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Mitosis in Allium cepa stained with brilliant cresyl blue Sweet, Harmon 1953

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MITOSIS IN ALLIUM CEPA STAINED WITH BRILLIANT CRESYL BLUE by H. SWEET  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ARTS i n the Department of BIOLOGY AND BOTANY •i  .  .  .  .  We accept this thesis as conforming to the standard required from candidates f o r the degree of MASTER OF ARTS  Members of the Department of Biology and Botany  The University of B r i t i s h Columbia A p r i l , 1953  ABSTRACT OP THESIS  B r i l l i a n t c r e s y l blue has been used but l i t t l e i n botanical histology.  The preparation and methods of  use of the s t a i n l n squash technique are described*  The  technique i s then used to follow the mitotic cycle i n Allium cepa. I t i s shown to be an excellent morphological s t a i n and i t has moreover some promise of being useful l n cytology.  Some seventy photomicrographs demonstrate i t s  possibilities*  # *• <»  ACKNOWLEDGMENTS  Sincere thanks i s due to the members of my committee Dr. A, H. Hutchinson, Dr. P. Dickson, Dr. V. C. Brink and Dr. K. M. Cole who were, whenever approached, generous of t h e i r time and suggestions*  In p a r t i c u l a r ,  I am most g r a t e f u l to Dr. A* H. Hutchinson who i n years past, afforded me opportunities f o r h i s t o l o g i c a l practice without which this study might not have been undertaken* Appreciation f o r h i s services i n a s s i s t i n g with the drawing of the genome of plate XVTI i s tendered to Mr. A. W. Lewis, and to Mr. R. Harris f o r the photography Involved and which was so painstakingly c a r r i e d out.  •» # •»  TABLE OP CONTENTS PAGE Introduction  . .1  Statement of the problem . . Historical  2  . .  3  Methods and Materials  4  B r i l l i a n t Cresyl Blue Technique He-statement of Problem  7  . . . . . . . . . . . . . .  Chromosome Structure Description  .10 10  . . . . . . . . . . . . .  Mitotic Movements of the Chromosomes  . . .14 . . . . . .  .14  Chromonemata  22  Coiling  25  Pairing  .27  Trabants  28  Segmentation  29  The P e l l i c l e . . .  .30  Ploidy  30  Colchicine mitosis Kinetochore  .-  30  . . . . . . . . . . . . . . . . . . .  Discussion Tassement Polaire  .31  .....31 . . . • • • « . . .  Interpretation of Images • • Duplication of Chromonemata Number of Chromonemata  . . . . . . . . . . . . .  31 ..34 35 .36  Page The S t a i n B r i l l i a n t C r e s y l Blue . . . . . 4 . . . . . 36 Suggested. F u r t h e r Summary •  37  . . . . . . . . . « • • » • • » •  BIBLIOGRAPHY APPENDIX  Investigations  . . • ••  * i •••'.»•...< .  . . . . . . . . . . . . .  . . • « • • • •  38 • 39  •  PLATES  43  INDEX TO PLATES.  6&  •it  *  »  MITOSIS IN ALLIUM CEPA STAINED WITH BRILLIANT CRESYL BLUE  Introduction.  I t i s an obvious truism that a proper understanding of somatic mitosis i s basic to the whole f i e l d of Biology, p a r t i c u l a r l y to Cytology and to Genetics (Vanderlyn 1948).  However, notwithstanding the f a c t that many  eminent b i o l o g i s t s have given attention to t h i s subject, there are s t i l l major problems to be solved l n the study of mitosis*  There i s no unanimity among investigators i n  regard to what impulse i n i a t e s the mitotic c y c l e , nor as to what forces d i r e c t the chromosomes from phase to phase. There are problems of chromosome structure, of the i n t e r n a l organization of the chromonemata, of the effects of conditions and reagents upon the chromaticity of chromosomal con-  2  stituents--these are but a few. The p r i n c i p a l aim of t h i s study was to f i n d and use a simple, s a t i s f a c t o r y technique applicable to the Investigation of chromosome structure and thus to a i d i n the solution of some of the undecided questions of cytology. There are numerous techniques i n current use f o r such work, but i t i s premature to conclude that the i d e a l technique and s t a i n has boon found. A c e t o - c a r m i n e has been i n common use by cytologists f o r many years, but some of i t s preparations are tedious to perform and results of i t s use may at times be uncertain. Materials f o r c y t o l o g i c a l investigations are r e a d i l y available.  The meristem of onion root-tips have  been i n almost constant use since Guignard Introduced this material i n 1884. Onion root-tips are of rapid growth a t taining a suitable length i n two or three days from the time the bulbs are f i r s t set i n water* well under ordinary cool conditions.  The bulbs also keep The chromosomes of  Allium cepa are large, measuring up to fourteen microns or more at late metaphase.  These are a few of the reasons f o r  the continued use of onion r o o t - t i p s i n cytology*  3  Historical  Guignard (1884) was the f i r s t to study mitosis In Allium cepa Koshy (1933).  Since that time the cytology of  the species has been under frequent i f not continuous i n vestigation.  Schaffner (1898) examined the formation of the  achromatic spindle i n A. cepa.Kellicott (1904), investigating p e r i o d i c i t y of c e l l d i v i s i o n i n root-tips of  cepa, found  there were two p r i n c i p a l waves of d i v i s i o n i n twenty-four hours, attaining crests at about 11 a.m.  and 1 a.m.  Gregoire  (1904) began a study of chromosome structure using A^ cepa and developed the alveolar theory which influenced chromosome theory f o r several years.  He was  i n s i s t e n t upon the d i s -  continuity of the prophase spireme.  Reed (1914) believed  the spireme to be continuous at f i r s t , segmenting  later.  A study of the prophase photomicrograph 4 of plate I w i l l show how d i f f i c u l t i t i s , i f not impossible, to decide the issue i n A^ cepa. Friesen (1920) Investigated the d a i l y p e r i o d i c i t y of c e l l d i v i s i o n i n iU cepa and found three cr©3ts of maximum  a c t i v i t y i n twenty-four hours, these being at 1  9 p.m.  and 5 a.m.  p.m.,  Taylor (1925) was more interested i n the  morphology of the chromosomes of A»_ cepa. He mentions a s a t e l l i t e on each of a pair of chromosomes, the attachment c o n s t r i c t i o n of these being sub-terminal.  Sharp (1929)  4  found the more chromatic  constituent of each chromosome to  persist throughout the mitotic cycle of A^ cepa, i n the form of two f i n e threads or chromonemata.  Pierre Martens (1929)  made a study of the l i v i n g c e l l s and found there i n the r e s t ing stage, the reticulum so often mentioned i n f i x e d material. Following the work of Blakeslee and Avery (1937) on Datura and Cosmos . Levah (1938) Investigated the effect of colcbJ&ie^ on r o o t - t i p c e l l s of A* cepa. Since 1940  there has been Increasing attention given  to the action of enzymes ribonuclease and upon chromosomes.  desoxyribonuclease  A recent paper by Kaufman7i(1951) reports  the use of these enzymes on the n u c l e i of A* cepa.  Thus i t  seems as Martens (1929) remarks In regard to the onion: Qn y revient toujours.  Methods and Materials  The onions recommended f o r use i n this study were of the larger sized s i l v e r - s k i n v a r i e t y .  These were grown  i n the Okanagan d i s t r i c t of the province and are e a s i l y obtainable i n season l o c a l l y .  They are of uniform  size  and remain i n usable condition f o r several months when s t o r ed under cool dry conditions.  They sprout r e a d i l y and  y i e l d a p l e n t i f u l supply of t i p s .  Supported by small  stones i n glass or enamelled dishes, they were sprouted i n  5 .  tap-water.  They were kept at room temperature under natural  day-and-night  conditions, since i t was believed that thereby  the d a i l y p e r i o d i c i t y of c e l l d i v i s i o n was most l i k e l y to occur (Priesner 1920). Various pre-treatments were attempted--Method Twenty (Sharp 1929)—the  cold treatment (Darlington 1 9 4 7 ) —  thorium n i t r a t e and ammonium hydroxide (Nebel 1933)—the  aim  b e i n g to achieve a clearer d e l i n e a t i o n o f the chromonemata. None of these however seemed to be successful.  Recently a  pre-treatment f o r t u i t o u s l y discovered by Mr. A. Unrau gives promise of being useful. the t i p s with Feulgen's for  This consists of f i r s t  staining  leuco-basic fuchsin then staining  a few minutes with b r i l l i a n t c r e s y l blue.  Time however  did not permit further testing of this method. To f a c i l i t a t e chromosome counts, colchaS^Siras used a f t e r the method of Levan (1938). ing  The bulb with grow-  tips attached, was transferred from tap-water to an  0,05%—0.08$  aqueous s o l u t i o n of colchiiie^for from one to  two hours immediately preceding the c l i p p i n g .  This proved  to be quite s a t i s f a c t o r y f o r the purpose, the chromosomes remaining spread out i n a delayed metaphase, usually undivided, but occasionally divided (photomicrographs 56 plate XIV).  54 and  6  Normally bulbs that had been started i n water at 1G a.m., would y i e l d s a t i s f a c t o r y  tips with numerous d i v i d -  ing c e l l s , when cut a few days l a t e r at 1 p.m.  But i n dark  cloudy winter days, this was not always the case, best tips then being obtainable i n mid-forenoon.  The time of induct-  ion of metabolic a c t i v i t y has some effect upon the timing of the maximum crest of the c e l l d i v i s i o n wave (Priesner 1920). Tips were cut d i r e c t l y into the k i l l i n g f l u i d , ' Parmer's alcohol acetic a c i d , when they were from one to three centimetres long.  Various other f i x a t i v e s were  t r i e d : Navishin's—B.C. f i x — L a Cour's 2BE—but these proved to be unsatisfactory, the tips f a l l i n g to stain properly, i f at a l l . About twenty-four hours was allowed f o r f i x a t i o n , although less time would probably have s u f f i c e d , but this period of f i x a t i o n yielded good  staining.  In much of the e a r l i e r work on root-tips techniques were used:  imbedding  Gregolre (1904), Praser and S n e l l  (1911), Gates (1912), Sharp (1929), Koshy (1933), a l l used embedding techniques.  In this study i t was thought  desir-  able to use a squash technique to reduce time and to gain the desirable results of squash methods.  With this i n mind,  smears were attempted using accepted aceto-carmine, Feulgen, and aceto-orceine techniques—.Warmke (1935), Mensinkai (1939), La Cour (1941).  None of these however proved e n t i r e l y  7  s a t i s f a c t o r y , but a search through Stain Technology resulted i n a t r i a l of b r i l l i a n t c r e s y l blue (Stewart and Schertiger 1949).  This stain has proved reasonably s a t i s f a c t o r y f o r  - t i p chromosomes when a squash technique i s used. not successful with embedded material.  It is  The staining solution  i s quickly prepared, the staining procedure short and simple. Very good spreading of the c e l l s generally r e s u l t s , and the s l i d e s can be made permanent. usable f o r a week o r more.  The temporary slides are  The s l i d e s photograph w e l l .  B r i l l i a n t c r e s y l blue, C.I.877, i s known chemic a l l y as aminodimethylamlnomethyldiphenazonium chloride.  It  has been used i n the past as a v i t a l s t a i n f o r blood corpuscles (Conn 1940).  I t has been but recently applied to  plant tissues (Stewart and Schertiger  1949).  For our pur-  poses i t i s used In two per cent strength, i n f o r t y - f i v e per cent aqueous acetic a c i d .  Staining;requires  from f i v e to ten  minutes. The technique as given by Stewart and Schertiger follows: 1.  F i x a t i o n i n Farmer's acetic-alcohol.  2.  Digest i n a solution of concentrated  hydrochloric  a c i d , and 95$ ethyl alcohol, equal parts, f o r 5-6  minutes.  3.  Transfer to Farmer's s o l u t i o n , 2 - 3  4.  Transfer  to 70$ alcohol.  minutes.  8  5.  Place t i p on a clean s l i d e coated with Haupt's solution.  6.  (See modifications)•  Section transversely, using about the f i r s t 3 millimetres of the t i p only.  7.  Add a drop or two of the 45#. aqueous acetic acid and tease out with clean, sharp needles.  8.  Add a drop or two of the s t a i n , and then apply a clean cover glass. .  9.  Stain f o r 3 - 10 minutes.  Then apply a paper,towel  to the cover glass, and with l i g h t pressure squeeze out the excess s t a i n . 10.  Seal with equal parts gum-mastic and p a r a f f i n .  Modifications Haupt's solution i n step 5 may i n t e r f e r e with staining.  Gum arable or a 3%-4% s o l u t i o n of sugar i s r e -  commended (Naithanl 1937). Step 7 i s a modification.  The Stain technology  a r t i c l e recommended adding the stain here, and teasing out i n the s t a i n .  But the s t a i n i s very dense.  Step 10 was not used.  Instead a temporary  of the mounting f l u i d was used here.  seal  I f i t runs under the  cover g l a s s , no harm i s done, although the blue i s decolorized.  The c e l l s are then amber colored.  9  The slides may he made permanent a f t e r a few days. The mounting f l u i d i s a mixture of lacto-phenol and p o l y v i n y l alcohol, prepared as follows: 1.  15 grams of p o l y v i n y l alcohol i s added slowly to 100 ml. of cold water.  2.  S t i r , and heat i n a water bath to 80°C. u n t i l i t has the consistency of clear molasses.  The mounting f l u i d consists of 56$ of the above, and 44$ lacto-phenol by volume. 1.  The cover glass i s f l o a t e d off i n 70$ alcohol i n a Petri dish.  2.  Excess alcohol i s removed from the s l i d e by touching the edges to a paper towel.  3.  A small drop of the p o l y v i n y l alcohol mixture i s then added to the smear, and the cover glass replaced as i t was before.  The s l i d e w i l l dry over night.  Such per-  manent slides have been found quite good a year l a t e r . In the temporary slides the c e l l s are stained darkly but i n the permanent preparations, the blue has been replaced by a l i g h t amber.  The temporary s l i d e s require strong  a r t i f i c i a l l i g h t when i n use. The p o l y v i n y l alcohol was obtained l o c a l l y from Canadian Industries Limited.  The s t a i n , b r i l l i a n t c r e s y l  blue, Is stocked by most supply houses. The photomicrographs (PMG) were taken with a Spencer microscope using an oil-immersion lens and a XIO  10  ocular.  The 4 mm. lens and a X15 ocular combination was  t r i e d but soon given up*  Most of the PMG's are magnified  about 2200 to 2300 times, this magnification being attained by extending the camera bellows* mercury vapor lamp.  The source of l i g h t was a  The prints commonly yielded more d e t a i l  than was v i s i b l e through a microscope. For the set of chromosomes on plate XVII a B. & L. camera-lucida was used, the mirror at 45 degrees and the drawings made at table l e v e l *  Re-atatement of Problem  As mentioned above, the aim of t h i s study was to f i n d and use a suitable technique f o r a c y t o l o g i c a l i n v e s t i gation of root-tips of A*_ cepa*  I t was considered p a r t i -  c u l a r l y desirable to reveal the chromonemata at the d i f ferent mitotic phases--also to make some study of chromosome morphology--to attempt to determine the time of chromonemata duplication i n A*_ cepa. I t was hoped, too, that some new and i n t e r e s t i n g features might be found. Chromosome Structure Before proceeding to a description of mitosis as seen with b r i l l i a n t cresyl'blue, a b r i e f consideration of chromosome structure i s desirable.  In PMG 24 plate V I ,  the anaphase chromosome i s seen to be a more or less  11  c y l i n d r i c a l body, here quite smooth iii o u t l i n e .  Where the  focussing has been more p r e c i s e — B I G 51 plate X I I I — t h e outl i n e i s undulating which denotes  or i t may be moniliform—PMG 27 plate V I I —  the c o i l i n g of chromonemal  threads embedded i n  the chromosome (Kaufmann 1936). Selecting chromosome (a) of PMG 24, plate VI f o r examination, i t i s seen to be composed of a p a i r of darkly staining or chromatic, p a r a l l e l , hooked rods. chromatids or half-ehromosomes  the two chromatids  the  (Nebel 1939)' They are seen  l y i n g i n a common, l e s s densely staining chromatic substance.  These are  In chromosome (b)  matrix, the  less  of this same group,  are evidently twisted about each  but i n (c) they are again more or l e s s p a r a l l e l .  other,  Abraham (1939)  states that such p a r a l l e l alignment of the anaphase  chromatic  halves has been reported by various authors. ai The p r i n c i p i a element of the chromatid i s the. chromosome  thread or ch'rombnema. often h e l i c a l l y  running  throughout  Abraham* 1937). (Nebel 1939)*  known  "chromatic substance"  proteins and nucleic  (Mensinkai be well to  1939^.  recognize the  helically  acid  Kaufmann (1926) terms  and "ehromonematic substance "  The chromonemata,  single  of chromatin, a chemical sub-  a combination of  that i t might  synonymous.  thread i s seen to be o p t i c a l l y  It Is composed  as nucleoprotein  mentions  and  the length of the chromatid (KaufmaOTfl.936)  This  stance which i s  coiled,  coiled,  may  as be  12  seen In the uppermost part of the f i g u r e , i n PMG Some disagreement  37 plate X.  s t i l l continues In regard to the number of  chromonemata i n the anaphase chromosome, the number being given as one, two, or four by d i f f e r e n t authors, two r e ceiving the most recognition (Kaufman?!1936)•  These chromo-  nemata l i e i n a less chromatic matrix (Abraham 1939)* (KaufmanYil948), which i n PMG 24 plate VI i s dimly seen between the two p a r a l l e l chromatids. In PMG 23 of plate VI the chromosome on the lower l e f t i s seen to be constricted near the upper end.  Such  a c o n s t r i c t i o n i s c a l l e d variously, the attachment c o n s t r i c t ion  (Kaufman*1936), the centromere (Darlington 1937) or the  klnetoohore (Levah 1939).  The spindle f i b r e s are s a i d to be  attached here as the chromosome moves toward one of the poles of the c e l l .  The kinetochore of the chromosome at the  lower right i s c e n t r a l l y located.  The kinetochore occupies  a d e f i n i t e and commonly constant p o s i t i o n with respect to' the ends of the chromosome, but this p o s i t i o n may however be altered (Kaufman^1948).  I t s essential c h a r a c t e r i s t i c i s  i n i t s reacting to the polar a t t r a c t i o n , so that i t i s of utmost importance i n movement of the metaphase chromosomes to  the poles (Matsura 1941). In plate XI PMG 42, the chromosome at the lower  l e f t i s seen to have a small, black, knob-like terminal appendage.  This i s the s a t e l l i t e or trabant attached to the  13  chromosome by a short filament. A s i m i l a r trabant i s seen attached to the central chromosome of PMG 41.  The trabant  i s believed to be the r o l l e d up ends o f the chromonemata (Menslnkai 1939), which pass along the filament to the knob* I t was discovered by Navashin In 1912 (Meninkai 1939).  In  A. cepa there are two chromosomes which have a trabant. The formation of the trabant i s now believed to be associated with the formation of the nucleolus (Menslnkai 1939), (Kaufraamil948). The nucleolus, an important feature of the nucleus and sharing i n the nuclear changes brought about by mitosis, should be mentioned at this time.  I t i s a viscous, semi-  s o l i d , spherical body (Menslnkai 1939) associated with the nucleus at metaphase, interphase and prophase stages. I t disappears during prophase but i s reformed again at t e l o phase (PMG € 5 plate VTII).  I t does not s t a i n with b r l l -  l i a n t cresyl/tbut i s recognized as a c l e a r colorless area somewhat enlarged by a halo, which by many i s regarded as an a r t i f a c t (Menslnkai 1939).  The halo i s not commonly  seen i n the Navishin-haematoxylin preparation PMG 2 plate I . k* a r t i f a c t Is an appearance that arises from treatment * 1  (Darlington 1937). Another term "daughter" chromosome should be explained at this time.  In PMG 54 plate XIV several of the  chromosomes are seen to be double.  Each half chromosome  14  i s of course a chromatid, by our previous d e f i n i t i o n *  Pre-  sently, however, the two halves w i l l he separated and move to opposite poles of the spindle.  Each h a l f * after the sep-  aration, then i s known as a daughter chromosome (Koshy 1933), (Abraham 1939). The spindle does not s t a i n with b r i l l i a n t c r e s y l blue.  I t i s a b a r r e l shaped structure best seen at late  anaphase stretching from one chromosome group across the c e l l centre to the other group.  Other f i b r e s appear to pass  from the central plate to the poles.  Commonly i t appears  to be composed of many f i n e spindle f i b r e s some of which are said to be attached to the kinetochores.  The f i b r e s are by  many believed to be a r t i f a c t s (Darlington 1937), (Robyns 1929).  Description  Anaphase In a d e t a i l e d account of mitosis i t i s convenient to s t a r t at anaphase because at this stage the chromosomes have a t r u l y i n d i v i d u a l i z e d and independent existence (Hedayetullah 1931).  The anaphase chromosome has already  been studied In PMG 24 plate VI.  We have noted the two  darkly staining rod-like chromatids l y i n g p a r a l l e l i n a common less chromatic matrix.  In PMG 2ft plate VII the  15  s e p a r a t i o n o f the s i s t e r chromosomes i s almost completed. In PMG 25 s e p a r a t i o n  i s complete with the e x c e p t i o n  of a  " b r i d g e " at ( b ) . Most o f the chromosomes are assuming or have assumed a U o r V form, but a J" o r shepherd's crook may be seen on the lower r i g h t o f PMG 26.  In PMG 25 two t r a b a n t s can be  d i s t i n g u i s h e d as dark s t a i n i n g g l o b u l e s near the lower end o f the f i g u r e .  In PMG 27 movement has progressed a  t h e r , toward the p o l e s .  The chromosomes  somewhat more extended as the loosened.  little  are now a p p a r e n t l y  c o i l s o f the chromonemata are  The a p p a r e n t l y i n t e r l a c i n g chromonemata are seen i n  i n the lower l e f t  chromosome (a) o f PMG 27.  I n PMG 28 p l a t e V I I — a p o l a r view o f l a t e the chromosomes the k i n e t o c h o r e s VIII since  fur-  have  reached the p o l e s , o r more  have a r r i v e d  i s a similar polar here the  anaphase--  clear  there.  PMG 30  view o f anaphase, areas dnote  The chromosomes are assembled  accurately,  of plate  but  rather l a t e r  the r e - f o r m i n g  nucleoli.  i n a hemishpere not u n l i k e the  r i b s o f an expanded u m b r e l l a . In PMG 29 and PMG 32 the chromosome appear to be t u r n i n g inwards to form cylinder.  extremities  an apparent  S i m i l a r f i g u r e s are found on p l a t e IX.  hollow In PMG 35  i t would appees that the c y l i n d e r i s now completely e n c l o s i n g the n u c l e o l i which appear w i t h i n the n u c l e u s . anaphase  formed  as c l e a r u n s t a i n e d areas  With the completion o f the c y l i n d e r the  ends and the telophase b e g i n s .  16  In anaphase then  i s seen, f i r s t  the s e p a r a t i o n o f  the s i s t e r chromatids of the metaphase chromosome to form independent  daughter  chromosomes which.then  move  to o p p o s i t e  p o l e s of the c e l l where th^y appear . to r o l l up i n t o a hollow c y l i n d e r e n c l o s i n g the n u c l e o l i .  Anaphase is. of s h o r t dur-  ation. Telophase begins w i t h the anaphase chromosomes r o l l e d i n t o an apparent hollow c y l i n d e r blue i s s t i l l h e a v i l y s t a i n e d . chromaticity of  is  p i a t e XVI, and  PMG  31  im  PMG  As telophase  much l e s s e n e d ,  as on the  proceeds left  that may  be t r a c e d  i n PMG  The lower nucleus of PMG  i s p r o b a b l y a very l a t e telophase  of  this 61  appear as 61 o f  35 of p l a t e IX, a l s o i n 33 and 34,  of plate VIII.  cresyl  o f PMG  the c o n s t i t u e n t chromonemata  a maze of emerging c o i l s , p l a t e XVI,  which w i t h b r i l l i a n t  or i n  32 p l a t e V I I I  a giant c e l l .  Such  giant c e l l s are found i n the c e n t r a l c y l i n d e r of the r o o t - t i p s of A. cepa. (Vanderlyn 1948), C  C o i n c i d e n t w i t h the chromosomal t r a n s f o r m a t i o n s ,  i s the appearance of the n u c l e a r  membrane (Vanderlyn  but t h i s membrance does not s t a i n w i t h b r i l l i a n t  1948)  c r e s y l blue,  and as noted, the n u c l e o l i reappear and s t e a d i l y i n c r e a s e i n s i z e , PMG  31 plate" V I I I .  I t i s suggested by Gates  n u c l e o l u s a r i s e s from the chromosomal m a t r i x , ance of  (1948) the  the d i s a p p e a r -  which r e v e a l s the chromonemata at t h i s phase.  17  The disappearance i n the Navashin-haematoxylin  of the matrix Is more marked PMG 3*1 plate IX where the ex-  tended chromonemata appear as f a i n t l y staining ladder-like structures* In telophase then, the chromosome cylinder of l a t e anaphase expands as the chromonemata are released from the chromosomal matrix and extend t h e i r c o l l s , f i n a l l y merging into the interphase as i n the lower nucleus of PMG plate VIII*  3&.  The formation of the c e l l - p l a t e also indicates  the approaching  end of telophase ((Hedayetullah 1931)  b r i l l i a n t c r e s y l blue does not s t a i n the plate*  hut  I t i s not  seen i n these preparations* The term interphase was f i r s t used by Lundegardh l n 1912  to denote the stage between telophase and the be-  ginning of the following prophase, i n r a p i d l y dividing tissue (Hedayetullah 1931)*  For the purposes of this paper the  term w i l l be used i n that sense*  ''Resting stage" w i l l be  used where necessary to denote the same stage i n less a c t i v e l y dividing c e l l s e s p e c i a l l y those i n older portions of the root-tip.  The interphase i s a quiescent period following  the telophase, i f a c e l l may be said ever to be quiescent. Such an interphase c e l l i s seen i n PMG The two lower n u c l e i are at interphase.  One distinguishing  feature of the interphase i s the spherical nucleolus (Hedayetullah 1931)*  p l a t e XV*  Another c h a r a c t e r i s t i c i s the  18  repeatedly mentioned "reticulum" or nuclear net, traces  of  which may be seen i n silhouette over the nucleolus i n these two c e l l s *  A. portion of a s i m i l a r nucleus i s seen i n the  upper part of PMG found i n PMG X1O00.  45 plate XII.  Other interphase c e l l s are  40 plate X under lower magnification of about  In such interphase nuclei"there i s reason to believe  that the Identity of the chromosomes has not been lost but i s only concealed  i n the reticulum." (Kaufman7?1926).  authors, including Kaufman?, b e l i e v e the intertwined  Some threads  of the chromosomes can be traced i n the interphase (Sharp 1929). (Abraham 1939).  In our material i n PMG  45 plate XII  the chromatic dots mentioned by Abraham are evident.  These  do sometimes follow a s p i r a l course and are connected by f a i n t l y s t a i n i n g threads which could be chromonematic threads on which the chromatic dots appear as minute beads.  Much  the same condition i s found i n the Interphase n u c l e i of PMG 40 plate X. The Navashin-haematoxylin interphase may be seen i n PMG  2 plate I.  There are found here chromatic p a r t i c l e s  also but often larger than i n those nuclei just studied. There are connecting  threads sometimes c o i l e d and a reticulum  the threads being somewhat coarser. In these c e l l s the nuclear membrane i s c l e a r l y defined, also the c e l l walls are marked. heavily stained.  The n u c l e o l i are  19  Interphase In the interphase then, the chromatic m a t e r i a l i n f i x e d c e l l s commonly appears  as d a r k l y s t a i n i n g  some minute, others l a r g e r , connected threads thus forming the r e t i c u l u m .  particles,  by f a i n t l y  staining  The n u c l e o l i ,  reformed  i n the telophase,  are present here.  In t h i s " q u i e s c e n t "  p e r i o d , i s reached  the maximum e x t e n s i o n o f the chromon-  emata (Koshy 1933). Prophase A f t e r a p e r i o d o f " r e s t " the nucleus a g a i n p r e pares f o r d i v i s i o n .  I n t h i s m a t e r i a l the e a r l i e s t prophase  i s marked by an e n l a r g i n g nucleus s i m i l a r to the uppermost nueleus o f PMG 36 p l a t e IX o r the uppermost nucleus o f PMG 14 p l a t e IV.  The r e - a p p e a r i n g chromatic  threads are f i r s t n o t i c e d  i n s i l h o u e t t e around the p e r i p h e r y o f the n u c l e o l u s .  Two Such  looped chromonemata may thus be d i s t i n g u i s h e d i n the upper nucl e u s o f PMG 21 p l a t e V I .  Others are seen  In PMG 4 o f p l a t e I the chromatic  i n PMG 3  of plate I.  threads are seen  as a maze o f tangled, smooth, s l e n d e r r i b b o n .  Often there are  found darker s t a i n i n g nodules a l o n g a l i g h t e r s t a i n e d t h r e a d . These may be small masses o f chromatin, they  may be  the chromomeres, o r  minor l o o p s i n the chromonemata.  A c a r e f u l search  20  w i l l show that the seemingly single thread i s double, r e vealed by clear loops along the thread.  But generally the  swelling of the thread by the acetic a c i d i n the f i x a t i v e serves to conceal the d u a l i t y .  I t would seem on close exam-  ination that this spireme thread i s discontinuous. II this i s undoubtedly true.  In plate  Here the thread i s c l e a r l y  double; c o l l i n g i s also much more evident often attested to by the undulations along the margins.  The increasing d i a -  meter and darker staining Indicate that considerable materi a l i s being deposited on the thread as a matrix. The e a r l i e s t chromonemal threads of the Navashinhaematoxylin  material are much looped, more so than the  Parmer's b r i l l i a n t c r e s y l blue preparation.PMG 10 of plate III. Plate IV shows further advances.  The  colled  chromonemata are d e f i n i t e l y forming into recognizable chromosomes. In 0  PMG  IS the nucleolus i s now In the centre  of a sphere, the darkly staining chromosome thread draped over It and i n close contact with the now contracting nuclear membrane.  In PMG  16 the parted ends of the two  threads prove that the thread i s s t i l l  double.  Prophase Is concluded as the chromosomes assemble on the equatorial p l a t e . Here they are found i n plate V PMG  17 and 18, at their greatest contraction, and swollen  21  with accumulations of chromatic material.  The commonly  achromatic matrix stains heavily at this time and also a t metaphase.  The swollen appearance of the chromosomes i n  PMG 18 may be due i n part to the action of the acetic a c i d i n the f i x a t i v e (Baker 1945). In prophase then the chromosomes reappear, at f i r s t as long, smooth threads which presently condense and assume t h e i r usually c o i l e d structure.  When f u l l y formed they  move to the equatorial plate as the ensuing metaphase approaches.  The n u c l e o l i  derlyn 1948).  and nuclear membrane disappear (Van-  The prophase i s the most common phase on any  s l i d e so I t i s presumed to be of rather long duration. Metaphase Metaphase begins as the chromosomes assemble on the equatorial plate—PMG 17 and 18 plate V.  The klneto-  chore^ i s observed to be attracted to the equatorial plate and held there, (Vanderlyn 1948) (Koshy 1933) to l i e l n the equatorial plane  but the arms of the chromosomes point out  at various angles--PMG 20 plate V.  That they are double  composed of two chromatids i s unmistakable—PMG 22 plate VI. This Is a c-ialtosls metaphase.  S i m i l a r l y each chromatid i s  composed of two apparently intert?/lning chromonemata.  Thus  the chromatids i n PMG 22 sometimes have a moniliform appearance due to this apparent intertwining.  The chromosomes  22  of l a t e metaphase PMG 21 p l a t e VI a r e remarkable i n t h a t there i s but l i t t l e matrix.  Rather the chromosomes a r e un-  u s u a l l y s l e n d e r even resembling  t h e long t w i s t i n g strands o f  the prophase* Metaphase terminates chromatids.  with separation of the s i s t e r  S e p a r a t i o n may b e g i n a t t h e k i n e t o c h o r e as i n  chromosome (a) o f PMG 19 but n o t always s o . In c - m i t o s i s of PMG 22 the ends may s e p a r a t e f i r s t *  I f the chromosomes  are s t i c k y , s e p a r a t i o n may b r i n g about the f o r m a t i o n o f chromosome bridges o f PMG 39 p l a t e X* Metaphase thus sees the assembling  o f the now  double chromosomes at the e q u a t o r i a l p l a t e , f o l l o w e d b y the s e p a r a t i o n o f the s i s t e r chromatids as they move along the s p i n d l e toward the p o l e s * Chromonemata A chromonema, p l u r a l chromonemata, i s the chromosome t h r e a d ( D a r l i n g t o n 1937).  According  to Nebel (1939),  the chromonema i s an o p t i c a l l y s i n g l e t h r e a d w i t h i n t h e chromosome.- The metaphase chromosome w i t h i t s two chromat i d s , a r e q u a d r i p a r t i t e w i t h r e s p e c t t o chromonemata (Kaufmamil948)*  The anaphase chromosome then would have o n l y two  chromonemata.  T h i s seems to be the most common i n t e r p r e t a t i o n  of the somatic  chromosome s t r u c t u r e , although Nebel (1936)  s t a t e s p l a i n l y t h a t the chromosomes o f both T r a d e s c a n t l a  23  and T r i l l i u m are quadripartite at a l l stages of mitosis, excepting metaphase, which i s eight p a r t i t e s . A search f o r chromonemata i s generally s u c c e s s f u l l y rewarded i n the meiotic chromosomes of T r i l l i u m erectum (Huskins and Smith 1935).  As f o r r o o t - t i p study observat-  i o n a l d i f f i c u l t i e s are great (Taylor 1925), since chromosomes do not reveal c o i l s l n mitosis (Nebel 1939), However, one purpose of this study was to reveal the mitotic chromonemata.  Even so, t h e i r presence Is often only  indicated by an undulating or monillform contour (KaufmanTi 1936)* to which attention has already been directed.  The  monillform contour, seen i n the anaphase chromosome, i n dicates two intertwined s p i r a l threads (Kaufmanml936)• Thus two chromonemata are indicated i n PMG 27 plate VII, anaphase chromosome ( a ) , and again i n chromatid (a) of PMG 54 plate XIV. In telophase the chromonemata are seen i n PMG 37 of plate X« where some of the chromosomes are massed i n 'tassement polaire* which denotes the beginning of telophase. At the upper part of this f i g u r e i s seen the s p i r a l c o i l s of presumably two intertwined chromonemata. In PMG 68 plate XVII the chromosomes appear to be r o l l i n g up into a cylinder; this denotes, i n this paper, the  beginning o f telophase s i n c e here the chromosomes are l o s i n g t h e i r m a t r i x and r e v e a l i n g the chromonemata p a r t i c u l a r l y a t (a).  The prophase PMG 3 and 4 o f P l a t e I show a maze o f  chroiaonemal  threads.  That  they are double  can a l s o be checked  but the thread more o f t e n appears s i n g l e , which would be due to the s w e l l i n g a c t i o n o f the a c e t i c a c i d i n the Farmer's f i x a t i v e s o l u t i o n used i n t h i s  study.  I n the metaphase PMG 19 o f p l a t e V the chromonemal c o i l s are e v i d e n t at (b) a p p a r e n t l y w i t h a d e f i c i e n t  supply  of m a t r i x . - A l s o i n the c o l c h i c i n e metaphase of PMG 53 p l a t e x T f i n t e r t w i n i n g chromonemata are noted a t ( a ) . I n c o n s i d e r i n g what may be found  i n the i n t e r p h a s e ,  the c o n t r o v e r s y o f f a c t v e r s u s a r t i f a c t i s encountered, f a c t b e i n g an appearance t h a t a r i s e s from treatment ton 1937).  Authors  arti-  (Darling-  do not agree as to how much o f the ap-  pearance o f the f i x e d i n t e r p h a s e nucleus i s a r t i f a c t .  This  w r i t e r , from the present study, r a t h e r f a v o r s the a r t i f a c t side of the q u e s t i o n , that i s , the n u c l e a r r e t i c u l u m so o f t e n mentioned as b e i n g found i n the interphase nucleus may be l a r g e l y a r t i f a c t due to the a c t i o n o f the f i x a t i v e .  A  genuine a r t i f a c t may be noted i n PMG 59 p l a t e XV a t ( a ) . This i s a p o r t i o n of a f i x e d interphase nucleus.  However  at (b) i n the same i l l u s t r a t i o n , there can be d e s c r i e d  very  f i n e i n t e r l a c i n g threads i n an i n t e r p h a s e n u c l e u s , which have been i n t e r p r e t e d as chromonemata (Koshy 1933) (Kaufmann 1936).  25  (Menslnkai 1939). XII•  They appear again at (a) i n PMG 45 plate  Chromonemata then, are revealed i n a l l mitotic phases  by the b r i l l i a n t cresyl blue technique. Coiling Cleveland (1949) c l a s s i f i e s thus the different types of c o i l i n g :  major, minor, super and r e l a t i o n a l .  major c o i l i s the simple h e l i x .  The  Minor c o i l s are minute  loops imposed on the turns of the major c o i l .  Super c o i l i n g  involves the c o i l i n g of the whole h e l i x into a giant compound helix.  Relational c o i l i n g involves whole s i s t e r chromatids,  each being Intertwined with the other, or s i m i l a r l y one chromonema may be twisted about another. An example of such r e l a t i o n a l c o i l i n g between two chromonemata i s found at (a) PMG 53 plate XIV. A close inspection of these chromonemata may reveal at least major c o l l s and even super c o i l s .  Super  c o i l s are found at (a) PMG 64 plate XVI. In this material i t i s scarcely possible to d i s tinguish minor c o i l s .  With Parmer's f i x a t i v e , such c o i l s  would have the appearance of black dot3.  Such dots could  also be merely chroraomeres, small accumulations of the chromatic substance. Such are seen at (a) PMG 48 plate XII along a chromonema where one might expect to f i n d minor c o i l s . They appear again i n chromosome (a) PMG 62 plate XVI and at (b) i n the same PMG.  26  Major c o i l s are found at (a) PMG  37 plate X,  where probably two chromonemata are Involved, also In  PMG  61 plate XVT at (a) where probably only one chromonema i s colled.  Other major c o i l loops are seen at (a*) PMG  19  plate V. In the prophases i l l u s t r a t e d on plates I I , I I I and IV one may f i n d a l l the types of c o i l i n g : at  relational  (a) PMG 9 plate I I I , the darker 'chromomere' dots which  may be minor c o i l s at (b)Jln the same PMG and major c o i l s at ( c ) . A common anaphase Indication of c o i l i n g Is found In chromosome (a) PMG 27 plate VII where the chromonemata appear as i f braided, one about the other. the actual s i t u a t i o n however. interpret the o p t i c a l section.  Such may not be  I t i s generally d i f f i c u l t to In t h i s case we have probably  two chromonemata with the major c o i l s of the h e l i x one h e l i x a l i t t l e below the other and viewed from the side (Abraham 1939). In order to get a better idea of the c o i l e d chromenemata i n the somatic chromosome, a study of wire models Is h e l p f u l . and Koshy (1933).  Such models were used by Mensinkai (1939) Two p a r a l l e l wires are c o i l e d into a h e l i x  on a wooden core and after the core has been removed the h e l i x may be allowed to lengthen by spreading the c o i l s as  27  i n Figure 22 of Menslnkai (1939). useful i n attempting XII•  Such a model could he  to i n t e r p r e t the 'vacuoles' of plate  These 'vacuoles' are apparently spaces between the  c o i l s of the double h e l i x .  The explanation of the two  loops at (a) PMG 39 plate X i s to be found i n Minsinkai's wire diagram 22* One advantage of the c o i l i n g of the chromonemata i s the great reduction In the length of the chromosomes, at metaphase*  By means of major, super and perhaps minor  c o i l s , the chromonemal thread can be made compact (Menslnkai 1939). This reduction In chromosomal length permits of f r e e r , more rapid movements of the chromosome*  The c o l l i n g  i s probably brought about by-molecular changes i n the chromosome (Menslnkai  1939), since mitosis includes a nucleic a c i d  cycle i n addition to the c o i l i n g cycle*  The charge of  nucleic acid upon the chromosomes changes as the mitotic cycle proceeds, charging during prophase and metaphase, but discharging at telophase and Interphase (Darlington 1947), Pairing Two PMG's 43 and 44 on plate XI i l l u s t r a t e p a i r i n g . In 43 p a i r i n g has taken place at metaphase since the chromosomes are at t h e i r greatest contraction, also they are heavily stained and swollen with accumulated nucleo-  28  proteins.  In the p r i n t some four pairs may be seen.  On the  s l i d e the p a i r i n g was more d e f i n i t e l y established by changing the focus. In PMG 44 the chromosomes are slender, that metaphase separation has taken place.  evidence  Pairing i s very  d e f i n i t e i n a t least ten of the p a i r s , the remaining s i x , however, have been disturbed by pressure on the cover glass. There are too many pairs here to be merely f o r t u i t o u s . Re|td (1914) found p a i r i n g on the equatorial p l a t e , *  n  ill  tids.  C  E  P  A  before separation of the metaphase s i s t e r chroma-  The p a i r i n g , moreover, could also be noted i n the ensuing  anaphase. Trabanta As already noted, i n iU Cepa there are two of the sixteen chromosomes that have a trabant or s a t e l l i t e .  This  p a i r of chromosomes have a sub-terminal kinetochore, as recorded by Taylor (1926) and Vanderlyn being attached to the shorter limb.  (1948), the trabants  In this study both  s a t e l l i t e d chromosomes were r a r e l y I f ever found i n any one complement of chromosomes.  Generally only one was found  often not any, one reason being that the trabant was not i n focus• I t w i l l be noted that the chromosome bearing the trabant i n PMG 42 plate XI has a median c o n s t r i c t i o n , which  29  i s unusual. Segmentation Segmentation of chromosomes may he noted on p l a t e XV.  Such segmentation i s produced by pressure exerted on the  cover glass i n squeezing out excess s t a i n . ed at w i l l *  I t may be produc-  The segmentations are always at r i g h t angles or  approximately so, to the long axis of the chromosome.  They  are regularly spaced but not equally so i n the d i f f e r e n t chromosomes.  There may be some s i g n i f i c a n c e In this regu-  l a r i t y of spacing and i n the right angle segmenting.  Some-  t!mes--PMG 60--it would appear that each segment has one of the major c o i l s .  However, c o i l i n g i s not generally so  regular along the chromosome,'but rather i t varies (Huskins and Smith 1935).  I t i s very remarkable that segmentations  so small should be so regular.  I t may have some bearing on  chromosome structure, p a r t i c u l a r l y so i f i t could  be  shown the segmentations do at times follow the c o i l i n g . I t i s also remarkable that i n no case i s a segment out of line. The P e l l i c l e The. existence of a membrane which delimits the condensed chromosome has been inferred from various aspects of chromosomal behavior (KaufmanTp.936)•  The same author  describes the membrane or p e l l i c l e as being Peulgen negative.  I t may be that the p e l l i c l e , here l i g h t l y stained, i s seen i n PMG  57 of plate XV at (a) between two segments.  this i s the p e l l i c l e seems the most probably In our PMG  1  That  explanation.  57 the p e l l i c l e i s evidently very t h i n .  Ploidy Occasionally as i n PMG  26 plate VII there ape more  than sixteen chromosomes found i n the chromosome complement* In this case twenty-four were counted so that there were probably as many as thirty-two i n the genome i n this case* This could happen i f the c e l l plate f a i l e d to mature or form after chromosome separation had taken place, i n the previous anaphase*  On two occasions  a group of eight were  found, but t h e i r photograph does not show the number c l e a r l y A search i n nearby areas f a i l e d to locate the other  missing  eight e o l l a . Colchicine mitosis As already mentioned colchicine was f a c i l i t a t e chromosome counting. on this material was  used to  The effects of c o l c h i c i n e  quite s i m i l a r to the r e s u l t s obtained  by Levan (1938), with one possible exception.  To quote  Levan (1938): "They are however not arranged into an equatorial p l a t e . " assembly.  Our PMG  54 plate XIV shows such an  The multipolar spindle of Levan, as recovery  gins to take place, i s i l l u s t r a t e d i n our PMG  be-  67 plate XVII*  31  In our e e l l four d i f f e r e n t groups of chromosomes would r e sult* Kinetochore In these preparations the kinetochore i s usually c l e a r l y defined.  Quite p o s s i b l y this i s partly due to the  pressure on the c o v e r s l i p increasing the gap between the two chromosome segments, into which the c o n s t r i c t i o n divides the chromosome*  In some instances as i n the chromosome on  the l e f t i n PMG 49 plate XIII the connecting chromonemal thread has snapped and i s no longer seen.  In many other  instances, the connecting thread remains^, Plate XIII. That i s In each chromosome, the chromonemal thread i s continuous, though probably somewhat modified at the kinetochore (Kauf mamil926).  I t w i l l be noted that thread i s  less densely stained at the kinetochore.  These preparations  do not show the details of kinetochore structure as revealed by Matsura (1940) i n h i s study on T r i l l i u m kamtschaticum Pall.  Discussion  Tassementpolaire I t w i l l be noted that i n the description of t e l o phase, the massing of the chromosomes which Gregoire c a l l s 'tassement p o l a i r e ' was not included*  However such a phase  32  is i l l u s t r a t e d i n PMG 38 plate X and possibly also i n PMG 37.  The f i r s t mentioned i l l u s t r a t i o n i s considered to have  rather more a r t i f a c t than usual.  One notes the close massing  of the chromosomes and the comparatively smooth contour of these two telophase masses.  Moreover the negative of t h i s  print shows two or three t r a i l i n g l i n e s of some apparently s t i c k y substance across the gap between the two masses.  These  ribbons are not actual chromosomes, they do not show chromosome structure.  For these reasons i t i s concluded that  these masses are p a r t l y the r e s u l t of f i x a t i o n a r t i f a c t . Probably something  of the same s t i c k y nature i s seen i n  PMG ^SfcV plate V as inosculations between some of the ehromosomes p a r t i c u l a r l y at Moreover a r t i f a c t has already been seen i n PMG 59 of plate XV.  On other occasions, i n this material  chromosomes have been seen at metaphase i n twisted s t i c k y masses which were evidently a r t i f a c t but are not available f o r photography.  now  Formation of such a r t i f a c t  masses when found i n a recent study under the d i r e c t i o n of George Weber, M. D. had to be prevented by suitable changes i n the pre-treatment.  Coincidental with these changes the  massing disappeared. A l s o , i n the sequence of telophase movements there seems no natural place f o r these masses.  The chromo-  somes move to the poles forming there the umbrella-like  33  structure of PMG 30, plate VIII.  Then as already recorded,  follows the formation of an apparent cylinder which however might possibly be rather more s p h e r i c a l , as the extended ends contract and appear to c u r l i n . I t should be admitted that the upper umbrella of PMG  30, plate V I I I , does i t s e l f show some tendency to mass-  ing but not to the extent shown i n Gregoire's (1903) photograph I. Moreover, chromosomes i n the mitotic cycle are subject to quite considerable movement forces.  In moving  pictures of the l i v i n g c e l l , one i s convinced of the great a c t i v i t y i n the c e l l of these forces.  Such massing as  shown i n Gregoire's (1904) photograph, would preclude movement at least temporarily, whereas i n this writer's description  of late anaphase and ensuing telophase there i s no such  hindrance to chromosome movement.  To sum up the evidence:  s i m i l a r masses of chromosomes have been found at metaphase, but when the pre-treatment of the toot-tlps was massing disappeared.  altered, the  In the e a r l y work of this study, a  i . m . ' i •» of similar a r t i f a c t was u  sometimes encountered but  which did not occur as experience improved the technique used.  The massing seems unnatural i n that i t precludes  movement whereas movement i s the outstanding c h a r a c t e r i s t i c of c e l l u l a r a c t i v i t i e s . massing was  Kaufmann(1926) concluded that the  either a r t i f a c t or o p t i c a l effect due to the  54  mass  of  (1904)  material does  mentions alcohol ion  not  that plus  which  fact.  the  now  on  the  w o u l d be  very  In  useful  a  phbtographs  are  of  in  the a  in  series  sections.  Care  photographs.  a  showing series  stained  XIII  and  others  interpret  mLM&pKoVffgraphy.  seen the  in  on p l a t e coils  should  be  of  XII,  interpreted  of  the  coils  as  as  In  a  is  a  artipositive  this  lowest  the  the  Figure  study  same  There  produced little  taken  detail  the is  than as  Matsura of  The  PMG diffi-  effects  "vacuoles" the  presence  (Koshy 1933), are  in  only  6 of  similar  of  that  negative  plate.  numerous  structure  is  chromosome  Indications  such.  part  may a p p e a r  the  spiral  there  and w i t h  more  There are  in  Often  and o t h e r  are  in  s h o u l d be  somewhat  dots  of  combinat-  photographs  such dots  which  a double  in  a  evidence  produce  respect  heavily  the  conditions  such  95$  was  Sections  magnification  Such  time  investigation.  high  50  of  used  further  (1899)  taking  at  seen  to  before  technique  Gregoire  seem  is  taken  may b e  is  would  type,  They  culty  It  Gregoire  acid,  polaire'  Optical  (1940). plate  that  this  Sometimes  of  such  at  but  of  of  this  print.  of  optical  interpretation helpful  in  used  study  focus.  effect  rarely.  The  stain.  hydrochloric  indicated  question.  the  fixative  'tassement is  of  fixative  of  but  the  Interpretation  depth  the  drops  used  that  density  preferred  Further study  stand  in  the  record  a few  is  probability  and  the  coils  of  and seen  in  35  PMG 19 plate V. Nebel (1939) Interprets the two rows of c o i l s here at (Jt)~as images of four threads appressed Into two p a i r s . Prom time to time one sees an end view of a chromosome as at (b) PMG 61 plat;e 16.  Kaufmannt 1926) interprets  this hollow r i n g with a narrow lumen, as the space between the two ends of the chromonemata which are here p e r i p h e r a l l y disposed.  Very good Interpretations of o p t i c a l sections  commonly met with are given also by Nebel (1933).  These  are worthy of study. Duplication of Chromonemata During this study, constant watchfulness was maintained to f i n d i f possible the exact time of the d u p l i cation of the two chromonemata i n each prophase chromosome. In early prophase two are seen.  In metaphase PMG 19 plate V  there are four chromonemata as we have just noted.  The  purpose was to f i n d just when the duplication occurred. Most attention was given to the group seen i n PMG 16 plate IV.  But although several pictures were taken at d i f f e r e n t  f o c i , i t appeared that there were as yet but two chromonemata present i n each chromosome.  At the upper r i g h t of this  i l l u s t r a t i o n are the forked ends of the two chromonemata only.  Thus the f i r s t evidence of reduplication appears  at metaphase i n PMG 19 plate V.  36  Number of Chromonemata I t was  hoped that at telophase evidence might be  found as to the actual number of chromonemal threads i n the somatic chromosome since at this time the matrix i n some manner loses i t chromaticlty  (Sharp 1929)  or i t dissolves  and becomes merged with the karyolymph (Koshy 1933), leaving the chromonemata much more e a s i l y v i s i b l e . (a) In PMG  61 plateXVT yields some evidence.  nemata (two  Chromosome The chromo-  ?) are seen l y i n g c o i l e d side by side.  chromonemata on the l e f t may  be double.  The  That on the r i g h t  appears single at f i r s t but a l i t t l e further along there i s possible evidence of d u a l i t y i n i t also--a rather heavy loop. I t would appear that there i s some evidence of d u a l i t y i n each of those two chromonemata.  This p a i r of chromonemata  are worthy of close scrutiny and a study should be made of the others also.  I t w i l l be remarked that i n this p a i r of  n u c l e i , the one i s further advanced than the other. The Stain B r i l l i a n t Cresyl Blue Stewart and Schertiger (1949) write that t h i s s t a i n shows a high degree of s p e c i f i c i t y f o r nuclear structures.  This study supports the view also.  I t has  been shown t h a ^ o n i y the nucleus i s stained nrrHnnrlily. A l l other struetures--nucleoli, c e l l w a l l s , cytoplasm, karyolymph—do not s t a i n o r d i n a r i l y .  Some further evidence  37  has been gained elsewhere erial,  as to i t s s p e c i f i a i t y f o r n u c l e a r mat-  but time does not permit i t s p r e s e n t a t i o n i n t h i s  i n t h i s study, b r i l l i a n t  paper,  c r e s y l blue has r e v e a l e d , without  the  use o f s p e c i a l pre-treatment, most o f the chromosomal s t r u c t u r e s d i s c u s s e d by eminent a u t h o r s .  I t i s probably u n e x c e l l e d at p r e -  sent as a m o r p h o l o g i c a l s t a i n f o r the r o o t - t i p s of A. cepa. u s i n g the pre-treatment  of Mr. Unrau, i t s value may  By  be enhanced  in cytological studies. Suggested It of  Further Investigations  i s the o p i n i o n o f the author that i n t e n s i v e  study  the telophase chromosome might r e v e a l i n t e r e s t i n g proof of  o p i n i o n s a l r e a d y c u r r e n t i n regard to chromosome s t r u c t u r e .  It  might f u r n i s h data to r e f u t e other a s s e r t i o n s , e s p e c i a l l y i n regard to the number o f chromonemata i n the telophase chromosome.  Such f u r t h e r i n v e s t i g a t i o n , u s i n g b r i l l i a n t  c r e s y l blue,  might a l s o decide the i s s u e h e r e i n r a i s e d once more i n regard to  the'"tassement  polaire'- of G r e g o i r e .  38  Summary By means o f the s t a i n b r i l l i a n t i n A l l i u m cepa  has been f o l l o w e d  The f o l l o w i n g c o n c l u s i o n s (1) B r i l l i a n t  c r e s y l blue,  mitosis  throughout one complete c y c l e .  are drawn:  c r e s y l blue  i s an e x c e l l e n t s t a i n f o r the  r o o t - t i p s o f A l l i u m cepa, a l l the common morphol o g i c a l features — c h r o m a t i d s , kinetochore, being c l e a r l y  trabant-  delineated.  (2) There are i n d i c a t i o n s that the s t a i n w i l l be usef u l i n the i n v e s t i g a t i o n o f c y t o l o g i c a l problems. (3) I t i s suggested that the ''tassement p o l a i r e ' o f telophase  may be i n p a r t , the product o f a r t i f a c t .  (4) There i s s t i l l  some u n c e r t a i n t y as t o the number  of chromonemata i n the telophase  o f A l l i u m cepa.  (5) In t h i s study, the f i r s t evidences o f d u p l i c a t i o n of the chromonemata are seen at metaphase. Abbreviations:for'photomicrograph*  PMG i s used.  for 'colchicine mitosis'  c-mitosis.  39  BIBLIOGRAPHY Abraham, A., Chromosome Structure and the Mechanics of Mitosis and Meiosis, Ann. Bot. n.s. 3: 545-568, 1939. Baker, J . R., Cytological Technique, Methuen and Co., 1951. Blakeslee, A. P., and Avery, A. G., Methods of Doubling of Chromosomes i n Plants, Jour., of Hered. 28:393-411j 1937. Caspersson, T., Jour. Roy. Micro. S o c , Determination of the Absorption Spectra of C e l l Structures, 60:80-25. Cleveland, L. R., The Whole L i f e Cycle of the Chromosomes and Their C o i l i n g System, Am. Phllos. Soc. Trans^ 1949. 39: 1-100, Conn, H. J . , B i o l . Stains, 5th ed., New York Agric. S t a . Geneva, N. Y., 1946. Darlington, C. D. , The Old Terminology and the New Analysis of Chromosome Behavior, Ann. Bot. 49:679-585, 1935. , Misdivision and Genetics Jour. Genet. 37:341-364, 1939.  of the Centromere,  , Recent Advances i n Cytology, 2nd ed., Blakiaton, 1937. , Nucleic Acid and the Chromosomes, Symposia of the Soc. f o r E x p t l . B i o l . I , Nucleic Acid, 252-269, 1949. and La Cour, L. P., The Handling of Chromosomes, G:. Allen and Unwin, London, 1947. Digby, L., The Somatic, Premeiotic and Melotlc Nuclear D i v i sions i n Gaitonia canadican. Ann. Bot. 24:127-757, 1910. Praser, H.C.I., and S n e l l , P., The Vegetative D i v i s i o n i n V i o l a faba, Ann. Bot. 25:845-55, 1911. Preisner, Ray C., Daily Rythms of Elongation and C e l l D i v i s i o n i n Certain Roots, Amer. Jour. Bot. 7:384-407, 1920. Gates, R.R., Pollen Formation i n Oenothera sigas. Ann. Bot. 25:909-940, 1911. , Somatic Mitosis i n Oenothera» Ann. Bot. 26:993-1010, 1912.,  40  , The Structure of the Chromosome, Jour. Roy. Micro. Soc. 58:97-111, 1938. Gregoire, V., Les Cineses P o l l l n i q u e Chez l e s L i l l a o e e s , La C e l l u l e 16:235-296, 1899. , Reconatitution du Moyau et Formation des Chromosomes dans l e s Cineses Somatiques, I , Racine T r i l l i u m Homoeotypique dans l e T r i l l i u m cernura, La C e l l u l e 21:7-74, 1904.  d o  , La Structure de l a Element Chroraosomique au Repos at en D i v i s i o n dans les C e l l u l e s Vegetales (Racines d»Allium), La C e l l u l e 23:311-53, 1906. Gulick, A., Chemistry of the Chromosomes, Bot. Rev. 1:433-457, 1941. Guignard, L., Heeherches sur l a Structure et l a D i v i s i o n du Moyau G e l l u l a i r e chez Vegetaux, Ann. des S c i . Nat. Bot., Ser. VI, 17:5-59, 1884. Hedayetullah, S. , Structure and D i v i s i o n of Somatic Chromosomes i n Narcissus, Jour. Roy. Micro. Soc. 51:349-377, 1931. Huskins, C. L., and Smith, S. G., Meiotlo Chromosome Structure i n T r i l l i u m erectum L., Ann. Bot.» 1 9 3 5 49:119-150. Eaufmann, B. P., Chromosome Structure and I t s Relation to the Chromosome Cycle, Amer. Jour. Bot. 13:59-80, 1926. , Chromosome Structure, Bot. Review 2:529-553, 1936. , Chromosome Structure i n Relation to the Chromosome Cycle, I I , Bot. Rev. 14:57-127, 1948. , Gray, H., McDonald, M. R., Nucleic Acid i n Fixed C e l l s , Jour, of C e l l and Comp. Phys., Supp. I , 71-94, July 1951. K e l l i c o t t , W. E., The Daily P e r i o d i c i t y of C e l l D i v i s i o n and of Elongation i n the Root of Allium, Torrey Bot. Club B u l l . 13, 529-550, 1904. Koshy, T. K., Chromosome Studies i n Allium I , Roy. Micro. Soc. 53:305-318, 1933. " , Chromosome Studies i n Allium I I , Jour. Roy. Micro. Soc. 54:104-20, 1934.  4&  , Number and Behavior of Chromosomes i n Aloe l i t o r a l i s , Ann* Bot. n.s* 1:43-53, 1937* La Cour, L., Acetic-ereein: A New S t a i n - f i x a t i v e f o r ehromosomes, Stain Teehn. 16:169-174, 1941* Levan, A., Cytological Studies i n Allium I I , Hereditas 16:257-84, 1932. . Heterochromaty i n Chromosomes During the Cont r a c t i o n Stage, Hereditas 32:449-468, 1946* , Effect of Colchicine on Hoot-mitosis i n Allium, Hereditas 24:471-486, 1938. Low, Jeanette, Root-tip Smears f o r Maize,Stain Techn. 21:127-28, 1926* Martens, P i e r r e , Nouvelles Eecherches Experimentales sur l a Cinese dans l a C e l l u l e Vivant, La C e l l u l e 39:169-215, 1930* Matsura, Hajime, Chromosome Studies on T r i l l i u m Kamtsohatioum P a l l . X I I I , The Structure and Behavior of the Kinetochore, Cytelogia XT:367-379, 1940. Mensinkai, S. W., The Conception of the S a t e l l i t e and the Nucleolus and the Behavior of These Bodies l n C e l l D i v i s i o n , Ann. Bot. n.s. 3:763-94, 1939. Naithani, S. P., Chromosome Studies i n Hyacinthus O r i e n t a l l s , Ann. Bot. 1:257-76, 1937. Nebel, B. R., Chromosome Structure i n Tradesoantiae IV, Cytelogia 5:1-14, 1933. , and Ruttie, M. L., Chromosome Structure, IX, Amer. Jour. Bot. 23:652-663, 1936. , Chromosome Structure, Bot. Rev. 5:563-626, 1939. Reed, T., The Nature of the Double Spireme i n Allium cepa, Ann. Bot. 28:271-81, 1914. Robyns, Walter, La Figure Achromatique sur Material P r a i a , dans l e s D i v i s i o n Somatlques des Phanerogams, La C e l l u l e 39:85-117, 1929. Schaffner, J . H., Karyoklnesis i n the Root-tips of Allium cepa. Bot. Gaz. 26:225-38, 1898.  Sharp, L. W., Structure of the Large Somatic Bot. Gaz. 88:349-377, 1929.  Chromosomes,  . Intro, to Cytology, McGraw H i l l , 1934. Stewart, Wilson N, and Schertiger, Ann M., B r i l l i a n t Cresyl Blue as a Stain f o r Plant Chromosomes, Stain Techn. 24:39-45, 1949. Vanderlyn, L., Somatic Mitosis In the Hoot-tip of Allium cepa, Bot. Rev. 14:270-318, 1949.  * #*  4-4.  4.5  PLATS 111  r (a)  (CM  4-1  PLATE V.  20  57  PLATE IX.  53  PLATE XI.  55  PLATE X I I I .  5 0  PLATE XIV.  57  PLATE XV.  PLATE XVI  50  PLATE XVIII  6/  1  u  INDEX TO PLATES PMG  PLATE I  1  Interphase nucleus. X2350.  Cresyl blue.  2  Interphase c e l l . X2400.  3  Early prophase nucleus. X 2400. mata and nucleolus*  4  Somewhat l a t e r than #3. Double. X2400.  Haematoxylin.  Sectioned*  Cresyl blue chromone-  Chromonemata mostly smooth*  PLATE I I 5  Prophase nucleus l a t e r . discontinuous. X2370.  Considerable c o l l i n g * Spireme  6  Mid-prophase nucleus l a t e r than #5*  7  Mid-prophase.  8  As above. X2340*  C o i l i n g various.  X2400*  X2400.  PLATE I I I 9 10  Mid-prophase nucleus. E a r l y prophase c e l l .  X2400. Haematoxylin.  C o i l i n g chromonemal  thread. X J2'Z>0  11  Mid-prophase nucleus.  X2400.  12  Mid-prophase nucleus.  X2340. PLATE IV  13  Later prophase--chromosomes becoming charged with nucleic acids. Chromosomes peripheral, nucleolus draped with chromosomes. X2400.  14  E a r l i e r than the l a s t n u c l e u s j c o i l l n g evident.  15  Late prophase—chromosomes with two chromonemata. Super c o i l s . X2230.  16  Same as l a s t . Different focus. some. X2230*  X2300.  Biforked end of chromo-  (o3 PLATE V 17  E a r l y metaphase--ehromosom.es on equatorial plane.  X2350.  18. Later metaphase. Chromosomes h e a v i l y charged with nucleic acids. X2430. Inosculations at ^Sj) Polar view. 19  Metaphase, l a t e r . Short chromosome dividing at kinetochore. Chromonemal c o i l s atCv, quadripartite. Lateral view. X2400.  20  Metaphase l a t e r a l view.  X2350.  PLATE VI 21  Metaphase and early prophase n u c l e i . Chromosomes l i g h t l y charged. Chromonemata i n various c o i l i n g . Leucobasic fuchsin s t a i n followed by c r e s y l blue. Slide by A. Unrau. X2400.  22  Colchicine mitosis—chromosomes i n delayed metaphase on equatorial plane: Quadripartite. X2350.  is  Metaphase~chromosomes undivided.  24  E a r l y anaphase—chromosomes b i p a r t i t e . y^ZdOO.  X2350.  PLATE VII 25  Anaphase—chromosomes heavily staining of chromatic and achromatic elements. Chromosome bridge due to s t i c k y heterochromatic substances. Trabant at lower r i g h t . X2500.  • , '•  26  E a r l i e r anaphase t e t r a p l o i d .  27  Later anaphase—chromosomes slender.  28  (a).  X2350.  X2450.  Later anaphase—polar view.  X2450.  Chromonemata at  Chromosomes i n polar areas.  PLATE VIII 29  Late anaphase.  Chromosomes beginning to r o l l up.  X2400.  go  Late anaphase polar view. bowl. N u c l e o l i . X2400.  31  Telophase. Chromosomes l o s i n g the achromatic substance. Chromonemata c o l l s appearing.Nucleoli. X2200.  Chromosomes as an inverted  PLATE VIII 32  Anaphase, l a t e .  (Cont'd.)  Also very late telophase nucleus.  X2200.  PLATE IX 33  Telophase—embedded, haematoxylin s t a i n . Chromosomes l o s i n g t h e i r achromatic substance. X2130.  3k  Early telophase.  X2250.  35* Late telophase.  Chromonemata evident.  X2300.  36 E a r l y telophase and interphase nuclei.  X235O.  PLATE X 37'Tassement p o l a i r e . 1  Chromonemata i n major c o i l s at (a).  X25OO.  3^  As above.  39  Early telophase 'sticky' chromosomes.  X2300.  4-0 Late telophase and interphase n u c l e i .  X1B00.  A r t i f a c t clumping.  X2350.  Plate XI  4-1 Metaphase—sub-terminal 4-2 As bbove—trabant  X2350.  chromosome with trabant.  X235O.  on chromosome with median kinetochore.  4-3  Metaphase p a i r i n g .  X24-00-  44  Pairing after metaphase separation.  X880';-  Plate XII. 4-5-4-3 Colchicine m i t o s i s — d e l a y e d metaphase. emata indicated by • vacuoles'. X235O. PLATE  4-9-52 Chromosomes at metaphase.  Coils of chromon-  XIII  X24-00.  PLATE XIV  53  Colchicine mitosis metaphase. ing of chromonemata.  X25OO.  Relational c o i l -  PLATE XIV  (Cont'd.)  54  Colchicine mitosis. ripartite. X2400.  Metaphase p l a t e .  Chromosomes quad  55  S c a t t e r e d metaphase l i g h t l y segmented.  56  Late c o l c h i c i n e m i t o s i s . ing. X2350.  X2200.  Metaphase d i v i s i o n proceed-  PLATE XV 57  Anaphase segmentation. X2500.  Chromosome membrane a t ( a ) .  58  E a r l y anaphase segmentation.  59  Anaphase segmentation.  X1300.  60  Anaphase segmentation.  X2500.  X2700.  PLATE XVI 61  Late anaphase p o l a r view and e a r l y i n t e r p h a s e . emata a t ( a ) . X 2300.  Chromon  62  Anaphase—chromonemata i n t e r t w i n i n g .  63  Metaphase—horseshoe chromosome at ( a ) . X2300.  64  Late prophase--chromosomes charged w i t h n u c l e i c a c i d . X2300.  X2200.  PLATE XVII ,66  Genome o f 16 chromosomes.  X1130.  67  Colchicine mitosis. X2200.  Recovery.  68  E a r l y telophase—chromonemata ( a ) . X2400.  69  Mid-prophase.  X2300.  Multipolar  On P l a t e X V I I I .  spindle.  

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