2019 Volume 84 Issue 3 Pages 227-231
The intraspecific euploid (2x, 4x) cytotypes have been reported in Tragopogon pratensis from the cold deserts of Ladakh division in Jammu and Kashmir. Both the cytotypes showed perfect bivalent formation, regular meiotic course and normal microsporogenesis leading to high pollen fertility. The 4x plants are more vigorous and growing much taller than the 2x plants and show gigantism in various vegetative and floral characters besides the increase in the size of stomata and pollen grains. Analysis of meiotic chromosome pairing and chromosome size in the 4x cytotype suggests its allopolyploid nature.
The genus Tragopogon L. (family Asteraceae) represented by about 150 species throughout the world, is mainly distributed in Eurasia with a center of diversification in the eastern Mediterranean basin. The genus consists of annual, biennial and mostly perennial herbs with entire and parallel-veined leaves. The main morphological characters used to distinguish species in the genus include fruit morphology, ligule color, a ratio of ligule/involucral bract lengths, number of involucral bracts and the thickness of peduncle (Suárez-Santiago et al. 2011). The genus is taxonomically very complex due to the high incidence of interspecific hybridization (Ownbey 1950, Krahulec et al. 2005). The presently analyzed species T. pratensis is an annual or perennial herb commonly growing along field margins and roadsides in the cold deserts of Ladakh division in Jammu and Kashmir. The species is characterized by having recurved to coiled leaf apices, peduncles usually small sized, involucres urceolate in bud and ray florets yellow in color. The plant is considered to be a useful remedy for liver and gallbladder problems (Chevallier 1996). It appears to have a detoxifying effect and stimulate the appetite and digestion. The high inulin content makes this herb a useful food for diabetics since inulin is a nutrient made of fructose rather than glucose units and therefore does not raise blood sugar levels (Chevallier 1996). The roots are astringent, depurative, diuretic, expectorant, nutritive and stomachic (Lust 1983, Chiej 1984). Syrup made from the roots gives great relief in cases of obstinate coughs and bronchitis (Chiej 1984).
The aims of the present study were (i) to work out the exact chromosome count on individual plant basis (ii) to segregate the cytotypes through morphometric analysis, study in detail the meiotic behaviour and (iii) to work out the pollen fertility.
Exploration surveys were conducted in May–September, 2014 to 2017. The materials for meiotic investigations and pollen fertility were collected from the four wild plants growing at Mulbekh (3250 m, 3300 m) and Sapi-La (3500 m). The duly identified and cytologically examined specimens were deposited in the Herbarium, Department of Botany, Punjabi University (PUN).
Meiotic analysis and pollen viabilityFor meiotic preparations, unopened capitula of suitable sizes from one plants of each locality were fixed in a freshly prepared Carnoy’s fixative (6 parts of ethanol, 3 parts of chloroform, a part of acetic acid) for 24 h. Materials were subsequently transferred to 70% ethanol and stored in a refrigerator. Meiotic preparations were made by squashing the young anthers from ray/disc florets through standard a squash method using 1% acetocarmine. Chromosomes were counted in the well-spread preparations by having the pollen mother cells (PMCs) at diakinesis, metaphase I (M I) and anaphase I (A I). PMCs were also observed carefully to examine the detailed meiotic course including microsporogenesis. Pollen fertility was estimated through a stainability test for which mature anthers from florets were squashed in a mixture (1 : 1) of glycerol and 1% acetocarmine. Well filled and fully stained pollen were treated as viable while shriveled and poorly/partially stained pollens were considered as sterile. Photomicrographs of PMCs with chromosomes, sporads and pollen grains were taken from the freshly prepared slides using a Leica Qwin Digital System.
MorphometryPlant height, number of internodes, length of internode, number and size of leaves, capitulum size, the size and frequency of stomata, and size of pollen grains were used for the analysis of 2x and 4x cytotypes. Stomatal studies were made from the abaxial epidermal peels obtained from the middle portion of mature leaves through treatment with 2% KOH. Thoroughly washed peels were mounted in glycerol and stained with safranin red. Stomatal index was calculated by using the formula (SI=S/S+E×100), where SI stands for the stomatal index, S is the number of stomata and E is the number of epidermal cells. The pollen grain size was measured through micrometry.
Wild individuals of T. pratensis gathered from two localities in the cold deserts of Ladakh were analyzed for gametic chromosomes number, pollen fertility, and morphometric analysis. The plants depicted two chromosome numbers (n=6, 12) of ploidy levels (2x, 4x) (Table 1). The 2x cytotype was reported for the first from India and 4x cytotype confirms the previous report (Koul and Gohil 1973). Further studies on the detailed meiotic course including microsporogenesis and pollen fertility were made separately for 2x and 4x cytotypes. The cytotypes were also studied for comparison of macro- and micro-parameters for their identification in the field as well as under laboratory conditions (Table 1).
| Sites of collection/parameters | Diploid cytotype (n=6) | Tetraploid cytotype (n=12) |
|---|---|---|
| Site of collection | Sapi-La, 3500 m | Mulbekh, 3250 m, 3300 m |
| (Voucher number) | (PUN 61982, 62179) | (PUN 63105, 62455) |
| Distribution | Rare | Rare |
| Habit | Perennial herb | Perennial herb |
| Habitat | Along roadsides | Along field margins |
| Plant height (cm) | 35–40 | 50–60 |
| Number of nodes per plant | 15–20 | 20–28 |
| Length of internode (cm) | 2.0–2.5 | 2.3–3.0 |
| Leaves | ||
| (i) No. of leaves/plant | 15–20 | 20–30 |
| (ii) Texture | Smooth | Smooth |
| (iii) Size Length (cm) | 10.0–11.0 | 14.5–16.0 |
| Width (cm) | 0.6–1.0 | 0.7–2.6 |
| Capitula | ||
| (i) Colour of ray florets | Yellow | Yellow |
| (ii) Size (cm) | 4.5–5.0 | 6.0–7.0 |
| (iii) Peduncle size (cm) | 2.0–2.2 | 1.4–1.6 |
| Stomata | ||
| (i) Size (µm) | Length×breadth | Length×breadth |
| 26.53–29.67×21.28–22.28 | 30.24–31.280×23.31–26.42 | |
| (27.10±0.98)×(21.83±0.50) | (30.73±0.52)×(24.61±1.61) | |
| (ii) Stomatal index (SI) | 21.13–34.12 (28.12±4.31) | 20.71–31.31 (26.50±3.18) |
| Pollen grains | ||
| (i) Size (µm) | Uniform sized | Heterogeneous sized |
| 39.73–43.36×37.07–41.13 | Large | |
| (41.85±1.52)×(39.08±1.67) | 53.53–62.45×49.37–59.49 | |
| (57.48± 3.85)×(53.0±4.42) | ||
| Small | ||
| 32.19–33.92×31.264–32.40 | ||
| (33.29±0.95)×(31.78±0.57) | ||
| (ii) Fertility (%) | 100 | 95–100 |
Two plants studied from Sapi-La (3550 m, PUN 61982, 62179) showed six bivalents in PMCs at diakinesis (Fig. 1A) and M I (Fig. 1B). The chromosome count of n=6 suggests that the plants were diploid. Further meiotic course including sporad formation in all the plants was perfectly normal (Fig. 1C) resulting in fertile and equal-sized pollen grains (Fig. 2E).
Two plants scored from Mulbekh (3250 m, PUN 63105, 3300 m, PUN 62455) revealed a tetraploid chromosome number of n=12 which was confirmed from the presence of 12 bivalents at diakinesis, out of which are six large-sized (Fig. 1D) and 12 : 12 segregation of chromosomes at A I (Fig. 1E). Further meiotic course and sporad formation in all the plants was found to be perfectly normal (Fig. 1F) resulting in high pollen fertility. The plants showed the presence of fertile pollen grains of two sizes as large (94.80%) and small (5.20%) (Fig. 2F).
Morphometric analysisMacroscopic characters analyzed presently in the two polyploid cytotypes included plant height, number of nodes/plant, length of internodes, leaf size, number of leaves/plant, size of capitulum and length of the pedicel (Table 1). Analysis of data on these parameters revealed that 4x plants grow taller (50–60 cm, Fig. 2B) than the 2x (35–40 cm, Fig. 2A). The number of nodes/plant, as well as the internodal length, was more in 4x cytotype than the 2x cytotype. Besides, the number of leaves, as well as their size, was noticed to be higher in 4x plants than 2x plants. The size of capitulum and pedicel length was also noticed to be larger in the case of tetraploid plants (Table 1). Two cytotypes can also be differentiated in micro-characters like stomata and pollen grains. Stomata were significantly larger in the 4x cytotype (30.73±0.52×24.61±1.61 µm, Fig. 2C) compared to the 2x (27.10±0.98×21.83±0.50 µm, Fig. 2D). On the other hand, SI in the two cytotypes was not significantly different. The fertile pollen grains in the 4x cytotype were noticed to be of two types, as large (53.53–62.45 µm×49.30–59.49 µm) (94.80%) and small (32.19–33.92 µm×31.26–32.40 µm) (5.20%) compared to the 2x plants where pollen grains are of uniform size (39.73–43.36 µm×37.07–41.13 µm).
The 4x cytotype (n=12) has already been reported by Koul and Gohil (1973) from other regions of Kashmir Himalayas. However, the 2x cytotype has been recorded for the first time from India. But the 2x cytotype has already been reported by a number of cytologists from Bulgaria (Kuzmanov et al. 1969), Italy (Gadella and Kliphuis 1970a), Netherland (Gadella and Kliphuis 1971), France (Gadella and Kliphuis 1970b), Canada (Tomkins and Grant 1978, Mulligan 1984), Germany (Albers and Pröbsting 1998) and U.S.A. (Kapoor and Gerrias 1982). The record of a chromosome number of 2n=14 from Grenada by Díaz de la Guardie and Blanca (1988) suggests the existence of intraspecific dysploidy.
On the basis of chromosome pairing allopolyploids behaved like diploids and are generally considered to be much more common than autopolyploids, mainly due to their adoptive superiority involving perfect chromosome pairing, segregation, and gametic fertility. A cursory look on the size of bivalents in two cytotypes reveals that diploid cytotype has overall larger bivalents as compared to the tetraploid cytotype where bivalents are smaller in size which is in agreement with the general rule according to which polyploid species have smaller chromosomes/bivalents as compared to diploids. In T. pratensis, the tetraploid individuals showed typical allopolyploid meiotic behavior with normal chromosome pairing leading to perfect 12 bivalents, regular segregation, and high pollen fertility. Furthermore, the presence of two genomes ascertained on the basis of six large+six small sized bivalents in a 4x cytotype of T. pratensis also indicates towards its allopolyploid nature.
The authors are grateful to the University Grant commission, New Delhi for providing financial assistance under the DRS, SAP I, II & III, ASSIST Programme and Juniour Research Fellowship to Younas Rasheed (Award letter no. 2121430298 12/8/2015). The authors are also thankful to Head, Department of Botany, Punjabi University, Patiala for providing necessary laboratory, herbarium and library facilities and IPLS-DBT (BT/PR 4548/INF/22/146/2012).
