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The cytogenetic consequences of spontaneous cell fusion in Zea mays L. Peeters, John P. 1984

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THE CYTOGENETIC CONSEQUENCES OF SPONTANEOUS CELL FUSION IN ZEA MAYS L. By JOHN P. PEETERS B.Sc.,The U n i v e r s i t y  of Massachusetts i n Boston,  A, THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in THE FACULTY OF GRADUATE STUDIES (Department o f Botany) (Genetics  Programme)  We accept t h i s t h e s i s as conforming to the r e q u i r e d standard  THE UNIVERSITY OF BRITISH COLUMBIA JANUARY 1 9 8 4 © J o h n P.Peeters, 1 9 8 4  I982.  In  presenting  requirements of  British  it  freely  agree for  this  thesis  f o r an a d v a n c e d  Columbia, available  that  I agree  degree that  f o r reference  permission  scholarly  i n partial  may  a t the University shall  and study.  I  copying  be g r a n t e d  o r by h i s o r h e r r e p r e s e n t a t i v e s .  understood  that  for  f i n a n c i a l gain  or publication  shall  n o t be a l l o w e d  permission.  Department  of  BOTANY  The University of British 2075 W e s b r o o k P l a c e Vancouver, Canada V 6 T 1W5  Date  7Q)  January 1 5 , 1 9 8 4  Columbia  make  further  of this  by t h e head  department  copying  of the  the Library  f o rextensive  purposes  fulfilment  thesis  o f my  I ti s  of this without  thesis my  written  ABSTRACT  The  o r i g i n of spontaneously o c c u r r i n g new  c o n t a i n i n g a d d i t i o n a l DNA  has  karyotypes  remained enigmatic, although  these genomic changes have been f r e q u e n t l y r e p o r t e d l i t e r a t u r e both i n v i v o and This study d e s c r i b e s  i n the  in vitro. the c y t o g e n e t i c  consequences of  spontaneous c e l l f u s i o n i n v i v o during m e i o s i s i n the p o l l e n mother c e l l s of Zea mays L. I t suggests t h a t spontaneous f u s i o n i s a n a t u r a l mechanism which can to the formation  c o n t r i b u t e not  of spontaneously o c c u r r i n g p o l y p l o i d s  a l s o to aneuploidy and  to the genesis  of new  only but  karyotypes  which c o n t a i n a d d i t i o n a l s t r u c t u r a l l y m o d i f i e d or B chromosomes.  cell  chromosomes,  iii TABLE OF CONTENTS  ABSTRACT  ••  i  L I S T OF TABLES  i v  L I S T OF FIGURES....  v i  ACKNOWLEDGEMENTS  •  i x•  INTRODUCTION  1  SPONTANEOUS ANEUPLOIDS AND POLYPLOIDS  3  B CHROMOSOMES  5  '.  CYTOMIXIS  9  I N VITRO C E L L FUSION AND PREMATURE CHROMOSOME CONDENSATION  13  SPONTANEOUS CELL FUSION  ..  1?  I N S I T U CHROMATIN MODIFICATION PURPOSE OF THIS STUDY  18 ,  19  MATERIALS AND METHODS  20  O r i g i n and h i s t o r y o f t h e samples  20  C y t o l o g i c a l methods  21  Karyotypes  27 .  Growth c o n d i t i o n s . .  28  Illustrations  28  RESULTS Spontaneous c e l l  '  31  fusion  The c y t o g e n e t i c c o n s e q u e n c e s o f s p o n t a n e o u s  31 cell,  fusion  *. . . .  37  The F l a n d F2 g e n e r a t i o n s  83  H y b r i d i t y and t h e e f f e c t s o f growth c o n d i t i o n s  92  iv DISCUSSION  96  Spontaneous c e l l f u s i o n  97  In s i t u chromosome m o d i f i c a t i o n and degradation....  99  B chromosome formation  104  Aneuploidy and p o l y p l o i d y  108  A d d i t i o n a l c y t o l o g i c a l observations  110  Conclusion  115  BIBLIOGRAPHY.  117  LIST OF TABLES  v  Table I: Area comparison between normal and p o s t f u s i o n cells  35  Table I I : M e i o t i c survey of a s i n g l e p a r e n t a l p l a n t with a high i n c i d e n c e of m e i o t i c a b n o r m a l i t i e s . . . . Table I I I : C h a r a c t e r i s t i c s of the F l g e n e r a t i o n  36 8?  vi LIST OF FIGURES  F i g u r e 1: Sikkim P r i m i t i v e ear samples  29  F i g u r e 2: Sikkim P r i m i t i v e karyotype  '. 29  F i g u r e 3: Sikkim P r i m i t i v e karyotype  '  29  F i g u r e 4: P o s t f u s i o n c e l l with i n s i t u a b o r t i o n o f one complement  41  F i g u r e 5: P o s t f u s i o n c e l l w i t h i n s i t u a b o r t i o n o f one complement  41  F i g u r e 6: C e l l f u s i o n a t d i a k i n e s i s F i g u r e 7: P o s t f u s i o n c e l l with m o d i f i e d  43 supernumerary  chromosomes F i g u r e 8:  Frequencies  43 o f c e l l f u s i o n and o f  p o s t f u s i o n products  stabilized  throughout m e i o s i s  i n one  parental plant  45  F i g u r e 9: F u s i n g c e l l a t e a r l y prophase 1 showing i n s i t u chromosome m o d i f i c a t i o n F i g u r e 10: P o s t f u s i o n c e l l with a p p a r e n t l y m e i o t i c a l l y i n a c t i v e , modified  47 stabilized, supernumerary  chromosomes  47  F i g u r e 11: P o s t f u s i o n c e l l with m e i o t i c a l l y a c t i v e modified  supernumerary chromosomes  F i g u r e 12: P o s t f u s i o n c e l l with a s i n g l e m o d i f i e d  49 super-  numerary chromosome with a m e i o t i c a l l y a c t i v e NOR  •  ^9  F i g u r e 13: M i c r o c e l l s produced by spontaneous p r o t o p l a s t fragmentation  5^  F i g u r e jLki  Postfusion aneuploid  F i g u r e 15: M u l t i c e l l  5^  cell  f u s i o n a t d i a k i n e s i s showing p a r t i a l  i n s i t u chromosome degradation F i g u r e 16: M u l t i c e l l  56  f u s i o n a t d i a k i n e s i s showing p a r t i a l  i n s i t u chromosome degradation  56  F i g u r e 17: P o l y p l o i d PMC  58  F i g u r e 18: P o l y p l o i d p o s t f u s i o n c e l l with two axes o f division F i g u r e 19: A b o r t i n g  58 c e l l showing chromatin  degradation  and a d d i t i o n a l RNA s y n t h e s i s F i g u r e 20: P a r t i a l l y aborted  63  cell  63  F i g u r e 21: A b o r t i n g metaphase 1 c e l l  65  F i g u r e 22: Aborted c e l l  65  F i g u r e 23: A r r e s t e d diplotene. c e l l  6?  Figure' 24: P o s t f u s i o n a b o r t i n g c e l l at metaphase 1  67  F i g u r e 25: Large s y n c y t i a o f PMCs and t a p e t a l c e l l s . . . . 73 F i g u r e 26: D e t a i l o f F i g u r e 25 showing f u s i o n between PMCs and nurse c e l l s F i g u r e 27: P o s t f u s i o n c e l l with two p a r a l l e l  73 independent  spindles  . 75  F i g u r e 28: P o s t f u s i o n c e l l with a m e i o t i c g r a d i e n t . . . . . 75 F i g u r e 29: PMC with i n c r e a s e d l e v e l s o f RNA s y n t h e s i s . . 77 F i g u r e 30: P o l y p l o i d somatic n u c l e i i n an anther  at mid  meiosis F i g u r e 31: Cytomixis  77 between a pachytene and an a b o r t i n g  cell F i g u r e 32: Nucleo-cytoplasmic  79 t r a n s f e r between two par-  t i a l l y fused e a r l y prophase 1 c e l l s  79  viii F i g u r e 33: Breaking  81  chromosome bridge  F i g u r e 34: A p o s s i b l e example o f n e o c e n t r i c a c t i v i t y . . .  81  F i g u r e 35: F l progeny with the presence i n the genome of a new m o d i f i e d supernumerary chromosome.. 88 F i g u r e 36: F l progeny with a new karyotype c o n t a i n i n g 3 a d d i t i o n a l chromosomes and a s m a l l fragment..88 • F i g u r e 3?:  Change i n B chromosome morphology through increased heterochromatinisation F l t o F2  from the  88  generation  F i g u r e 3 8 : F2 somatic karyotype o f stock A3X2 with 4 B  90  chromosomes. F i g u r e 39 and F i g u r e 40: D i f f e r e n t karyotypes o f two c l o n a l c e l l s showing chromosome as a r e s u l t o f a temperature  fragmentation  shift  94  ix ACKNOWLEDGMENTS  I wish to express my deep a p p r e c i a t i o n t o Dr. H.G. Wilkes f o r s t i m u l a t i n g me t o s t a r t t h i s p r o j e c t while I was an undergraduate a t the U n i v e r s i t y o f Massachusetts and f o r the continuous guidance and support he granted  me throughout  the e v o l u t i o n of t h i s work. I am g r e a t l y indebted  t o Dr. A.J.F. G r i f f i t h s f o r  h i s s u p e r v i s i o n and h e l p f u l c r i t i c i s m . I t i s a l s o my p l e a s u r e  t o extend thanks t o the members  of my committee, t o C a r o l i n e Bouloc and t o my f a m i l y f o r t h e i r h e l p and encouragement. P r e l i m i n a r y r e s u l t s o f t h i s work, i n c l u d i n g F i g u r e s 4,6,12,15,17t19i23 and 40 were i n c o r p o r a t e d presented  i n a thesis  f o r the honors degree a t the U n i v e r s i t y o f  Massachusetts. These F i g u r e s have been i n c l u d e d here i n order to make the present  t h e s i s complete.  1 INTRODUCTION  The  spontaneous appearance of new  t a i n i n g a d d i t i o n a l DNA p l a n t s and  has  k a r y o t y p e s con  -  been f r e q u e n t l y observed i n b o t h  a n i m a l s i n v i v o but a l s o i n v i t r o , i n c e l l  cul-  t u r e s . K a r y o t y p e s which appear de novo w i t h a d d i t i o n a l show extreme d i v e r s i t y i n terms of t o t a l DNA  DNA  c o n t e n t as w e l l  as chromosomal arrangements. E x p l a i n i n g the o r i g i n of these karyotypes represents  an i m p o r t a n t and  difficult  challenge.  A l t h o u g h v a r i o u s models have been proposed t o account f o r t h e s e spontaneous changes, t h e s e models have i n g e n e r a l mained h y p o t h e t i c a l . One  of t h e s e models i n v o l v e s  re-  spontaneous  c e l l f u s i o n as a p o s s i b l e c o n t r i b u t o r t o s p o n t a n e o u s l y o c c u r ing  polyploids. Spontaneous c e l l f u s i o n has  been d e t e c t e d  occasionally  i n both p l a n t s and humans but the causes and g e n e t i c  conse-  quences of spontaneous c e l l f u s i o n a r e e s s e n t i a l l y unknown. E x p e r i m e n t a l i n v i t r o c e l l f u s i o n can, however , p r o v i d e some c l u e s as t o the g e n e t i c s  o f p o s t f u s i o n . I t i s known  t h a t , f o l l o w i n g i n v i t r o c e l l f u s i o n and under c e r t a i n c o n d i t i o n s , a spectrum of p r o d u c t s can be o b t a i n e d p l o i d s , aneuploids,  modified  including poly-  chromosomes, i n a c t i v a t e d c h r o -  mosomes and genomic r e a r r a n g e m e n t s . Some of t h e s e p r o d u c t s a r e the r e s u l t of i n s i t u c h r o m a t i n m o d i f i c a t i o n and mosome l o s s . Chromatin m o d i f i c a t i o n i n s i t u has reported  f o r t r a n s f e c t e d DNAs and  f o r c l o n a l DNA,  chro-  a l s o been following  cytomixis. This study describes  the c y t o g e n e t i c  consequences of  2  spontaneous , c e l l f u s i o n i n v i v o , d u r i n g m e i o s i s o f p o l l e n mother c e l l s i n Zea mays L. The r e s u l t s suggest t h a t c h r o m a t i n m o d i f i c a t i o n and d e g r a d a t i o n i n s i t u can a l s o o c c u r f o l l o w i n g spontaneous c e l l f u s i o n , g e n e r a t i n g a n e u p l o i d s and c e l l s w i t h m o d i f i e d supernumerary chromosomes. F o l l o w i n g s e l f i n g , new k a r y o t y p e s were r e c o v e r e d c o n t a i n i n g a d d i t i o n a l DNA i n t h e form o f m o d i f i e d supernumerary chromosomes. These u s u a l l y h e t e r o c h r o m a t i c chromosomes, which a r e a l s o c a l l e d a c c e s s o r y o r "B".chromosomes, a r e e x t r e m e l y common i n both p l a n t s and a n i m a l s , b u t a r e o f unknown o r i g i n . T h i s i s t h e f i r s t s t u d y where spontaneous c e l l i s d i r e c t l y i m p l i c a t e d i n p r o d u c i n g new k a r y o t y p e s t h a n p o l y p l o i d y and t h a t demonstrates  cytologically  d u c t i o n o f B chromosomes from t h e A chromosomes.  fusion  other the pro-  3 SPONTANEOUS ANEUPLOIDS AND POLYPLOIDS  P o l y p l o i d s which appear spontaneously i n v i v o have been f r e q u e n t l y r e p o r t e d i n p l a n t s (Karpechenko, 1 9 2 7 ; Babcock" and Navashin, 1 9 3 0 ; Smith, 1 9 4 1 ; P r i c e , 1955;. M i l l e r , 1963;  Grant, 1 9 6 5 ; S a l e s s e s , 1 9 7 0 ; T a i and V i c k e r y , 1 9 7 2 ;  Sarbhoy, I 9 8 O ) and t e t r a p l o i d or near t e t r a p l o i d c e l l s  have  a l s o been r e p o r t e d r e c e n t l y i n human:tissues, both of mal i g n a n t and non malignant o r i g i n s .  ( A t k i n , 1 9 7 9 ; Otto and  Therman, 1982). Reports on the spontaneous i n v i v o appearance of t r i somies or o f a n e u p l o i d s a r e a l s o widespread i n p l a n t s  (Babcock  and Navashin, 1 9 3 0 ; Takagi, 1 9 3 5 ; Nandi, 1 9 3 7 ; Smith, 1 9 4 1 ; McGinnis, 1 9 6 2 ; Hacker and R i l e y , 1 9 6 3 ; K o h e l , 1 9 6 6 ;  Haunold,  I 9 6 8 ; Khush, 1 9 7 3 ) • In maize, spontaneous t r i p l o i d s  have  been r e p o r t e d by M c C l i n t o c k ( 1 9 2 9 ) and Beadle ( 1 9 3 2 ) , and more r e c e n t l y the occurrence o f spontaneous aneuploids i n maize was s t u d i e d by G h i d o n i et a l . ( 1 9 8 2 ) . Aneuploidy i n humans i s by no means uncommon ( G r i f f i t h s , 1982) and'the d r a s t i c e f f e c t s o f trisomy 1 3 , 18 and  .21 are w e l l known. Furthermore, the a s s o c i a t i o n of an-  e u p l o i d y and cancer has been w e l l e s t a b l i s h e d . (German, 1 9 7 4 ) . Recent s t u d i e s show, however, t h a t a very h i g h frequency o f the spontaneous aneuploids ifchat a r e d e t e c t e d i n humans a r e not v i a b l e . Spontaneous  aneuploids and p o l y p l o i d s have a l s o been  found i n v i t r o i n t i s s u e c u l t u r e of both p l a n t and animal  4 cells  ( S c a l e t t a et a l . ,  et a l . ,  1980;  1967;  W e i s s a n d G r e e n , 1967;  McCoy a n d P h i l l i p s ,  Ting  1982).  Aneuploidy generally affects f e r t i l i t y  (Khush,  1973)  a n d , as c l e a r l y shown i n t h e c a s e o f humans, i t s e f f e c t s c a n be e x t r e m e l y d e t r i m e n t a l t o t h e g e n e t i c b a l a n c e . I t i s likely  t h a t a h i g h percentage of the spontaneous a n e u p l o i d s  i n b o t h p l a n t s and a n i m a l s a r e n e v e r even d e t e c t e d  because  o f e a r l y a b o r t i o n . H o w e v e r , t r i p l o i d s and p o l y p l o i d s sometimes  can  be a s s o c i a t e d w i t h more v i g o r o u s g r o w t h a n d c h a r -  acteristics  ( M c C l i n t o c k , 1929), a n d t h i s a s s o c i a t i o n  could  a l s o p r o v e t r u e f o r some c a n c e r s w h i c h show a n e u p l o i d o r polyploid patterns Liu,  I98I;  (German,1974; R o w l e y ,  Reichman and  Levin,  I98O;  Kulick  and  I98I).  V a r i o u s h y p o t h e s e s have been p r o p o s e d . t o a c c o u n t f o r t h e a p p e a r a n c e o f t h e s e new  k a r y o t y p e s . Spontaneous  p l o i d s have been a t t r i b u t e d  e i t h e r t o spontaneous c e l l  f a i l u r e of c y t o k i n e s i s  ( B e a d l e , 1932;  Ramanna, 1973)  polyfusion,  or t o  s p i n d l e f a i l u r e s , w h i l e s p o n t a n e o u s a n e u p l o i d s a r e most g e n e r a l l y t h o u g h t o f as a r i s i n g f r o m n o n d i s j u n c t i o n .  These  d i f f e r e n t models have remained i n g e n e r a l t h e o r e t i c a l , the d i f f i c u l t y  o f d e t e c t i n g s u c h spontaneous changes  they occur i n s i t u .  given  when  5  •  B CHROMOSOMES  Spontaneous genomic changes i n v o l v i n g s m a l l and u s u a l l y h e t e r o c h r o m a t i c s u p e r n u m e r a r y chromosomes, a l s o c a l l e d a c c e s s o r y o r B chromosomes, h a v e b e e n known t o o c c u r i n a huge number o f p l a n t a n d a n i m a l s p e c i e s a n d p o s s i b l y e v e n i n humans ( R i d l e r e t a l . , 1970) ( f o r reviews see Muntzing,  197^ a n d J o n e s , 1975).  Although  so f r e q u e n t l y s t u d i e d a n d r e p o r t e d o n , B chromosomes have remained  a perpetual curiosity i nthat neither  t h e i r o r i g i n n o r even t h e i r p r e c i s e f u n c t i o n a r e understood. The tive  most g e n e r a l c o n s e n s u s  i s t h a t Bs h a v e n e g a -  e f f e c t s by r e d u c i n g both growth  and f e r t i l i t y , as  w e l l as reducing t h e g e n e t i c s t a b i l i t y chromosome complement ( M e l a n d e r ,  o f t h e normal  1950; M u n t z i n g , 197^;  J o n e s , 1975; B o u g o u r d a n d P a r k e r , 1979; L a t a , 1982; P a r k e r e t a l . , 1982)-.  I n maize,  where Bs w e r e  first  r e p o r t e d i n 1915 "by Kuwada, t h e s e e f f e c t s a r e s i m i l a r (see Randolph, to  19^1).  Bs h a v e a l s o b e e n shown i n m a i z e  induce l o s s o f knobbed A h e t e r o c h r o m a t i n  (Rhoades and  Dempsey, 1973) b e c a u s e o f d e l a y e d k n o b h e t e r o c h r o m a t i n replication  ( P r y o r e t a l . , 1980).  B chromosomes w e r e  found t o a f f e c t m e i o t i c r e c o m b i n a t i o n o f t h e autosomes e i t h e r by i n c r e a s i n g i t (Barlow and Vosa, 1973;  1970; Ward,  F l e t c h e r and H e w i t t , I98O) o r by d e c r e a s i n g i t  ( V i i n i k k a , 1973)•  6 Accessory  chromosomes a r e known t o be a b l e t o m a i n -  t a i n t h e i r l e v e l s i n t h e genome by a number o f n o n - M e n d e l i a n modes o f i n h e r i t a n c e .  In the primary  oocyte  of  Bs a r e p r e f e r e n t i a l l y d i s t r i b u t e d on t h e egg t h a n t h e p o l a r b o d y s i d e ( H e w i t t , 1976). accumulation  1981)  .  side rather  I n maize  m e c h a n i s m s f o r Bs i s u n i q u e .  undergo m i t o t i c n o n d i s j u n c t i o n a t v e r y h i g h (up t o 98%)  grasshoppers  the  B chromosomes frequencies  i n t h e s e c o n d p o l l e n m i t o s i s ( C a r l s o n and  This event i s subsequently  f e r t i l i z a t i o n o f t h e egg mosomes (Roman, 1948).  f o l l o w e d by  preferential  by s p e r m c o n t a i n i n g two Beckett  (I982) has  Chou,  B  also  chro-  suggested  t h a t m e i o t i c d r i v e i s an a d d i t i o n a l m e c h a n i s m by w h i c h are maintained  i n maize.  Numerous s t u d i e s show t h a t B chromosomes extremely  Bs  polymorphic  and H e w i t t , 1972;  even w i t h i n p o p u l a t i o n s  P a t t o n , 1977;Bougourd and  are  (Gibson  Parker,  1979;  V o l o b u e v , I 9 8 O ; C a r r and  C a r r , 1982? H e n r i q u e s - G i l e t a l . ,  1982)  been a s u b s t a n t i a l  .  P o l y m o r p h i s m has  c o n t r i b u t i n g to the d i f f i c u l t y as t o t h e o r i g i n and t h e genome.  No  i n formulating a  f u n c t i o n of accessory  a d d i t i o n a l l i g h t has  q u e s t i o n s by t h e m o l e c u l a r  factor  chromosomes i n  b e e n s h e d on  a n a l y s i s o f Bs.  (Gupta,  r e v e a l any  DNA  these  Although  chromosomes a r e known n o t t o p a i r w i t h t h e As meiosis  hypothesis  B  during  I 9 8 I ) , p r e v i o u s r e p o r t s have f a i l e d  to  component s p e c i f i c t o B chromosomes  ( C h i l t o n and M c C a r t h y , 1973;  D o v e r , 1975)-  I n some  cases  s p e c i f i c g e n o m i c s e q u e n c e s a r e f a r more a b u n d a n t i n t h e  7 Bs than i n the chromosomes o f the normal complement ( K l e i n and E c k h a r d t , 1976).  A c c o r d i n g t o Amos and  Dover (1981), B chromosomes c o n t a i n r e p r e s e n t a t i v e sequences 'of the spectrum  o f f a m i l i e s o f DNA  t h a t make up the A  chromosome complement and t h e r e i s no de_ novo o r i g i n o f B chromosome sequences. S i n c e a c c e s s o r y chromosomes a r e g e n e r a l l y viewed as h a v i n g no n e c e s s a r y f u n c t i o n f o r the s u r v i v a l or g e n e t i c b a l a n c e o f an organism but appear t o have, on the c o n t r a r y , an o v e r a l l n e g a t i v e impact on growth and s u r v i v a l and t h a t they appear t o have e v o l v e d s e l f - s u r v i v a l mechanisms, B chromosomes a r e sometimes r e f e r r e d t o as p a r a s i t i c g e n e t i c elements.  (Rhoades and Dempsey, 1972;  P a r k e r et a l . , I982)/ DNA  C a r l s o n , 1977?  A more r e c e n t d e f i n i t i o n f o r t h e s e  sequences c o u l d be s e l f i s h DNA  ( D o o l i t t l e and  Sapienza,  1980). A number o f hypotheses o r i g i n o f B chromosomes.  have been proposed  f o r the  I n maize, f o r example, B c h r o -  mosomes have been suggested t o o r i g i n a t e from the c h r o mosome K10  (Ward, 1979)-  Although i t i s only l o g i c a l to  assume t h a t a c c e s s o r y chromosomes d e r i v e somehow from normal complement ( G i b s o n and H e w i t t , 1972; Volobuev,  Patton,  the 1977;  I98O; Amos and Dover, I98I), n e i t h e r t h e p r e c i s e  mechanism n o r the t i m i n g o f t h e s e genomic changes a r e known.  G i v e n t h e i r h e t e r o c h r o m a t i c and v a r i a b l e  DNA  c o m p o s i t i o n , Bs c o u l d i n i t i a l l y o r i g i n a t e from an A f r a g ment and then e v o l v e by a m p l i f i c a t i o n and sequence  8 transposition  (Amos and Dover, 1981).  have been proposed  f o r the t r a n s p o s i t i o n and  of s p e c i f i c c l a s s e s  of DNA  and the genome can  be viewed as being very " f l u i d " 1982;  D'Eustachio  Various mechanisms amplification presently  (see Dover, 1982;  Lewin,  and Ruddle, 1983).  I t i s c l e a r that e x p l a i n i n g  the de novo appearance  of an a d d i t i o n a l A fragment i n the genome has  represented  the major stumbling block i n forming a hypothesis on the o r i g i n of B chromosomes.  T h i s study suggests how  a d d i t i o n a l fragments can a r i s e de novo.  such  9 CYTOMIXIS  C y t o m i x i s was d e f i n e d by Gates (1911) as "an e x t r u s i o n o f chromatin  from t h e n u c l e u s  c e l l through cytoplasmic  connections,  of an a d j a c e n t m o t h e r - c e l l " .  o f one mother-  i n t o t h e cytoplasm  The term c y t o m i x i s has  s i n c e been used i n a b r o a d e r r a t h e r t h a n narrower sense i n the l i t e r a t u r e .  Some r e s e a r c h e r s have indeed used t h e  term c y t o m i x i s t o d e s c r i b e t h e t r a n s f e r o f o r g a n e l l e s between c e l l s ( W e i l i n g , 1965) o r even f o r spontaneous c e l l f u s i o n ( S a l e s s e s , 19?0). Although  c y t o m i x i s has been f r e q u e n t l y r e p o r t e d ,  n e i t h e r i t s s i g n i f i c a n c e n o r i t s g e n e t i c consequences a r e w e l l defined.  Some i n v e s t i g a t o r s have c o n s i d e r e d  cyto-  m i x i s as an a r t i f a c t (Tarkowska, 19^5. 1966), but most view c y t o m i x i s as a t r u e and w i d e s p r e a d phenomenon. . C y t o m i x i s has been m o s t l y  d e s c r i b e d i n angiosperms  d u r i n g m e i o s i s o f p o l l e n mother c e l l s (PMCs) ( r e v i e w e d by Kamra, i960). somatic  I t has been r e p o r t e d i n f r e q u e n t l y i n  t i s s u e s , r o o t s , f o r example (Bowes, 1973). and  may even occur between t a p e t a l c e l l s and t h e PMCs a c c o r d i n g t o Cooper (1952).  U n f o r t u n a t e l y , no c o r r e l a t i o n  has been e s t a b l i s h e d between a somatic  and a m e i o t i c  event,  i n o t h e r words, i t remains t o be determined i f c y t o m i x i s i s expressed  l o c a l l y or i n d i f f e r e n t parts of a plant.  However, some l i g h t on t h i s q u e s t i o n has been shed by M o r i s s e t ' s study. (1978), which showed t h a t some a n t h e r s  can be a f f e c t e d by, c y t o m i x i s , w h i l e o t h e r a n t h e r s  of the  same p l a n t can undergo c o m p l e t e l y normal m e i o s i s . Most r e p o r t s on c y t o m i x i s d e s c r i b e what Bobak (I966) has  d e f i n e d as "chromosomes s t r e t c h i n g • f r o m c e l l t o  c e l l " , f o r m i n g t y p i c a l and  easily identifiable figures.  Such f i g u r e s would o b v i o u s l y o n l y be p o s s i b l e i n the presence of l a r g e i n t e r c e l l u l a r communication E.M.  channels.  s t u d i e s have c l e a r l y shown the presence of l a r g e h o l e s  (2 m i c r o n s ) i n the c a l l o s e w a l l s o f some angiosperms and a p o s i t i v e c o r r e l a t i o n between these channels and  cytomixis  was  1974;  established. (Heslop-Harrison,  Whelan et a l . , 197^; The  1966;  Whelan,  Cheng et a l . , 1975).  d e s c r i p t i o n o f c y t o m i x i s and of i t s a f t e r e f f e c t s  are e x t r e m e l y  conflicting.  t h a t p a r t i a l DNA  The  consensus o b s e r v a t i o n i s  t r a n s f e r occurs from a "donor" t o a  " r e c i p i e n t " c e l l , through " c y t o m i c t i c channels". 1929;  Cooper, 1952;  S a r v e l l a , 1958;  1964;  Bobak, 1966;  S a l e s s e s , 1970;  1971;  Omara, 1976;  Lakshmi and Rao,  (Church,  Kamra, 19.60; B e l l , Romanov and 1978;  Orlova,  M o r i s s e t , 1978).  I f t h i s t r a n s f e r occurs when the chromosomes are h i g h l y c o n t r a c t e d ( i . e . a t metaphase), two c o u l d r e s u l t (see Omara, 1976).  i n t a c t aneuploid  When p a r t i a l DNA  occurs d u r i n g i n t e r p h a s e , t h i s would a l s o g e n e r a t e unbalanced c e l l s , but i n v o l v i n g chromatin  cells  transfer two  fragments.  The v i a b i l i t y o f the above groups o f c e l l s has questioned.  S e v e r a l r e s e a r c h e r s have suggested t h a t  been  11 c y t o m i x i s i s a p o s s i b l e cause o f a n e u p l o i d y ( S a r v e l l a , 1957; O r l o v a , 1971;  B e l l , 1964;  and p o l y p l o i d y  S a l e s s e s , 1970;  T a i and V i c k e r y , 1972;  Romanov and  Omara, 1976).  In  a d d i t i o n , Kamra (i960) and Chen et a l . (1975) have suggested t h a t t h e c e l l s w i t h a d d i t i o n a l chromatin a l s o g i v e r i s e t o f e r t i l e gametes.  the m i g r a t e d  chromatin  the r e c i p i e n t c e l l s .  may  These fragments would  t h e n o b v i o u s l y be d e f i n e d as a c c e s s o r y I n s i t u chromatin  fragments  chromosomes.  m o d i f i c a t i o n and  e l i m i n a t i o n of  has' been d e s c r i b e d f r e q u e n t l y i n A c c o r d i n g t o S h k u t i n a and  (1974) : " i f the a d d i t i o n a l chromatin i n t o the microsporocyte  t h a t has  Kozlovskaya penetrated  i s l y z e d before the t e t r a d stage,  a c e l l t h a t has r e t a i n e d the b a s i c s e t o f chromosomes  may  a p p a r e n t l y remain v i a b l e " . Most s t u d i e s on c y t o m i x i s r e v e a l t h a t a h i g h centage of t h e c h i m e r i c c e l l s a b o r t and, as one  per-  could  p r e d i c t , the i n c i d e n c e o f c y t o m i x i s i t s e l f i s h i g h l y v a r i a b l e and always c o r r e l a t e d w i t h reduced According  t o Lakshmi and Rao  (1978), the  a s s o c i a t e d w i t h c y t o m i x i s can r e a c h 95% complete s t e r i l i t y r e s u l t s .  fertility.  abnormalities of the PMCs and  I n some c a s e s , a n t h e r s were  found w i t h a l l the c e l l s connected one t o the corresponding  t o 100%  cytomixis.  other,  A h i g h p e r c e n t a g e of  c y t o m i x i s can p r o b a b l y be a s s o c i a t e d w i t h complete l a c k of c e l l i n d i v i d u a l i z a t i o n and hence s t e r i l i t y .  Therefore  c y t o m i x i s can o n l y have some s i g n i f i c a n c e when i t occurs below a c e r t a i n frequency.  12 Cytomixis has been mainly (de Nettancourt  and Grant,  1963;  described i n hybrids S a l e s s e s , 19?0~; T a i and  V i c k e r y , 19?2), i n t r i p l o i d maize (McClintock, 1929)  and  a l s o i n "normal" p l a n t s . Hypotheses concerning the o r i g i n of cytomixis are numerous and i n c l u d e the g e n e t i c b a r r i e r , the growth r a t e s due  to h y b r i d i t y , the c o m p e t i t i o n f o r space  or f i n a l l y delayed c e l l w a l l formation  Morisset, 19?8).  uncoordinated  (reviewed  by  13 I N VITRO CELL FUSION AND  The  PREMATURE CHROMOSOME CONDENSATION  p o t e n t i a l of somatic  immediately  n o t i c e d i n 1965  c e l l h y b r i d i z a t i o n was  when H a r r i s and  produced i n t e r s p e c i f i c heterokaryons Sendai v i r u s e s .  Since  this  Watkins  by u s i n g i n a c t i v a t e d  time i n v i t r o c e l l  e x p a n d e d i n t o an i n d e p e n d e n t f i e l d  of research.  C a r l s o n et a l . produced the f i r s t  parasexual  p l a n t h y b r i d t h r o u g h p r o t o p l a s t f u s i o n and by t i s s u e c u l t u r e , s t i m u l a t i n g i m p o r t a n t to the r e a l  s i g n i f i c a n c e of t h i s  Although significant  in vitro  i m p l i c a t i o n s and  number o f q u e s t i o n s cesses  cell  (reviewed  f u s i o n has  e t a l . , 1982;  stability In t y p i c a l  interspecific  division  and  new  i n s i g h t s as  already yielded a  cytogenetic  I98I;  Savage,  hybrids  1976)  G l e b a e t a l . , 1982;  e t a l . , 1983). i s not  fusion  However g e n e t i c a general  (syncaryons),  the  chromosomes a r e p r o g r e s s i v e l y l o s t  ( B l a u e t a l . , 1983)•  The  rule. parental during  complement w h i c h  l o s e s chromosomes i s r e f e r r e d t o as t h e d o n o r , w h i l e complement w h i c h r e m a i n s i n t a c t (D'Eustachio  and  pro-  have been  cell  R i n g e r t z and  of p o s t f u s i o n products  n u c l e i f u s e and cell  Blau  regeneration  heterokaryons  ( s e e a l s o E v a n s e t a l . , I98O and Wilson  1972  interspecific  answers i n g e n e t i c s ,  following in vitro  by H a r r i s , 1970;  In  has  unsolved.  S t a b l e h o m o k a r y o n s and frequently reported  fusion  science.  on t h e m o l e c u l a r  of p o s t f u s i o n remain  first  R u d d l e , 1983).  the  i s the r e c i p i e n t The  mechanism f o r  this  14 e l i m i n a t i o n has remained p o o r l y  understood.  I f s e l e c t i v e p r e s s u r e i s a p p l i e d on donor genes, the chromosome b e a r i n g t h e s e genes w i l l be r e t a i n e d .  After a  c e r t a i n time these chromosomes w i l l s t a b i l i z e i n the h y b r i d c e l l and s e l e c t i v e p r e s s u r e i s no l o n g e r n e c e s s a r y .  Genomic  s t a b i l i t y i s sometimes a c h i e v e d a f t e r a s h o r t p e r i o d  (Kao  et a l . , 1976).  Chromosome l o s s e s a r e not l i m i t e d t o i n t e r s p e c i f i c h y b r i d s but a l s o occur i n i n t r a s p e c i f i c h y b r i d s by R i n g e r t z and Savage, I976).  As a r e s u l t o f t h i s  e l i m i n a t i o n mechanism, h y b r i d c e l l s w i t h an  aneuploid  number o f "donor" chromosomes a r e f r e q u e n t l y ( S c h i e d e r , 1982; 1983).  Tamaki, 1982;  (reviewed  observed  D'Eustachio and  Ruddle,  Chromosome l o s s e s can happen r a p i d l y a t f i r s t  and t h e n , as s t a b i l i z a t i o n i s a c h i e v e d , a t a s l o w e r r a t e (Weiss and Green, 1967;  Nabholz et a l . , I969; Kao  et a l ,  1976).  Of p a r t i c u l a r i n t e r e s t i s the f a c t t h a t h y b r i d c e l l p o p u l a t i o n s o f t e n show c o n s i d e r a b l e v a r i a t i o n i n t h e i r chromosomal c o n s t i t u t i o n .  I n some l i n e s , no two  cells  seem t o have e x a c t l y the same chromosome complement.  Great  v a r i a t i o n can a l s o be found i n c l o n e d p o p u l a t i o n s d e r i v i n g from a s i n g l e f u s i o n event ( R i n g e r t z and Savage, 1976). D e s p i t e t o t a l l o s s o f donor chromosomes, donor genes may  be r e t a i n e d (Schwartz  D u d i t s e t a l . , 1979;  et a l . , 1971;  Power et a l . ,  Shepard et a l . , 1983).  This  1975;  probably  i m p l i e s gene t r a n s l o c a t i o n s which have indeed been d e t e c t e d  following  c e l l f u s i o n (see Boone et a l . , 19?2; Douglas  et a l . , 1973; F r i e n d et a l . , 19?6). M o r p h o l o g i c a l changes i n c l u d i n g changes i n chromosome c h a r a c t e r i s t i c s have been r e p o r t e d i n v i t r o c e l l f u s i o n (Ringertz  1977).  In a d d i t i o n , r e v e r t a n t s  following  and Savage, 1976; Kao, (Melton, I98I) and  chromosome i n a c t i v a t i o n (Hotchkiss and Gabor, I98O; Sanchez-Rivas et a l . , 1982) were a l s o found. Various hypotheses have been proposed t o account for  chromosome l o s s e s  i n c e l l hybrids.  A c c o r d i n g to  Bennett et a l . (1976) changes i n the chromosome-spindle interaction i s a c r i t i c a l involves the  factor.  However t h i s hypothesis  the l o s s o f whole chromosomes and does not e x p l a i n  s t r u c t u r a l changes which have been r e p o r t e d  in v i t r o c e l l fusion.  following  R i n g e r t z and Savage (1976) have  suggested t h a t n u c l e a r asynchrony could generate premature chromosome condensation or P.C.C. which i n t u r n may be a f a c t o r causing chromosome breaks and t r a n s l o c a t i o n s , as w e l l as e l i m i n a t i o n . P.C.C. occurs immediately a f t e r an i n t e r p h a s e nucleus i s c o n f r o n t e d w i t h the cytoplasm o f a m i t o t i c c e l l , and was f i r s t d e s c r i b e d  i n 1970 by Johnson and Rao.  these same authors r e p o r t e d  In 1972,  t h a t f u s i o n of mammalian  m i t o t i c and S phase c e l l s causes the s t a t e o f "chromosome p u l v e r i z a t i o n " and t h a t t h i s r e s u l t s i n g r e a t e r  chromosome  l o s s e s than f u s i o n i n v o l v i n g c e l l s i n G l or G2. (Rao and Johnson, 1972).  A p r e c i s e d e s c r i p t i o n o f the morphology  of P.C.C. induced chromosomes i s given by Hittelman et a l . ( I 9 8 O ) .  P.C.C. also occurs i n plants (Szabados  and Dudits, I 9 8 O ) , and even Xenopus oocytes were found to induce P.C.C. i n i n j e c t e d somatic plant n u c l e i (Von der Haar et a l . , I 9 8 I ) .  The c o n t r i b u t i o n of P.C.C. to  chromosome e l i m i n a t i o n f o l l o w i n g i n v i t r o c e l l f u s i o n remains unclear.  17 SPONTANEOUS CELL FUSION  Reports o f spontaneous c e l l f u s i o n i n v i v o a r e extremel y uncommon. Spontaneous hybrids  c e l l f u s i o n has been found i n p l a n t  ( S a l e s s e s , 1970), i n h a p l o i d s  under "normal c o n d i t i o n s "  (Levan, 1941) o r  ( P r i c e , 1956; Sarbhoy, 1980). I n  maize, spontaneous c e l l f u s i o n has been r e p o r t e d normal g e n e t i c  conditions  i n asynaptic  and maize-Tripsacum h y b r i d s  (Chaganti,  under ab-  maize ( M i l l e r ,  1963)  1965)- On t h e b a s i s  of P.C.C. o r E.M. e v i d e n c e , a few s t u d i e s suggest t h a t spontaneous c e l l f u s i o n can a l s o o c c u r i n human carcinomas and cancers ( A t k i n , 1979; Reichmann and L e v i n , I98I; H a r r i s , 1982) o r i n Bloom's syndrome ( O t t o and Therman, I982). Spontaneous c e l l f u s i o n has a l s o been r e p o r t e d  i n v i t r o i n mixed  t i s s u e c u l t u r e s (Weiss and Green, 1967; R i n g e r t z  and Savage,  1976). These few r e p o r t s i n d i c a t e t h a t spontaneous c e l l  fusion  i s p r o b a b l y a v e r y r a r e event which o c c u r s m a i n l y under abnormal g e n e t i c  or environmental c o n d i t i o n s . Furthermore, i t  appears t h a t n e i t h e r t h e e t i o l o g y n o r even t h e t r u e c y t o g e n e t i c consequences of- spontaneous c e l l f u s i o n a r e c u r r e n t l y understood.  18 IN SITU CHROMATIN MODIFICATION  S t u d i e s on i n v i t r o c e l l f u s i o n have shown that  struc-  t u r a l chromatin changes as w e l l as chromosome l o s s e s can occur i n s i t u without a f f e c t i n g c e l l v i a b i l i t y . These changes u s u a l l y only take p l a c e i n the "donor"  complement. Changes  i n chromatin s t r u c t u r e and i n s i t u d e g r a d a t i o n have a l s o been r e p o r t e d f o r c l o n a l DNA ly,  i n s t u d i e s on c y t o m i x i s . Recent-  Calos et a l . (1983) and Razzaque et a l . (1983) have shown  that s t a b l e plasmids that are t r a n s f e c t e d i n t o mammalian c e l l s are e q u a l l y s u b j e c t to a h i g h frequency of i n s i t u m o d i f i c a t i o n i n the form of d e l e t i o n s , d u p l i c a t i o n s complex rearrangements,  and  such as the i n s e r t i o n of genomic  sequences. The r e l a t i o n s h i p between these d i f f e r e n t o b s e r v a t i o n s i s not c l e a r but these o b s e r v a t i o n s a l l appear to i n d i c a t e t h a t the s t a b i l i t y of chromatin which i s added to the  normal  genome i s not always achieved and that t h i s chromatin can be s u b j e c t t o s t r u c t u r a l m o d i f i c a t i o n s and even d e g r a d a t i o n i n s i t u . Razzaque et a l . (I983) have suggested i n t h e i r study that chromatin can be " f o r e i g n " to a c e l l and p o s s i b l y be r e c o g n i z e d as such.  PURPOSE OF THIS STUDY  The purpose of t h i s study i s t o : a) . Describe m e i o t i c a b n o r m a l i t i e s t h a t were detected i n anthers  o f Zea mays L., i n p a r t i c u l a r the d e t a i l e d c y t o -  l o g i c a l consequences o f i n v i v o c e l l  fusion.  b) . Study the k a r y o t y p i c and phenotypic  e v o l u t i o n of the  F l and F2 g e n e r a t i o n s , f o l l o w i n g s e l f i n g o f the p a r e n t a l p l a n t s with the abnormal m e i o s i s .  c) . Discuss the p o s s i b l e i m p l i c a t i o n s spontaneous f u s i o n has i n the f o r m a t i o n of karyotypes de novo with  additional:DNA.  cell  which appear  MATERIALS  AND METHODS  1. O r i g i n and h i s t o r y o f the samples.  Samples o f Zea mays L. of the I n d i a n l a n d r a c e M u r l i or Sikkim P r i m i t i v e were obtained by Dr. H.G. Wilkes  of the  U n i v e r s i t y o f Massachusetts i n Boston during h i s s a b b a t i c a l year t o I n d i a i n 1978/79 through Dr. J.K.S. Sachan of the Indian A g r i c u l t u r a l Research I n s t i t u t e  (Wilkes, 1981).  T h i s s m a l l popcorn i s grown i n the Sikkim and Darj e e l i n g r e g i o n s of the E a s t e r n Himalayas a t m i d - e l e v a t i o n  (6000-8000 f e e t ) i n moist t r o p i c a l c l o u d f o r e s t c o n d i t i o n s and  i s used only f o r ceremonial  peoples  o f f e r i n g s by the B u d d h i s t i c  o f the r e g i o n . Little  i s known about the o r i g i n or h i s t o r y o f M u r l i  which has been claimed to be a p r i m i t i v e s o r t o f a n c e s t r a l maize.  The s m a l l ear ( F i g u r e 1) resembles the r e c o n s t r u c t e d  a n c e s t o r o f P.C. Mangelsdorf, but Sikkim P r i m i t i v e could a c t u a l l y be a l o c a l a d a p t a t i o n t o s h o r t day c o n d i t i o n s of a commercial popcorn such as L a d y f i n g e r (Wilkes, The  I98I).  samples were f i r s t grown i n 1980 a t the Waltham  Suburban Experiment S t a t i o n (Massachusetts/U.S.A.) but the p l a n t s f a i l e d to f l o w e r before the k i l l i n g f r o s t because o f t h e i r pronounced s h o r t day requirements  1982).  (Wilkes and P e e t e r s ,  In I98I under i d e n t i c a l c o n d i t i o n s the p l a n t s d i d  flower i n e a r l y September and c y t o l o g i c a l m a t e r i a l was obtained.  In an attempt to f i n g e r p r i n t t h i s l a n d r a c e , the  21 karyotypes were e s t a b l i s h e d ( F i g u r e s 2 and 3 ) . during  t h i s process t h a t the presence o f some s i g n i f i c a n t  meiotic abnormalities  2.  2.1.  Itis  was  discovered.  C y t o l o g i c a l methods  G e n e r a l f i x a t i o n and s t a i n i n g procedure used f o r  meiotic  products. E n t i r e or p a r t i a l t a s s e l s were c o l l e c t e d a t the  appropriate 3:1  or 4 : 1  time and f i x e d i n e t h a n o l / a c i d i c a c i d e i t h e r vol/vol.  A l t e r n a t i v e l y , Carnoy's mixture was  A f t e r 24 hours o f f i x a t i o n a t room temperature, the  used.  m a t e r i a l was t r a n s f e r r e d t o ? 0 % ethanol  and s t o r e d i n a  deep-freeze a t - 2 0 degrees C. u n t i l i t was The  studied.  c l a s s i c p r o p i o n i c s t a i n was found t o g i v e the  best and most c o n s i s t e n t r e s u l t s f o r pachytene a n a l y s i s as w e l l as r o u t i n e study, f o l l o w i n g the standard technique.  squashing  The s t a i n was prepared from" carmine  S c i e n t i f i c Co., F a i r Lawn, New J e r s e y ) (Sigma, S t . L o u i s , MO) a c c o r d i n g  j(Fisher  and p r o p i o n i c a c i d  t o the method o f Burnham  (1982).  2.2.  Slide  preparation.  Clean dust  f r e e s l i d e s were obtained  by washing the  s l i d e s i n d i v i d u a l l y i n soap and hot water, r i n s i n g i n d i s t i l l e d water and wrapping each s l i d e i n l e n s paper where they were allowed  t o dry f o r a few days before  being  used.  22 Although time consuming, t h i s method was improve a l l the p r e p a r a t i o n s and study. using  2.3.  Coverslips lens  f o r s p e c i a l p r e p a r a t i o n s were cleaned  preparation.  method of L i n (1977) f o r maize endosperms  m o d i f i e d i n the f o l l o w i n g way a) S e l e c t e d  f o r root  r o o t s were p r e f i x e d  naphthalene ( A l d r i c h Chem. Co., dimethyl s u l f o x i d e Sallee b) The  for  was  tips:  i n a s o l u t i o n of 1-bromoMilwaukee, WIS)  and  (Sigma) f o l l o w i n g the procedure of  (1982). r o o t s were then t r a n s f e r r e d through 3 r i n s e s  d i s t i l l e d water (5 min. c) The  used throughout t h i s  paper.  Somatic c e l l The  was  found to g r e a t l y .  each).  r o o t s were h y d r o l y s e d in-IN HCL  10 min.  and  of  were s t a i n e d  at 60 degrees  i n Feulgen f o r 1-2  C.  hours a t  room temperature. The min.  samples were then d e s t a i n e d i n d i s t i l l e d water f o r and  d) The  t r a n s f e r r e d to 50% a c i d i c a c i d f o r another 10  r o o t s were t r a n s f e r r e d to a f i l t e r  d i g e s t i o n s o l u t i o n c o n s i s t i n g of 0.9 0.025 M sodium c i t r a t e (Sigma) and phosphate (Sigma) a t pH e) The  10 min.  sterilized  M s o r b i t o l (Sigma),  0.025 M.  potassium  6.  samples were then t r a n s f e r r e d to a f i l t e r  steril-  i z e d d i g e s t i o n s o l u t i o n w i t h Beta-Glucuronidase (Sigma # G-O876)', 1 ml.  d i g e s t i o n s o l u t i o n to 0.1  ml.  enzyme  and  23 were t r e a t e d overnight  a t room temperature.  f ) Using a pasteur p i p e t t e , the r o o t s t i p s were t r a n s f e r r e d to a f i l t e r s t e r i l i z e d s o l u t i o n of 50% a c i d i c a c i d 50% d i s t i l l e d water and the c e l l s were allowed t o s w e l l f o r 20 min. g) The c e l l s were then g e n t l y and  sucked i n t o a pasteur  t r a n s f e r r e d t o a c l e a n s l i d e i n a mixture of 45% f i l t e r  s t e r i l i z e d d i s t i l l e d water-acidic  acid.  h) The i n d i v i d u a l c e l l s were g e n t l y c o v e r s l i p was a p p l i e d . mum  pipette  separated and a c l e a n  S u f f i c i e n t pressure to o b t a i n maxi-  c e l l expansion was determined by m o n i t o r i n g under the  microscope and then the s l i d e s were t r a n s f e r r e d  immediately  to dry i c e . i>) A f t e r c o v e r s l i p removal the s l i d e s were a i r ' d r i e d i n a dust f r e e chamber and then the p r e p a r a t i o n s  ,~  were t r a n s f e r r e d  to xylene ( F i s h e r ) and mounted i n Permount or Clearmount. This technique was found t o g i v e  extremely good r e s u l t s w i t h  no v i s i b l e cytoplasmic background and optimum spreading.  Magnifications  chromosome  of up t o 4500X were r e a d i l y  achieved.  2.4. P o l l e n The  sterility. 12-KI method (Burnham, 1982) was used t o e s t i -  mate p o l l e n s t e r i l i t y on the b a s i s o f s t a r c h content and size.  24  2.5« Microscope and f i l m s . The microscope used was a L e i t z Orthoplan with an optovar.  equipped  The f i l m s used were Kodak T e c h n i c a l Pan  # 2415 developed a t maximum c o n t r a s t (index 2.4) or Kodakcolor 6 4 or 25 ASA (processed by Kodak).  2.6.  In vivo karyotypic determination.  a) M e i o t i c karyotypes  : M e i o t i c samples were taken f o r  k a r y o t y p i c a n a l y s i s e i t h e r from s i d e t i l l e r s , when present or from p a r t i a l t a s s e l s e c t i o n s .  I n the l a t t e r case, the  i n c i s i o n made on the p l a n t was s t e r i l i z e d with ethanol and c l o s e d w i t h tape. b) Somatic karyotypes  : Karyotypes were a l s o  successfully  determined from young germinating seeds by t a k i n g  side  r o o t s and then a p p l y i n g the method d e s c r i b e d f o r somatic c e l l preparation.  2.7. S e l f i n g . The standard breeding method d e s c r i b e d by N e u f f e r (1982) was used.  2.8. A d d i t i o n a l methods. The f o l l o w i n g c y t o l o g i c a l methods were used i n t h i s study but photographic r e s u l t s from these techniques have not been i n c l u d e d i n t h i s  thesis:  25 RNA s p e c i f i c s t a i n s . Two g e n e r a l RNA s p e c i f i c s t a i n i n g methods were used.  P r e p a r a t i o n s were made "by f i r s t  squashing the c e l l s  i n 45% a c i d i c a c i d and- then removing the c o v e r s l i p s "by the. dry i c e method (Conger and F a i r c h i l d , 1953). drying,  the s l i d e s were e i t h e r s t a i n e d  at pH 4.9 i n 0.1M c i t r i c " a c i d b u f f e r nitrate  ( F i s h e r ) with a p r o t e c t i v e  After a i r  i n hot t o l u i d i n e blue  (Fisher)  colloidal  or i n s i l v e r developer  (Howell and Black, I98O). Both methods were met only with moderate success and  could not be improved beyond a c e r t a i n l e v e l because  of the cytoplasmic background.  DNA s p e c i f i c s t a i n . The  Feulgen s t a i n i n g procedure o f D a r l i n g t o n and  La Cour (I966) was found t o g i v e r e s u l t s as DNA s p e c i f i c s t a i n . by  e x c e l l e n t and c o n s i s t e n t Basic f u c h s i n was p r o v i d e d  Fisher.  Combined RNA and DNA s t a i n i n g . Acridine  orange as f l u o r e s c e n t  s t a i n was found ear-  l i e r t o g i v e good r e s u l t s on maize samples (Dr. R.S.K. Chaganti, pers. communication).  Acridine  orange has a l s o  been s u c c e s s f u l l y used as s t a i n f o r s p e c i a l by v a r i o u s  applications  groups (Bhaduri and Bhanja, 1962; S t o c k e r t and  L i s a n t i , 19?2; F r a n k l i n and F i l i o n ,  I98I).  26 From t h i s i n f o r m a t i o n  the f o l l o w i n g procedure was  S l i d e s were immersed i n 0.1 a t e i t h e r pH 6.1,  6.2  t r a n s f e r r e d to 0.125 2 min.  or 6.3  citrate buffer  f o r 10 min.  and  developed: (Fisher)  then were  mg./ml. a c r i d i n e orange (Sigma) f o r  at the s e l e c t e d pH.  The  s l i d e s were d e s t a i n e d  successive  baths of b u f f e r at the same g i v e n pH  each.  s l i d e s were then mounted i n the b u f f e r and  ved  The  immediately under e p i f l u o r e s c e n c e  F r a n k l i n and The  Filion  pH was  DNA  found to be c r i t i c a l and  pH u n i t s was  always  by  sample.  giving  experimentally.  enough to a l t e r  considerably  Under optimal s t a i n i n g was  red.  obtained but the r e s u l t s were not  thick sections.  To demonstrate c e l l f u s i o n E.M. were prepared.  Meiotic  i n epoxy (Pease, 1964)  "thick"  sections  samples were f i x e d and  embedded  and  were cut u s i n g a R e i c h e r t with a glass k n i f e .  1961).  obser-  consistent.  E.M.  i n hot  the pH  f l u o r e s c e d deep green w h i l e the RNA  High r e s o l u t i o n was  min.  (I98I).  the r e s u l t s f o r a g i v e n conditions  for 5  as"described  the best r e s u l t s could only be determined A change of 0.1  in 3  The  "thick" sections  of 2 microns  0MU3 ultramicrotome equipped preparations  b a s i c t o l u i d i n e blue a t pH 11.1  were then s t a i n e d (Trump et a l . ,  2? Chromosome  banding.  C-banding was performed u s i n g p i n a c y a n o l c h l o r i d e ( A l d r i c h ) a c c o r d i n g t o the p r o t o c o l of Narayan (I98O). P o s i t i v e C-banding was obtained u s i n g t h i s method, but a very h i g h percentage  o f c e l l s were c o n s i s t e n t l y l o s t i n  t h e - p r o c e s s , and f o r t h i s reason banding  o f one p a r t i c u l a r  abnormal c e l l i n a p r e p a r a t i o n appeared very The  C-banding procedure  developed  difficult.  by Ward (I98O) f o r  maize m a t e r i a l u s i n g Leishman's s t a i n a l s o r e s u l t e d i n high c e l l  loss.  3-  Karyotypes  In the. 3 ear samples which were g i v e n t o Dr. Wilkes two d i f f e r e n t karyotypes" 3).  were found.  Among the 30 p a r e n t a l karyotypes  from these l i n e s a l l karyotypes chromosomes.  ( F i g u r e 2 and F i g u r e i n i t i a l l y established  were 2n=20 with no B  These r e s u l t s were l a t e r confirmed by f u r t h e r  k a r y o t y p i c analyses o f the i n i t i a l seed s t o c k s .  Furthermore  the r e s u l t s of Pande et a l . (I983) i n s t u d i e s on the d i f f e r e n t Sikkim P r i m i t i v e s .confirm t h a t these stocks do not possess  B chromosomes.  Their- study a l s o shows very  l a r g e k a r y o t y p i c and phenotypic' polymorphisms among the Sikkim P r i m i t i v e s , which makes the p r e c i s e i d e n t i f i c a t i o n of  these stocks  difficult.  28 4. Growth  conditions.  Most samples were grown a t the Waltham Experiment S t a t i o n (Waltham, MA, U.S.A.) during the summers o f 1980, ±981 and 1982.  A l t e r n a t i v e l y some specimens were grown  i n the green-houses o f the U n i v e r s i t y o f MA i n Boston or under c o n t r o l l e d c o n d i t i o n s i n a growth chamber.  In the  l a t t e r case, the u s u a l growth c o n d i t i o n s were s e t a t 30/25 degrees s h i f t s  (day/night)  f o r 12 hour p e r i o d s .  The e f f e c t s  of d i f f e r e n t temperatures (ranging from 16 t o 30° C.) on the samples were a l s o s t u d i e d u s i n g the growth chamber.  5. I l l u s t r a t i o n s .  The m a g n i f i c a t i o n o f the photographs,when g i v e n i n the c a p t i o n s .  The estimated  g i v e n f o r the other photographs.  known, i s  magnification i s  29  F i g u r e 1 : Sikkim P r i m i t i v e ear samples. One ears which were g i v e n to Dr. H.G.  of the s m a l l Wilkes by  Dr.  J.K.S. Sachan as Sikkim P r i m i t i v e and used i n t h i s study ( t o p ) . The bottom ear i s the s e l f e d F l p r o d u c t . • N o t i c e the s i z e d i f f e r e n c e due  to  h y b r i d i t y (see s e c t i o n 4 ) .  F i g u r e s 2 and 3 s Sikkim P r i m i t i v e karyotypes. The karyotypes which,were e s t a b l i s h e d from  two the  i n i t i a l samples from I n d i a , showing knob patt e r n s and r e l a t i v e arm r a t i o s . The are arranged from # 1 to 10 The  (top to  chromosomes bottom).  observed v a r i a n c e i n chromosome s t r u c t u r e  and knob d i s t r i b u t i o n appears  to be t y p i c a l of  the Sikkim P r i m i t i v e types (see Pande, et a l . ,  1983).  30  3  RESULTS 1. Spontaneous c e l l f u s i o n . While e s t a b l i s h i n g the karyotypes  o f the samples  of Sikkim P r i m i t i v e , a s m a l l number o f abnormal m e i o t i c cells  were n o t i c e d .  The i n c i d e n c e and d i v e r s i t y of these  a b n o r m a l i t i e s appeared q u i t e v a r i a b l e from p l a n t t o p l a n t ranging from  near absence t o approximately  t o t a l number o f m e i o t i c c e l l s .  The most p u z z l i n g obser-  v a t i o n r e p e a t e d l y made was the occurrence t y p i c a l l y normal c e l l s ,  30% o f the  of c e l l s  among  karyo-  which contained  super-  numerary chromosome fragments and a d d i t i o n a l n u c l e o l i ( P e e t e r s , 1982).  During  the i n i t i a l phase o f t h i s  the o r i g i n and s i g n i f i c a n c e of these c e l l s  study,  were unknown  and . d i f f i c u l t t o e x p l a i n . The  i n i t i a l hypothesis  on o b s e r v a t i o n s  t h a t was formulated,  o f some abnormal t a p e t a l c e l l s ,  based  was t h a t  the tapetum was somehow i n v o l v e d i n the p r o d u c t i o n of these to  chimeric c e l l s .  Although  t h i s hypothesis  proved  be wrong, a t t e n t i o n had been focused on c e l l t o c e l l •  contacts and i t was noted t h a t c e l l f u s i o n occurred a t a low frequency  i n these samples ( i n the range o f 0 to 1%).  On the b a s i s o f these p r e l i m i n a r y o b s e r v a t i o n s , I decided t o c h a r a c t e r i z e spontaneous c e l l f u s i o n c y t o l o g i cally.  The f i r s t obvious  task was to search among the  parental population f o r plants containing a higher frequency  o f m e i o t i c a b n o r m a l i t i e s and then to determine  !  32  how r e p r e s e n t a t i v e these a b n o r m a l i t i e s were. To detect c e l l f u s i o n i n a c y t o l o g i c a l  preparation,  a s c r e e n i n g procedure had t o be developed to d e t e c t r a r e event.  I t was n o t i c e d t h a t a t a l l m e i o t i c stages ab-  normal anthers malities  this  c o n t a i n i n g a very high frequency  o f abnor-  (up t o 100%), mainly o f a b o r t i n g or aborted  could o c c a s i o n a l l y be found i n otherwise  cells  normal t a s s e l s .  In such anthers- i t was not uncommon to f i n d f u s i n g c e l l s , e s p e c i a l l y a t the e a r l i e r stages  o f meiosis  and the  a s s o c i a t i o n of conspicuous m e i o t i c a b n o r m a l i t i e s  such as  c e l l a b o r t i o n with c e l l f u s i o n proved t o be a g e n e r a l rule.  However i t was l a t e r found t h a t anthers  appeared a t f i r s t  which  t o be t o t a l l y normal could a l s o c o n t a i n  f u s i n g or fused c e l l s , which were then o b v i o u s l y difficult  very  to detect.  C e l l f u s i o n appeared t o be caused by absence of c e l l w a l l formation around the PMCs. w a l l s a r e normally of meiosis  present  around the PMCs a t a l l stages  i n maize (Warmke and Lee, 1977)•  when pr esent,  These w a l l s ,  are very easy to d e t e c t i n a c y t o l o g i c a l  p r e p a r a t i o n because during squashing u s u a l l y separated  from t h e i r c e l l  remain as very v i s i b l e separate example F i g u r e 9 ) . at  Thick c a l l o s e c e l l  the p r o t o p l a s t s a r e  w a l l s which then  s t r u c t u r e s (see f o r  In the present  study  i n some  anthers  e a r l y prophase 1, no v i s i b l e c e l l w a l l s could be  detected.  However, a n a l y s i s o f other anthers  of the  same t a s s e l showed t h a t a t l a t e r m e i o t i c stages, w a l l s were normal.  cell  As a consequence, the f o r m a t i o n of  33  the t h i c k c a l l o s e w a l l s which normally  forms pre-  m e i o t i c a l l y around the PMCs i n h i g h e r p l a n t s H a r r i s o n , 1966; be delayed and  Ledbetter  and  P o r t e r , 1970)  only at e a r l y meiosis  (Heslopappeared to  i n some p a r e n t a l p l a n t s  t h i s i s b e l i e v e d to have been the primary f a c t o r  causing  c e l l fusion. P o s t f u s i o n products could be i d e n t i f i e d  i n c r e a s e d c e l l volume, c e l l shape and  by  the presence of  a d d i t i o n a l chromosomes or chromosome fragments.  Aborting  c e l l s could a l s o sometimes serve as an i n d i c a t o r of a previous  f u s i o n event.  P o s t f u s i o n c e l l s were  c a n t l y l a r g e r than the normal c e l l s on the time c e l l f u s i o n occurred  (Table I ) .  a postfusion c e l l .  a f t e r c e l l f u s i o n , the c e l l had shape which became l e s s and c e l l regained  i t s shape.  to the observations al.,  Depending  p r i o r to f i x i n g ,  shape c o u l d be another c r i t e r i o n a l l o w i n g the of a. normal and  signifi-  cell  distinction  During or s h o r t l y  a distinct  "bilobed"  l e s s accentuated  as the  new  These observations  are s i m i l a r  of f u s i n g c e l l s i n v i t r o  (Shepard et  1983). A f t e r t a s s e l s from 30 p a r e n t a l p l a n t s were s t u d i e d ,  one  r e p r e s e n t a t i v e p a r e n t a l p l a n t found to c o n t a i n a h i g h  p r o p o r t i o n of a b n o r m a l i t i e s analysis.  was  selected for detailed  Over 17,000 i n d i v i d u a l c e l l s were observed  the r e s u l t s are summarized i n Table l a r g e number of c e l l s was  II.  and  Although such a  s t u d i e d , no r e a l p a t t e r n emerged  as to the d i s t r i b u t i o n of a p a r t i c u l a r abnormality  in a  given anther.  I n t h e p l a n t t h a t was s t u d i e d  m a t e l y 50% of t h e a n t h e r s were abnormal.  approxi-  The c y t o g e n e t  consequences o f spontaneous c e l l f u s i o n a r e d e s c r i b e d below.  35 Table I : Area comparison between normal and p o s t f u s i o n c e l l s  Stage *  Diameter  E a r l y Pro. I II  Pachytene E a r l y Met.  I  (mu  )  Area (mp  2  )•  N  105.9^ 155.8  8815.41 19064.47  17630.82 Exp. 19064.47 Obs.  N  110.6 155.8  9610.40 19064.47  19220.80 Exp. 19064.47 Obs.  N T  112.33 153.85  9910.49 18590.83  19820.98 Exp. 18590.83 Obs.  rji  rp  II  (mp.) Area  Expected/Observed  p  * Observations of each stage taken from the same p r e p a r a t i o n for uniformity. N : Normal c e l l  ( s i z e i s the averaged  T : Late p o s t f u s i o n c e l l complements.  v a l u e from 10 o b s e r v a t i o n s ) .  (no b i l o b e d shape) w i t h two  full  Table I I M e i o t i c survey o f a s i n g l e p a r e n t a l p l a n t with a h i g h i n c i d e n c e o f m e i o t i c a b n o r m a l i t i e s (From 1 7 , 1 3 2  Pachytene D i p l o t e n e D i a k i n e s i s Meta. I& Pro. I I Meta. I I Ana. I I Prop, o f Early Pro. I Ana. I Abnor.  STAGE # CELLS NORMAL EMPTY OR ABORTED ABORTING HAVING FUSED  1  1566  1912  680  562  877  ^95  528  286  162  69  215  187  81  7  10  2T,8A  IIT.I7A  1498  2100  1329  357  661  720  21  101  23^5  SYNCYTIUM MICROCELL  7*  3  2T*,3A*  6T,l4A  5T»,7A»  k%  h*  7*  0.5%  2k*  17  6  7  5  2  0  0  Q.h%  53  22  7  29  22  12  5  3  0.9%  2  k  13  21  15  I  0  0  0.396  3522  2310  982  744  1962  2910  2154  25^8  * Not a l l countable 1 2 3 k  23.9#  2  FUSING  TOTAL  observations)  I n c l u d i n g fused c e l l s P o l y p l o i d s , a n e u p l o i d s , and c e l l s with m e i o t i c a l l y a c t i v e supernumerary chromosomes A b o r t i n g c e l l mass from 3 o r more fused c e l l s C e l l s having between I and 9 chromosomes  T A u t o t e t r a p l o i d w i t h apparent m e i o t i c s t a b i l i t y A C e l l w i t h m e i o t i c a l l y a c t i v e a d d i t i o n a l chromosomes ( i n c l u d i n g chromosome fragments)  3Q#  2. The c y t o g e n e t i c consequences o f spontaneous c e l l f u s i o n .  2.1. Chromatin mo'dification and degradation  i n situ.  Approximately 0.2% of the c e l l s s t u d i e d were postf u s i o n products  i n which one complement was  clearly  "becoming d i s o r g a n i z e d and was d i s i n t e g r a t i n g i n situ"'" without  apparently  a f f e c t i n g the other complement which  appeared m o r p h o l o g i c a l l y 4 and 5)•  and m e i o t i c a l l y normal ( F i g u r e s  These i n i t i a l observations  were confirmed  the c y t o l o g i c a l a n a l y s i s o f f u s i n g c e l l s . difficult  by  Although very  t o d e t e c t , c e l l s t h a t were i n the process of  f u s i n g showed c l e a r l y i n some cases  t h a t the chromosomes  of one complement were u n c o i l i n g and d i s i n t e g r a t i n g i n the presence of the other complement which showed no or only minor changes i n chromosome s t r u c t u r e ( F i g u r e 6 ) . The  chromosomes t h a t were u n c o i l i n g had the " p u l v e r i z e d "  apprearance s i m i l a r to some P.C.C. chromosomes observed f o l l o w i n g i n v i t r o c e l l f u s i o n . (Matsui et a l . , 1972; Szabados and Dudits, 1980). As a r e s u l t of t h i s chromosome degradation  process  which was t a k i n g p l a c e i n one complement of some of the f u s i n g c e l l s d u r i n g c e l l d i v i s i o n , a spectrum o f postf u s i o n products contained  was obtained.  P o s t f u s i o n c e l l s which  one normal complement and only p a r t s o f the  "*" Used here t o s t r e s s the f a c t t h a t these  changes were  o c c u r r i n g i n s i d e one s i n g l e p o s t f u s i o n c e l l .  38 other chromosome complement ( F i g u r e ?) was  the most  frequent c l a s s of s t a b i l i z e d products of c e l l  fusion  (Table I I ) . The frequency of c e l l f u s i o n a t d i f f e r e n t m e i o t i c stages i s shown on F i g u r e 8.  C e l l f u s i o n was  obviously  .much more frequent at e a r l y prophase 1 and was a l s o o c c u r r i n g before the onset of m e i o s i s . are d i f f i c u l t  possibly  These stages  to analyse i n maize (Rhoades, 1950).  However some c l u e s as to the c y t o g e n e t i c s of p o s t f u s i o n at  these stages c o u l d be obtained by o b s e r v i n g the  n u c l e o l i . . As expected p o s t f u s i o n c e l l s were always found to c o n t a i n the n u c l e o l i of both complements.  Most  p o s t f u s i o n c e l l s were found w i t h more than two l a r g e n u c l e o l i and i n d i v i d u a l i z e d chromatin fragments a s s o c i a t e d with n u c l e o l i could a l s o be d e t e c t e d i n these c e l l s ( F i g u r e 9).  Although  i t was  not p o s s i b l e to determine  i f the  fragments i n these c e l l s were o r i g i n a t i n g from o n l y complement, t h i s was  one  i n f e r r e d from the r e c o v e r y at  pachytene (where i n d i v i d u a l chromosomes are h i g h l y v i s i b l e ) of  p o s t f u s i o n c e l l s c o n t a i n i n g one normal complement and  chromatin fragments from the other complement. o  F o l l o w i n g p a r t i a l i n s i t u degradation of  one  chromosome complement, c e l l s were found i n which the a d d i t i o n a l s t r u c t u r a l l y m o d i f i e d chromosomes a p p a r e n t l y had s t a b i l i z e d .  The  f a t e and morphology of these  ments was. very wide ranging.  frag-  In some c e l l s the fragments  f a i l e d t o f o l l o w the c y t o p l a s m i c cues f o r chromosome condensation which were f u n c t i o n a l f o r the normal complement ( F i g u r e 10).  Such fragments were s t a b i l i z e d  because, d e s p i t e t h e i r i n a b i l i t y t o condense, they showed no signs o f f u r t h e r d i s i n t e g r a t i o n and p a c h y t e n e - l i k e fragments were observed i n c e l l s as l a t e as telophase 1. However these fragments were not found beyond 1.  interphase  These u n c o i l e d chromosomes were never i n v o l v e d  with  the s p i n d l e and t h e r e f o r e appeared t o be a c e n t r i c .  Most  s t a b i l i z e d a d d i t i o n a l chromatin fragments r e s u l t i n g from p a r t i a l i n s i t u d e g r a d a t i o n appeared t o be m e i o t i c a l l y active, despite s t r u c t u r a l modification. i n d i c a t e d e i t h e r by c e n t r i c a c t i v i t y  T h i s was  ( F i g u r e 11) or i n one  e x c e p t i o n a l case by RNA s y n t h e s i s , i n a p o s t f u s i o n c o n t a i n i n g a m o d i f i e d supernumerary  clearly  cell  chromosome w i t h a  n u c l e o l u s o r g a n i z e r r e g i o n (NOR) ( F i g u r e 12).  I t can be  i n f e r r e d from observations o f t h i s c e l l along w i t h o b s e r v a t i o n s of numerous other c e l l s c o n t a i n i n g a spectrum of m o r p h o l o g i c a l l y m o d i f i e d supernumerary  chromosomes t h a t  the fragments which could s t a b i l i z e were fragments from random A chromosomes.  Stabilized centric  fragments  appeared to be always i n c l u d e d i n the anaphases reorganizing n u c l e i i n telophase.  and i n the  The r e s u l t s obtained  l a t e r i n t h i s study suggest that these c e l l s , which were m o d i f i e d k a r y o t y p i c a l l y and appeared s t a b i l i z e d , gave to f e r t i l e gametes.  rise  Genomic s t a b i l i t y o f the fragments r e s u l t i n g  from  i n s i t u chromosome degradation was not always achieved. M i c r o n u c l e i r e s u l t i n g from the supernumerary elements were observed,  as w e l l as c e l l a b o r t i o n . I t i s not  known i f t o t a l e l i m i n a t i o n of one chromosome complement could occur f o l l o w i n g c e l l f u s i o n without a f f e c t i n g the v i a b i l i t y of the c e l l . F i g u r e 8 suggests t h a t metaphase 1 r e p r e s e n t e d a c r i t i c a l stage f o r the s t a b i l i t y o f post f u s i o n c e l l s ' and i n a d d i t i o n t h a t p o s t f u s i o n c e l l s i n which no chromosome degradation occurred were more s t a b l than the c e l l s with p a r t i a l degradation. The reasons why metaphase 1 appears  t o be such a c r i t i c a l stage f o r  the s t a b i l i t y o f p o s t f u s i o n c e l l s are not known.  41  F i g u r e 4 s P o s t f u s i o n c e l l w i t h i n s i t u a b o r t i o n of one complement. E a r l y metaphase 1 c e l l  showing  m o d i f i c a t i o n and degradation of one chromosome complement (arrow) i n the presence of the other normal complement (two b i v a l e n t s are f u s e d ) . Estimated m a g n i f i c a t i o n X 1600.  F i g u r e 5 : P o s t f u s i o n c e l l w i t h i n s i t u a b o r t i o n of one  ,  complement. 10 normal b i v a l e n t s are present and the other complement i s showing v e r y  clearly  i n s i t u degradation. The p a c h y t e n e - l i k e s t a t e o f c o n t r a c t i o n of the a b o r t i n g complement  indicates  t h a t f u s i o n probably occurred a t or b e f o r e pachytene.  Metaphase 1 (X 1250).  42  ^3  Figure 6 : Cell fusion at diakinesis.  Chromosome u n c o i l i n g  and e a r l y " p u l v e r i z a t i o n " i s a p p a r e n t i n one complement ( a r r o w ) w h i l e o n l y m i n o r c h a n g e s i n chromosome  s t r u c t u r e are detectable i n the other  complement. Estimated  m a g n i f i c a t i o n X 700.  Figure ? : Postfusion c e l l with modified  supernumerary  chromosomes. One c o m p l e t e complement i s p r e s e n t with a d d i t i o n a l modified from the other  complement  of p a r t i a l  in situ  Diakinesis  (X 1 2 5 0 ) .  chromosome  fragments  (arrow) as a r e s u l t  degradation.  45  F i g u r e 8 : Frequencies  of c e l l f u s i o n and of s t a b i l i z e d  p o s t f u s i o n products throughout  m e i o s i s i n one  p a r e n t a l p l a n t . S t a n d a r d i z e d values from Table I I showing the d i s t r i b u t i o n of c e l l f u s i o n as w e l l as s t a b l e p o s t f u s i o n c e l l s  (CF)  throughout  m e i o s i s . T r e p r e s e n t s the p r o p o r t i o n of c e l l s which d i d not show any chromosome d e g r a d a t i o n of  e i t h e r complement while A r e p r e s e n t s the  p r o p o r t i o n of c e l l s was  partially  i n which one  complement  incomplete as a r e s u l t of  partial  i n s i t u chromosome d e g r a d a t i o n (aneuploids and  cells  w i t h s t a b l e a d d i t i o n a l m o d i f i e d supernumerary chromosomes). T h i s graph suggests t h a t the s t a bility  of p o s t f u s i o n c e l l s was  at metaphase 1, and furthermore cells  i n which there was  not always achieved that postfusion  no chromosome d e g r a d a t i o n  were more s t a b l e than c e l l s with p a r t i a l tion.  degradai-  (Values s t a n d a r d i z e d to 2000 o b s e r v a t i o n s  per s t a g e ) .  46  N U M B E R OF C E L L S A  F I G U R E S  F i g u r e 9 : Fusing c e l l a t e a r l y prophase 1 showing i n s i t u chromosome m o d i f i c a t i o n . Three l a r g e n u c l e o l i are present  and an a d d i t i o n a l s m a l l  on a separate  nucleolus  chromosome fragment (arrow) i n d i -  cates t h a t chromosome m o d i f i c a t i o n i s o c c u r r i n g probably  i n one complement only. Note the volume  of the fused c e l l i n comparison to the normal adjacent  c e l l and a l s o the presence of a c e l l  w a l l (next t o the arrow). Estimated  m a g n i f i c a t i o n X 300.  F i g u r e 10 : P o s t f u s i o n c e l l with a p p a r e n t l y m e i o t i c a l l y i n a c t i v e , modified  stabilized, supernumerary  chromosomes. The i n a b i l i t y of such fragments to  respond t o the cytoplasmic  mosome condensation  cues f o r chro-  which were o b v i o u s l y  f u n c t i o n a l f o r the normal complement i n d i c a t e s c l e a r l y t h a t these  chromosomes (arrow) were  modified.  (Black dots are b a c t e r i a l  Estimated  m a g n i f i c a t i o n X 1600.  contaminants).  48  49  F i g u r e 11  : P o s t f u s i o n c e l l with m e i o t i c a l l y a c t i v e m o d i f i e d supernumerary chromosomes. This e a r l y anaphase 1 c e l l suggests t h a t s t a b i l i z a t i o n of the chromosome fragments has  taken p l a c e . T e l o c e n t r i c a c t i v i t y  i s v i s i b l e i n one  o f these fragments  (arrow).  Such fragments were found to be i n c o r p o r a t e d i n the r e o r g a n i z i n g n u c l e i a t the telophases t h i s type of PMC Estimated  F i g u r e 12  may  generate f e r t i l e  magnification X  and  gametes.  1250.  : P o s t f u s i o n c e l l with a s i n g l e m o d i f i e d  super-  numerary chromosome with a m e i o t i c a l l y a c t i v e NOR.  S t r u c t u r a l m o d i f i c a t i o n of t h i s a d d i t i o n a l  chromosome 6 ( arrow ) i s very obvious and t h i s case i s f u r t h e r confirmed s y n t h e s i s (as evidenced  in  by low ribosome  by the s i z e of the  n u c l e o l u s ) which could i n d i c a t e e i t h e r a s m a l l e r number of repeats  or s t r u c t u r a l gene m o d i f i c a t i o n s .  D i a k i n e s i s (X 1250).  50  51 2.2.  Aneuploidy Aneuploid  and P o l y p l o i d y . c e l l s , d e f i n e d here as c e l l s  containing  e i t h e r a l a r g e r or a s m a l l e r number of i n t a c t chromosomes i n comparison t o t h e normal genomic c e l l s , were o c c a s i o n a l l y observed i n approx. 0.55%  of the c e l l s s t u d i e d .  Two  d i s t i n c t l y d i f f e r e n t mechanisms were n o t e d i n the of t h i s  production  aneuploidy. I n some a n t h e r s  i n which the PMCs d i d not appear to  have c e l l w a l l s (see s e c t i o n 1 ) , spontaneous of the p r o t o p l a s t s i n t o m i c r o c e l l s was  fragmentation  detected  (Figure  13).  These s m a l l a n e u p l o i d c e l l s appeared s t a b l e m e i o t i c a l l y and i n r a r e cases (0.017%) f u s e d w i t h normal c e l l s ,  generating  a n e u p l o i d c e l l s w i t h an i n c r e a s e d chromosome number. F i g u r e 14 shows an a n e u p l o i d p o s t f u s i o n c e l l which a p p e a r s , on the b a s i s of c e l l volume, shape and  chromosome mor-  p h o l o g y , t o have been d e r i v e d from such a f u s i o n . I n a d d i t i o n t o t h i s f i r s t mechanism f o r p a r t i a l a b o r t i o n o f one  aneuploidy,  complement i n s i t u f o l l o w i n g  spontaneous c e l l f u s i o n c o u l d a l s o g e n e r a t e e i t h e r m i c r o c e l l s or c e l l s w i t h some a d d i t i o n a l chromosomes which appeared m o r p h o l o g i c a l l y normal. c e l l s c o n t a i n i n g o n l y one  Examples o f p o s t f u s i o n  i n t a c t complement and  fragments  from the o t h e r complement have a l r e a d y been g i v e n . c e l l s r e s u l t e d from chromosome d e g r a d a t i o n  of  complement i n s i t u f o l l o w e d by s t a b i l i z a t i o n fragments.  The  These  one of the  c e l l s s u b j e c t t o t h i s p r o c e s s would be  expected t o c o n t a i n o n l y m o r p h o l o g i c a l l y a l t e r e d  chromosomes. few  However some o f these c e l l s  contained a  supernumerary chromosomes which appeared to be  normal i n a d d i t i o n to the abnormal chromosomes or chromosome fragments ( F i g u r e s 15 and 16).  I f the abnormal  chromosome fragments were e l i m i n a t e d i n such c e l l s , f o r i n s t a n c e i n the case of a c e n t r i c fragments, an a n e u p l o i d c e l l with a d d i t i o n a l chromosomes with no or only minor d e f e c t s could probably  be  generated.  M i c r o c e l l s could a l s o be d e r i v e d from  partial  chromosome e l i m i n a t i o n i n the normal c e l l adjacent to a fusing c e l l frequency  (see s e c t i o n 2 . 3 0 '  The t o t a l m i c r o c e l l  i n one p a r e n t a l p l a n t i s g i v e n i n Table I I .  The mechanism f o r the s e l e c t i v e degradation some chromosomes i s not understood  of only  but could be r e l a t e d  to the degree of i n d i v i d u a l chromosome  condensation,  g i v e n t h a t c e l l f u s i o n could take p l a c e a t stages where the chromosomes were h i g h l y c o n t r a c t e d ( F i g u r e 8 ) . As source  expected,  spontaneous c e l l f u s i o n could be the  o f s t a b l e p o l y p l o i d c e l l s which contained two  i n t a c t chromosome complements and i n which no chromosome m o d i f i c a t i o n could be d e t e c t e d  (Figure 1?).  appeared s t a b l e m e i o t i c a l l y and probably  Such  cells  could produce  unreduced gametes (see F i g u r e 8 ) . However m e i o t i c c o m p l i c a t i o n s o f p o s t f u s i o n c e l l s which showed no i n s i t u chromosome degradation  of either  complement were detected i n one s p e c i a l case.  Itis  b e l i e v e d t h a t when c e l l f u s i o n occurred l a t e ,  just  prior  53 t o o r a t e a r l y metaphase 1, t h e axes o f c e l l d i v i s i o n were a l r e a d y determined i n both c e l l s and t h a t t h i s r e s u l t e d i n a p o s t f u s i o n c e l l w i t h a double p o l a r i t y ( F i g u r e 1 8 ) . These c e l l s , which f u r t h e r demonstrated t h e o c c u r r e n c e of spontaneous c e l l f u s i o n , were q u i t e r a r e s i n c e t h e i n c i d e n c e o f c e l l f u s i o n a t metaphase 1 was a l r e a d y much s m a l l e r t h a n a t e a r l y prophase 1 ( F i g u r e 8 ) .  i t was  q u i t e i n t e r e s t i n g t o d i s c o v e r i n such p o s t f u s i o n  cells  t h a t t h e f o r m a t i o n o f t h e two s p i n d l e s c o u l d be unequal w i t h a "main" s p i n d l e ( w h i c h i n v o l v e d most chromosomes) and a minor s p i n d l e ( F i g u r e 1 8 ) .  54  F i g u r e 13  : M i c r o c e l l s produced by spontaneous p r o t o p l a s t fragmentation.  Both c e l l volume and  number i n d i c a t e t h a t these two  chromosome  m i c r o c e l l s at  l a t e d i a k i n e s i s were d e r i v e d from one  initial  PMC,  These  probably  by p r o t o p l a s t cleavage.  m i c r o c e l l s were always w e l l i n d i v i d u a l i z e d and appeared to be s t a b l e m e i o t i c a l l y . Estimated  F i g u r e 14  magnification X  : Postfusion aneuploid  300.  c e l l . This c e l l at d i a k i n e s i s  contains 3 m o r p h o l o g i c a l l y chromosomes (arrow) and  normal a d d i t i o n a l  i s b e l i e v e d to have  been generated by f u s i o n with a m i c r o c e l l . T h i s i s i n f e r r e d from c e l l volume, shape of . the p o s t f u s i o n c e l l and  l a c k of chromosome  m o d i f i c a t i o n . Note the a d d i t i o n a l n u c l e o l u s . Estimated  magnification X  550.  55  1  4  56  F i g u r e 15 : P o s t f u s i o n c e l l with a d d i t i o n a l chromosomes which appear m o r p h o l o g i c a l l y normal. Three a d d i t i o n a l fused, normal l o o k i n g chromosomes are present  i n t h i s e a r l y metaphase I c e l l  ( l a r g e arrow) w h i l e a d d i t i o n a l fused chromosomes which appear m o d i f i e d and abnormal are a l s o present  ( s m a l l arrows).. I f these abnormal  mosomes are e l i m i n a t e d , an aneuploid r e s u l t . Note the l a t e p e r s i s t i n g  cell  chrocould  nucleolus.  Estimate m a g n i f i c a t i o n X -1300.  F i g u r e 16 : M u l t i c e l l f u s i o n a t d i a k i n e s i s showing i n s i t u chromosome degradation.  Thirteen  a d d i t i o n a l chromosomes a r e present 6 chromosome fragments. Estimated  m a g n i f i c a t i o n X 350'  partial  as w e l l as  57  58  F i g u r e 17 : P o l y p l o i d PMC. T h i s c e l l appeared on the b a s i s of i t s s i z e t o be d e r i v e d from from the spontaneous f u s i o n o f two PMCs. Twenty normal b i v a l e n t s a r e present  i n s t e a d o f the u s u a l 10. No  signs of chromosome degradation  are v i s i b l e .  Metaphase 1 (X 1850).  F i g u r e 18 : P o l y p l o i d p o s t f u s i o n c e l l with two axes of d i v i s i o n . Such metaphase 1 c e l l s a r e b e l i e v e d to r e s u l t from l a t e c e l l f u s i o n when c e l l p o l a r i t y was a l r e a d y determined and a r e an a d d i t i o n a l proof f u s i o n . Notice  o f the occurrence o f c e l l  the unequal s p i n d l e s with an  uneven chromosome d i s t r i b u t i o n . Such c e l l s may be i n v o l v e d i n the p r o d u c t i o n gametes. Estimated  m a g n i f i c a t i o n X 600.  o f aneuploid  59  17  IS  6o 2.3' C e l l  abortion  Table  II shows t h a t c e l l a b o r t i o n was  most frequent  c l a s s of m e i o t i c abnormality  presence of a b o r t i n g or aborted  c e l l s was  by f a r the and  the  found i n a  very l a r g e p r o p o r t i o n of the anthers t h a t were s t u d i e d . As mentioned p r e v i o u s l y , there was  usually a positive  c o r r e l a t i o n between the presence of a f u s i n g c e l l c e l l abortion i n neighboring c a u s a l i t y between the two  cells.  The  degree of  events i s not known but was  complete i n the sense t h a t c e l l f u s i o n and could be observed independently.  cell  Obviously  a b o r t i o n was a).  not achieved.  The  cell  cytogenetics  not  abortion abortion  could f o l l o w c e l l f u s i o n when the balance of the f u s i o n c e l l was  and  postof  cell  q u i t e v a r i a b l e , as shown below.  Abortion  of non  Chromatin The most frequent  fused  cells.  degradation c l a s s of a b o r t i n g c e l l s  c e l l s i n which only chromatin degradation p l a c e , while RNA  was  On the c o n t r a r y , RNA i n these c e l l s  was  was  taking  not a f f e c t e d (as seen by the  s y n t h e s i s appeared g r e a t l y enhanced  ( F i g u r e 19) and  only n u c l e o l i with no v i s i b l e  a b o r t i n g c e l l s .containing chromosome fragments were  o c c a s i o n a l l y observed.  However p a r t i a l  appeared to be achieved  i n some of t h e s e - a b o r t i n g  which then contained  nucleoli).  stabilization  an abnormal complement and  cells,  i n which  the chromosomes were i n phase m e i o t i c a l l y w i t h the  other  normal c e l l s  cells  (Figure 2 0 ) .  These p a r t i a l l y  were only seen at or a f t e r metaphase 1.  aborted  61 RNA degradation Some r a r e cases o f c e l l a b o r t i o n appeared, on the b a s i s of o b s e r v a t i o n s o f the nucleolus,, t o show RNA degradation only and a n u c l e o l a r c e l l s with a normal complement were found. Simultaneous  RNA and chromatin  degradation  The two p a t t e r n s of c e l l a b o r t i o n which have j u s t been d e s c r i b e d a r e b e l i e v e d to i n v o l v e mostly c e l l s t h a t were a t e a r l y a b o r t i n g s t a g e s .  When s t a b i l i z a t i o n c o u l d  not be achieved, both DNA and RNA appeared  t o be degraded  s i m u l t a n e o u s l y , r e s u l t i n g sometimes i n a c e l l w i t h no v i s i b l e n u c l e o l u s or chromatin..  These l a t e stages of c e l l  a b o r t i o n were easy t o i d e n t i f y because of s i g n i f i c a n t m e i o t i c a b n o r m a l i t i e s ( F i g u r e 21).  I n a d d i t i o n these  cells  s t a r t e d t o l o s e t h e i r volume and shape with the cytoplasm becoming v e r y dense and t h i c k , u l t i m a t e l y forming a s t r u c t u r e resembling a p o l l e n g r a i n w i t h a s h r i v e l e d  cell  w a l l ( F i g u r e 22). Arrested  cells  C e l l s which f a i l e d t o continue meiosis and were a r r e s t e d a t one p a r t i c u l a r m e i o t i c stage were a l s o among PMCs a t l a t e r stages.  observed  These c e l l s d i d not appear  g e n e t i c a l l y i n a c t i v e because a d d i t i o n a l n u c l e o l i were produced  ( F i g u r e 23).  l a t e m e i o t i c stages.  These a r r e s t e d c e l l s aborted a t  b). A b o r t i o n Following  of p o s t f u s i o n  cells  spontaneous c e l l f u s i o n , some of  the  p o s t f u s i o n c e l l s aborted, p o s s i b l y as a r e s u l t of l a c k synchrony or because of l a c k of s t a b i l i z a t i o n  following  the i n i t i a t i o n of i n s i t u chromosome m o d i f i c a t i o n d e g r a d a t i o n i n one considered  complement (which o b v i o u s l y  as a p a r t i a l  of  and  can  abortion)^. These a b o r t i n g  be post-  f u s i o n c e l l s seemed to have g r e a t e r s t a b i l i t y than the other a b o r t i n g l a t e meiotic two  cells  ( s i n c e they were a l s o observed at  s t a g e s ) , p o s s i b l y because of the presence of  complements.  In these c e l l s , the a b i l i t y of  chromosomes to condense c o u l d vary from one the other  ( F i g u r e 24)  which i l l u s t r a t e d  individual  chromosome to  again t h a t  the  degree of i n d i v i d u a l chromosome " m o d i f i c a t i o n i n s i t u appeared to vary and  a l s o t h a t the a b o r t i n g  s u b j e c t to the s i g n a l s f o r c e l l  synchrony.  c e l l s were  63  F i g u r e 19 : A b o r t i n g c e l l showing chromatin  degradation  and a d d i t i o n a l RNA s y n t h e s i s . Three n u c l e o l i are p r e s e n t , two on a fragment o f chromosome 6  (arrow).  Pachytene ( X 1250).  F i g u r e 20 : P a r t i a l l y aborted c e l l . T h i s c e l l was to  be i n synchrony  and appeared  found  with the other normal c e l l s  m e i o t i c a l l y s t a b i l i z e d despite  s t r o n g s t r u c t u r a l chromosome m o d i f i c a t i o n s .  Late D i a k i n e s i s ( X 1250).  SO  65  F i g u r e 21  : A b o r t i n g metaphase 1 c e l l s m a l l s p i n d l e and Estimated  Figure  22  Aborted Estimated  . N o t i c e the abnormally  chromatin fragments.  magnification X  1250.  cell. magnification X  600.  66  67  F i g u r e 23 : A r r e s t e d d i p l o t e n e c e l l . T h i s c e l l was found i n an anther a t telophase RNA s y n t h e s i s (arrow). tually  1 and shows a d d i t i o n a l  Such a r r e s t e d c e l l s  even-  aborted.  Estimated  m a g n i f i c a t i o n X 1250.  F i g u r e 24 : P o s t f u s i o n a b o r t i n g c e l l a t metaphase 1. Seven n u c l e o l i a r e present  as w e l l as a g r a d i e n t o f  chromosome c o n t r a c t i o n ranging from pachytene (arrow) to d i a k i n e s i s , which suggests t h a t the degree o f i n d i v i d u a l chromosome m o d i f i c a t i o n i n an a b o r t i n g c e l l  could be v a r i a b l e . The  v i a b i l i t y o f p o s t f u s i o n a b o r t i n g c e l l s appeared h i g h e r than a b o r t i n g c e l l s with only one complement. Estimated  m a g n i f i c a t i o n X 450.  68  2.4.  A d d i t i o n a l meiotic Syncytium  abnormalities  formation  A syncytium i s d e f i n e d as "a l a r g e  multinucleate  c e l l a r i s i n g from c e l l f u s i o n " ( R i n g e r t z and 1976).  The  term Plasmodium was  Savage,  a l s o used p r e v i o u s l y i n  the l i t e r a t u r e i n place of syncytium but p r e s e n t l y r e f e r s to the s t r u c t u r e s formed by slime molds. . S y n c y t i a were observed r a t h e r f r e q u e n t l y l a t e r meiotic  stages (Table  even at  II) but were common only i n  the anthers c o n t a i n i n g a very h i g h p r o p o r t i o n c e l l s and were u s u a l l y absent i n the other  of.aborting  anthers.  These g i a n t c e l l s were formed as a r e s u l t of 3 types of c e l l f u s i o n , e i t h e r f u s i o n between PMCs, f u s i o n between nurse c e l l s or f u s i o n between PMCs and nurse ( F i g u r e s 25 and was  26).  cells  While f u s i o n between only two  g e n e r a l l y found to form s t a b l e p o s t f u s i o n  cells  products,  the s t a b i l i t y of m u l t i c e l l f u s i o n products (3 or more c e l l s ) seemed very l i m i t e d s i n c e the s y n c y t i a appeared always to be a b o r t i n g .  These observations  are  by i n v i t r o c e l l f u s i o n , where m u l t i n u c l e a t e are known to have l i t t l e v i a b i l i t y  1976).  mouse and  Savage,  ( I 9 8 I ) have shown  i n v i t r o f u s i o n products between  Chinese hamster c e l l s can be v i a b l e .  In the present have no  protoplasts  ( R i n g e r t z and  However Marin and L a n f r a n c h i  that t r i p a r e n t a l hybrids  confirmed  other  viable pollen.  study s y n c y t i a were considered  e f f e c t than to decrease the number of  to  C e l l p o l a r i t y and The which had  cytoplasmic  70  determinants.  consequences of c e l l f u s i o n i n v o l v i n g c e l l s t h e i r a x i s of d i v i s i o n determined has  d e s c r i b e d i n s e c t i o n 2.2.  C e l l f u s i o n i n such  o b v i o u s l y .caused major m e i o t i c c o m p l i c a t i o n s , p o s s i b l y when the two  c e l l s had  the same o r i e n t a t i o n ( F i g u r e  cases except  t h e i r axes i n p r e c i s e l y  27).  In a d d i t i o n to c e l l p o l a r i t y , evidence presence of cytoplasmic  teen  determinants was  f o r the  found once i n  a p o s t f u s i o n c e l l where a g r a d i e n t of m e i o t i c f i g u r e s ranging  from metaphase 1 to telophase  ( F i g u r e 28).  found  T h i s o b s e r v a t i o n seems to be d i s t i n c t from  the observations study  1 was  t h a t were r e p o r t e d p r e v i o u s l y i n t h i s  of chromosomes at d i f f e r e n t stages  i n aborting c e l l s  of c o n t r a c t i o n  (see f o r example F i g u r e 24)  because i n  t h i s case the g r a d i e n t of m e i o t i c f i g u r e s appeared to a c r o s s - c e l l g r a d i e n t whereas i n a b o r t i n g c e l l s arrangement of the chromosomes was Abnormal RNA The (NOR)  and DNA  be  the  random.  levels  s t r u c t u r e of the n u c l e o l u s  organizer  region  and the n u c l e o l a r c y c l e i n maize are w e l l known  (Givens  and P h i l l i p s , 1976; De La Torre and  C o l i n a s , 1978).  In t h i s study a d d i t i o n a l n u c l e o l i were f r e q u e n t l y observed i n aborting c e l l s  (see F i g u r e s 19 and 24)  but a l s o  o c c a s i o n a l l y i n "normal" c e l l s adjacent to f u s i n g c e l l s , at stages with NOR RNA  s y n t h e s i s was  a c t i v i t y as w e l l as at stages where no expected ( F i g u r e 29).  71 In a d d i t i o n to i n c r e a s e d RNA  l e v e l s , abnormal  DNA  l e v e l s d e r i v e d by mechanisms other than those p r e v i o u s l y d e s c r i b e d i n t h i s p r o j e c t were a l s o found once i n an abnormal anther as s o m a t i c - l i k e n u c l e i t h a t were c l e a r l y polyploid  ( F i g u r e 30).  These n u c l e i may  have o r i g i n a t e d  e i t h e r by n u c l e a r f u s i o n or by abnormal DNA  replication.  Cytomixis The  presence of c e l l s undergoing cytomixis i s  o b v i o u s l y very easy to d e t e c t i n a c y t o l o g i c a l Only two  c e l l p a i r s showing what has  cytomixis were found i n t h i s study c e l l f u s i o n combined with delayed  been d e s c r i b e d  as  ( F i g u r e s 31 and 32) c e l l w a l l formation  not appear to r e s u l t i n c y t o m i x i s . indeed  preparation.  One  and did  could e n v i s i o n  t h a t c e l l w a l l formation around c e l l s at e a r l y  f u s i o n stages cytomixis.  could produce two  However, except f o r one  ( F i g u r e 32), c e l l f u s i o n was It  p a r t i a l l y fused c e l l s or two  always massive and  i s c l e a r that i f cytomixis had  p l a c e i n these anthers,  rare  and  exceptions complete.  been t a k i n g  t h i s could have been the o r i g i n o f  the c e l l s with a d d i t i o n a l chromosome fragments which were described e a r l i e r i n section Other  2.1.  observations  In a d d i t i o n to the a b n o r m a l i t i e s a l r e a d y  described  as a consequence of c e l l f u s i o n , other s i g n i f i c a n t n o r m a l i t i e s were found d u r i n g meiosis plants.  One  of these  parental  i s chromosome b r i d g e f o r m a t i o n which  c o u l d take p l a c e i n 100% ( F i g u r e 33)  i n the  ab-  of the PMCs of some anthers  while being t o t a l l y absent i n s i s t e r  anthers  •  from t h e same t a s s e l .  These b r i d g e s were d i c e n t r i c .  A n o t h e r group o f v e r y u n u s u a l m e i o t i c a b n o r m a l i t i e s was d i s c o v e r e d i n a p p r o x i m a t i v e l y 5 metaphases which a p p r e a r e d t o show n e o c e n t r i c a c t i v i t y ( F i g u r e 3*0 •  72  73  F i g u r e 25 - . L a r g e s y n c y t i a o f PMCs a n d t a p e t a l  cells.  C e l l f u s i o n o f "both t h e PMCs a n d t h e n u r s e is very v i s i b l e  cells  i n t h e s e two e a r l y p r o p h a s e 1  syncytia. Abortion i s s t a r t i n g i n the nurse  cell  syncytium. E s t i m a t e d m a g n i f i c a t i o n X 200.  F i g u r e 26 : D e t a i l o f F i g u r e 25 s h o w i n g f u s i o n PMCs a n d n u r s e c e l l s .  Transfer of chromatin  fragments from t h e t a p e t a l c e l l the  between  syncytium into  PMC s y n c y t i u m ( a r r o w ) i n d i c a t e s t h a t  fusion  b e t w e e n t h e two g i a n t c e l l s h a s o c c u r r e d . Notice the characteristic structures of aborting somatic n u c l e i  (some o f w h i c h a p p e a r t o h a v e  been i n d u c e d i n t o  division).  E s t i m a t e d m a g n i f i c a t i o n X 4-00.  7k  75  F i g u r e 27  : P o s t f u s i o n c e l l with two  p a r a l l e l independent  s p i n d l e s . T h i s metaphase 1 p o s t f u s i o n c e l l i s b e l i e v e d to have o r i g i n a t e d from a f u s i o n between two  c e l l s with the a x i s of d i v i s i o n  already  determined. Such c e l l s were u s u a l l y s u b j e c t to m e i o t i c complications may  but i n t h i s case  telophase  have been normal because of the o r i e n t a t i o n  of the s p i n d l e s . Estimated  F i g u r e 28  magnification X  200.  : P o s t f u s i o n c e l l with a m e i o t i c T h i s c e l l has  a clear c r o s s - c e l l meiotic  g r a d i e n t from telophase Estimated  gradient.  1 to metaphase 1.  magnification' X  450.  76  77  F i g u r e 29  : PMC This  with increased  l e v e l s of RNA  c e l l with a normal complement was  adjacent to a f u s i n g c e l l and RNA  synthesis  found  shows a d d i t i o n a l  (arrow) at a. stage were RNA  t h e s i s normally has Late d i a k i n e s i s  F i g u r e 30  synthesis.  (X  syn-  ceased. 1880).  : P o l y p l o i d somatic n u c l e i i n an anther at m e i o s i s . The  normal s i z e d n u c l e i  large ploidy variations were probably d e r i v e d from abnormal DNA appear to be  i n the  synthesis.  other n u c l e i which  therefore  may  from p r e - m e i o t i c c e l l s which  (X  560).  or  These n u c l e i d i d  of t a p e t a l o r i g i n ( s i n c e the  to enter m e i o s i s . Telophase 1  (arrow) i n d i c a t e  from n u c l e a r f u s i o n  c e l l s appeared normal) and originated  mid  not  tapetal have  failed  30  79  F i g u r e 31  : Cytomixis cell.  between a pachytene and an a b o r t i n g  Chromosome t r a n s f e r from the pachytene  c e l l i n t o the a b o r t i n g c e l l  is clearly  visible  and must be the r e s u l t of a,communication channel between the two only two  cases  cells  (arrow). T h i s i s one  of  of cytomixis t h a t were found  d u r i n g t h i s study. N o t i c e the fragmented pachytene chromosomes i n the a b o r t i n g Estimated  F i g u r e 32  magnification X  : Nucleo-cytoplasmic  cell. 1350.  t r a n s f e r between two  partially  fused e a r l y prophase 1 c e l l s . T h i s other p o s s i b l e example of cytomixis  shows t r a n s f e r of the  nucle-  olus through a l a r g e contact area between the cells. Estimated  magnification X  250.  two  81  F i g u r e 33 : Breaking chromosome b r i d g e . The fragment which i s sometimes  formed (arrow) can be l o s t and form  a micronucleus. Anaphase 1 (X 1880).  Figure  34  A p o s s i b l e example Metaphase  of n e o c e n t r i c  1 (X 1880).  activity.  82  3. The F l and F2 Generations F o l l o w i n g the d i s c o v e r y and c y t o g e n e t i c  analysis  of spontaneous c e l l f u s i o n i n the p a r e n t a l meiosis  i t was  a n t i c i p a t e d t h a t , e v e n t u a l l y , new karyotypes could be found i n the next g e n e r a t i o n . * Four ears  ( l a b e l l e d A l to  A4) were c o l l e c t e d f o l l o w i n g s e l f i n g o f the p a r e n t a l p l a n t s showing the abnormal meiosis  and o f which the  karyotypes were known (see M a t e r i a l s and Methods).  Most  of the seeds from these ears appeared normal (9^*3%) and the p o l l e n samples c o l l e c t e d from the p a r e n t a l had  shown an estimated  plants  f e r t i l i t y range o f 5^ to 91% (which  r e f l e c t e d the p r o p o r t i o n of abnormal meioses i n the p a r e n t a l population). Seeds from these 4 stocks were grown under i d e n t i c a l c o n d i t i o n s the next summer and the karyotypes o f 36 F l p l a n t s were e s t a b l i s h e d .  The r e s u l t s are summarized i n  Table I I I . By chance, stock A3 was the f i r s t to' flower and i n t h i s stock one p l a n t c o n t a i n i n g a s m a l l a d d i t i o n a l and independent chromosome was d i s c o v e r e d chromosome was present  ( F i g u r e 35)-  This  i n every c e l l and t h e r e f o r e was  karyotypic. As a d d i t i o n a l p l a n t s came .to m a t u r i t y ,  further  c y t o l o g i c a l examination o f stock A3 l e a d to the d i s c o v e r y of 3 a d d i t i o n a l p l a n t s w i t h accessory III).  chromosomes  These chromosomes were a l l d i f f e r e n t  (Table  morphologically  d e s p i t e the f a c t t h a t they had o r i g i n a t e d from the same  s e l f - p o l l i n a t e d plant. accessory  L i n e A3K14 had 3 independent  chromosomes as w e l l as what appeared to be  an a d d i t i o n a l fragment which could a l s o be independent but was u s u a l l y h y b r i d i z e d t o an A chromosome ( F i g u r e 36). The  c y t o l o g i c a l appearance and m e i o t i c  behavior  of a l l these chromosome fragments was extremely v a r i a b l e not  only between the 4 stocks  one  c l o n a l c e l l t o the other.  only one o f these accessory  bearing  them but a l s o from  In the 3 p l a n t s  chromosomes, t h i s  which was c l e a r l y a u n i v a l e n t ,  changed  containing fragment,  morphologically  i n t h a t i t could h y b r i d i z e to i t s e l f t o v a r i o u s  degrees  but more s i g n i f i c a n t l y , i t s s t a t e of c o n t r a c t i o n was q u i t e d i f f e r e n t from c e l l t o c e l l a t a g i v e n stage. example a t mid-pachytene i n some c e l l s i t was  For  completely  c o i l e d and appeared very heterochromatic but t h i s degree of c o n t r a c t i o n could range t o a completely u n c o i l e d i n other  cells.  apparently  U s u a l l y i t had a "beaded" appearance,  c o n s i s t i n g of a l t e r n a t i n g eu and heterochromatin.  M e i o t i c a l l y a l s o , t h i s chromosome showed a very behavior.  state  erratic  Although u s u a l l y independent, i t sometimes  h y b r i d i z e d t o the A chromosomes, causing complications.  meiotic  In a d d i t i o n , the chromosome appeared to  show e a r l y s e g r e g a t i o n  i n up to 5 0 % o f the c e l l s a t  metaphase/anaphase 1.  In the p l a n t s w i t h 3 a d d i t i o n a l  chromosome fragments t h i s was not the case because these chromosomes were u s u a l l y p a i r e d  ( F i g u r e 36) which  85 a p p a r e n t l y made them behave as t h e s t a b l e b i v a l e n t s . The  cytological  observations  t h a t were made o f t h e  s i n g l e u n i v a l e n t were s i m i l a r between t h e 3 p l a n t s b e a r i n g t h i s fragment but s i z e d i f f e r e n c e s were n o t i c e d between t h e s e 3 i n d i v i d u a l  fragments.  The degree o f  homology between t h e s e fragments i s n o t known. On t h e b a s i s o f chromosome morphology and number, i t was c l e a r t h a t t h e s e chromosomes were n o t Tripsacum contaminants (Dr. C.V. P a s u p u l e t i , p e r s .  communication)  which c o u l d have been i n t r o d u c e d from o t h e r s t o c k s i n the f i e l d d u r i n g p o l l i n a t i o n .  I n a d d i t i o n , no s i g n s  were found i n t h e p l a n t morphology o f t h e presence i n the genome o f Tripsacum genes. modified "B"  Although these a d d i t i o n a l  chromosomes c o u l d be d e f i n e d as " a c c e s s o r y " o r  chromosomes, i n t h e F l they d i d n o t have t h e t y p i c a l  maize B chromosome morphology o r b e h a v i o r . The  s e v e r e m e i o t i c a b n o r m a l i t i e s which had been  found i n many o f t h e p a r e n t a l p l a n t s were o n l y v e r y  scarce  i n t h e i r d i s t r i b u t i o n among t h e F l p l a n t s (Table I I I ) . I n t h e F l p l a n t s which c o n t a i n e d  such a b n o r m a l i t i e s , t h e  f r e q u e n c y o f abnormal PMCs was a l s o reduced.  I t i s not  known i f t h i s i s due t o an a d a p t i v e response and/or t o different  summer growth c o n d i t i o n s .  One s t o c k i d e n t i f i e d w i t h an a c c e s s o r y  chromosome  ( s t o c k A3K2) was s e l f f e r t i l i z e d and t h e F2 g e n e r a t i o n ( l a b e l l e d A3X2) was grown i n t h e growth chamber under "normal" c o n d i t i o n s (see M a t e r i a l s and Methods).  S u r p r i s i n g l y , pachytene a n a l y s i s r e v e a l e d t h a t heterochromatinisation ( F i g u r e 37)  extensive  o f the chromosome had occurred  and t h a t , i n a d d i t i o n , the chromosome had  become m o r p h o l o g i c a l l y  and m e i o t i c a l l y q u i t e s i m i l a r to  a maize B chromosome.  I t was a l s o found by somatic  a n a l y s i s o f a l a r g e number o f seeds that t h i s had  chromosome  s i g n i f i c a n t l y i n c r e a s e d i n number i n some l i n e s  one up to a maximum o f k ( F i g u r e 3 8 ) .  As expected these  supernumerary chromosomes were a l l i d e n t i c a l This observation represents p o s s i b i l i t y o f having not a m p l i f y  from  morphologically.  another c r i t e r i o n a g a i n s t the  a Tripsacum contaminant (which would  but on the c o n t r a r y be e l i m i n a t e d , see  Chaganti,  1965). The  p r e c i s e mechanism f o r t h i s r a p i d a m p l i f i c a t i o n  i s not known but may have been m i t o t i c s i n c e some mosaics were found i n the somatic  karyotypes.  A p l a n t c o n t a i n i n g 4 o f these chromosomes was found to have slower growth and some s h r i v e l e d l e a v e s .  Somatic  and m e i o t i c a n a l y s i s showed t h a t these supernumerary chromosomes had evolved  and s t a b i l i z e d and were c l e a r l y  B chromosomes. No aneuploids F l plants studied.  or t r i p l o i d s were found among the 36 In stock A l , two dwarf p l a n t s were  found (Table I I I ) and had abnormal karyotypes with l o c a t i o n s and chromosome breakage.  trans-  Table I I I : C h a r a c t e r i s t i c s o f the F l g e n e r a t i o n SEED STOCK PLANT#  A3 A3 A3 A3  A3 A3  A3  A4 A4 A3 A3 A3 A3 A3 A4 A3 A3 A4 AI AI AI AI A2 A2 A2 AI . A2 A2 A2 A2 A2 Ak A3 A3 A2 . A3  KI K2 K3  Kk  K5 K6 K7 K8  K9  KIO KII KI2 KI3  KI4  KI5 KI6 KI7 KI8  KI9  K20 K2I K22 K23  K24 K25  K26  K27 K28 K29 K30  K3I K32 K33 K34  K35 K36  KARYOTYPE  MEIOTIC ABNORMALITIES  2n:20 2n:20*I 2n:20*I 2ni20 2n:20 2n:20  Some c e l l f u s i o n s Some c e l l f u s i o n s Some c e l l f u s i o n s None None None C o l l e c t e d too e a r l y None None None Some c e l l f u s i o n s None None A b o r t i o n s & Fusions None None None None None Translocations* None None None None None Translocations* None None None None None None None None None None  2m20 2n:20 2m 20  2ni20-M 2n:20 2m 20  2ni20-l-3  2n:20 2m 20 2n:20 2n:20 2n:20 2n:20 2nj20 2n:20 2n:20 . 2n:20 2m 20 2n:20 2n:20 2n:20 2n: 20 2m20 2n:20 2n:20 2m20 2m20 2ni20 2m 20  PLANT MORPHOLOGY Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Dwarf Normal Normal Normal Normal Normal Dwarf Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal  * I n c l u d i n g spontaneous chromosome fragmentation and r e s u l t i n g i n 100% s t e r i l i t y i n l a t e m e i o s i s . SUMMARY Stock (ear) AI A2 A3 A4 Total  Nb. o f p l a n t s studied  5 9 17 5 36  Nb. o f abnormal karyotypes  Nb. o f a c c e s s o r y chromosomes  2  0 0 6  1,1,1  0 0  and 3  0 6  88  F i g u r e 35 '• F l progeny with the presence i n the genome o f a new m o d i f i e d  supernumerary chromosome  (arrow).  Both F i g u r e s 35 and 36 a r e c e l l s a t d i a k i n e s i s . Estimated  m a g n i f i c a t i o n X350.  F i g u r e 36 : F l progeny with a new karyotype c o n t a i n i n g 3 a d d i t i o n a l chromosomes and a s m a l l fragment. The  3 chromosomes i n t h i s c e l l are p a i r e d  together  ( l a r g e arrow) w h i l e , what appeared t o be an e x t r a fragment ( s i n c e i t could be i n d i v i d u a l i z e d ) i s h y b r i d i z e d t o a b i v a l e n t ( s m a l l arrow). Estimated  m a g n i f i c a t i o n X 800.  F i g u r e 37 s Change i n B chromosome morphology through i n creased h e t e r o c h r o m a t i n i s a t i o n F2.generation.  from the F l to.  The i n s e r t shows the chromosome  at mid-pachytene i n F l while the arrow shows the F2 chromosome a t the same stage. N o t i c e the "beaded" appearence o f the F l chromosome which may be due to an a l t e r n a t i o n o f euchromatin and heterochromatin. Estimated  m a g n i f i c a t i o n X 1200.  89  3 7  90  F i g u r e 38 : F2 s o m a t i c k a r y o t y p e s  o f s t o c k A3X2 w i t h 4 B  chromosomes. T h e s e a c c e s s o r y chromosomes  (bottom  r i g h t ) w e r e a l l d e r i v e d f r o m one i n i t i a l  chro-  mosome, a n d showed a r a p i d  evolution (see t e x t ) .  From a s o m a t i c m e t a p h a s e (X 4450).  II  « -Ji-  lt  II  II  II  n  it  4. H y b r i d i t y and the e f f e c t s of growth c o n d i t i o n s . When the o r i g i n a l samples from I n d i a were grown under d i f f e r e n t c o n d i t i o n s i n the green-house no or few examples of abnormal meiosis were found but t h e r e  was  g e n e t i c v a r i a t i o n i n p l a n t morphology i n comparison to the  field.  One s t r i k i n g example was the f o r m a t i o n o f  s e l f - p o l l i n a t i n g ears (having male i n f l o r e s c e n c e s formed at  the t i p of the e a r ) , a t r a i t  some o f the Sikkim P r i m i t i v e s  which has been found i n  (Sachan and Sarkar, 1982).  These s p e c i a l ears were never found i n the f i e l d could have been due to h y b r i d i t y combined with growth c o n d i t i o n s .  and  different  Although synapsis was always complete,  f u r t h e r evidence of h y b r i d i t y was found as knob h e t e r o z y g o s i t y , F l ear morphology  ( F i g u r e 1 ) and s e g r e g a t i o n of  two ear markers i n the F2 (Dr. G a l i n a t , p e r s . communication). I t appears that the samples were outcrossed between  two  d i f f e r e n t Sikkim P r i m i t i v e s or a Sikkim P r i m i t i v e and another popcorn but were not from n a t u r a l ,  open-pollinated  conditions. The e f f e c t s of d i f f e r e n t temperatures on the samples from I n d i a was s t u d i e d u s i n g the growth chamber.  Seven  p l a n t s were-grown a t 16 degrees C. (day/night) and, out of  these, two p l a n t s were dwarf and m o r p h o l o g i c a l l y  abnormal (one being almost t o t a l l y a l b i n o and the other having r e d pigmented l e a v e s ) .  These two p l a n t s d i d not  93 reach m a t u r i t y .  I n 3 o f the remaining  p l a n t s , which were  v e r y s m a l l too, m e i o t i c samples were taken and a l l showed approx. 80% empty or aborted PMCs w h i l e  chromosome  breakage was o c c u r r i n g i n the remaining  PMCs ( F i g u r e 39  and F i g u r e 40) w i t h no v i a b l e p o l l e n being formed a t l a t e meiosis. Another temperature s e t t i n g o f 23/18 degrees C. (day/night) showed no major type o f a b n o r m a l i t i e s , e i t h e r morphological  or c y t o l o g i c a l .  S i m i l a r r e s u l t s were obtained  i n experiments a n t e r i o r to those d e s c r i b e d and o f which no accurate records were kept.  These experiments,  q u i t e l i m i t e d , i n d i c a t e d t h a t these p l a n t s were s e n s i t i v e t o changes i n growth c o n d i t i o n s .  although extremely  94  F i g u r e s 39 and 40 : D i f f e r e n t karyotypes  o f two c l o n a l  cells  showing chromosome fragmentation as a • r e s u l t o f a temperature  s h i f t . N o t i c e the  f u z z y appearance o f both the chromosomes and the n u c l e o l i . The two c e l l s are a t diakinesis  and are a t the same m a g n i f i c a -  t i o n . (X 1250).  95  96 DISCUSSION  Although a number of questions have, remained unanswered, the s i g n i f i c a n c e of t h i s work i s to show t h a t spontaneous c e l l f u s i o n can be d i r e c t l y i n v o l v e d i n v i v o genesis of a number of d i f f e r e n t new containing  a d d i t i o n a l DNA.  karyotypes  In the l i t e r a t u r e ,  the  c o r r e l a t i o n between spontaneous c e l l f u s i o n and genomic changes has polyploids.  i n the  been l i m i t e d only to cle novo  spontaneous occurring  However t h i s study shows that spontaneous  cell  f u s i o n can a l s o generate aneuploidy and more s i g n i f i c a n t l y , new  karyotypes c o n t a i n i n g  s t a b l e new  a d d i t i o n a l DNA  and m o d i f i e d supernumerary chromosomes. DNA  was  This  found to d e r i v e from p a r t i a l i n s i t u  degradation of one  sequences  additional chromatin  complement, f o l l o w i n g c e l l fusion..  Some  of these a d d i t i o n a l chromosome fragments appeared to have been i n h e r i t e d ; ' and  evolved as t r u e B chromosomes.  This,  i s the f i r s t time that a mechanism f o r the formation of these sequences i n the genome has (Peeters  and  been demonstrated  Wilkes1983).  Spontaneous c e l l f u s i o n was c y t o l o g i c a l l y and reported  t h i s could  so i n f r e q u e n t l y .  difficult  e x p l a i n why  to  i t has  detect been  I t i s c l e a r however t h a t  spontaneous c e l l f u s i o n between normally i n d i v i d u a l i z e d c e l l s could be v e r y widespread. bution  The  extent of the c o n t r i -  of spontaneous c e l l f u s i o n to de novo  karyotypes both i n v i v o and investigated.  occurring  i n v i t r o remains to  be  97 Spontaneous c e l l f u s i o n  C e l l f u s i o n can be caused by a number of f a c t o r s both i n v i v o and  in vitro  ( R i n g e r t z and Savage, 1976).  In p l a n t s , c e l l w a l l s o b v i o u s l y r e p r e s e n t an a d d i t i o n a l n a t u r a l b a r r i e r to c e l l f u s i o n and must be removed before membrane f u s i o n can be induced  in vitro.  In t h i s  study  the PMCs of some anthers a t e a r l y m e i o t i c stages were found not to have c a l l o s e c e l l w a l l s , which were only formed l a t e r .  The  delay i n c e l l w a l l formation, p o s s i b l y  combined with underdeveloped anthers, appears to have been the main f a c t o r causing spontaneous c e l l f u s i o n . h y b r i d i t y and the s t r e s s generated i n environmental to  h y b r i d i t y was  by the r a d i c a l change  growth c o n d i t i o n s , from s h o r t day  temperate, may Although  Both  have been i n v o l v e d i n t h i s  synapsis was  found  tropical  delay.  always complete, evidence  ( s e c t i o n 4) and h y b r i d i t y ,  for  especially  under s t r e s s f u l c o n d i t i o n s , can be d e t r i m e n t a l to the g e n e t i c balance.  Numerous examples of u n c o o r d i n a t i o n due to h y b r i d -  i t y have been g i v e n i n the l i t e r a t u r e f o r both i n t e r i n t r a s p e c i f i c hybrids 1972;  (see S t e b b i n s , 1958;  Bennett et a l . , 1976  T a i and  and the I n t r o d u c t i o n ) .  r e c e n t l y , c r o s s e s i n grasshoppers  and  Vickery, More  have a l s o shown s i g n i f -  i c a n t f r e q u e n c i e s of chromosome mutations a f t e r h y b r i d i z a t i o n ( P e e t e r s , 1982;  Shaw et a l . , 1983)  and s i m i l a r  observations  were made i n . c r o s s e s between c e r t a i n p o p u l a t i o n s D r o s o p h i l a melanogaster i n the so c a l l e d  of  "hybrid.dysgenesis"  98 syndrome which i s now known to be caused by t r a n s p o s a b l e elements  (Ish-Horowicz,  In  1982).  the present study h y b r i d i t y by i t s e l f  d i d not  appear to be s u f f i c i e n t t o cause a delay i n c e l l w a l l f o r m a t i o n , s i n c e under some growth c o n d i t i o n s the p l a n t s did  not show these a b n o r m a l i t i e s . Experimental evidence showing the s e n s i t i v i t y o f  these p l a n t s t o temperature 4) and the responses  was g i v e n p r e v i o u s l y ( s e c t i o n  o f maize t o photoperiod changes i s  w e l l documented (Rood and Major, 1981; Rood and Major,  I98O; Canard  and Ledent,  I98I).  As i n d i c a t e d e a r l i e r , the p l a n t s f a i l e d to f l o w e r the f i r s t year ( i n I98O) because o f t h e i r pronounced s h o r t day requirements.  The second year the p l a n t s  flowered extremely l a t e i n the summer and these were the p l a n t s i n which the a b n o r m a l i t i e s were observed. i n daylength and temperature  A shift  i n t h e l a t e summer induced  these p l a n t s v e r y r a p i d l y i n t o meiosis and a n t h e s i s .  This  s t r e s s , combined with h y b r i d i t y , are b e l i e v e d to have been the two main f a c t o r s which caused delayed c e l l w a l l f o r mation  i n the PMCs and hence spontaneous c e l l  fusion.  These o b s e r v a t i o n s c o u l d i n d i c a t e t h a t spontaneous c e l l f u s i o n can be induced as a r e s u l t of s t r e s s caused by changes i n t h e environmental  c o n d i t i o n s and t h e r e f o r e be a  s i g n i f i c a n t c o n t r i b u t o r to spontaneous genomic changes.  If  t h i s h y p o t h e s i s i s c o r r e c t , t h i s c o n t r i b u t i o n c o u l d be important both i n v i v o and i n v i t r o s i n c e environmental parameters  can o b v i o u s l y be q u i t e v a r i a b l e i n both cases.  In s i t u chromosome m o d i f i c a t i o n and  degradation  The most s i g n i f i c a n t o b s e r v a t i o n t h a t was  made during  t h i s study and which does not appear to have been d e s c r i b e d p r e v i o u s l y i n the l i t e r a t u r e i s t h a t , f o l l o w i n g spontaneous c e l l f u s i o n , p a r t i a l chromatin degradation  of one  complement  could take p l a c e i n s i t u and g i v e r i s e to a range of d i f f e r e n t s t a b l e new  karyotypes c o n t a i n i n g a d d i t i o n a l  This important o b s e r v a t i o n  DNA.  is especially d i f f i c u l t  to e x p l a i n g i v e n the c l o n a l nature of the c e l l s Somewhat s i m i l a r observations degradation  The  of i n s i t u m o d i f i c a t i o n  of n a t u r a l l y t r a n s f e c t e d c l o n a l DNA  however r e p o r t e d  i n s t u d i e s on cytomixis  and  were  (Introduction).  f o l l o w i n g hypotheses could account f o r these  a) Genetic  involved.  observations:  variability  Although the occurrence of spontaneous mutations has  been known f o r a l o n g time, i t i s only r e c e n t l y through  p l a n t c e l l propagation  and  regeneration  t h a t the extent  the g e n e t i c v a r i a b i l i t y of c l o n a l l i n e s has established  i t y has  been t r u l y  (Reviewed by L a r k i n and Scowcroft, l ° 8 l ;  a l s o Screenivasan  and J a l a j a , 1 8 2 ) .  been coined  Q  This genetic  "somaclonal v a r i a t i o n " .  some c a u t i o n as to the r e a l frequency of t h i s must be taken. (Evans and  of  see  variabil-  However variability  Indeed c e l l c u l t u r e i t s e l f can be mutagenic  Sharp, 1 9 8 3 ) .  In the present  study g e n e t i c v a r i a b i l i t y could have  100 contributed conditions  to some extent to unbalanced  e s p e c i a l l y i f i t s n a t u r a l frequency was  because of s t r e s s and (see t h i s  postfusion increased  the presence of a transposable  element  discussion).  b) C e l l synchrony Studies  of i n v i t r o c e l l f u s i o n have c l e a r l y shown  t h a t , at l e a s t under some c o n d i t i o n s , synchrony can generate P.C.C. and  differences in c e l l  a l s o l o s s of chromatin  (Introduction). Despite the f a c t t h a t most PMCs i n maize are i n synchrony, minor d i f f e r e n c e s of phase can o c c a s i o n a l l y detected  and  furthermore, i n t h i s study c e l l s t h a t were  a r r e s t e d were a l s o observed ( s e c t i o n 2.3-)• between two  Cell  fusion  out of phase c e l l s could perhaps cause, i n  t h i s environment the l o s s of phase of one f o l l o w i n g c e l l f u s i o n , bearing were i n d i v i s i o n .  This l o s s of phase could  a drop i n the p r o p o r t i o n  c e l l s around t h i s stage ( F i g u r e  c) R e c o g n i t i o n  complement  i n mind t h a t these  c r i t i c a l stages such as metaphase and there was  be  of n o n - s e l f  cells  occur at  t h i s may  of " s t a b l e "  explain  why  postfusion  8).  DNA  Recent molecular a n a l y s i s of t r a n s f e c t e d DNAs has shown that s t a b l e plasmids become u n s t a b l e i n a f o r e i g n host c e l l  (Calos  et a l . , 1983'; Razzaque et a l . , 1983)-  This l a c k of balance could be due  to DNA  chromatin s t r u c t u r e and/or the a s s o c i a t e d  differences i n proteins.  The  101 presence of such a nucleocytoplasmic"imbalance i s very u n l i k e l y i n t h i s case because of the c l o n a l nature of the c e l l s  involved.  d) F u s i o n between a normal and The hypothesis the observations  an a b o r t i n g  cell  which appears best to account f o r  of i n s i t u chromatin degradation  of  one  complement i s t h a t c e l l f u s i o n could i n v o l v e a normal  and  an a b o r t i n g c e l l . Aborting  '  c e l l s were q u i t e frequent  (Table II)  and  were u s u a l l y a s s o c i a t e d with f u s i n g c e l l s . . Furthermore, a b o r t i n g c e l l s were not t o t a l l y unstable  genetically  since  p a r t i a l l y aborted  c e l l s were observed ( F i g u r e 20).  quite conceivable  t h a t , i f c e l l f u s i o n could take p l a c e  between a normal and  It i s  an a b o r t i n g c e l l , the degree of s t a b i -  l i z a t i o n of the a b o r t i n g cytoplasm could be i n c r e a s e d by the cytoplasmic  significantly  c o n d i t i o n s of the normal  c e l l and beyond a c e r t a i n t h r e s h o l d .  Moreover the  fact  t h a t these c e l l s were s u b j e c t to the e x t e r n a l m e i o t i c cues f o r d i v i s i o n and  synchrony may  a d d i t i o n a l element i n t h i s  have represented  stabilization.  Evidence of the presence of a n u c l e a r s t a b i l i z i n g chromatin has  an  component  been found i n Xenopus l a e v i s  oocytes.(Wyllie  et a l . , 1977)  the cytoplasmic  c o n d i t i o n s at metaphase i n Rana p i p i e n s  was  and  a component  a l s o found by Meyerhof and Masui (1979) •  stabilizing  This model would e x p l a i n most, i f not a l l of observations  o f i n s i t u chromatin degradation.  i n i t i a t i o n of c e l l a b o r t i o n could be induced to c e l l f u s i o n and  my  The just  prior  the degree of m o d i f i c a t i o n made to  a b o r t i n g complement c o u l d vary g r e a t l y depending on t i m i n g of t h i s i n i t i a t i o n . a b i l i t y was  a b o r t i o n were found themselves to be q u i t e  achieved  I f cytoplasmic  s t a b i l i z a t i o n could  c e l l s , the combination of these two  was  observed i n t h i s The  a few  recovery  cell  diversified  f o l l o w i n g the f u s i o n between normal and  e x p l a i n the c y t o g e n e t i c  the  A further factor for vari-  the f a c t t h a t the c y t o g e n e t i c paths o f  ( s e c t i o n 2.3.)•  the  be  aborting  v a r i a b l e s could  d i v e r s i t y of p o s t f u s i o n c e l l s  that  study. of p o s t f u s i o n c e l l s with  only one  or  " i n t a c t " a d d i t i o n a l chromosomes ( F i g u r e s 12, 15,  i s d i f f i c u l t t o e x p l a i n but may  be due  to s l i g h t d i f f e r e n c e s  i n chromosome s t r u c t u r e ( f o r i n s t a n c e i n c o i l i n g t r a n s c r i p t i o n a l a c t i v i t y ) i n the p r e - a b o r t i n g the i n i t i a t i o n of a b o r t i o n .  or  cell,  others not.  at  In other words, depending  on t h e i r i n d i v i d u a l s t a t e , some chromosomes would p r o t e c t e d but  be  Changes i n the nucleosome  t h e i r r e l a t i o n to nuclease  16)  and  s e n s i t i v i t y have been reviewed  by Weisbrod (1982). I f the hypothesis of one  complement was  between a normal and may  t h a t i n s i t u chromatin  indeed  degradation  the r e s u l t of the f u s i o n  an a b o r t i n g c e l l ,  then t h i s mechanism  be only very i n f r e q u e n t i n other cases o f spontaneous  c e l l fusion.  However s i n c e spontaneous c e l l f u s i o n must  "be the r e s u l t of s t r o n g environmental or g e n e t i c  causes  (such as a v i r u s i n f e c t i o n ) even i f these c o n d i t i o n s a r e very l o c a l i z e d , they probably frequency o f c e l l a b o r t i o n .  c o n t r i b u t e e q u a l l y t o the Furthermore as a l r e a d y  i n d i c a t e d p r e - f u s i o n c o n d i t i o n s probably induce c e l l a b o r t i o n i n some cases.  can, by themselves  104 B chromosome f o r m a t i o n  In  the p o s t f u s i o n c e l l s which contained s t a b i l i z e d  c e n t r i c a d d i t i o n a l chromosome fragments, these fragments were c l e a r l y i n c o r p o r a t e d i n t o the anaphases and t e l o phases, d e s p i t e s t r u c t u r a l m o d i f i c a t i o n ( F i g u r e 11). These PMCs appeared completely s t a b l e and t h e r e f o r e probably produced unbalanced gametes.  These gametes  appeared to be v i a b l e as i n f e r r e d from the r e c o v e r y o f 5 d i s t i n c t l y d i f f e r e n t karyotypes (one normal and 4 d i f f e r e n t abnormal karyotypes w i t h a c c e s s o r y chromosomes) from one s e l f p o l l i n a t e d F l ear, f o l l o w i n g s e l f i n g o f the p a r e n t a l p l a n t s showing f u s i o n o f PMCs.  I t i s , not  i m p o s s i b l e to exclude, however, that the a c c e s s o r y chromosomes that were observed i n the F l o r i g i n a t e d from spontaneous  c e l l f u s i o n and p a r t i a l i n s i t u chromatin  degradation during ovule f o r m a t i o n . C l e a r evidence was found that these new chromosomes were not contaminant Tripsacum chromosomes ( s e c t i o n 3) and on the b a s i s o f F l morphology and.behavior, these chromosomes were a l s o c l e a r l y not B contaminants. As demonstrated i n t h i s study, a c c e s s o r y chromosomes could d e r i v e d i r e c t l y from c e l l f u s i o n and f u r t h e r more the f a c t that these fragments were found t o have evolved from the F l t o F2  g e n e r a t i o n as t r u e B chromo-  somes, c l e a r l y shows that c e l l f u s i o n i s one mechanism  which can g i v e r i s e to these sequences i n the genome. Although  i t i s unanimously accepted t h a t Bs evolve  somehow from the normal complement (Gupta,  1981),  the p r e c i s e mechanism g e n e r a t i n g m o d i f i e d A fragments i n the genome has remained t o t a l l y unknown ( I n t r o d u c t i o n ) . Since B chromosomes are extremely p l a n t s and animals  widespread i n "both  (Miintzing, 1974j Jones,  1975) t h i s  could be an i n d i c a t i o n o f the t r u e frequency of spontaneous c e l l f u s i o n i n v i v o . I t i s not known i f , f o l l o w i n g spontaneous f u s i o n , some B chromosomes were p r e f e r e n t i a l l y  cell  lost  while others with d i f f e r e n t sequences were r e t a i n e d . The answer t o t h i s q u e s t i o n would shed more l i g h t on t h e controversy which e x i s t s as t o the nature of B chromosomes.  Some authors have indeed considered  these  exogenous sequences as p a r a s i t i c elements (Rhoades and Dempsey, 1972) , g i v e n t h e i r a p p a r e n t l y p a s s i v e c o n t r i b u t i o n i n the genome.  A more r e c e n t d e f i n i t i o n c o u l d be s e l f i s h  DNA ( D o o l i t t l e and Sapienza, I98O). known to be extremely  polymorphic  B chromosomes are  but a t l e a s t a p a r t  of t h i s polymorphism i s due to e v o l u t i o n and spontaneous morphological  changes (Bougourd and Parker, 1979)'  Some  types o f Bs, such as Bs with a NOR, are extremely uncommon and only two or three examples o f such B chromosomes have been g i v e n i n the l i t e r a t u r e T h i s o b s e r v a t i o n was confirmed one  case o f NOR B  (Carr and Carr,1980).  i n t h i s study, where only  chromosome was found  ( F i g u r e 12)  106 d e s p i t e the r e l a t i v e l y h i g h number o f p o s t f u s i o n c e l l s w i t h accessory chromosomes t h a t Were observed  (Table I I ) .  T h i s may i n d i c a t e t h a t some A sequences have a g r e a t e r c a p a c i t y t o evolve as s t a b l e B chromosomes. important  t o c o n s i d e r here that c e l l f u s i o n occurred  mostly at stages where the NOR was active.  Itis  transcriptionally  T h e r e f o r e t h i s d i d not appear to be a f a c t o r  which f a v o r e d the r e t e n t i o n of chromosome 6 (which  bears  the NOR) d u r i n g i n s i t u degradation, p o s s i b l y because a c t i v e chromatin  i s more s e n s i t i v e t o nucleases  (Weisbrod,  1982). The  i n h e r i t e d fragments were found t o evolve  m o r p h o l o g i c a l l y from the F l t o F2 g e n e r a t i o n  through  i n c r e a s e d h e t e r o c h r o m a t i n i s a t i o n , i n d i c a t i n g t h a t these sequences became i n a c t i v a t e d . to  I t was q u i t e s u r p r i s i n g  f i n d that t h i s morphological  e v o l u t i o n occurred so  r a p i d l y , although t h e r e o b v i o u s l y are q u i t e a few m i t o t i c c y c l e s from the zygote t o the l a s t cell  division.  pre-meiotic  I t i s a l s o i n t e r e s t i n g t o note here t h a t  i n maize stocks c a r r y i n g Bs, a n e g a t i v e between the presence  correlation  o f knob heterochromatin  B chromosome was e s t a b l i s h e d (Longley, 1938).  and the T h i s may  be due to the asynchronous r e p l i c a t i o n o f the knob and B heterochromatin  (Pryor et a l . ,  I98O).  The mechanism d e s c r i b e d here on the f o r m a t i o n o f B chromosomes accounts  f o r most of the o f t e n c o n f l i c t i n g  molecular  and  c y t o g e n e t i c s t u d i e s on Bs  (see I n t r o d u c t i o n ) .  There appears to have teen a discrepancy the r e l a t i v e l y  h i g h p r o p o r t i o n of p l a n t s i n the F l with  a d d i t i o n a l chromosomes and f u s i o n c e l l s w i t h accessory meiosis  (Figure 8).  the frequency  of s t a b l e post-  chromosomes i n t h e . p a r e n t a l  However t h i s could be e x p l a i n e d  p r e f e r e n t i a l f e r t i l i z a t i o n by sperm c o n t a i n i n g accessory  between  by  these  chromosomes, which i s a w e l l e s t a b l i s h e d B  chromosome p r o p e r t y  (Introduction).  This  hypothesis  i m p l i e s t h a t the s e l e c t i v e advantage of c a r r y i n g these fragments f o r f e r t i l i z a t i o n had  a l r e a d y been a c q u i r e d .  Furthermore, d e s p i t e the l a r g e number of m e i o t i c s t u d i e d (17,000) t h i s r e p r e s e n t s  only a s m a l l  p o r t i o n of the PMCs s i n c e 50,000,000  cells  pro-  or so p o l l e n are  formed by a medium-sized maize p l a n t (Paton, 1921).  108 Aneuploidy and p o l y p l o i d y .  In the 36 F l p l a n t s t h a t were examined, no a n e u p l o i d or  t r i p l o i d ( r e s u l t i n g from the f u s i o n of an egg with an  unreduced  sperm) were d i s c o v e r e d but, g i v e n that a n e u p l o i d  and p o l y p l o i d PMCs which appeared  completely s t a b l e were  observed d u r i n g the p a r e n t a l m e i o s i s , i t i s very l i k e l y t h a t these products of spontaneous c e l l f u s i o n can a l s o c o n t r i b u t e t o de novo o c c u r r i n g karyotypes.  Numerous  authors have shown that a n e u p l o i d and 2 or 4n p o l l e n i n p l a n t s can be v i a b l e (Karpechenko,  1927; M c C l i n t o c k , 1928;  P r i c e , 1956; M i l l e r , 1963; Grant, 1965; Ramanna, 1973; Pfeiffer  and Bingham, 1982).  Different,mechanisms  l e a d i n g to the f o r m a t i o n of  a n e u p l o i d c e l l s were found d u r i n g t h i s study. mechanisms were p a r t i a l  These  e l i m i n a t i o n and d e g r a d a t i o n of  only some chromosomes f o l l o w i n g c e l l f u s i o n and the f o r m a t i o n of m i c r o c e l l s which c o u l d fuse with normal (see  s e c t i o n 2.2.).  cells  I n a d d i t i o n , p o s t f u s i o n c e l l s with '  two axes of d i v i s i o n and no i n s i t u chromatin  degradation  ( F i g u r e 18) probably c o u l d a l s o c o n t r i b u t e t o the p r o d u c t i o n of a n e u p l o i d gametes by uneven t e t r a d f o r m a t i o n . Among these mechanisms f o r aneuploidy, the spontaneous fragmentation of PMCs i n t o m i c r o c e l l s and subsequent  f u s i o n o f these m i c r o c e l l s to normal c e l l s i s  probably the most s i g n i f i c a n t i n terms of g e n e t i c s t a b i l i t y .  The s t a b i l i t y o f m i c r o c e l l s i s indeed w e l l e s t a b l i s h e d and these c e l l s have been found t o d i v i d e n o r m a l l y i n p l a n t t i s s u e c u l t u r e ( T i n g et a l . , I 9 8 O ) . can be produced e x p e r i m e n t a l l y ,  Such c e l l s , which  are o f great  interest  f o r chromosome t r a n s f e r and the f o r m a t i o n of i n v i t r o aneuploids ( M c N e i l l and Brown, I 9 8 O ) . Cytogenetic mechanisms  evidence f o r the e x i s t e n c e  o f these  f o r aneuploidy does not seem t o have been  g i v e n p r e v i o u s l y i n the l i t e r a t u r e .  I  110 A d d i t i o n a l c y t o l o g i c a l observations  During t h i s study a few very unusual  additional  o b s e r v a t i o n s were made d u r i n g meiosis i n the p a r e n t a l plants.  I t i s only p o s s i b l e to s p e c u l a t e on the nature  and i m p l i c a t i o n s of these o b s e r v a t i o n s . I n a c t i v e supernumerary chromosomes F i g u r e 10 shows an example o f a p o s t f u s i o n c e l l with supernumerary chromosome fragments  which f a i l e d  to f o l l o w the cytoplasmic cues f o r chromosome condensation. C e l l s c o n t a i n i n g such " i n a c t i v e " chromosomes have a l s o been r e p o r t e d f o l l o w i n g c h e m i c a l l y induced abnormal meiosis (Rajendra and Bates, l 8 l ) and the presence of Q  cytoplasmic cues r e g u l a t i n g chromosome condensation i n d i v i d i n g c e l l s i s w e l l e s t a b l i s h e d (Meyerhof  1979).  Numerous hypotheses  and Masui,  can account f o r the o b s e r v a t i o n s  t h a t were made here o f supernumerary chromosome  fragments  f a i l i n g t o condense f o l l o w i n g p a r t i a l m o d i f i c a t i o n and d e g r a d a t i o n of one complement. Rungger (1979) has shown t h a t a c t i n i s present and i s i n v o l v e d i n chromosome condensation o f Xenopus chromosomes w h i l e Matsumoto et a l . (1980) have shown t h a t HI h i s t o n e p h o s p h o r y l a t i o n i s a l s o i n v o l v e d i n chromosome condensation.  More r e c e n t l y Otto et a l . (1981), s t u d y i n g  a l l o c y c l i c chromosomes i n Bloom's syndrome have suggested that chromosomes may possess a " c o i l i n g c e n t e r " which i s  Ill l o c a t e d near the centromere.  My observations would agree  with such a hypothesis s i n c e these u n c o i l e d chromosomes were never found to be i n v o l v e d with the s p i n d l e and thus appeared t o be a c e n t r i c .  Since the chromosomes o f the  i n t a c t complement were c o n t r a c t i n g normally, the o b s e r v a t i o n s t h a t were made o f p o s t f u s i o n c e l l s with i n a c t i v e supernumerary chromosomes confirms t h a t these chromosomes were s t r u c t u r a l l y m o d i f i e d as a r e s u l t o f p a r t i a l in' s i t u degradation.  I t was  a l s o q u i t e i n t e r e s t i n g t o n o t i c e t h a t these chromosomes were at l e a s t p a r t i a l l y s t a b l e (see s e c t i o n 2.1.)• Neocentric  activity  A few cases o f n e o c e n t r i c a c t i v i t y were ( F i g u r e 34).  observed  A c c o r d i n g t o Peacock et a l . (1981), n e o c e n t r i c  a c t i v i t y i n maize i s due only to a very h i g h number of copies o f a 185 bp. repeat i n chromosome K10 although repeat i s a l s o present i n knob heterochromatin.  this  The degree  of p r e f e r e n t i a l s e g r e g a t i o n i s d i r e c t l y p r o p o r t i o n a l t o the number o f copies o f the repeat c a r r i e d on K10. Since K10 was absent  i n these samples, my  observations  of p r e f e r e n t i a l s e g r e g a t i o n may i n d i c a t e a case of amplif i c a t i o n o f t h i s 185 bp. repeat from knob  heterochromatin  i n some o f the PMCs. D i c e n t r i c bridges  •  D i c e n t r i c bridges were sometimes observed i n 100% o f the PMCs w h i l e being t o t a l l y absent  and occurred  from s i s t e r  anthers of the same t a s s e l . be an i n d i c a t i o n  T h i s unusual  o f the presence  observation could  o f a t r a n s p o s a b l e element  i n the samples which was being induced i n some p r e - m e i o t i c anthers but not i n o t h e r s . In t h i s work, other examples were a l r e a d y g i v e n which showed t h a t m e i o t i c a b n o r m a l i t i e s c o u l d be l o c a l i z e d to some anthers w h i l e g e n e t i c a l l y  i d e n t i c a l s i s t e r anthers  were t o t a l l y normal and i n d i c a t i n g degree o f g e n e t i c  individualization.  Bridge formation as a r e s u l t posable  t h a t anthers show a h i g h  of a displaced trans-  element has been d e s c r i b e d by F e d o r o f f (1982) and  once formed, these b r i d g e s are perpetuated termed the chromatid by McClintock posable  (1942).  i n a cycle  type o f breakage-fusion-bridge Furthermore the presence  cycle  of trans-  elements i n maize i s w e l l e s t a b l i s h e d (Marx, 1983)  and some o f t h e i r important  g e n e t i c p r o p e r t i e s have been  s t u d i e d (Dooner and Nelson, 1979; K e r m i c l e , I98O; Burr and Burr, 1982).  The i n d u c t i o n o f t r a n s p o s a b l e elements  i n maize can occur a t an extremely  localized level.  example t h e i r a c t i v i t y can be l i m i t e d  For  only t o a s p e c i f i c  p a r t o f the k e r n e l ( F e d o r o f f , 1982) and what has been d e s c r i b e d as paramutation  probably r e p r e s e n t s  another  example o f l o c a l i z e d , t r a n s p o s a b l e element mediated genetic v a r i a t i o n  i n maize as w e l l as i n other p l a n t s  ( B r i n k , 1973; Hagemann, 1978).  113 I f the d i c e n t r i c bridge a transposable  formations were induced  element i t i s p o s s i b l e that the  by  element(s)  was  also mobilized  by the s t r e s s caused by c e l l  fusion  and  consequently c o n t r i b u t e d to p o s t f u s i o n genomic  and  chromosomal m o d i f i c a t i o n s . Meiotic  determinants.  A p o s t f u s i o n c e l l with a c l e a r c r o s s - c e l l g r a d i e n t was  observed ( F i g u r e 28).  This p o s t f u s i o n  appeared to i n d i c a t e that the m e i o t i c were present  made by other r e s e a r c h e r s ,  cell  determinants that  i n the c e l l s could be independent from  e x t e r n a l s i g n a l s f o r c e l l synchrony. was  meiotic  A similar  the  observation  following i n v i t r o  cell  f u s i o n i n l a r g e d i v i d i n g polykaryons where " m i t o t i c waves" were observed and  "suggesting  t h a t the c o n c e n t r a t i o n  m i t o t i c t r i g g e r s formed a g r a d i e n t ( R i n g e r t z and The  Savage, 19?6,  p.  key  determinants i n d i v i d i n g  (see J a c k i e and  In t h i s study a c l e a r example c o n f i r m i n g  presence o f " m e i o t i c " ( F i g u r e 28) and  cell"  these determinants p l a y a  r o l e i n developmental b i o l o g y  1980).  the  70).  presence of cytoplasmic  c e l l s i s w e l l e s t a b l i s h e d and  across  and  of  determinants was  Kalthoff, the  only found once  the r e l a t i o n s h i p between these f a c t o r s  c e l l synchrony i n the anthers i s not known.  Obviously  c e l l f u s i o n c o u l d d i s r u p t s e v e r e l y the balance, created cytoplasmic critical  f a c t o r s and  t h i s appeared to be e s p e c i a l l y  i n cases i n v o l v i n g c e l l p o l a r i t y ( F i g u r e  18).  by  114 Abnormal presence o f n u c l e o l i . The s y n t h e s i s o f a d d i t i o n a l n u c l e o l i was observed i n both "normal" and a b o r t i n g c e l l s a t stages where RNA s y n t h e s i s was expected as w e l l as a t stages a t which RNA s y n t h e s i s normally had ceased  ( F i g u r e 29).  o b s e r v a t i o n s were made i n cancer c e l l s  Similar  (Sheldon and  Lehman, 1981). Some o f these c e l l s may have shown rDNA a m p l i f i c a t i o n , as a response to the s t r e s s induced by the i n i t i a t i o n o f c e l l abortion.  Examples o f rDNA a m p l i f i c a t i o n are w e l l  known i n animal c e l l s  ( f o r a review see T o b l e r , 1975; Long  and Dawid, 1980) and, although p o s s i b l e examples have been g i v e n (Avanzi et a l . , 1973)> c o n t r o v e r s y as t o the e x i s t e n c e of rDNA a m p l i f i c a t i o n i n p l a n t s s t i l l  exists.  115 Conclusion  • Spontaneous c e l l f u s i o n i s d i f f i c u l t  to detect but  could prove t o be a s i g n i f i c a n t f o r c e i n both the i n v i v o and  i n v i t r o genesis o f spontaneous a n e u p l o i d s ,  and  new karyotypes c o n t a i n i n g  polyploids  a d d i t i o n a l DNA. The exact  prevalence and s i g n i f i c a n c e of t h i s mechanism i n the gene s i s of new karyotypes remains t o be i n v e s t i g a t e d . One mechanism g i v i n g r i s e to B chromosomes has been demonstrated f o r the f i r s t time. These chromosomes were found t o r e s u l t from p a r t i a l i n s i t u chromatin d e g r a d a t i o n of one complement, f o l l o w i n g spontaneous c e l l f u s i o n as w e l l as a s t r u c t u r a l e v o l u t i o n o f the r e s u l t i n g s t a b i l i z e d fragments, through The  heterochromatinisation.  f a c t t h a t spontaneous c e l l f u s i o n can generate  de novo s t a b l e a d d i t i o n a l gene sequences i n the genome i s of s e l f evident This  importance f o r e v o l u t i o n and gene dosage.  importance i s even g r e a t e r  i n l i g h t o f the f a c t t h a t  some of these s t a b i l i z e d sequences were probably modified It  as.a r e s u l t o f i n s i t u  extensively  degradation.  i s c l e a r t h a t some o f the s m a l l e r  stabilized  sequences generated by c e l l f u s i o n and p a r t i a l i n s i t u chromatin degradation could perhaps t r a n s p o s e . t o the A chromosomes and a l s o g i v e r i s e t o new genomic arrangements. Such t r a n s p o s i t i o n s  could be enhanced by the genomic s t r e s s  of c e l l f u s i o n and chromatin m o d i f i c a t i o n and degradation as w e l l as by the presence o f a c t i v a t e d t r a n s p o s a b l e  elements.  116 These transposed  sequences would probably  not then be s u b j e c t  to the extensive h e t e r o c h r o m a t i n i s a t i o n which was  found  to occur during B chromosome e v o l u t i o n (see a l s o Amos and Dover, 1981) and hence remain t r a n s c r i p t i o n a l l y a c t i v e . Spontaneous  c e l l f u s i o n t h e r e f o r e could a l s o be  i n v o l v e d i n the p r o d u c t i o n I t may  of g e n e t i c a l l y u n s t a b l e  cells.  p l a y a major r o l e i n k a r y o t y p i c e v o l u t i o n i n neo-  p l a s i a i f p o s t f u s i o n mechanism s i m i l a r to those t h i s study  can a l s o occur  mitotically.  described i n  117  BIBLIOGRAPHY  Amos, A. and Dover, G. I 9 8 I . 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