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Hi>reclitas 72: 129-148


Chromosome studies in Alliurn sativurn

Institute for Experimental Botany C S A V , Olomorrc. Czechoslovakia
Institute of Genetics, Lund, Sweden

(Received March 29, 1972)

The chromosomes were studied in 4 strains of garlic, Allium sativum, belonging to 2 different
morphologic types, 3 strains to the so-called H type and I strain to the U type. Since Allium
sativum never sets any seed, each strain can be regarded as a clone. One aim of the study
was to define whether their genetic isolation had resulted in the accumulation of chromosomal differences between the clones. It was demonstrated by measurements of all chromosomes of 5 5 root mitoses that the karyotypes of the clones, although generally maintaining
their differentiation in 2 homologous sets, exhibited a significantly higher variation between
clones than between cells within clones, the parameters studied being total chromosome
length, arm ratio and 2 satellite indices. The differences were especially pronounced in the
2 satellite-carrying chromosome pairs. The results of the measurements were born out by
observations on meiosis: even though normal bivalent formation characterized the 2 clones
of the H type studied (the U type could not be studied, because it very rarely forms flowers),
meiotic disturbances were frequent, indicating small structural differences between the
homologues. In one of the clones a large structural change, a ring of 4 chromosomes, was
observed regularly at first meiosis. The implications of our observations for karyotypic
evolution in Alliitm are discussed.
The garlic, Allium sativum L., is an old cultivated
plant, valued since ancient times as a vegetable
and a spice. It occurs in a great variety of morphologic types, all completely seed sterile. Among
the different clones, the vegetative phase is
variably predominant; in some, inflorescences
are formed only exceptionally and after heavy
vernalization, in others inflorescences are formed,
containing bulbils mainly or exclusively, in
others, again, the inflorescences contain a high
proportion of flowers, although always completely
Like all species of Allium, the garlic has very
favorable chromosomes, inviting detailed analysis. Roots tips sprout rapidly, when cloves of
bulbs are placed in water, making prefixative
treatments and Allium-tests easy. In the flowering
types, meiosis, both male and female, is readily
available. In these types the numbers of welldeveloped flowers will increase if the bulbils
are removed, but even so pollen will not develop
beyond the one-nucleate stage and seeds will not

In spite of the advantages of Allium sativirm

for chromosome work, only little such work has
been done, especially in comparison with the
classical chromosome material of Allium cepa.
It has been widely utilized for Allium-tests by
and collaborators (see review, DEYSSON
1968). The chromosomes of Allium sativirm have
been described in the literature repeatedly
et al. 1960; BATTA(MENSINKAI
GLIA 1963), the most detailed study of their morphology during mitosis being that of BATTAGLIA.
When in the summer of 1968, the present writers
happened to find a garlic type with an interchange ring at meiosis, it was tempting to examine the chromosomes of some garlic clones.
The fact that each garlic clone represents an
isolated genetic system added specific interest to
our study, which could be expected to elucidate
questions such as: how efficient are conservative
forces in maintaining a common karyotype in
all clones? How far has each clone reached in its
individual karyotypic evolution? With these
questions in mind, 2 representatives of widely
Hcreditas 72. I972



(3) The Lund clone, an H type, referred to

below as LH or No. 3. It was obtained in 1960
from the Moscow botanic garden and has been
grown since then at the institute of genetics in
Lund. It differs from the preceding types by
having an abundance of flowers together with
small bulbils in the inflorescences. Professor
Material and methods
Per Wendelbo, Gothenburg, has kindly made a
On the basis of important morphologic and preliminary taxonomic determination of this
physiologic characters the species of Allium type and found it to correspond to Allium
which according to VVEDENSKY
sativum may be divided into 3 main types longicuspis REGEL,
(1935) is a wild predecessor of Allium sativum.
( H R U Band
(1) The H type has bulbs with violet bulb This is the type that forms a chromosome ring
coats and with cloves in 2 groups. Flower stems at meiosis.
(4) The Olomouc U T clone, referred to below
develop regularly. The leaves are broad and are
formed in a definite number. The H type com- as OU or No. 4. This clone, derived from the
prises late forms, which require much water and farm of Mr. T. Konvi;ka, Kuncice, Walachei,
not far from the birth place of Gregor Mendel,
remain upright when ripe.
(2) The U type and (3) the A type both have was included in 1967 into the type collection of
bulbs with white bulb coats and with cloves in the Olomouc institute. The bulbs are fairly large
several groups. Flower stems develop rarely and with big cloves.
In the statistical treatment below, comparisons
only after heavy vernalization. Leaves are formed
in indefinite number. Both types are lying down will be made with the chromosome measurements
(1963) in Allium sativum material
when ripe. They differ from each other in the of BATTAGLIA
following respects: The U type comprises late purchased in Pisa, Italy. Although BATTAGLIA
forms with broad leaves and requires much did not discuss the genetic status of his material,
water, whereas the A type comprises early forms it will be referred to below as the Pisa clone or
No. 5.
with narrow leaves and requires less water.
Mitotic chromosomes were examined in root
In the present investigation only the H and
the U types were represented with 3 and 1 clone, meristems. Root tips were obtained by dipping
respectively. Here follows a brief description of cloves through a plastic net into running tap
water bubbled through with air and kept at
our material:
(1) The Olomouc No. 15K,an H type, referred 16C. Meiosis was studied in anthers and to a
to below as O H or No. 1. This clone, derived lesser degree in ovules in flower buds taken in
from the farm of Mr. K. Kontny, Olomouc, the field. For temporary preparations, root tips
and commonly grown in the village of ternovir and flower buds were fixed in 60% acetic acid
near Olomouc, was included 1965 in the type with 0.1 n hydrochloric acid, macerated by
collection of the Olomouc institute. It has large warming the fixative to 60C and squashed in 2%
bulbs with medium-sized cloves. The inflorescen- orcein in 60% acetic acid. Permanent slides were
ces contain almost exclusively bulbils of medium made after various fixations followed by Feulgen
size. It belongs to the variety ophioscorodon staining, occasionally by Feulgen and light green.
(LK.) D ~ L L .characterized
by the flower stem Very good results were obtained with the fixafollowed by the
tive of ~ S T E R G R E Nand HENEEN
being twisted into a spiral in its upper part.
(2) The Uppsala clone, an H type, referred to schedule worked out by these authors (1962).
below as UH or No. 2. This clone was kindly For chromosome measurements, chromosomes
presented by Professor Nils Hylander, Uppsala. were drawn in the center of the viewfield with
It has been grown in the Uppsala botanic garden the aid of a camera lucida at a magnification of
since 1951, when it was obtained from Bergen, 7000 times.
Norway. According to Dr. Hylander, this is a
typical garden variety, ordinarily completely
without flowers. It also belongs to the variety

diverging morphologic types were selected for

analysis from the type collection of the Olomouc

Hereditas 72, 1972


Fig. 1 . MetaDhase chromosomes of root mitosis in clone OH, at arrow: nucleolar organizing body.

1 . Mitotic chromosomes

The chromosomes of the 4 clones of the present

paper agree very well with the fundamental
karyotype of Allium sativum, as established by
(1963). The general appearance of
the chromosomes is seen in Fig. 1. All 4 clones
have chromosomes easily arranged into pairs
(Fig. 2). Of these pairs the 3 shortest ones are
recognizable individually, whereas the 5 longest
ones can be identified only as a group. The 5
longest pairs have approximately median centromeres (m chromosomes according to LEVANet
al. 1964). Their variation in size makes it possible
with some degree of certainty to distinguish the
longest pair, No. 1, and the shortest pair, No. 5,
whereas the 3 intermediate pairs, Nos. 2-4 are
indistinguishable. The 3 shortest pairs consist of
2 longer pairs, Nos. 6 and 7, both with satellites,
and 1 shorter pair, No. 8, with clearly asymmetric arms. No. 8 is on the border between m
and sm chromosomes.
The 2 satellited pairs have very characteristic

13 1


features. The longer of them, No. 6, is the most

asymmetric pair of the karyotype with an arm
ratio of 2.2 to 2.9, thus clearly an sm chromosome.
The shorter, No. 7, is an m chromosome but
more asymmetric than any of the longer m chromosomes and thus usually identifiable, even when
the satellite constriction does not show. The 2
satellited pairs have essentially the same gross
organization: their short arm being divided into
a small proximal segment and a big satellite.
This satellite chromosome differs from the type
most common in Allium, viz. with a small satellite
at the end of the short arm. The sativum-type
was assumed by VED BRAT(1965) to have originated from the normal type by inversion in
the satellite arm.
The 2 satellited pairs are easily distinguishable:
the larger pair, No. 6, has both the proximal
segment and the satellite smaller than No. 7,
while the long arm of No. 6 is much longer.
Both satellited pairs are involved in nucleolus
(I.c. Fig.
formation. As illustrated by BATTAGLIA
7, p. 37), somatic cells have a maximum of 4
nucleoli. The early formation of nucleoli
during anaphase-telophase is illustrated in Fig. 3
Hereditas 72, I972



Fig. 2. a-d: karyotypes of the 4 clones studied, a: clone OH, b: UH, c: LH, d: OU; e: chromosomes separately
drawn from a second meiotic anaphase in clone LH. - Feulgen, x2500.

Hereditas 72, 1972




Fig. 3. Clone L H , a: root mitosis, early telophase showing formation of nucleoli; b: sister nuclei
during interphase, 3 and 4 nucleoli, respectively. - Feulgen and light green, x4100.

a of the present paper. The location of the nucleoli

indicates that their centers of formation are close
to the centromeres, thus well compatible with
their being formed at the connecting fiber of the
satellites. In Fig. 3b, a pair of more advanced
telophase nuclei show 4 smsll nucleoli in one and
1 big fusion nucleolus 2 small nucleoli in the
The satellites are usually more evident in No. 6,
the attachment fiber often being very long at
least in one member of this pair. Fig. I is a typical
instance: in one member the satellite is removed
from the chromosome by a distance 3 times the
satellite length, in the other member, the satellite
is in direzt contact with the proximal segment.
As in Viciu fubu and in many other materials
a small granule is often seen on the satellite

attachment thread of No. 7, presumably corresponding to the nucleolar organizer. This was
clesrly visible in the original of Fig. I on the
extended satellite fiber at about 4/5of the distance
from the proximsl segment to the satellite (Fig. I
arrow). The condition of the satellite fiber,
whether extended or not, was analyzed in 55
cells from the 4 clones studied and in the 5 cells
(I.c. Fig. 3, p. 18-19). It is seen
of the cells
from Table 1 that from 60 to 100c~O
of the 5 materials had the thread of one chromosome more extended than of the other, and
most often (74%) the most extended thread was
in the longest member of pair 6. The LH clone
differs by having this situation in only 2 out of
10 cells.
Pair No. 7 shows considerably more variation

72, I972



Table 1. Relation between satellite-fiber length

and total chromosome length in pair No. 6


Case (see below)







1 4

Case 1:
Case 2:
Case 3 :
Case 4:









satellite fiber extended in both members of pair

Table 2. Visible satellite in the two members of

pair No. 7



Visible satellite in











Hereditas 72, 1972




Satellite on










satellite fiber extended in longest member of pair

satellite fiber extended in shortest member of pair
satellite fiber extended in unspecified member of




in appearance between the 2 members. Commonly 1 satellite only is clearly visible and only
rarely is the satellite removed far from the proximal segment of the arm. In cells with just 1
apparently satellited No. 7, the other member
may show a faint, hardly visible constriction on
the short arm (Fig. 2a) or no trace of a constriction in 1 member of the pair (Fig. 2b) or in both
(Fig. 2c). With regard to this character there
were clear differences between the clones (Table
2). Whereas satellites were seen only occasionally
and with difficulty in LH, all cells of OU showed
clear and well-defined satellites in both partners.
The clones OH and UH were intermediate with
either 1 or 2 satellites in pair No. 7. The analysis
whether the same regularity existed as in No. 6,
viz. with a more pronounced separation between
chromosome and satellite in the longest member



Table 3. Relation between satellite appearance

and chromosome length in pair No. 7


of the pair, gave no clear indication that this was

the case (Table 3).
Including the Pisa clone of BATTAGLIA,
measurements were available of all chromosomes of
60 cells from 5 clones. The total absolute chromosome length per cell is given in Table 4, including
50 cells from all clones processed with Feulgen
staining and 10 cells from the clones OH and UH
with orcein staining. The total chromosome
length varies from 127 to 219 p. The latter value,
from the Pisa clone, is not directly comparable
with the other values from the Feulgen series,
since the technique was different. Excepting the
Pisa clone, the Feulgen material had consistently
smaller chromosomes than the orcein material,
both in length and thickness. In OH a persistent
difference was noticed: after Feulgen, the long
chromosomes were relatively longer and the
short chromosomes shorter than after orcein.
This regularity was not seen in clone UH.
The measurements were used for 2 purposes.
Since all clones had roughly the same karyotype,
and since meiosis showed generally good pairing
into 8 bivalents, it was considered permissible
to combine all measurements into an average
idiogram. The second purpose of the measurements was to investigate whether any statistically
significant differences existed between the measurements of individual chromosomes of different clones. This would be expected a priori because of the clonal nature of the material, and
also in view of the frequent meiotic disturbances
indicating structural differences between the 2
haploid sets.
Because of the impossibility to distinguish the
5 longest pairs individually, they were arranged
strictly in size order. It would have been preferable also to consider the location of the
centromere in determining the place of each



Table 4. Absolute length in p of total diploid set






of cells









chromosome in a specific pair, and this was

actually attempted but found impracticable at
present. It was noticed, however, that arranging
the chromosomes in size order brought about
certain regularities also in the other parameter.
Pairs Nos. 6-8 presented no difficulty of this
kind, since they were always identifiable. The
idiogram resulting from all measurements is
presented in Fig. 4. The length unit is percentage of the total haploid set. It is seen that the
long arm of No. 6 is the second longest arm of
the set, after the long arm of No. 1. Shortest
arms are the short arms of Nos. 7, 8 and 6 in
decreasing order. The short arms of the satellited
chromosomes exhibit certain regularities, as in-

of cells








dicated above. In both No. 6 and 7, the satellite

takes about 75% of the arm length, the exact
values in our measurements being:
No. 6:73.9 f 0.97
No. 7: 75.9f 0.82
Looking at the idiogram as a whole, it is seen
that the satellite chromosomes break the regularity of the nonsatellite chromosomes, in which
both total length of the long arm fall upon nearly
straight lines. The slope of the former is more
pronounced than that of the latter, which means
that the ratio long arm : short arm increases with
decreasing total length. The actual values for
total lengths and arm ratios are given in Table 5,
which is based on the variance analyses described
below, from which the 10 orcein cells were
To illustrate the variation between the different clones, averages for each of the 16 chromosomes were calculated separately for each clone
and compared. The results in regard to chromosome length are given diagrammatically in Fig. 5
and in regard to arm ratio in Fig. 6. These
figures give a strong impression of variability,

Table 5. Total length and arm ratio

Chromosome Total length in %
pair No.
Mean Standard

Fig. 4. Averag idiogram of the 4 clones studied, x

axis: the 8 chromosome types in order of decreasing
size, y axis: length expressed in % of total haploid
chromosome length.



Arm ratio




Hereditas 72, 1972





Fig. 5. Average dimensions of the 16 mitotic chromosomes; in each group of 5 bars, from left to right: clones
OH,UH,LH,OU and Pisa; length unit: % total haploid length.

and it is interesting to find that OU, which

morphologically is most distant from the others,
often shows deviating values (No. 4 from the
left in each group of 5 in Fig. 5 and 6). The LH
type (No.2 from the left), which we know is an
interchange heterozygote for 2 large chromosome pairs, gave very few signs of deviation from
the other clones of H type (Nos. 1 and 3). The
Pisa clone (No. 5 from the left) often seems to
follow the OU and deviate from the 3 H types.
In the hope of obtaining somewhat more precise information concerning the differences observed, whether accidental or significant, the
entire material was submitted to variance analysis. The 2 levels of variation, between clones
and between cells within clones, were investigated.
It is true that objections may be raised against
this procedure because of the nature of the present
data, especially concerning the 5 longest chromosome pairs. At least in the 3 shortest pairs,
however, which are individually identifiable, the
analysis should be permissible. In addition, all
Hereditas 72, 1972

the means were further tested by t analysis

between pairs of clones. Because of the rather
good agreement between the 2 homologues of
each pair in a cell, they were represented in the t
analysis by only one value each. Even though a
preliminary test indicated that there was no
significant variation in the relative values between
the Feulgen and the orcein materials, the latter
were excluded from this analysis. The total
number of cells in these calculations was 50,
distributed as follows: OH, 15 cells; UH, LH
and OU, each LO cells; Pisa, 5 cells. The 3 properties studied were total chromosome length,
ratio long arm: short arm and the dimensions of
the satellite chromosomes.
The variance analysis and the t tests were
undertaken to explore the probability that structural chromosome differences existed among the
clones. As summarized in Fig. 7 the 2 methods
of analysis gave concordant results. In this diagram, probabilities that accidental causes underlie
the variation are recorded by 1, 2 and 3 asterisks








Fig. 6. Ratio long to short arm, same arrangement of diagram as in Fig. 5.

for probabilities below 5, 1 and 0.1 yo, respective- In the satellited chromosomes, Nos. 6 and 7,
ly. For each of the 8 chromosome types, the result significant differences abounded, including both
of the variance analysis is given in the single chromosome length and arm ratio. Especially
square on top, and the result of the t tests in No. 6 exhibited many significant differences acthe triangular arrangement of squares below. In tually in 9 of the 10 comparisons performed, 4
each square of the diagram, asterisks in the upper of the differences with a significance of 0.1%.
part refer to chromosome length and the lower Chromosome No. 7 had significant differences
part to arm ratio. Blank squares indicate that no in 6 comparisons, all involving either clone 4 or 5.
significant difference was established in the Chromosome No. 8 had 4 significant differences,
none of which reached a P value of 0.1.
comparison in question.
Altogether, the results of this rather crude
Among the 5 longer chromosomes with nearly
median centromere, No. 5 was without any signi- analysis gave a very good indication that structurficant difference, Nos. 1 and 3 had single differ- al differences exist among the chromosomes of
ences, significant on the 5 % level, whereas Nos. 2 the 5 clones compared. Significant differences
and 4 had 2 differences each significant on the were found in 42 of the 160 t tests. Each differ0.1% level. In chromosomes 1 and 3, 5 of the 7 ence involved 2 clones, and the distribution of
significant differences were in arm ratio, 4 of them the 84 involvements among the 5 clones is reinvolving clones 1 , 2 and 3; in chromosomes 2 corded in Table 6. From this it is seen that 52
and 4, all 9 significant differences were in chro- involvements referred to chromosome length and
mosome length, all except 2 involving clone 4. 32 to arm ratio. Generally speaking, the involveHereditas 72, 1972



Chromosome No.






** ***


*** **


Fig. 7. Comparison between the 5 clones analyzed of the total length (upper asterisks in each square) and arm
ratio (lower asterisks) of the 8 chromosome pairs; for each chromosome the upper single square shows
differences between clones as determined by variance analysis; the lower squares arranged into triangles show
the results of reciprocal t tests between pairs of clones; 1 to 3 asterisks indicate differences with probabilities
of 5, 1 and 0.1%, respectively; clones Nos. 1-5: OH, U H , LH, OU and Pisa.

ments were scattered fairly equally over the 5

clones. Clone 4, however, was clearly involved
more often than the others in chromosome length
variation. Since clone 4 (OU) was the only representative of the U type, it was taxonomically
well separated from clones 1-3, which were all
of the H type. The lower rate of significant
differences in clone 5 is undoubtedly due to the
low number of measurements - only from 5
cells - being available from this clone.
The heavy involvement of the satellited chromosomes, Nos. 6 and 7, called for a specific
analysis. In all measurements of these chromosomes the proximal segment of the short arm and
the satellite had been measured individually.
The averages of the long arms, proximal segments
and satellites were expressed in relative values in
order to eliminate differences in contraction
among cells. It was found that, if the clones were
ordered according to increasing total length of
chromosome 6, the order became 4-1-5-3-2, and
of chromosome 7, 5-4-3-1-2. Within each of
these series the 3 different elements, long arm,
Herediias 72, 1972

proximal segment and satellite, showed a general

increase in length. As seen in Fig. 8, in which
the length of each element is expressed as percentage of the mean of the 5 clones, there are
certain deviations from what would be expected,
if the increase in total chromosome length were
the sum of a gradual and equal increase of all 3
elements. Especially the proximal segment exhibited considerable fluctuations, indicating heterogeneity among the clones. This method of
analysis is another aspect of the average dimensions given in Fig. 5 and is in good agreement
with the values underlying this figure. Some differences between them may be due to the fact that
the orcein-stained materials were included in the
present calculations but not in those of Fig. 5.
The satellite lengths of chromosomes 6 and 7
were also expressed in relation to the long arm
and to the proximal segment of the short arm
in the same chromosome. The 2 ratios, long arm
to satellite (q : sat) and satellite to proximal
segment (sat : prox) were used by BATTAGLIA
(1963) in Allium sativum and by BOTHMER
( I 970)





Fig. 8. Relative length of long arm (L. a.), proximal

segment of short arm (Prox.) and satellite (Sat.) in
chromosome 6 (above) and 7 (below); each length
value expressed as percentage of the mean value of all
clones; the clones are ordered from left to right
according to increasing total length of chromosome 6
and 7, respectively.

in Allium ampelopmsum, respectively. Both these

indices have the shorter chromosome element as
denominator. They were calculated in the present
material, and also the inverted ratios, sat : q
and prox : sat. In the entire material, the following averages for these indices were obtained:

Chromosome No.

3.49 f 0.041 I
0.29 f 0.0033

1.77 f 0.0237
0.57 f 0.0075

2.80 & 0.0449

0.37 f 0.0058

3.18 5 0.0543
0.32 & 0.0054


straight (q : sat)
inverted (sat : q)
straight (sat : prox)
inverted (Drox : sat)

The differences between the indices of the

individual clones were tested by variance and t
Hereditas 72, I972



C hromorome N o .


Clone No.



Fig. 9. Comparison between the satellite indices of the 5 clones studied

upper part, BOTHMER:
lower part, chromosomes 6; left part,
chromosome 7: right part); in each square, upper asterisks represent the
straight, and lower the inverted indices; clone 3, showing satellite
constriction only in I of the cells analyzed, was excluded in right part of
diagram; meaning of asterisks and clone numbering same as in Fig. 7.

analyses in the same way as the differences in

chromosome length and arm ratio, and the
results are presented in Fig. 9. In the upper part
of the figure, Battaglias index is given, and in the
lower half Bothmers index; in both cases the
original, straight indices are represented by
asterisks in the upper part of each square, and
the inverted indices in the lower part. In chromosome No. 6, the 2 indices gave largely concordant
results, in chromosome No. 7, however, there
were no significant differences using Battaglias
index, whereas Bothmers index resulted in 3
differences with a significance of 5-1 . In
chromosome No. 6, the variance analysis gave
good evidence of significantly more variation
among the clones than among cells within clones,
and the t analysis indicated significant differences
in all comparisons except 3. In 2 comparisons,
clones 1-3 and 3-4 exhibited highly significant
differences in both Battaglias and Bothmers
indices. Comparing the straight with the inverted
indices, the latter gave somewhat higher signiHerrditas 72. I972

ficance in 6 comparisons,
in 7, thus indicating that
having the bigger value
were slightly more efficient
P values.

lower in 2, and same

the inverted indices,
in the denominator,
in securing significant

2. Meiotic chromosomes
Observations on meiosis were made in the 2
clones OH and LH. It would have been valuable
to study meiosis in OU, but this was not feasible,
since flowers never develop in this clone.
CIorie OH. This clone forms 8 bivalents at
meiosis, and the majority of the cells d o not
show any obvious irregularities. The appearance
of the 8 bivalents of 1 cell at metaphase is presented in Fig. IOd. Ring bivalents are most
common, but usually 1 or 2 rods are present in
each cell. Exceptionally as many as 5 rod bivalents
have been seen, and even univalents may occur at
metaphase. Casual observations in other clones
of the Olomouc type collection have revealed



Fig. 10. First meiosis in a -c: clone L H , d: clone OH; a- b: diakinesis, c

Orcein, ~ 2 4 0 0 .
amphibivalent and 6 bivalents, d : 8 bivalents.

d: metaphase I ; a

c I

Hereditas 72. I972



occasional cells, in which all chromosomes are

present as univalents at the first meiotic division,
but no such cells were seen in the present materials.
Scanning through a great number of cells,
frequent disturbances were noted in the meiosis
of the OH clone. The most common deviation
was the presence of chromatic bodies outside of
the spindle both during the first and the second
division. They were especially striking during the
second division, when they often formed spherical
stained bodies in the periphery of the cells both
during metaphase and anaphase. In the pollen
tetrads they often formed small extra pollen cells.
Since they were decidedly more frequent during
the second division, they probably represented
small acentric fragments formed during the first
division and becoming free during interkinesis.
Ordinary acentric fragments were often seen
between the anaphase groups at first meiosis,
and conventional inversion bridges with acentric
fragments were common enough to suggest the
presence of rather big inversions between homologues. In one slide the following irregularities
were noted in some 50 cells scanned at anaphase
Inversion bridge with acentric fragment
1 acentric fragment
1 lagging univalent
2 lagging univalents


and in the same slide some 100 cells at metaphase

- anaphase I1 had 10 cells with 1 or more. chromatic bodies outside the spindle.
In another slide the following counts were made
of irregularities, observed in 92 cells at anaphase
Inversion bridge with acentric fragment
Bridge, no acentric seen
I acentric fragment
1 lagging univalent
2 lagging univalents


These rather casual observations give fair

evidence that the clone OH has developed structural differences between the 2 homologous
haploid sets.
Clone LH. This is the other clone, in which
meiotic observations were made. As mentioned,
the meiosis of this clone has a very striking feature: 2 of the bivalents are involved in an interchange ring of 4 chromosomes, an amphibivalent
Hereditas 72, 1972

according to the nomenclature of HAKANSSON

(1931). This ring is very obvious from diplotene
through diakinesis and metaphase I. Its appearance may be seen in Fig. 1Oa-c and Fig. 1 la-e.
It is possible to see that the ring is constituted
by 2 of the biggest chromosome pairs. Judging
from the large size of 2 of the chromosomes of
the ring and the difference in size between them
and the other 2 chromosomes involved, we
assume that the chromosome pairs in question
are Nos. 1 and 3, but it is difficult to be quite
sure, since the shape of the chromosomes is
rather irregular during first meiosis. That the
chromosomes of the ring belong to 2 pairs of
rather different size is especially clear during
diakinesis (Fig. 1 la, d). The general character of
the ring suggests that the 8 chromosome arms
involved behave much as usual, forming 1 or 2
chiasmata with each other. This would mean
that the point of translocation most likely is
close to or in the centromere. This is compatible
with the fact that no difference in mitotic chromosome morphology was noticed in the two
chromosome pairs most likely involved. If the
point of translocation was near the centromere in
both chromosomes and the interchange took
place in such a way that one of the translocation
chromosomes obtained both the long arms and
the other both the short arms, the change from
the normal somatic karyotype would be negligible
as far as chromosome size is concerned. Instead
of the normal chromosomes 1 and 3 with the
following measurements:
No. 1 : 8.3+7.5=15.8; arm ratio: 1.10
No. 3: 7.4+6.4= 13.8; arm ratio: 1.15
we would obtain the 2 translocation chromosomes:
Long arms: 8.3 + 7 . 4 = 15.7; arm ratio: 1.12
Short arms: 7.5 6.4= 13.9; arm ratio: 1.17.

The ring was analyzed in 40 cells, 24 at diakinesis and 16 at metaphase I. The number of
chiasmata per ring varied as follows:
Number of chiasmata:
Number of cells:

4 5
18 18



Most configurations were rings, 1 was an open

chain. The orientation at metaphase I was
usually nondisjunctional, a big open ring with
neighboring centromeres facing towards the same


Fig. 1 I . First meiosis in clone LH, a. d: diakinesis, b, c, e: metaphase 1.

pole. Among the 16 configurations analyzed,

14 were open rings, 2 were zigzag orientations.
During diplotene and diakinesis, nucleoli were
often visible in the orcein slides (Fig. Ila, d).
There were normally 1 or 2 nucleoli per cell,
attached to a specific region of 2 bivalents,
easily recognizable as Nos. 6 and 7 (Fig. 12).
Even though chromosomal phenotype varied a
great deal from mitotic appearance, there was
little difficulty to identify the long arm, the centromere, sometimes apparent as a deep constriction, the proximal segment, the satellite fiber and
the satellite. Often the proportion between the




satellites of Nos. 6 and 7 was the same as in

mitotic cells, but at other times these structures
were distorted, either overcondensed or the
opposite. The satellite fiber sometimes appeared
thick and swollen and of another constitution
than the rest of the chromosome (Fig. 12a). The
nucleoli were often huge, usually bigger in pair
6 than in pair 7 (Fig. 10b is an exception). Sometimes the 2 nucleoli fused, still with the 2 chromosome pairs attached (Fig. 10a), sometimes one
nucleolus or both were divided into 2 lobes, 1
for each chromosome (Fig. 12e, f).
The first meiotic division in this clone proceeds
Hereditas 72, I972




F g. 12. Bivalents Nos. 6 and 7 from 6 cells at diakinesis, showing nucleolar attachments. Orcein, x 2400.

fairly regularly. Usually 8 chromosomes go to

each pole. The ring causes some disturbances,
and lagging univalents were frequent at first
anaphase. Among 92 cells analyzed at this stage,
49 were apparently quite regular, 3 I had 1 lagging
univalent and 4 cells had 2. Among these cells
were also 4 with 1 bridge and 1 acentric, 2 cells
with only a bridge and 1 cell with only a fragment.
The second division is usually regular (Fig.
le). In 1 slide 120 cells at second metaphase were
analyzed; 114 were apparently normal (in 10
cells the chromosome number was determined to
8), 6 cells had a chromatin body outside the
Hcwdiitas 72. I972

spindle, in one of these cases the body was in a

small separate cell in the dyad.
Cursory examination of meiosis in a number of
other clones has been made by one of us (Konvifka). In all of them, most cells show a generally
good pairing into 8 bivalents, and in all of them
a certain proportion of cells with irregularities
occur. In conjunction with the experiences from
the 2 clones OH and LH reported here, the results
indicate that each clonc has its own characteristic
pattern of abnormalities. The genome of AIlium
surivum is evidently in the process of differentiating into a variety of slightly different forms,
characterized by their structural chromosome



organization. The materials studied by us showed

more meiotic disturbances than the 3 populations
of KOUL and GOHIL(1970), in which meiosis
was completely regular.

diately become established independently of

structural hybridity, triploidy, aneuploidy or
other obstacles to fertility. In AIlium sativum,
the sterility is undoubtedly more deeply rooted
than in Allium carinatum; the few experiments we
made removing the bulbils were without effect,
and in AIlium sativum the pollen development was
never seen to reach the first pollen mitosis,
In the present paper, the chromosomes of 4 whereas in Allium carinatum mature binucleate
strains of Allium sativum are described and pollen was formed regularly.
compared. Since, according to all experience,
Another diploid Allium form, AIlium cepa
this species never produces any seed, each of the proliferum, the so-called Egyptian or tree onion,
strains studied may be considered a clone, is similar to sativum in propagating exclusively
completely isolated from the other clones of the vegetatively and in having no pollen developing
species. Each clone had a characteristic external beyond the one-nucleate stage in the solitary
morphology and growth habit, and the clones flowers formed. It differs from Allium sativum,
examined represented 2 of the 3 main types however, in being clearly of hybrid origin: its
recognized within Allium sativum.
mitotic chromosomes form 2 groups of 8,
In spite of the certainly long isolation of the between which the chromosomes differ in size,
clones, their karyotypes were generally con- form and heterochromatic properties, and at
cordant both in number and gross morphology meiosis univalents predominate, 0-7 bivalents
of the chromosomes. Also the morphologic being formed per cell (LEVAN,LEVANand
homology between the individual bivalents was KONVIEKA
unpubl.). Judging from the satellite
preserved, the mitotic chromosomes being easily chromosomes, one of which is similar to the
arranged into 8 pairs and meiosis normally satellite chromosome of AIlium jistulosum and
showing good bivalent pairing. These facts show the other to that of Allium cepa but without
that Allium sativum is derived originally from a satellite, these 2 species are involved in the origin
diploid sexual species, which lost the capacity of of Allium cepa proliferum, even though the
sexual reproduction. This is a situation common hybrids so far produced artificially between them
in the genus Allium, even though in many types do not develop bulbils. In the case of Allium
a certain capacity for sexual reproduction is sativum it would be valuable to search for fertile
maintained also in those mainly propagated by forms in nature. It should also be attempted by
bulbils. Thus, in triploid forms of AIlium carina- the application of environmental stimuli to move
tum that were normally completely seed sterile the balance over onto the sexual side.
removal of the bulbils resulted in some seed
As is well known, meiosis in sexual organisms
setting (LEVAN1937), and in diploidiforms seed functions as an efficient mechanism to maintain
are produced along with bulbils under natural structural homology within the bivalent pairs.
conditions (DIANNELIDES
1944, 1947). Still, Conversely, release from the meiotic control is
even in the diploid forms the vegetative mode expected to lead to the accumulation of strucof propagation often predominates as shown by tural abnormalities in the karyotype. Striking
(1 962) and by although distant examples of the latter are the
(1 947, 1964). These authors long-term cell cultures of both animal and plant
demonstrated by means of chromosomal criteria origin. Especially in animal cell lines it is
that giant clones of diploid and triploid now a common experience that the stemline
Allium carinatum, reaching an extension of 28 karyotype undergoes extensive evolutionary
kilometers and more, existed in the eastern alps changes from the normal karyotype of the origiof Europe. The combination of vegetative and nal species. Usually the changes are no more
sexual propagation, common in many apomicts extreme than to make it possible to recognize
(cf. Poa alpha, MUNTZING and MUNTZING what species the culture comes from, but cases
1971), is evidently ideal for rapid genetic adapta- are known, where even this is difficult. Anyhow,
tion to the environment: new successful segre- most cell cultures rapidly undergo chromosome
gants from sexual recombination can imme- changes of quite another magnitude than those

Hereditas 72, 1972



observed in the ANium sativum clones. What is

said here about permanent cell cultures is true
also with the parasitically growing tumor cells.
Both these materials undergo evolutionary
changes and show similarities karyotypically
with vegetatively reproducing organisms. In
comparison with plant species as Allium sativum
there is an essential difference: cells in tissue culture, in addition to the release from meiotic
control, have become exposed to a radically
changed environment, and tumor cells have undergone a fundamental change in their reaction
to the environment; moreover, in the case of both
materials there is no longer the requirement to
build up a complex multicellular organism:
the entire evolutionary potential can be concentrated on cell growth and cell proliferation. The
many extremely successful permanent cell lines,
as WL11 as the tumor stemlines with their extremely high competitive capacity, constitute good
evidence that in their case the vegetative mode of
propagation is by no means the evolutionary
cul-de-sac it is in the multicellular organisms.
It is of considerable interest that significant
differences were demonstrated among the Allium
sativum clones in 3 chromosomal parameters.
The differences were of the same kind and were
established in the same way as the differences in
marker chromosomes among 8 natural populations of the Allium ampeloprasum complex in
the Aegean area (BOTHMER
1970). Thus, in both
materials the differences were usually demonstrable only by detailed chromosome measurements;
in other words, they belonged to the category of
cryptostructural variation. Chromosome length,
arm ratio and satellite index were slightly different and in several cases the differences were
statistically significant. In Allium ampeloprasum
geographical isolation had had the same effect
as clonal isolation in Allium sativum. In the allogamous species Secale cereale, HENEEN( 1 962)
demonstrated that inbred lines were characterized
by significant cryptostructural differences. In this
case inbreeding had been enforced for some 30
generations, and the chromosome differences
among the inbred lines primarily illustrates
individual features of the pool of structural
variation normally occurring in the original
population and made homozygous by the inbreeding (cf. MUNTZING1939).
In Allium sativum the satellite chromosomes
were affected by structural deviations strikingly
Herediditas 72, 1972

more often than the other chromosomes. In

A Ilium ampeloprasum the satel lite chromosomes
were the only ones, in which the differences were
tested statistically, but at least in arm ratio (I.c.,
Table 7, p. 530) the means showed considerably
higher variation in the satellited chromosomes
No. 7 and 8 than in the others. In the Allium
carinatum material quoted above, only the satellite chromosomes showed very obvious variability. Thus, while all other chromosomes remained
m and sm, at least 1 satellite chromosome had
changed into a clear st type. It should be noted
that also in the genus Allium as a whole, the
satellite chromosomes show much more variation
in arm ratio than the other chromosomes. In
the rye material of HENEEN(1962) the satellite
chromosome No. 7 was the only one showing
significant differences in arm ratio at the 1 and
0.1% levels in all comparisons between the 3
inbred lines tested. These results are compatible
with results from many other organisms, including man, indicating that satellite chromosomes are liable to greater hazards leading to
breaks and translocations than other chromosomes. This situation is well illustrated by the
results of our variance and t analyses in Allium
sativum (Fig. 7 ) .
In sexual organisms those cryptostructural
changes that are successful in passing the meiotic
filter have much better chances to spread rapidly
in the population than similar changes in vegetatively propagating organisms. In the latter even
potentially favorable changes will easily become
lost in the multicellular meristems, and only
those which succeed in gradually infiltrating a
big fraction of the clonal germline, i.e. the
meristems producing side bulbs or bulbils in the
inflorescences, will have evolutionary significance. This must take much time, and the advantage these forms have gained in escaping the
meiotic control will be counteracted by the slowness with which a change comes to expression.
That this type of changes is of evolutionary
importance has long been appreciated by A. K.
and his associates. Their idea is founded
on numerous investigations in plants with mainly
or exclusively vegetative propagation, belonging
to Marantaceae, Liliaceae, Amaryllidaceae, Araceae and many others. Chromosome variation,
numerical and structural, was demonstrated on
all levels: between cells in the meristems of the
individual plants, between clones within the



species and between related species. It was conIn addition to the differences of cryptostruccluded that speciation in vegetatively reproduc- tural nature, certain of the Allium sativum
ing plants is effected through the entrance of clones had undergone major chromosomal
somatic alterations of chromosomes into the changes. Thus, the American material of Allium
growing tip of the daughter shoots (SHARMA sativum, studied by BATTACLIA
(1963) exhibited
1956, p. 188), or alterations in number and gross alterations in 4 chromosome pairs, among
morphology of chromosomes in somatic tissues them the 2 satellite pairs. Our clone LH had
are, therefore, responsible for speciation in the undergone an interchange, probably involving
genus, which is brought into effect through vege- the entire arms of 2 of the biggest pairs. The
1964a, latter change, showing as a ring or a chain of 4
tative propagation (SHARMA
p. 180).
at first meiosis, has been observed repeatedly in
It is evident that the chromosome variation various AIlium species (LEVAN 1935, 1939;
observed among the clones is not exclusively due KATAYAMA
1936; KOUL 1963, 1966; RICKARDS
to changes in the somatic cells of the clones. 1964). It will be interesting to study a greater
Part of the variation probably represents rem- number of different sativum clones to look for
nants from the structural variation that existed more patterns of structural variation and to try
in the original sexual population, from which and correlate them with other properties of the
the clones descended, thus the same type of clones. The chromosomal variability is compatchanges as among the inbred Secale lines. Since ible with observations of morphologic deviations
it is difficult to estimate the age of the clones, appearing in clonal materials. In our clone of
the question as to the relative significance of the Allium cepa proliferum most bulbils have darkly
2 factors cannot be determined; probably both purple external bulb coats, but on several occafactors have participated. The fact that in Allium sions plants have appeared completely lacking
sativum pairwise structural homology has been the purple color. It is known that different garden
preserved to a predominant extent indicates either varieties of Allium cepa proliferum differ conthat more profound changes of the basic AIiium siderably in morphology and include triploid
karyotype cannot exist with maintained viability, types (KOUL and GOHIL1970).
or that the period without meiotic control has
Another property that may be a sign of genetic
been too short to let more extensive changes differences is the different phenotypic manifestaaccumulate. The fact that in many of the clones tion of the satellite constriction in chromosome
studied by the SHARMA
group even somatic No. 7 in Allium sativum. Our clone LH showed
numbers with apparently homologous pairs pre- rarely any constriction in chromosome 7, and
dominated, although the mechanisms of chromo- when it appeared it was always very indistinct,
some variation in the meristems should give rise whereas clone OU had a very clear constriction
to both even and odd deviant numbers, is in in both homologs in every cell. The other 2
line with the conditions in our Allium sativum clones, OH and UH, were intermediate, usually
clones. Thus, 7 clones of Colocasia antiquorum having a clear constriction in one of the homologs
had 2n=22, 26, 28, 38 and 42 (SHARMA
and and often in both. A similar situation was found
(1970) in the Allium ampeloprasum
1963), and 40 clones of Caladium bicolor by BOTHMER
had 2n=22, 26, 28, 30 and 32 (SHARMA
and complex. Two diploid populations of Allium
1964b; SARKAR1971), and no clones bourgaei, belonging to this complex, had, in
with odd somatic numbers were found in these addition to the 2 satellite pairs Nos. 7 and 8,
2 materials. It seems that the preservation of seen in all ampeloprasum forms, a third satellite
homologous pairs is associated with better via- chromosome, No. 6, which was not seen in any
bility even in some materials in which meiosis other of the forms studied. Similar observations
has been circumvented. As is well known, there were made by ISING(1969) in collections from
do occur odd somatic numbers in many spon- natural populations of Cyrtanthus breviflorus, in
taneous materials with vegetative propagation, which a tetraploid type had a constriction in the
as in the apomictic species Poa alpina, in which middle of the long arm of the second longest
especially at polyploid levels odd and even chromosome, while other tetraploid and diploid
numbers often are equally viable (MUNTZING collections were without this constriction.
1954; MUNTZINC and MUNTZING 1971).
In conclusion, the significant karyotypic differHtreditas 72, I972



ences demonstrated between the clones of Allium

sativum have at least partially developed after
the loss of sexual propagation of this species.
In conjunction with the morphologic and
physiologic differentiation among the clones, the
karyotypic variability indicates that even with
complete lack of sexual recombination a species
is capable of evolutionary adaptation to the

- The present work, belonging to a

project for the development of cytogenetic test systems,

was supported by grants from the John and Augusta
Perssons Foundation. We are also grateful to the late
Professor Nils Hylander for material, to Professor Per
Wendelbo for control determination of the LH clone,
to Doctors Roland von Bothmer, Gunnar lsing and
Goran Levan and to Professor Arne Miintzing for valuable help with the manuscript, and to Miss Kerstin
Nyholm for skillful technical assistance.

KOUL,A. K. 1963. A spontaneously occurring translocation heterozygote of Allium cepa. - J . Ind. Bot. Soc.
42: 416-418.

- 1966. Structural hybridity in AIIiurn atropurpureum

WALDST.and KIT. - J . Cytol. Genet. I: 1-5.
KOUL,A. K. and GOHIL,R. N. 1970. Causes averting
sexual reproduction in Allium sativum LINN.- CytoIogia 35: 197-202.

LEVAN,A. 1935. Cytological studies in Allium, VI. The

chromosome morphology of some diploid species of
Allium. - Hereditas 20: 289-330.

- 1937. Cytological studies in

the Allium paniculatuni

group. Ibid, 23: 317-370.
- 1939. Amphibivalent formation in Allium cernctrtm
and its consequences in the pollen. - Bot. Not. p.

A. A. 1964.
Nomenclature for centromeric position on chromosomes. - Hereditas 52: 201-220.
S . W. 1939. Cytogenetic studies in the genus
Allium. - J . Genet. 39: 1-45.
MUNTZING,A. 1939. Chromosomenaberrationen bei
Pflanzen und ihre genetische Wirkung. - Z. Indukt.
Abstamm.- Vererbungsl. 76: 325-350.

- 1954.

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R. v. 1970. Cytological studies in AIlium 1.
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von diploidem Allium carinatum. - Wiener Bot. Z. 9 3 :

- 1947.

Cytologische Untersuchungen an spontanem

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Inr. Rev.

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A. 1931. u b e r Chromosomenverkettung in

The cytological basis of polymorphism in

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~ S T E R G R E N ,G. and HENEEN,W. K. 1962. A squash
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G. K. 1964. Some theoretical aspects of selective segregation in interchange complexes. - Chromosoma 15: 140-155.

A. K. 1971. Mode of evolution in Caladium
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A. K. 1963. Cytological
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- 19646.

Studies on the cytology of Caladium bicolor

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inbred rye. - Ibid. 48: 182-200.
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K. and K O N V I ~ K0.
A ,1954. Polni pokusy (Field TSCHERMAK-WOESS,
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S-223 62 Lund, Sweden

- Hereditas I S : 17-61.

Hereditas 72, 1972