allium sativum

Hi>reclitas 72: 129-148 (I9721

Chromosome studies in Alliurn sativurn 0. KONVICKA A N D ALBERT LEVAN

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 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 sterile.

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 well- developed flowers will increase if the bulbils are removed, but even so pollen will not develop beyond the one-nucleate stage and seeds will not form.

implications of our observations for karyotypic

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 DEYSSON and collaborators (see review, DEYSSON 1968). The chromosomes of Allium sativirm have been described in the literature repeatedly (MENSINKAI 1939; KHOSHOO et al. 1960; BATTA- 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 exa- mine 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

9 Hcreditas 72. I972

130 0. KONVIEKA AND ALBERT LEVAN

diverging morphologic types were selected for analysis from the type collection of the Olomouc institute.

Material and methods On the basis of important morphologic and physiologic characters the species of Allium sativum may be divided into 3 main types ( H R U B ~ and KONVIEKA 1954):

(1) The H type has bulbs with violet bulb coats and with cloves in 2 groups. Flower stems develop regularly. The leaves are broad and are formed in a definite number. The H type comprises late forms, which require much water and remain upright when ripe.

(2) The U type and (3) the A type both have bulbs with white bulb coats and with cloves in several groups. Flower stems develop rarely and only after heavy vernalization. Leaves are formed in indefinite number. Both types are lying down when ripe. They differ from each other in the following respects: The U type comprises late forms with broad leaves and requires much water, whereas the A type comprises early forms with narrow leaves and requires less water.

In the present investigation only the H and the U types were represented with 3 and 1 clone, respectively. Here follows a brief description of our material:

(1) The Olomouc No. 15K, an H type, referred to below as OH or No. 1 . This clone, derived from the farm of Mr. K. Kontny, Olomouc, and commonly grown in the village of ternovir near Olomouc, was included 1965 in the type collection of the Olomouc institute. It has large bulbs with medium-sized cloves. The inflorescences contain almost exclusively bulbils of medium size. It belongs to the variety ophioscorodon (LK.) D~LL., characterized by the flower stem being twisted into a spiral in its upper part.

(2) The Uppsala clone, an H type, referred to below as UH or No. 2. This clone was kindly presented by Professor Nils Hylander, Uppsala. It has been grown in the Uppsala botanic garden since 1951, when it was obtained from Bergen, Norway. According to Dr. Hylander, this is a typical garden variety, ordinarily completely without flowers. It also belongs to the variety ophioscorodon.

(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 many small bulbils in the inflorescences. Professor Per Wendelbo, Gothenburg, has kindly made a preliminary taxonomic determination of this type and found it to correspond to Allium longicuspis REGEL, which according to VVEDENSKY (1935) is a wild predecessor of Allium sativum. This is the type that forms a chromosome ring at meiosis.

(4) The Olomouc U T clone, referred to below as OU or No. 4. This clone, derived from the farm of Mr. T. Konvi;ka, Kuncice, Walachei, not far from the birth place of Gregor Mendel, was included in 1967 into the type collection of the Olomouc institute. The bulbs are fairly large with big cloves.

In the statistical treatment below, comparisons will be made with the chromosome measurements of BATTAGLIA (1963) in Allium sativum material purchased in Pisa, Italy. Although BATTAGLIA did not discuss the genetic status of his material, it will be referred to below as the Pisa clone or No. 5.

Mitotic chromosomes were examined in root meristems. Root tips were obtained by dipping cloves through a plastic net into running tap water bubbled through with air and kept at 16°C. Meiosis was studied in anthers and to a lesser degree in ovules in flower buds taken in the field. For temporary preparations, root tips and flower buds were fixed in 60% acetic acid with 0.1 n hydrochloric acid, macerated by warming the fixative to 60°C and squashed in 2% orcein in 60% acetic acid. Permanent slides were made after various fixations followed by Feulgen staining, occasionally by Feulgen and light green. Very good results were obtained with the fixative of ~ S T E R G R E N and HENEEN followed by the schedule worked out by these authors (1962). For chromosome measurements, chromosomes were drawn in the center of the viewfield with the aid of a camera lucida at a magnification of 7000 times.

Hereditas 72, 1972

CHROMOSOMES OF ALLIUM SATIVUM 13 1

Fig. 1 . MetaDhase chromosomes of root mitosis in clone OH, at arrow: nucleolar organizing body. ~ Feulgen, ~ 1 7 0 0 .

Observations 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 BATTACLIA (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 LEVAN et 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

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 ori- ginated 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 formation. As illustrated by BATTAGLIA (I.c. Fig. 7, p. 37), somatic cells have a maximum of 4 nucleoli. The early formation of nucleoli during anaphase-telophase is illustrated in Fig. 3

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132 0. KONVIeKA A N D ALBERT LEVAN

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

CHROMOSOMES OF ALLIUM SATIVUM 133

L 1 ou I

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 other.

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 corre- sponding to the nucleolar organizer. This was clesrly visible in the original of Fig. I on the extended satellite fiber at about 4/5 of 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 of BATTAGLIA (I.c. Fig. 3, p. 18-19). It is seen from Table 1 that from 60 to 100c~O of the cells 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

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134 0. K O N V I ~ K A AND ALBERT LEVAN

Table 1. Relation between satellite-fiber length and total chromosome length in pair No. 6

Table 3. Relation between satellite appearance and chromosome length in pair No. 7

No. Clone Case (see below) Total No. Clone Satellite on Total pairs

1 2 3 4 longest shortest member member

1 O H 1 4 5 1 - 20 2 U H 13 1 -- 1 15 1 OH 7 4 I I 3 LH 2 3 1 4 10 2 UH 5 4 9

6 1 1 2 10 Total 12 8 20

4 ou 5 Pisa 4 I - - 5

Total 39 I I 3 7 60

Case 1: satellite fiber extended in longest member of pair Case 2: satellite fiber extended in shortest member of pair Case 3 : satellite fiber extended in unspecified member of pair Case 4: satellite fiber extended in both members of pair

in appearance between the 2 members. Com- monly 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 2. Visible satellite in the two members of pair No. 7

No. Clone Visible satellite in Total pairs

both one none

20 1 OH 9 I I 15 2 UH 6 9

3 LH I 9 10 10 4 ou 10 5 5 Pisa 5

Total 31 20 9 60

- -

- - - - -

Hereditas 72, 1972

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 pre- ferable also to consider the location of the centromere in determining the place of each


Table 4. Absolute length in p of total diploid set

No. Clone Feulgen Orcein

Number Mean Standard Number Mean Standard of cells error of cells error

I OH 15 148.6 5.0 5 179.0 9.0 2 U H 10 152.3 5.2 5 174.3 17.0 3 LH 10 127.2 2.9 4 ou 10 133.7 3.0 5 Pisa 5 218.6 17.9 .-

- - .~

- - ~

~ ~

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-

- 1 2 3 4 5 6 7 0

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.

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: 75.9 f 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 excluded.

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 % Arm ratio pair No.

Mean Standard Mean Standard error error

15.78 0.081 14.67 0.048 13.83 0.055 12.79 0.052 11.74 0.052 11.35 0.075 10.52 0.066 9.34 0.059

1.10 0.009 1.13 0.011 1.15 0.012 1.20 0.013 1.19 0.011 2.59 0.035 1.32 0.014 1.67 0.017

Hereditas 72, 1972


15

10

5

1 3 4 5 6 7 8 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 pre- cise 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


3.0

2.5

2.0

15

1D 1 2 3 4 5 6 7 a

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

for probabilities below 5, 1 and 0.1 yo, respectively. For each of the 8 chromosome types, the result of the variance analysis is given in the single square on top, and the result of the t tests in the triangular arrangement of squares below. In each square of the diagram, asterisks in the upper part refer to chromosome length and the lower part to arm ratio. Blank squares indicate that no significant difference was established in the comparison in question.

Among the 5 longer chromosomes with nearly median centromere, No. 5 was without any significant difference, Nos. 1 and 3 had single differences, significant on the 5% level, whereas Nos. 2 and 4 had 2 differences each significant on the 0.1% level. In chromosomes 1 and 3, 5 of the 7 significant differences were in arm ratio, 4 of them involving clones 1 , 2 and 3; in chromosomes 2 and 4, all 9 significant differences were in chromosome length, all except 2 involving clone 4.

In the satellited chromosomes, Nos. 6 and 7, significant differences abounded, including both chromosome length and arm ratio. Especially No. 6 exhibited many significant differences actually in 9 of the 10 comparisons performed, 4 of the differences with a significance of 0.1%. Chromosome No. 7 had significant differences in 6 comparisons, all involving either clone 4 or 5. Chromosome No. 8 had 4 significant differences, none of which reached a P value of 0.1.

Altogether, the results of this rather crude analysis gave a very good indication that structural differences exist among the chromosomes of the 5 clones compared. Significant differences were found in 42 of the 160 t tests. Each difference involved 2 clones, and the distribution of the 84 involvements among the 5 clones is recorded in Table 6. From this it is seen that 52 involvements referred to chromosome length and 32 to arm ratio. Generally speaking, the involve-

Hereditas 72, 1972

138 0. KONVIEKA AND ALBERT LEVAN

Chromosome No.

1

r;l CloneNo.

2 3 4 5

2

3

4

6 I:::I :q$ ** ***

*** ** 3

4

5 zoiance

7

pJ 8

2 3 4 5 2 3 4 5 1 1 I **l*r*l

U '2

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 repre- sentative 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,

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 he- terogeneity 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)

Herediias 72, 1972


Prox.

11’’ I

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:

Index Chromosome No.

6 7

BATTACLl.4 straight (q : sat) 1.77 f 0.0237 inverted (sat : q) 0.29 f 0.0033 0.57 f 0.0075

straight (sat : prox) 2.80 & 0.0449 3.18 5 0.0543 inverted (Drox : sat) 0.37 f 0.0058 0.32 & 0.0054

3.49 f 0.041 I

BOTHMER

The differences between the indices of the individual clones were tested by variance and t

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140 0. KONVICKA A N D ALBERT LEVAN

C hromorome N o .

6 7 Battaglia

Bothme r

Clone No. 2 3 4 5 2 4 5

2 3 4 5

4 U

nce

-test

U Fig. 9. Comparison between the satellite indices of the 5 clones studied (BATTACLIA: 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, Battaglia’s index is given, and in the lower half Bothmer’s 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 Battaglia’s index, whereas Bothmer’s 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 Battaglia’s and Bothmer’s indices. Comparing the straight with the inverted indices, the latter gave somewhat higher signi-

ficance in 6 comparisons, lower in 2, and same in 7, thus indicating that the inverted indices, having the bigger value in the denominator, were slightly more efficient in securing significant P values.

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 a t 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

Herrditas 72. I972


n

Fig. 10. First meiosis in a -c: clone L H , d: clone OH; a- b: diakinesis, c amphibivalent and 6 bivalents, d : 8 bivalents. ~ Orcein, ~ 2 4 0 0 .

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 I: Inversion bridge with acentric fragment 5 1 acentric fragment 2 1 lagging univalent 14 2 lagging univalents 2

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 I: Inversion bridge with acentric fragment 4 Bridge, no acentric seen 2 I acentric fragment 2

2 lagging univalents 4 1 lagging univalent 31

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 fea- ture: 2 of the bivalents are involved in an interchange ring of 4 chromosomes, an amphibivalent

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: 4 5 6 Average Number of cells: 18 18 4 4.65

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

Hereditas 72, 1972


Fig. 1 I . First meiosis in clone LH, a. d: diakinesis, b, c, e: metaphase 1. - Orcein, X 2200.

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). Some- times 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


d e f ? IOU

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

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 (Kon- vifka). 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 differentiat- ing into a variety of slightly different forms, characterized by their structural chromosome

Hcwdiitas 72. I972


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.

Discussion In the present paper, the chromosomes of 4 strains of Allium sativum are described and compared. Since, according to all experience, this species never produces any seed, each of the strains studied may be considered a clone, completely isolated from the other clones of the species. Each clone had a characteristic external morphology and growth habit, and the clones examined represented 2 of the 3 main types recognized within Allium sativum.

In spite of the certainly long isolation of the clones, their karyotypes were generally concordant both in number and gross morphology of the chromosomes. Also the morphologic homology between the individual bivalents was preserved, the mitotic chromosomes being easily arranged into 8 pairs and meiosis normally showing good bivalent pairing. These facts show that Allium sativum is derived originally from a diploid sexual species, which lost the capacity of sexual reproduction. This is a situation common in the genus Allium, even though in many types a certain capacity for sexual reproduction is maintained also in those mainly propagated by bulbils. Thus, in triploid forms of AIlium carinatum that were normally completely seed sterile removal of the bulbils resulted in some seed setting (LEVAN 1937), and in diploidiforms seed are produced along with bulbils under natural conditions (DIANNELIDES 1944, 1947). Still, even in the diploid forms the vegetative mode of propagation often predominates as shown by GEITLER and TSCHERMAK-WOESS (1 962) and by TSCHERMAK-WOES (1 947, 1964). These authors demonstrated by means of chromosomal criteria that “giant clones” of diploid and triploid Allium carinatum, reaching an extension of 28 kilometers and more, existed in the eastern alps of Europe. The combination of vegetative and sexual propagation, common in many apomicts (cf. Poa alpha, MUNTZING and MUNTZING 1971), is evidently ideal for rapid genetic adaptation to the environment: new successful segre- gants from sexual recombination can imme-

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, whereas in Allium carinatum mature binucleate pollen was formed regularly.

Another diploid Allium form, AIlium cepa proliferum, the so-called Egyptian or tree onion, is similar to sativum in propagating exclusively vegetatively and in having no pollen developing beyond the one-nucleate stage in the solitary flowers formed. It differs from Allium sativum, however, in being clearly of hybrid origin: its mitotic chromosomes form 2 groups of 8, between which the chromosomes differ in size, form and heterochromatic properties, and at meiosis univalents predominate, 0-7 bivalents being formed per cell (LEVAN, LEVAN and KONVIEKA unpubl.). Judging from the satellite chromosomes, one of which is similar to the satellite chromosome of AIlium jistulosum and the other to that of Allium cepa but without satellite, these 2 species are involved in the origin of Allium cepa proliferum, even though the hybrids so far produced artificially between them do not develop bulbils. In the case of Allium sativum it would be valuable to search for fertile forms in nature. It should also be attempted by the application of environmental stimuli to move the balance over onto the sexual side.

As is well known, meiosis in sexual organisms functions as an efficient mechanism to maintain structural homology within the bivalent pairs. Conversely, release from the meiotic control is expected to lead to the accumulation of structural abnormalities in the karyotype. Striking although distant examples of the latter are the long-term cell cultures of both animal and plant origin. Especially in animal cell lines it is now a common experience that the stemline karyotype undergoes extensive “evolutionary” changes from the normal karyotype of the original species. Usually the changes are no more extreme than to make it possible to recognize what species the culture comes from, but cases are known, where even this is difficult. Anyhow, most cell cultures rapidly undergo chromosome changes of quite another magnitude than those

10 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 concen- trated on cell growth and cell proliferation. The many extremely successful permanent cell lines, as WL 11 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 demonstra- ble 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 allo- gamous 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. MUNTZING 1939).

In Allium sativum the satellite chromosomes were affected by structural deviations strikingly

more often than the other chromosomes. In A Ilium ampeloprasum the sat el li te 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 ad- vantage these forms have gained in escaping the meiotic control will be counteracted by the slow- ness with which a change comes to expression. That this type of changes is of evolutionary importance has long been appreciated by A. K. SHARMA and his associates. Their idea is founded on numerous investigations in plants with mainly or exclusively vegetative propagation, belonging to Marantaceae, Liliaceae, Amaryllidaceae, Ara- ceae 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

Herediditas 72, 1972


species and between related species. It was con- cluded that “speciation in vegetatively reproducing plants is effected through the entrance of somatic alterations of chromosomes into the growing tip of the daughter shoots” (SHARMA 1956, p. 188), or “alterations in number and morphology of chromosomes in somatic tissues are, therefore, responsible for speciation in the genus, which is brought into effect through vegetative propagation” (SHARMA and SARKAR 1964a, p. 180).

It is evident that the chromosome variation observed among the clones is not exclusively due to changes in the somatic cells of the clones. Part of the variation probably represents rem- nants from the structural variation that existed in the original sexual population, from which the clones descended, thus the same type of changes as among the inbred Secale lines. Since it is difficult to estimate the age of the clones, the question as to the relative significance of the 2 factors cannot be determined; probably both factors have participated. The fact that in Allium sativum pairwise structural homology has been preserved to a predominant extent indicates either that more profound changes of the basic AIiium karyotype cannot exist with maintained viability, or that the period without meiotic control has been too short to let more extensive changes accumulate. The fact that in many of the clones studied by the SHARMA group even somatic numbers with apparently homologous pairs pre- dominated, although the mechanisms of chromosome variation in the meristems should give rise to both even and odd deviant numbers, is in line with the conditions in our Allium sativum clones. Thus, 7 clones of Colocasia antiquorum had 2n=22, 26, 28, 38 and 42 (SHARMA and SARKAR 1963), and 40 clones of Caladium bicolor had 2n=22, 26, 28, 30 and 32 (SHARMA and SARKAR 1964b; SARKAR 1971), and no clones with odd somatic numbers were found in these 2 materials. It seems that the preservation of homologous pairs is associated with better viability even in some materials in which meiosis has been circumvented. As is well known, there do occur odd somatic numbers in many spon- taneous materials with vegetative propagation, as in the apomictic species Poa alpina, in which especially at polyploid levels odd and even numbers often are equally viable (MUNTZING 1954; MUNTZINC and MUNTZING 1971).

In addition to the differences of cryptostructural nature, certain of the Allium sativum clones had undergone major chromosomal changes. Thus, the American material of Allium sativum, studied by BATTACLIA (1963) exhibited gross alterations in 4 chromosome pairs, among them the 2 satellite pairs. Our clone LH had undergone an interchange, probably involving the entire arms of 2 of the biggest pairs. The latter change, showing as a ring or a chain of 4 at first meiosis, has been observed repeatedly in various AIlium species (LEVAN 1935, 1939; KATAYAMA 1936; KOUL 1963, 1966; RICKARDS 1964). It will be interesting to study a greater number of different sativum clones to look for more patterns of structural variation and to try and correlate them with other properties of the clones. The chromosomal variability is compatible with observations of morphologic deviations appearing in clonal materials. In our clone of Allium cepa proliferum most bulbils have darkly purple external bulb coats, but on several occa- sions plants have appeared completely lacking the purple color. It is known that different garden varieties of Allium cepa proliferum differ considerably in morphology and include triploid types (KOUL and GOHIL 1970).

Another property that may be a sign of genetic differences is the different phenotypic manifesta- tion of the satellite constriction in chromosome No. 7 in Allium sativum. Our clone LH showed rarely any constriction in chromosome 7, and when it appeared it was always very indistinct, whereas clone OU had a very clear constriction in both homologs in every cell. The other 2 clones, OH and UH, were intermediate, usually having a clear constriction in one of the homologs and often in both. A similar situation was found by BOTHMER (1970) in the Allium ampeloprasum complex. Two diploid populations of Allium bourgaei, belonging to this complex, had, in addition to the 2 satellite pairs Nos. 7 and 8, seen in all ampeloprasum forms, a third satellite chromosome, No. 6, which was not seen in any other of the forms studied. Similar observations were made by ISING (1969) in collections from natural populations of Cyrtanthus breviflorus, in which a tetraploid type had a constriction in the middle of the long arm of the second longest chromosome, while other tetraploid and diploid collections were without this constriction.

In conclusion, the significant karyotypic differ-

Htreditas 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 environment.

Acknowledgements. - 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.

Literature cited BATTAGLIA, E. 1963. Mutazione cromosomica e cariotipo fondamentale in Allium sativum L. - Caryologia 16: 1 4 6 .

BOTHMER, R. v. 1970. Cytological studies in AIlium 1. Chromosome numbers and morphology in Allium Sect. Allium from Greece. - Bot. Not. 123: 518-550.

DIANNELIDIS, T. 1944. uber das spontane Vorkommen von diploidem Allium carinatum. - Wiener Bot. Z. 93:

- 1947. Cytologische Untersuchungen an spontanem diploidem Allium carinatum. - osterreich. Bot. Z. 94: 74-98.

DEYSSON, G . 1968. Antimitotic substances. - Inr. Rev. Cytol. 24: 99-148.

GEITLER, L. and TSCHERMAK-WOESS, E. 1962. Chromo- somale Variation, strukturelle Hybriditat und ihre F o l p n bei Allium carinatum. - Osterreich. Bot. Z. 109: 150-167.

HAKANSSON, A. 1931. uber Chromosomenverkettung in Pisum. - Hereditas IS : 17-61.

HENEEN, W. K. 1962. Chromosome morphology in inbred rye. - Ibid. 48: 182-200.

HRUB~., K. and K O N V I ~ K A , 0. 1954. Polni pokusy (Field trials). - Olomouc, p. 213-217.

ISING, G. 1969. Cytogenetic studies in Cyrtanthus. IV. Chromosome morphology in Cyrtanthus luteus BAKER (Anoiganthus luteus BAKER) and Cyrtanthus breviflorus HARVEY (Anoiganthus brevijlorus BAKER). - Hereditas

KATAYAMA, Y. 1936. Chromosome studies in some Alliums. - J. Coll. Agr., Tokyo Imp. Univ. 13: 431-441.

KHOSHOO, T. N., ATAL, C. K. and SHARMA, V. B. 1960. Cytotaxonomical and chemical investigations on the north-west Indian garlics. - Res. Bull. ( N . S.) Panjab Univ. 11: 37-47.

66-72.

63: 352-384.

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. - Cyto-

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. 2 5 6 2 5 8 .

LEVAN, A., FREDGA, K. and SANDBERG, A. A. 1964. Nomenclature for centromeric position on chromosomes. - Hereditas 52: 201-220.

MENSINKAT, 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. The cytological basis of polymorphism in Poa alpina. - Hereditas 40: 459-516.

MUNTZING, A. and MUNTZING, G. 1971. An apomictic biotype of Poa alpina in the Koster islands of Sweden.

~ S T E R G R E N , G. and HENEEN, W. K. 1962. A squash technique for chromosome morphological studies. -

RICKARDS, G. K. 1964. Some theoretical aspects of selec- tive segregation in interchange complexes. - Chromo- soma 15: 140-155.

SARKAR, A. K. 1971. Mode of evolution in Caladium bicolor. - Thesis, Calcutta, 54 p.

SHARMA, A. K. 1956. A new concept of a means of speciation in plants. - Caryologia 9 : 93-130.

SHARMA, A. K. and SARKAR, A. K. 1963. Cytological analysis of different cytotypes of Colocasia antiquorum. - Bull. Bot. Soc. Bengal 17: 16-22. - 1964a. A study on the structure and behaviour of

chromosomes in different species of Yucca. - Bot. Tidskr. 60: 180-190. - 19646. Studies on the cytology of Caladium bicolor

with special reference to the mode of speciation. - Genet. iber. 16: 2 1 4 7 .

TSCHERMAK-WOESS, E. 1947. Uber chromosomale Plastizi- tat bei Wildformen von Allium carinaturn und anderen Allium-Arten aus den Ostalpen. - Chromosoma 3: 66-87. - 1964. Weitere Untersuchungen zum chromosomalen Polymorphismus von Allium carinatum. - osterreich.

VED BRAT, S. 1965. Genetic systems in Allium. 1. Chro-

VVEDENSKY, A. 1. 1935. Allium. - Flora URSS 4: 112-

Iogia 35: 197-202.

- Ibid. 67: 143-144.

Ibid. 48: 332-341.

Bot. Z . 111: 159-165.

mosome variation. - Chromosoma 16: 486-499.

280. Albert Levan Institute of Genetics S-223 62 Lund, Sweden

Hereditas 72, 1972

allium sativum

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