CHI~,OMOSOME STUDIES IN THE SCILLEAE. II.

BY S. O. S. ])ARK.

(John Inqws Horticultural Institution, Merton.)

(With Eleven Text-figures.)

T]~E presen~ study is concerned with mitosis in the root tips and pollen grains and meiosis in pollen mother cells of two species of the Seilleae, Bellevalia romana Sweet (Hyacinthus ~'oma'nus L.) and 5'cilla itcdica Linn., which are especially favourable for conclusions as to the properties of individual cln'omosomes on account of the small numbers and wide range of size.

METXODS.

Root tips were fixed in 2 BE. Pollen mother cells were prepared by the smear technique, Medimn Flemming and Benda being use/[ respec- tively for fixing Bellevalia and ~%illa.

BELLEVALIA ~r¢OIIlA.~VA.

Somatic chromosomes. These were described by ])arlington (1926), and their lengths deter-

mined from observations of side views of somatic anaphases. Fig. 1 shows a complete complement at somatic metaphase in polar view. Lewitsky (1931) has illustrated the complements of other species.

Meiosis. The very early prophase stages were found to fix extremely well,

hut later stages were unsatisfactory nntiI metaphase was reached. As there were only fern' bivalents, it was possible to trace out the entire length of the ehromatin threads in a nucleus at paehytene. Fig. 2 shows this pachytene nucleus with the threads corresponding to the four bi- valents drawn separately, and the outlines of the two nucleolar masses a.re drawn in ~o give t]te orientation. Each bivalent can be seen to consist of two closely paired threads which consist of a series of paired bodies (ehromomcrcs) connected by fine drawn-out portions. In Zea Mays paehytene pairing has been illustrated by McClintock (1931).

By the time metaphase is reached, these threads have shortened and thickened to form bivalcnts held together by two to four chiasmata each, as shown by Fig. 3 and Table I.

86 Chromosome Studi~s in the Ncilleae. I I

Chromoso~ne ~neasurements.

A comparison of the chromosome lengths at various stages was made. In Liliu,m such a comparison has been made very completely by Belling (1%8).

Pachytene. The length of each bivalent in the drawing was measured by means

of a thread. These lengths were then multiplied by all arbitrary :factor in order to compensate for the drawings being the projceeion upon a plane

C

Fig. 1. Net~phase of mitosis in roo~ tip of Bellevalia roma~ta. × 5000.

of chromosomes arranged in three-dimensional space. The factor chosen as 2~rr 2

giving the most probable approximation was - - i.e. 2><,. The values

obtained are given in Table II.

Meta, phase of meiosis. The lengths were measured by thread from the drawings. This stage

is unsatisfactory, owing to the varying relations of ~he attach-merit con- strietion causing w~rying amounts of stretching of the chromatin material

88 Chromosome Studie~ in the Scilleae. I I

composing the bivale]lts (see Fig. 3). Late diakhlesis would probably be a better stage at which to make these measurements. Each half of the b i v a l e n t w a s m e a s u r e d s e p a r a t ) e l y a n d the m e a n t a k e n h i order to

reduce errors introduced by the foreshori;ening of curves or asymmetrical

ch~awing.

A B C D Fig. 3. Bellevalia romana. Side view of metaphase bivalents in t w o nuclei, x 5000.

T A B L E I.

B e l l e v a l i a r o m a n a . Twenty nuclei at metap]tase I. No. of eh iasmata per bivMen~

zk

2 3 ~ Tot~1 I~o. of bivalents 0 29 4=2 9 80 inc. of c h i a s m a ~ 0 58 126 30 220 No. of fe rmium ehiasmata 0 16 33 9 58 TerminM chiasmat~ per b ivalent - - 0"55 0-79 1'00 - -

TABLE II. B e ] l e w H a r o m a n a . Le~gth of bivcde~ts cg 2cw]~ylene.

Leng th on Computed length

94= 66.7

1,1] 149

4=50.7

Chromosome drawing (1) 2 × (1) pair ram. ram. 1 C 155 310 2 D 110 220 3 B 233 4=66 4= A 24=5 490

Total

Average per

bivMent

2"75 0'73

S. O. S. DAmK

TABLE III.

Betlevalia romana. Length of bivale'nts. 1Kitosis

r ~ h'iciosis An~]?hase r ~ -~

M[et~phaso ( ] )~ r l ing ton) Pachy6ono ]Kotaphaso ]3 iva len t s i~ l* ~ / z

A 13'9 21 149 13 J9 10.3 16-5 141 11.6 C 10.3 13.4 94:.0 8.7 D 9.8 12.2 66.7 7-8

T o t a l 44 '3 63"1 ,t50'7 4 t ' l

£' omatic chromosomes.

89

Measurements of the chromosome lengths were taken from drawings of metaphase plates. These are unsatisfactory because of the curvature of' the chromosomes in the cells. Both members of each pair were measured and the greatest length taken.

The lengths were also calculated from the drawings raade from anaphasc by Burlington (1926). These latter values give a more accurate idea of the lengths of the somatic chromosomes.

JS~ C I L L A f f T i l L f C ' A .

Somatic chromosomes.

At metaphasc the chromosomes are very crowded on the plate, owing to theh' large size relative to the nucleus. Each of the eight pairs can be distingt6shed in favourable divisions, while at meiosis the bivalents can be separated into three main classes.

Below is a description of the chromosome types : Long class (four pairs}:

(1) Lt ha~dng a snbterminal attachment constriction and a large trabant.

(2) L~' with a subterminal attachment constriction, the short arm being almost spherical.

(3) L the attachment constriction is subterminal, with the short arm about 1/5th ~he length of the long arm.

(4) L,s' Laving a submedian attachment constriction. Medium class (two pairs):

(5) M with a median attachment constriction. (6) Ms' with a submediau attachment constriction.

Short class (two pub's): (7) S~" with a subtenninal attachment constriction. (8) ~q,s with a snbmedian attachment constriction.

90 Uh~'omosome Studies in the Ncilleae. I I

Many of the individual pairs within classes can be recognised at metaphase I of meiosis, but they cannot be made out regularly enough to permit the application of quantitative methods.

Fig. 6 shows the meta,phase plate in a root tip division, Nitosis was also examined in pollen grain divisions. As only the

haploid number is present the chromosomes are less curled than in root tip divisions.

Fixation is probably less rapid in pollen grains, as the ct~romosomes appear bloated (Fig. 5). However the relative lengths of the members

2

;s

~T

OT L T

Fig. 6. 1%{etaphase of mitosis in root5 tip of ,S'cilla italica. × 5000.

of the complement could be determined and these were checked against the lengths that could be measured in drawings of root tip divisions. The following values were obtained for the average lengths of the members of the chromosome groups:

long 10"7/~; medium 6.5/.~; short 3.8/~.

Meiosis.

Early prophase stages were unsuitable for statistical treatment be- oause Cite chromosomes were so crowded in the nucleus that only occa-

S. O. S. DAr~I~ 91

sionally could the whole complement be interpreted. In Fig. 6 is shown an entire nucleus at diakinesis.

L ~ Lt,

Ss LT ST Ls Ms L M Lt

Fig. g. Pollen grMn divisions in Scilla italicct. Top left: prophase. Top right: metaphase. :Bol~om: side ~dew of anaphase, x 2500.

3 o 31 3 o 4 o 1 o !2 o 11 1 o 1 8 2

]Pig, 6. Comptementl of biv~len~s from & pollen mo~her cell ~ di~khlesis, x 2500.

At me taphase eight bivalents are usual ly formed, their s inuous out- lines giving a characterist ic appearance to the nuclei (Fig. 7). Occa- sionally univalents are seen, owing to the failure of one of the shor~ pairs of chromosomes 4o form a chiasma (Fig. 7, middle drawing).

9 o 3 2

I

4 9 o 91 11 61 22197

2 o 81 f ~ 0 o 21 8 0 ] 7 5 2 2 21 0 o 8 o

{, 8 2 1 0 91 2 o 2 2 41 3 2 1118 9

~ig. 7, Side views o1" met&pha, se ]: in t&rco pollen moi~her cell lmclei. Top: showing all biv,Mcnts 1)a.ired. Centre : f~ilure of' chiasm~ format ion in one biv&lent. ZBo~tom : inter- locking of two biv&Ients, x 2500.

S. O. S. DAgK 93

At anaphase the um'avdting of the ohromatids from the chiasmata can be seen, and in the olu'omosomes that have aotually separated, the

Fig. 8. Au~phase of firs~ division ill ~hreo nuclei, showing the opmfing-out of chiasma~.

ohromatids still retain the twists imposed on them while associated in successive chiasmata. The closed loops produced by "compensating"

94 Chromos'ome 5't,udies i'n the Seilleae. I I

chiasmata are parlicularly noticeable (Fig. 8). Usually ~hese loops inter- lock. Univalents have been seen te~ ~imes at metaphase and four times at anaphase-telophase. Their fate depends upon the position they happen

@

Fig. 9. Telophase of the first division, showing a lagging univalent, dividing, i ts mate having passed whole Go one pole. × 5000.

to occupy in the spindle; they may be in eluded one iu each daughter nucleus, both in one daughter nucleus or one can be included in a daughter nucleus while the other divides upon ~he equatorial plate (Fig. 9). No case has been seen with both univalents dividing in the first division.

S. O. S. DAm¢ 95

The chiasma~a of ~wen~y-five nuclei were analysed, ~he results being shown in Table IV. From ~he s~agisbical daga ~he graphs shown in Figs. 10 and 11 were consgruel~ed. I~ will be observed ~hag ghe occurrence o f u n i v a l e n g s is hOg i n t~he p r e s e n g c a s e n e c e s s a r i l y a s y m p g o m o f

T A B L E IV .

Sci t la igal ica analysis of 25 metal)base 1 nuclei. h ' I o d i u m

2 bivMon~s A

Long 4 bivMents

~h 3 ,i 5 6 To(,M 1

,13 24 i I i00 129 96 5 6 295 ,14 30 i I 99 0.29 0.32 0.2 0.17 0.34 1-02 1.25 1 I - - 2.95

Short 2,

,b't

Chiasm¢~t~ pot biwlenl; 2 To~M bivalengs 28 2 45 3 50 To~M ohi~sma, ta~ 3 56 2 90 9 I01 TerminM c h i a s m t ~ 0 23 2 ,1:9 4 55 TerminMis~gion coetticicnt~ 0 0.4I i 0.54 0.44 0.54 TerminM chiasm~g~ per bivMeng 0 0.82 1 1.I 1.3 - - Av. No. chiasm~La per blwlenl; 2.02

Chiasmaga per bivMeug r-O 1 2 3 TogM ) l 2 TogM bivMengs 2 15 7 i 25 7 17 To~M ohiasmM;a 0 15 I4: 3 32 7 34 TerminM chiasmaL~ 0 12 10 2 2~ 2 15 TerminMisM~ion coeilioient~ - - 0.8 0.7I 0.67 0.75 0.29 TerminM ohiasmM;~ per bivMong 0.8 1.4 2 - - 0.29 Av. No. chiasmM~ per bivMenl~ 1.28 - -

/ / Short .S'r/ i

/ i i's I i i / /'~"

/ / / , , N / ~

/ . Medium//~o n-

i . " ~ / / : / : ," //" o.~ i . I

2 3 4

Number of ehiasm~¢% per blvMon~

2 3 To~al

Fig. I0. Graph showing l~crminMisMSon ill l~he four recognisMJle meiotic chromosome groups in Scilla italica.

® ®

I 5

05

,.Q

¢g

4 4~

~8

5%, - - - ' ~ - - - - ~ Shor~

3 To~M totM 1 25 50 3 44 76 2 19 43

0.,14 0.67 0.43 0.57 0.88 2 - - - -

1.76 1.52

96 Ch~'omosome ~t~edie8 in the Seilleae. H

hybridity, but is merely due to the failure of chiasma formation in a chromosome pah' with low chiasma frequency.

Iq~te~'loeki,ng.

Meiosis in ,S'eilla, italiec~ is extremely regular. An abnormalRy is shown however in the bottom right-hand drawing of Fig. 7. Here one of the "medium" length bivalents has been interlocked in a " long" bivalent during zygotene pairing. Chiasma ibrmation in the "medium" bivalent has been interfered wRh, one chiasma only being formed instead

12 X®

/'~ Scitla ira{lea Lon~% / li)

m

~, Me

G '% ./ ~o Short,'~' ~0 l I t

i I

,~"~ I I I

I / i I

V I I I J 2 3 4

Average nmnber of ch i~sm~ per bivalon~

Fig. 11. Graph showing rela, tion of ohi~sma frequency to chromosome length in Stiller, italica. The corresponding values for ~lenobothru~ ~)arallel'us (,S') and Cllorthipln~s elege~ns (C) ~re ~lso marked.

of the two or three found in these bivalents in every other case but one. Also the mechanism of the spindle repulsions has been upset, the attach- ment constrictions have been forced out of theJz true alignment on the spindle and as a result are unable to exert their mutual repulsion. The lower atgachmen~ consOrietion of the medium bivalen~ happens go be aligned with the attachments of the interlocking "long" bivalent and some repulsion is exerted, as shown by the straight, and apparently stretched, appearance of the lower chromosome.

S. O. S. DAl~x 97

6%asma frequency.

In Fig. 11 is plotted the graph connecting average chromosome length with the average number of chiasmata per bivalent in the groups. For reference there are also plo~ted the values obtained ibr Steno- both,rus pa,ralld, us (Darling~on and Dark, 1.932) aud Chorthippus elegans (Darlinggon, 1.932, ~hc chromosome lengths having been calculated by Dr Darlinggon from au unpublished drawing of mi~osis). All flu'co series of values fall approximately on the same curve, indicating that Ncilla itaZica is also an organism in which chiasma frequency is an indirect function of ~h.c length of the bivMent. The Scilla graph cmphasiscs, more than did the orighlal Stcnoboth,rus w~,lues, that the curve approaches the onc-chiasma abscissa for organisms in which regular pairing has been developed in conjunction with large size variation in the chromosomes.

Terminalisation.

In Fig. I0 the number of terminM ehiasmata per bivMent has been plotted against the total chiasmata per bivMcnt for the four distinguish- able chromosome groups. The vMues represented by a dot within a circle were obtMned from single observations and so are nob significant. The steepness of the graphs before their turning over to the horizontal is an indication of the intensity of terminMisation. The four groups cannot be directly compared because the variation in relative lengths of the arms means that the forces acting in terminMisation are different in each case. The theoretical curve should rise from the orighl and flatten out eventually along the ordinate 2. The steepness of the rise and rapidity of tttrnhlg over should depend largely upon the completeness of tcr- minalisation, but other factors come in to complicate its form, e. 9. the mechanical differences introduced when a large number of ehiasmata are present on one side only of the attachment constriction and when the same number arc distributed between arms on either side of the attachment constriction. This means that an intricate formula con- gaining a large number of factors wend be necessary to express the curve hi terms of the forces acting in chiasma movement. In the main however tcrminMisatiou is more complete in the shorter than in the longer bivMcnts.

SUNIKAP~Y.

1. The chromosomes of Belleval,ia ,re,mann have been illustrated and their lengths measured aC various stages of mitosis and meiosis. They

Journ . of Clonegics xx~x 7

98 Chromosome S~udies in the Scilleae. H

cont~racg in ]engtfll to one-elevengh between pachygene and metaphase (el. Belling).

2. The somagie cln'omosomes of Nc,ilh~ italiccb consist of eighg morpho- logically dist, ine~ pairs. At~ meiosis four classes of bivalent~s can be recog- niscd. @hiasma formabion is an indirec~ fmm~ion of lenggh as in Sle,no- bothrus and Hyacinthus amethystinus. Terminalisat~ion is more complege in each chiasma class in ghe short~ chromosomes than in the long ones.

My thanks arc due to Dr Darling~on and Prof. Haldane for crigicism and advice.

i%EFEI~ENCES.

]~ELLINC, J. (1928). "Nodes and ehiasmas in the bivMents of Lilium with regard to sogmolltM interchange." Biol. Bull. 54, 465-70.

D~mi~c~'o=% C. D. (1926). "Chromosome studies in the Seiltetm." Jour~. Gen. i6 , 237-51.

- - (1932). "The origin and beh~vionr of ehis.sm~t~. VI. Hyavh~thus amethy- ~tinus." ~iol. Bull. 63, 368-71.

DA~LI~C-TO~, C. D. ~nd DAlai(, S. O. S. (1932). "The origin and beh~viour of ehias- mata. II. Stenobothrus2Jarallcl.~,s." Cytologia, 3, 169-85.

L~WlTSKY, G. A. (1931). "The morphology of chromosomes." Bull. AiJpl. Bet. 27 (1), 19-174.

h'ICCL~TOCK, ]3. (1931). "Cytological observations of deficiencies involvh~g l~mwn genes, ~ransloeaf~ions ~nd inversions in Zea d]fa.ys." Univ. dJfi~souri Agr. l~es. Sta. .Bull. No. 163, 3-30.