2019 Volume 84 Issue 1 Pages 9-13
Meiotic studies were carried out on 26 accessions for the evaluation of genetic diversity of Cyanotis cristata and C. vaga from various geographical areas of Una, Hamirpur, Kangra, Palampur, Sirmaur (Himachal Pradesh), Dehradun and Mussoorie (Uttrakhand) in India. All accessions were diploids with n=12. The meiosis was found to be mostly normal in both species. In C. cristata, some meiotic cells showed abnormal figures such as early disjunction, bridges, laggards, interbivalent connections, vagrant or unoriented bivalents. All accessions of C. vaga showed normal meiotic course with low frequency of interbivalent connections. These species showed high pollen fertility in spite of presence of some meiotic abnormalities.
Cyanotis D. Don. is a cosmopolitan genus belonging to family Commelinaceae and includes 56 species. The species are distributed throughout tropical and subtropical Africa, Asia and Australia. The genus is represented by 16 species in India (Karthikeyan et al. 1989). A large number of species of this genus have been investigated cytologically from various parts of the world (Islam and Baten 1952, Sharma 1955, Kammathy and Rao 1961, Guervin and Le Coq 1966, Morton 1967, Heitz 1968, Jones and Jopling 1972, Bhattacharya 1975). The 16 indian species of Cyanotis have been recorded the chromosome number of n=12 or 2n=24 by several workers (Sharma 1955, Kammathy and Rao 1961, 1964, Raghavan and Rao 1961, 1965, Shetty and Subramanyam 1961, 1962, Mitra and Datta 1967, Rao et al. 1968, 1970, Bhattacharya 1975, Mehra and Sachdeva 1976, Sarkar et al. 1980, Renugadevi and Sampathkumar 1983, 1986, Alam and Sharma 1984, Bai et al. 1984, Lalithambika and Kuriachan 1996, 1997). In C. vaga, 2n=24 and 34 were reported (Bhattacharya 1975, Zheng et al.1989). Other chromosome number of 2n=48 in C. adscendens (Raghavan and Rao1961, Lalithambika and Kuriachan 1997), n=16 in C. papilionacea (Lalithambika and Kuriachan 1996), 2n=20 in C. axillaris and C. cucullata (Shetty and Subramanyam 1962, Lalithambika and Kuriachan1996, 1997, Rao et al. 1970, Renugadevi and Sampathkumar 1983, 1986, Bai et al. 1984) and 2n=26 in C. villosa (Faden and Suda 1980) were recorded. The Cyanotis is polybasic with x=8, 10, 11, 12, 13, 14, 15 and 17, and common basic chromosome number of x=12 was proposed previously (Shetty and Subramanyam 1962, Lewis 1964, Raghavan and Rao 1965, Guervin and Le Coq 1966, Heitz 1968, Rao 1970, Jones and Kukkonen 1971, Chimphamba 1973).
The present study was conducted to investigate cytological status of two species of Cyanotis, namely C. cristata and C. vaga from various localities of North-West India. The present investigations are aimed chromosome number, meiotic chromosome behavior, and pollen fertility to provide basic cytogenetic and cytogeographical information in two Indian Cyanotis species for their genetic variability and phylogeny.
Plant materials were collected from 26 localities growing in different regions of North-West India. Collected flower buds were fixed in a Carnoy’s fixative (ethanol : chloroform : glacial acetic acid=6 : 3 : 1) for 24 h at room temperature, and then transferred in 70% ethanol and stored at 4°C until use. Voucher specimens were deposited at the Herbarium, Department of Botany, Punjabi University, Patiala and accession numbers were obtained (PUN) in Table 1. Meiotic chromosomes were obtained from anthers of fixed floral buds by squashing in 2% acetocarmine. Microphotographs of chromosome counts were taken from freshly prepared slides using a Nikon Eclipse 80i microscope. Pollen fertility was investigated using glycerol–acetocarmine (1 : 1) technique (Marks 1954). The well-stained pollens were considered as fertile, while the unstained and shrunken pollens were considered as sterile. To study pollen morphology, mature flower buds were fixed in 70% ethanol and pollens were processed using an acetolysis technique (Erdtman 1954). The morphology of pollens was described as per the terminology is given by Nair (1964). The average size of pollens was estimated on the basis of measurements along the polar diameter (P) and equatorial diameter (E) using a micrometer. The microphotographs of pollen were taken by using a Magnus MLX Plus microscope.
| Taxa | Locality with altitude | Accession number (PUN) | Chromosome number (n) | Pollen fertility (%) | Pollen size (µm) | |
|---|---|---|---|---|---|---|
| P | E | |||||
| C. cristata | Dehradun, UK (637 m) | 62139 | 12 | 94.44 | 63.05±2.13 | 41.71±0.91 |
| Mussoorie, UK (2006 m) | 62140 | 12 | 91.78 | 24.25±0.01 | 17.46±0.57 | |
| Palampur, HP (1472 m) | 62141 | 12 | 88.88 | 59.17±17 | 42.22±0.74 | |
| Nagrota, HP (733 m) | 62142 | 12+(0-1)B | 94.89 | 62.71±1.63 | 38.46±1.03 | |
| Bhompur, HP (738 m) | 62143 | 12+(0-1)B | 93.67 | 67.56±1.76 | 44.28±1.55 | |
| Chintpurni, HP (940 m) | 62144 | 12 | 92.00 | 59.17±1.50 | 39.77±1.12 | |
| Banganga, HP (733 m) | 62145 | 12 | 94.49 | 58.49±1.00 | 41.03±1.03 | |
| Nagarkot, HP (733 m) | 62146 | 12 | 94.73 | 46.56±1.04 | 35.40±0.89 | |
| Gupt Ganga, HP (733 m) | 62147 | 12 | 93.82 | 52.38±1.27 | 36.52±0.72 | |
| Tirsoo, HP (746 m) | 62148 | 12 | 91.22 | 63.05±2.63 | 37.83±1.33 | |
| Nadaun, HP (478 m) | 62149 | 12 | 84.84 | 45.59±1.93 | 35.89±0.91 | |
| Kufalu, HP (932 m) | 62150 | 12 | 92.07 | 69.16±2.68 | 43.94±1.70 | |
| Ponta Sahib, HP (389 m) | 62151 | 12 | 93.42 | 73.04±2.94 | 43.31±1.83 | |
| Rajban, HP (1552 m) | 62152 | 12 | 91.52 | 60.14±1.40 | 37.83±1.52 | |
| Shillai, HP (1900 m) | 62153 | 12 | 95.31 | 68.19±1.67 | 48.79±1.23 | |
| Chamunda, HP (1662 m) | 62154 | 12 | 90.56 | 25.70±0.57 | 19.40±0.01 | |
| Rajban road, HP (1552 m) | 62155 | 12 | 91.66 | 25.70±0.57 | 14.55±0.01 | |
| Nahan, HP (932 m) | 62156 | 12 | 92.06 | 56.74±1.12 | 36.86±0.63 | |
| Basoli, HP (393 m) | 62157 | 12 | 92.40 | 63.68±1.92 | 44.28±1.12 | |
| Jawalamukhi, HP (610 m) | 62158 | 12 | 97.64 | 57.23±0.67 | 40.40±0.99 | |
| Gagret, HP (439 m) | 62159 | 12+(0-1)B | 90.16 | 56.55±1.06 | 38.12±1.24 | |
| C. vaga | Nagrota, HP (733 m) | 62137 | 12 | 83.87 | 50.44±1.52 | 40.25±1.24 |
| Nagarkot, HP (733 m) | 62136 | 12 | 85.93 | 50.92±1.29 | 40.74±0.73 | |
| Palampur, HP (1472 m) | 62138 | 12 | 91.02 | 50.44±1.44 | 40.74±0.63 | |
| Mussorie, UK (2006 m) | 62134 | 12 | 90.81 | 46.56±0.50 | 36.86±1.11 | |
| Mussorie, UK (2006 m) | 62135 | 12 | 91.96 | 44.62±0.70 | 32.98±1.06 | |
Meiotic analysis of 21 accessions of species of C. cristata showed chromosome number of n=12 of pollen mother cells (PMCs) (Figs. 1, 2). This number is in accordance with previous reports of 2n=24 (Sharma 1955, Kammathy and Rao 1961, Raghavan and Rao 1961, Shetty and Subramanyam 1962, Fotedar and Roy 1969, Bhattacharya 1975, Mehra and Sachdeva 1976, Sarkar et al. 1980, Alam and Sharma 1984, Renugadevi and Sampathkumar 1986, Lalithambika and Kuriachan 1997). Previous reports of 2n=30 (Guervin and Le Coq 1966) and 2n=26 (Jones and Jopling 1972) were not detected. Three accessions from Una and Kangra (HP) showed a B chromosome along with 12 bivalents at diakinesis (Fig. 3) which is a coincidence with the previous report of 2n=24+0-1B from Bangladesh (Islam and Baten 1952). Meiotic course studied in five accessions of C. vaga collected from two localities of Kangra and Mussoorie illustrated chromosome number of n=12 (Figs. 10, 11). The present chromosome number in C. vaga is in agreement with the previous reports of Morton (1967) and Bhattacharya (1975). Chromosome number of 2n=20 and 34 (Morton 1967, 1993, Thulin 1970, Zheng et al.1989) were not observed in this study. Cytological studies revealed that C. cristata and C. vaga exhibit a common meiotic count of n=12 (Figs. 1, 10) thus both species are diploid based on x=12.
Abnormal meiotic behavior was observed at various stages of meiosis in almost all the accessions of C. cristata and C. vaga (Table 2). In C. cristata, in spite of normal bivalent formation and equal distribution at anaphase I (A I) (Fig. 2), some PMCs showed meiotic abnormalities which included unoriented bivalents, an early disjunction of the chromosome, chromatin bridges, laggards and interchromosomal connections (Figs. 4–8). In C. vaga all the accessions under present study showed the presence of interbivalent connections (Fig. 12).
Spindle apparatus plays an important role in the orientation of bivalents at metaphase I (M I). Bivalents positioned away from the equatorial plate are said to be un-oriented bivalents. The accessions of C. cristata collected from 11 localities (Table 1) showed the presence of un-oriented bivalents at M I (Fig. 4). Lack of coordination between chromosomes and spindle leads to non-synchronous disjunction of bivalents (Sharma 1976).
Early disjunction of bivalents has been observed in 13 accessions of C. cristata, out of which accession collected from Kufalu (932 m) showed the highest frequency (16.66%) of early disjunction at M I (Fig. 8) followed by accessions from Dehradun (637 m) and Chamunda (1662 m) with frequency of 15.38% and 14.28% respectively. Accessions scored from Banganga (733 m) showed the lowest frequency (2.85%) of this phenomenon.
In the present study, chromatin bridges have been observed in three plants of C. cristata collected from Palampur (1472 m), Banganga (733 m) and Rajban road (1552 m) (Fig. 5). Out of which maximum percentage (10.00%) of chromatin bridges have been observed in accession from Rajban road and minimum (2.06%) in Palampur. Anaphasic bridges may arise either due to interlocking of chromosomes or failure of chiasmata in a bivalent to terminalize and chromosomes get stretched between the two poles (Saylor and Smith 1966).
Six accessions of C. cristata showed the presence of laggards at A I (Fig. 6). This phenomenon has been observed with the highest frequency (20.00%) in Rajban road (1552 m) accession and the lowest frequency (2.06%) in Palampur (1472 m) accession. The failure of chromosome movement or delayed terminalization, asynapsis and premature disjunction of bivalents have been suggested as possible causes of laggard formation (Gupta and Priyadarshan 1982, Jayabalan and Rao1987, Soheir et al. 1989).
Interchromosomal connections were observed in all accessions of C. cristata (Fig. 7) and two accessions of C. vaga (Table 2, Fig. 12). Interbivalent connections may be attributed to the formation of chromatic knots, resulted from the fusion of heterochromatic regions of some chromosomes during early stages of first meiotic division (Viinikka and Nokkala 1981). These may also be responsible for cytomixis or chromosomal stickiness (Thomas and Revell 1946, Singhal and Gill 1985).
| Species | PUN number | No. of PMCs observed | Abnormal PMCs (%) | ||||
|---|---|---|---|---|---|---|---|
| Early disjunction | Bridges | Laggards | Interbivalent connections | Unoriented bivalents | |||
| C. cristata | 62139 | 145 | 15.38 | — | 2.56 | 35.89 | 2.56 |
| 62140 | 90 | 9.45 | — | 5.40 | 74.32 | 8.10 | |
| 62141 | 99 | 6.20 | 2.06 | 2.06 | 16.55 | 2.42 | |
| 62142 | 120 | 7.69 | — | — | 42.30 | — | |
| 62143 | 103 | — | — | — | 71.42 | — | |
| 62144 | 140 | 7.69 | — | — | 69.23 | — | |
| 62145 | 160 | 2.85 | — | 5.71 | 45.71 | 5.71 | |
| 62146 | 100 | — | — | — | 8.10 | — | |
| 62147 | 96 | 8.10 | — | — | 32.43 | 5.40 | |
| 62148 | 79 | 8.10 | 8.10 | — | 35.13 | 2.70 | |
| 62149 | 125 | — | — | — | 61.53 | — | |
| 62150 | 136 | 16.66 | — | — | 33.33 | — | |
| 62151 | 100 | — | — | — | 75.00 | — | |
| 62152 | 98 | — | — | — | 41.66 | 8.33 | |
| 62153 | 142 | 8.00 | — | 4.00 | 34.00 | 2.00 | |
| 62154 | 89 | 14.28 | — | — | 71.42 | — | |
| 62155 | 123 | 6.66 | 10.00 | 20.00 | 26.66 | — | |
| 62156 | 156 | — | — | — | 40.00 | — | |
| 62157 | 98 | — | — | — | 20.00 | 5.00 | |
| 62158 | 112 | — | — | — | 17.07 | 9.7 | |
| 62159 | 130 | 7.69 | — | — | 38.46 | 11.53 | |
| C. vaga | 62137 | 80 | — | — | — | 29.41 | — |
| 62136 | 100 | — | — | — | 13.33 | — | |
| 62138 | 110 | — | — | — | — | — | |
| 62134 | 120 | — | — | — | 29.03 | — | |
| 62135 | 99 | — | — | — | 34.21 | — | |
Out of the accessions of C. cristata under present study, those from Mussoorie showed a higher percentage of meiotic abnormalities (8.84%), followed by accession from Chamunda (7.38%) and the rest of the accessions showed a lower frequency of irregular meiosis (0.69–6.15%) (Table 2). The fertility of pollen grains was found to vary from 83.87 to 91.96% among accessions of C. cristata, in C. vaga the pollen fertility varied from 84.84 to 97.64% in the studied accessions (Table 1).
In C. cristata, heterogenous sized and apparently fertile pollen grains (Fig. 9) are showed large variation in size from 24–75 µm. In the case of C. vaga, pollen grains show medium size between 44–50 µm (Fig. 13). In the present case, pollen size did not cause a reduction in pollen fertility.
There remain two possibilities that the observed meiotic abnormality is the result of an artifact or normal phenomenon in meiosis and the method used for evaluation of pollen fertility relies on the stainability of pollens and is not a complete test of its germinating abilities. Therefore, it is necessary to use different methods to investigate the relationship between meiosis and pollen fertility.
The authors are grateful to the University Grants Commission, New Delhi for providing facilities under DRS SAP III and ASSIST programme. Financial support to Poonam Rani under JRF programme of CSIR is greatly acknowledged. Thanks are also due to Head, Department of Botany, Punjabi University, Patiala for providing the necessary infrastructural facilities.
