Chromosome Variation in the Common Garlic, Allium sativum L.

Transcription

1 Cytologia 43: , 1978 Chromosome Variation in the Common Garlic, Allium sativum L. Received November 11, 1976 S. C. Verma and R. K. Mittal Department of Botany, Panjab University, Chandigarh, India The common garlic, Allium sativum L., is exclusively vegetatively reproduced. Barring mutations, the only source of variation in such species is the occurrence of structural changes in chromosomes. This implies karyotypic heterogeneity and some evidence in favour of this exists for the nucleolar chromosomes of A. sativum (Battaglia 1963). However, as pointed out by Konvicka and Levan (1972), karyotype studies on garlic have not been made as extensively as in Allium cepa although chromosomes of A. sativum have been described a number of times in literature (Mensinkai 1939, Khoshoo et al. 1960, Battaglia 1963). There are three basic types in A. sativum, namely H, U and A types, distinguishable on the basis of morphological characters (Table 1, Hruby and Konvicka 1954). Out of these three types, H and U types have been studied in detail by Konvicka and Levan (1972). It was, therefore, intended to study the A type, commonly available in India, and to compare the same with H and U types. Besides, A. sativum is consistently used in our laboratory as a classwork material, along with A. cepa, which suggested its investigation initially limited to the local material. The preliminary studies carried out on five bulbs have only impressed upon the need to undertake extensive studies of the material from several sources. Material and method In Northern India only A type of garlic is available (Table 1). Healthy bulbs were purchased from the local market. Actively dividing root tips were obtained by placing the "cloves" (bulblets) of five different bulbs in distilled water at room temperature (24 }2 Ž). Three days old root tips were pretreated for 3 hours with a 1:1 mixture of 0.2% colchicine and 0.002M 8-hydroxyquinolene, and then fixed in 1:3 acetic-alcohol. The fixed root tips were hydrolysed in IN-HCI at 60 Ž for 8 minutes, stained with Feulgen and squashed either in 45% acetic acid or in aceto carmine (to intensify stain). The squashes were made permanent following dehydra tion in grades of acetic acid and butanol and mounted in euparal (Bhaduri and Ghosh 1954). Karyotype analyses of chromosomes at metaphase are based on drawings traced from enlarged photomicrographs so as to minimise errors of measurements from camera-lucida drawings. The terminology for chromosome morphology and chro mosome classification are according to Levan, Fredga and Sandberg (1964).

2 384 S. C. Verma Results and R. K. Mittal Cytologia 43 and comments Variation in chromosome size and form has been studied from five dimfferent bulbs of Allium sativum and in each case, although 15 cells were studied, a detailed analysis is given for the best differentiated cell at metaphase. This has been done purposely to make it a case study to discover if there is any necessity for extensive observations on a wide variety Fi gs 1-3. Somatic of material. complement of Allium chromosomes sativum have L. secondary showing 2n=16, ~1000. Arrowed constrictions. Table 1. Characteristics of the three basic types in Allium sativum L. (adapted from Konvicka and Levan 1972) * Pollen development ** Single cloved The flowering never (or shoot simple) reaches first bulbs terminates or in pollen with bulbils; mitosis few cloves there are (Konvicka are reported no flowers. and by Levan Khoshoo 1972). et al. (1960). õ

3 1978 Chromosome Variation in the Common Garlic, Allium sativum L. 385 The somatic chromosome complement of A. sativum comprises 16 chromosomes (Figs. 1-3). Notwithstandiqg variation in chromosome metrics, the sixteen chro mosomes can be arranged into 8 pairs (Figs. 4-8) by the combination of characters Figs Idiogrammed karyotypes, in decreasing order of length, of cases 1-5 respectively,

4 386 S. C. Verma and R. K. Mittal Cytologia 43 like, i) total length (chromosomes 1-8 being arranged in decreasing order of their lengths), ii) arm-ratios and iii) cytological markers (satellited chromosome Nos. 6 and 7). The variation within individuals is insignificant, when compared with Fig. 9. Comparative idiograms of Nucleolar chromosomes (Nos. 6 and 7) in the five cases (1-5). Fig. 10, Generalised idiogram of Allium sativum L. (A type) based on means of 5 cases analysed. Relative length of chromosome pairs is given. ~4000. variation between individuals (clones). In all the five cases, the eight pairs of chromosomes tend to fall into three distinct groups, A) 5 "long" pairs, B) 2 satel lited pairs, and C) the shortest pair.

5 1978 Chromosome Variation in the Common Garlic, Attium sativum L. 387 A. Five "long" pairs (Chrs. 1-5) They have nearly median centromeres (m-chromosomes) with the means of 1/s arm ratio ranging from (Table 10). The metrics of the chromosomes matched as a "homologous" pair, are generally not identical (Tables 2-6), but the Table 2. Chromosome metrics in case 1 (Fig. 4) * Unequal sized chromosome pairs, where difference exceeds 0.5ƒÊ õ Heteromorphic chromosome pairs, where difference in arm ratio exceeds 0.1. Table 3. Chromosome metrics in case 2 (Fig. 5) * Unequal sized chromosome pairs, where difference exceeds 0.5ƒÊ. õ Heteromorphic chromosome pairs, where difference in arm ratio exceeds 0.1.

6 388 S. C. Verma and R. K. Mittal Cytologia 43 dissimilarity is particularly conspicuous in case of the third pair, which, in 4 out of 5 cases is constituted of distinctly unequal chromosomes (Figs. 4, 6-8; Tables 2, 4-7). Between the 5 cases, the third pair also shows the highest variation in respect of relative length and arm ratio (Fig. 11). Table 4. Chromosome metrics in case 3 (Fig. 6) * Unequal sized chromosome pairs, where difference exceeds 0.5ƒÊ õ Heteromorphic chromosome pairs, where difference in arm ratio exceeds 0.1. Table 5. Chromosome metrics in case 4 (Fig. 7) * Unequal sized chromosome pairs, where difference exceeds 0.5ƒÊ. õ Heteromorphic chromosome pairs, where difference in arm ratio exceeds 0.1.

7 1978 Chromosome Variation in the Common Garlic, Allium sativum L. 389 Table 6. Chromosome metrics in case 5 (Fig. 8) * Unequal sized chromosome pairs, where difference exceeds 0.5ƒÊ. õ Heteromorphic chromosome pairs, where difference in arm ratio exceeds 0.1. Fig. 11. Composite karyogram to reveal the extent of variability of all the 8 chromosomes of the haploid complement. Relative length is given for one chromosome of a pair, out of total of 16 chromosomes, based on total means.

8 390 S. C. Verma and R. K. Mittal Cytologia 43 B. Two satellited pairs (Chrs. 6, 7) The second group comprising two satellited pairs is very characteristic. The larger of the two pairs (No. 6) is the most asymmertic pair of the complement with a mean arm ratio of It is, therefore, a sm-chromosome. The smaller satellited pair (No. 7) is classifiable as a m-chromosome (mean arm ratio, 1.47), but it is more asymmetric than any of the five "long" m-chromosomes (Figs. 4-8, 10). Both the satellited chromosome pairs have essentially the same gross morphology of the satellite-bearing arm, the short arm in each case. It consists of a small proximal segment and a large satellite (Fig. 9). The two nucleolar pairs are easily distingu ishable, the larger pair (No. 6) has both the porximal segment and the satellite smaller than the smaller pair (No. 7) and the long arm much longer (Figs. 4-10). Of the two satellited pairs, pair No. 6 is more variable in respect of relative length and arm ratio (Fig. 11). Also, pair No. 6 shows variation in the appearance of the Table 7. Analysis of unequal sized/heteromorphic chromosomes õ Differ either in arm ratio only or both in size and arm ratio (see Tables 2-6). Table 8. Total chromosome length and chromosome volume satellite. In 3 out of 5 cases, satellite is visible on only one member of the pair (Fig. 9), but being the most asymmetric pair of the complement, its component chromosomes are easily distinguishable from rest of the complement. It is possible that the inability to reveal the proximal segment is due to its loss or deletion, causing heteromorphism of the 6th pair (see Discussion). Heterozygosity for size and arm ratio is evidenced in either the 6th pair (cases 1, 2; Fig. 9; Table 7), or the 7th pair (case 4; Fig. 9; Table 7), or in both the satellited pairs (cases 3, 5; Fig. 9; Table 7). Chromosome pair No. 6 is generally heteromor phic (Table 7). C. Shortest pair (Chr. 8) It is readily distinguishable from all others, being the smallest pair of the com plement. It has clearly asymmetric arms and borders m and sm-chromosomes

9 1978 Chromosome Variation in the Common Garlic, Atlium sativum L. 391 (mean arm ratio 1.67). There is evidence of heterozygosity for size, and in at least 3 out of 5 cases, it is constituted of clearly unequal chromosomes with different arm ratios (Figs. 5, 6, 8; Tables 3, 4, 6). In fact all the five cases are different, the most asymmetric chromosome in this pair occurs in case 1 (Table 2). Figure 10 summarises the idiogram for the A type garlic, based on the mean values of eight pairs of chromosomes. It shows particularly the characteristic features of the two satellited pairs (No. 6 and 7), and that the long arm of chromo some No. 6 is the second longest in the complement being only shorter than the long arm of No. 1. Short arms of chromosome Nos. 7, 6 and 8 (satellite+proximal segment in 6 and 7) are comparatively shorter than others and form a series in the decreasing order. Discussion The common garlic, Allium sativum L., exists in three basic types-h, U and A (Table 1), and chromosome information is now available for all the three types. Therefore, a comparison can be made to reveal differences, if any, between the types, and to analyse the variation within types. It is profitable to consider first the clearly marked nucleolar chromosomes. 1. The Nucleolar chromosomes Types of nucleolar chromosomes There are two satellited pairs and the present work agrees well with the results of Khoshoo et al. (1960) and Battaglia (1963), that in both cases the secondary constrictions are located proximally on the short arm. Konvicka and Levan (1972) have also reported the occurrence of 2 satellited pairs in the chromosome comple ments of H and U types, and in both the pairs, the secondary constriction is near the centromere, very similar to the present observations on the A type. Such nu cleolar chromosomes in Allium are named as the Sativum type (Ved Brat 1965). The data contrast, however, with the report of Mensinkai (1939) that only one of the pairs has proximal secondary constriction (Chr. No. 6), whereas, the other pair (Chr. No. 7) resembles Allium scordoprasum type (see Ved Brat 1965), in possessing the secondary constriction sub-medianly on the nucleolar arm. This situation is not usual in A. sativum and in fact has not been reported by any one else so far. There is obviously preponderance of the Sativum type of nucleolar pairs. But, it may be noted that the segment between the centromere and the secondary con striction in chromosome number 7 is comparatively larger than that of chromosome number 6 (Tables 2-6, Figs. 9, 10), the latter chromosome is otherwise also well marked, being distinctly asymmetrical. Origin of Sativum type Ved Brat (1965) has suggested the origin of the Sativum type of nucleolar chro mosome by a process of paracentric inversion in the nucleolar arm of the m-sm chromosome, originally of A. paniculatum type. Whereas this could be one of the ways of origin, the involvement of pericentric inversions and/or deletions cannot

10 392 S. C. Verma and R. K. Mittal Cytologia 43 be completely ruled out. The distinctly asymmetrical chromosome number 6 lends support to such a process, if A. paniculatum type is to be considered as the ancestral type (see Ved Brat 1965). Heterozygosity The nucleolar chromosomes, being marked can easily reveal structural alter ations, if present. Battaglia (1963) reported heterozygosity in either one or both the nucleolar pairs in respect of their lengths and arm ratios. In the present sample, there is evidence of heterozygosity in both the nucleolar pairs (see Tables 2-6, 7; Fig. 9) suggesting structural alterations or rearrangements in these chromosomes. Table 7 reveals that chromosome pair No. 6 is heteromorphic in all the five cases, whereas No. 7 is heteromorphic in only three cases. However, chromosome pair No. 7 is comparatively more distinctly heteromorphic than No. 6 (excepting case 1). In case of the 6th pair, particularly case 3 and 5, (Figs. 6 and 8), there is posibility of the origin of heterozygosity due to loss of the intermediary segment in the short arm. But, the probability of unequalness of the homologues due to differences in condensation cannot be completely ruled out. The problem can perhaps be settled by employing the N-banding technique of Matsui (1974), improved by Funaki et al. (1975), to ascertain whether such heterozygosity is due to deletion of the inter mediary segment and thereby of the nucleolus organising region. Another point regarding heterozygosity concerns the frequency of revealing all the 4 nucleolar chromosomes at metaphase. Khoshoo et al. (1960) stated having observed all the 4 secondary constrictions in their collections of A-type from several sources. In our case, only 3 secondary constrictions are revealed in 3 out of the 5 samples, and this "inability" is confined to chromosome number 6 (Fig. 9). As suggested earlier, there is some possibility of deletion of the intermediary segment, in some cases at least (cases 3, 5; Figs. 6, 8 and 9) causing heterozygosity. It is very interesting to find that Konvicka and Levan (1972) reported this property for chromosome number 7 of the H and U types which implies variations in the structure of the nucleolar pairs. Furthermore, comparison of the metrics of chr. Nos. 6 and 7 of A type with H and U types also suggest variation. The chromosome number 6 in the A type is comparatively longer and more symmetrical, whereas the chromo some number 7 is comparatively smaller and more asymmetrical than the correspond ing pairs of H and U types (Table 10). An interpretation Excepting mutations, the only source of variation in vegetatively reproduced species, like A. sativum, is the structural rearrangements. Heterozygosity of the nucleolar chromosomes substantiates it. Besides, we consider it a signifiocant genetic variation. Nucleolus is an important organelle and its functional role or efficiency is probably adaptively related to its location in the nucleolar chromosome. It is probable that the variation in the site of N. O. region in the nucleolar chromosomes is the principle source of variability between clones of A, sativum adapted to different climate and soil conditions. It is noteworthy that the mean arm ratios of the non nucleolar chromosomes of the three types nearly resemble each other, but there are considerable differences between the nucleolar chromosomes.

11 1978 Chromosome Variation in the Common Garlic, Allium sativum L Heterozygosity of non-mucleolar chromosomes It has been emphasised earlier that the two nucleolar pairs are the most variable members of the complement of A. sativum. The remaining chromosomes of the complement also reveal heterozygosity as reflected in the difference between the lengths and arm ratios of constituent chromosomes of the pairs (Tables 2-6). Mark ed differences are observed between the constituent chromosomes of pair Nos. 3 and 8 (see Tables 2-7). The rest of the chromosome pairs are, comparatively, rather conservative. The occurrence of heterozygosity per se is not unexpected in exclusively vegetatively reproduced organisms. These are due to structural al terations and rearrangements. Besides in some cases chromosomes of a pair differ only in size and not in arm ratio (Table 7) which aspect needs to be investigated fully to determine the possibility of deletions, translocations and/or unequal condensation of the homologous chromosomes. 3. Chromosome metrics When the total chromosome length of the complement and the chromosome volumes of the five cases are compared with each other (Table 8), wide differences are observed. In case 2, chromosome volume is more than 11/2 times of that in Table 9. Comparison of total chromosome length of the diploid set (in microns) * Taken from Konvicka and Levan (1972). ** Chandigarh A type, present work. cases 1, 4 and 5 (Table 8). These differences are not due to differences in age of roots or procedure, as in all the cases roots of same age were processed similarly under identical conditions. When the present data are compared with that of Bat taglia (1963) for the Pisa clone and with that of Konvicka and Levan (1972) for 4 clones of H and U types (Table 9), it is observed that the values for the H and U types are considerably lower than for the A type. The clone from Pisa (Italy) shows a higher value, nearly comparable to case 2 of present study (Tables 8, 9). The observed difference between the total chromosome lengths of the complements of A. sativum from several sources can be due to a combination of any of the following six factors falling under two categories: a. Extrinsic factors i) differences in the procedure including pretreatment, fixative used and stain ing (e.g. Orcein staining schedule of Konvicka and Levan, 1972, produced com

12 394 S. C. Verma and R. K. Mittal Cytologia 43 paratively longer chromosomes, Table 9), presumably affecting chromosome coiling and condensation (contraction). ii) differences in the age of the roots studied by different workers, because age affects the lengths of the chromosomes and also chromosome volume (Bennett and Rees 1969). iii) the nature of stored food material in the cloves, perhaps affected by the nutritive conditions of the soil where the particular clones are cultivated. Positive effect of the phosphorus content on chromosome size and volume are known, owing mainly to differences in protein content (Bennett and Rees 1969). b. Intrinsic factors iv) difference in the DNA content, v) difference in the protein component,* vi) difference in both, DNA and proteins.* (*depends on nutritional conditions as well, see iii).) The first 2 points can be ruled out in the presently observed variation within the A type. There is some probability of differences in DNA, where deletions are suggested. The total chromosome length and chromosome volume in case 2 are significantly larger than the rest (Table 8). Here particularly, and otherwise in general, we are inclined to suggest that nature of the stored food material in the cloves may be an important factor affecting chromosome lengths and it needs to be verified. Besides, studies on the DNA and protein content of the chromosome complement from several sources are desirable, coupled with karyotype studies and degree of metabolic activity including cell division. Bennett and Rees (1969) have suggested a close correlation between the amount of the 'non-permanent' component of the chromosomes in meristem and the rate of cell metabolism. 4. The karyotype In general the karyotype is similar to the one reported by Khoshoo et al. (1960). I:t agrees with the fundamental karyotype proposed by Battaglia (1963). Barring the nucleolar chromosomes, the rest of the karyotype is more or less similar to the observations of Konvicka and Levan (1972) on H and U Types (Table 10). Des pite variation in chromosome metrics between bulbs and the demonstration of he terozygosity within the complements of 5 cases, the 16 chromosomes can be con veniently grouped into 8 pairs, each pair constituted of comparable chromosomes. This fact has been emphasised by Konvicka and Levan (1972) too for H and U types. Such a feature is characteristic of sexual species, where meiosis controls conservation of general similarity of the homologous chromosomes. Therefore, it should not be impossible to discover the sexual relatives of the cultivated garlic, as also expressed by Konvicka and Levan (1972). There is broad agreement in the relative lengths of chromosomes and their arm ratios when compared with the data on H and U types by Konvicka and Levan (1972), (Table 10). The differences are marked only in respect of the nucleolar chro mosomes (Nos. 6 and 7). The nucleolar chromosomes are also comparatively more variable within the material studied (Fig. 11) as compared to the non-nucleolar

13 1978 Chromosome Variation in the Common Garlic, Allium sativum L. 395 chromosomes. We are inclined to accept the suggestion of Ved Brat (1965) that nucleolar chromosomes in Allium, including A. sativum are presumably far more susceptible to spontaneous mutations. There is one more instructive comparison, between total chromosome lengths and arm ratios. The total chromosome lengths from various data reveal consider able differences, whereas the arm ratios show a general correspondence (see Tables 9, 10). These observations can be accommodated in the hypothesis advanced earlier that differences in chromosome length (and volume) are primarily the outcome of the probable differences in the stored food material in the cloves, affected by nutritive conditions of the soil, including the type of fertilizers used. Table 10. Comparison of relative lengths and arm ratios of chromosomes of type A with H and U types * Taken from Konvicka and Levan (1972). ** Present work. õ Arm ratios difference of chromosome 6 and 7, between Hand U and A types, exceeds 0.1. Summary The common garlic, Allium sativum L. (2n=16), occurs basically as three mor photypes-h, U and A types (Table 1), all exclusively vegetatively reproduced. Five karyotypes from five bulbs of the A type have been analysed in detail to make a case study to discover if there is any necessity for extensive observations on a wide variety of material. The chromosome information thus revealed is compared also with that of Konivicka and Levan (1972) for H and U types. This study has amply impressed upon the need to undertake extensive chromosome studies of A. sativum. Within A type and between H, U and A types, wide variations occur in respect of the chromosome metrics, including arm ratios, particularly involving the nucleo lar chromosomes. There are invariably two nucleolar pairs, and both are of the Sa tivum type, and generally, either one or both the pairs are heterozygous. Amongst the non-nucleolar chromosomes, chromosome nos. 3 and 8 are usually heterozygous. The probable sources of variation are discussed, and it is proposed that, besides structural alterations, general variability in respect of size and total chromosome length may be caused by differences in the stored food in the cloves, which in turn is affected by nutritive conditions of the soil. The proposal needs to be verified ex perimentally.

14 396 S. C. Verma and R. K. Mittal Cytologia 43 Literature cited Battaglia, E Mutazione chromosomica e cariotipo fondamentale in Allium sativum L. Caryologia 16: Bennett, M. D. and Rees, H Induced and developmental variation in chromosomes of meristematic cells. Chromosoma (Berl.) 27: Bhaduri, P. N. and Ghosh, P. N Chromosome squashes in cereals. Stain Tech. 29: Funaki, K., Matsui, S. and Sasaki, M Location of nucleolar organizers in animal and plant chromosomes by means of an improved N-banding technique. Chromosome (Berl.) 49: Hruby, K. and Konvicka, O PoIni pokusy (Field trials). Olomouc, p Khoshoo, T. N., Atal, C. K. and Sharma, V. B Cytotaxonomical and chemical investi gations on the North-West Indian garlics. Res. Bull. (N. S.) Panjab Univ. 11: Konvicka, O. and Levan, A Chromosome studies in Allium sativum. Hereditas 72: Levan, A., Fredga, K. and Sandberg, A. A Nomenclature for centromeric position on chromosomes. Hereditas 52: Matui, S Nucleolus organizer of Vicia faba revealed by the N-banding technique. Jap. J. Genetics 49: Mensinkai, S. W Cytogenetic studies in the genus Allium. J. Genet. 39: Ved Brat, S Genetic systems in Allium I. Chromosome variation. Chromosoma (Berl.) 16: