Caryologia

International Journal of Cytology, Cytosystematics and Cytogenetics

ISSN: 0008-7114 (Print) 2165-5391 (Online) Journal homepage: https://www.tandfonline.com/loi/tcar20

Studies on the Comparative Effects of Certain

Chemicals on the Polyploidising Efficiency of
Colchicine in Trigonella Foenum-Graecum L.

S. S. Raghuvanshi & Sheila Joshi

To cite this article: S. S. Raghuvanshi & Sheila Joshi (1965) Studies on the Comparative Effects
of Certain Chemicals on the Polyploidising Efficiency of Colchicine in Trigonella�Foenum-Graecum
L., Caryologia, 18:1, 69-84, DOI: 10.1080/00087114.1965.10796156

To link to this article: https://doi.org/10.1080/00087114.1965.10796156

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 STUDIES ON THE COMPARATIVE EFFECTS OF CERTAIN
CHEMICALS ON THE POLYPLOIDISING EFFICIENCY
OF COLCHICINE IN TRIGONELLA FOENUM-GRAECUM L.

S. S. RAGHUVANSHI and SHEILA JOSHI

Cytogenetics Laboratory, Botany Department, Lucknow University, Lucknow, India

Received: 28th March 1964

INTRODUCTION

The importance of colchicine as a polyploidising agent has been long since

emphasised (EIGSTI 1938, DERMAN 1940) and work has also been carried out
where its effects in combination with X-rays has been studied (BRUES and JACKSON
1940, LEVINE 1945, 1946a, 1946b). The present investigation on the effect of
colchicine in combination with other chemicals was undertaken with the view
to see if its capacity to induce polyploid cells increases or decreases in the
presence of certain other chemicals. Amongst the chemicals chosen to be used
in combination with colchicine most of them are known to be pretreatment
chemicals for the clarification of karyotype (SHARMA and BHATIACHARYYA 1960).
The work of D'AMATO (1949), LEVAN (1949), EIGSTI and DusTIN (1957) show
that a number of chemicals are capable of causing chromosomal doubling. Since
polyploidy can be induced through spindle breakdown it was postulated that
the use of chemical which causes a change in the viscosity of plasma thereby
bringing about spindle disturbance may accelerate the effectivity of colchicine
in the production of polyploid cells.
The use of chemicals in combination with colchicine may bring about
differential change in the viscosity of plasma thereby leading to different degrees
of solidification ultimately providing different conditions in every combination
for the effectivity of colchicine.

MATERIAL AND METHODS

In order to study the comparative effects of the chemicals, solutions of the

following chemicals were prepared in distilled water in the usual way.

69] [Caryologia, Vol. 18, n. 1, 1965

70 RAGHUVANSHI and JOSHI

1. Paradichlorobenzene 7. Gammexane
2. Coumarine 8. Aesculine
3. Saponin 9. Acenapthene
4. a-bromonapthalene 10. 8-hydroxyquinoline and
5. ~-bromonapthol 11. Phenol
6. Phloroglucinol

To find the minimum duration of treatment to induce polyploidy at a certain

concentration the seeds of Trigonella foenum-graecum were treated with .2% aqueous
colchicine for 15, 30, 45, 60, 75, 90, 105 and 120 minutes. Since the seeds
germinated within 24 hours, root tips were fixed 50 hours after treatment. The
root tips of various seedlings were squashed and it was observed that upto 75 minutes
of treatment there is only an assemblage of arrested metaphases accompanied by a
few tetraploid cells, whereas in longer durations of treatment (90, 105 and 120 minutes)
the percentage of tetraploid cells increases and the octaploid and high ploid cells
make their appearance. So ultimately 80 minutes of duration was considered optimum
in the case of Trigonella seeds.
Three sets of experiments were carried out. In one only the effect of chemicals
was observed. In the bther in 2.5 cc of the various solutions 5 mg. of colchicine was
dissolved in order to obtain solutions of .2% strength with respect to colchicine.
A control set of experiment was set up in which the seeds were soaked in distilled
water and a few in .2% colchicine alone for the same duration as in the case of the
chemicals. After 80 minutes of treatment the seeds were washed in sterile wate~·
and germinate-d in petridishes lined with filter paper moistened in sterile water in
order to prevent fungal infection. Three sets of root tips were fixed 50, 7 4 and 98
hours after treatment in acetic alcohol (1: 3) saturated with iron oxide. Twenty four
hours after fixation the root tips were heated in 1N HCl for two minutes in order to
facilitate cell separation during squashing. Then washed in acetic alcohol and
transferred in a crucible containing acetocarmine which was heated for 1-2 minutes,
cooled .11nd corked the material in the stain in tubes until 24 hours which improved
the staining. The next day the root tips were slightly heated and squashed in a
drop of acetocarmine, sealed the coverslip with sealing mixture and later on made
permanent by the ethanol-butanol schedule. Cytological studies were carried out on
five root-tips in each case.

OBSERVATION

The rate of root growth shows great variation in the different chemicals
(Fig. 18). As far as the chemicals are considered alone in comparison to water
the rate has been accelerated in gammexane, paradichlorobenzene and ace-
napthene, while phloroglucinol shows no change and there is a decrease in the
rest of the chemicals. At the time when the measurements were taken there was
no germination in coumarine and phenol, however germination did take place
 COMPARATIVE EFFECTS OF CERTAIN CHEMICALS IN TRIGONELLA 71

in coumarine but phenol treated set did not germinate. In combination with
colchicine there is decrease in every chemical excepting in the case of saponin
and ~-bromonapthol which show an increase. Aesculine and 8-hydroxyquinoline
show no change in the growth rate in the chemical alone and in combination
with colchicine. In the case of phloroglucinol and gammexane combinations
growth has been the same as in colchicine alone ~-bromonapthol presents a
very interesting relationship, in the case of chemical alone it is 3.7 em. being
half of that met in the combination (.67 em.), which itself is half in comparision
to the growth rate in water.
An account of the mitotic activity as indicated by frequency of metaphase
plates per root tip is summarised in Fig. I8. The control set kept in water
shows the lowest extreme while in combination saponin reaches the maximum
of 462, followed by cx-bromonapthalene, aesculine, phloroglucinol, gammexane,
8-hydroxyquinoline, acenapthene, paradichlorobenzene, ~-bromonapthol and col-
chicine in the descending order. In combination with colchicine the accumulation
of the metaphase plates increases in all the sets except in paradichlorobenzene
which shows a clear decrease. Hence an examination of the Figs. I8 and I9
shows that the rate of root growth and that of karyokinetic activity are not
interrelated to each other.
As far as the ploidy level of the root tips is concerned, in the chemicals
alone no polyploid cells were encountered in any treatment whatsoever, however,
arrested metaphases were found in the case of cx-bromonapthalene. In combi-
nation every root tip shows an assemblage of 2n, 4n, and 8n cells while 3n, 6n
and IOn cells were rather rare (Table I). As far as the 2n cells are concerned in
every case there is a decrease from the first to the second set, except in the
case of paradichlorobenzene combination. The 4n cells increase in every combi-
nation from the first to the second set except in cx-bromonapthalene, ~-bromo­
napthol and gammexane in which there is appearance of 8n cells in the second
set mostly for the first time, hence the decrease in the 4n cells is well understood.
However, in paradichlorobenzene, saponin and aesculine almost a static condi-
tion of the 4n cells is seen in the two sets. The 8n cells invariably increase in
the second set except in the case of paradichlorobenzene. In paradichlorbenzene
combination although the mitotic activity decreases considerably from the first
to the second set there is an increase in the cell size which may simply indicate
high ploid nature of the cells which have entered the resting stage and do not
undergo division. This will also explain the evident decrease of the 8n cells
from the first to the second set. The 3n cells are found in colchicine, cx-bromo-
napthalene and ~-bromonapthol combinations while 6n cells in 8-hydroxyquino-
line and IOn cells in gammexane and saponin only. Excepting in the ~-bromo­
napthol combination there is decrease in the frequency of metaphase plates
in the second set.
72 RAGHUVANSHI and JOSHI

In combinations the increase in the capacity of colchicine to induce polyploid

cells becomes clear by the following points. In the first set the percentage of
the 2n cells is the highest in colchicine while the 4n cells is the lowest in com-

TABLE I

Table showing frequency of cells of various ploidy and frequency of metaphase plates 50
and 70 hours after treatment with colchicine alone and in combination.

Frequency
Chemicals 4n 8n 3n 6n IOn of metaphase
per root tip.

I 100 12
I. Water 100 12
II
I 75 22.9 2 37
2. Colchicine II 24.5 63.6 11.6 45
I 39.5 53 7.4 236
3. Col. + 8-hydrox. II 6.9 65.5 25.9 1.7 88
I 29.7 67.8 1.3 1.2 239
4. Col. + gammex. II ll.8 63 25 81
I 27.3 53.1 19.5 120
5. Col. + paradichloro II 37.8 55.1 6.9 33
I 40.5 54.3 5.1 141
6. Col. + acenap. II 12 60 28 128
I 37.4 60.7 1.8 372
7. Col. + a.-bromo. II 31.8 50 18.1 19
I 43.4 54.2 2.1 83
8. Col. + tl-bromo. II 20.1 48.5 30.8 319
I 21.7 50 28.2 291
9. Col. + aescul. II 10 50 40 28
I 55 42.5 2.3 260
10. Col. + phloro. II 4.4 70 25.5 73
I 30.5 63.8 5.5 463
11. Col. + sapo. II 3.1 63.5 28.5 4.7 75
I
I2. Col. + coum. II 34 59 6.8 96

parison to the combinations. In the first set no Bn cells were observed in colchi-
cine alone, however examination of the data in the second set indicates that
the frequency of the Bn cells also increases in every combination.

The distribution of the various chromosomal aberrations in the different

combinations of the chemicals is shown in Table II. As far as the chemicals
 COMPARATIVE EFFECTS OF CERTAIN CHEMICALS IN TRIGONELLA 73

are considered alone the only anomaly came across is fragmentation in gamme-
xane and paradichlorobenzene (Fig. 1). The maximum number of the fragments
observed being two. However in combination with colchicine the following
anomalies were observed. Tripolar spindle was seen in 8-hydroxyquinoline com-
bination (Fig. 2), it was also observed in aesculine combination. Arrested me-

TABLE II
Table showing distribution of chromosomal abe"ations found in various
colchicine-chemical combinations.

0... 0 0 :a.8 P.
~
Ill
"0 §... § -3 "0 8 0...
Anomalies Ill
.s.8
>.
~I..."" "">=
0 CJ 0
8
.-9
()()i "'"" +
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1. Curved spindle + +
2. Tripolar spindle + +
3. Arrested metaphase + +
4. Two spindles in one cell + + +
5. Unequal anaphasic separation of chs. + +
6. 'Somatic reduction + + +
7. Stray chromosomes at metaphase + + +
8. Multinucleate cells + + + +
9. Split spindles +
10. Fragmentation + +
11. Somatic groupings + + + + + + +
12. Well spread chs. + + + +
13. Clear constrictions + +

taphases characteristic of colchicine were abundant in <X-bromonapthalene. The

groupings of the chromosomes was a chracteristic feature of paradichlorobenzene.
In Fig. 3 the split nature of the chromosomes is very clear. However the
occurrence of chromosomes in groups was found in 8-hydroxyquinoline, ~-bro­
monapthol (Fig. 4), saponin, gammexane (Fig. 5), phloroglucinol and aesculine
(Fig. 6). The figure 6 shows a compact group of 16 chromosomes and a rather
loose grouping of 8 chromosomes which show clear splitting indicating thereby
that probably the division of the chromosomes in the first group has been
completed while in the latter group the chromosomes simply show splitting
but their chromatids have yet to undergo separation. The functioning of 2
independent spindles within a single cell was commonly observed in saponin
 ,.
/~I'.',
••••
••••
'\,'''
"!"

9
10

Fig. 1. - A fragment in 2n cell following paradichlorobenzene treatmeut.

Fig. 2. - Late anaphase showing a tripolar configuration in 8-hydroxyquinoline combination.
Fig. 3. - Somatic grouping in a 2n cell in paradichlorobenzene combination, the chromosomes
clearly show the split nature.
Fig. 4. - Three groups of chromosomes in a cell, two groups of 4 chromosome each and the
central larger of 24 ~-bromonapthol combination
Fig. 5. - A cell showing groups of varying number of chromosomes in gammexane combi-
nation.
Fig. 6. - A compact group of 16 chromosomes and a rather loose grouping of 8 chromosomes
which show splitting in aesculine combination.
Fig. 7. - Anaphase in two very unequal spindles in a cell with one stray chromosome in
saponin combination.
Fig. 8. - Two independent metaphase plates in a single cell in ~-bromonapthol combination.
Fig. 9. - Unequal anaphasic separation of chromosomes (8 and 24) in ~-bromonapthol
combination.
Fig. 10. - Somatic reduction in 2n cell in saponin combination. (Figs. 1-10: 1480 x ).
 COMPARATIVE EFFECTS OF CERTAIN CHEMICALS IN TRIGONELLA 75

..
'
~
8 ~1····
t~\i \u~1
ooooo ••••
ol •. •

Fig. 11. - A 16 nucleate cell in ~-bromonapthol combination.

Fig. 12. - A cell with 8 nuclei in gammexane combination.
Fig. 13. - A cell with 19 nuclei in gammexane combination.
Figs. 14 and 15. - Two nuclei lying side by side within a single cell in acenapthene
combination.
Fig. 16. - Split spindle in phloroglucinol combination.
Fig. 17. - A cell having a spindle with 10 and 6 chromosomes and a separate group of
16 chromosomes in phloroglucinol combination. (Figs. 11-17: 1480 X).
76 RAGHUVANSHI and JOSHI

(Fig. 7), ~-bromonapthol (Fig. 8) and phloroglucinol. Unequal anaphasic sepa-

ration was common in ~-bromonapthol (Fig. 9) and to a lesser degree in phloro-
glucinol. Somatic reduction played an important role in saponin (Fig. 10),
8-hydroxyquinoline and phloroglucinol. The occurrence of clear constrictions
observed in colchicine alone is shared by acenapthene. Stray chromosomes at
metaphase were observed in saponin, aesculine and acenapthene combinations.
The chromosomes were well spread in colchicine alone and in combination with
saponin, acenapthene and paradichlorobenzene. Curved spindles were observed
in ~-bromonapthol and aesculine.
Multinucleate cells were a special feature of gammexane in which 4,
8 to 19 nuclei were observed (Figs. 12, 13). This anomaly was also found in
~-bromonapthol (Fig. 11). In 8-hydroxyquinoline and acenapthene (Figs. 14, 15)
two nuclei were abserved lying side by side although the frequency was rather
low. Split spindles were found in phloroglucinol (Fig. 16). A cell observed in
phloroglucinol (Fig. 17) shows six and ten chromosomes on one spindle and a
separate group of sixteen chromosomes. The split nature of chromosomes was
clear in paradichlorobenzene and aesculine. In colchicine the karyotype was
very well clarified.
DISCUSSION

The study of the effects of various chemicals in combination with colchicine

indicate certain pecularities which need comment. Amongst the various che-
micals tried in combination some like acenapthene, gammexane, 8-hydroxy-
quinoline and cx-bromonapthalene are known as polyploidising agents. With
acenapthene and cx-bromonapthalene SOHMUK (1938) obtained polyploids of
wheat and barley. SIMONET and GuiNOCHET (1939) reported the production of
polyploids in flex seedlings by paradichlorobenzene treatment. In the present
studies the duration of treatment proved to be rather short for them to be
effective. In the case where the seeds were treated with the chemicals alone,
arrested metaphase plates were met with in ~-bromonapthol only. No polyploid
cells were observed in chemicals. The rate of growth exceeded that of water
in the case of gammexane, paradichlorobenzene and acenapthene alone. While
with colchicine there was retardation in every combination tried (Fig. 18). In
the case of coumarine and phenol no germination took place when the measu-
rements were taken 50 hours after treatment however later on seeds treated
with coumarine did germinate while in phenol no germination was observed.
It may be said that the effectiveness of various chemicals in inhibiting growth
in part at least results from the damage inflicted on chromosomal complements,
as for instance maximum somatic aberrations were met with in [3-bromonapthol
and saponin combinations and the examination of Fig. 18 shows that in these
two chemicals the growth has been much retarded.
 COMPARATIVE EFFECTS OF CERTAIN CHEMICALS IN TRIGONELLA 77

The growth rate of root an the frequency of metaphase plates in the

roots do not show any relationship to each other. Hence our observations indicate
that the effect of the chemicals on the frequency of cell division is not coincident
with its effect on total growth. Similar results have been reported by CoMPTON
(1952) in the case of malic hydrazide treatment of Pisum sativum seeds. Although
the rate of growth in the case of phloroglucinol, gammexane combinations and

a aB
l:l !:zl
:z.
....
~ ·~ ~
...:.:
u

~ Ill ..:I
Q
.:I
u
~
3 ~ < 0
u
~::r ....
:t:
...
u u
s
~
.s
~
<
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..
c!

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....
~
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;:! g
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...0 ..."'::>
z
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s 0I
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.:I
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<
i
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?:
~
!>:
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~ iS
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00

:!:
0
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... I

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.... ..:1

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:z.
"":X::
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CHEMICALS

Fig. 18. - Graphic representation of the average length of root 50 hours after treatment

colchicine it is practically same there is great variation in the frequency of

metaphase plates per root tip, being 260, 239 and 37 respectively (Fig. 11). In
the second set in every combination there is a marked drop in the activity
of the cells except in the ~-bromonapthol combination, where the frequency
of metaphase plates has become almost four times from 83 to 319. However
it may be pointed out that in colchicine alone also there is increase in the
frequency of metaphase plates (37-45) but this increase is far lower than in the
combination. Hence the combined effects of the two chemicals (colchicine and
~-bromonapthol) accelerate the rate of mitotic activity in the roots of Trigonella
considerably.
78 RAGHUVANSHI and JOSHI

As far as the ploidy level of the cells is concerned (Table I) every root
tip shows an assemblage of 2n, 4n and 8n cells while 3n, 6n, 8n and IOn cells
are rather exceptional cases. The 3n cells form minority of the total cells being
met in ~-bromonapthol and oc.-bromonapthalene combinations. The IOn cells
were observed in gammexane and saponin combinations only, in the former
being 1.2% while the latter had 4.7% of the total assemblage of the cells. In
every combination there is decrease of 2n cells from the first to the second day
excepting in paradichlorobenzene combination which shows an increase from
27.3 to 37.8%. In the 4n cells there is increase in the percentage from the first
to the second set in the case of acenapthene, paradichlorobenzene, 8-hydroxy-
quinoline and phloroglucinol combinations; while aesculine and saponin show
no change. In the case of oc.-bromonapthelene, ~-bromonapthol and gammexane
there is a fall in the percentage of the 4n cells in the second set, perhaps by
further action of the chemicals they become 8n which is evident by the increase
of 8n cells in every set excepting paradichlorobenzene combination. This de-
crease may be explained on the basis that either the Bn cells undergo somatic
reduction or they may be due to high ploidy form resting nuclei thus inhibiting
further division.

The distribution of various aberrations in the various combinations has

been shown in Table II. As far as gammexane is concerned it is worthy to note
that even in the short period of treatment fragments were produced. The only
other chemical which induced fragments is paradichlorobenzene. SHARMA and
BHAITACHARYYA (1960) also observed fragments in paradichlorobenzene treatment.
The use of gammexane as polyploidising and chromosome breaking agent has
been studied by KUNDU and RAo (I949), D'AMATO (I949, I950) and SHARMA and
CHATTERJI (I960). Work of PRAKHAR and SwAMINATHAN (I951) has confirmed
that 8-hydroxyquinoline has c-mitotic effect but they failed to produce polyploids
in Lactuca sativa and Spinacia oleracia. In the present study no polyploid cells
were found after 80 minutes of treatment, probably this duration may have
been too short. It is interest that although 8-hydroxyquinoline in combination
with colchicine and alone has the same growth retarding effect on the roots,
there is a marked difference in the frequency of metaphase plates in the two
cases i.e. 236 and 10 respectively. D'AMATO et al. (I955) has reported chromo-
somal breakage and other physiological and structural effects following couma-
rine treatment, but in the present study we did not come across any aberration,
perhaps as in the case of other chemicals the duration of treatment was too
short to have any significant effect on mitosis. Our observations have confirmed
that it is an inhibitor of mitotic activity.
The instability of chromosome behaviour consists of one or any combination
~f the following phenomena occurring in the cells. 1. Subdivision of the chro-
 COMPARATIVE EFFECTS OF CERTAIN CHEMICALS IN TRIGONELLA 79

mosome complement into two or more groups that function independently

within the cell; 2. nondisjunction of the chromosomes; 3. unequal distribution
of the choromosomes to the two polar regions at anaphase and 4. somatic reduc-
tional division of the chromosomes. Subdivision of the chromosome complement
into independently operating groups in the cells is considered to be the same

...
liS
X
~
0 '~""
~
500 II) "'~
p;

!0
~ Ill 1<1

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100

CHEMICALS

Fig. 19. - Comparison of the frequency of metaphase plates per root tip in the various
treatments.

phenomena as that in Rubus (BRITTON and HuLL 1957) and in colchiploids of

Capsicum frutescens var. 'Oshkhosh' (RAGHUVANSHI and JosHI 1964).
Such figures were termed split spindles' by UPcorr (1939) and VAARMA
(1949). In order to explain the groupings of chromosomes within a cell it may be
assumed that the chromosomes are merely pawns of an abnormal spindle which
sometimes splits before anaphasic movement is completed, although in the be-
ginning it is a single structure. Alternatively it may be assumed that the chromo-
somes have the capacity to aggregate into two or more groups capable of forming
their own spindles which function independently. Evidence in favour of the latter
80 RAGHUVANSHI and JOSHI

possibility is seen in Fig. 3 which shows chromosomes in three groups (although

it is diploid cell), these may give rise to three separate spindles. The binucleate
cells have been observed at early prophase but it is not possible to determine
whether binucleate condition is the fore-runner or whether it is the pro-
duct of chromosome groupings. The tripartite separation has been interpreted
as incomplete splitting of the spindle (VAARMA 1953, GorrSCHAUC 1958). Une-

I (]] -~'-::!>··
·<I>-·
-01·--~(.~_)
•I
c:~)
Ia
I• (.:~·.·) (.~.)
...~

~~#c:·-,D-( <~> )~ r·l

~ ~- 16 ) <.::~~>· 161
I",.. -e<~·j
. 16 ··::r ,, ·.
....... .I ·...· · ..... ·...
! ,, ': •

...
.......
=: a ··=
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- ,
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....... ·"·· ..
: s ·: =. a :
··..... ·.... ·

\ IT]r··~~---
. , ~<.:~::.-.... riOI
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-
...-:.:§::::.-.
-
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(6
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. ·,0··.\c.·~.::
......... ._.:> -: ....
:·.·.~_:: <. ~.~--·;
Fig. 20. - Diagramatic representation of the three possible modes of origin of multinucleate
condition in the cells following various treatments.

qual mitotic anaphasic distribution as met in the case of ~-bromonapthol com-

bination has been reported in Triticum timopheevi and Aegilops bicornis by LI
and Tu (1947), in apple by HEGWOOD and HouGH (1958) and in Rubus by HULL
and BRITTON (1958). In a few cells at stages from prophase to metaphase there
was strong tendency for the whole chromosomes to separate into groups. This
when followed by a more or less normal anaphasic separation of chromatids must
have resulted in the formation of multinucleate cells and if cytokinesis does take
place there will be assemblage of cells with less than the diploid number of
chromosomes. Similar observations have been reported by HUSKINS (1947, 1948)
 COMPARATIVE EFFECTS OF CERTAIN CHEMICALS IN TRIGONELLA 81

following treatment of meristmatic regions of plants with sodium nucleate. The

occurrence of more than one spindle per cell or the socalled split spindles may
result in the cells having greater number of nuclei than the usual ones. If the
spindles happened to be close to gather and in the same plane the telophase
following the anaphasic separation of individual spindles may lead to usual
binucleate condition and there will be no indication of the multiple spindle
organisation in the end products. But occasionally the two spindles may be
slanted to each other which will ultimately result into tri or quadrinucleate cells.
Although it is not clear whether such ' split figures ' have a tendency to separate
homologous chromosomes, WILSON and CHENG (1949) found more cases of ho-
mologous separation in Triticum than a chance hit can explain. Since such sepa-
ration of chromosomes which may be called ' split figures ' are found before the
onset of spindle organisation (Fig. 3) it may be said that it is the result of chance
alignment of spindle fibers rather than from multiplication of spindle loci.
GLAss (1956, 1958) was able to identify complete genome complement separated
from rest of the chromosomes within liver cells of rat. Recently SAUNDERS et al.
(1962) have been successful in obtaining diploid mutants after colchicine treat-
ment of 4n seedlings of a certain genotype of Sorghum. If somehow apex of
the plant can be proliferated by cells which have undergone somatic reduction
into its n-chromosome complements then this will be the induction of immediate
homozygosity and its importance in plant breeding is well understood; for it
could be a potent tool to facilitate selection of immediately true breeding lines
and so speed up the production of new varieties. In biological processes the
importance of somatic reduction of chromosomes may perhaps be very great.
The factor responsible for somatic reduction has been claimed to be nucleic
acid supply of the cell (SHARMA and MooKERJEA 1954). The reductional separation
of the chromosomes in the somatic tissue may be the result of increased abnormal
synthesis of nucleic acids. The chemicals applied may bring about a disbalance
in the cell metabolism causing an abnormal increase of nucleic acid in the chro-
mosomes, ultimately being responsible for reductional separation. Hence the
occurrence of somatic reduction in the polyploid as well as the diploid cells is
due to disbalance in nucleic acids in the cell caused by treatment with various
chemicals.
In order to explain the multinucleate condition found in ~-bromonapthol,
gammexane and certain other chemical combinations there are different views.
KolLAR (1947) mentioned that binucleate and multinucleate cells with synchro-
nised division of nuclei occur in tumour cells. However CROKER (1933) has re-
ported the breakdown of the nucleus by a budding off of the chromatin as
small buds or irregular projections following treatment with 2,4-D and 2,4-5-T in
Allium cepa. These cells thus contained deficient nuclei or only scattered mi-

6
82 RAGHUVANSHI and JOSHI

cronuclei. Since in the present study we did not come across any nucleus giving
indication of budding this explanation is not valid.
It is proposed that the multinucleate condition in the present material could
arise in three ways (Fig. 20):
1. Lack of cytokinesis. - There may be normal division and separation
but no cytokinesis with the result the two nuclei remain in the same cell. These
may divide again thus increasing the number of nuclei per cell.
2. Reductional separation. - At anaphase there may be equal or unequal
reductional separation without being followed by cytokinesis. Then these resul-
tant nuclei with reduced number of chromosomes may undergo division again.
3. Split spindle. - There may be splitting of the spindle ultimately re-
sulting in the formation of four groups of chromosomes.
The failure of cytokinesis appears to be the most dominant factor in the
three possibilities. Reductional separation is clearly seen in figure 10 and split
spindle in Fig. 16. Due to lack of cytokinesis coupled with random orientation
of spindles multinucleate cells arise with highly variable number of chromosomes
(Figs. 5, 13). The varying size of nuclei is indirect proof of different chromo-
somal constitution of nuclei. Such multinucleate cells have been met with in
acenapthene, gammexane, ~-bromonapthol and 8-hydroxyquinoline combinations.
Table I clearly shows that the polyploidising capacity of colchicine increases
in the presence of these chemicals. Hence it may be concluded that the higher
efficiency in the production of polyploid cells in combination justifies the recom-
mendation of the usage of colchicine in combination rather than separately. The
significance of the use of colchicine in combination with the chemicals rather
than alone in the cases of plants where fruitful results are not obtained is great.
In plants where colchicine alone is not effective it may become so in combination
with certain chemicals which can antagonise the substances present in the plants
which inhibit colchicine activity.

Acknowledgements. - This work has been supported by the Council of Scientific and
Industrial Research of India. One of us (S.J.) is holder of C.S.I.R. followship. We are thankful
to Prof. A. R. RAo for providing the laboratory facilities.

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0
Original not consulted.

SUMMARY

The comparative effects of colchicine in combination with 11 chemicals (1 paradichloro-

benzene, 2 coumarine, 3 saponin, 4 a-bromonapthalene, 5 phloroglucinol, 6 8-hydroxy-
quinoline, 7 gammexane, 8 aesculine, 9 acenapthene, 10 ~-bromonapthol and 11 phenol) on
the production of polyploid cells in Trigonella foenum-graecum has been studied. In the
chemicals alone in comparison to water the root growth has been accelerated in (1), (7) and
(9) while (5) shows no change and in the rest there is a decrease. In combination with
colchicine every chemical shows decrease except in (3), and 10. The frequency of metaphase
plates per root tip is lowest in water while it reaches the maximum in (3) followed by (4),
(8), (5), (7), (6), (9), (1), (10) and colchicine in decending order. In combination with colchicine
the accumulation of metaphase plates increases in (1) while in the rest there is a decrease.
The rate of root growth and karyokinetic activity are not interrelated to each other. There
is some correlation between frequency of chromosomal aberrations and retardation of growth
rate of roots.
In chemicals alone only arrested metaphases were observed in (4) rest had all diploid
cells. In combination every root tip showed assemblage of 2n, 4n and 8n cells wh'le 3n, 6n
and IOn cells were rather rare. The highest percentage of !On cells is in saponin combination.
Although with the passing of time the ploidy level increases there is marked decrease in the
frequency of metaphase plates per root tip. The principal chromosomal aberrations encounter-
ed during the study are tripolar spindles, arrested metaphase plates, groups of chromosomes
within a cell, two independent spindles in a cell, unequal separation at anaphase, somatic
reduction, stray chromosomes at metaphase, multinucleate cells and ' split spindles '. The
percentage of polyploid cells increases in combinations as compared to the set treated with
colchicine alone. It is suggested that in plants where colchicine alone is not effective it may
become so in combination with certain chemicals which can antagonise the substances present.
in the plant which inhibit the activity of colchicine.