2019 Volume 84 Issue 1 Pages 89-92
Cytological investigations have been carried out in one wild accession of Anemone rupicola Cambess. collected from the cold deserts of Ladakh division of Jammu & Kashmir India. The chromosome count of 2n=16 was observed and is the first chromosome record for the species from India. Meiotic analysis showed structural heterozygosity for reciprocal translocations as indicated by the presence of multiple associations of four chromosomes. Detailed meiotic analysis showed Pollen Mother Cells (PMCs) with laggards (32.87%) and chromatin bridges (21.91%) at anaphases I, II (A I, II)/telophases I, II (T I, T II), leading to abnormal microsporogenesis. Consequently, abnormal sporads such as triads (37.14%) and polyads (28.57%) were formed. Structural heterozygosis and other associated abnormal meiotic irregularities in the species showed considerable pollen sterility (32.20%) which could be attributed to the presence of ring/chain shaped multivalent in PMCs.
Anemone rupicola Cambess. (Ranunculaceae) called as ‘Rock anemone’ ‘Wood anemone’ or ‘Wild swan’ is an erect, silky-pubescent, perennial herb characterized by large showy white flowers with pinkish undersides and a central boss of yellow stamens. A very common species grows on rocks, stream sides, stone cliffs and gravelly slopes between elevations 2400–4400 m in India. It is a hardy species and can withstand temperatures as low as −20°C. The seeds of plant are used against the number of ailments such as stomach worms, sharp pain, snake bites, tumor, indigestion, and gonorrhoea (Hao et al. 2017). The sap of crushed leaves is also used in treating of ears with pus.
As a part of a project to explore the cytogenetical diversity in the plants of Indian cold deserts, the present studies have been carried out in A. rupicola on an individual plant basis from the Ladakh Division of Jammu and Kashmir. The individual accession exists at the diploid level but showed evidence of reciprocal translocations. The present communication covers chromosome count, detailed meiotic course, microsporogenesis, and pollen fertility in the studied individual.
For male meiotic studies, individuals were collected from a wild accession in the Sapi, Kargil District of Ladakh Division (elevation 4300 m) during July 2017. The young floral buds of various sizes of each individual were fixed in freshly prepared Carnoy’s fixative (6 ethanol : 3 chloroform : 1 acetic acid, v/v/v) for 24 h and stored in 70% ethanol in a refrigerator at 4°C. Young anthers of each plant were squashed in 1% acetocarmine and slides of PMCs were prepared. PMCs were carefully examined for chromosome counts during late prophase 1 (P I), metaphase I (M I) and A I/A II. A total of 113 PMCs were examined for determining the chromosome counts while 20–30 slides were prepared from different anthers of each plant for analysis of chromosomal associations. The sporad analysis was made by examining 400–500 PMCs of the plant by squashing the anthers in 1% acetocarmine. Pollen fertility was estimated through a stainability test, for which mature anthers were squashed in glycerol–acetocarmine (1 : 1) and 1% aniline blue dye. The pollen grains with fully stained cytoplasm and nuclei were counted as fertile and those with partially stained or unstained cytoplasm were as sterile. The voucher specimen was deposited d as PUN 62197 in the Herbarium, Department of Botany, Punjabi University, Patiala (PUN).
Meiotic studies carried out in the wild accession of the species revealed the chromosome count of n=8, as confirmed from the presence of eight large-sized bivalents at diakinesis, M I and 8 : 8 chromosome segregation at A I (Fig. 1A–C). This chromosome count is the first record for the species from India. Further detailed meiotic studies revealed the phenomenon of a reciprocal translocation, which is characterized by the presence of a ring or zig-zag shaped quadrivalents at diakinesis (Fig. 1D, E) and chain shaped at M I (Fig. 1F). Analysis of various chromosomal associations on the basis of well-spread chromosomes in 113 PMCs (Table 1) revealed that 63 PMCs (55.75%) showed eight regular bivalents while the remaining 49 PMCs (43.36%) depicted the presence of quadrivalents (ring, chain or Zig-Zag type). Analysis of data revealed that the average chromosomal associations per PMC in the accession work out to 0.43IV+7.12II+0.01I and the most common type of chromosomal configuration consisting of 1IV (ring type)+6II, which was observed in 18 PMCs (18.58%), followed by IIV (chain type)+6II (14.15%) in 16 PMCs and least in IIV (Zig-Zag type)+6II (10.61%) in 12 PMCs.
| Chromosomal associations | ||||||
|---|---|---|---|---|---|---|
| PMCs observed | Quadrivalents | Bivalents | Univalents | |||
| Chain | Ring | Zig-Zag | ||||
| 63 | — | — | — | 8 | — | |
| 1 | — | — | — | 7 | 2 | |
| 12 | 1 | — | — | 6 | — | |
| 18 | — | 1 | — | 6 | — | |
| 6 | — | — | 1 | 6 | — | |
| 4 | 1 | — | — | 6 | — | |
| 3 | — | 1 | — | 6 | — | |
| 6 | — | — | 1 | 6 | — | |
| Total (%) | 113 | 16 (0.14%) | 21 (0.18%) | 12 (0.10%) | 805 (7.12%) | 2 (0.01%) |
Subsequently, detailed meiotic studies revealed the chromatin masses organized into separate units and at later stages of meiosis form multiple poles (Fig. 1G). Other associated meiotic abnormalities in the PMCs, include the presence of chromatin bridges (21.91%) (Fig. 1H) and laggards at A I, A II/T I, T II (32.87%) (Fig. 1I). Of 140 sporads analyzed, 65.71% were found to be abnormal, which included triads (Fig. 1J), and polyads (Fig. 1K) (Table 2). Consequence to the presence of these irregular meiotic abnormalities, the studied individual showed reduced pollen fertility 67.80% (Fig. 1L).
| Meiotic course | Sporad analysis | |||
|---|---|---|---|---|
| Meiotic stage | PMC with laggards (%) | PMC with Chromatin bridges (%) | Sporads | No. of sporads |
| A I | 24/146 (16.43%) | 12/146 (8.21%) | Triads | 52/140 (37.14%) |
| T I | 6/146 (4.10%) | 8/146 (5.47%) | Tetrads | 48/140 (34.28%) |
| T II | 18/146 (12.32%) | 12/146 (8.21%) | Polyads | 40/140 (28.57%) |
| Total | 48/146 (32.87%) | 32/146 (21.91%) | ||
A. rupicola an accession scored from Ladakh has been worked out for the first time for chromosome counts in India. The species has been worked out chromosomally quite extensively from different regions of the world and depicts polyploidy of chromosome number series, 2n=16, 32, 48 (Langlet 1927, Moffet 1932, Heimburger 1959, Baumberger 1971). However, the presently studied accessions showed the diploid chromosome number, 2n=16. This chromosome number confirms previous reports from outside India (Heimburger 1959). Besides, some populations in other localities are known to show the existence of tetraploid (2n=32) and hexaploid (2n=48) cytotypes (Langlet 1927, Baumberger 1971) based on x=8.
Reciprocal translocation is the phenomenon of chromosomal rearrangement between two non-homologous chromosomes, resulting in structural heterozygotes. Such structural heterozygotes occasionally produce duplications or deficiencies in the genome of PMCs, resulting in non-viable and sterile male gametes (Ghosh and Datta 2006). Such a phenomenon of reciprocal translocation leading to sterile pollen grains has also been reported in a number of plants, namely Citrus jambhiri (Singhal and Gill 1981), Chrysanthemum zawadskii (Kim et al. 2008), Artemisia parviflora (Gupta et al. 2010), Astragalus chlorostachys (Rana et al. 2012), Tradescantia spathacea (Koul et al. 2013), Saxifraga diversifolia (Kumar and Singhal 2013) and Achillea millefoilium (Singhal et al. 2014). Kumar and Singhal (2013) have reported multivalent and univalent in the varieties of S. diversifolia. In the present case, 38.41% of PMCs showed quadrivalent formation at diakinesis and M I. Occurrence of quadrivalents in the wild diploid individuals seems to be the result of the reciprocal translocations (Mahama and Palmer 2003).
Lopez et al. (2012), while studying the chiasma frequency in different ecotypes of Arabidopsis thaliana, suggested that depending upon the type of segregation, translocations significantly affect the pollen fertility. During adjacent type 1 and type 2 cases of segregation, homozygotes and translocated chromosomes go to one pole, producing unstable or non-viable gametes. In alternate segregation, homozygote chromosomes go to one pole, and translocated chromosomes go to another, thus producing viable or stable gametes. It has been reported that species showing structural heterozygosity either produce semi-sterile gametes (Ghaffari et al. 2009) or complete sterility due to reciprocal translocations in Allium consaguineum (Gohil and Koul 1978) and Allium roylei (Sharma and Gohil 2003). Reduction in some pollen fertility in the presently studied accessions might be attributed to the phenomenon of reciprocal translocation.
The authors are grateful to the University Grants Commission, New Delhi for providing financial assistance under the DRS SAP I and II and ASIST programme and CSIR for providing Junior Reserch Fellowship to Mr. Nissar Ahmad Khan (Award letter no. 121731348/Research-29/11/2018). Thanks are also due to the Head, Department of Botany for necessary laboratory facilities.
