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Polyploidy and its application in forestry and a preliminary study of aberrant Douglas-fir seedlings Bolotin, Moshe 1958

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POLYPLOIDY AND I T S A P P L I C A T I O N I N FORESTRY AND A PRELIMINARY STUDY OF ABERRANT DOUGLAS-FIR  SEEDLINGS  by MOSHE BOLOTIN B.S.F., U n i v e r s i t y o f W a s h i n g t o n ,  A THESIS SUBMITTED  1951  I N P A R T I A L FULFILMENT OF  THE REQUIREMENTS FOR THE DEGREE OF Master  of Forestry  i n t h e Department of Forestry We a c c e p t t h i s t h e s i s a s c o n f o r m i n g r e q u i r e d standard.  THE UNIVERSITY OF B R I T I S H February,  1958  to the  COLUMBIA  I ABSTRACT The paper reviews the l i t e r a t u r e on p o l y p l o i d y I n r e s p e c t to i t s p o s s i b l e a p p l i c a t i o n i n f o r e s t breeding.  The occurrence o f spontaneous  tree  polyploidy i n  v a r i o u s s p e c i e s i s enumerated and i t s q u a l i t i e s  outlined.  An account i s presented o f the success and f a i l u r e  thus  f a r a t t a i n e d i n the s e a r c h f o r improved v a r i e t i e s o f f o r e s t t r e e s p e c i e s through p o l y p l o i d y .  F i n a l l y , the  p o t e n t i a l i t i e s of p o l y p l o i d y i n f o r e s t r y a r e summarized and some recommendations concerning f u t u r e l i n e s o f research are outlined. The experimental work i n c o n n e c t i o n w i t h t h i s  thesis  was the p r e l i m i n a r y i n v e s t i g a t i o n of Douglas f i r (Pseudotsuga m e n z i e s i i  (Mirob) Franco) a b e r r a n t s found  each year among the s e e d l i n g s a t the B.C. F o r e s t S e r v i c e n u r s e r y i n Duncan. to be spontaneous  These abberrants have been thought a u t o p o l y p l o i d s because  they  resemble  such p o l y p l o i d s found among n u r s e r y stock of other . c o n i f e r o u s s p e c i e s elsewhere.  This study, however,  i n d i c a t e d beyond any doubt that these abberrants a r e not p o l y p l o i d s .  Other p o s s i b i l i t i e s which might have  caused the aberrant form a r e d i s c u s s e d , .  In presenting the  r e q u i r e m e n t s f o r an  of B r i t i s h it  this  freely  agree t h a t for  thesis in partial  advanced degree at the  Columbia, I agree that available  the  f o r r e f e r e n c e and  permission for extensive  s c h o l a r l y p u r p o s e s may  D e p a r t m e n t o r by  be  study.  I  s h a l l not  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, C a n a d a . D a t e F e b r u a r y , 1958  Columbia,  Head o f  thesis my  I t i s understood  gain  Forestry  further  copying of t h i s  copying or p u b l i c a t i o n of t h i s t h e s i s  Department o f  University s h a l l make  that  a l l o w e d w i t h o u t my  of  Library  g r a n t e d by t h e  his representative.  be  fulfilment  for  written  financial  permission.  ACKNOWLEDGEMENTS The w r i t e r i s indebted to D r . P. G. Haddock and Dean G. S. A l l e n f o r advice and c r i t i c i s m of the present thesis.  To Dr. A. H. Hutchinson  f o r a d v i c e and h e l p i n  t a k i n g most of the microphotographs. f o r a d v i c e on microtechnique  work.  To Mr. H. E . Sweet To Mr. W. V. Hancock  f o r advice and h e l p i n t a k i n g of microphotographs, t o Mr. K. A. Helium f o r t a k i n g the photographs of s e e d l i n g s , and  t o Miss L. Bowers f o r t y p i n g t h e s i s .  II TABLE OF CONTENTS I  Abstract List  of i l l u s t r a t i o n s  I l l 1  Introduction PART A:, REVIEW OF LITERATURE  3  Meaning of p o l y p l o i d y Causes of p o l y p l o i d y  . . . . . . . . . . . . . . . . .  8  Changes caused by p o l y p l o i d y Limitations of polyploidy  12  .  . 13  V a r i a b i l i t y of p o l y p l o i d s  1*+  P o l y p l o i d s and environment T r i p l o i d y i n woody p l a n t s  5  16  ,\  A l l o p o l y p l o i d y i n woody p l a n t s  19  .  P o l y p l o i d s among the c o n i f e r s  20  Spontaneous c o n i f e r o u s p o l y p l o i d s i n n u r s e r y stock •  23  P o t e n t i a l i t i e s of polyploidy i n f o r e s t r y  25  .  PART B: PRELIMINARY STUDY OF ABERRANTS IN DOUGLAS FIR Introduction  27  M a t e r i a l s and methods  29  Results •  33  Discussion  35  Illustrations  37  Bibliography  *+8  Acknowledgements  56  Ill LIST OF ILLUSTRATIONS F i g . 1. Normal and a b e r r a n t  s e e d l i n g s one-year o l d . . 37  F i g . 2. Normal and a b e r r a n t  s e e d l i n g s two-year o l d . . 37  F i g . 3« Stem d i f f e r e n c e s i n * n o r m a l and a b e r r a n t  seed-  lings F i g . k. An a b e r r a n t  38 s e e d l i n g w i t h a normal b r a n c h . .  F i g . 5» Pro-metaphase i n an a b e r r a n t  seedling. . . .  39  F i g . 6. Pro-metaphase i n a normal s e e d l i n g F i g . 7« Metaphase i n an a b e r r a n t  39  seedling  *f0 ho  F i g . 8. Metaphase i n a normal s e e d l i n g F i g . 9, E a r l y anaphase i n an a b e r r a n t  38  seedling. . .  >+l  F i g . 10. E a r l y anaphase i n a normal s e e d l i n g  'fl  F i g . 1 1 . Pro-metaphase i n a normal t w i g on ah aberrant,h2 F i g . 12. Metaphase i n a normal W i g F i g . 1 3 . Anaphase i n an a b e r r a n t  on ah a b e r r a n t .  . .if2  seedling  F i g . l * f . Anaphase i n a normal s e e d l i n g . . . F i g . 15.  Anaphase i n an a b e r r a n t  seedling  F i g . 16. Pro-metaphase i n a normal s e e d l i n g F i g . 1 7 . Telophase i n an a b e r r a n t  seedling. . . . . .  ^3 h'Q Mf hh '.^5  F i g . 18. Telophase i n a normal s e e d l i n g  ^5  F i g . 1 9 . Telophase i n an a b e r r a n t  *+6  seedling.'  F i g . 2 0 . Telophase i n a normal s e e d l i n g  ^6  F i g . 2 1 . Telophase i n an a b e r r a n t  A7  seedling. . . . . . .  F i g . 2 2 . Telophase i n a normal s e e d l i n g  k-J  PART A REVIEW OF LITERATURE ON POLYPLOIDY I N FOREST TREES  1 INTRODUCTION During the past few decades c o n s i d e r a b l e  work has  been  c a r r i e d out i n the i n v e s t i g a t i o n of p o l y p l o i d y i n p l a n t s . Many s u p e r i o r p o l y p l o i d s t r a i n s have been developed e i t h e r by s e l e c t i n g them from n a t u r a l p o p u l a t i o n s by a r t i f i c i a l  induction.  of d i p l o i d s or  With the d i s c o v e r y  of the e f f e c t  of c o l c h i c i n e i n the i n d u c t i o n of p o l y p l o i d y i n p l a n t s , a new  t o o l has  been added by which many more p o l y p l o i d s have  been produced.  These p o l y p l o i d s have r e p l a c e d  d i p l o i d s i n cases where t h e i r s u p e r i o r i t y or has  the  former  usefulness  been demonstrated. In f o r e s t r y the i n v e s t i g a t i o n of p o l y p l o i d y  considerably breeding.  behind other  lags  f i e l d s which d e a l w i t h p l a n t  T h i s l a g i s understandable because there i s  much l e s s o p p o r t u n i t y  f o r i n t r o d u c i n g new  p l a n t s i n f o r e s t r y than i n a g r i c u l t u r e .  s t r a i n s of Forestry i s a  l e s s i n t e n s i v e form of plant, c u l t u r e than h o r t i c u l t u r e or the r a i s i n g of f i e l d  crops;  the l i f e span of the ..'  i n d i v i d u a l p l a n t before  harvest, i s much, longer,,.and  p r a c t i c e of a r t i f i c i a l methods of r e g e n e r a t i o n common.  not  the as.  Another reason f o r the l a c k , o f i n t e n s i v e r e - .  search on p o l y p l o i d y i n f o r e s t t r e e s may  be the  fact  t h a t only a l i m i t e d amount of success has been a t t a i n e d i n f i n d i n g superior The field  polyploids.  purpose of the present  of p o l y p l o i d y r e s e a r c h  study i s to survey  i n plants i n general  the and i t s  2 a p p l i c a t i o n i n f o r e s t r y , w i t h p a r t i c u l a r emphasis on work c a r r i e d out on c o n i f e r o u s t r e e s .  The p r a c t i c a l work  c o n s i s t e d of the study of abnormal D o u g l a s - f i r (Pseudotsuga menziesii  (Mirob.) Franco) s e e d l i n g s v h i c h were suspected  to be a u t o p o l y p l o i d s . During the course of the study a schedule f o r squash technique i n v e g e t a t i v e buds of D o u g l a s - f i r was  developed  and a s o l u t i o n to the  of breaking dormancy i n t h a t s p e c i e s was  problem  undertaken.  3 MEANING OF POLYPLOIDY Most animals and p l a n t s are d i p l o i d s .  A diploid  organism c a r r i e s two homologous sets o f chromosomes i n each c e l l n u c l e u s ,  one s e t having  come from each  Reproductive c e l l s  (gametes) are h a p l o i d i . e . , they  c a r r y only one s e t o f chromosomes.  In r a r e  parent.  instances  the somatic n u c l e i o f p l a n t s may c o n t a i n only one s e t of chromosomes. and  Such monoploid p l a n t s a r e u s u a l l y  do not become e s t a b l i s h e d i n n a t u r e .  sterile  L i v i n g organisms  w i t h n u c l e i c o n t a i n i n g three or more s e t s of chromosomes are known as p o l y p l o i d s .  A p o l y p l o i d w i t h three  sets of  chromosomes i n the somatic nucleus i s c a l l e d a t r i p l o i d , w i t h f o u r s e t s a t e t r a p l o i d , and so on.  An a u t o p o l y p l o i d  i s a p o l y p l o i d which o r i g i n a t e d from one p a r e n t a l  species.  An a l l o p o l y p l o i d on the other hand, has i t s o r i g i n i n the h y b r i d i z a t i o n o f two or more s p e c i e s . one  A e u p l o i d p l a n t has  or more complete s e t s of chromosomes.  An  aneuploid  p l a n t has a normal complement (genome) p l u s some e x t r a chromosomes or with some chromosomes  missing.  P o l y p l o i d s may be s t a b i l i z e d and capable o f s e l f perpetuation,  or u n s t a b i l i z e d , i n which cases  r e p r o d u c t i o n i s the only p o s s i b l e means of  vegetative  propagation.  P a r t i c u l a r l y among u n s t a b i l i z e d and a r t i f i c i a l l y  induced  p o l y p l o i d s , s e v e r a l forms o f r e t r o g r e s s i o n t o d i p l o i d y may occur. alongside  In mixoploids, polyploid c e l l s .  diploid  c e l l s a r e encountered  Chimeras are p l a n t s composed  of t i s s u e s of d i f f e r e n t chromosomal count.  In p e r i c l i n a l  h chimeras s e v e r a l l a y e r s of t i s s u e are p o l y p l o i d whereas adjacent l a y e r s are d i p l o i d .  I n s e c t o r i a l chimeras c e r t a i n  s e c t o r s of the p l a n t are p o l y p l o i d and other s e c t o r s a r e diploid. and  The wide d i s t r i b u t i o n of m i x o p l o i d s , chimeras,  a n e u p l o i d s , p a r t i c u l a r l y among a r t i f i c i a l l y  induced  p o l y p l o i d s i n d i c a t e s that c y t o l o g i c a l examination of the various  t i s s u e s of p l a n t s i s necessary b e f o r e these  can be i d e n t i f i e d as t r u e  polyploids.  plants  5 CAUSES OF POLYPLOIDY The  occurrence o f spontaneous p o l y p l o i d y has been  a t t r i b u t e d t o extreme temperature changes and other sudden and severe changes i n the environment.  Experim-  e n t a l evidence i n d i c a t e s t h a t p o l y p l o i d y may be induced by r a t h e r moderate temperature changes i n some genera, but t h a t sudden changes a r e more e f f e c t i v e .  Extreme  temperature changes encountered by d i p l o i d races  that  extend i n t o unfavourable t e r r i t o r y , or a change i n the l o c a l c l i m a t e , might cause p o l y p l o i d y .  A survey o f  the n a t u r a l p o l y p l o i d s i n d i c a t e s t h a t they have a more n o r t h e r l y d i s t r i b u t i o n or a more a l p i n e h a b i t a t t h e i r d i p l o i d ancestors. subjected  to g r e a t e r  than  In such h a b i t a t s p l a n t s a r e  and sharper temperature v a r i a t i o n s  which might be the cause of p o l y p l o i d y  (Sax,1936).  Love and Love (1957) b e l i e v e that the higher frequency of p o l y p l o i d s i n a r c t i c r e g i o n s  i s an i n d i c a -  t i o n o f the a b i l i t y of p o l y p l o i d s t o s u r v i v e under adverse c o n d i t i o n s .  T h i s theory,  however, does n o t  support the f i n d i n g s o f Bowden (19^0) who i n v e s t i g a t e d the hardiness  o f p o l y p l o i d s as compared to that o f  d i p l o i d s and d i d not f i n d any g r e a t e r hardiness  i n the  polyploids. Several abnormalities asyndesis  i n meiosis,  such as  (Andersson, 19^-7) have been observed t o favour  the formation  of:.polyploidy i n p l a n t s  B  Abnormalities  in  6 the r e p r o d u c t i v e production  organs of p l a n t s might a l s o favour  o f unreduced gametes and  T h i s suggestion  thus cause p o l y p l o i d y .  f i n d s support i n a study made by  (195*+) on a hermaphrodite clone of grey poplar canescens Smith) w i t h a b n o r m a l i t i e s of the f l o w e r i n g produced by  parts.  One  the  Seitz  (Populus  i n the development  per cent of the progeny  s e l f p o l l i n a t i o n i n such p l a n t s were t r i p l o i d .  Upon c y t o l o g i c a l examination of the anthers i t was  found  that some of the p o l l e n mother c e l l s remained i n the d i p l o i d stage due and  i n t h i s way  a r i s e and  to f a i l u r e i n the r e d u c t i o n  division,  d i p l o i d p o l l e n g r a i n s are b e l i e v e d  produce p o l y p l o i d progeny.  that s e l f i n g i n p l a n t s i n c r e a s e s  to  It i s possible  the chances of  occurrence of p o l y p l o i d s s i n c e there  also  the  i s more chance f o r  the accumulation of chromosome a b n o r m a l i t i e s  due  to a  lower v a r i a b i l i t y i n the g e n e t i c make-up i n such a method of f e r t i l i z a t i o n . conifers  The  f a c t that s e l f i n g i s r a r e among the  (Allen,19^2) might e x p l a i n i n p a r t the r a r i t y  p o l y p l o i d y i n those  species.  Andersson (19^7) considered nature through the formation  p o l y p l o i d y to a r i s e i n  of unreduced gametes due  f a i l u r e i n the r e d u c t i o n d i v i s i o n . the other hand, b e l i e v e d doubling  Kiellander  and  to  (1950),  on  t h a t , at l e a s t i n the c o n i f e r s ,  of the chromosome number i s more l i k e l y to take  p l a c e a f t e r f e r t i l i z a t i o n than before i n  of  Datura stramonium by B l a k e s l e e  i t . T h i s was  found  (Vide Muntzing,1936),  might a l s o be true i n the gymnosperms.  7 S e v e r a l workers (Namikawa,193^) reported seedlings  a higher  (Johnsson,19^6) have  percentage of p o l y p l o i d s among t w i n  o f s e v e r a l genera than i n the corresponding  normal s e e d l i n g  populations.  It i s a l s o conceivable  that, i n nature, polyploids  may a r i s e through the spontaneous development o f chimeras and  the subsequent development of i n f l o r e s c e n c e s on the  polyploid sectors  (Andersson,19 +7) • !  8 CHANGES CAUSED BY POLYPLOIDY P o l y p l o i d s may  d i f f e r from d i p l o i d s i n the f o l l o w i n g  ways: a) v i g o r of v e g e t a t i v e  growth (may be g r e a t e r  or  lower),  b) s i z e of c e l l s and c e l l n u c l e i (may be l a r g e r ) , c) f r u i t  s i z e and shape (may be s h o r t e r and s t o u t e r ) ,  d) chemical and p h y s i c a l p r o p e r t i e s  (may be q u i t e  different), e) s i z e and number o f stomata (may be l a r g e r and f e w e r ) , f ) f e r t i l i t y of p o l l e n g r a i n and seed (may be lower i n autopolyploids  and higher  i n allopolyploids  than i n the corresponding parent s p e c i e s ) . g) c o l o u r and shape of l e a v e s  (may be darker and  thicker). These changes are extremely v a r i a b l e however, and, when p o l y p l o i d y i s to be induced a r t i f i c i a l l y , cannot be  i t s effects  predicted.  In f o r e s t r y a p p l i c a t i o n s d e s i r a b l e changes would i n c l u d e g r e a t e r r a t e o f growth and an improvement  i n the  chemical and p h y s i c a l p r o p e r t i e s of the woody p a r t s . increased  r a t e o f growth has been reported  Angiosperm-plants as poplar  An  i n such  (Johnsson,19^2,1953? and  o t h e r s ) , and b i r c h (Johnsson,19^6,1956, and o t h e r s ) . Among the c o n i f e r s , good r e s u l t s have been obtained allotriploid  l a r c h which was obtained  with  as the only progeny  9 from the c r o s s o f two s p e c i e s  1938),  (Larsen and Westergaard,  and a g i a n t t e t r a p l o i d fprm has been r e p o r t e d i n  s e e d l i n g s o f Cryptomeria .iaponica ( C h i b a , 1 9 5 2 ) . A l l other i n s t a n c e s of p o l y p l o i d y r e p o r t e d i n f o r e s t t r e e s p e c i e s r e s u l t e d i n r e t a r d e d growth r a t e s compared those  of the corresponding  with  diploids.  As f a r as s t r u c t u r a l changes i n the wood a r e concerned, Kanezawa  (1951)  r e p o r t e d an i n c r e a s e i n f i b e r  l e n g t h o f 2,0% and i n f i b e r t h i c k n e s s o f h0% i n induced p o l y p l o i d s of Japanese cypress  (Chamaecyparis obtusa E n d l ) .  S e v e r a l r e p o r t s have been made o f an i n c r e a s e d water i n a u t o p o l y p l o i d s as compared w i t h d i p l o i d s . made a study  (1950)  o f the wood i s lower i n the t r i p -  than i n the c o n t r o l and the s p e c i f i c weight of 'the  wood i n the d r y s t a t e i s lower. shrinkage two  Johnsson  o f t h i s problem i n Alnus g l u t i n o s a and found  t h a t dry matter content loid  content  No d i f f e r e n c e i n the  p r o p e r t i e s o f the wood was observed between the  strains.  On the b a s i s of the measured data t h e pore  volume was determined and found to be  70.13$  volume i n the t r i p l o i d s as compared with diploids.  o f the t o t a l  68.1*+$  i n the  T h i s d i f f e r e n c e i n pore volume i s e x p l a i n e d by  Johnsson as r e s u l t i n g from the l a r g e r elements i n the wood of the t r i p l o i d s . S e v e r a l e x t e r n a l f e a t u r e s have been suggested by v a r i o u s workers as a i d s i n i d e n t i f y i n g p o l y p l o i d s and  10 d i s t i n g u i s h i n g them from d i p l o i d s o f the same s p e c i e s . None o f these has proved chromosomal Sax  t o be so r e l i a b l e as a d i r e c t  count.  (1938) s t u d i e d the number o f stomata per  m i l l i m e t e r o f l e a f s u r f a c e i n herbarium number o f p l a n t genera condition.  square  material of a  i n the d i p l o i d and p o l y p l o i d  He found, with some e x c e p t i o n s , a c o r r e l a t i o n  between chromosomal counts and the s i z e o f stomata.  In  h i s opinion,.:stomatal s i z e cannot be used as an a b s o l u t e index o f p o l y p l o i d y , but i n many cases i t may be o f use i n p r e l i m i n a r y surveys.  Johnsson (19^0) d i d not f i n d a  d i r e c t c o r r e l a t i o n between stomatal l e n g t h and chromosome number i n the genus Populus  but, on the average,  aneuploids  seemed t o have l a r g e r stomata than d i p l o i d s , and t e t r a p l o i d s l a r g e r stomata than aneuploids and d i p l o i d s . The  study o f p o l y p l o i d y i n f o r e s t t r e e s up t o now  d e a l t only w i t h the Co (Co i s the g e n e r a t i o n t r e a t e d w i t h c o l c h i c i n e ) and C l ( C l i s the f i r s t progeny g e n e r a t i o n o f p l a n t s t r e a t e d w i t h c o l c h i c i n e ) g e n e r a t i o n s and consequently l i t t l e i s known a t present about the p r o p e r t i e s of subsequent generations i n f o r e s t t r e e s .  The f i n d i n g s o f  W e t t s t e i n (1937) i n p o l y p l o i d r a c e s o f mosses might throw l i g h t on the s u b j e c t .  F o l l o w i n g the development o f these  mosses f o r s e v e r a l g e n e r a t i o n s , he found  t h a t there was a  decrease i n s i z e of c e l l s and o f stomata with the passage  11 of generations so t h a t , a f t e r 11 y e a r s , the p o l y p l o i d s had the  same c e l l and stomata s i z e s as the d i p l o i d s .  was a l s o g r a d u a l l y  restored  Fertility  y e t the chromosome number rem-  ained the same and the p o l y p l o i d races d i d not cross the o r i g i n a l r a c e s .  Kanezawa (19*+8) considered t h a t a  s i m i l a r development i s l i k e l y to take p l a c e and  with  i n forest  that w i t h the passage o f g e n e r a t i o n s , as the p o l y p l o i d s  become adapted t o the environment, d i f f e r e n c e s stomatal s i z e s w i l l tend to be The  i n c e l l and  eliminated.  s i z e and f e r t i l i t y o f the p o l l e n g r a i n s  have been  suggested by some as a i d s i n d i s t i n g u i s h i n g between p l o i d y and d i p l o i d y . considerably  poly-  The p o l l e n g r a i n s u s u a l l y tend to be  l a r g e r and to have a much lower f e r t i l i t y i n  p o l y p l o i d r a c e s than i n the corresponding d i p l o i d s . (1938) i n h i s r e s e a r c h e s on spontaneous t r i p l o i d a r r i v e d a t the c o n c l u s i o n  the q u a l i t y of the p o l l e n .  polyploids fertility  Peto  poplars  that no c o r r e l a t i o n e x i s t e d  between chromosome number, the s i z e o f the p o l l e n and  trees  grain,  Furthermore, i n one o f the  (Populus a l b a v a r . a u e r o i n t e r t e x t a )  he found the  of the p o l l e n t o be as h i g h as 9*+ per c e n t .  12 LIMITATIONS OF POLYPLOIDY The  f i n d i n g s o f v a r i o u s workers i n the f i e l d o f  p o l y p l o i d y are summarized below as' t o i t s e f f e c t on the vigour  of p l a n t s .  Plants polyploidy.  show a v a r y i n g  degree o f s e n s i t i v i t y t o  F o r every s p e c i e s there i s an optimal  of chromosomes w i t h which i t e x h i b i t s i t s h i g h e s t and  r a t e o f growth.  vigor  When t h i s optimal number i s exceeded,  the growth r a t e and v i t a l i t y d e c r e a s e . considered  number  (1937)  Darlington  that the optimal number i s c o n d i t i o n e d  by the  s i z e of the metaphase p l a t e and the a v a i l a b i l i t y o f e x t r a space to permit an i n c r e a s e i n the number of chromosomes. According  t o him, p l a n t s w i t h a r e l a t i v e l y s m a l l number  of chromosomes and w i t h small chromosomes a r e , g e n e r a l l y speaking., more s u i t a b l e t o p o l y p l o i d i n d u c t i o n than- p l a n t s w i t h a l a r g e b a s i c chromosome number i n t h e i r genome or w i t h l a r g e chromosomes.  For t h i s reason he  considered  the angiosperms more l i k e l y t o r e a c t f a v o u r a b l y d u c t i o n than the other Pauley  to i n -  c l a s s e s of p l a n t s .  (19*+9) s t a t e d that t r i p l o i d y seems t o be the  n a t u r a l l i m i t i n the genus Populus and, t h e r e f o r e , the optimal number.  L i e l l a n d e r (1950) b e l i e v e d t h a t the  optimal number o f chromosomes i n Norway spruce Abies  (Picea  (L) K a r s t ) i s 2n = 2h and any i n c r e a s e o f the  number o f chromosomes above t h i s number would i n poor growth and lowering  of v i t a l i t y .  result  13 VARIABILITY OF POLYPLOIDS P o l y p l o i d y , -which i s e s s e n t i a l l y the increase  quantitative  of the -chromosome complement of p l a n t s , produces  v a r i o u s r e s u l t s i n d i f f e r e n t i n d i v i d u a l s of the same s p e c i e s . T h i s i s p a r t i c u l a r l y true i n c r o s s - f e r t i l i z e d p l a n t s  be-  cause i n such cases there tends to be an e f f e c t i v e r e combination of the g e n e t i c m a t e r i a l .  One  increased v a r i a t i o n i n autopolyploids  can be sought i n the  disturbances  l e a d t o numerical  aberrants ility  during m e i o s i s which may  and  aneuploids.  source of  There i s , however, the  that the e f f e c t s of such d i s t u r b a n c e s  eliminated  may  the  possib-  be  to a g r e a t degree at the gametic stage.  source of i n c r e a s e d v a r i a t i o n i n p o l y p l o i d s may i n c r e a s e d number of l o c i and  consequently the  p o s s i b i l i t y of recombination a f f o r d e d .  I f the  Another  be  the  greater character-  i s t i c i n v e s t i g a t e d i s determined by s e v e r a l genes s i m u l t aneously w i t h extreme f a c t o r i a l combinations, an v a r i a t i o n i s doubtless ploids  increased  a l s o to be expected i n the  poly-  (Johnsson,1950).  That the v a r i a b i l i t y  of p o l y p l o i d s i s g r e a t e r  that of the corresponding d i p l o i d s i n v a r i o u s p l a n t s i s a f a c t s t a t e d by many workers.  than  types of  Genetic  erences of d i p l o i d p l a n t s are accentuated i n the  diffpolyploids.  a  In order  to produce/superior s t r a i n of p o l y p l o i d s , s e l e c t i o n  i s necessary p r i o r to i n d u c t i o n as w e l l as a f t e r i t .  Ih POLYPLOIDS AND ENVIRONMENT I t i s widely  accepted  of chromosome d o u b l i n g  a t present  t h a t the process  i n a l l o p o l y p l o i d s has played a  great r o l e i n the e v o l u t i o n o f p l a n t s p e c i e s . however, s t i l l  There i s  a g r e a t d i v e r s i t y o f o p i n i o n among the  v a r i o u s workers w i t h regard to the e v o l u t i o n a r y  signific-  ance of chromosome d o u b l i n g  Muntzing  i n autopolyploids.  (1933) considered a u t o p o l y p l o i d y t o be o f importance i n p l a n t e v o l u t i o n because i t ; a) a f f e c t s both t h e morphology and the p h y s i o l o g y of plants. b) causes sexual i s o l a t i o n of p o l y p l o i d s . c) may g i v e r i s e t o p o l y p l o i d races and s p e c i e s i n nature. Stebbins  (19^7), on the other hand, considered  the e v o l u t i o n a r y processes  i n nature  that  are l i k e l y t o l i m i t  the importance o f a u t o p o l y p l o i d y because o f the reduced fertility  and the i n a b i l i t y t o g i v e r i s e t o anything new  among the d e r i v a t i v e s o f a u t o p o l y p l o i d s .  According  t o him,  the importance of chromosome d u p l i c a t i o n i n the e v o l u t i o n of p l a n t s i s , i n the main, i n f i x i n g and spreading  hybrid  combinations, e i t h e r i n t e r v a r i a t e o r i n t e r s p e c i f i c . The  f a t e o f a spontaneous a u t o p o l y p l o i d w i t h i n a  p o p u l a t i o n o f a d i p l o i d prototype  from which i t arose has  not been i n v e s t i g a t e d f o r f o r e s t t r e e s but two s t u d i e s conducted on b a r l e y s p e c i e s throw l i g h t on the problem. Sakai and Suzuki  (1955a,b) have found that, a u t o t e t r a p l o i d  15 s t r a i n s of b a r l e y were almost always poor competitors a g a i n s t the d i p l o i d s .  On the other hand, a l l o p o l y p l o i d s  were found to be s u p e r i o r to both p a r e n t a l s t r a i n s . f i n d i n g s might e x p l a i n the s u r v i v a l o f Sequoia an a u t o a l l o p o l y p l o i d w i t h 2n = M+  These  sempervirens,  (Stebbins,19 +8), i  and  Aesculus carnea, an a l l o p o l y p l o i d with 2 n = 80 (Upcott,  1936). I f the above f i n d i n g s are u n i v e r s a l l y true f o r p l a n t s , it  i s apparent that t h e r e are good p r o s p e c t s f o r f u t u r e  a l l o p o l y p l o i d s which could be developed a r t i f i c i a l l y naturally.  It i s also l i k e l y  or  that a u t o p o l y p l o i d s would  have to be propagated v e g e t a t i v e l y f o r each g e n e r a t i o n i n cases where they do not produce f e r t i l e  seeds.  16  TRIPLOIDY IN WOODY PLANTS T r i p l o i d s o f s e v e r a l woody p l a n t s e x h i b i t an i n c r e a s e d growth r a t e i n comparison *n the genus Populus  w i t h d i p l o i d s of the same s p e c i e s .  both spontaneous and  induced  triploids  are much f a s t e r growing and b i g g e r than the corresponding diploids  (Muntzing, 1 9 3 6 ,  and  others). Triploidy also  duced good r e s u l t s i n l n u s g l u t i n o s a A  (L) G e r t n ; young  to be 50% t a l l e r on the average  seed-  a  l i n g s of t h i s s p e c i e s were s t u d i e d by Johnsson found  pro-  (1950)  and  than the d i p l o i d s .  r e a l d i f f e r e n c e i n h e i g h t a t t r i b u t a b l e to t r i p l o i d y was bably h i g h e r s i n c e the t r i p l o i d  The pro-  s e e d l i n g s i n c l u d e d many aneu-  p l o i d s w i t h r e t a r d e d growth, i n B e t u l a verucosa  triploidy  produced  diameter  r o b u s t s e e d l i n g s w i t h a l a r g e r average  than t h a t of the d i p l o i d s "but w i t h a somewhat reduced (Johnsson, In  height  1956).  the c o n i f e r s , examples of spontaneous a u t o t r i p l o i d y  are unknown. In these the p o t e n t i a l i t i e s of induced  autotrip-  l o i d y ' are a t p r e s e n t unknown. In a p r i v a t e communication to the author, K i e l l a n d e r "artificial  (1957)  expressed  the o p i n i o n t h a t :  t r i p l o i d s o f such hardwoods as B e t u l a , Populus,  Alnus, and Ulmus possess good growing c a p a c i t i e s . A u t o t r i p l - r o i d s among the c o n i f e r s have not, however, been produced yet;  consequently we know nothing about a u t o t r i p l o i d Allotriploid larch  was  as  conifers".  (Larix decidua/Larix pccidentalis)  o b t a i n e d by L a r s e n and Westergaard  (1938).  This i s  17 the only mention found i n t h i s l i t e r a t u r e survey t r i p l o i d y of any k i n d i n the c o n i f e r s .  This  of  allotriploid  was  the only progeny obtained from a cross between the  two  s p e c i e s of l a r c h .  (1957)  Soegard  r e p o r t e d on  the  development of the above a l l o t r i p l o i d :  " i t has an  ex-  c e l l e n t r a t e of growth and  1957  i n January,  l*+.5  was  metres t a l l which i s almost the h e i g h t of a h y b r i d l a r c h L a r i x d e c i d u a / L a r i x l e p t o l e p i s of the same The main d i f f i c u l t y of both hardwoods and  age."  i n the p r o d u c t i o n of t r i p l o i d s  c o n i f e r s i s t h a t they cannot  induced d i r e c t l y from d i p l o i d s .  The  case of the  allo-  t r i p l o i d l a r c h c i t e d above i s uncommon and probably p l a c e because the two compatible cross.  and  parent  could not produce a d i p l o i d progeny of the to be produced through a  c r o s s between a t e t r a p l o i d and a d i p l o i d . t e t r a p l o i d s bear f r u i t at an o l d e r age  The  f a c t that  than d i p l o i d s makes  the problem more s e r i o u s i n woody p e r e n n i a l s . overcoming t h i s d i f f i c u l t y Larson,  development of new orchard work, found  One  way  i s to g r a f t t e t r a p l o i d s  on  (1956)  techniques  and  others, with  i n connection w i t h  the seed  age.  T r i p l o i d s , owing to t h e i r degree of s t e r i l i t y , difficulty  of  i n r e c e n t years that g r a f t i n g i s con-  ducive to f r u i t bearing a t a younger  another  took  s p e c i e s were mutually i n -  Normally a t r i p l o i d has  d i p l o i d stock.  be  present  i n the f a c t t h a t they are i n c a p a b l e  b e a r i n g a progeny t r u e to type.  At l e a s t two  types  of  of  gametes, u n i v a l e n t s and b i v a l e n t , and i n many cases unreduced t r i v a l e n t gametes, have been r e p o r t e d i n t r i p -  18  loids.  The high frequency of occurrence of aneuploids, i n  a d d i t i o n to the v a r i e t y of euploids w i t h d i f f e r e n t chromosome count, which have been reported as the progeny of t r i p l o i d s (Johnsson,19^2) makes i t c l e a r that v e g e t a t i v e propagation would be the only p r a c t i c a l means of reproducing t r i p l o i d s .  The e x t r a cost i n v o l v e d i n such propa-  g a t i o n would be a l i m i t i n g f a c t o r i n undertaking of t r i p l o i d s .  cultivation  I n the case of t r i p l o i d clones of poplar  such a means of propagation was found worthwhile and has been employed i n Sweden f o r some time.  Whether such a  method a l s o would be worthwhile i n other species w i l l have to be decided i n d i v i d u a l l y f o r each species a f t e r the added value of the t r i p l o i d crop has been compared w i t h the e x t r a cost involved i n v e g e t a t i v e propagation.  19 ALLOPOLYPLOIDY IN WOODY PLANTS A l l o p o l y p l o i d y i s d i r e c t l y e f f e c t i v e i n producing new s p e c i e s s i n c e such p o l y p l o i d s r e s u l t from  species  h y b r i d i z a t i o n f o l l o w e d by a chromosomal i n c r e a s e .  The  d u p l i c a t i o n o f each o f the p a r e n t a l genomes r e s t o r e s fertility  and the h y b r i d breeds t r u e to type.  a l l o p o l y p l o i d i s a constant  Thus the  s p e c i e s h y b r i d which has  c h a r a c t e r i s t i c s o f a t r u e s p e c i e s , and i n the case o f (Sax,1936).  g e n e r i c c r o s s e s may m e r i t a g e n e r i c rank A l l o p o l y p l o i d s are known to occur  i n nature and  under c u l t i v a t i o n , and have been produced in  several different f a m i l i e s .  Examples o f n a t u r a l  p o l y p l o i d woody p e r e n n i a l s are Sequoia (2n  experimentally allo-  sempervirens  = k-k) among the c o n i f e r s (Stebbins,19 +8) and Aesculus l  carnea ( 2 n = 8 0 ) among the hardwoods A l l o p o l y p l o i d s with  (TJpcott,1936).  s u p e r i o r growth q u a l i t i e s have  been produced a r t i f i c i a l l y i n f e r t i l e crosses f e r t i l e .  and i n numerous cases rendered I t i s s i g n i f i c a n t t h a t many of  the most important c u l t i v a t e d p l a n t s today are o f known or supposed a l l o p o l y p l o i d o r i g i n .  Very l i t t l e use has been  made i n f o r e s t r y thus f a r o f a l l o p o l y p l o i d y i n the propagat i o n of hybrid v a r i e t i e s of t r e e s .  20 POLYPLOIDY AMONG THE CONIFERS Changes i n the chromosome number have been of l i t t l e importance i n the d i f f e r e n t i a t i o n between families and genera of most coniferous trees.  Sax and Sax (1933)  believe that the s t a b i l i t y of the conifers with respect to polyploidy indicates that evolution i n t h i s group has passed i t s climax and that the existing forms are survivors of long natural s e l e c t i o n .  In another study, Sax (1932)  stated that the comparatively high number of chiasmata with the prevalence of i n t e r s t i t i a l chiasmata account f o r the great uniformity of chromosome number and the general s t a b i l i t y of that group.  He attributes the r a r i t y of  polyploids to the large number of chiasmata i n the d i p l o i d stage.  There i s an average of 2 A i n t e r s t i t i a l chiasmata  per bivalent and t h i s number seems to be remarkably constant i n a l l the conifers covered i n h i s study. Any autopolyploids  produced would be l i k e l y to form closely  paired tetravalents and the segregation  of homologous  chromosomes would be too i r r e g u l a r to produce a high degree of f e r t i l i t y .  Muntzing (1933) explained  the r a r i t y of  polyploids among the conifers by the absence of double f e r t i l i z a t i o n i n t h i s group and the fact that d i p l o i d and polyploid races can cross readily and,  consequently,  the polyploid forms are not i s o l a t e d and developed independently.  This hypothesis might also explain the  d i f f e r e n t i a t i o n of r e l a t i v e l y few species and genera  21 among t h e c o n i f e r s .  The m e c h a n i s m o f e v o l u t i o n i n t h e  c o n i f e r s h a s i n v o l v e d a g a i n o r l o s s o f one chromosome, s t r u c t u r a l r e a r r a n g e m e n t , a n d gene m u t a t i o n s , b u t p o l y p l o i d y has played  a small role.  has  been r e s p o n s i b l e  and  Cupressaceae.  The l o s s o f a chromosome  f o r t h e e v o l u t i o n o f T a x o d i a c e a e and  This  involved  a l o s s of a centromere  w h i c h f o l l o w s t r a n s l o c a t i o n s o f a l l e s s e n t i a l genes t o t h e r e s t of the centromeres.  Instances  some a r e f e w and o c c u r o n l y i n the family Araucariaceae.  o f a g a i n o f a chromo-  i n t h e genus P s e u d o t s u g a and This  l i c a t i o n o f a c e n t r o m e r e and c o u l d  always i n v o l v e s dup- , be a c h i e v e d by a (1937)•  s y s t e m o f t r a n s l o c a t i o n s a s p r o p o s e d by D a r l i n g t o n The m a j o r i t y haploid  of t h e c o n i f e r s a r e thus found t o have a  chromosome number o f 12  ( n = 12).  The o n l y  known  g e n e r a w i t h d e v i a t i n g chromosome n u m b e r s a r e T a x o d i u m , T h u j a , and J u n i p e r u s ,  e a c h o f w h i c h has. a h a p l o i d  l e m e n t o f 11  and P s e u d o t s u g a and A r a u c a r i a  ( n = 11)  a h a p l o i d number o f 13  (n =  Spontaneous s t a b i l i z e d are very  rare.  The o n l y  compwith  13).  p o l y p l o i d s among t h e c o n i f e r s  c a s e s known a r e t h a t o f  Juniperus  c h i n e s i s v a r . P f i t z e i a n i a , a n a u t o t e t r a p l o i d w i t h a 2n = hh number o f c h r o m o s o m e s , a n d S e q u o i a s e m p e r v i r e n s , a n a u t o a l l o p o l y p l o i d a l s o w i t h a chromosome c o m p l e m e n t o f 2n = Occasional  o c c u r r e n c e o f s p o n t a n e o u s p o l y p l o i d s among t h e  conifers i s reported has  M+.  f r o m t i m e t o t i m e b u t none o f t h e s e  p r o v e d t o be s t a b i l i z e d .  The o l d e s t r e p o r t e d  p o l y p l o i d i s t h a t f o u n d by C h r i s t i a n s e n  such  (1952) i n Denmark.  22 T h i s a u t o t e t r a p l o i d , L a r i x d e c i d u a M i l l e r , w h i c h was "between 56 and 58 y e a r s o l d a t t h e time o f d i s c o v e r y , was 15.2  metres t a l l and 97.5  cms. a t b r e a s t h e i g h t .  The  t e t r a v a l e n t chromosome complement was found t o a c t v e r y i r r e g u l a r l y d u r i n g m e i o s i s and most of the seeds produced were found t o be h o l l o w .  23 SPONTANEOUS CONIFEROUS POLYPLOIDS IN NURSERY STOCK The o c c u r r e n c e  of spontaneous p o l y p l o i d s among  c o n i f e r o u s n u r s e r y s t o c k has been r e p o r t e d f o r s e v e r a l species.  K i e l l a n d e r (1950)  r e p o r t e d such an o c c u r r e n c e  i n two-year-old Picea Abies.  The seed m a t e r i a l f o r t h e s e  s e e d l i n g s was g a t h e r e d from a l a r g e number o f t r e e s and i t was found t h a t t h e p o l y p l o i d a b e r r a n t s were a r a r e o c c u r r e n c e among t h e progeny o f many d i f f e r e n t The c a l c u l a t e d frequency  of occurrence f o r P i c e a Abies  was 8 per 1 0 0 , 0 0 0 s e e d l i n g s . resembled  trees.  The p o l y p l o i d  aberrants  t h e p o l y p l o i d s o f t h e same s p e c i e s w h i c h were  produced by means o f c o l c h i c i n e t r e a t m e n t . the same slowness  They e x h i b i t e d  o f growth and t h i c k n e e d l e s .  of t h e t o t a l h e i g h t o f t h e s e t w o - y e a r - o l d  The r a t i o  seedlings to  the t o t a l h e i g h t o f normal s e e d l i n g s o f t h e same age was found t o be 0 . 3 . Z i n n a i (1955) made a study o f spontaneous p o l y p l o i d y i n nursery stock of Cryptomeria  .iaponica and found, an  o c c u r r e n c e o f 5 . 1 5 p e r 1 0 , 0 0 0 i n unthinned o n l y 1.6*+ i n t h i n n e d seedbeds.  seedbeds and  Z i n n a i d i d n o t g i v e any  f i g u r e s on l e n g t h r a t i o o f p o l y p l o i d and normal s e e d l i n g s , but s t a t e d t h a t among s e e d l i n g s w i t h h e i g h t s from 6 . 5 t o 7 . 5 cm. t h e r e were 2 . 2 3 p o l y p l o i d s compared w i t h 1 . 6 5 p e r  1 0 , 0 0 0 s e e d l i n g s w i t h a h e i g h t o f 7 . 5 t o 9 . 0 cm, and o n l y 0 , 5 6 per 1 0 , 0 0 0 i n t h e 9 . 0 t o 1 5 . 0 cm. h e i g h t group.  He  a l s o found t h a t t h e r e was a h i g h o c c u r r e n c e o f p o l y p l o i d s  2h among t h e s e e d l i n g s removed by t h i n n i n g .  T h i s agrees w i t h  K i e l l a n d e r ' s explanation for the r a r i t y of p o l y p l o i d spruce and t h e r e a s o n t h a t p o l y p l o i d s o f t h i s and o t h e r c o n i f e r s have h i t h e r t o escaped o b s e r v a t i o n .  Apparently  a u t o t e t r a p l o i d s i n the c o n i f e r s are poor c o m p e t i t o r s o f the d i p l o i d s and i n a d d i t i o n , they do not reproduce t r u e to  type. Chiba and Watanabe (1952) have i n v e s t i g a t e d t h e  occurrence o f a u t o t e t r a p l o i d s i n nursery  stock of L a r i x  k a e m p f e r i and found t h a t they l a c k e d t h e g i a n t form o f the a u t o t e t r a p l o i d C r y p t o m e r i a .iaponica s e e d l i n g s ,  reported  e a r l i e r by Chiba (1950). No account has been found i n the l i t e r a t u r e o f t h e o c c u r r e n c e o f spontaneous p o l y p l o i d y i n Douglas f i r . Only one i n s t a n c e , (Meyer,195D i s known o f i n d u c t i o n o f polyploidy i n that species.  Meyer immersed seeds f o r  f o u r days i n a 0.2% s o l u t i o n o f c o l c h i c i n e i n water and obtained  s e e d l i n g s which proved t o be t e t r a p l o i d s .  Dean  George; S. A l l e n , F a c u l t y o f F o r e s t r y , U n i v e r s i t y o f B r i t i s h Columbia, and Mr. R. R. S i l e n e , U. S. F o r e s t S e r v i c e , P a c i f i c N. ¥. F o r e s t Experiment S t a t i o n , C o r v a l l i s , Oregon, t r i e d t o i n d u c e p o l y p l o i d y i n Douglas f i r by t r e a t i n g seed w i t h c o l c h i c i n e and have  obtained  abnormal s e e d l i n g s s i m i l a r t o those d e s c r i b e d  f o r other  coniferous  species.  25 P O T E N T I A L I T I E S OF P O L Y P L O I D Y I N In said  that,  induce all is  concluding despite  superior  the  this the  review  believed that  of  attempts  polyploids  polyploidy  in  c a n be u s e f u l  detail. might  It  is  species  in  varieties.  polyploidy  is  developing  diploid  of  their  of  the  plant  colchicine  or  of  the  in  polymore  characteristics  corresponding  to  to  In  which the  any  polyploid  and  of  entail  event,  population  varieties. be  selection the  resulting  the  in  increased  to  as i n  varieties  abandoned.  be f o u n d  produce better  and w e i g h e d a g a i n s t  would  have  in  The a d d e d , v a l u e  methods  field  chromosomes w o u l d have  superior  production  Improved  of  would  polyploids  a polyploid  forms.  be d e t e r m i n e d in  induction  each s p e c i e s .  of  production  those  in  is  number  essential in  place  this  chromosomes tends  as  take  hybrids.  before  true  for  fertile  necessary  produce  determined  Allopolyploidy,  these modified  than  by  investigation  order  The o p t i m a l  a good means  It  much more  of  of  trees,  whole,  methods  number  or  On t h e  Improved to  find  s h o u l d be i n v e s t i g a t e d  possible that  be more d e s i r a b l e  diploid of  various  can he  forest  c a n be i m p r o v e d .  The c h e m i c a l and p h y s i c a l c h a n g e s t h a t of  to  in  can provide  varieties  ploids  it  h a v e by no means b e e n e x h a u s t e d .  which e x i s t i n g particular,  literature,  many f u t i l e  varieties  possibilities  of  FORESTRY  variants  from  the  would have  extra  cost  is  to  involved  cultivation.  inducing  polyploidy  different  a combination of  this  ih  applications and o t h e r  all  tissues  of  chemicals  to  26 e n s u r e p e n e t r a t i o n . I n t h e p a s t , c o l c h i c i n e t r e a t m e n t was c o n f i n e d t o g e r m i n a t i n g s e e d s and s h o o t s . One a t t e m p t t o induce polyploidy  i n e x c i s e d embryos o f p i n e was  o u t w i t h o u t s u c c e s s , by Hyun (195^). I n e x c i s e d  carried embryos  the  m e r i s t e m a t i c t i s s u e i s more e x p o s e d  and  t r e a t m e n t o f e x c i s e d emryos i n c u l t u r e s h o u l d g i v e  b e t t e r r e s u l t s . Haddock ing  than i n seeds  (195*+) d e s c r i b e d a method o f g r o w -  e x c i s e d embryos o f s u g a r p i n e i n c u l t u r e , and s u c h a  m e t h o d , i f i t p r o v e s t o be s u c c e s s f u l i n o t h e r c o u l d be u s e d i n p o l y p l o i d a l i n d u c t i o n  conifers,  work.  The f a c t t h a t t h e r e i s no d o u b l e f e r t i l i z a t i o n i n the of  c o n i f e r s c o u l d be u s e d i n a t t e m p t s t o i n d u c e d o u b l i n g t h e chromosomes  i n t h e g a m e t i c s t a g e and m i g h t p r o v e t o  be u s e f u l i n t h e p r o d u c t i o n o f c o n i f e r o u s In  autotriploids.  t h e a n g i o s p e r m s , on t h e o t h e r hand, d o u b l i n g o f t h e  chromosomes  i n the gametic stage i s l e s s l i k e l y  since double f e r t i l i z a t i o n  and t h e d i f f e r e n t  to succeed  chromosomal  c o u n t i n t h e embryo and t h e e n d o s p e r m t i s s u e s m i g h t difficulties  i n the development  o f the seed.  cause  PART B A PRELIMINARY STUDY OF ABERRANT SEEDLINGS IN DOUGLAS F I R  27  INTRODUCTION The occurrence of a b e r r a n t s i n nursery s t o c k a t the B.C.  F o r e s t S e r v i c e nursery a t Duncan has been n o t i c e d  for several years.  Mr. Jack Long, superintendent o f the  n u r s e r y , has separated some of these a b e r r a n t s and p l a n t e d them i n two rows on the nursery grounds.  The o l d e s t o f  these a b e r r a n t s i s now t e n years o l d , but none has p r o duced any cones thus f a r . The a b e r r a n t s a r e c h a r a c t e r i z e d by t h i c k e r , darker needles than those i n normal s e e d l i n g s and by a r e t a r d e d growth.  The buds of the a b e r r a n t s a r e more round and  s m a l l e r than the normal buds ( F i g s . 1,2,3>^)» The frequency of occurrence o f the a b e r r a n t s has been estimated by Mr. Long as from 6 to 10 per m i l l i o n lings.  seed-  I t i s p o s s i b l e t h a t some were unnoticed and the  frequency o f occurrence may a c t u a l l y be h i g h e r .  The  d i f f e r e n t phenotypes t h a t a r e found among the a b e r r a n t s and t h e i r presence each year suggest t h a t they a r e o f r a r e occurrence among the progeny o f v a r i o u s t r e e s . e x t e r n a l f e a t u r e s of these a b e r r a n t s and t h e i r and r a r e occurrence every year s t r o n g l y suggest polyploidy.  They resemble  spontaneous auto-  a u t o p o l y p l o i d s which have been  d e s c r i b e d f o r other c o n i f e r o u s s p e c i e s ( K i e l l a n d e r , Chiba, 1952;  and o t h e r s ) .  The  1950;  The presence o f normal twigs on  some of the a b e r r a n t s ( F i g . h) resemble  sectorial  chimeras  i n autopolyploids. The i n v e s t i g a t i o n t o determine  whether these were  28  r e a l l y p o l y p l o i d s was undertaken i n the summer 1957> f o l l o w i n g a suggestion Orr-E\ri.ng.  by Dr. P. G. Haddock and Dr. A.  The study c o n s i s t e d o f :  a) The measurement of the l e n g t h of aberrant l i n g s and comparing them t o normal b) The embedding of c r o s s - s e c t i o n s " o f  seedlings. aberrant  needles and d e t e c t i n g any a b n o r m a l i t i e s  i n them.  c) The comparison of- stomata and guard c e l l i n the aberrants d) The breaking seedlings  and i n normal  seed-  lengths  seedlings.  of dormancy i n aberrant  and normal  t o f a c i l i t a t e a c y t o l o g i c a l study.  e) The f i n d i n g of a s u i t a b l e squash technique for  examining v e g e t a t i v e buds of Douglas f i r .  f ) The p r e p a r a t i o n  of s l i d e s of chromosomes i n  the d i v i d i n g c e l l s  of buds i n aberrant  normal Douglas f i r s e e d l i n g s .  and  29 MATERIALS AND METHODS The s t u d y was c o n f i n e d m o s t l y t o 22 a b e r r a n t s e e d l i n g s (5 O n e - y e a r - o l d and 17 t w o - y e a r - o l d ) a n d a n e q u a l number of normal  s e e d l i n g s o f t h e same a g e g r o u p s w h i c h w e r e  shipped t oVancouver,  t h a n k s t o t h e c o u r t e s y o f Mr. J .  £ong. T h e n o r m a l s e e d l i n g s w e r e p i c k e d a t random  from  among n u r s e r y s t o c k . A m e a s u r e m e n t o f t h e l e n g t h o f t h e a b e r r a n t s and n o r m a l planting  s e e d l i n g s was c a r r i e d o u t p r i o r t o  them i n p o t s i n t h e U n i v e r s i t y o f B r i t i s h  C o l u m b i a g r e e n - h o u s e o n November  25, 1957*  Needles o f a b e r r a n t and normal ded  i n paraffin,  s e e d l i n g s w e r e embed-  c u t i n t o c r o s s - s e c t i o n s , mounted, and  s t a i n e d w i t h s a f r a n i n and f a s t  green.  f o u n t s o f s t o m a t a f r o m a b e r r a n t and n o r m a l  needles  were p r e p a r e d by p e e l i n g o f f t h e l o w e r e p i d e r m i s o f needles a f t e r b o i l i n g inserting  i n water f o r a few m i n u t e s , t h e n  i n cold water  ( J o h a n s s e n , 19^0). T h i s method  o f p e e l i n g o f f t h e e p i d e r m i s was f o u n d t o be more  satis-  f a c t o r y t h a n t h e method e m p l o y i n g c o l l o d i o n f i l m s  (Long .  and e l e m e n t s , 193*+) • The s t o m a t a w e r e s t a i n e d w i t h a c e t o a c a r m i n e . The l e n g t h o f t h e g u a r d - c e l l s i n t h e a b e r r a n t and  i n t h e n o r m a l s e e d l i n g s was m e a s u r e d a n d c o m p a r e d . I n o r d e r t o b r e a k t h e dormancy o f t h e s e e d l i n g s t h e  following  t r e a t m e n t s were  a) C h i l l i n g planting  tried:  t h e s e e d l i n g s f o r 2h h o u r s p r i o r t o i n t h e g r e e n - h o u s e by p l a c i n g  the seed-  30 l i n g s i n a r e f r i g e r a t o r w i t h t h e i r r o o t s covered w i t h wet  (6  s o i l contained i n a p l a s t i c bag.  a b e r r a n t s and 6 normal s e e d l i n g s ) . b) Coating some of the buds of s e e d l i n g s grown i n the green house w i t h g i b e r e l l i c a c i d i n l a n o l i n paste (Marth e t a l , 1956). 0+ a b e r r a n t s and k normal s e e d l i n g s ) . c) I n c r e a s i n g the photoperiod i n the green house by 2 hours d a i l y .  (5 a b e r r a n t s and 7 normal s e e d l i n g s ) .  d) I n c r e a s i n g the photoperiod i n the green house by h hours d a i l y .  (5 a b e r r a n t s and 7 normal s e e d l i n g s ) .  e) T r e a t i n g p l a n t s grown i n d o o r s , i n a heated l a b o r a t o r y , w i t h a r t i f i c i a l l i g h t from o r d i n a r y t a b l e lamps f o r a l t e r n a t e p e r i o d s of 2>+ hours, i n a d d i t i o n to f e r t i l i z i n g w i t h s m a l l amounts of ammonium s u l f a t e .  0+ aberrant s e e d l i n g s ) .  During the course of the study i t was was fir.  found t h a t there  no completely s a t i s f a c t o r y squash technique f o r Douglas N e i t h e r the c o n s e r v a t i v e method w i t h  aceto-carmine  (Johanssen,19 +0) nor the method suggested by Mergen and 1  Novotny (1956), using c r y s t a l v i o l e t s t a i n , gave good r e s u l t s . By a process of t r i a l and e r r o r , v a r i o u s other were used and i t was  found t h a t an a d a p t a t i o n of the a c e t o -  lacmoid squash method suggested by M i k a e l s o n s t a i n i n g spruce and b i r c h was fir.  schedules  (1952) f o r  the most s u i t a b l e f o r Douglas  The f i x a t i v e used by him, however, was  found to  r e s u l t sometimes i n p l a s m o l y s i s of Douglas f i r c e l l s and d i d not g i v e as good r e s u l t s as a c e t i c a l c o h o l .  Only  temporary  s l i d e p r e p a r a t i o n s , s e a l e d w i t h n a i l p o l i s h , were used. M i k a e l s o n suggested  t h a t these s l i d e s c o u l d be made permanent  31 by removing the s e a l a f t e r four days and mounting i n w e l l aged ( a c i d ) e u p a r a l . The  f o l l o w i n g schedule  has been t r i e d i n chromosome  s t a i n i n g o f v e g e t a t i v e buds o f Douglas f i r and found to be most s u i t a b l e : Step 1, A c t i v e l y d i v i d i n g buds a r e cut o f f the s e e d l i n g or t r e e and d i s s e c t e d l o n g i t u d i n a l l y . Step 2. (This step i s c a r r i e d out only when an i n h i b i t i o n of the s p i n d l e formation and shortening d e s i r e d ; otherwise  of the chromosomes a r e  i t may be o m i t t e d ) .  D i s s e c t e d buds a r e  t r e a t e d as f o l l o w s : a) Placed i n a s o l u t i o n o f 1%> c o l c h i c i n e i n water f o r one hour under a r t i f i c i a l l i g h t from an o r d i n a r y t a b l e lamp.  (Mergen and Novotny, 1957)  b) Placed i n a 0.002M s o l u t i o n of 8 i n water f o r 2k hours. ( l i l i e s ,  hydro-oxyquinoline 1952)  c) Placed i n a s a t u r a t e d s o l u t i o n of p a r a d i c h l o r o benzene i n water f o r 2k hours. (Hyun, 195*0 • Step 3 . D i s s e c t e d buds a r e k i l l e d and f i x e d i n a c e t i c a l c o h o l 3*1  ( G l a c i a l a c e t i c a c i d t o 96% ethanol i n  the r a t i o of 3:1)  (Darlington,1950).  Step k. S i n g l e needles a r e e x c i s e d and h y d r o l i z e d i n a mixture of 96% ethanol and concentrated i n the r a t i o of 1:1  hydrochloric acid  f o r 5 to 15 minutes, depending on  hardness o f the m a t e r i a l . (Mikaelson,195*0 • Step 5. H y d r o l i z i n g f l u i d  i s washed o f f with water.  (Mikaelson, 195*0. Step 6. S i n g l e needles a r e placed on a s l i d e and s t a i n e d  32 w i t h standard a c e t i c lacmoid s t a i n \%  acetic acid).  (1% r e s o r c i n blue i n  (Mikaelson, 195*+)•  Step 7. Needles a r e squashed with a cover g l a s s .  The s l i d e  i s p l a c e d i n a b i b u l o u s book and pressure i s a p p l i e d t o i t . Tapping  o f the s l i d e may h e l p t o o b t a i n p o l a r views and  s c a t t e r the chromosomes. ( D a r l i n g t o n and La Cour, 1950). Step 8. S l i d e i s examined under a microscope  and, i f under-  s t a i n e d more s t a i n i s a p p l i e d , ( D a r l i n g t o n and La Cour,1950).  33 RESULTS The measurements of average l e n g t h of a b e r r a n t s normal s e e d l i n g s are presented i n Table TABLE 1.  No.of seedlings  and  1.  Comparison of stem and root l e n g t h s of one-and two-year-old a b e r r a n t s with those of an equal number of normal s e e d l i n g s picked at random.  Type  Length, inches Stem Root T o t a l  Range of Totalf Length^ inches'  R a t i o of Length Aberrant/ normal  One-year-old  5  Normal  h»9  *+.7  9.6  8.0-10.1  5  Aberrant  2.9  3.0  5*9  ^.5-6.0  0.6  13.5-25.*+ 8.5-21,5  0.6  Two-year-old  I B N o r m a l 12.5 17 Aberrant 6.6  9.0 21.5 6.0 12.6  From the t a b l e i t can be seen t h a t the r a t i o of the t o t a l l e n g t h of a b e r r a n t s to t h a t of normal s e e d l i n g s r e mained constant on the average,  f o r the one-and the  two-  year-old seedlings. The ranges  of the one-year-old  overlap and, i n the two-year-old aberrants overlapped  s e e d l i n g s d i d not  s e e d l i n g s , only three  the range of the normal s e e d l i n g s .  A " t " t e s t conducted  on the two-year-old  seedlings  i n d i c a t e d t h a t the normal and aberrant s e e d l i n g s were from d i f f e r e n t p o p u l a t i o n s w i t h a confidence l e v e l of much l e s s than 1 per cent.  3^ No i r r e g u l a r i t i e s i n the epidermal  l a y e r and  the  size  of the mesophyll c e l l s were noted i n the c r o s s - s e c t i o n s of the aberrant n e e d l e s .  Such i r r e g u l a r i t i e s were des-  c r i b e d i n a u t o p o l y p l o i d s of pine the f i r s t  (Hyun, 195k)•  This  i n d i c a t i o n t h a t the a b e r r a n t s might not  was  be  polyploids. A subsequent study  of the s i z e of stomata and  c e l l s f a i l e d to r e v e a l any the aberrants and  s i g n i f i c a n t d i f f e r e n c e between  the normal s e e d l i n g s .  The  the average guard c e l l i n both the aberrants normal s e e d l i n g was  guard  around 57  l e n g t h of and  the  microns.  Of the f i v e v a r i o u s treatments to break the dormancy of the s e e d l i n g s a l l the f o u r aberrant i n d o o r s and  s e e d l i n g s grown  f e r t i l i z e d w i t h ammonium s u l f a t e (treatment  resumed t h e i r growth ten days a f t e r the s t a r t of treatment.  Of the 5 a b e r r a n t s and  grown under i n c r e a s e d photoperiod only two  a b e r r a n t s and  e)  the  7 normal s e e d l i n g s of k hours (treatment  one normal s e e d l i n g resumed  d)  their  growth three weeks a f t e r the s t a r t of the treatment.  The  other three treatments f a i l e d to break dormancy i n any  of  the s e e d l i n g s t r e a t e d . C y t o l o g i c a l study  of s i x a b e r r a n t s and f o u r normal  s e e d l i n g s f a i l e d to show any complement of 26 This f i n d i n g  i n c r e a s e above the normal  chromosomes (Zenke, 1953  and  others).  ( F i g s . 5,7,9,11,13,15,17,19,21) was  f o r a l l p l a n t s s t u d i e d and  consistent  i n various t i s s u e s of the  bud.  35 DISCUSSION In s p i t e of the f a c t that the e x t e r n a l f e a t u r e s of aberrant  seedlings  obvious t h a t the than p o l y p l o i d y .  the  s t r o n g l y suggest p o l y p l o i d y i t i s  cause f o r the aberrant lilies  shape must be  other  (1952) d e s c r i b e d a s i m i l a r pheno-  menon i n dwarf spruce w i t h a l l the e x t e r n a l f e a t u r e s of a p o l y p l o i d but w i t h a r e g u l a r d i p l o i d number of chromosomes i n the somatic t i s s u e . such cases must be due  The  cause of the aberrant  to some s o r t of a mutation and  would r e q u i r e a f u r t h e r study. aberrants  form i n  The  this  f a c t t h a t some of  have normal branches below the aberrant  suggests t h a t a t l e a s t i n some o f the aberrants  the  ones the  appearance of the mutation took p l a c e i n the somatic t i s s u e s . Some of the o l d e r aberrants  i n the Duncan nursery  normal branch whorls above aberrant  have  whorls which suggests  t h a t the mutation can be normalized as w e l l as formed i n the somatic t i s s u e s of the  plant.  I t i s of i n t e r e s t to note that the aberrants s t r o n g l y resemble the f o l i a g e of t r e e s expressing phenomenon of brooming which was Douglas f i r t r e e s  (Buckland and  described  studied the  i n older  K u i j t , 1957)*  T h i s broom-  i n g appears a t a l a t e stage i n the development of the The  p o s s i b i l i t y t h a t the  same causes u n d e r l i e the  of brooming as w e l l as the aberrant be  tree.  formation  form under.study cannot  excluded. During the course of the f o r e g o i n g  study i t has  been  36 noted that a t anaphase only the h a p l o i d number o f chromosomes i s d i s c e r n i b l e i n a l l Douglas f i r bud c e l l s and  t h i s p e c u l i a r i t y would r e q u i r e f u r t h e r study.  studied  37  ' F i g . 1.  Normal and a b e r r a n t o n e - y e a r - o l d s e e d l i n g s :  a, b, a b e r r a n t s e e d l i n g s ; c, d, normal s e e d l i n g s  F i g . 2.  Normal and a b e r r a n t t w o - y e a r s - o l d seedl i n g s : a, d, normal s e e d l i n g s ; b, c, aberrants  38  Fig. 3  otera d i f f e r e n c e s i n a b e r r a n t and normal s e e d l i n g s a, normal s e e d l i n g ; b, a b e r r a n t s e e d l i n g . (Note d i f f e r e n c e i n green hue of the n e e d l e s and i n t h e shape of buds)  Fig.A  A b e r r a n t s e e d l i n g w i t h a normal branch: a, normal branch; b, c, a b e r r a n t branches  Fig.  6.  Prometaphase i n a normal s e e d l i n g .  (X23C0)  F i g . 7.  F i g . 8.  M e t a p h a s e i n an a b e r r a n t s e e d l i n g .  Metaphase (X2500)  ( p o l a r v i e w ) i n a normal  (X3000)  seedling.  hi  *ig.  10.  ^ a r l y anaphase i n a normal s e e d l i n g .  (X2900)  P i g . 12.  Metaphase i n a normal tv.dg on an a b e r r a n t ling.  (X1350)  seed-  Fig.  16.  fro-metaphase i n a normal s e e d l i n g . (X2900)  Fig.  17.  Telophase ( t i l t e d p o l a r view) i n an a b e r r a n t seedling.  Fig.  18.  (X2020)  Telophase ( t i l t e d p o l a r view-' i n a normal seedling.  (X1950)  20.  Telophase i n a normal s e e d l i n g .  (X2730)  F i g . 22.  Telophase i n a normal s e e d l i n g .  (X1900)  »+8 BIBLIOGRAPHY A l l e n , G. S.,  19*+2.  Parthenocarpy,  self-sterility  A l l e n , G. S.,  of Douglas f i r .  19*+6.  parthenogenesis, and Jour. F o r e s t r y  Emhryogeny and development of the  a p i c a l meristem  of Pseudotsuga.  Amer. 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