Regular Article
Morphometric Analysis and Meiotic Behavior in 4x and 6x Cytotypes of Achyranthes aspera L. from North India
2019 Volume 84 Issue 4 Pages 373-377
Details
2019 Volume 84 Issue 4 Pages 373-377
Achyranthes aspera, known as Latjeer, is a medicinally as well as an economically important plant and shows a lot of cytomorphological diversity. Keeping this status in consideration, the present attempt was made to study the detailed meiotic course in 12 plants of A. aspera collected from different areas of North India. The meiotic analysis depicts the presence of two cytotypes with 2n=28 and 42. The chromosome number of 2n=42 confirms the previous reports whereas the 2n=28 is a new chromosome number. The meiotic behavior in 4x and 6x plants may suggest their allopolyploid nature.
The genus Achyranthes L. (family Amaranthaceae) is represented by 15 species worldwide (Shu et al. 2003). In India, the genus has only three species, A. aspera, A. bidentata and A. coynei, of which two are found in North India, i.e., A. aspera and A. bidentata. Due to the wide distribution of the genus coupled with the chromosomal diversity (2n=14, 34, 35, 38, 42, 84) (Rice et al. 2015). The genus presents an interesting cytotaxonomic problem. The presently investigated species A. aspera is an erect perennial herb characterized by having opposite ovate/obovate leaves, hermaphrodite flowers arranged in terminal spikes and spinous bracteoles with free long adnate membranous wings. Shu et al. (2003) recognized four infraspecific taxa of A. aspera, viz., var. argentae, var. rubrofusca, var. indica, and var. aspera, based on the leaf shape and the pubescens of stem and leaves.
The aim of the present study was to carry out detailed meiotic analysis on different plants of the species along with pollen fertility collected from different regions of North India and also to mark out the morphometric analysis which can be used to identify the individuals of the 4x and 6x cytotypes in the field.
For cytomorphological analysis, wild plants of A. aspera were collected from Punjab (Pb), Haryana (HR) and Rajastan (RJ) belonging to North India (Table 1). For meiotic and pollen grain studies, young flower buds of the appropriate size of each plant were fixed in Carnoy’s fixative (6 parts ethanol : 3 parts chloroform : 1 part acetic acid) for 24 h. The material was transferred to 70% ethanol and stored in a refrigerator until use.
| Plants | Locality with altitude (m) | Accession number | Chromosome number (2n) | Ploidy level | Pollen fertility (%) |
|---|---|---|---|---|---|
| P-1 | Nabha, Pb. 254 | 57445 | 28 | 4x | 71±2.11 |
| P-2 | Sunam, Pb. 230 | 57435 | 42 | 6x | 91±5.31 |
| P-3 | Rajpura, Pb. 258 | 57436 | 28 | 4x | 87±3.22 |
| P-4 | Jalander, Pb. 228 | 57437 | 42 | 6x | 92±4.21 |
| P-5 | Khanna, Pb. 250 | 57438 | 28 | 4x | 69±4.11 |
| P-6 | Patiala, Pb. 250 | 57440 | 28 | 4x | 68±1.33 |
| P-7 | Samana, Pb. 238 | 57441 | 28 | 4x | 70±1.45 |
| P-8 | Ablowal, Pb. 250 | 57442 | 28 | 4x | 93±2.55 |
| P-9 | Bhadson, Pb. 250 | 57443 | 28 | 4x | 94±3.55 |
| P-10 | Patiala, Pb. 250 | 57449 | 42 | 6x | 73±3.41 |
| P-11 | Sirsa, HR. 205 | 57448 | 42 | 6x | 90±4.14 |
| P-12 | Udaipur, RJ. 600 | 60351 | 42 | 6x | 91±3.88 |
For chromosomal analysis, anthers were crushed and squashed in 1% acetocarmine. Complete observations of exact chromosome counts and meiotic abnormalities, if any, were made from freshly prepared slides. In each case, 400 pollen mother cells (PMCs) were observed at different meiotic stages of prophase I, metaphase I/II (M I/II), anaphase 1/II (A I/II), telophases 1/II (T I/II) and sporads. Pollen fertility was estimated through a stainability test by squashing the mature anthers in glycerol and acetocarmine (1 : 1) mixture. Well stained pollen grains with stained nuclei were taken as fertile and apparently viable, whereas shriveled or unstained nuclei were taken as sterile. The photographs of the PMCs and pollen grains were obtained by a Leica Qwin Digital Imaging System and a Nikon 80i Digital Imaging System. The identified voucher specimens were deposited in the Herbarium, Department of Botany, Punjabi University Patiala (PUN).
The seven individual plants designated as P-1, P-3, P-5, P-6, P-7, P-8, and P-9 collected from different localities of Punjab were found to be at tetraploid level as confirmed from the presence of 14 bivalents at M I (Fig. 1A) and 14 : 14 equal segregation of chromosomes at A I (Fig. 1B). The meiotic course in some of the plants (P-1, P-5, P-6, and P-7) was found to be abnormally characterized by chromatin bridges (Fig. 1C), laggards, (Fig. 1D) and unoriented bivalents (Fig. 1E). These meiotic abnormalities ultimately lead to reduction in pollen fertility (Fig. 1F). In rest of the plants, the meiotic behavior was found to be perfectly normal leading to high pollen fertility.
The meiotically analyzed plants (P-2, P-4, P-10, P-11, P-12) collected from localities including Sunam, Jalandher, Patiala in PB, Sirsa region in HR and Udaipur region in RJ were found to exist at hexaploid level as confirmed by the presence of 21 bivalents at M I (Fig. 1G) and equal segregation of 21 : 21 chromosomes at A I (Fig. 1H). Further meiotic course and microsporogenesis in these plants were found normal with high pollen fertility (Fig. 1I).
Morphometric analysisBoth the tetraploid and hexaploid cytotypes grow erect and are mostly found on the roadsides, wastelands, open areas in the altitudinal range of 250–600 m. Macroscopic characters studied presently in the two cytotypes included, area of plant spread, plant height, the diameter of stem, color of stem, number of leaves/branches, leaf size and length and number of spikes/plant (Table 2). Analysis of data on these characters revealed that 4x plants grow much taller (50–70 cm) than the 6x plants (40–60 cm) (Table 2, Fig. 2). The area of plant spread and width of stem was also observed to be more in 4x than 6x plants (Table 2). Besides, number of leaves/branches, leaf size, and length of spike were also noted to be higher in 4x plants then 6x plants. However, the number of spikes per plant was observed to be higher in 6x plants than 4x (Table 2). Among the 4x plants, further two clear cut morphotypes have been identified on the basis of leaf shape, i.e., an obovate leaf with the round apex (morphotype I) and an obovate leaf with the acuminate apex (morphotype II) (Fig. 2A, B). Morphologically, there is a lot of variation between the cytomorphotypes (Table 2). Among two morphotypes of tetraploid cytotypes, morphotype I is taller (50–70 cm) than morphotype II (50–65 cm) and have large leaves (4.70×7.44 cm) as compared to morphotype II (4.08×6.10 cm). Maximum plant spread area is also more in morphotype I (196×207 cm), while it is (99×90 cm) in morphotype II. The color of a mature spike is green in tetraploids and reddish in hexaploids. As far as the pubecenec of leaves and stem is concerned we did not find any marked difference in the two morphotypes as well as in hexaploid cytotype.
| Characters | Tetraploid (n=14) | Hexaploid (n=21) | |
|---|---|---|---|
| Morphotype I | Morphotype II | ||
| PUN 57445, 57436, 57438 | PUN 57440 57441, 57442, 57443 | ||
| Area of plant spread (cm2) | 196×207 | 99×90 | 100×60 |
| Height of plant (cm) | 50–70 | 50–65 | 40–60 |
| Diameter of stem (cm) | 2.52±.066 | 2.26±0.11 | 2.36±0.09 |
| Color of young stem | Purplish | Light green | Light green |
| No. of leaves/branch | 21.00±1.92 | 9.80±0.58 | 20±1.00 |
| Leaf color | Light green | Whitish green | Dark green |
| Leaf shape | Obovate round apex | Obovate acuminate | Obovate |
| Leaf lamina | Broad | Broad | Narrow |
| Leaf width (cm±SD) | 4.70±0.50 | 4.08±0.07 | 2.80±0.40 |
| Leaf length (cm±SD) | 7.44±0.27 | 6.10±0.04 | 3.1±0.82 |
| Length of spike (cm± SD) | 41.00±1.30 | 39.80±0.86 | 30±2.07 |
| Color of mature spike | Purplish/green | Whitish, shiny | Green |
| No. of spikes/plant | 9.60±1.99 | 8.20±0.37 | 21.60±4.02 |
| Texture of spike | Hard | Soft | Hard |
| Pollen size (µm) | 23.2×22.2 | 20.38×20.20 | 24.70×23.01 |
As per the review of literature, the genus Achyranthes has a basic number of x=7. Taxonomically, 15 species are known under this genus, out of which the chromosome data is available only for eight species at world level and two species from India. The chromosome numbers are diverse in the genus, ranging from 2n=14–84 with a well-known ploidy series (2x, 3x, 4x, 6x, and 12 x) (Bala et al. 2011).
Presently we have worked out two cytotypes (4x and 6x) of A. aspera collected from different areas of North India covering states of Punjab, Haryana, and Rajasthan (Table 1). The chromosomal literature reveal that the hexaploid cytotype has been worked out quite extensively from India as well as from outside India (Ahuja 1955, Pal 1964, Sharma and Banik 1965, Gill and Vasudeva 1970, Sharma 1970, Behera and Patnaik 1974, Sidhu and Bir 1983, Morton 1993 and de Lange et al. 2004). However, the chromosome number of 2n=28 (4x) has been reported for the first time. Besides, the species is also known to have intraspecific octaploid with 2n=56 (Kumar 1982), decaploid with 2n=70 (Romano et al. 1986) and dodecaploid with 2n=84 (Van Loon 1974) cytotypes, thus the species shows a lot of chromosomal diversity.
In the genus, the lowest chromosome number is reported to be 2n=14 (Bir and Sidhu 1980). The presently as well as previously worked out euploids are all the multiple of x=7. In the present study, all the plants of hexaploid cytotypes show the normal course of meiosis with high pollen fertility (Table 1). However, the maximum plants of the tetraploid cytotypes show the abnormal course of meiosis with low pollen fertility. The bridges and laggards were observed at A I, which may be due to paracentric inversions (Sinha and Godward 1972), or due to interlocking of bivalents (Bhattacharjee 1953). The laggards observed may be due to presence of abnormal spindle formation or non-synchronization of bivalents (Pagliarini 2000). These abnormalities either may remain as such or form micronuclei and further result in the formation of unbalanced gametes or pollen grains (Ranjbar et al. 2011). This abnormality along with the other abnormalities results in the formation of hyperploids which ultimately results in the origin of polyploid cytotypes (Sheidai et al. 2009). Cytology of this species has been reported in the number of temperate regions of the world. But the existence of two cytotypes of 4x and 6x have been found in India only.
New polyploids may arise either due to autopolyploidization or allopolyploidization. Consequences of both types of polyploidization have important and significant evolutionary and ecological implications (Soltis et al. 2014). 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. In presently analyzed A. aspara, the tetraploid and hexaploid individuals showed typical allopolyploid meiotic behavior with normal chromosome pairing leading to perfect bivalents and regular segregation. However, the presence of only bivalents in a polyploid taxon must not be confirmation of its allopolyploid nature. So on the basis of chromosome pairing and pollen fertility, the 4x and 6x individuals of A. aspara could be tentatively considered as allopolyploid on meiotic analysis, till confirmation through classical and molecular genome analyses.
Department of Biotechnology (DBT), Govt of India is duly acknowledged for providing facilities under DBT/IPLS program (BT/PR 4548/INF/22/146/2012) at Punjabi University Patiala. Thanks are also to the Head, Department of Botany, Punjabi University Patiala, for necessary laboratory facilities.
