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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 University of Massachusetts i n Boston, I 9 8 2 . A, THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Botany) (Genetics Programme) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA JANUARY 1 9 8 4 © J o h n P.Peeters, 1 9 8 4 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e he a d o f my d e p a r t m e n t 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 . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f BOTANY The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1W5 Date January 1 5 , 1 9 8 4 7Q) ABSTRACT The o r i g i n of spontaneously occurring new karyotypes containing additional DNA has remained enigmatic, although these genomic changes have been frequently reported i n the l i t e r a t u r e both i n vivo and i n v i t r o . This study describes the cytogenetic consequences of spontaneous c e l l fusion i n vivo during meiosis i n the pollen mother c e l l s of Zea mays L. I t suggests that spontaneous c e l l fusion i s a natural mechanism which can contribute not only to the formation of spontaneously occurring polyploids but also to aneuploidy and to the genesis of new karyotypes which contain additional s t r u c t u r a l l y modified chromosomes, or B chromosomes. i i i TABLE OF CONTENTS ABSTRACT •• i i LIST OF TABLES v LIST OF FIGURES.... v i ACKNOWLEDGEMENTS • i x • INTRODUCTION 1 SPONTANEOUS ANEUPLOIDS AND POLYPLOIDS 3 B CHROMOSOMES '. 5 CYTOMIXIS 9 IN VITRO CELL FUSION AND PREMATURE CHROMOSOME CONDEN-SATION 13 SPONTANEOUS CELL FUSION . . 1? IN SITU CHROMATIN MODIFICATION 18 PURPOSE OF THIS STUDY , 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 K a r y o t y p e s 27 . Growth c o n d i t i o n s . . 28 I l l u s t r a t i o n s 28 RESULTS ' 31 Spontaneous c e l l f u s i o n 31 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 *. . . . 37 The F l and 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 i v DISCUSSION 96 Spontaneous c e l l fusion 97 In s i t u chromosome modification and degradation.... 99 B chromosome formation 104 Aneuploidy and polyploidy 108 Additional 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 postfusion c e l l s 35 Table I I : Meiotic survey of a single parental plant with a high incidence of meiotic abnormalities.... 36 Table III: Characteristics of the F l generation 8? LIST OF FIGURES v i Figure 1: Sikkim Primitive ear samples 29 Figure 2: Sikkim Primitive karyotype '. 29 Figure 3: Sikkim Primitive karyotype ' 29 Figure 4: Postfusion c e l l with i n s i t u abortion of one complement 41 Figure 5: Postfusion c e l l with i n s i t u abortion of one complement 41 Figure 6: C e l l fusion at diakinesis 43 Figure 7: Postfusion c e l l with modified supernumerary chromosomes 43 Figure 8: Frequencies of c e l l fusion and of s t a b i l i z e d postfusion products throughout meiosis i n one parental plant 45 Figure 9: Fusing c e l l at early prophase 1 showing i n s i t u chromosome modification 47 Figure 10: Postfusion c e l l with apparently s t a b i l i z e d , m e i o t i c a l l y i n a c t i v e , modified supernumerary chromosomes 47 Figure 11: Postfusion c e l l with m e i o t i c a l l y active modified supernumerary chromosomes 49 Figure 12: Postfusion c e l l with a single modified super-numerary chromosome with a m e i o t i c a l l y active NOR • ^9 Figure 13: Microcells produced by spontaneous protoplast fragmentation 5^ Figure jLki Postfusion aneuploid c e l l 5^ Figure 15: M u l t i c e l l fusion at diakinesis showing p a r t i a l i n s i t u chromosome degradation 56 Figure 16: M u l t i c e l l fusion at diakinesis showing p a r t i a l i n s i t u chromosome degradation 56 Figure 17: Polyploid PMC 58 Figure 18: Polyploid postfusion c e l l with two axes of d i v i s i o n 58 Figure 19: Aborting c e l l showing chromatin degradation and additional RNA synthesis 63 Figure 20: P a r t i a l l y aborted c e l l 63 Figure 21: Aborting metaphase 1 c e l l 65 Figure 22: Aborted c e l l 65 Figure 23: Arrested diplotene. c e l l 6? Figure' 24: Postfusion aborting c e l l at metaphase 1 67 Figure 25: Large syncytia of PMCs and tapetal c e l l s . . . . 73 Figure 26: D e t a i l of Figure 25 showing fusion between PMCs and nurse c e l l s 73 Figure 27: Postfusion c e l l with two p a r a l l e l independent spindles . 75 Figure 28: Postfusion c e l l with a meiotic gradient..... 75 Figure 29: PMC with increased lev e l s of RNA synthesis.. 77 Figure 30: Polyploid somatic n u c l e i i n an anther at mid meiosis 77 Figure 31: Cytomixis between a pachytene and an aborting c e l l 79 Figure 32: Nucleo-cytoplasmic transfer between two par-t i a l l y fused early prophase 1 c e l l s 79 v i i i Figure 33: Breaking chromosome bridge 81 Figure 34: A possible example of neocentric a c t i v i t y . . . 81 Figure 35: F l progeny with the presence i n the genome of a new modified supernumerary chromosome.. 88 Figure 36: F l progeny with a new karyotype containing 3 additional chromosomes and a small fragment..88 • Figure 3?: Change i n B chromosome morphology through increased heterochromatinisation from the Figure 3 8 : F2 somatic karyotype of stock A3X2 with 4 B Figure 39 and Figure 40: Different karyotypes of two clonal c e l l s showing chromosome fragmentation F l to F2 generation 88 chromosomes. 90 as a r e s u l t of a temperature s h i f t 94 i x ACKNOWLEDGMENTS I wish to express my deep appreciation to Dr. H.G. Wilkes f o r stimulating me to st a r t t h i s project while I was an undergraduate at the University of Massachusetts and for the continuous guidance and support he granted me throughout the evolution of t h i s work. I am greatly indebted to Dr. A.J.F. G r i f f i t h s f o r his supervision and h e l p f u l c r i t i c i s m . It i s also my pleasure to extend thanks to the members of my committee, to Caroline Bouloc and to my family for t h e i r help and encouragement. Preliminary r e s u l t s of this work, including Figures 4,6,12,15,17t19i23 and 40 were incorporated i n a thesis presented for the honors degree at the University of Massachusetts. These Figures have been included here i n order to make the present thesis complete. INTRODUCTION 1 The spontaneous appearance of new karyotypes con -t a i n i n g a d d i t i o n a l DNA has been f r e q u e n t l y observed i n both p l a n t s and animals i n v i v o but a l s o i n v i t r o , i n c e l l c u l -t u r e s . Karyotypes which appear de novo w i t h a d d i t i o n a l DNA show extreme d i v e r s i t y i n terms of t o t a l DNA content 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 important and d i f f i c u l t challenge. Although various models have been proposed to account f o r these spontaneous changes, these models have i n general r e -mained h y p o t h e t i c a l . One of these models i n v o l v e s 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 to spontaneously occur-i n g p o l y p l o i d s . Spontaneous c e l l f u s i o n has been detected o c c a s i o n a l l y 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 are e s s e n t i a l l y unknown. Experimental i n v i t r o c e l l f u s i o n can, however , provide some clues as to the genetics of 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 condi-t i o n s , a spectrum of products can be obtained i n c l u d i n g poly-p l o i d s , aneuploids, modified chromosomes, i n a c t i v a t e d chro-mosomes and genomic rearrangements. Some of these products are the r e s u l t of i n s i t u chromatin m o d i f i c a t i o n and chro-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 a l s o been 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, f o l l o w i n g c y t o m i x i s . This study describes the cytogenetic consequences of 2 spontaneous , c e l l f u s i o n i n v i v o , during meiosis of 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 that chro-matin m o d i f i c a t i o n and degradation i n s i t u can a l s o occur f o l l o w i n g spontaneous c e l l f u s i o n , generating aneuploids and c e l l s w i t h modified supernumerary chromosomes. Foll o w i n g s e l f i n g , new karyotypes were recovered c o n t a i n i n g a d d i t i o n a l DNA i n the form of modified supernumerary chromosomes. These u s u a l l y heterochromatic chromosomes, which are a l s o c a l l e d accessory or "B".chromosomes, are extremely common i n both p l a n t s and animals, but are of unknown o r i g i n . This i s the f i r s t study where spontaneous c e l l f u s i o n i s d i r e c t l y i m p l i c a t e d i n producing new karyotypes other than p o l y p l o i d y and that demonstrates c y t o l o g i c a l l y the pro-duction of B chromosomes from the A chromosomes. 3 SPONTANEOUS ANEUPLOIDS AND POLYPLOIDS Polyploids which appear spontaneously i n vivo have been frequently reported i n plants (Karpechenko, 1927; Babcock" and Navashin, 1930; Smith, 1941 ; Price, 1955;. M i l l e r , 1 9 6 3 ; Grant, 1965; Salesses, 1970; Tai and Vickery, 1972; Sarbhoy, I98O) 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 also been reported recently i n human:tissues, both of ma-lignant and non malignant o r i g i n s . (Atkin, 1979; Otto and Therman, 1982). Reports on the spontaneous i n vivo appearance of t r i -somies or of aneuploids are also widespread i n plants (Babcock and Navashin, 1930; Takagi, 1 9 3 5 ; Nandi, 1937; Smith, 1 9 4 1 ; McGinnis, 1 9 6 2 ; Hacker and Riley, 1963; Kohel , 1 9 6 6 ; Haunold, I 9 6 8 ; Khush, 1973)• In maize, spontaneous t r i p l o i d s have been reported by McClintock (1929) and Beadle ( 1 9 3 2 ) , and more recently the occurrence of spontaneous aneuploids i n maize was studied by Ghidoni 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 dr a s t i c effects of trisomy 1 3 , 18 and .21 are well known. Furthermore, the association of an-euploidy and cancer has been well established. (German, 1 9 7 4 ) . Recent studies show, however, that a very high frequency of the spontaneous aneuploids ifchat are detected i n humans are not viable. Spontaneous aneuploids and polyploids have also been found i n v i t r o i n tissue culture of both plant and animal 4 c e l l s ( S c a l e t t a et a l . , 1967; Weiss and Green, 1967; T i n g et a l . , 1980; McCoy and P h i l l i p s , 1982). A n e u p l o i d y g e n e r a l l y a f f e c t s f e r t i l i t y (Khush, 1973) and, as c l e a r l y shown i n t h e case of humans, i t s e f f e c t s can 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 l i k e l y t h a t a h i g h p e r c e n t a g e o f t h e 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 of e a r l y a b o r t i o n . However, t r i p l o i d s and p o l y p l o i d s can sometimes be a s s o c i a t e d w i t h more v i g o r o u s growth and c h a r -a c t e r i s t i c s ( M c C l i n t o c k , 1929), and t h i s a s s o c i a t i o n c o u l d a l s o prove t r u e f o r some c a n c e r s which show a n e u p l o i d o r p o l y p l o i d p a t t e r n s (German,1974; Rowley, I98O; K u l i c k and L i u , I 9 8 I ; Reichman and L e v i n , I 9 8 I ) . V a r i o u s hypotheses have been p r o p o s e d . t o account f o r th e appearance o f t h e s e new k a r y o t y p e s . Spontaneous p o l y -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 u s i o n , f a i l u r e o f c y t o k i n e s i s ( B e a d l e , 1932; Ramanna, 1973) or t o s p i n d l e f a i l u r e s , w h i l e spontaneous a n e u p l o i d s a r e most g e n e r a l l y t hought o f as a r i s i n g from 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 , g i v e n t h e d i f f i c u l t y o f d e t e c t i n g such spontaneous changes when t h e y o c c u r i n s i t u . 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 supernumerary chromosomes, a l s o c a l l e d a c c e s s o r y o r B chromosomes, have been known t o oc c u r i n a huge number o f p l a n t and a n i m a l s p e c i e s and p o s s i b l y even i n humans ( R i d l e r et a l . , 1970) ( f o r r e v i e w s see M u n t z i n g , 197^ and J o n e s , 1975). A l t h o u g h so f r e q u e n t l y s t u d i e d and r e p o r t e d on, B chromosomes have remained a p e r p e t u a l c u r i o s i t y i n t h a t n e i t h e r 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 under-s t o o d . The most g e n e r a l consensus i s t h a t Bs have nega-t i v e e f f e c t s by r e d u c i n g b o t h growth and f e r t i l i t y , as w e l l as r e d u c i n g t h e g e n e t i c s t a b i l i t y o f t h e normal chromosome complement (Me l a n d e r , 1950; M u n t z i n g , 197^; Jo n e s , 1975; Bougourd and P a r k e r , 1979; L a t a , 1982; P a r k e r et a l . , 1982)-. I n maize, where Bs were f i r s t 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, 19^1). Bs have a l s o been shown i n maize t o i n d u c e 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) because o f d e l a y e d knob h e t e r o c h r o m a t i n r e p l i c a t i o n ( P r y o r e t a l . , 1980). B chromosomes were 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 the autosomes e i t h e r by i n c r e a s i n g i t ( B a r l o w and Vosa, 1970; Ward, 1973; F l e t c h e r and H e w i t t , I98O) or by d e c r e a s i n g i t ( V i i n i k k a , 1973)• 6 A c c e s s o r y chromosomes a r e known t o be a b l e t o main-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 non-Mendelian modes o f i n h e r i t a n c e . I n t h e p r i m a r y o o c y t e o f g r a s s h o p p e r s 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 the egg s i d e r a t h e r t h a n t h e p o l a r body s i d e ( H e w i t t , 1976). I n maize the a c c u m u l a t i o n mechanisms f o r Bs i s u n i q u e . B chromosomes 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 f r e q u e n c i e s (up t o 98%) i n t h e second p o l l e n m i t o s i s ( C a r l s o n and Chou, 1981) . T h i s event i s s u b s e q u e n t l y f o l l o w e d by 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 o f t h e egg by sperm c o n t a i n i n g two B c h r o -mosomes (Roman, 1948). B e c k e t t (I982) has a l s o s u g g e s t e d 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 mechanism by which Bs a r e m a i n t a i n e d i n maize. Numerous s t u d i e s show t h a t B chromosomes a r e e x t r e m e l y p o l y m o r p h i c even w i t h i n p o p u l a t i o n s ( G i b s o n and H e w i t t , 1972; P a t t o n , 1977;Bougourd and P a r k e r , 1979; Volobuev, I98O; 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) . Polymorphism has been a s u b s t a n t i a l f a c t o r c o n t r i b u t i n g t o t h e d i f f i c u l t y i n f o r m u l a t i n g a h y p o t h e s i s as t o t h e o r i g i n and f u n c t i o n o f a c c e s s o r y chromosomes i n the genome. No a d d i t i o n a l l i g h t has been shed on t h e s e q u e s t i o n s by t h e m o l e c u l a r a n a l y s i s o f Bs. A l t h o u g h B chromosomes a r e known n o t t o p a i r w i t h t h e As d u r i n g m e i o s i s (Gupta, 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 t o r e v e a l any DNA component s p e c i f i c t o B chromosomes ( C h i l t o n and McCarthy, 1973; Dover, 1975)- I n some cases s p e c i f i c genomic sequences a r e f a r more abundant i n t h e 7 Bs than i n the chromosomes of the normal complement ( K l e i n and Eckhardt, 1976). According to Amos and Dover (1981), B chromosomes contain r e p r e s e n t a t i v e sequences 'of the spectrum of f a m i l i e s of DNA that make up the A chromosome complement and there i s no de_ novo o r i g i n of B chromosome sequences. Since accessory chromosomes are g e n e r a l l y viewed as having no necessary f u n c t i o n f o r the s u r v i v a l or genetic balance of an organism but appear to have, on the contrary, an o v e r a l l negative impact on growth and s u r v i v a l and t h a t they appear to have evolved s e l f - s u r v i v a l mechanisms, B chromosomes are sometimes r e f e r r e d to as p a r a s i t i c genetic elements. (Rhoades and Dempsey, 1972; Carlson, 1977? Parker et a l . , I982)/ A more recent d e f i n i t i o n f o r these DNA sequences could be s e l f i s h DNA ( D o o l i t t l e and Sapienza, 1980). A number of hypotheses have been proposed f o r the o r i g i n of B chromosomes. In maize, f o r example, B chro-mosomes have been suggested to o r i g i n a t e from the chro-mosome K10 (Ward, 1979)- Although i t i s only l o g i c a l to assume that accessory chromosomes derive somehow from the normal complement (Gibson and Hewitt, 1972; Patton, 1977; Volobuev, I98O; Amos and Dover, I98I), n e i t h e r the p r e c i s e mechanism nor the t i m i n g of these genomic changes are known. Given t h e i r heterochromatic and v a r i a b l e DNA composition, Bs could 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 evolve by a m p l i f i c a t i o n and sequence 8 transposition (Amos and Dover, 1981). Various mechanisms have been proposed for the transposition and amplification of s p e c i f i c classes of DNA and the genome can presently be viewed as being very " f l u i d " (see Dover, 1982; Lewin, 1982; D'Eustachio and Ruddle, 1983). It i s clear that explaining the de novo appearance of an additional 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. This study suggests how such additional fragments can a r i s e de novo. 9 CYTOMIXIS Cytomixis was defined by Gates (1911) as "an ex t r u s i o n of chromatin from the nucleus of one mother-c e l l through cytoplasmic connections, i n t o the cytoplasm of an adjacent m o t h e r - c e l l " . The term cytomixis has since been used i n a broader r a t h e r than narrower sense i n the l i t e r a t u r e . Some researchers have indeed used the term cytomixis to describe the t r a n s f e r of organelles between c e l l s ( W e i l i n g , 1965) or even f o r spontaneous c e l l f u s i o n (Salesses, 19?0). Although cytomixis has been f r e q u e n t l y reported, n e i t h e r i t s s i g n i f i c a n c e nor i t s genetic consequences are w e l l defined. Some i n v e s t i g a t o r s have considered cyto-mixis as an a r t i f a c t (Tarkowska, 19^5. 1966), but most view cytomixis as a true and widespread phenomenon. . Cytomixis has been mostly described i n angiosperms during meiosis of p o l l e n mother c e l l s (PMCs) (reviewed by Kamra, i960). I t has been reported i n f r e q u e n t l y i n somatic 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 the PMCs according to Cooper (1952). Un 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 other words, i t remains to be determined i f cytomixis i s expressed l o c a l l y or i n d i f f e r e n t p a r ts of a p l a n t . However, some l i g h t on t h i s q u e stion has been shed by Moris s e t ' s study. (1978), which showed th a t some anthers can be a f f e c t e d by, c y t o m i x i s , while other anthers of the same p l a n t can undergo completely normal meiosis. Most repo r t s on cytomixis describe what Bobak (I966) has defined as "chromosomes s t r e t c h i n g • f r o m c e l l to c e l l " , forming t y p i c a l and e a s i l y i d e n t i f i a b l e f i g u r e s . Such f i g u r e s would obviously only 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 channels. E.M. s t u d i e s have c l e a r l y shown the presence of l a r g e holes (2 microns) i n the c a l l o s e w a l l s of 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 e s t a b l i s h e d . (Heslop-Harrison, 1966; Whelan, 1974; Whelan et a l . , 197^; Cheng et a l . , 1975). The d e s c r i p t i o n of cytomixis and of i t s a f t e r e f f e c t s are extremely c o n f l i c t i n g . The consensus observation i s that p a r t i a l DNA t r a n s f e r occurs from a "donor" to 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". (Church, 1929; Cooper, 1952; S a r v e l l a , 1958; Kamra, 19.60; B e l l , 1964; Bobak, 1966; S a l e s s e s , 1970; Romanov and Orlova, 1971; Omara, 1976; Lakshmi and Rao, 1978; 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 contracted ( i . e . at metaphase), two i n t a c t aneuploid c e l l s could r e s u l t (see Omara, 1976). When p a r t i a l DNA t r a n s f e r occurs during interphase, t h i s would a l s o generate two unbalanced c e l l s , but i n v o l v i n g chromatin fragments. The v i a b i l i t y of the above groups of c e l l s has been questioned. Several researchers have suggested t h a t 11 cytomixis i s a p o s s i b l e cause of aneuploidy and p o l y p l o i d y ( S a r v e l l a , 1957; B e l l , 1964; Salesses, 1970; Romanov and Orlova, 1971; Tai and V i c k e r y , 1972; Omara, 1976). In a d d i t i o n , Kamra (i960) and Chen et a l . (1975) have suggested that the c e l l s w i t h a d d i t i o n a l chromatin fragments may a l s o give r i s e to f e r t i l e gametes. These fragments would then obviously be defined as accessory chromosomes. In s i t u chromatin 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 the migrated chromatin has' been described f r e q u e n t l y i n the r e c i p i e n t c e l l s . According to Shkutina and Kozlovskaya (1974) : " i f the a d d i t i o n a l chromatin that has penetrated i n t o the microsporocyte 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 set of chromosomes may apparently remain v i a b l e " . Most s t u d i e s on cytomixis r e v e a l that a high per-centage of the chimeric c e l l s abort and, as one could p r e d i c t , the incidence of cytomixis 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 f e r t i l i t y . According to Lakshmi and Rao (1978), the abnormalities a s s o c i a t e d w i t h cytomixis can reach 95% of the PMCs and complete s t e r i l i t y r e s u l t s . In some cases, anthers were found w i t h a l l the c e l l s connected one to the other, corresponding to 100% c y t o m i x i s . A high percentage of cytomixis can probably 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 cytomixis can only 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 described i n hybrids (de Nettancourt and Grant, 1963; Salesses, 19?0~; Tai and Vickery, 19?2), i n t r i p l o i d maize (McClintock, 1929) and also i n "normal" plants. Hypotheses concerning the o r i g i n of cytomixis are numerous and include the genetic b a r r i e r , the uncoordinated growth rates due to hybridity, the competition f o r space or f i n a l l y delayed c e l l wall formation (reviewed by Morisset, 19?8). 13 IN VITRO CELL FUSION AND PREMATURE CHROMOSOME CONDENSATION The p o t e n t i a l of s o m a t i c c e l l h y b r i d i z a t i o n was i m m e d i a t e l y n o t i c e d i n 1965 when H a r r i s and Watkins f i r s t produced i n t e r s p e c i f i c h e t e r o k a r y o n s by u s i n g i n a c t i v a t e d S e n d a i v i r u s e s . S i n c e t h i s t i me i n v i t r o c e l l f u s i o n has expanded i n t o an independent f i e l d o f r e s e a r c h . I n 1972 C a r l s o n e t a l . produced th e f i r s t p a r a s e x u a l i n t e r s p e c i f i c 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 r e g e n e r a t i o n 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 new i n s i g h t s as t o t h e r e a l s i g n i f i c a n c e o f t h i s s c i e n c e . A l t h o u g h i n v i t r o c e l l f u s i o n has a l r e a d y y i e l d e d s i g n i f i c a n t i m p l i c a t i o n s and answers i n g e n e t i c s , a number o f q u e s t i o n s on t h e m o l e c u l a r and c y t o g e n e t i c p r o -c e s s e s o f p o s t f u s i o n r e m a i n u n s o l v e d . S t a b l e homokaryons and h e t e r o k a r y o n s have been f r e q u e n t l y r e p o r t e d 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 ( r e v i e w e d by H a r r i s , 1970; R i n g e r t z and Savage, 1976) (see a l s o Evans et a l . , I98O and I 9 8 I ; G l e b a e t a l . , 1982; W i l s o n e t a l . , 1982; B l a u e t a l . , 1983). However g e n e t i c s t a b i l i t y o f p o s t f u s i o n p r o d u c t s i s n o t a g e n e r a l r u l e . I n t y p i c a l i n t e r s p e c i f i c h y b r i d s ( s y n c a r y o n s ) , t h e p a r e n t a l n u c l e i f u s e and chromosomes a r e p r o g r e s s i v e l y l o s t d u r i n g c e l l d i v i s i o n ( B l a u e t a l . , 1983)• The 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 donor, w h i l e t h e complement which remains i n t a c t i s t h e r e c i p i e n t ( D ' E u s t a c h i o and R u d d l e , 1983). The mechanism f o r t h i s 14 e l i m i n a t i o n has remained poorly understood. I f s e l e c t i v e pressure i s a p p l i e d on donor genes, the chromosome bearing these genes w i l l be r e t a i n e d . A f t e r 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 pressure i s no longer necessary. Genomic s t a b i l i t y i s sometimes achieved a f t e r a short p e r i o d (Kao et a l . , 1976). Chromosome lo s s e s are not l i m i t e d to i n t e r s p e c i f i c hybrids but a l s o occur i n i n t r a s p e c i f i c hybrids (reviewed by Ringertz and Savage, I976). As a r e s u l t of 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 of "donor" chromosomes are f r e q u e n t l y observed (Schieder, 1982; Tamaki, 1982; D'Eustachio and Ruddle, 1983). Chromosome lo s s e s can happen r a p i d l y at f i r s t and then, as s t a b i l i z a t i o n i s achieved, at a slower 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 populations o f t e n show considerable 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 c e l l s seem to 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 cloned populations d e r i v i n g from a s i n g l e f u s i o n event (Ringertz and Savage, 1976). Despite t o t a l l o s s of donor chromosomes, donor genes may be r e t a i n e d (Schwartz et a l . , 1971; Power et a l . , 1975; Dudits et a l . , 1979; Shepard et a l . , 1983). This 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 detected following c e l l fusion (see Boone et a l . , 19?2; Douglas et a l . , 1973; Friend et a l . , 19?6). Morphological changes including changes i n chromosome ch a r a c t e r i s t i c s have been reported following i n v i t r o c e l l fusion (Ringertz and Savage, 1976; Kao, 1977). In addition, revertants (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 also found. Various hypotheses have been proposed to account for chromosome losses i n c e l l hybrids. According to Bennett et a l . (1976) changes i n the chromosome-spindle in t e r a c t i o n i s a c r i t i c a l factor. However t h i s hypothesis involves the loss of whole chromosomes and does not explain the s t r u c t u r a l changes which have been reported following i n v i t r o c e l l fusion. Ringertz and Savage (1976) have suggested that nuclear asynchrony could generate premature chromosome condensation or P.C.C. which i n turn may be a factor causing chromosome breaks and translocations, as well as elimination. P.C.C. occurs immediately a f t e r an interphase nucleus i s confronted with the cytoplasm of a mitotic c e l l , and was f i r s t described i n 1970 by Johnson and Rao. In 1972, these same authors reported that fusion of mammalian mitotic and S phase c e l l s causes the state of "chromosome pulverization" and that t h i s results i n greater chromosome losses than fusion involving c e l l s i n Gl or G2. (Rao and Johnson, 1972). A precise description of 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 injected somatic plant nuclei (Von der Haar et a l . , I 9 8 I ) . The contribution of P.C.C. to chromosome elimination following i n v i t r o c e l l fusion remains unclear. 17 SPONTANEOUS CELL FUSION Reports of spontaneous c e l l f u s i o n i n v i v o are extreme-l y uncommon. Spontaneous c e l l f u s i o n has been found i n p l a n t hybrids (Salesses, 1970), i n haploids (Levan, 1941) or under "normal c o n d i t i o n s " ( P r i c e , 1956; Sarbhoy, 1980). In maize, spontaneous c e l l f u s i o n has been reported under ab-normal genetic c o n d i t i o n s i n asynaptic maize ( M i l l e r , 1963) and maize-Tripsacum hybrids (Chaganti, 1965)- On the basis of P.C.C. or E.M. evidence, a few studie s suggest that spon-taneous c e l l f u s i o n can a l s o occur 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) or i n Bloom's syndrome (Otto and Therman, I982). Spon-taneous c e l l f u s i o n has a l s o been reported 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; Ringertz 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 f u s i o n i s probably a very r a r e event which occurs mainly under ab-normal gene t i c or environmental c o n d i t i o n s . Furthermore, i t appears that n e i t h e r the e t i o l o g y nor even the true cytoge-n e t i c consequences of- spontaneous c e l l f u s i o n are c u r r e n t l y understood. 18 IN SITU CHROMATIN MODIFICATION Studies on i n v i t r o c e l l fusion have shown that struc-t u r a l chromatin changes as well as chromosome losses 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 usually only take place i n the "donor" complement. Changes i n chromatin structure and i n s i t u degradation have also been reported for clonal DNA i n studies on cytomixis. Recent-l y , Calos et a l . (1983) and Razzaque et a l . (1983) have shown that stable plasmids that are transfected into mammalian c e l l s are equally subject to a high frequency of i n s i t u modification i n the form of deletions, duplications and complex rearrangements, 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 observations i s not clear but these observations a l l appear to indicate that 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 subject to s t r u c t u r a l modifications and even degradation 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 "foreign" to a c e l l and possibly be recognized as such. PURPOSE OF THIS STUDY The purpose of t h i s study i s to : a) . Describe meiotic abnormalities that were detected i n anthers of Zea mays L., i n p a r t i c u l a r the detailed cyto-l o g i c a l consequences of i n vivo c e l l fusion. b) . Study the karyotypic and phenotypic evolution of the F l and F2 generations, following s e l f i n g of the parental plants with the abnormal meiosis. c) . Discuss the possible implications spontaneous c e l l fusion has i n the formation of karyotypes which appear de novo with additional:DNA. MATERIALS AND METHODS 1. Origin and hist o r y of the samples. Samples of Zea mays L. of the Indian landrace Murli or Sikkim Primitive were obtained by Dr. H.G. Wilkes of the University of Massachusetts i n Boston during his sabbatical year to India 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 Inst i t u t e (Wilkes, 1981). This small popcorn i s grown i n the Sikkim and Dar-jeel i n g regions of the Eastern Himalayas at mid-elevation (6000-8000 feet) i n moist t r o p i c a l cloud forest conditions and i s used only f o r ceremonial offerings by the Buddhistic peoples of the region. L i t t l e i s known about the o r i g i n or hi s t o r y of Murli which has been claimed to be a primitive sort of ancestral maize. The small ear (Figure 1) resembles the reconstructed ancestor of P.C. Mangelsdorf, but Sikkim Primitive could ac t u a l l y be a l o c a l adaptation to short day conditions of a commercial popcorn such as Ladyfinger (Wilkes, I98I). The samples were f i r s t grown i n 1980 at the Waltham Suburban Experiment Station (Massachusetts/U.S.A.) but the plants f a i l e d to flower before the k i l l i n g f r o s t because of th e i r pronounced short day requirements (Wilkes and Peeters, 1982). In I98I under i d e n t i c a l conditions the plants did flower i n early September and cy t o l o g i c a l material was ob-tained. In an attempt to fi n g e r p r i n t t h i s landrace, the 21 karyotypes were established (Figures 2 and 3). I t i s during this process that the presence of some s i g n i f i c a n t meiotic abnormalities was discovered. 2 . Cytological methods 2 . 1 . General f i x a t i o n and staining procedure used for meiotic products. Entire or p a r t i a l tassels were col l e c t e d at the appropriate time and fixed i n ethanol/acidic acid either 3 : 1 or 4 : 1 v o l / v o l . A l t e r n a t i v e l y , Carnoy's mixture was used. After 24 hours of f i x a t i o n at room temperature, the material was transferred to ? 0 % ethanol and stored i n a deep-freeze at - 2 0 degrees C. u n t i l i t was studied. The c l a s s i c propionic s t a i n was found to give the best and most consistent r e s u l t s f o r pachytene analysis as well as routine study, following the standard squashing technique. The s t a i n was prepared from" carmine j(Fisher S c i e n t i f i c Co., F a i r Lawn, New Jersey) and propionic acid (Sigma, St. Louis, MO) according to the method of Burnham ( 1 9 8 2 ) . 2 . 2 . S l i d e preparation. Clean dust free s l i d e s were obtained by washing the sl 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 lens paper where they were allowed to dry f o r a few days before being used. 22 Although time consuming, th i s method was found to greatly . improve a l l the preparations and was used throughout t h i s study. Coverslips for s p e c i a l preparations were cleaned using lens paper. 2.3. Somatic c e l l preparation. The method of L i n (1977) for maize endosperms was modified i n the following way f o r root t i p s : a) Selected roots were prefixed i n a solution of 1-bromo-naphthalene (Aldrich Chem. Co., Milwaukee, WIS) and dimethyl sulfoxide (Sigma) following the procedure of Sallee (1982). b) The roots were then transferred through 3 rinses of d i s t i l l e d water (5 min. each). c) The roots were hydrolysed in-IN HCL at 60 degrees C. for 10 min. and were stained i n Feulgen f o r 1-2 hours at room temperature. The samples were then destained i n d i s t i l l e d water for 10 min. and transferred to 50% a c i d i c acid f o r another 10 min. d) The roots were transferred to a f i l t e r s t e r i l i z e d digestion solution consisting of 0.9 M s o r b i t o l (Sigma), 0.025 M sodium c i t r a t e (Sigma) and 0.025 M. potassium phosphate (Sigma) at pH 6. e) The samples were then transferred to a f i l t e r s t e r i l -ized digestion solution with Beta-Glucuronidase (Sigma # G-O876)', 1 ml. digestion solution to 0.1 ml. enzyme and 23 were treated overnight at room temperature. f) Using a pasteur pipette, the roots t i p s were transferred to a f i l t e r s t e r i l i z e d solution of 50% a c i d i c acid 50% d i s t i l l e d water and the c e l l s were allowed to swell for 20 min. g) The c e l l s were then gently sucked into a pasteur pipette and transferred to a clean 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 gently separated and a clean coverslip was applied. S u f f i c i e n t pressure to obtain maxi-mum c e l l expansion was determined by monitoring under the microscope and then the s l i d e s were transferred immediately to dry i c e . i>) After coverslip removal the s l i d e s were air' dried i n a , ~ dust free chamber and then the preparations were transferred to xylene (Fisher) and mounted i n Permount or Clearmount. This technique was found to give extremely good re s u l t s with no v i s i b l e cytoplasmic background and optimum chromosome spreading. Magnifications of up to 4500X were r e a d i l y achieved. 2.4. Pollen s t e r i l i t y . The 12-KI method (Burnham, 1982) was used to e s t i -mate pollen s t e r i l i t y on the basis of starch content and si z e . 24 2.5« Microscope and fil m s . The microscope used was a L e i t z Orthoplan equipped with an optovar. The films used were Kodak Technical Pan # 2415 developed at maximum contrast (index 2.4) or Kodakcolor 6 4 or 25 ASA (processed by Kodak). 2 . 6 . In vivo karyotypic determination. a) Meiotic karyotypes : Meiotic samples were taken for karyotypic analysis either from side t i l l e r s , when present or from p a r t i a l t a s s e l sections. In the l a t t e r case, the i n c i s i o n made on the plant was s t e r i l i z e d with ethanol and closed with tape. b) Somatic karyotypes : Karyotypes were also successfully determined from young germinating seeds by taking side roots and then applying the method described f o r somatic c e l l preparation. 2.7. S e l f i n g . The standard breeding method described by Neuffer (1982) was used. 2.8. Additional methods. The following c y t o l o g i c a l methods were used i n t h i s study but photographic results from these techniques have not been included i n thi s thesis: 25 RNA s p e c i f i c stains. Two general RNA s p e c i f i c staining methods were used. Preparations were made "by f i r s t squashing the c e l l s i n 45% a c i d i c acid and- then removing the coverslips "by the. dry ice method (Conger and F a i r c h i l d , 1953). After a i r drying, the s l i d e s were either stained i n hot toluid i n e blue at pH 4.9 i n 0.1M c i t r i c " acid buffer (Fisher) or i n s i l v e r n i t r a t e (Fisher) with a protective c o l l o i d a l developer (Howell and Black, I98O). Both methods were met only with moderate success and could not be improved beyond a certa 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 staining procedure of Darlington and La Cour (I966) was found to give excellent and consistent results as DNA s p e c i f i c s t a i n . Basic fuchsin was provided by Fisher. Combined RNA and DNA staining. Acridine orange as fluorescent s t a i n was found ear-l i e r to give good results on maize samples (Dr. R.S.K. Chaganti, pers. communication). Acridine orange has also been successfully used as s t a i n for s p e c i a l applications by various groups (Bhaduri and Bhanja, 1962; Stockert and L i s a n t i , 19?2; Franklin and F i l i o n , I98I). 26 From th i s information the following procedure was developed: Slides were immersed i n 0.1 c i t r a t e buffer (Fisher) at either pH 6.1, 6.2 or 6.3 f o r 10 min. and then were transferred to 0.125 mg./ml. acridine orange (Sigma) fo r 2 min. at the selected pH. The s l i d e s were destained i n 3 successive baths of buffer at the same given pH f o r 5 min. each. The s l i d e s were then mounted i n the buffer and obser-ved immediately under epifluorescence as"described by Franklin and F i l i o n (I98I). The pH was found to be c r i t i c a l and the pH giving the best results could only be determined experimentally. A change of 0.1 pH units was enough to a l t e r considerably the r e s u l t s f o r a given sample. Under optimal staining conditions DNA fluoresced deep green while the RNA was red. High resolution was obtained but the r e s u l t s were not always consistent. E.M. thick sections. To demonstrate c e l l fusion E.M. "thick" sections were prepared. Meiotic samples were fixed and embedded i n epoxy (Pease, 1964) and "thick" sections of 2 microns were cut using a Reichert 0MU3 ultramicrotome equipped with a glass knife. The preparations were then stained i n hot basic t o l u i d i n e blue at pH 11.1 (Trump et a l . , 1961). 2? Chromosome banding. C-banding was performed using pinacyanol chloride (Aldrich) according to the protocol of Narayan (I98O). Positive C-banding was obtained using this method, but a very high percentage of c e l l s were consistently l o s t i n the-process, and f o r th i s reason banding of one p a r t i c u l a r abnormal c e l l i n a preparation appeared very d i f f i c u l t . The C-banding procedure developed by Ward (I98O) f o r maize material using Leishman's s t a i n also resulted i n high c e l l l o s s . 3- Karyotypes In the. 3 ear samples which were given to Dr. Wilkes two d i f f e r e n t karyotypes" were found. (Figure 2 and Figure 3). Among the 30 parental karyotypes i n i t i a l l y established from these l i n e s a l l karyotypes were 2n=20 with no B chromosomes. These re s u l t s were l a t e r confirmed by further karyotypic analyses of the i n i t i a l seed stocks. Furthermore the r e s u l t s of Pande et a l . (I983) i n studies on the di f f e r e n t Sikkim Primitives .confirm that these stocks do not possess B chromosomes. Their- study also shows very large karyotypic and phenotypic' polymorphisms among the Sikkim Primitives, which makes the precise i d e n t i f i c a t i o n of these stocks d i f f i c u l t . 28 4. Growth conditions. Most samples were grown at the Waltham Experiment Station (Waltham, MA, U.S.A.) during the summers of 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 of the University of MA i n Boston or under controlled conditions i n a growth chamber. In the l a t t e r case, the usual growth conditions were set at 30/25 degrees s h i f t s (day/night) f o r 12 hour periods. The effects of d i f f e r e n t temperatures (ranging from 16 to 30° C.) on the samples were also studied using the growth chamber. 5. I l l u s t r a t i o n s . The magnification of the photographs,when known, i s given i n the captions. The estimated magnification i s given f o r the other photographs. 29 Figure 1 : Sikkim Primitive ear samples. One of the small ears which were given to Dr. H.G. Wilkes by Dr. J.K.S. Sachan as Sikkim Primitive and used i n th i s study (top). The bottom ear i s the selfed F l product.•Notice the size difference due to hybridity (see section 4). Figures 2 and 3 s Sikkim Primitive karyotypes. The two karyotypes which,were established from the i n i t i a l samples from India, showing knob pat-terns and r e l a t i v e arm r a t i o s . The chromosomes are arranged from # 1 to 10 (top to bottom). The observed variance i n chromosome structure 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 Primitive types (see Pande, et a l . , 1983). 30 3 RESULTS 1. Spontaneous c e l l fusion. While establishing the karyotypes of the samples of Sikkim Primitive, a small number of abnormal meiotic c e l l s were noticed. The incidence and d i v e r s i t y of these abnormalities appeared quite variable from plant to plant ranging from near absence to approximately 30% of the t o t a l number of meiotic c e l l s . The most puzzling obser-vation repeatedly made was the occurrence among karyo-t y p i c a l l y normal c e l l s , of c e l l s which contained super-numerary chromosome fragments and additional n u c l e o l i (Peeters, 1982). During the i n i t i a l phase of th i s study, the o r i g i n and sign i f i c a n c e of these c e l l s were unknown and . d i f f i c u l t to explain. The i n i t i a l hypothesis that was formulated, based on observations of some abnormal tapetal c e l l s , was that the tapetum was somehow involved i n the production of these chimeric c e l l s . Although t h i s hypothesis proved to be wrong, attention had been focused on c e l l to c e l l • contacts and i t was noted that c e l l fusion occurred at a low frequency i n these samples ( i n the range of 0 to 1%). On the basis of these preliminary observations, I decided to characterize spontaneous c e l l fusion cyt o l o g i -c a l l y . The f i r s t obvious task was to search among the parental population f o r plants containing a higher frequency of meiotic abnormalities and then to determine ! 3 2 how representative these abnormalities were. To detect c e l l fusion i n a cy t o l o g i c a l preparation, a screening procedure had to be developed to detect t h i s rare event. I t was noticed that at a l l meiotic stages ab-normal anthers containing a very high frequency of abnor-malities (up to 100%), mainly of aborting or aborted c e l l s could occasionally be found i n otherwise normal tassels. In such anthers- i t was not uncommon to f i n d fusing c e l l s , e s p e c i a l l y at the e a r l i e r stages of meiosis and the association of conspicuous meiotic abnormalities such as c e l l abortion with c e l l fusion proved to be a general rul e . However i t was l a t e r found that anthers which appeared at f i r s t to be t o t a l l y normal could also contain fusing or fused c e l l s , which were then obviously very d i f f i c u l t to detect. C e l l fusion appeared to be caused by absence of c e l l wall formation around the PMCs. Thick callose c e l l walls are normally present around the PMCs at a l l stages of meiosis i n maize (Warmke and Lee, 1977)• These walls, when present, are very easy to detect i n a cyt o l o g i c a l preparation because during squashing the protoplasts are usually separated from t h e i r c e l l walls which then remain as very v i s i b l e separate structures (see f o r example Figure 9). In the present study i n some anthers at early prophase 1, no v i s i b l e c e l l walls could be detected. However, analysis of other anthers of the same t a s s e l showed that at l a t e r meiotic stages, c e l l walls were normal. As a consequence, the formation of 33 the thick callose walls which normally forms pre-m e i o t i c a l l y around the PMCs i n higher plants (Heslop-Harrison, 1966; Ledbetter and Porter, 1970) appeared to be delayed only at early meiosis i n some parental plants and t h i s i s believed to have been the primary factor causing c e l l fusion. Postfusion products could be i d e n t i f i e d by increased c e l l volume, c e l l shape and the presence of additional chromosomes or chromosome fragments. Aborting c e l l s could also sometimes serve as an indicator of a previous fusion event. Postfusion c e l l s were s i g n i f i -cantly larger than the normal c e l l s (Table I ) . Depending on the time c e l l fusion occurred p r i o r to f i x i n g , c e l l shape could be another c r i t e r i o n allowing the d i s t i n c t i o n of a. normal and a postfusion c e l l . During or shortly a f t e r c e l l fusion, the c e l l had a d i s t i n c t "bilobed" shape which became less and less accentuated as the new c e l l regained i t s shape. These observations are s i m i l a r to the observations of fusing c e l l s i n v i t r o (Shepard et a l . , 1983). Af t e r tassels from 30 parental plants were studied, one representative parental plant found to contain a high proportion of abnormalities was selected f o r detailed analysis. Over 17,000 i n d i v i d u a l c e l l s were observed and the r e s u l t s are summarized i n Table I I . Although such a large number of c e l l s was studied, no r e a l pattern 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 i n a given anther. I n the p l a n t that was s t u d i e d approxi-mately 50% of the anthers were abnormal. The cytogenet consequences of spontaneous c e l l f u s i o n are described below. 35 Table I : Area comparison between normal and postfusion c e l l s Stage * Diameter (mp.) p Area (mu ) Expected/Observed 2 Area (mp ) • Early Pro. I N II rji Pachytene N II rp Early Met. I N T 105.9^ 155.8 110.6 155.8 112.33 153.85 8815.41 19064.47 9610.40 19064.47 9910.49 18590.83 17630.82 Exp. 19064.47 Obs. 19220.80 Exp. 19064.47 Obs. 19820.98 Exp. 18590.83 Obs. * Observations of each stage taken from the same preparation for uniformity. N : Normal c e l l (size i s the averaged value from 10 observations). T : Late postfusion c e l l (no bilobed shape) with two f u l l complements. Table II Meiotic survey of a single parental plant with a high incidence of meiotic abnormalities (From 1 7 , 1 3 2 observations) STAGE Early Pro. I Pachytene Diplotene Diakinesis Meta. I& Ana. I Pro. II Meta. II Ana. II Prop, of Abnor. # CELLS NORMAL 1 4 9 8 2 1 0 0 1 3 2 9 2 3 ^ 5 1566 1 9 1 2 6 8 0 562 EMPTY OR ABORTED 3 5 7 6 6 1 720 877 ^ 9 5 5 2 8 286 162 2 3 . 9 # ABORTING 1 2 1 101 6 9 2 1 5 187 81 7 10 k% HAVING 2 FUSED 7* 2T * , 3 A * 2T,8A IIT.I7A 6T,l4A 5T» , 7 A » h* 7* 0.5% FUSING 2k* 17 6 7 5 2 0 0 Q.h% SYNCYTIUM 3 53 2 2 7 2 9 22 12 5 3 0.9% MICROCELL 2 k 13 21 15 I 0 0 0.396 TOTAL 1 9 6 2 2 9 1 0 2 1 5 4 3 5 2 2 2 3 1 0 2 5 ^ 8 9 8 2 7 4 4 3Q# * Not a l l countable 1 Including fused c e l l s 2 Polyploids, aneuploids, and c e l l s with meiotically active supernumerary chromosomes 3 Aborting c e l l mass from 3 or more fused c e l l s k C e l l s having between I and 9 chromosomes T Autotetraploid with apparent meiotic s t a b i l i t y A C e l l with m e i o t i c a l l y a c t i v e additional chromosomes (including chromosome fragments) 2. The cytogenetic consequences of spontaneous c e l l fusion. 2.1. Chromatin mo'dification and degradation i n s i t u . Approximately 0.2% of the c e l l s studied were post-fusion products i n which one complement was c l e a r l y "becoming disorganized and was disintegrating i n situ"'" without apparently a f f e c t i n g the other complement which appeared morphologically and m e i o t i c a l l y normal (Figures 4 and 5)• These i n i t i a l observations were confirmed by the c y t o l o g i c a l analysis of fusing c e l l s . Although very d i f f i c u l t to detect, c e l l s that were i n the process of fusing showed c l e a r l y i n some cases that the chromosomes of one complement were uncoiling and dis 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 structure (Figure 6). The chromosomes that were uncoiling had the "pulverized" apprearance s i m i l a r to some P.C.C. chromosomes observed following i n v i t r o c e l l fusion. (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 taking place i n one complement of some of the fusing c e l l s during c e l l d i v i s i o n , a spectrum of post-fusion products was obtained. Postfusion c e l l s which contained one normal complement and only parts of the "*" Used here to stress the fac t that these changes were occurring inside one single postfusion c e l l . 38 other chromosome complement (Figure ?) was the most frequent class 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 fusion at d i f f e r e n t meiotic stages i s shown on Figure 8. C e l l fusion was obviously .much more frequent at early prophase 1 and was possibly also occurring before the onset of meiosis. These stages are d i f f i c u l t to analyse i n maize (Rhoades, 1950). However some clues as to the cytogenetics of postfusion at these stages could be obtained by observing the n u c l e o l i . . As expected postfusion c e l l s were always found to contain the n u c l e o l i of both complements. Most postfusion c e l l s were found with more than two large 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 associated with n u c l e o l i could also be detected i n these c e l l s (Figure 9). Although i t was not possible to determine i f the fragments i n these c e l l s were orig i n a t i n g from only one complement, t h i s was infe r r e d from the recovery at pachytene (where i n d i v i d u a l chromosomes are highly v i s i b l e ) of postfusion c e l l s containing one normal complement and chromatin fragments from the other complement. o Following 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 addi t i o n a l s t r u c t u r a l l y modified chromosomes apparently had s t a b i l i z e d . The fate and morphology of these frag-ments was. very wide ranging. In some c e l l s the fragments f a i l e d to follow the cytoplasmic cues f o r chromosome condensation which were functional for the normal complement (Figure 10). Such fragments were s t a b i l i z e d because, despite t h e i r i n a b i l i t y to condense, they showed no signs of further di s i n t e g r a t i o n and pachytene-like 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 interphase 1. These uncoiled chromosomes were never involved with the spindle and therefore appeared to be acentric. Most s t a b i l i z e d additional 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 degradation appeared to 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. This was c l e a r l y indicated either by centric a c t i v i t y (Figure 11) or i n one exceptional case by RNA synthesis, i n a postfusion c e l l containing a modified supernumerary chromosome with a nucleolus organizer region (NOR) (Figure 12). It can be inferred from observations of this c e l l along with observations of numerous other c e l l s containing a spectrum of morphologically modified supernumerary chromosomes that the fragments which could s t a b i l i z e were fragments from random A chromosomes. S t a b i l i z e d centric fragments appeared to be always included i n the anaphases and i n the reorganizing nuclei i n telophase. The res u l t s obtained l a t e r i n th i s study suggest that these c e l l s , which were modified karyotypically and appeared s t a b i l i z e d , gave r i s e to f e r t i l e gametes. Genomic s t a b i l i t y of the fragments r e s u l t i n g from i n s i t u chromosome degradation was not always achieved. Micronuclei r e s u l t i n g from the supernumerary elements were observed, as well as c e l l abortion. It i s not known i f t o t a l elimination of one chromosome complement could occur following c e l l fusion 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 . Figure 8 suggests that metaphase 1 represented a c r i t i c a l stage for the s t a b i l i t y of post fusion cells' and i n addition that postfusion c e l l s i n which no chromosome degradation occurred were more stabl than the c e l l s with p a r t i a l degradation. The reasons why metaphase 1 appears to be such a c r i t i c a l stage for the s t a b i l i t y of postfusion c e l l s are not known. 41 Figure 4 s Postfusion c e l l with i n s i t u abortion of one complement. Early metaphase 1 c e l l showing modification and degradation of one chromosome complement (arrow) i n the presence of the other normal complement (two bivalents are fused). Estimated magnification X 1600. Figure 5 : Postfusion c e l l with i n s i t u abortion of one , complement. 10 normal bivalents are present and the other complement i s showing very c l e a r l y i n s i t u degradation. The pachytene-like state of contraction of the aborting complement indicates that fusion probably occurred at or before pachy-tene. Metaphase 1 (X 1250). 42 ^3 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 . 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 apparent i n one complement (arrow) w h i l e o n l y minor changes i n chromosome s t r u c t u r e a r e d e t e c t a b l e i n t h e o t h e r complement. E s t i m a t e d m a g n i f i c a t i o n X 700. F i g u r e ? : P o s t f u s i o n c e l l w i t h m o d i f i e d supernumerary chromosomes. One complete complement i s p r e s e n t w i t h a d d i t i o n a l m o d i f i e d chromosome fragments from t h e o t h e r complement (arrow) as a r e s u l t o f p a r t i a l i n s i t u d e g r a d a t i o n . D i a k i n e s i s (X 1250). 45 Figure 8 : Frequencies of c e l l fusion and of s t a b i l i z e d postfusion products throughout meiosis i n one parental plant. Standardized values from Table I I showing the d i s t r i b u t i o n of c e l l fusion (CF) as well as stable postfusion c e l l s throughout meiosis. T represents the proportion of c e l l s which did not show any chromosome degradation of either complement while A represents the proportion of c e l l s i n which one complement was p a r t i a l l y incomplete as a r e s u l t of p a r t i a l i n s i t u chromosome degradation (aneuploids and c e l l s with stable additional modified supernumerary chromosomes). This graph suggests that the sta-b i l i t y of postfusion c e l l s was not always achieved at metaphase 1, and furthermore that postfusion c e l l s i n which there was no chromosome degradation were more stable than c e l l s with p a r t i a l degradai-ti o n . (Values standardized to 2000 observations per stage). 4 6 N U M B E R F I G U R E S O F C E L L S A Figure 9 : Fusing c e l l at early prophase 1 showing i n s i t u chromosome modification. Three large n u c l e o l i are present and an additional small nucleolus on a separate chromosome fragment (arrow) i n d i -cates that chromosome modification i s occurring 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 also the presence of a c e l l wall (next to the arrow). Estimated magnification X 300. Figure 10 : Postfusion c e l l with apparently s t a b i l i z e d , m e i o t i c a l l y i n a c t i v e , modified supernumerary chromosomes. The i n a b i l i t y of such fragments to respond to the cytoplasmic cues f o r chro-mosome condensation which were obviously functional f o r the normal complement indicates c l e a r l y that these chromosomes (arrow) were modified. (Black dots are b a c t e r i a l contaminants). Estimated magnification X 1600. 48 49 Figure 11 : Postfusion c e l l with m e i o t i c a l l y active modified supernumerary chromosomes. This early anaphase 1 c e l l suggests that s t a b i l i z a t i o n of the chromosome fragments has taken place. Telocentric 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 incorporated i n the reorganizing n u c l e i at the telophases and th i s type of PMC may generate f e r t i l e gametes. Estimated magnification X 1250. Figure 12 : Postfusion c e l l with a single modified super-numerary chromosome with a m e i o t i c a l l y active NOR. Structural modification of t h i s a dditional chromosome 6 ( arrow ) i s very obvious and i n th i s case i s further confirmed by low ribosome synthesis (as evidenced by the s i z e of the nucleolus) which could indicate either a smaller number of repeats or s t r u c t u r a l gene modifications. Diakinesis (X 1250). 50 51 2.2. Aneuploidy and P o l y p l o i d y . Aneuploid c e l l s , defined here as c e l l s c o n t a i n i n g e i t h e r a l a r g e r or a smaller number of i n t a c t chromosomes i n comparison to the 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 noted i n the production of t h i s aneuploidy. In some anthers 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 fragmentation 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 detected (Figure 1 3 ) . These s m a l l aneuploid 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%) fused w i t h normal c e l l s , generating aneuploid c e l l s w i t h an increased chromosome number. Figure 14 shows an aneuploid p o s t f u s i o n c e l l which appears, on the basis of c e l l volume, shape and chromosome mor-phology, to have been derived from such a f u s i o n . In a d d i t i o n to t h i s f i r s t mechanism f o r aneuploidy, p a r t i a l a b o r t i o n of one 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 could a l s o generate e i t h e r micro-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 mo r p h o l o g i c a l l y normal. Examples 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 only one i n t a c t complement and fragments from the other complement have already been given. These c e l l s r e s u l t e d from chromosome degradation of one 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 of the fragments. The c e l l s subject to t h i s process would be expected to contain only morphologically a l t e r e d chromosomes. However some of these c e l l s contained a few supernumerary chromosomes which appeared to be normal i n addition to the abnormal chromosomes or chromosome fragments (Figures 15 and 16). I f the abnormal chromosome fragments were eliminated i n such c e l l s , f o r instance i n the case of acentric fragments, an aneuploid c e l l with additional chromosomes with no or only minor defects could probably be generated. Microcells could also be derived from p a r t i a l chromosome elimination i n the normal c e l l adjacent to a fusing c e l l (see section 2 . 3 0 ' The t o t a l microcell frequency i n one parental plant i s given i n Table II. The mechanism f o r the selec t i v e degradation of only some chromosomes i s not understood but could be related to the degree of i n d i v i d u a l chromosome condensation, given that c e l l fusion could take place at stages where the chromosomes were highly contracted (Figure 8). As expected, spontaneous c e l l fusion could be the source of stable polyploid c e l l s which contained two intact chromosome complements and i n which no chromosome modification could be detected (Figure 1?). Such c e l l s appeared stable m e i o t i c a l l y and probably could produce unreduced gametes (see Figure 8). However meiotic complications of postfusion c e l l s which showed no i n s i t u chromosome degradation of either complement were detected i n one spe c i a l case. I t i s believed that when c e l l fusion occurred l a t e , just p r i o r 53 to or at e a r l y metaphase 1, the axes of c e l l d i v i s i o n were already determined i n both c e l l s and that 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 (Figure 1 8 ) . These c e l l s , which f u r t h e r demonstrated the occurrence 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 the incidence of c e l l f u s i o n at metaphase 1 was already much smal l e r than at e a r l y prophase 1 (Figure 8 ) . i t was q u i t e i n t e r e s t i n g to discover i n such p o s t f u s i o n c e l l s that the formation of the two sp i n d l e s could be unequal wi t h a "main" s p i n d l e (which i n v o l v e d most chromosomes) and a minor s p i n d l e (Figure 1 8 ) . 54 Figure 13 : Microcells produced by spontaneous protoplast fragmentation. Both c e l l volume and chromosome number indicate that these two microcells at late diakinesis were derived from one i n i t i a l PMC, probably by protoplast cleavage. These microcells were always well i n d i v i d u a l i z e d and appeared to be stable m e i o t i c a l l y . Estimated magnification X 300. Figure 14 : Postfusion aneuploid c e l l . This c e l l at diakinesis contains 3 morphologically normal additional chromosomes (arrow) and i s believed to have been generated by fusion with a microcell. This i s inferred from c e l l volume, shape of . the postfusion c e l l and lack of chromosome modification. Note the additional nucleolus. Estimated magnification X 550. 55 1 4 56 Figure 15 : Postfusion c e l l with additional chromosomes which appear morphologically normal. Three additional fused, normal looking chromosomes are present i n t h i s early metaphase I c e l l (large arrow) while additional fused chromosomes which appear modified and abnormal are also present (small arrows).. I f these abnormal chro-mosomes are eliminated, an aneuploid c e l l could r e s u l t . Note the la t e p e r s i s t i n g nucleolus. Estimate magnification X -1300. Figure 16 : M u l t i c e l l fusion at diakinesis showing p a r t i a l i n s i t u chromosome degradation. Thirteen additional chromosomes are present as well as 6 chromosome fragments. Estimated magnification X 350' 57 58 Figure 17 : Polyploid PMC. This c e l l appeared on the basis of i t s size to be derived from from the spon-taneous fusion of two PMCs. Twenty normal biva-lents are present instead of the usual 10. No signs of chromosome degradation are v i s i b l e . Metaphase 1 (X 1850). Figure 18 : Polyploid postfusion c e l l with two axes of d i v i s i o n . Such metaphase 1 c e l l s are believed to r e s u l t from l a t e c e l l fusion when c e l l p o l a r i t y was already determined and are an additional proof of the occurrence of c e l l fusion. Notice the unequal spindles with an uneven chromosome d i s t r i b u t i o n . Such c e l l s may be involved i n the production of aneuploid gametes. Estimated magnification X 600. 59 1 7 I S 6o 2.3' C e l l abortion Table II shows that c e l l abortion was by f a r the most frequent class of meiotic abnormality and the presence of aborting or aborted c e l l s was found i n a very large proportion of the anthers that were studied. As mentioned previously, there was usually a p o s i t i v e co r r e l a t i o n between the presence of a fusing c e l l and c e l l abortion i n neighboring c e l l s . The degree of causality between the two events i s not known but was not complete i n the sense that c e l l fusion and c e l l abortion could be observed independently. Obviously c e l l abortion could follow c e l l fusion when the balance of the post-fusion c e l l was not achieved. The cytogenetics of c e l l abortion was quite variable, as shown below. a). Abortion of non fused c e l l s . Chromatin degradation The most frequent class of aborting c e l l s was c e l l s i n which only chromatin degradation was taking place, while RNA was not affected (as seen by the n u c l e o l i ) . On the contrary, RNA synthesis appeared greatly enhanced i n these c e l l s (Figure 19) and aborting c e l l s .containing only n u c l e o l i with no v i s i b l e chromosome fragments were occasionally observed. However p a r t i a l s t a b i l i z a t i o n appeared to be achieved i n some of these-aborting c e l l s , which then contained an abnormal complement and i n which the chromosomes were i n phase m e i o t i c a l l y with the other normal c e l l s (Figure 2 0 ) . These p a r t i a l l y aborted c e l l s were only seen at or a f t e r metaphase 1. 61 RNA degradation Some rare cases of c e l l abortion appeared, on the basis of observations of the nucleolus,, to show RNA degradation only and anucleolar c e l l s with a normal complement were found. Simultaneous RNA and chromatin degradation The two patterns of c e l l abortion which have just been described are believed to involve mostly c e l l s that were at early aborting stages. When s t a b i l i z a t i o n could not be achieved, both DNA and RNA appeared to be degraded simultaneously, r e s u l t i n g sometimes i n a c e l l with no v i s i b l e nucleolus or chromatin.. These l a t e stages of c e l l abortion were easy to i d e n t i f y because of s i g n i f i c a n t meiotic abnormalities (Figure 21). In addition these c e l l s started to lose t h e i r volume and shape with the cytoplasm becoming very dense and thick, ultimately forming a structure resembling a pollen grain with a shriveled c e l l wall (Figure 22). Arrested c e l l s C e l l s which f a i l e d to continue meiosis and were arrested at one p a r t i c u l a r meiotic stage were also observed among PMCs at l a t e r stages. These c e l l s did not appear ge n e t i c a l l y inactive because additional n u c l e o l i were produced (Figure 23). These arrested c e l l s aborted at late meiotic stages. b). Abortion of postfusion c e l l s Following spontaneous c e l l fusion, some of the postfusion c e l l s aborted, possibly as a r e s u l t of lack of synchrony or because of lack 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 modification and degradation i n one complement (which obviously can be considered as a p a r t i a l abortion)^. These aborting post-fusion c e l l s seemed to have greater s t a b i l i t y than the other aborting c e l l s (since they were also observed at late meiotic stages), possibly because of the presence of two complements. In these c e l l s , the a b i l i t y of i n d i v i d u a l chromosomes to condense could vary from one chromosome to the other (Figure 24) which i l l u s t r a t e d again that the degree of i n d i v i d u a l chromosome "modification i n s i t u appeared to vary and also that the aborting c e l l s were subject to the signals f o r c e l l synchrony. 63 Figure 19 : Aborting c e l l showing chromatin degradation and additional RNA synthesis. Three n u c l e o l i are present, two on a fragment of chromosome 6 (arrow). Pachytene ( X 1250). Figure 20 : P a r t i a l l y aborted c e l l . This c e l l was found to be i n synchrony with the other normal c e l l s and appeared m e i o t i c a l l y s t a b i l i z e d despite strong s t r u c t u r a l chromosome modifications. Late Diakinesis ( X 1250). SO 65 Figure 21 : Aborting metaphase 1 c e l l . Notice the abnormally small spindle and chromatin fragments. Estimated magnification X 1250. Figure 22 Aborted c e l l . Estimated magnification X 600. 66 67 Figure 23 : Arrested diplotene c e l l . This c e l l was found i n an anther at telophase 1 and shows additional RNA synthesis (arrow). Such arrested c e l l s even-t u a l l y aborted. Estimated magnification X 1250. Figure 24 : Postfusion aborting c e l l at metaphase 1. Seven n u c l e o l i are present as well as a gradient of chromosome contraction ranging from pachytene (arrow) to diakinesis, which suggests that the degree of i n d i v i d u a l chromosome modification i n an aborting c e l l could be variable. The v i a b i l i t y of postfusion aborting c e l l s appeared higher than aborting c e l l s with only one complement. Estimated magnification X 450. 68 2.4. Additional meiotic abnormalities Syncytium formation A syncytium i s defined as "a large multinucleate c e l l a r i s i n g from c e l l fusion" (Ringertz and Savage, 1976). The term Plasmodium was also used previously i n the l i t e r a t u r e i n place of syncytium but presently refers to the structures formed by slime molds. . Syncytia were observed rather frequently even at l a t e r meiotic stages (Table II) but were common only i n the anthers containing a very high proportion of.aborting c e l l s and were usually absent i n the other anthers. These giant c e l l s were formed as a r e s u l t of 3 types of c e l l fusion, either fusion between PMCs, fusion between nurse c e l l s or fusion between PMCs and nurse c e l l s (Figures 25 and 26). While fusion between only two c e l l s was generally found to form stable postfusion products, the s t a b i l i t y of m u l t i c e l l fusion products (3 or more c e l l s ) seemed very l i m i t e d since the syncytia appeared always to be aborting. These observations are confirmed by i n v i t r o c e l l fusion, where multinucleate protoplasts are known to have l i t t l e v i a b i l i t y (Ringertz and Savage, 1976). However Marin and Lanfranchi ( I 9 8 I ) have shown that t r i p a r e n t a l hybrids i n v i t r o fusion products between mouse and Chinese hamster c e l l s can be viable. In the present study syncytia were considered to have no other effect than to decrease the number of viable pollen. - 70 C e l l p o l a r i t y and cytoplasmic determinants. The consequences of c e l l fusion involving c e l l s which had t h e i r axis of d i v i s i o n determined has teen described i n section 2.2. C e l l fusion i n such cases obviously .caused major meiotic complications, except possibly when the two c e l l s had t h e i r axes i n p r e c i s e l y the same orientation (Figure 27). In addition to c e l l p o l a r i t y , evidence f o r the presence of cytoplasmic determinants was found once i n a postfusion c e l l where a gradient of meiotic figures ranging from metaphase 1 to telophase 1 was found (Figure 28). This observation seems to be d i s t i n c t from the observations that were reported previously i n t h i s study of chromosomes at d i f f e r e n t stages of contraction i n aborting c e l l s (see f o r example Figure 24) because i n th i s case the gradient of meiotic figures appeared to be a c r o s s - c e l l gradient whereas i n aborting c e l l s the arrangement of the chromosomes was random. Abnormal RNA and DNA lev e l s The structure of the nucleolus organizer region (NOR) and the nucleolar cycle i n maize are well known (Givens and P h i l l i p s , 1976; De La Torre and Colinas, 1978). In this study additional n u c l e o l i were frequently observed i n aborting c e l l s (see Figures 19 and 24) but also occasionally i n "normal" c e l l s adjacent to fusing c e l l s , at stages with NOR a c t i v i t y as well as at stages where no RNA synthesis was expected (Figure 29). 71 In addition to increased RNA l e v e l s , abnormal DNA l e v e l s derived by mechanisms other than those previously described i n t h i s project were also found once i n an abnormal anther as somatic-like nuclei that were c l e a r l y polyploid (Figure 30). These nuclei may have originated either by nuclear fusion or by abnormal DNA r e p l i c a t i o n . Cytomixis The presence of c e l l s undergoing cytomixis i s obviously very easy to detect i n a c y t o l o g i c a l preparation. Only two c e l l pairs showing what has been described as cytomixis were found i n t h i s study (Figures 31 and 32) and c e l l fusion combined with delayed c e l l wall formation did not appear to r e s u l t i n cytomixis. One could envision indeed that c e l l wall formation around c e l l s at early fusion stages could produce two p a r t i a l l y fused c e l l s and cytomixis. However, except f o r one or two rare exceptions (Figure 32), c e l l fusion was always massive and complete. It i s clear that i f cytomixis had been taking place i n these anthers, t h i s could have been the o r i g i n of the c e l l s with additional chromosome fragments which were described e a r l i e r i n section 2.1. Other observations In addition to the abnormalities already described as a consequence of c e l l fusion, other s i g n i f i c a n t ab-normalities were found during meiosis i n the parental plants. One of these i s chromosome bridge formation which could take place i n 100% of the PMCs of some anthers (Figure 33) while being t o t a l l y absent i n s i s t e r anthers • 72 from the same t a s s e l . These bridges were d i c e n t r i c . Another group of very unusual m e i o t i c a b n o r m a l i t i e s was discovered i n approximatively 5 metaphases which appreared to show neoce n t r i c a c t i v i t y (Figure 3*0 • 73 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 . C e l l f u s i o n o f "both t h e PMCs and t h e nu r s e c e l l s i s v e r y v i s i b l e i n t h e s e two e a r l y prophase 1 s y n c y t i a . A b o r t i o n i s s t a r t i n g i n t h e n u r s e c e l l s y n c y t i u m . 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 showing f u s i o n between PMCs and nu r s e c e l l s . T r a n s f e r o f c h r o m a t i n fragments from t h e t a p e t a l c e l l s y n c y t i u m i n t o t h e PMC s y n c y t i u m (arrow) i n d i c a t e s t h a t f u s i o n between the two g i a n t c e l l s has o c c u r r e d . N o t i c e t h e c h a r a c t e r i s t i c s t r u c t u r e s o f a b o r t i n g s o m a t i c n u c l e i (some o f whi c h appear t o have been i n d u c e d i n t o d i v i s i o n ) . 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 Figure 27 : Postfusion c e l l with two p a r a l l e l independent spindles. This metaphase 1 postfusion c e l l i s believed to have originated from a fusion between two c e l l s with the axis of d i v i s i o n already determined. Such c e l l s were usually subject to meiotic complications but i n t h i s case telophase may have been normal because of the orientation of the spindles. Estimated magnification X 200. Figure 28 : Postfusion c e l l with a meiotic gradient. This c e l l has a clear c r o s s - c e l l meiotic gradient from telophase 1 to metaphase 1. Estimated magnification' X 450. 76 77 Figure 29 : PMC with increased l e v e l s of RNA synthesis. This c e l l with a normal complement was found adjacent to a fusing c e l l and shows additional RNA synthesis (arrow) at a. stage were RNA syn-thesis normally has ceased. Late diakinesis (X 1 8 8 0 ) . Figure 30 : Polyploid somatic n u c l e i i n an anther at mid meiosis. The normal sized nuclei (arrow) indicate large ploidy variations i n the other nuclei which were probably derived from nuclear fusion or from abnormal DNA synthesis. These nuclei did not appear to be of tapetal o r i g i n (since the tapetal c e l l s appeared normal) and therefore may have originated from pre-meiotic c e l l s which f a i l e d to enter meiosis. Telophase 1 (X 560). 3 0 79 Figure 31 : Cytomixis between a pachytene and an aborting c e l l . Chromosome transfer from the pachytene c e l l into the aborting c e l l i s c l e a r l y v i s i b l e and must be the r e s u l t of a,communication channel between the two c e l l s (arrow). This i s one of only two cases of cytomixis that were found during t h i s study. Notice the fragmented pachytene chromosomes i n the aborting c e l l . Estimated magnification X 1350. Figure 32 : Nucleo-cytoplasmic transfer between two p a r t i a l l y fused early prophase 1 c e l l s . This other possible example of cytomixis shows transfer of the nucle-olus through a large contact area between the two c e l l s . Estimated magnification X 250. 81 Figure 33 : Breaking chromosome bridge. 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 possible example of neocentric a c t i v i t y . Metaphase 1 (X 1880). 82 3. The F l and F2 Generations Following the discovery and cytogenetic analysis of spontaneous c e l l fusion i n the parental meiosis i t was anticipated that, eventually, new karyotypes could be found i n the next generation. * Four ears ( l a b e l l e d A l to A4) were col l e c t e d following s e l f i n g of the parental plants showing the abnormal meiosis and of which the karyotypes were known (see Materials and Methods). Most of the seeds from these ears appeared normal (9^*3%) and the pollen samples collected from the parental plants had shown an estimated f e r t i l i t y range of 5^ to 91% (which r e f l e c t e d the proportion of abnormal meioses i n the parental population). Seeds from these 4 stocks were grown under i d e n t i c a l conditions the next summer and the karyotypes of 36 F l plants were established. The re 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 th i s stock one plant containing a small additional and independent chromosome was discovered (Figure 35)- This chromosome was present i n every c e l l and therefore was karyotypic. As additional plants came .to maturity, further c y t o l o g i c a l examination of stock A3 lead to the discovery of 3 additional plants with accessory chromosomes (Table I I I ) . These chromosomes were a l l d i f f e r e n t morphologically despite the fact that they had originated from the same s e l f - p o l l i n a t e d plant. Line A3K14 had 3 independent accessory chromosomes as well as what appeared to be an additional fragment which could also be independent but was usually hybridized to an A chromosome (Figure 36). The c y t o l o g i c a l appearance and meiotic behavior of a l l these chromosome fragments was extremely variable not only between the 4 stocks bearing them but also from one clonal c e l l to the other. In the 3 plants containing only one of these accessory chromosomes, th i s fragment, which was c l e a r l y a univalent, changed morphologically i n that i t could hybridize to i t s e l f to various degrees but more s i g n i f i c a n t l y , i t s state of contraction was quite d i f f e r e n t from c e l l to c e l l at a given stage. For example at mid-pachytene i n some c e l l s i t was completely co i l e d and appeared very heterochromatic but t h i s degree of contraction could range to a completely uncoiled state i n other c e l l s . Usually i t had a "beaded" appearance, apparently consisting of alternating eu and heterochromatin. M e i o t i c a l l y also, t h i s chromosome showed a very e r r a t i c behavior. Although usually independent, i t sometimes hybridized to the A chromosomes, causing meiotic complications. In addition, the chromosome appeared to show early segregation i n up to 50% of the c e l l s at metaphase/anaphase 1. In the plants with 3 additional chromosome fragments th i s was not the case because these chromosomes were usually paired (Figure 36) which 85 apparently made them behave as the s t a b l e b i v a l e n t s . The c y t o l o g i c a l observations that were made of the s i n g l e u n i v a l e n t were s i m i l a r between the 3 pl a n t s bearing 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 these 3 i n d i v i d u a l fragments. The degree of homology between these fragments i s not known. On the basis of chromosome morphology and number, i t was c l e a r that these chromosomes were not Tripsacum contaminants (Dr. C.V. P a s u p u l e t i , pers. communication) which could have been introduced from other stocks i n the f i e l d during p o l l i n a t i o n . I n a d d i t i o n , no signs were found i n the pl a n t morphology of the presence i n the genome of Tripsacum genes. Although these a d d i t i o n a l modified chromosomes could be defined as "accessory" or "B" chromosomes, i n the F l they d i d not have the t y p i c a l maize B chromosome morphology or behavior. The severe m e i o t i c abnormalities which had been found i n many of the p a r e n t a l p l a n t s were only very scarce i n t h e i r d i s t r i b u t i o n among the F l pl a n t s (Table I I I ) . In the F l p l a n t s which contained such a b n o r m a l i t i e s , the frequency of abnormal PMCs was al s o reduced. I t i s not known i f t h i s i s due to an adaptive response and/or to d i f f e r e n t summer growth c o n d i t i o n s . One stock i d e n t i f i e d w i t h an accessory chromosome (stock A3K2) was s e l f f e r t i l i z e d and the F2 generation ( l a b e l l e d A3X2) was grown i n the 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). Surprisingly, pachytene analysis revealed that extensive heterochromatinisation of the chromosome had occurred (Figure 37) and that, i n addition, the chromosome had become morphologically and mei o t i c a l l y quite s i m i l a r to a maize B chromosome. It was also found by somatic analysis of a large number of seeds that t h i s chromosome had s i g n i f i c a n t l y increased i n number i n some l i n e s from one up to a maximum of k (Figure 3 8 ) . As expected these supernumerary chromosomes were a l l i d e n t i c a l morphologically. This observation represents another c r i t e r i o n against the p o s s i b i l i t y of having a Tripsacum contaminant (which would not amplify but on the contrary be eliminated, see Chaganti, 1 9 6 5 ) . The precise mechanism for this rapid amplification i s not known but may have been mitotic since some mosaics were found i n the somatic karyotypes. A plant containing 4 of these chromosomes was found to have slower growth and some shriveled leaves. Somatic and meiotic analysis showed that 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 or t r i p l o i d s were found among the 36 F l plants studied. In stock A l , two dwarf plants were found (Table III) and had abnormal karyotypes with trans-locations and chromosome breakage. Table III : Characteristics of the F l generation SEED STOCK PLANT# KARYOTYPE MEIOTIC ABNORMALITIES PLANT MORPHOLOGY A3 KI 2n:20 Some c e l l fusions Normal A3 K2 2n:20*I Some c e l l fusions Normal A3 K 3 2n:20*I Some c e l l fusions Normal A3 Kk 2ni20 None Normal A3 K5 2n:20 None Normal A3 K6 2n:20 None Normal A3 K7 Collected too early Normal A4 K8 2m20 None Normal A4 K9 2n:20 None Normal A3 KIO 2m 20 None Normal A3 KII 2ni20-M Some c e l l fusions Normal A3 KI2 2n:20 None Normal A3 K I 3 2m 20 None Normal A3 KI4 2ni20-l-3 Abortions & Fusions Normal A4 KI5 2n:20 None Normal A3 KI6 2m 20 None Normal A3 KI7 2n:20 None Normal A4 KI8 2n:20 None Normal AI KI9 2n:20 None Normal AI K20 2n:20 Translocations* Dwarf AI K2I 2nj20 None Normal AI K22 2n:20 None Normal A2 K 2 3 2n:20 . None Normal A2 K24 2n:20 None Normal A2 K 2 5 2m 20 None Normal AI K26 2n:20 Translocations* Dwarf . A2 K27 2n:20 None Normal A2 K28 2n:20 None Normal A2 K29 2n: 20 None Normal A2 K 3 0 2m20 None Normal A2 K3I 2n:20 None Normal Ak K32 2n:20 None Normal A3 K33 2m20 None Normal A3 K34 2m20 None Normal A2 . K35 2ni20 None Normal A3 K36 2m 20 None Normal * Including 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 meiosis. SUMMARY Stock (ear) Nb. of plants studied Nb. of abnormal karyotypes Nb. of accessory chromosomes AI 5 2 0 A2 9 0 0 A3 17 1,1,1 and 3 A4 5 0 0 Total 36 6 6 88 Figure 35 '• F l progeny with the presence i n the genome of a new modified supernumerary chromosome (arrow). Both Figures 35 and 36 are c e l l s at diakinesis. Estimated magnification X350. Figure 36 : F l progeny with a new karyotype containing 3 additional chromosomes and a small fragment. The 3 chromosomes i n th i s c e l l are paired together (large arrow) while, what appeared to be an extra fragment (since i t could be individualized) i s hybridized to a bivalent (small arrow). Estimated magnification X 800. Figure 37 s Change i n B chromosome morphology through i n -creased heterochromatinisation from the F l to. F2.generation. The inse r t shows the chromosome at mid-pachytene i n F l while the arrow shows the F2 chromosome at the same stage. Notice the "beaded" appearence of the F l chromosome which may be due to an al t e r n a t i o n of euchromatin and heterochromatin. Estimated magnification 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. These a c c e s s o r y chromosomes (bottom r i g h t ) were a l l d e r i v e d from one i n i t i a l c h r o -mosome, and showed a r a p i d e v o l u t i o n (see t e x t ) . From a s o m a t i c metaphase (X 4450). II « -Ji-lt I I II I I n it 4. Hybridity and the effects of growth conditions. When the o r i g i n a l samples from India were grown under di f f e r e n t conditions i n the green-house no or few examples of abnormal meiosis were found but there was genetic v a r i a t i o n i n plant morphology i n comparison to the f i e l d . One s t r i k i n g example was the formation of s e l f - p o l l i n a t i n g ears (having male inflorescences formed at the t i p of the ear), a t r a i t which has been found i n some of the Sikkim Primitives (Sachan and Sarkar, 1982). These s p e c i a l ears were never found i n the f i e l d and could have been due to hybridi t y combined with d i f f e r e n t growth conditions. Although synapsis was always complete, further evidence of hybridi t y was found as knob heterozy-gosity, F l ear morphology (Figure 1 ) and segregation of two ear markers i n the F2 (Dr. Galinat, pers. communication). It appears that the samples were outcrossed between two d i f f e r e n t Sikkim Primitives or a Sikkim Primitive and another popcorn but were not from natural, open-pollinated conditions. The effects of d i f f e r e n t temperatures on the samples from India was studied using the growth chamber. Seven plants were-grown at 16 degrees C. (day/night) and, out of these, two plants were dwarf and morphologically abnormal (one being almost t o t a l l y albino and the other having red pigmented leaves). These two plants did not 93 reach maturity. In 3 of the remaining plants, which were very small too, meiotic samples were taken and a l l showed approx. 80% empty or aborted PMCs while chromosome breakage was occurring i n the remaining PMCs (Figure 39 and Figure 40) with no viable pollen being formed at late meiosis. Another temperature se t t i n g of 23/18 degrees C. (day/night) showed no major type of abnormalities, either morphological or c y t o l o g i c a l . Similar r e s u l t s were obtained i n experiments anterior to those described and of which no accurate records were kept. These experiments, although quite l i m i t e d , indicated that these plants were extremely sensitive to changes i n growth conditions. 94 Figures 39 and 40 : Different karyotypes of two clonal c e l l s showing chromosome fragmentation as a • r e s u l t of a temperature s h i f t . Notice the fuzzy appearance of both the chromosomes and the n u c l e o l i . The two c e l l s are at diakinesis and are at the same magnifica-t i o n . (X 1250). 95 96 DISCUSSION Although a number of questions have, remained unanswered, the significance of this work i s to show that spontaneous c e l l fusion can be d i r e c t l y involved i n the i n vivo genesis of a number of d i f f e r e n t new karyotypes containing additional DNA. 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 fusion and spontaneous genomic changes has been l i m i t e d only to cle novo occurring polyploids. However th i s study shows that spontaneous c e l l fusion can also generate aneuploidy and more s i g n i f i c a n t l y , new karyotypes containing stable new additional DNA sequences and modified supernumerary chromosomes. This additional DNA was found to derive from p a r t i a l i n s i t u chromatin degradation of one complement, following c e l l fusion.. Some of these additional chromosome fragments appeared to have been inherited; ' and evolved as true B chromosomes. This, i s the f i r s t time that a mechanism fo r the formation of -these sequences i n the genome has been demonstrated (Peeters and W i l k e s 1 9 8 3 ) . Spontaneous c e l l fusion was d i f f i c u l t to detect c y t o l o g i c a l l y and this could explain why i t has been reported so infrequently. I t i s clear however that spontaneous c e l l fusion between normally i n d i v i d u a l i z e d c e l l s could be very widespread. The extent of the c o n t r i -bution of spontaneous c e l l fusion to de novo occurring karyotypes both i n vivo and i n v i t r o remains to be investigated. 97 Spontaneous c e l l fusion C e l l fusion can be caused by a number of factors both i n vivo and i n v i t r o (Ringertz and Savage, 1976). In plants, c e l l walls obviously represent an additional natural b a r r i e r to c e l l fusion and must be removed before membrane fusion can be induced i n v i t r o . In t h i s study the PMCs of some anthers at early meiotic stages were found not to have callose c e l l walls, which were only formed l a t e r . The delay i n c e l l wall formation, possibly combined with underdeveloped anthers, appears to have been the main factor causing spontaneous c e l l fusion. Both hybridit y and the stress generated by the r a d i c a l change i n environmental growth conditions, from short day t r o p i c a l to temperate, may have been involved i n t h i s delay. Although synapsis was always complete, evidence f o r hybridi t y was found (section 4) and hybridity, especially under s t r e s s f u l conditions, can be detrimental to the genetic balance. Numerous examples of uncoordination due to hybrid-i t y have been given i n the l i t e r a t u r e f o r both i n t e r and i n t r a s p e c i f i c hybrids (see Stebbins, 1958; Tai and Vickery, 1972; Bennett et a l . , 1976 and the Introduction). More recently, crosses i n grasshoppers have also shown s i g n i f -icant frequencies of chromosome mutations a f t e r hybridization (Peeters, 1982; Shaw et a l . , 1983) and s i m i l a r observations were made in.crosses between certain populations of Drosophila melanogaster i n the so c a l l e d "hybrid.dysgenesis" 98 syndrome which i s now known to be caused by transposable elements (Ish-Horowicz, 1982). In the present study hybridity by i t s e l f did not appear to be s u f f i c i e n t to cause a delay i n c e l l wall formation, since under some growth conditions the plants did not show these abnormalities. Experimental evidence showing the s e n s i t i v i t y of these plants to temperature was given previously (section 4) and the responses of maize to photoperiod changes i s well documented (Rood and Major, I98O; Canard and Ledent, 1981; Rood and Major, I98I). As indicated e a r l i e r , the plants f a i l e d to flower the f i r s t year ( i n I98O) because of t h e i r pronounced short day requirements. The second year the plants flowered extremely l a t e i n the summer and these were the plants i n which the abnormalities were observed. A s h i f t i n daylength and temperature i n the la t e summer induced these plants very rapi d l y into meiosis and anthesis. This stress, combined with hybridity, are believed to have been the two main factors which caused delayed c e l l wall f o r -mation i n the PMCs and hence spontaneous c e l l fusion. These observations could indicate that spontaneous c e l l fusion can be induced as a r e s u l t of stress caused by changes i n the environmental conditions and therefore be a s i g n i f i c a n t contributor to spontaneous genomic changes. I f this hypothesis i s correct, t h i s contribution could be important both i n vivo and i n v i t r o since environmental parameters can obviously be quite variable i n both cases. In s i t u chromosome modification and degradation The most s i g n i f i c a n t observation that was made during t h i s study and which does not appear to have been described previously i n the l i t e r a t u r e i s that, following spontaneous c e l l fusion, p a r t i a l chromatin degradation of one complement could take place i n s i t u and give r i s e to a range of d i f f e r e n t stable new karyotypes containing additional DNA. This important observation i s especially d i f f i c u l t to explain given the clonal nature of the c e l l s involved. Somewhat si m i l a r observations of i n s i t u modification and degradation of n a t u r a l l y transfected clonal DNA were however reported i n studies on cytomixis (Introduction). The following hypotheses could account f o r these observations: a) Genetic v a r i a b i l i t y Although the occurrence of spontaneous mutations has been known for a long time, i t i s only recently through plant c e l l propagation and regeneration that the extent of the genetic v a r i a b i l i t y of clonal l i n e s has been t r u l y established (Reviewed by Larkin and Scowcroft, l ° 8 l ; see also Screenivasan and J a l a j a , 1 Q 8 2 ) . This genetic v a r i a b i l -i t y has been coined "somaclonal v a r i a t i o n " . However some caution as to the r e a l frequency of t h i s v a r i a b i l i t y must be taken. Indeed c e l l culture i t s e l f can be mutagenic (Evans and Sharp, 1 9 8 3 ) . In the present study genetic v a r i a b i l i t y could have 100 contributed to some extent to unbalanced postfusion conditions especially i f i t s natural frequency was increased because of stress and the presence of a transposable element (see this discussion). b) C e l l synchrony Studies of i n v i t r o c e l l fusion have c l e a r l y shown that, at least under some conditions, differences i n c e l l synchrony can generate P.C.C. and also loss of chromatin (Introduction). Despite the fact that most PMCs i n maize are i n synchrony, minor differences of phase can occasionally be detected and furthermore, i n t h i s study c e l l s that were arrested were also observed (section 2.3-)• C e l l fusion between two out of phase c e l l s could perhaps cause, i n t h i s environment the loss of phase of one complement following c e l l fusion, bearing i n mind that these c e l l s were i n d i v i s i o n . This loss of phase could occur at c r i t i c a l stages such as metaphase and this may explain why there was a drop i n the proportion of "stable" postfusion c e l l s around th i s stage (Figure 8). c) Recognition of non-self DNA Recent molecular analysis of transfected DNAs has shown that stable plasmids become unstable i n a foreign host c e l l (Calos et a l . , 1983'; Razzaque et a l . , 1983)-This lack of balance could be due to DNA differences i n chromatin structure and/or the associated proteins. The 101 presence of such a nucleocytoplasmic"imbalance i s very u n l i k e l y i n this case because of the clonal nature of the c e l l s involved. d) Fusion between a normal and an aborting c e l l The hypothesis which appears best to account for the observations of i n s i t u chromatin degradation of one complement i s that c e l l fusion could involve a normal and an aborting c e l l . ' Aborting c e l l s were quite frequent (Table II) and were usually associated with fusing c e l l s . . Furthermore, aborting c e l l s were not t o t a l l y unstable g e n e t i c a l l y since p a r t i a l l y aborted c e l l s were observed (Figure 20). It i s quite conceivable that, i f c e l l fusion could take place between a normal and an aborting c e l l , the degree of s t a b i -l i z a t i o n of the aborting cytoplasm could be s i g n i f i c a n t l y increased by the cytoplasmic conditions of the normal c e l l and beyond a cer t a i n threshold. Moreover the fact that these c e l l s were subject to the external meiotic cues for d i v i s i o n and synchrony may have represented an additional element i n t h i s s t a b i l i z a t i o n . Evidence of the presence of a nuclear component s t a b i l i z i n g chromatin has been found i n Xenopus laevis oocytes.(Wyllie et a l . , 1977) and a component s t a b i l i z i n g the cytoplasmic conditions at metaphase i n Rana pipiens was also found by Meyerhof and Masui (1979) • This model would explain most, i f not a l l of my observations of i n s i t u chromatin degradation. The i n i t i a t i o n of c e l l abortion could be induced just p r i o r to c e l l fusion and the degree of modification made to the aborting complement could vary greatly depending on the timing of t h i s i n i t i a t i o n . A further factor f o r v a r i -a b i l i t y was the fact that the cytogenetic paths of c e l l abortion were found themselves to be quite d i v e r s i f i e d (section 2.3.)• I f cytoplasmic s t a b i l i z a t i o n could be achieved following the fusion between normal and aborting c e l l s , the combination of these two variables could explain the cytogenetic d i v e r s i t y of postfusion c e l l s that was observed i n t h i s study. The recovery of postfusion c e l l s with only one or a few " i n t a c t " additional chromosomes (Figures 12, 15, 16) i s d i f f i c u l t to explain but may be due to s l i g h t differences i n chromosome structure (for instance i n c o i l i n g or 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 pre-aborting c e l l , at the i n i t i a t i o n of abortion. In other words, depending on t h e i r i n d i v i d u a l state, some chromosomes would be protected but others not. Changes i n the nucleosome and t h e i r r e l a t i o n to nuclease s e n s i t i v i t y have been reviewed by Weisbrod (1982). I f the hypothesis that i n s i t u chromatin degradation of one complement was indeed the r e s u l t of the fusion between a normal and an aborting c e l l , then t h i s mechanism may be only very infrequent i n other cases of spontaneous c e l l fusion. However since spontaneous c e l l fusion must "be the r e s u l t of strong environmental or genetic causes (such as a virus infection) even i f these conditions are very l o c a l i z e d , they probably contribute equally to the frequency of c e l l abortion. Furthermore as already indicated pre-fusion conditions probably can, by themselves induce c e l l abortion i n some cases. B chromosome formation 104 In the postfusion c e l l s which contained s t a b i l i z e d centric additional chromosome fragments, these fragments were c l e a r l y incorporated into the anaphases and t e l o -phases, despite s t r u c t u r a l modification (Figure 11). These PMCs appeared completely stable and therefore probably produced unbalanced gametes. These gametes appeared to be viable as inferred from the recovery of 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 di f f e r e n t abnormal karyotypes with accessory chromosomes) from one s e l f p o l l i n a t e d F l ear, following s e l f i n g of the parental plants showing fusion of PMCs. I t is, not impossible to exclude, however, that the accessory chromosomes that were observed i n the F l originated from spontaneous c e l l fusion and p a r t i a l i n s i t u chromatin degradation during ovule formation. Clear evidence was found that these new chromosomes were not contaminant Tripsacum chromosomes (section 3) and on the basis of F l morphology and.behavior, these chromosomes were also c l e a r l y not B contaminants. As demonstrated i n th i s study, accessory chromo-somes could derive d i r e c t l y from c e l l fusion and further-more the fact that these fragments were found to have evolved from the F l to F2 generation as true B chromo-somes, c l e a r l y shows that c e l l fusion i s one mechanism which can give r i s e to these sequences i n the genome. Although i t i s unanimously accepted that Bs evolve somehow from the normal complement (Gupta, 1981), the precise mechanism generating modified A fragments i n the genome has remained t o t a l l y unknown (Introduction). Since B chromosomes are extremely widespread i n "both plants and animals (Miintzing, 1974j Jones, 1975) t h i s could be an in d i c a t i o n of the true frequency of spontaneous c e l l fusion i n vivo. It i s not known i f , following spontaneous c e l l fusion, some B chromosomes were p r e f e r e n t i a l l y l o s t while others with d i f f e r e n t sequences were retained. The answer to thi s question would shed more l i g h t on the controversy which exists as to the nature of B chromo-somes. Some authors have indeed considered these exogenous sequences as p a r a s i t i c elements (Rhoades and Dempsey, 1972) , given t h e i r apparently passive contribution i n the genome. A more recent d e f i n i t i o n could be s e l f i s h DNA ( D o o l i t t l e and Sapienza, I98O). B chromosomes are known to be extremely polymorphic but at least a part of t h i s polymorphism i s due to evolution and spontaneous morphological changes (Bougourd and Parker, 1979)' Some types of Bs, such as Bs with a NOR, are extremely un-common and only two or three examples of such B chromo-somes have been given i n the l i t e r a t u r e (Carr and Carr,1980). This observation was confirmed i n this study, where only one case of NOR B chromosome was found (Figure 12) 106 despite the r e l a t i v e l y high number of postfusion c e l l s with accessory chromosomes that Were observed (Table I I ) . This may indicate that some A sequences have a greater capacity to evolve as stable B chromosomes. I t i s important to consider here that c e l l fusion occurred mostly at stages where the NOR was t r a n s c r i p t i o n a l l y active. Therefore t h i s did not appear to be a factor which favored the retention of chromosome 6 (which bears the NOR) during i n s i t u degradation, possibly because active chromatin i s more sensitive to nucleases (Weisbrod, 1982). The inherited fragments were found to evolve morphologically from the F l to F2 generation through increased heterochromatinisation, in d i c a t i n g that these sequences became inactivated. It was quite surprising to f i n d that t h i s morphological evolution occurred so rapidly, although there obviously are quite a few mitotic cycles from the zygote to the l a s t pre-meiotic c e l l d i v i s i o n . It i s also i n t e r e s t i n g to note here that i n maize stocks carrying Bs, a negative c o r r e l a t i o n between the presence of knob heterochromatin and the B chromosome was established (Longley, 1938). This may be due to the asynchronous r e p l i c a t i o n of the knob and B heterochromatin (Pryor et a l . , I98O). The mechanism described here on the formation of B chromosomes accounts f o r most of the often c o n f l i c t i n g molecular and cytogenetic studies on Bs (see Introduction). There appears to have teen a discrepancy between the r e l a t i v e l y high proportion of plants i n the F l with additional chromosomes and the frequency of stable post-fusion c e l l s with accessory chromosomes i n the.parental meiosis (Figure 8). However t h i s could be explained by 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 containing these accessory chromosomes, which i s a well established B chromosome property (Introduction). This hypothesis implies that the s e l e c t i v e advantage of carrying these fragments f o r f e r t i l i z a t i o n had already been acquired. Furthermore, despite the large number of meiotic c e l l s studied (17,000) t h i s represents only a small pro-portion of the PMCs since 50,000,000 or so pollen are formed by a medium-sized maize plant (Paton, 1921). Aneuploidy and polyploidy. 108 In the 36 F l plants that were examined, no aneuploid or t r i p l o i d ( r e s u l t i n g from the fusion of an egg with an unreduced sperm) were discovered but, given that aneuploid and polyploid PMCs which appeared completely stable were observed during the parental meiosis, i t i s very l i k e l y that these products of spontaneous c e l l fusion can also contribute to de novo occurring karyotypes. Numerous authors have shown that aneuploid and 2 or 4n pollen i n plants can be viable (Karpechenko, 1927; McClintock, 1928; Price, 1956; M i l l e r , 1963; Grant, 1965; Ramanna, 1973; P f e i f f e r and Bingham, 1982). Different,mechanisms leading to the formation of aneuploid c e l l s were found during t h i s study. These mechanisms were p a r t i a l elimination and degradation of only some chromosomes following c e l l fusion and the formation of microcells which could fuse with normal c e l l s (see section 2.2.). In addition, postfusion 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 (Figure 18) probably could also contribute to the production of aneuploid gametes by uneven tetrad formation. Among these mechanisms f o r aneuploidy, the spontaneous fragmentation of PMCs into microcells and sub-sequent fusion of these microcells 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 genetic s t a b i l i t y . The s t a b i l i t y of microcells i s indeed well established and these c e l l s have been found to divide normally i n plant tissue culture (Ting et a l . , I 9 8 O ) . Such c e l l s , which can be produced experimentally, are of great interest f o r chromosome transfer and the formation of i n v i t r o aneuploids (McNeill and Brown, I98O). Cytogenetic evidence for the existence of these mechanisms f o r aneuploidy does not seem to have been given previously i n the l i t e r a t u r e . I 110 Additional c y t o l o g i c a l observations During t h i s study a few very unusual additional observations were made during meiosis i n the parental plants. It i s only possible to speculate on the nature and implications of these observations. Inactive supernumerary chromosomes Figure 10 shows an example of a postfusion c e l l with supernumerary chromosome fragments which f a i l e d to follow the cytoplasmic cues for chromosome condensation. C e l l s containing such "i n a c t i v e " chromosomes have also been reported following chemically induced abnormal meiosis (Rajendra and Bates, l Q 8 l ) and the presence of cytoplasmic cues regulating chromosome condensation i n dividing c e l l s i s well established (Meyerhof and Masui, 1979). Numerous hypotheses can account f o r the observations that were made here of supernumerary chromosome fragments f a i l i n g to condense following p a r t i a l modification and degradation of one complement. Rungger (1979) has shown that a c t i n i s present and i s involved i n chromosome condensation of Xenopus chromosomes while Matsumoto et a l . (1980) have shown that HI histone phosphorylation i s also involved i n chromosome condensation. More recently Otto et a l . (1981), studying 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 center" which i s I l l located near the centromere. My observations would agree with such a hypothesis since these uncoiled chromosomes were never found to be involved with the spindle and thus appeared to be acentric. Since the chromosomes of the intac t complement were contracting normally, the observations that were made of postfusion c e l l s with inactive supernumerary chromosomes confirms that these chromosomes were s t r u c t u r a l l y modified as a re s u l t of p a r t i a l in' s i t u degradation. I t was also quite i n t e r e s t i n g to notice that these chromosomes were at least p a r t i a l l y stable (see section 2.1.)• Neocentric a c t i v i t y A few cases of neocentric a c t i v i t y were observed (Figure 34). According to Peacock et a l . (1981), neocentric a c t i v i t y i n maize i s due only to a very high number of copies of a 185 bp. repeat i n chromosome K10 although t h i s repeat i s also present i n knob heterochromatin. The degree of p r e f e r e n t i a l segregation i s d i r e c t l y proportional to the number of copies of the repeat carried 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 segregation may indicate a case of ampli-f i c a t i o n of th i s 185 bp. repeat from knob heterochromatin i n some of the PMCs. Dicentric bridges • Dicentric bridges were sometimes observed and occurred i n 100% of the PMCs while being t o t a l l y absent from s i s t e r anthers of the same t a s s e l . This unusual observation could be an in d i c a t i o n of the presence of a transposable element i n the samples which was being induced i n some pre-meiotic anthers but not i n others. In this work, other examples were already given which showed that meiotic abnormalities could be l o c a l i z e d to some anthers while 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 indi c a t i n g that anthers show a high degree of genetic i n d i v i d u a l i z a t i o n . Bridge formation as a r e s u l t of a displaced trans-posable element has been described by Fedoroff (1982) and once formed, these bridges are perpetuated i n a cycle termed the chromatid type of breakage-fusion-bridge cycle by McClintock (1942). Furthermore the presence of trans-posable elements i n maize i s well established (Marx, 1983) and some of t h e i r important genetic properties have been studied (Dooner and Nelson, 1979; Kermicle, I98O; Burr and Burr, 1982). The induction of transposable elements i n maize can occur at an extremely l o c a l i z e d l e v e l . For example t h e i r a c t i v i t y can be li m i t e d only to a s p e c i f i c part of the kernel (Fedoroff, 1982) and what has been described as paramutation probably represents another example of l o c a l i z e d , transposable element mediated genetic v a r i a t i o n i n maize as well as i n other plants (Brink, 1973; Hagemann, 1978). 113 I f the d i c e n t r i c bridge formations were induced by a transposable element i t i s possible that the element(s) was also mobilized by the stress caused by c e l l fusion and consequently contributed to postfusion genomic and chromosomal modifications. Meiotic determinants. A postfusion c e l l with a clear c r o s s - c e l l meiotic gradient was observed (Figure 28). This postfusion c e l l appeared to indicate that the meiotic determinants that were present i n the c e l l s could be independent from the external signals for c e l l synchrony. A s i m i l a r observation was made by other researchers, following i n v i t r o c e l l fusion i n large dividing polykaryons where "mitotic waves" were observed and "suggesting that the concentration of mitotic triggers formed a gradient across the c e l l " (Ringertz and Savage, 19?6, p. 70). The presence of cytoplasmic determinants i n dividing c e l l s i s well established and these determinants play a key role i n developmental biology (see Jackie and Kalthoff, 1980). In t h i s study a clear example confirming the presence o f " m e i o t i c " determinants was only found once (Figure 28) and the r e l a t i o n s h i p between these factors and c e l l synchrony i n the anthers i s not known. Obviously c e l l fusion could disrupt severely the balance, created by cytoplasmic factors and t h i s appeared to be especially c r i t i c a l i n cases involving c e l l p o l a r i t y (Figure 18). 114 Abnormal presence of n u c l e o l i . The synthesis of additional n u c l e o l i was observed i n both "normal" and aborting c e l l s at stages where RNA synthesis was expected as well as at stages at which RNA synthesis normally had ceased (Figure 29). Similar observations were made i n cancer c e l l s (Sheldon and Lehman, 1981). Some of these c e l l s may have shown rDNA amplification, as a response to the stress induced by the i n i t i a t i o n of c e l l abortion. Examples of rDNA amplification are well known i n animal c e l l s ( for a review see Tobler, 1975; Long and Dawid, 1980) and, although possible examples have been given (Avanzi et a l . , 1973)> controversy as to the existence of rDNA amplification i n plants s t i l l exists. Conclusion 115 • Spontaneous c e l l fusion i s d i f f i c u l t to detect but could prove to be a s i g n i f i c a n t force i n both the i n vivo and i n v i t r o genesis of spontaneous aneuploids, polyploids and new karyotypes containing additional DNA. The exact prevalence and significance of this mechanism i n the gen-esis of new karyotypes remains to be investigated. One mechanism giving r i s e to B chromosomes has been demonstrated for the f i r s t time. These chromosomes were found to re s u l t from p a r t i a l i n s i t u chromatin degradation of one complement, following spontaneous c e l l fusion as well as a s t r u c t u r a l evolution of the r e s u l t i n g s t a b i l i z e d fragments, through heterochromatinisation. The fact that spontaneous c e l l fusion can generate de novo stable additional gene sequences i n the genome i s of s e l f evident importance for evolution and gene dosage. This importance i s even greater i n l i g h t of the fact that some of these s t a b i l i z e d sequences were probably extensively modified as.a r e s u l t of i n s i t u degradation. It i s clear that some of the smaller s t a b i l i z e d sequences generated by c e l l fusion and p a r t i a l i n s i t u chromatin degradation could perhaps transpose.to the A chromosomes and also give r i s e to new genomic arrangements. Such transpositions could be enhanced by the genomic stress of c e l l fusion and chromatin modification and degradation as well as by the presence of activated transposable elements. 116 These transposed sequences would probably not then be subject to the extensive heterochromatinisation which was found to occur during B chromosome evolution (see also Amos and Dover, 1981) and hence remain t r a n s c r i p t i o n a l l y active. Spontaneous c e l l fusion therefore could also be involved i n the production of gen e t i c a l l y unstable c e l l s . 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