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Karyomorphological Study in Ledebouria botryoides (Asparagaceae)
2023 年 88 巻 1 号 p. 15-19
詳細
2023 年 88 巻 1 号 p. 15-19
Cytogenetical studies were conducted on Ledebouria botryoides. We reported 2n=60 (15m+10sm+5st) chromosomes. The karyotype was asymmetrical and fell into the 4B category of Stebbins’ asymmetry classes. Two groups of chromosomes were observed, i.e. 15 pairs of long chromosomes (2.82–6.65 µm) and 15 pairs of short chromosomes (1.67–2.80 µm). Two chromosome pairs had a satellite on the short arms. The meiotic course was found to be normal with n=30 bivalents.
Ledebouria Roth (Asparagaceae) is a genus distributed in India, Madagascar, and sub-Saharan Africa, mainly in southern Africa (Manning et al. 2002). It comprises more than 78 species and four infraspecific taxa (modified after Manning et al. 2004 and Giranje et al. 2019). The genus has a high degree of multiformity in morphology. Blotches on the leaves, bulbils at the leaf tip, and shape and size of the leaves considerably vary at the species level under different climatic and geographical conditions (Giranje and Nandikar 2016, Deshmukh et al. 2022). Accordingly, these characters are of little use in understanding the taxonomy of Ledebouria. The genus Ledebouria needs a thorough biosystematic approach to understand species limits as cytotypes exist for many species (Goldblatt et al. 2012).
In India, the genus comprises three species (modified after Giranje and Nandikar 2016). Ledebouria botryoides (Baker) J.C. Manning & Goldblatt, an East African species is grown in Indian nurseries as an ornamental. It is a succulent plant with blotched leaves. Cytogenetically, it is a complex with various chromosome counts on record, viz. 2n=80 (Matsuura and Suto 1935); 2n=68 (Mahalakshmi and Sheriff 1970); 2n=66 (Sharma 1970); 2n=66, 68, 126 (Sen 1973); 2n=60 (Vij et al. 1982); 2n=44, 55 (Stedje and Nordal 1987); 2n=30 (Vijayavalli and Mathew 1988); 2n=44, 55, 66 (Stedje 1994) and 2n=66 (Haque and Ghosh 2016). Chromosome number is the most frequently documented cytological character. Chromosome number, size, symmetry, position of the centromere, secondary constriction, and heterochromatic regions are the mainly used diagnostic chromosomal landmarks used in comparative analysis (Vimala et al. 2021). Expensive techniques including chromosome banding and fluorescence in situ hybridization (FISH) may be necessary to establish a relationship between taxa with uniform (symmetric) karyotypes; however, taxa with asymmetric karyotypes can be studied with simple squashing and conventional staining with orcein/carmine (Haque and Ghosh 2016). The structure and behavior of plant chromosomes during reduction division is important because the genetic variation of both gametes and the future sporophyte depends on it (Haque and Ghosh 2016). Polyploidy is common among plant species, especially in monocots and this raises special issues for the analysis of meiosis in many plants (Haque and Ghosh 2016).
The available data on the cytogenetics of L. botryoides is poorly discussed except for Haque and Ghosh (2016). Therefore, a re-evaluation of the karyotype and meiotic studies is highly needed to know the relationship with other Ledebouria species. In this paper, a detailed karyotype along with the meiotic behavior of chromosomes in L. botryoides is discussed.
The plant materials for the present investigations were obtained from the nursery and cultivated in the Lead Botanical Garden, Shivaji University, Kolhapur, and the herbarium specimen was prepared and deposited in the Herbarium of Department of Botany, Shivaji University, Kolhapur (acronym SUK). Mitotic preparations were made from the root tips. The well-grown root tips (6–10 mm long) were excised and pre-treated with a saturated solution of para-dichlorobenzene for 4–5 h at 9±3°C. Further, the root tips were hydrolyzed in 1 M HCl and squashed in 2% propionic-orcein. For meiotic studies, appropriately sized flower buds were fixed in Carnoy’s fixative (3 : 1, absolute ethanol and acetic acid) and smears of anthers from floral buds were stained using 2% propionic–orcein. Suitable somatic and meiotic plates from freshly prepared slides were photographed with a camera mounted on a fluorescence microscope (Leica DM 2000, Germany). Ten plates with well-separated somatic chromosomes were selected for karyotype analysis by following the method of Levan et al. (1964). The degree of karyotype asymmetry was determined using the categories of Stebbins (1971) and the CVCL (coefficient of variation of chromosome length) and MCA (mean centromeric asymmetry) as proposed by Peruzzi and Eroglu (2013).
Ledebouria botryoides exhibited 2n=60 chromosomes (Fig. 1a). Based on centromeric position, it had three types of chromosomes, i.e. five pairs of chromosomes with subterminal region centromeres, 10 pairs of chromosomes with submedian region centromeres and 15 pairs of chromosomes with median region centromeres, and hence the karyotype formula 15m + 10sm + 5st. Karyogram is shown in Fig. 1b. The karyotype was asymmetrical and fell into the 4B category of Stebbins’ asymmetry classes. The total haploid chromosome length (THCL) was 101.09 µm. The mean chromosome length (MCL) was found to be 3.37±1.41 µm (Table 1). The length of the longest chromosome was 6.65±1.06 µm whereas the length of the shortest chromosome was 1.67±0.21 µm. The karyotype was bimodal with two groups of chromosomes, i.e. 15 pairs of long chromosomes (2.82–6.65 µm) and 15 pairs of short chromosomes (1.67–2.80 µm) (Fig. 1b). Two chromosome pairs (No. 20 and 25) had a satellite on short arms. The meiotic course was found to be normal with n=30 bivalents (Fig. 2c). Pollen mother cells at different stages such as pachytene, diplotene, diakinesis, metaphase-I, anaphase-I, metaphase-II, anaphase-II, and telophase-II were observed to be normal (Fig. 2).
| Chromosome pair | Long arm (l) (µm)±SD | Short arm (s) (µm)±SD | Total length (c=l+s) (µm)±SD | ‘d’ value (l−s) (µm)±SD | ‘r’ value l/s±SD | ‘i’ value (s/c×100)±SD | Centromeric position |
|---|---|---|---|---|---|---|---|
| 1 | 5.24±1.15 | 1.40±0.57 | 6.65±1.06 | 3.84±1.47 | 3.74±2.17 | 21.09±9.02 | st |
| 2 | 4.62±1.27 | 1.38±0.44 | 5.99±0.99 | 3.24±1.63 | 3.35±1.81 | 22.99±10.14 | st |
| 3 | 4.50±1.08 | 1.22±0.37 | 5.72±1.05 | 3.28±1.23 | 3.69±1.88 | 21.34±7.57 | st |
| 4 | 4.40±0.94 | 1.09±0.21 | 5.49±0.99 | 3.32±0.93 | 4.06±1.36 | 19.77±4.35 | st |
| 5 | 3.61±1.15 | 1.50±0.35 | 5.11±0.98 | 2.10±1.40 | 2.40±1.58 | 29.43±10.30 | sm |
| 6 | 3.76±0.98 | 1.17±0.24 | 4.93±1.05 | 2.60±0.97 | 3.22±0.90 | 23.69±4.73 | st |
| 7 | 3.24±1.12 | 1.43±0.22 | 4.67±1.07 | 1.81±1.21 | 2.26±0.95 | 30.68±8.56 | sm |
| 8 | 3.29±1.26 | 1.13±0.25 | 4.42±1.07 | 2.16±1.48 | 2.92±2.14 | 25.54±9.32 | sm |
| 9 | 2.87±0.73 | 1.32±0.28 | 4.19±0.86 | 1.56±0.69 | 2.18±0.65 | 31.42±5.99 | sm |
| 10 | 2.58±0.76 | 1.41±0.06 | 3.98±0.74 | 1.17±0.78 | 1.83±0.58 | 35.32±5.85 | sm |
| 11 | 2.53±0.62 | 1.22±0.27 | 3.75±0.76 | 1.31±0.58 | 2.08±0.50 | 32.49±6.59 | sm |
| 12 | 2.00±0.36 | 1.23±0.18 | 3.23±0.40 | 0.78±0.40 | 1.63±0.38 | 37.97±5.67 | m |
| 13 | 2.02±0.35 | 1.05±0.21 | 3.07±0.49 | 0.97±0.32 | 1.92±0.46 | 34.20±4.80 | sm |
| 14 | 1.91±0.36 | 1.13±0.16 | 3.04±0.50 | 0.78±0.24 | 1.69±0.17 | 37.13±2.31 | sm |
| 15 | 1.74±0.20 | 1.08±0.30 | 2.82±0.42 | 0.66±0.30 | 1.61±0.50 | 38.33±6.01 | m |
| 16 | 1.77±0.44 | 1.03±0.06 | 2.80±0.42 | 0.74±0.48 | 1.72±0.50 | 36.78±5.81 | sm |
| 17 | 1.78±0.45 | 0.96±0.19 | 2.75±0.45 | 0.82±0.52 | 1.85±0.62 | 35.07±7.77 | sm |
| 18 | 1.58±0.19 | 1.08±0.25 | 2.65±0.30 | 0.50±0.32 | 1.46±0.42 | 40.60±6.83 | m |
| 19 | 1.60±0.25 | 0.99±0.15 | 2.60±0.31 | 0.61±0.28 | 1.61±0.37 | 38.29±4.97 | m |
| 20 | 1.45±0.18 | 1.00±0.16 | 2.46±0.30 | 0.45±0.16 | 1.45±0.18 | 40.84±3.17 | m |
| 21 | 1.34±0.14 | 1.06±0.14 | 2.40±0.24 | 0.29±0.15 | 1.27±0.19 | 44.02±3.39 | m |
| 22 | 1.29±0.18 | 1.00±0.13 | 2.29±0.29 | 0.29±0.12 | 1.29±0.13 | 43.68±2.43 | m |
| 23 | 1.26±0.21 | 1.00±0.13 | 2.26±0.31 | 0.26±0.17 | 1.26±0.17 | 44.30±3.56 | m |
| 24 | 1.25±0.19 | 0.96±0.15 | 2.22±0.30 | 0.29±0.17 | 1.30±0.18 | 43.44±3.74 | m |
| 25 | 1.24±0.20 | 0.98±0.12 | 2.22±0.30 | 0.26±0.12 | 1.27±0.12 | 44.14±2.27 | m |
| 26 | 1.22±0.23 | 0.83±0.04 | 2.05±0.22 | 0.39±0.25 | 1.47±0.33 | 40.45±5.05 | m |
| 27 | 1.03±0.13 | 0.93±0.16 | 1.96±0.25 | 0.10±0.15 | 1.11±0.20 | 47.35±4.07 | m |
| 28 | 1.06±0.20 | 0.85±0.05 | 1.91±0.18 | 0.21±0.22 | 1.24±0.29 | 44.56±5.14 | m |
| 29 | 0.97±0.14 | 0.83±0.05 | 1.79±0.13 | 0.14±0.16 | 1.16±0.21 | 46.22±4.25 | m |
| 30 | 1.02±0.22 | 0.65±0.10 | 1.67±0.21 | 0.38±0.26 | 1.58±0.47 | 38.79±6.42 | m |
The previous study on chromosomes of L. botryoides was restricted to only somatic counts [(2n=80 (Matsuura and Suto 1935); 2n=66 (Sharma 1970); 2n=66, 68, 126 (Sen 1973); 2n=60 (Vij et al. 1982); 2n=44, 55 (Stedje and Nordal 1987); 2n=30 (Vijayavalli and Mathew 1988); 2n=44, 55, 66 (Stedje 1994)]. Haque and Ghosh (2016) conducted a detailed karyotype analysis and reported 2n=66 chromosomes. However, our findings revealed 2n=60 chromosomes and asymmetrical karyotype which agrees with the findings of Vij et al. (1982). Vij et al. (1982) found that the chromosome ranged from 6.62–1.98 µm in length and can be grouped as 20 large and 40 medium to small sized. In the present study, chromosome length ranged from 6.65–1.67 µm which is comparable to the findings of Vij et al. (1982). However, we observed 30 large and 30 medium to small-sized chromosomes. Dixit et al. (1989) studied the cytogenetics of Ledebouria revoluta (L.f.) Jessop [formerly Scilla hyacinthina (Roth) Macbr.] and reported polyploidy, i.e. 2n=30, 45 and 60. In their study, one of the populations from Diva Ghat, Pune district, Maharashtra exhibited 2n=60 chromosomes. The chromosome length ranged from 4.69–1.08 µm and the karyotype was moderately symmetrical (2C category). The karyotype of L. botryoides in the present study differed from the Diva Ghat population (2n=60) in the chromosome length and two terminal satellites on the two pairs of chromosomes. Chromosomes were larger (6.65–1.67 µm) in L. botryoides and smaller (4.69–1.08 µm) in L. revoluta studied by Dixit et al. (1989). Giranje et al. (2019) studied the karyotype of L. revoluta and reported a hexaploid population with 2n=6x=90 chromosomes with median, submedian and subterminal centromeres. The karyotype was moderately asymmetrical (2C category) and the chromosome length ranged from 6.33–0.86 µm. L. botryoides showed similar chromosomes but the karyotype was more asymmetrical (4B category) and the chromosomes were larger than the L. revoluta population studied by Giranje et al. (2019). In the present investigation, bimodal karyotype was observed in L. botryoides, i.e. 15 pairs of long chromosomes (2.82–6.65 µm) and 15 pairs of short chromosomes (1.67–2.80 µm). Goldblatt et al. (2012) reported bimodal karyotype in L. nossibeensis (H. Perrier) J.C. Manning & Goldblatt (2n=30). The species has two large pairs and 13 small pairs of chromosomes. Stedje (1996) also reported the bimodal karyotype consisting of four long and 24 short chromosomes in L. somaliensis (Baker) Stedje & Thulin. Matsuura and Sato (1935) reported 2n=80 chromosome number in Drimiopsis botryoides (now L. botryoides) which suggests the basic chromosome number as x=8. It is on this basis that Darlington and Wylie (1956) have mentioned the basic number for the genus as x=8 but other reports, i.e. 2n=20 in L. humifusa (Baker) J.C. Manning & Goldblatt (formerly D. saundersiae Baker) (de Wet 1957) and 2n=60 in L. petiolata J.C. Manning & Goldblatt (formerly D. maculata Lindl. & Paxton) (Fernandes and Neves 1962) suggest x=10 is the basic number in the genus. Based on x=10, L. botryoides could be a hexaploid. Stedje and Nordal (1987) suggested three different basic numbers, i.e. 10, 11, and 12 distributed exclusively in the southern, eastern, and western areas of Africa, respectively. According to Stedje and Nordal (1987), the ancestor has been a southern x=10 species. Stedje and Nordal (1987) noticed that increase in basic numbers is accompanied by increasing asymmetry. Manning et al. (2004) suggested x=11, 10, and 5 as the basic numbers. Based on the present study and previous works it can be concluded that L. botryoides is a polyploid species in which karyotypically dissimilar forms are observed. To establish the primary basic number for the genus, further studies are needed.
We thank the Head, Department of Botany, Shivaji University, Kolhapur for providing the necessary facilities during this study. PVD thanks Chhatrapati Shahu Maharaj Research Training and Human Development Institute (SARTHI), Pune, for providing a Senior Research Fellowship vide letter dated 17/09/2019.
