tapetum and the epidermis, but the number of layers i s usually not d i s t i n c t . Several zygotene stages can be seen i n the PMCs i n anthers treated by the acetocarmine squash technique ( F i g . 15, B).  PMCs at diakinesis show only bivalents,  as f a r as can be seen, although no accurate counts are obtainable.  In the c e l l at the upper right i n F i g . 15, C  the bivalents are separating at anaphase I.  In t h i s and  -94-  F i g u r e 15:  A.  Microsporogenesis Poa c o n f i n i s  i n staminate p l a n t s o f  S y n i z e s i s i n PMCs. (x 1610)  B.  Zygotene i n PMC. (x 3220)  C.  Metaphase I i n PMC a t l e f t ; anaphase I i n PMC a t upper r i g h t . (x 3220)  -95other c e l l s a t anaphase and t e l o p h a s e ( F i g * 16, m e i o t i c i r r e g u l a r i t i e s can be d i s c e r n e d .  A ) , no  S e v e r a l chromo-  some counts i n PMCs a t f i r s t d i v i s i o n metaphase and anaphase show numbers approximating 2n are  •  40.  No exact  counts  Dyad f o r m a t i o n i s f o l l o w e d r a p i d l y by the  second  p o s s i b l e i n the c e l l s examined.  d i v i s i o n and the f o r m a t i o n of the spore t e t r a d s ( F i g . 16, B).  By t h i s  pletely  stage the t a p e t a l l a y e r has been almost com-  d i g e s t e d by the a r c h e s p o r i a l c e l l s , and  a l i n e of d i s i n t e g r a t e d c e l l s .  In F i g . 16,  shows as  C several  microspores can be seen l y i n g a g a i n s t the anther w a l l , a day b e f o r e a n t h e s i s .  The l a y e r o f dark t i s s u e  directly  beneath them i s the tapeturn. The percentage of good p o l l e n produced  i n the  staminate c o n f i n i s p l a n t s i s e v i d e n t l y h i g h , judging from the s t a i n i n g p r o p e r t i e s of the p o l l e n g r a i n s and the percentage of shrunken 5.  low  grains.  Ovule development i n staminate f l o w e r s (a)  M a t e r i a l s and methods  Ovule development i n staminate f l o w e r s was  ex-  amined i n the permanent s l i d e s prepared f o r the study of microsporogenesis; as i n the study of anther development i n the p i s t i l l a t e p l a n t s , no s l i d e s were prepared c i a l l y for this  purpose.  espe-  -96-  r,  F i g u r e 16:  A.  M i c r o s p o r o g e n e s i s i n staminate p l a n t s o f Poa c o n f i n i s  Telophase I i n PMCs. (x  B.  1610)  Spore t e t r a d s . (x 1300')  C.  M i c r o s p o r e s , one day b e f o r e a n t h e s i s ; note the d i g e s t e d t a p e t a l l a y e r j u s t below the microspores* (x  1610)  -97(b) Due  Observations  to the r e l a t i v e l y s m a l l number of s l i d e s p r e -  pared, o n l y a few  stages of development can be seen,  A  number of EMCs, t y p i c a l "umbrella" stages, and s e v e r a l e a r l y embryo sacs are seen i n some ovules i n the male florets.  One  anomaly observed  i s the presence  EMCs, both i n s y n i z e s i s , i n one o v u l e . female  of  two  Development on the  s i d e i s w e l l behind t h a t i n the a n t h e r s , and i n  s e v e r a l f l o r e t s i n which the anthers are almost embryo sacs are o n l y t w o - c e l l e d .  No  embryo sacs can  seen which are beyond the t w o - c e l l e d stage. the developmental g r e s s e s , appears  r i p e the be  Although  p i c t u r e i n the o v u l e s , as f a r as i t p r o e s s e n t i a l l y l i k e t h a t i n the  p l a n t s , i n a l l but the very young p i s t i l s  pistillate  the s t y l e s  and  stigmas are v e r y much s h r i v e l l e d . C.  H y b r i d i z a t i o n techniques and attendant  macroscopic  observations 1.  Techniques  used t o secure c r o s s e s  P l a n t s of P. c o n f i n i s . P. p r a t e n s i s . P. compressa , P. macrantha and P. annua were brought  i n from  the  f i e l d a t v a r i o u s dates d u r i n g February, March and A p r i l o f 1955.  Except f o r the P. annua, which was  brought  i n as a  s l a b of sod i n a wooden f l a t , a l l p l a n t s were p o t t e d i n  -98-  beach sand and put under 14-hour day l e n g t h c o n d i t i o n s w i t h the a i d of a r t i f i c i a l l i g h t . The s o u r c e s o f t h e s e f i v e s p e c i e s were as follows: (1)  Poa c o n f i n i s : the  P i s t i l l a t e p l a n t s were o b t a i n e d from  o r i g i n a l W h i f f i n S p i t b i o t y p e on t h e U n i v e r s i t y  farm, and a l s o from t h e P o i n t R o b e r t s m a t e r i a l c o l l e c t e d on A p r i l 1 7 t h and A p r i l 23rd.  A l l t h e stam-  i n a t e p l a n t s used were brought from P o i n t R o b e r t s . (2)  Poa.pratensis:  The p l a n t s used b e l o n g e d t o a  strain  o b t a i n e d from an abandoned g r a s s n u r s e r y s i t e on the  U n i v e r s i t y farm.  The o r i g i n o f t h i s s t r a i n i s  unknown. (3)  Poa compressa:  These p l a n t s were a l s o from a  strain  of unknown d e r i v a t i o n , o b t a i n e d from the same abandoned n u r s e r y as t h e P. p r a t e n s i s p l a n t s . (4)  Poa macrantha:  These p l a n t s were from a .number c o l -  l e c t e d on the sand dunes a t Twin Harbours S t a t e P a r k , Washington, i n November 1954.  They had been  i n the U n i v e r s i t y p l o t s u n t i l t h e y were brought i n t o t h e greenhouse i n March. i n proved t o be (5)  Poa annua:  A l l p l a n t s brought  pistillate.  A sod composed c h i e f l y o f P. annua was  dug up from a waste s i t e c l o s e t o t h e U n i v e r s i t y greenhouses.  -99The f i r s t  s e r i e s o f p o l l i n a t i o n s were made from  A p r i l 1 3 t h t o 1 5 t h between p i s t i l l a t e P. c o n f i n i s ( W h i f f i n S p i t ) and P. p r a t e n s i s g e t h e r on March 1 5 t h .  plants  plants  brought i n t o -  A t o t a l o f seven P. c o n f i n i s i n -  f l o r e s c e n c e s were p o l l i n a t e d .  Two o f t h e p l a n t s used i n  these c r o s s i n g s a r e seen i n F i g . 1 7 .  F i g u r e 1 7 : P l a n t s used i n t h e a t t e m p t e d c r o s s P. c o n f i n i s x P. p r a t e n s i s . Note t h e sparse flowering of the p i s t i l l a t e c o n f i n i s parent. A second s e r i e s of p o l l i n a t i o n s were made from May 1 s t t o 1 1 t h , i n v o l v i n g a l l f i v e Poa s p e c i e s .  In this  s e r i e s a l l t h e P. c o n f i n i s used was from t h e m a t e r i a l lected at Point Roberts.  col-  -1001  The f o l l o w i n g  c r o s s i n g s were attempted*.  (1) P. c o n f i n i s x P. p r a t e n s i s  (16)  (2) P. c o n f i n i s x P. annua"  (3 )  (3) P. c o n f i n i s x P. compressa  (8 )  (4) P. macrantha x P. p r a t e n s i s  (1 )  (5) P. macrantha x P. compressa  (2 )  (6) P. macrantha x P. c o n f i n i s  (2 )  (7) P. macrantha x P. annua  (1 )  P o l l i n a t i o n o f two P. c o n f i n i s  inflorescences  w i t h c o n f i n i s p o l l e n was a l s o  c a r r i e d out.  Two  panicles  each o f P. c o n f i n i s and P. macrantha were kept bagged w i t h out  any p o l l i n a t i o n .  A s t a r t was made t o emasculate P.  compressa i n f l o r e s c e n c e s  by the hot water treatment d e v e l -  oped by Stephens and Quinby (99), but t h e r e proved t o be v e r y few staminate c o n f i n i s p l a n t s  available f o r recipro-  c a l c r o s s e s and t h e emasculations were not c a r r i e d on. There was j u s t enough ations  c o n f i n i s p o l l e n f o r the four  pollin-  which were made. The P. c o n f i n i s p l a n t s  used as male and female  p a r e n t s and the P. macranths p l a n t s were bagged w i t h g l a s s i n e anthesis.  bags one to s e v e r a l  P. annua i n f l o r e s c e n c e s  to t h e i r shedding p o l l e n .  used as female parents days b e f o r e  were a l s o bagged  The p r a t e n s i s  prior  and compressa  * Numbers i n b r a c k e t s r e f e r t o number o f i n f l o r e s cences p o l l i n a t e d .  -101p a n i c l e s were not bagged, but the two s p e c i e s were kept i n separate s e c t i o n s of the greenhouse d u r i n g t h a t t h e i r f l o w e r i n g times  the few days  coincided.  Most p o l l i n a t i o n s were c a r r i e d out between 7:00 and  8:00 a.m., as i t was found t h a t t h e P. p r a t e n s i s and  P. compressa p l a n t s , p a r t i c u l a r l y , shed t h e i r p o l l e n e a r l y , and t h e r e was o f t e n very l i t t l e p o l l e n l e f t  very  later  i n the day a f t e r the greenhouse v e n t i l a t o r s had been opened up.  The s t i l l a i r o f the e a r l y morning hours a l s o  reduced the amount o f f o r e i g n p o l l e n that might have been f l o a t i n g around. P o l l e n was c o l l e c t e d from t h e p l a n t s o f the species being used as t h e male parent by tapping p a n i c l e s over a c l e a n g l a s s microscope s l i d e .  their  The g l a s -  sine bags were then removed from the p a n i c l e s o f the female parent or p a r e n t s and the p o l l e n on t h e s l i d e dusted l i g h t l y over the stigmas with t h e a i d o f a f i n e camel-hair brush.  The g l a s s i n e bags were then  replaced.  P o l l i n a t i o n s were u s u a l l y made on a t l e a s t t h r e e  consec-  u t i v e mornings, and o c c a s i o n a l l y f i v e or s i x , as the f l o w e r i n g o f the c o n f i n i s and macrantha p l a n t s p r o g r e s s e s downward from the top s p i k e l e t s o f the p a n i c l e s and o f t e n takes s e v e r a l days t o be completed.  The g l a s s i n e bags were  removed three or f o u r days a f t e r the l a s t p o l l i n a t i o n s had been made.  -1022.  Observations f o l l o w i n g  pollinations  Seed began to form a f t e r a l l o f the i n t e r s p e c i f i c c r o s s e s except the two  involving  attempted P. annua;  however, none of the seed from these c r o s s e s reached m a t u r i t y without s h r i v e l l i n g ,  A s i n g l e , s h r i v e l l e d seed  found on the P. macrantha i n f l o r e s c e n c e annua p o l l e n may  have been the r e s u l t of a c c i d e n t a l  l i n a t i o n by one of the other Poa Healthy seed formed florescences formed  p o l l i n a t e d with  species.  on the two P. c o n f i n i s i n -  p o l l i n a t e d with c o n f i n i s p o l l e n .  No  on e i t h e r the P. c o n f i n i s or P. macrantha  inflorescences  pol-  which were bagged and l e f t  seed pistillate  unpollinated.  The seed which formed a f t e r the i n t e r s p e c i f i c c r o s s i n g s appeared  to f i l l  out and develop q u i t e  u n t i l about t h r e e weeks a f t e r p o l l i n a t i o n . from the c r o s s e s c o n f i n i s x p r a t e n s i s  seeds  and c o n f i n i s x com-  pressa were compared under the b i n o c u l a r a m a g n i f i c a t i o n of x 40, w i t h s e v e r a l  A few  normally  microscope, a t  seeds from the con-  f i n i s x c o n f i n i s p o l l i n a t i o n two weeks a f t e r the p o l l i n a t i o n s were made:  no v i s i b l e d i f f e r e n c e s  c o u l d be d e t e c t e d  between these seeds. The number of seeds per s p i k e l e t which began to form a f t e r the i n t e r s p e c i f i c c r o s s e s , i n which P. was  the female p a r e n t , was  confinis  o n l y s l i g h t l y l e s s than the  number found on P. c o n f i n i s p l a n t s  c o l l e c t e d i n nature.  - 1 0 3 -  In nature, two  seeds per s p i k e l e t  seem to form r e g u l a r l y ;  a f t e r the crosses i n the greenhouse  the average  s l i g h t l y over l j seeds per s p i k e l e t . s p i k e l e t s c o n t a i n i n g the s h r i v e l l e d  was  D i s s e c t i o n of the seed r e v e a l e d t h a t  almost a l l the primary f l o r e t s bore seeds, but o n l y about 50  per cent of the secondary f l o r e t s c o n t a i n e d a seed.  T h i s accounts f o r the d i f f e r e n c e i n the percentage o f seed developing i n nature and the percentage d e v e l o p i n g a f t e r the c r o s s e s .  There d i d not appear to be any  difference  between the percentage o f seed forming a f t e r the c r o s s c o n f i n i s x p r a t e n s i s and the c r o s s c o n f i n i s x In the c r o s s e s where P. macrantha about two  was  compressa.  the female parent  seeds per s p i k e l e t formed, a l t h o u g h not enough  i n f l o r e s c e n c e s were p o l l i n a t e d t o make v e r y a c c u r a t e estimates.  Comparable f i g u r e s from nature were not a v a i l -  a b l e f o r P.  macrantha.  All  seeds which were not used f o r embryo  ex-  c i s i o n (below) were p l a c e d on moist f i l t e r paper i n P e t r i d i s h e s , but not a s i n g l e seed germinated out of the seve r a l hundred o b t a i n e d from the v a r i o u s i n t e r s p e c i f i c crosses.  The s h r i v e l l e d seeds absorbed moisture and looked  plump and h e a l t h y a f t e r about t h r e e days i n the P e t r i d i s h e s , but c l o s e r examination r e v e a l e d t h a t the maternal t i s s u e s of the seed formed a hollow s h e l l , devoid o f normal endosperm t i s s u e .  A p p a r e n t l y , most o f the seeds  -104had a l r e a d y been i n f e c t e d w i t h mould organisms a t the time 0  they were p l a c e d i n the P e t r i d i s h e s . not  Very few seeds were  mouldy a f t e r a week. The seeds from the c r o s s macrantha x c o n f i n i s  were somewhat plumper  than those from any o f the o t h e r  crosses, although s t i l l being s l i g h t l y s h r i v e l l e d . any of the seeds from the o t h e r i n t e r s p e c i f i c number of those from the macrantha  Unlike  crosses, a  x c o n f i n i s cross  still  contained f a i r l y l a r g e amounts o f endosperm when p l a c e d i n the P e t r i p l a t e .  However, none o f these seeds germin-  ated. At  the time o f w r i t i n g (two weeks a f t e r the  germination t e s t s were s t a r t e d ) , none of the seeds from the  c o n f i n i s x c o n f i n i s p o l l i n a t i o n s have germinated,  although most of the seeds have not y e t been touched by mould  growths. 3,  Embryo c u l t u r e (a)  M a t e r i a l s and methods  A f t e r the seed from the f i r s t  group o f c r o s s e s  s h r i v e l l e d i t was d e c i d e d t o t r y some embryo e x c i s i o n bef o r e the seed from the l a t e r c r o s s e s a l s o began to s h r i v e l . As f a r as p o s s i b l e , o n l y seed t h a t had not begun to  s h r i v e l was used.  The seeds were f i r s t  soaked i n .1  -105-  per cent mercury b i c h l o r i d e and then r i n s e d seed was  s o l u t i o n f o r twenty minutes  i n d i s t i l l e d water f o r f i v e minutes.  p l a c e d immediately  on the flamed g l a s s p l a t e o f  the stage of a R e i c h e r t b i n o c u l a r d i s s e c t i o n A r e t o r t r i n g at a height of two of the microscope  microscope.  inches above the  enabled the hands to be kept  d u r i n g the e x c i s i o n procedure.* w i t h the a i d of two  The  stage  steady  embryos were e x c i s e d  v e r y s h a r p - p o i n t e d , flamed  under a m a g n i f i c a t i o n of x 4 0 . out with as l i t t l e  Each  Each embryo was  scalpels dissected  a s s o c i a t e d t i s s u e as p o s s i b l e a d h e r i n g  to i t . The Randolph and  embryos were c u l t u r e d on media used Cox  ( 8 8 ) f o r growing I r i s embryos.  by Sodium  hexametaphosphate (Calgon) i s the source o f phosphorus in this 0 . 7  per cent agar medium c o n t a i n i n g 2 per cent  cane sugar, t o g e t h e r with o t h e r m i n e r a l s a l t s . prepared  i n 1 2 5 c c . Erlenmeyer  f l a s k s , and  were p l a c e d t o g e t h e r i n each f l a s k .  The  This  s e v e r a l embryos  constant tempera-  t u r e room i n which the embryo c u l t u r e s were p l a c e d was at 2 5 °  was  kept  C.  * T h i s idea was suggested to the w r i t e r by E n g e l b e r t ( 3 7 ) , w h o used a r e t o r t r i n g f o r s t e a d y i n g her hands w h i l e emasculating Poa f l o r e t s . The i n f l o r e s c e n c e o f the p l a n t being emasculated was put through the r e t o r t r i n g .  -106(b)  O b s e r v a t i o n s and r e s u l t s  Embryos were s u c c e s s f u l l y e x c i s e d from seeds which formed a f t e r the f o l l o w i n g  crosses: No. Embryos E x c i s e d  P. c o n f i n i s x P. p r a t e n s i s  (5)  P. c o n f i n i s x P. compressa  (10)  x P. compressa  (7)  P. macrantha x P. c o n f i n i s  (7)  P. macrantha  In some o f the seeds a t the time o f e x c i s i o n the beginning  o f endosperm f a i l u r e could be seen, as e v i d -  enced by a brown c o l o u r o f the outer  endosperm t i s s u e and  the r e t r a c t i o n o f the endosperm from the i n n e r w a l l o f the seed  coat. Some o f the embryos were undoubtedly e x c i s e d a  few days too l a t e and were probably with fungus spores.  a l r e a d y contaminated  There was v e r y l i t t l e  differentiation  of embryonic t i s s u e s n o t i c e a b l e i n any o f the seeds which were d i s s e c t e d , except one or two of those from the c r o s s macrantha x c o n f i n i s . At the time o f w r i t i n g ( e i g h t e e n days a f t e r the e x c i s i o n s were made and the culture.s s t a r t e d ) s i x o f the embryos from the macrantha and growing w e l l .  x c o n f i n i s cross a r e s t i l l  alive  There a r e s i g n s o f r o o t growth and t i s -  sue d i f f e r e n t i a t i o n i n some o f them and a l l have  enlarged  -107considerably.  Only three other embryos have not been  contaminated by b a c t e r i a l or fungus growths. are  from the c r o s s c o n f i n i s  the  c r o s s macrantha x compressa.  Two  of these  x compressa and the t h i r d from These t h r e e embryos ap-  pear t o be s t i l l a l i v e , but as y e t t h e r e i s l i t t l e or no s i g n o f growth i n them.  -108VII  DISCUSSION On the b a s i s of both the herbarium and  s t u d i e s i t seems evident tinct  t h a t P.  field  confinis i s a rather  species, a conclusion already  dis-  (68).  drawn by Marsh  Although i t grows i n many l o c a t i o n s i n c l o s e a s s o c i a t i o n w i t h P. p r a t e n s i s , P, a b l y other Poa  compressa, P. macrantha and  species, there  prob-  i s no obvious i n d i c a t i o n  t h a t h y b r i d s are formed between these s p e c i e s and finis  i n nature.  However, i t should  d i f f e r e n t f l o w e r i n g times of P.  the other bluegrasses for  con-  be emphasized t h a t  t h i s c o n c l u s i o n i s based on somewhat l i m i t e d The  P.  observations.  c o n f i n i s and  probably reduces the  some of  opportunity  i n t e r s p e c i f i c c r o s s i n g to o c c u r j furthermore, as  sug-  gested by the r e s u l t s from the h y b r i d i z a t i o n attempts, any  seed t h a t does form a f t e r i n t e r s p e c i f i c  v o l v i n g P.  c o n f i n i s i s probably i n v i a b l e . The  out  crossing i n -  extreme u n i f o r m i t y  of dune bluegrass  i t s range of upward of 600 m i l e s along  the  Pacific  Coast i s very n o t i c e a b l e ; as p r e v i o u s l y noted, any a b i l i t y that does e x i s t i n a few  characters  duces by  The  f a c t t h a t P.  vari-  seems to bear  no r e l a t i o n s h i p to the geographic d i s t r i b u t i o n of species.  through-  the  c o n f i n i s probably r a r e l y r e p r o -  seed undoubtedly c o n t r i b u t e s to the l a c k of  a t i o n ; many of the b i o t y p e s  have p o s s i b l y e x i s t e d f o r  vari-  -109-  hundreds of y e a r s , m a i n t a i n i n g themselves e n t i r e l y means of the rhizomatous r o o t system.  Evidence  i s p a r t i c u l a r l y s t r i k i n g a t P o i n t Roberts,  by  of t h i s  where the  f a i r l y e x t e n s i v e , i n d i v i d u a l c l a n s of the grass on  the  sand banks a r e composed of e i t h e r a l l p i s t i l l a t e or a l l staminate  plants.  Quite l i k e l y a l l the p l a n t s w i t h i n each  group are members of a s i n g l e Both staminate and  clone. p i s t i l l a t e c l o n e s seem t o  e x i s t a t most l o c a l i t i e s , although t h i s f e a t u r e has been checked c l o s e l y .  not  At any r a t e , the s i t u a t i o n i n  P. c o n f i n i s c e r t a i n l y does not p a r a l l e l t h a t a l r e a d y ment i o n e d (page 47) i n P. nervosa,  which has only p l a n t s w i t h  a b o r t i v e anthers over most of i t s range. The problems concerned with dioecism i n p l a n t s —its  sporadic occurrence  throughout the p l a n t kingdom, i t s  p o s s i b l e advantages, and  other f e a t u r e s — a r e of c o n s i d e r -  able i n t e r e s t .  the p r e s e n t  Although  study has not c o n t r i b -  uted much to the g e n e r a l knowledge of dioecism, the mere f a c t t h a t P. c o n f i n i s i s a d i o e c i o u s s p e c i e s i s of some special  interest. E x a c t l y what b e n e f i t s dioecism c o n f e r s upon dune  b l u e g r a s s , growing as i t does i n such a r e s t r i c t e d h a b i t a t , i s hard to imagine. Poa  I t i s i n t e r e s t i n g t h a t the two  other  s p e c i e s which are c o n f i n e d s o l e l y to the sandy areas  -110of the P a c i f i c Coast o f North America, P. D o u g l a s i i and P. macrantha, are a l s o d i o e c i o u s . to suggest  The w r i t e r has  beyond Darwin's ( 3 4 ) i d e a t h a t under  c o n d i t i o n s the s e p a r a t i o n of the sexes may  little  severe  avoid  the  s t r a i n on the r e p r o d u c t i v e powers of a p l a n t which velops both male and  female organs s i m u l t a n e o u s l y .  f u r t h e r p o s s i b i l i t y i s t h a t the s e p a r a t i o n of the may  de-  a l l o w the accumulation  A sexes  of g r e a t e r amounts of  hidden  v a r i a b i l i t y w i t h i n the s p e c i e s than would be p o s s i b l e i f the p l a n t s were h e r m a p h r o d i t i c .  Presumably, i n herma-  p h r o d i t i c p l a n t s , mutations a f f e c t , and are t r a n s m i t t e d by both male and female elements a l i k e .  In a d i o e c i o u s  s p e c i e s , on the other hand, d i f f e r e n t mutations w i l l cumulate independently two  sexes.  In t h i s way  ac-  of each other i n the p l a n t s of the a g r e a t e r amount of hidden  vari-  a b i l i t y i s available for release following crossing. a s p e c i e s such as dune b l u e g r a s s , which probably reproduces  by seed,  In  rarely  " f l e x i b i l i t y " ' i s s a c r i f i c e d somewhat  f o r "immediate f i t n e s s " ( 9 7 ) .  But d i o e c i s m may  provide a  means of m a i n t a i n i n g g r e a t e r f l e x i b i l i t y or c a p a c i t y f o r v a r i a t i o n through the independent accumulation  of mutations  i n male arid i n female p l a n t s . The a d a p t a t i o n of P. c o n f i n i s to sandy areas a p a r t i c u l a r type  i s i n i t s e l f rather interesting,  t h e r e i s much room f o r c o n j e c t u r e about the p l a n t ' s  of  and ecology.  -IllSuch f a c t o r s as s o i l a e r a t i o n and  p l a n t competition  have much to do w i t h the a p p a r e n t l y t a t i o n of dune b l u e g r a s s . it  has  may-  narrow range of adap-  I t i s rather s u r p r i s i n g that  grown so w e l l on a s o i l w i t h f i n e aggregates a t  U n i v e r s i t y farm, where i t shows' i t s e l f ing  r a p i d l y and  ous  l e a f growth and  capable of  forming a t h i c k sod, m a i n t a i n i n g flowering profusely.  On the  the  spreada vigorother  hand, the p l a n t s growing i n s o i l i n the greenhouse d i d not do as w e l l as those i n sand. The  sand dunes and  t h r i v e s are o b v i o u s l y growth as one finis  beach f l a t s where P. c o n f i n i s  not as bare and  unsuitable  might i n i t i a l l y suppose, although P.  c e r t a i n l y e x i s t s under more s t r i n g e n t  than most p l a n t s p e c i e s .  A c e r t a i n amount of  from what i s blown by the wind.  pointed  out, too,  "...  despite t h e i r proximity rapid.  organic driftwood  S a l i s b u r y (91)  dune s o i l s are normally not to the  heavy l e a c h i n g must be  more, the m o i s t u r e - h o l d i n g s o i l must be The  has  saline  sea." because l e a c h i n g i s  Since most of the P a c i f i c Coast r e c e i v e s a  heavy r a i n f a l l ,  i n P.  As  con-  conditions  matter i s always a v a i l a b l e from the decay of and  for plant  expected.  c a p a c i t y of the  fairly Further-  sand or sandy  low. experiments on the i n d u c t i o n of  flowering  c o n f i n i s mostly p o i n t out the need f o r f u r t h e r study  -112on t h i s aspect of the problem.  The f a c t t h a t P.  confinis  p l a n t s brought i n t o the greenhouse from the f i e l d i n December d i d not f l o w e r , whereas those brought i n . d u r i n g  Feb-  r u a r y (and l a t e r ) d i d f l o w e r , suggests the need f o r a d e f i n i t e p e r i o d of dormancy and/or low temperature b e f o r e f l o w e r i n g can be i n i t i a t e d .  F u r t h e r experiments u s i n g  c o l d treatments might enable f l o w e r i n g to be  initiated  even e a r l i e r i n the w i n t e r or perhaps a t o t h e r times o f the y e a r .  The l a c k o f c o n t r o l over the temperatures i n  the greenhouse makes f l o w e r i n g experiments somewhat d i f ficult.  The l i g h t c o n d i t i o n s i n the greenhouses a t The  U n i v e r s i t y of B r i t i s h Columbia are u s u a l l y poor d u r i n g the w i n t e r , and t r o u b l e i s a p p a r e n t l y o f t e n encountered i n i n d u c i n g p l a n t s to flower i n the greenhouses at t h i s time.  F l o w e r i n g i s f r e q u e n t l y sparse i f i t does occur a t  all. Almost a l l the c y t o l o g i c a l d e t a i l s of both male and female gametophyte  development  seem t o i n d i c a t e t h a t  P. c o n f i n i s reproduces s e x u a l l y , and a l l the s l i d e s amined  have been i n t e r p r e t e d i n the l i g h t o f t h i s  ex-  final  conclusion. A c a r e f u l study of a l l stages of female gametophyte development  has f a i l e d to r e v e a l any  irregularities  t h a t might l e a d to a p o m i c t i c embryo sac and seed f o r m a t i o n . I t should be emphasized that' the r e g u l a r behaviour o f the  -113EMC  at m e i o s i s i s not i n i t s e l f  p r o d u c t i o n , although  evidence  of sexual r e -  i t does p r e c l u d e the p o s s i b i l i t y of  any form of d i p l o s p o r y o c c u r r i n g . sporous b i o t y p e s of other Poa q u i t e normal (44, 68, 84,  However, i n some apo-  s p e c i e s the EMC  100).  The  t e g r a t i o n of the m e i o t i c products  meiosis i s  subsequent  disin-  i n such p l a n t s , accom-  panied by the development of the aposporous embryo sac or sacs, i s t h e r e f o r e a p p a r e n t l y not r e l a t e d t o the r e g u l a r ity  or i r r e g u l a r i t y of the EMC  meiosis.  On t h i s b a s i s i t  i s p o s s i b l e to assume t h a t P. c o n f i n i s might be aposporous --the embryo sacs developing from somatic being d i p l o i d .  c e l l s and  I t becomes v e r y important,  thus  therefore>  e s t a b l i s h the o r i g i n o f the embryo sac i n i t i a l  cell  to  that  can be seen below the d i s i n t e g r a t i n g megaspores i n the c h a r a c t e r i s t i c "umbrella"  stages.  U n f o r t u n a t e l y , no  chromosome counts have been o b t a i n a b l e i n any cell,  initial  or i n any l a t e r embryo sac stages; however, t h e r e  are other i n d i c a t i o n s t h a t the embryo sac i n i t i a l ,  or  o n e - c e l l e d embryo sac, a c t u a l l y i s a h a p l o i d megaspore d e r i v e d from the normal m e i o s i s . The  f a c t t h a t the i n i t i a l  cell  i s always found  d i r e c t l y i n l i n e with the d i s i n t e g r a t i n g c e l l s of the t r i a d or t e t r a d , i n the l o n g i t u d i n a l a x i s of the o v u l e , i s not suggestive of aposporous development as i t occurs in  other t y p i c a l aposporous apomicts.  In most aposporous  -114Poa  s p e c i e s , f o r i n s t a n c e , the somatic  not u s u a l l y have such a d e f i n i t e and o r i g i n , and  initial  cells  constant p o i n t of  commonly a r i s e a t v a r i o u s p l a c e s i n the  c e l l u s or chalaza  (1,  5,  63,  84,  100).  A second  t i o n which l e s s e n s the l i k e l i h o o d of aposporous  i n an o v u l e , and  s i n g l e embryo sac (with two mentioned).  develop-  e x c e p t i o n s , as p r e v i o u s l y  T h i s , a g a i n , i s u n l i k e the commonly encount-  c e l l s o f t e n develop  sev-  simultaneously and  pete with each o t h e r , or where a s e x u a l embryo sac one  embryo  i n l a t e r stages of only a  ered s i t u a t i o n i n other aposporous apomicts, where e r a l somatic  nu-  observa-  ment i s the appearance i n e a r l y stages of only one sac i n i t i a l  do  comand  or more aposporous embryo sacs compete with each o t h e r .  As Akerberg (1)  first  should consequently polyembryony and  proposed f o r P. p r a t e n s i s . t h e r e  be a p o s i t i v e a s s o c i a t i o n between  apospory.  On the other hand, t h e r e i s no  a p r i o r i reason f o r assuming t h a t the development of a s i n g l e aposporous c e l l ,  always at the b a s a l end of the  l i n e of megaspores, i s not t h e o r e t i c a l l y p o s s i b l e i n P.  confinis. A f u r t h e r probable  i n d i c a t i o n o f sexual embryo  sac development i s t h a t four d i s i n t e g r a t i n g megaspores have never been seen i n any o v u l e .  Admittedly,  t h e r e are  a great many s l i d e s i n which the number of d i s i n t e g r a t i n g megaspores i s d i f f i c u l t  or i m p o s s i b l e to count, but some  -115s l i d e s d e f i n i t e l y showing can be seen.  o n l y two or t h r e e such  cells  T h i s suggests that one of the megaspores,  r a t h e r than a somatic c e l l , always f u n c t i o n s as the embryo sac i n i t i a l .  Where o n l y two  disintegrated c e l l s  appear  the second m e i o t i c d i v i s i o n i n one of the dyad c e l l s l i k e l y f a i l e d ; where t h r e e d i s i n t e g r a t e d c e l l s the second d i v i s i o n has presumably  has  appear  been completed.  In  e i t h e r case, a f u n c t i o n a l megaspore completes the t r i a d or t e t r a d .  The i n t e r p r e t a t i o n s of the r a t h e r  stages at t h i s p e r i o d of development on the f o r e g o i n g presumptions.  critical  have a l l been based  The f a i r l y f r e q u e n t oc-  currence o f t r i a d s i n P. c o n f i n i s i s a l s o found i n o t h e r Poa s p e c i e s , and may  be taken as a normal phenomenon.  Tinney (100) and Akerberg ( 4 ) ,  working w i t h a p o m i c t i c .  s t r a i n s of P. p r a t e n s i s . found t h a t the f o r m a t i o n of a t r i a d was more frequent than the f o r m a t i o n of a tetrad.  T h i s was  due to the p a r t i a l or complete  complete failure  of the second d i v i s i o n i n the m i e r o p y l a r dyad c e l l .  Hak-  ansson (50) noted the same t h i n g i n s e x u a l s t r a i n s o f P. a l p i n a . The o r i g i n s of the twin embryo sacs seen i n two s l i d e s a r e unknown. one EMC  Although the presence of more than  i n a s i n g l e ovule has never a c t u a l l y been shown  i n the p i s t i l l a t e p l a n t s , two EMCs do occur i n one of the n o n - f u n c t i o n a l p i s t i l s of a male f l o r e t , as p r e v i o u s l y  -116noted.  T h e r e f o r e , t h e r e i s good reason t o b e l i e v e t h a t  two EMCs c o u l d o c c a s i o n a l l y develop a l s o i n the ovule o f a p i s t i l l a t e f l o r e t , g i v i n g r i s e to two spore t r i a d s or t e t r a d s and two f u n c t i o n a l megaspores.  A second  source of twin embryo sacs i s t h e simultaneous  possible  develop-  ment of two megaspores d e r i v e d from a s i n g l e EMC. it  That  i s not e x c l u s i v e l y t h e c h a l a z a l megaspore which i s  f u n c t i o n a l i s evident from F i g . 9,  which shows the  second megaspore from the c h a l a z a l end d e v e l o p i n g as the embryo sac i n i t i a l .  I t a l s o seems p o s s i b l e , then, t h a t  both a c h a l a z a l megaspore and one of t h e other spores could be f u n c t i o n a l . Fig.  T h i s may a c t u a l l y be o c c u r r i n g i n  9, D, although the b a s a l megaspore i s not as w e l l  developed as i t s neighbor. The d i v i s i o n and growth o f the a n t i p o d a l in  cells  the l a t e r stages o f embryo sac development i s c e r t a i n l y  remarkable, but probably has l i t t l e  or no b e a r i n g on the  problem o f determining the method o f r e p r o d u c t i o n i n P. c o n f i n i s . are  A c c o r d i n g t o Andersen  (10) "The Gramineae  conspicuous f o r t h e i r s t r o n g l y developed a n t i p o d a l s . "  and Maheshwari (67) a l s o s t a t e s t h a t the presence o f many a n t i p o d a l s i s common i n the g r a s s e s .  A number o f  workers have r e p o r t e d the occurrence o f unusual a n t i p o d a l behaviour i n Poa s p e c i e s . of  Andersen  (10) found a n t i p o d a l s  great s i z e i n P. compressa and P. p r a t e n s i s b i o t y p e s  -117which. she s t u d i e d .  They had dense protoplasm, and per-  s i s t e d u n t i l l a t e i n t o endosperm f o r m a t i o n .  Tinney (100)  r e p o r t e d the presence o f up t o f i v e or s i x a n t i p o d a l s i n some o f h i s a p o m i c t i c  pratensis biotypes, while  Nielsen  (71) noted the enlargement and the d e n s e l y - s t a i n i n g prope r t y of the a n t i p o d a l s i n sexual p l a n t s o f the same species. Akerberg ( 5 ) , i n v e s t i g a t i n g apomictic bluegrass,  s t r a i n s o f Kentucky  found the a n t i p o d a l c e l l s almost f i l l e d the  whole of the n u c e l l a r chamber, and Hakansson (51) observed a s i m i l a r phenomenon i n sexual s t r a i n s o f P. a l p i n a . particularly i n unfertilized  embryo s a c s .  The w r i t e r i s un-  aware o f any e x p l a n a t i o n f o r e x c e s s i v e a n t i p o d a l development; p o s s i b l y such t i s s u e may f u n c t i o n as a temporary source of n u t r i t i o n f o r the egg c e l l and f u s i o n nucleus when p o l l i n a t i o n and f e r t i l i z a t i o n a r e delayed  f o r some  reason. There i s no s i g n o f parthenogenetic  embryo  development i n P. c o n f i n i s . even i n f l o r e t s which have been kept u n p o l l i n a t e d f o r some days a f t e r a n t h e s i s . apomictic  In other  Poa s p e c i e s , as has a l r e a d y been seen, preco-  cious embryo formation  i s q u i t e common, and i s not c o n t i n -  gent upon p o l l i n a t i o n , even though endosperm u s u a l l y r e q u i r e s p o l l i n a t i o n and (probably) t i o n o f the f u s i o n The  formation the f e r t i l i z a -  nucleus.  l a c k of m a t e r i a l and shortage of time d i d  - 1 1 8 -  not permit  a very  extensive  genesis t o be made, a l t h o u g h for  examination o f microsporoi t was s t u d i e d c l o s e l y enough  the w r i t e r t o conclude t h a t i t i s q u i t e r e g u l a r .  Niel-  sen (77) says t h a t "Among the grasses most o f the apomictic s p e c i e s examined have e x h i b i t e d a c o n s i d e r a b l e amount o f m e i o t i c d i s t u r b a n c e , p a r t i c u l a r l y during microsporogenesis."  Once a g a i n , although  of microsporogenesis  t h e evidence  from t h e study  i s not i n i t s e l f c o n c l u s i v e , i t pro-  vides a further i n d i c a t i o n that reproduction i s probably  i n P. c o n f i n i s  sexual.  The  c y t o l o g i c a l study of embryo and endosperm  development f o l l o w i n g t h e p o l l i n a t i o n of female P. c o n f i n i s f l o r e t s with p r a t e n s i s p o l l e n i s of c o n s i d e r a b l e ance.  The evidence  import-  c e r t a i n l y , p o i n t s t o the formation of  t r u e h y b r i d embryos between the two s p e c i e s , although t h e possibility  t h a t the p r a t e n s i s p o l l e n merely s t i m u l a t e s  the development o f h a p l o i d ( o r d i p l o i d ) egg c e l l s i n t o embryos cannot be e n t i r e l y r u l e d out.  The p e r i o d o f delay  between p o l l i n a t i o n and both endosperm and embryo format i o n i s c e r t a i n l y long enough f o r double f e r t i l i z a t i o n to c  take p l a c e .  On $he other hand, the f e r t i l i z a t i o n of the  f u s i o n nucleus  o n l y may be i n v o l v e d .  U n f o r t u n a t e l y , be-  cause of the small s i z e of the o v u l e s , P. c o n f i n i s i s particularly  unfavourable f o r t h e o b s e r v a t i o n o f f e r t i l i z a t i o n . The f a c t t h a t embryos and seeds form i n a l l the  -119c r o s s e s i n v o l v i n g P. c o n f i n i s and P. macrantha  (except  those w i t h P. annua), and t h a t i n a l l cases the seeds subsequently s h r i v e l a t about the same stage of m a t u r i t y , shows t h a t whatever the e x p l a n a t i o n of embryo and sperm formation may cases.  endo-  be i t i s probably the same i n a l l  The p r o d u c t i o n of a p p a r e n t l y normal seed f o l l o w i n g  the a r t i f i c i a l p o l l i n a t i o n of female P. c o n f i n i s with c o n f i n i s p o l l e n i s a l s o important;  florets  i t shows almost  d e f i n i t e l y t h a t the s h r i v e l l i n g o f the seed from the i n terspecific  c r o s s e s i s not due merely to  environmental  c o n d i t i o n s i n the greenhouse, aphid i n f e s t a t i o n s , e t c . The formation of a p p a r e n t l y normal seed, f o l l o w e d by a b o r t i o n of the seed p r i o r to i t s maturation, common development a f t e r c e r t a i n i n t e r s p e c i f i c other genera (67)•  is a  the  fairly  crosses i n  From the o b s e r v a t i o n s made d u r i n g  the  embryo e x c i s i o n i t appears t h a t the seed a b o r t i o n f o l l o w i n g the i n t e r s p e c i f i c c r o s s i n g s i s due to some form o f endosperm f a i l u r e i n the l a t e r stages of development. There has been c o n s i d e r a b l e study c a r r i e d out on the d e v e l opment and r o l e of the endosperm, i t s p h y s i o l o g i c a l  rela-  t i o n s h i p , and other f e a t u r e s .  study  Further h i s t o l o g i c a l  of the endosperm f a i l u r e f o l l o w i n g the c r o s s i n g s made i n the present  study might be  worthwhile.  The very b r i e f attempts at embryo e x c i s i o n and c u l t u r e show t h a t t h e r e i s hope of o b t a i n i n g h y b r i d p l a n t s  -120between P. c o n f i n i s and o t h e r Poa  s p e c i e s by t h i s method  (assuming t h a t the embryos which form are a c t u a l l y s p e c i f i c h y b r i d embryos, and not h a p l o i d s ) . counts to  inter-  Chromosome  i n these p u t a t i v e h y b r i d s , i f they can be  raised  a s u f f i c i e n t l y advanced stage, should p r o v i d e the  f i n a l proof of the nature of t h e i r o r i g i n .  I t i s obvious  t h a t more work needs to be done on embryo c u l t u r e ;  the  w r i t e r has o n l y been a b l e to show t h a t i t i s d e f i n i t e l y practical.  Since a method f o r i n d u c i n g f l o w e r i n g i n P.  c o n f i n i s and known, and fairly  other b l u e g r a s s e s a t the same time i s  the embryo e x c i s i o n and  now  c u l t u r e shown to be  easy, the c u l t u r e of q u i t e a l a r g e number of  bryos should not be a very l o n g or d i f f i c u l t The  em-  task.  f o r m a t i o n of a higher percentage  l o o k i n g seed from the cross P. macrantha x P.  of healthierconfinis.  together w i t h the success of the embryo c u l t u r e f o l l o w i n g t h i s c r o s s , i s p o s s i b l e evidence of a c l o s e r between these two  relationship  s p e c i e s than between any other  two  s p e c i e s e n t e r i n g i n t o the c r o s s e s . On the b a s i s of the r e s u l t s from the and h y b r i d i z a t i o n a l work i t i s obvious  t h a t any  work of the type c a r r i e d out i n the Carnegie of Any  cytological breeding  Institution  Washington program w i l l not be a p p l i c a b l e to P.  confinis.  h y b r i d s between P. c o n f i n i s and other s p e c i e s would  -121presumably reproduce s e x u a l l y and would not give r i s e to apomictic l i n e s i n l a t e r generations, parent was  apomictic.  even i f the  T h i s c o n c l u s i o n i s based on  other the  f a c t t h a t so f a r a p o m i c t i c h y b r i d l i n e s have o n l y been o b t a i n a b l e f o l l o w i n g c r o s s i n g s i n which both p a r e n t a l s t r a i n s or s p e c i e s are themselves predominantly  apomictic.  Through b a c k c r o s s i n g of h y b r i d s w i t h apomicts as r e c u r r e n t parents  i t might be p o s s i b l e to o b t a i n  hybrid l i n e s eventually. s p e c u l a t i v e and of  apomictic  However, t h i s reasoning  probably very i m p r a c t i c a l .  The  i s very  chances  o b t a i n i n g economically worthwhile h y b r i d l i n e s i n  t h i s manner are c e r t a i n l y r a t h e r remote, and the economic use of dune b l u e g r a s s per se i s much more l i k e l y .  Never-  t h e l e s s , f u r t h e r attempts at i n t e r s p e c i f i c h y b r i d i z a t i o n i n v o l v i n g P. c o n f i n i s should be c a r r i e d out i f p o s s i b l e ; any  hybrids w i l l be of c o n s i d e r a b l e s c i e n t i f i c  even i f they are of no p r a c t i c a l  interest  value.  S e v e r a l aspects of the P. c o n f i n i s study which might have been i n v e s t i g a t e d are worth mentioning as of  f u r t h e r p o s s i b l e work.  One  of these  lines  i s a study of the  range of chromosome numbers i n dune b l u e g r a s s .  Despite  the u n i f o r m i t y of the s p e c i e s throughout i t s d i s t r i b u t i o n range, d i f f e r e n c e s i n chromosome numbers may b i o t y p e s from separate l o c a l i t i e s . p r e v i o u s l y recorded  The  exist i n  f a c t t h a t the o n l y  chromosome count (by Hartung (53)  made  -122on a female p l a n t from Tillamook Bay, Oregon) and t h a t determined by the w r i t e r are both the same (2n - 42)  does  not mean t h a t other e u p l o i d or a n e u p l o i d numbers may  not  exist. Another problem which has not been covered i s that of the r a t i o of male to female p l a n t s i n the progenies of P. c o n f i n i s p l a n t s .  I f P. c o n f i n i s reproduces  s e x u a l l y the sex r a t i o should be approximately 1:1 i n s e e d l i n g progenies (assuming t h a t o t h e r f a c t o r s , such as unequal v i a b i l i t y of male and female s e e d l i n g s , do not enter i n t o the p i c t u r e ) .  I f i t reproduces predominantly  by apomixis (which, as we have now  seen, i s v e r y  improb-  a b l e ) , the sex r a t i o would be c o n s i d e r a b l y m o d i f i e d . Because apomixis i m p l i e s the p r o d u c t i o n of progeny t i c a l w i t h the maternal parent one would m i c t i c a l l y formed  expect a l l apo-  seed to g i v e r i s e to female p l a n t s .  Male p l a n t s could presumably sexually.  iden-  o n l y a r i s e from seed formed  T h i s q u e s t i o n o f d i o e c i s m and apomixis has not  been d i s c u s s e d e a r l i e r because i t seems to have no d i r e c t b e a r i n g on the study of dune b l u e g r a s s , but i t should be mentioned  t h a t a number o f d i o e c i o u s p l a n t s i n s e v e r a l  genera are a l s o a p o m i c t i c ( 4 5 ) .  However, the w r i t e r i s  not aware of any papers on d i o e c i o u s apomicts i n which the problem of the sex r a t i o and apomixis i s f u l l y cussed.  dis-  -123-  The  s m a l l s i z e and l a c k o f heteromorphism of the  chromosomes of P. c o n f i n i s makes any d e t e c t i o n of chromosomes very u n l i k e l y .  In any  sex  event, A l l e n (9)  points  out t h a t "A v i s i b l e d i f f e r e n c e between the chromosomes of a p a i r does not n e c e s s a r i l y accompany dioecism." t h e r e are a p p a r e n t l y no e f f e c t i v e s t a i n i n g which enable the i n e r t , heterochromatic  As y e t ,  techniques  r e g i o n s of  sex  chromosomes to be p i c k e d out. A number of P. c o n f i n i s p l a n t s from s e v e r a l b i o types have been grown from seed and are now e s t a b l i s h e d i n the U n i v e r s i t y n u r s e r y .  These w i l l be examined f o r  the d e t e r m i n a t i o n of sex r a t i o s when they f l o w e r . F u r t h e r study of dune b l u e g r a s s under t i o n needs to be made; a t the moment, the  cultiva-  potentialities  of the s p e c i e s have s t i l l not been f u l l y i n v e s t i g a t e d . The development of more t r i a l p l o t s and t h e i r maintenance under c o n d i t i o n s of standard t u r f management should  be  attempted.  dif-  The o p p o r t u n i t y f o r adequate t e s t i n g of  f e r e n t t u r f s p e c i e s and  s t r a i n s now  seems to be p o s s i b l e  f o l l o w i n g the f o r m a t i o n of the U n i v e r s i t y of B r i t i s h Columbia P r e s i d e n t ' s Committee on Sports T u r f  Research.  A f i n e t u r f nursery has been e s t a b l i s h e d on the U n i v e r s i t y farm, and funds are a v a i l a b l e f o r i t s upkeep.  The  estab-  lishment of P. c o n f i n i s on a sandy area of the P o i n t Grey G o l f and  Country  Club i s a l s o  contemplated.  -124VIII  SUMMARY Dune b l u e g r a s s , Poa  c o n f i n i s Vasey, i s a d i o e -  c i o u s grass n a t i v e to the P a c i f i c Coast of North America. I t has a very r e s t r i c t e d h a b i t a t , being  c o n f i n e d almost  e n t i r e l y to the s e m i - s t a b i l i z e d , porous sand areas of coast.  Herbarium s t u d i e s , and  the  f i r s t - h a n d observations  of  a number of P. c o n f i n i s s i t e s i n d i c a t e t h a t i t i s a rather d i s t i n c t  species.  Under c u l t i v a t i o n on The  U n i v e r s i t y of B r i t i s h  Columbia farm P. c o n f i n i s grows v i g o r o u s l y and t h i c k sod. tem and nature  forms a  I t s f i n e - l e a v e d growth, rhizomatous root  r a p i d l y - s p r e a d i n g h a b i t , together  with the  sys-  sandy  of i t s n a t i v e h a b i t a t , a l l suggest t h a t i t may  f i n d use as a t u r f s p e c i e s f o r sandy g o l f courses s i m i l a r areas along the  and  coast.  A survey of the l i t e r a t u r e on r e p r o d u c t i o n i n the bluegrasses Poa,  r e v e a l s t h a t apomixis i s widespread i n  and t h a t apospory or d i p l o s p o r y f o l l o w e d by pseudo-  gamous embryo development i s the u s u a l form of reproduction.  Breeding  procedures w i t h apomictic  grasses must be c o n s i d e r a b l y m o d i f i e d , but techniques  apomictic  the  standard  of improvement are s t i l l t h e o r e t i c a l l y  able to the b l u e g r a s s  blue-  avail-  breeder.  A c y t o l o g i c a l study of embryo sac development  -125-  i n p i s t i l l a t e p l a n t s i n d i c a t e s t h a t i t f o l l o w s the scheme, and  that reproduction  t a i n s a s i n g l e EMC  i s sexual.  "normal"  Each ovule  con-  which undergoes a r e g u l a r m e i o t i c  div-  i s i o n , g i v i n g r i s e to a t r i a d or t e t r a d of megaspores, of which forms the h a p l o i d embryo sac. numbers of 2 n • 4 2 are found .in two The  presence of twin  embryo sacs  one  Somatic chromosome  EMCs i n d i a k i n e s i s .  i s observed i n two  ovules.  Very marked a n t i p o d a l development, with an i n c r e a s e i n s i z e and cell,  number of c e l l s , and  i n the number of n u c l e i per  i s c h a r a c t e r i s t i c of the mature female gametophyte* Microsporogenesis  appears "normal." ets and  ovules  i n the  staminate p l a n t s a l s o  S t u d i e s of anthers i n p i s t i l l a t e  i n staminate f l o r e t s show t h a t t h e i r  opment proceeds normally  up to a c e r t a i n p o i n t and  flordevelthen  breaks down* Embryo and  endosperm development can be seen i n  s e c t i o n s of ovules prepared a f t e r the p o l l i n a t i o n of P_* c o n f i n i s by P. p r a t e n s i s .  The  embryos are b e l i e v e d to  the product of t r u e h y b r i d i z a t i o n . the c r o s s i n g s among P. sis  and  P.  Seed forms a f t e r a l l  c o n f i n i s . P. macrantha. P.  compressa u s i n g the f i r s t  be  two  praten-  s p e c i e s as female  parents,  but the seed i n v a r i a b l y s h r i v e l s s h o r t l y b e f o r e  reaching  maturity. The  e x c i s i o n of h y b r i d  embryos from seeds formed  a f t e r some of the i n t e r s p e c i f i c c r o s s e s , and  the.culture  -126of these  embryos on agar media have been s u c c e s s f u l l y c a r -  r i e d out.  T i s s u e s of p u t a t i v e macrantha .x c o n f i n i s h y b r i d s 1  are growing, and showing some d i f f e r e n t i a t i o n . The r e s u l t s i n d i c a t e t h a t h y b r i d s between P. c o n f i n i s and other b l u e g r a s s e s  can probably be o b t a i n e d  through embryo c u l t u r e t e c h n i q u e s . considerable s c i e n t i f i c  These would be o f  i n t e r e s t ; a t the moment, however,  t h e i r p r a c t i c a l v a l u e , i f any, i s not known.  -127LITERATURE CITED Akerberg, E. Apomictic and sexual seed formation i n Poa p r a t e n s i s . Hereditas 25:357-370, 1939. The a p p l i c a t i o n of c y t o l o g y to herbage p l a n t breeding, i n The b r e e d i n g o f herbage p l a n t s i n Scandanavia and F i n l a n d , Imp. A g r i c . Bur. J o i n t Pub. No. 3 : 5 2 - 6 1 , 1940. Bastard m e l l a n Poa p r a t e n s i s L. x Poa a l p i n a L. a r t i f i c i e l l t f r a m s t S l l d , Bot. N o t i s e r 1936:563-566. 1936. [An a r t i f i c i a l h y b r i d between Poa p r a t e n s i s and Poa a l p i n a . Review by R. Peter Jones i n Imp. Bur. Pasture & Forage Crops, Herb. Rev. 4 : 3 2 - 3 3 , 1936.] Cytogenetic s t u d i e s i n Poa p r a t e n s i s and i t s h y b r i d w i t h Poa a l p i n a . Hereditas 2_8:1-126, 1942 F u r t h e r s t u d i e s of the embryo and endosperm development i n Poa p r a t e n s i s , H e r e d i t a s 29:199-201 1943. "~ Seed p r o d u c t i o n of the Poa s p e c i e s , Svensk F r o t i d n i n g 7:79-83, 1938.. [ E n g l i s h t r a n s l a t i o n by R. Peter Jones i n Imp. Bur. Pasture and Forage Crops, Herb. Rev. 6:228-233.] Studien \iber d i e Samenbildung b e i Poa p r a t e n s i s L., Bot. N o t i s e r 1936:213-280. 1936. [English summary, p. 268.) , and B i n g e f o r s , S. Progeny s t u d i e s i n the h y b r i d Pod p r a t e n s i s x Poa a l p i n a . Hereditas 3_£:125-136, 1953. A l l e n , C. E. The genotypic b a s i s of sex-expression sperms, Bot. Rev. 6:227-300, 1940.  i n Angio-  -128(10)  A n d e r s e n , A l i c e M. Development o f the female gametophyte and caryops i s o f Poa p r a t e n s i s and Poa compressa. J o u r . A g r i c . Res. 3±i1001-1018, 1927.  (11)  A n d e r s o n , J . P. F l o r a o f A l a s k a and a d j a c e n t p a r t s o f Canada, I I Typhaceae t o Poaceae, Iowa S t a t e C o l l e g e J o u r , of S c i . 18:38-445, 1944.  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A g r o n . 2J5:233-234, 1933.  York,  (100)  Tinney, F. W.. C y t o l o g y o f p a r t h e n o g e n e s i s i n Poa p r a t e n s i s . J o u r . A g r i c . Res. 60:351-360, 1940.  (101)  and Aamodt, G. S. The progeny t e s t as a measure o f the t y p e s o f seed development i n Poa p r a t e n s i s L., J o u r . Hered. 3.1:457-464, 1940.  (102)  T r i p l e t t , E. L. C y t o l o g y of Poa h y b r i d s , Carnegie I n s t . "Wash., Year Book £2:173-174, 1953.  (103)  and C l a u s e n , J . Chromosome numbers o f h y b r i d Poa l i n e s , Carnegie I n s t . Wash., Year Book j£: 15.6-157, 1954.  (104)  Vasey, G. Grasses o f the P a c i f i c S l o p e — i n c l u d i n g A l a s k a and t h e a d j a c e n t i s l a n d s , U. S. Dept. A g r i c , D i v . of B o t . , B u l l . No. 13, P a r t I I , Washington, U. S. Govt. P r i n t i n g O f f i c e , 1892.  (105)  V i n a l l , H. N. and H e i n , M. A. B r e e d i n g m i s c e l l a n e o u s g r a s s e s , U. S. Dept. A g r i c , Year Book, 1937, pp. 1032-1102.  (106)  W i n k l e r , H. F o r t p f l a n z u n g der Gewachse, 7 A p o m i x i s , Handw. d. N a t u r w i s s . 4:451-461, 1934. [Cited after S t e b b i n s , 1950"j  (107)  _  Tiber den B i o n t e n w e c h s e l und d i e Abswichungen von seinem normalen V e r l a u f , P l a n t a 33:1-90. 1942. ^Cited a f t e r Gustafsson, 1946J  -137(108)  Winkler, H. Uber Parthenogenesis und Apogamie im P f l a n z e n r e i c h e , Progr. r e i Bot. 2:293-454, 1908. p i t e d a f t e r Gustafsson, 1946^  (109)  . V e r b r e i t u n g und Ursache der Parthenogenesis im P f l a n z e n - und T i e r r e i o h , Jena, G. F i s c h e r , 166 pp., 1 9 2 0 . (.Cited a f t e r S t e b b i n s , 19507J  -138X  APPENDIX F u l l b o t a n i c a l nomenclature f o r a l l p l a n t  and  subspecies mentioned i n the Grindelia Poa  text:  squarrosa (Pursh) Dunal.  abbreviata  R.  "  alpina  "  ampla Merr.  "  annua L.  Br.  L.  11  arachnifera  "  a r c t i c a R.  Torr. Br.  subsp. c a e s p i t a n s  (Simra.) Nannf.  "  depauperata ( F r . ) Nannf.  "  elongata  "  microglumis Nannf.  "  stricta  ( B l . ) Nannf.  (Lindeb.) Nannf.  "  a r i d a Vasey  "  atropurpurea  "  bulbosa  "  caespitosa  "  Canbyj ( S c r i b n . )  "  compressa  "  c o n f i n i s Vasey  "  c u r t a Rydb.  "  cuspidata  "  D o u g l a s i i Nees.  Scribn.  L. Hk.  L.  Nutt.  Piper  species  -139Poa  eminens P r e s l .  "  glauca  Vahl.  "  g l a u c i f o l i a S c r i b n . and  "  g r a c i l l i m a Vasey  "  her.iedalica H. Smith=(P. a l p i n a L. v a r . v i v i p a r a L. x P. p r a t e n s i s L. subsp. alpigena (Fr.) H i i t . )  "  -Iemtlandica (Almqu.) Richt.=(P. a l p i n a var v i v i p a r a L. x P. l a x a Hke. subsp. f l e x u o s a (Sm.) Hyl.)  "  K e l l c - g g i i Vasey  "  l a b r a d o r i c a Steud.  "  laxa  Hke.  subsp. f l e x u o s a Poa  Will.  laxiflora  (Sm.^  Hyl.  Buckl.  "  macrantha Vasey  "  nemoralis  "  nervosa (Hook.) Vasey  "  nevadensis Vasey  "  p a l u s t r i s L.  "  Piperi Hitch.  "  pratensis  L.  L.  subsp. e u p r a t e n s i s  Hiit.  "  a n g u s t i f o l i a ( L . ) Lindb. F i l .  "  irrigata  (Lindm.) Lindb. F i l .  "  alpigena  (Fr.) H i i t .  L.  -140Poa P r i n g l e i  Scribn.  "  rhizomata  "  scrabrella  "  trivialis  Hitch. (Thurb.) L.  R6sa gymnocarpa N u t t .  Benth.  

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