2022 Volume 87 Issue 4 Pages 323-330
Comprehensive studies on meiotic behavior and pollen biology of a species are crucial for its conservation strategies. The presence of meiotic bottlenecks directly or indirectly influences the reproductive success of a plant species. The present study records chromosome number, male meiotic behavior, seed set, pollen viability, and pollen germination in two populations of Swertia thomsonii, an important medicinal plant of Himalaya. The chromosome count recorded in both accessions was 2n=2x=26, however; chromosomal abnormalities (21.23 and 4.31%) were recorded in population 1 (Pop-1) and population 2 (Pop-2), respectively. These include chromosomal stickiness, out of the plate, formation of laggards, abnormal spindles, bridges, multipolarity, and polysporads. These meiotic irregularities compromise pollen viability, pollen germination, and seed set of species. The possible cause for these recorded meiotic abnormalities was cold stress as the species grows in temperate or sub-alpine regions with cold climatic conditions. Another possible reason might be the presence of heavy metals in the soil more than permissible limits, which was determined by soil sample analysis. Therefore, these findings along with other reproductive and ecological studies can aid in the development of successful conservation and management strategies for this medicinally important plant species.
Himalaya harbors a rich repository of medicinal plants. Among these, Swertia (Gentianaceae) holds a distinct position as many species of this genus have their medicinal usage in the Indian pharmaceutical codex, British and American pharmacopeias, and other traditional medicinal systems of India viz Ayurveda, Unani, and Siddha. Swertia species have been reported to act as a potential source of bioactive compounds and have been used to treat different ailments such as hepatitis, inflammation, diabetes, cancer, bacillary dysentery, jaundice, fever, gastric troubles, pneumonia, and other diseases (Kaur et al. 2021). S. thomsonii is used as folklore medicine in the treatment of headache and malarial fever (Brahmachari et al. 2004). Phytochemical analysis of the whole plant has shown the presence of 7-hydroxy-1-O-[β-dxylopyranosyl-1dxylopyranosyl],3,dimethoxyxanthone, gentiakochianin or swertianin and gentiacaulien (Ahmad et al. 2002).
Swertia is extremely polymorphic with so many misperceptions in its taxonomy. This genus is polybasic with chromosome number ranging from 14–26 (2n=14, 16, 18, 20, 24, 26) (Kaur et al. 2021). Chromosome counts of 58 species of Swertia are known all over the world, out of which only 21 species were reported from India. Khoshoo and Tandon (1963) are considered to be pioneers in determining chromosome counts in several Indian species. S. paniculata (2n=2x=16) and S. iberica (2n=7x=42) represent the lowest and highest chromosome numbers, respectively, of the genus documented so far. However, a comprehensive cytological study on important Swertia species from various geographical regions is still wanting.
The cellular events that occur in meiosis are evolutionary conserved, genetically controlled, and comprise highly coordinated pairing of homologous chromosomes, crossing over, reduction in chromosome number, and gamete formation. The normal and harmonious course of meiosis thus ensures normal cell division and gamete viability, securing the propagation of cells and hence the survival of species (Shabir et al. 2013). Microsporogenesis is thus an ideal process for studying meiotic mutations affecting the steps involved in the meiotic reduction of diploid pollen mother cells to form haploid spores. The presence of heavy metals in the soil more than permissible limits is toxic and the toxicity of these elements leads to meiotic abnormalities and therefore developmental disturbances which affect pollen and ovule viabilities (Seregin et al. 2001, Zohair et al. 2012). The present study was planned to study the male meiotic behavior of the species and the following questions were addressed in the present study (i) What is the male meiotic behavior of the species? (ii) How do meiotic irregularities impair seed set, pollen viability, and pollen germination in the selected plant? (iii) What is the correlation between the prevalence of cold conditions in alpine habitats and the possible cause of meiotic abnormalities and reduction in pollen fertility in the species? (iv) What is the relation between the presence of heavy metals in the soil and the meiotic behavior of the target plant species?
S. thomsonii C. B. Clarke (Gentianaceae) locally named as, ‘Tikta,’ ‘Chuck theek karpoh,’ ‘Momram,’ and ‘Kodi-jad’ is a perennial rhizomatous herb, 60–100 cm tall; the stem is simple, erect, hollow, round and glabrous; basal leaves are spathulate and long-stalked, cauline leaves are connate, narrow, lanceolate, acute and stalk less; inflorescence cyme with dense panicles; fruit capsule; seeds angularly winged.
Meiotic studiesFlower buds at an ideal stage were collected for meiotic studies from wild populations viz Doodhpathri population (Pop-1, 2,700 MASL) and Gulmarg population (Pop-2, 2,550 MASL) of Kashmir Himalaya. The herbarium specimens of the plant species collected from various populations were submitted to Kashmir University Herbarium (KASH). The floral buds were collected in the early morning from the two aforementioned populations, at each population, the buds were fixed in Carnoy’s fixative (ethanol : acetic acid in 3 : 1 ratio) for 24 h and then floral buds were washed with 70% ethanol and then stored in it under refrigeration at 4°C until use. For meiotic studies, slides were prepared by squashing the anthers in 2% acetocarmine. Chromosome number was determined at diakinesis, metaphase, and anaphase. All meiotic phases were examined and abnormalities if any were recorded. Pollen fertility was estimated by squashing the anthers in 2% acetocarmine. Completely round and well-stained pollen grains were considered fertile or viable, and those that were unstained and distorted in shape were taken as non-viable. Photomicrographs were taken from freshly prepared slides using a Magnus MLX LED Microscope.
Seed setPercentage seed set of species was calculated by the mean number of seeds produced per capsule to the mean number of ovules born per flower following Lubbers and Christensen (1986).
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Further in vitro pollen germination was carried out by using different germination media by following Brewbaker and Kwack (1963) methods with some modifications. Percentage pollen germination was calculated by the number of pollen grains germinated to the number of pollen grains observed.
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For heavy metals analysis, soil samples (0–15 cm) from respective sites were collected. Soil samples were dried at room temperature and ground by pestle and mortar and passed through a 2 mm sieve. The concentration of heavy metals was determined by following the digestion method US Environmental Protection Agency (1996).
Metrological data collectionTo understand the effect of temperature on pollen fertility and seed set, metrological data was obtained from the Indian metrological department Rambagh Srinagar, India. Information regarding mean maximum and minimum temperatures was collected over a period of one decade (2011–2020) for months July and August which represents the flowering period of the sampled plant species.
Statistical analysisStatistical analysis was carried out in R version 4.0.3 (R Core team 2020). The linear model of regression (significance level of 5%) was employed between meiotic abnormality and seed set by using the basic Trend line R package (Mei and Yu 2020: https://rdrr.io/cran/basicTrendline/). The relationship between meiotic abnormality, pollen fertility, in-vitro pollen germination, and seed set were carried out by using R package cormorant (Roman, M.: https://github.com/r-link/corrmorant).
In the present study, a total of 1973 and 1198 PMCs from two populations (Pop-1) and (Pop-2) were analyzed. Both accessions showed 13 bivalents at diakinesis and metaphase I confirming diploid chromosome number 2n=2x=26 (Fig. 1a). Diplotene/ diakinesis showed the presence of a linear, ring, and eight-shaped bivalents with single nucleolus (Fig. 1b). Anaphase I showed 13 chromosomes at each pole (Fig. 1c). A good percentage of PMCs (78.77 and 95.69%) showed normal meiotic behavior, however, c. 21.23 and 4.31% PMCs showed meiotic irregularities in Pop-1 and Pop-2, respectively. These irregularities include chromosomal stickiness, 42.48 and 14.28% (Fig. 1d–f), Formation of laggards, 16.66 and 3.53% (Fig. 1g–i), out of the plate, 11.02 and 3.53% (Fig. 2a, b) and formation of bridges; 18.94 and 21.12% (Fig. 2c–e). Most of the PMCs showed normal anaphase II and telophase II (Fig. 2f, g) with less percentage of multipolarity at telophase II, 8.06 and 0% (Fig. 2h), tetrads with micronuclei, 3.08 and 0% (Fig. 2i) in Pop-1 and Pop-2, respectively. Telophase II is followed by the formation of monads, dyads, triads, tetrads, and finally pollen grains (Fig. 3a–d). The meiotic abnormalities recorded at various stages of meiosis in both these populations are depicted in Table 1.
| Population | Geo-coordinates | Altitude (m-asl) | Total number PMCs analysed | Meiotic abnormalities | |||||
|---|---|---|---|---|---|---|---|---|---|
| Stickiness at M-I/A-I/T-I/ M-II/A-II/T-II | Laggards A-I/A-II | Out of plate M-I/M-II | Bridges A-I/A-II | Multipolarity at T-II | Tetrads with micronuclei | ||||
| Doodhpathri (Pop-1) | 33.84N 74.55E | 2,700 | 1,973 | 291/685 (42.48%) | 51/306 (16.66%) | 42/381 (11.02%) | 36/190 (18.94%) | 13/151 (8.60%) | 8/260 (3.08%) |
| Gulmarg (Pop-2) | 34.04N 74.38E | 2,550 | 1,198 | 30/210 (14.28%) | 8/226 (3.53%) | 7/217 (3.22) | 4/188 (2.12%) | 0/133 (0%) | 0/224 (0%) |
The most frequent abnormality in two meiotic divisions i.e., meiosis I and meiosis II, were related to abnormal chromosome segregation, stickiness at metaphase, formation of laggards and bridges at anaphase that result in the formation of micronuclei at telophase. These abnormalities are mostly due to abnormal spindle formation and failure of proper segregation of chromosomes that lead to late disjunction. Due to abnormal spindle and bridge formation, chromosomes fail to reach the respective poles which result in multipolarity at telophase II. The formation of laggards at anaphase is the outcome of mild or intense stickiness at metaphase that led to the formation of micronuclei in tetrads. Consequent to stickiness and associated meiotic abnormalities, inadequate pollen viability and germination were found, which may be probable cause for a low seed set of species. The pollen fertility (Fig. 3e) recorded in the two populations was 75.86 and 89.83% respectively. Pollen germination (Fig. 3f–h) estimated in the in vitro studies were 29.79 and 70.51% in Pop-1 and Pop-2, respectively which suggests that all pollen grains were not physiologically active. The percentage seed set (Fig. 3i) in the two populations was 43.64 and 81.11, respectively. The mean percentage of meiotic abnormality, pollen fertility, in-vitro pollen germination, and seed set recorded in both populations respectively is depicted in Table 2. From pollen fertility and seed set results it was obvious that both of them decrease with an increase in the percentage of meiotic abnormalities in the two studied populations.
| Population | Meiotic abnormality (Mean %) | Pollen fertility (Mean %) | In vitro pollen germination (Mean %) | Seed set (Mean %) |
|---|---|---|---|---|
| Pop-1 | 21.23 | 75.86 | 29.79 | 43.64 |
| Pop-2 | 4.31 | 89.83 | 70.51 | 81.11 |
The result of the linear regression between meiotic abnormality (%) and seed set (%) showed a (significant (p＜0.001) decline of −2.15% in the seed set with the increasing unit percentage of meiotic abnormality (Fig. 4). The results also revealed that meiotic abnormality shows a high degree of negative correlation with pollen fertility (r=−0.85), in vitro pollen germination (r=−0.97) and seed set (r=−0.96). Furthermore, seed set showed a positive correlation with pollen fertility (r=0.85) and in vitro pollen germination (r=0.97) (Fig. 5).
In the case of Pop-1 which is located relatively higher elevation (2,700 masl) as compared with Pop-2 (2,550 masl), the pollen fertility and seed set were relatively less. This is possibly due to the presence of differential environmental stress along the altitudinal gradient. For the Doodhpatri population, the mean maximum temperature for one decade (2011–2020) for months of flowering (July and August) was estimated to be 18.6–19.9°C, and the mean minimum temperature was 11–11.5°C while as for Gulmarg population it was found to be 19.9–20.6°C and 10.49–10.99°C, respectively.
Soil samples analysis of both sites showed the presence of heavy metals viz. lead (Pb) 154–163, nickel (Ni) 65–65.9, cobalt (Co) 28–36, chromium (Cr) 10–16.2, zinc (Zn) 3.46–7.34 and mercury (Hg) 4.6–48 mg kg−1 of soil.
The present study reported the gametophyte chromosome count (2n=2x=26) for S. thomsonii which is in agreement with previous records (Khoshoo et al. 1963, Vasudevan 1975), however; the present study demonstrated the meiotic behavior of the species and in present study, meiotic abnormalities were also recorded. These meiotic abnormalities include chromosomal stickiness, out of the plate, formation of laggards, abnormal spindles, bridges, multipolarity, and tetrads with polysporads. During meiotic studies, 13 bivalents were recorded at diakinesis showing balanced segregation (13 : 13) at anaphase and telophase in both populations in most of the PMCs. The most frequent abnormality in the two meiotic divisions was related to chromosome segregation, such as stickiness at metaphase 42.48 and 14.28% in Pop-1 and Pop-2, respectively. It was followed by the formation of bridges at 18.94 and 21.12% and laggards at 16.66 and 3.53% at anaphase, which resulted in the formation of micronuclei and polysporads at telophase. Chromosomal stickiness observed in the present study was either mild or intense. Chromosomal stickiness was reported by the number of workers in various flowering plants (Tripathi and Kumar 2010, Kaur and Singhal 2012, 2014, Rana et al. 2013, Rashid et al. 2021). Late disjunction of chromosomes was also observed in the present study, which might be due to abnormal spindle formation. Spindle abnormalities are reported to occur due to environmental factors and incompatible gene interaction (Nirmala and Rao 1996, Baptista-Giacomelli et al. 2000). Formation of laggards/fragments as meiotic configurations during the meiotic course is considered a meiotic syndrome that portrays reduced control over the meiotic course (Jones and Brumpton 1971). The presence of laggards and micronuclei is possibly due to abnormal spindle formation, disturbed cytoskeleton, and other cellular changes (Vasek 1962). The abnormal meiotic course often leads to disturbances in microsporogenesis, thus resulting in pollen malformation or sterility and negatively influencing the reproductive success of the species in the wild (Lattoo et al. 2006, Singhal and Kumar 2008, Kumar et al 2010). It has been reported that meiosis is the most sensitive stage in the life cycle of plants and has a role in the progression of gametes and natural selection but this cellular phenomenon is influenced by both genetic and environmental factors (Ahmad et al. 1984, Viccini and Carvalho 2002, Sun et al. 2004, Bajpai and Singh 2006, Rezaei et al. 2010). The basic information and biochemical bases are still unknown, however; some workers attributed it to genetic and environmental factors (Caetano-Pereira et al. 1995, Bione et al. 2000). Some workers suggest that these abnormalities are largely due to the presence of mutant genes (Kaul and Murthy 1985) and sometimes by abiotic factors such as high temperatures, herbicides (Caetano-Pereira et al. 1995) and/ or by cold stress (Singh et al. 2020). These meiotic bottlenecks lead to pollen sterility and low seed sets in this species. The low pollen germination rates in this species as recorded on in vitro germination trials possibly impairs the successful fertilization which eventually results from low seed counts per capsule and hence the reproductive success of this species. Pollen germination and pollen tube growth are prerequisites for fertilization in seed-bearing plants (Tiwari et al. 2017). From the results of statistical analysis, it was evident that an increase in meiotic abnormality results in a decrease in pollen fertility, pollen germination, seed set, and vice-versa. Based on the results, the present study revealed a clear effect of meiotic abnormality on pollen fertility, in vitro pollen germination, seed set, and therefore reproductive success of presently studied species.
Prevalence of cold conditions and meiotic abnormalitiesIt is a consensus that there is a temperature lapse with increasing altitude due to a decrease in atmospheric pressure. In the present study, it was observed that both mean maximum and minimum temperature decreased with increasing altitude. These findings conform with previous studies which had shown that approximately (−0.5 to −0.7°C) temperature lapse occurs with every 100 m increase in altitude in mountain regions (Moser et al. 2010, Holden and Rose 2011, Chen et al. 2018). The mean maximum and minimum temperature recorded in the present study during the flowering season was found to be below the normal range. Plant growth and development are strongly influenced by in situ environmental conditions such as temperature, light intensity, water availability, and soil nutrition (Williams and Mazer 2016). Both low and high temperatures beyond optimum can negatively affect the meiotic course of a plant species thereby impeding pollen fertility and performance which finally results in low seed sets (Williams and Mazer 2016). Microsporogenesis and megasporogensis (i.e., development of male and female gametophyte), pollen germination, stigma receptivity, and fertilization are temperature-sensitive phenomena that influences ovule development and seed formation (Rosbakh et al. 2018). Temperature extremes beyond optimum (25±5°C) adversely affect key steps involved in meiosis such as spindle formation, synaptonemal complex organization, chromosome segregation, and cytokinesis that finally leads to developmental defects (Hatfield and Prueger 2015, De Storme and Geelen 2020). Chromosome segregation needs normal polymerization of microtubules to ensure proper meiotic spindle formation, however; its stability is sensitive to lower temperatures (Liu et al. 2003). Low temperatures as recorded in the present study might be responsible for posing such stressful conditions, eventually leading to meiotic abnormalities and therefore resulting in low pollen fertility and seed set. Kaur and Singhal (2019) also reported erratic meiosis and abnormal pollen development in various plant species growing in different cold regions of the Himalaya. It needs further investigation so that trends in changing global climate change can be predicted from such regions.
Relation between the presence of heavy metals in the soil and meiotic abnormalitiesPlants are sensitive to both deficiency and added values of some micronutrients including heavy metals. Nowadays heavy metals are considered important environmental pollutants and their toxicity is a problem of great concern from environmental, ecological, and nutritional perspectives (Gjorgieva Ackova 2018). In the present study, the estimated heavy metal concentration in the accessed soil samples at two sites was more than the permissible limits given by Word Health Organization (WHO). The presence of these metals in soil may have added to the cause of meiotic abnormalities reported in presently studied species. Heavy metals can exert their toxic effect at different levels such as cellular, sub-cellular, and/or molecular. Sathaiah and Reddy (1984) evaluated the clastogenic, mitotoxic, and cytotoxic effects of Hg2+, Pb2+, and Cd2+ and they found the maximum degree of mutagenesis caused by lead. It has been found in previous studies that heavy metal toxicity leads to genotoxicity and therefore meiotic disturbances which impede pollen and ovule fertility. Pb and Zn are mostly widespread heavy metals and their higher concentration in soil has harmful biological and ecological consequences (Biskup and Izmailow 2004, Ismael et al. 2018, Hajmoradi and Taleb Beydokhti 2019). Plants can tolerate lower concentrations of these metals but higher concentrations cause significant effects on cytomorphological characters due to their genotoxic and mutagenic effects. Kumar and Gupta (2008) reported a gradual decrease in pollen fertility in Capsicum, when treated with heavy metals. Several other studies have found a significant correlation between meiotic abnormality with increasing doses of heavy metals such as Hg and Cd (Rai and Kumar 2010, Choudhary et al. 2012). Likewise similar results were obtained in Cichorium intybus, Trigonella foenum-graecum and Capsicum annum (Jafri et al. 2011, Choudhary et al. 2012, Gulfishan et al. 2012). The presence of heavy metals in soil, the prevalence of cold climate conditions, and high environmental stress along altitudinal gradient collectively may be responsible for the observed meiotic abnormality and low seed set for the presently studied species.
Most species conservationist believes that before setting up a conservation program for a threatened species it is better to know about its intrinsic reproductive constraints (Renner 1999, Williams 2008, Friedman and Ryerson 2009). Meiotic abnormalities are one of the constraints in the sexual reproduction of plants. The presently investigated plant species are already facing threats such as habitat fragmentation, overexploitation, herbivory, grazing, and lack of agro-techniques for mass cultivation, therefore, the presence of meiotic bottlenecks can add to the factors that lead to a decrease in its population size. Therefore, keeping in view meiotic abnormalities and anthropogenic threats sustainable conservation strategies are needed to devise for this valuable plant species.
We are thankful to the Head, Department of Botany, the University of Kashmir for providing the necessary lab facilities. We also extend our sincere thanks to the Indian metrological department Rambagh Srinagar, for providing the required metrological information. The Authors also acknowledge the funding under MANF-2018-19-JAM-100111 in favor of BAW.
