Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 76(3): 29-37, 2023 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-2154 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Sofi, I.I., Verma, S., Gan- ie, A.H., Sharma, N., & Shah, M.A. (2023). Meiotic behavior and its implica- tions on the reproductive success of Arnebia euchroma (Royle ex Benth.) I.M.Johnst. (Boraginaceae), an important medicinal plant of Trans- Himalaya. Caryologia 76(3): 29-37. doi: 10.36253/caryologia-2154 Received: July 4, 2023 Accepted: November 13, 2023 Published: February 29, 2024 Copyright: © 2023 Sofi, I.I., Verma, S., Ganie, A.H., Sharma, N., & Shah, M.A. This is an open access, peer- reviewed article published by Firenze University Press (http://www.fupress. com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Meiotic behavior and its implications on the reproductive success of Arnebia euchroma (Royle ex Benth.) I.M.Johnst. (Boraginaceae), an important medicinal plant of Trans- Himalaya Irfan Iqbal Sofi1,*, Shivali Verma2, Aijaz H. Ganie1, Namrata Sharma2, Manzoor A. Shah1 1 University of Kashmir, Srinagar-19006, Jammu and Kashmir, India 2 University of Jammu, Jammu Tawi-18006, Jammu and Kashmir, India *Corresponding author. E-mail: sofi.irfan98@gmail.com Abstract. The present study reports the chromosome number, meiotic behavior and its relation with pollen fertility and seed set of Arnebia euchroma (Royle ex Benth.) I.M.Johnst. The species shows a chromosome count of 2n = 2x = 14. The meiotic abnormalities such as chromatin stickiness, cytomixis, laggard formation, chromosom- al bridges, were also observed in the Pollen Mother Cells (PMCs) of the target plant species. The linear model of regression showed a significant reduction of seed set with increasing meiotic abnormality and correlation analysis highlighted positive relation- ship between pollen viability and seed set. Meiotic abnormalities within the species hinder its reproductive process, causing a decline in reproductive efficiency. This study highlights the importance of addressing these intrinsic factors in future conservation programs to prevent a decline in the species population in nature. Keywords: chromosome number, meiotic abnormalities, pollen viability, seed set. INTRODUCTION Reproduction is an essential and vital stage in the life history of plants and a necessary natural process for survival, multiplication and evolution (Wani et al. 2022). The meiotic abnormalities are one of the factors that affect the reproductive success of plant species (Wani et al. 2022). The decline in seed set and loss of genetic variability, are some of the repercussions of the meiotic depression (Cohen et al. 2021). The studies on reproduction, meiotic behaviour and seed biology may aid in identifying the key factors that affect the reproductive success of species as well as sustenance or survival of its population (Gan et al. 2013). The study is also critical for developing strate- gies for sustainable utilisation and effective conservation measures of threat- ened species (Rashid et al. 2022a). http://www.fupress.com/caryologia https://doi.org/10.36253/caryologia-2154 https://doi.org/10.36253/caryologia-2154 http://www.fupress.com/caryologia http://www.fupress.com/caryologia mailto:sofi.irfan98@gmail.com 30 Irfan Iqbal Sofi et al. The restricted distribution pattern of Arnebia euchroma (Royle ex Benth.) I.M.Johnst., in Himalaya is further declining and this species is at risk of disap- pearing because of habitat deterioration, fragmentation and climate change (Lal et al. 2020; Sofi et al. 2022a). Therefore, understanding of the meiotic behavior may improve the knowledge about reproduction and inher- ent bottlenecks of the species. The foundation of con- servation biology, reintroduction, and mass produc- tion and multiplication rely on quality of germplasm. Understanding the biological characteristics of the spe- cies will unveil the attributes of the germplasm (Ma et al. 2022). Therefore, the present study was conducted to study the meiotic behaviour and its impact on pollen fertil- ity and seed set. Understanding the aspects of meiotic behaviour, pollen fertility and seed production may provide vital clues for sustainable development of this important medicinal plant species of the Trans-Hima- laya. MATERIAL AND METHODS Study area The study was conducted in the four studied sites (Table 1) of the trans-Himalayan range of Ladakh, India. The area is dominated by mountains and harsh climatic conditions, however, regarded as cold biodiversity hot- spot because of presence of rich species diversity (Sofi et al. 2022b). Analysis of pollen mother cell (PMC) meiosis During the present investigation, floral buds of A. euchroma from four different natural populations were fixed for meiotic studies. Young, unopened flower buds of suitable sizes were randomly collected from various plants within each studied population. The collected buds were preserved for 24 hours in Carnoy’s fixative, which is a mixture of ethanol, chloroform, and glacial acetic acid (6: 3: 1 v/v). The materials were then trans- ferred to 70% ethanol and kept at 4°C under refrigera- tion until use. Anthers were squashed in 1% propio- carmine and slides were observed under microscope. Pollen mother Cells (PMCs) were observed to count the chromosome number and meiotic abnormalities if any. Photomicrographs of chromosomes were taken from freshly prepared slides using an EVOS XL micro- scope. Chiasmata number was counted for cells at diplotene. Pollen fertility estimation Pollen fertility was estimated by collecting 10-15 fresh f loral buds with dehiscing anthers followed by squashing in 2% acetocarmine and glycerol (Marks 1954). Well-filled pollen grains with uniformly stained Table 1. Proportion of Meiotic abnormalities in PMCs of Arnebia euchroma. Population /Site Meiotic stage No. of PMCs Normal PMCs PMCs with stickiness/ clumping PMCs with laggards PMCs with chromatin bridges PMCs with cytoplasmic channels PMCs with satellite chromosomes Matayen Diplotene Diakinesis Metaphase-I Anaphase-I 52 47 93 20 52 47 56 - - - 7 11 - - 9 6 - - 7 3 - - 14 - - - - - Karpokhar Diplotene Diakinesis Metaphase-I Anaphase-I Telophase-I 24 17 54 13 6 24 13 20 - - - - 6 3 - - - - - - - - - 10 - - - 11 - - - - 4 - - Changoyal Diplotene Metaphase-I Anaphase-I 34 42 16 34 16 - - 9 16 - 6 - - - - - - - - - - Rungdum Diplotene Metaphase-I Anaphase-I Telophase-I Telophase-II 19 29 21 7 5 19 21 - 7 5 - 8 20 - - - - - - - - - 1(6:3) - - - - - - - - - - - - 31Meiotic behavior and its implications on the reproductive success of Arnebia euchroma cytoplasm were considered fertile while shrivelled and unstained pollen grains were counted as sterile. Percent- age pollen fertility/viability was calculated as follows: Pollen viability = Number of fertile pollen grains Total number of pollen grains observed ×100 Seed set calculation Individual plants were randomly chosen, labelled, and tallied according to the Lubbers and Christensen (1986) technique for the quantity of seeds produced per plant in order to estimate the seed set. Seed set = Total number of seeds produced per flower Total number of ovules borne per flower × 100 Statistical analysis Statistical analysis including linear model regression and correlation analysis was used to depict relationship between different parameters; the analysis was carried using the software r. RESULTS All the four populations of A. euchroma matched in having 14 chromosomes in their PMCs revealing 2n = 14 (diploid chromosome count) and x=7 (haploid chromo- some count), i.e., 2n = 2x = 14. The PMCs were analysed at diplotene, diakinesis, metaphase-I, anaphase-I and telophase I. In Matayen population, a total of 212 cells were scanned with 52 cells (24.52%) at diplotene (Fig. 1 A), 47 cells (22.16%) at diakinesis (Fig. 1 B), 93 cells (43.86%) at metaphase and 20 cells (9.43%) at anaphase I (Table 1). At diakinesis, we found perfect 7IIs and 6 cells had 2IVs+4IIs (Fig. 1 B). At metaphase-I, studied PMCs with6 IIs (Figs. 1 C, D) perfect 7IIs (Figs. 1 E, F), clump- ing (Fig. 1 G), laggards, chromatin bridges and cytoplas- Figure 1. (A) A PMC at diplotene, (B) A PMC at diakinesis, (C & D) A PMC at metaphase with 6 IIs, (E & F) A PMC at metaphase with 7 IIs, (G) A PMC at anaphase showing clumping of chromosomes, (H, & I) PMCs at metaphase showing migration of chromatin material. Scale bars =10 µm. 32 Irfan Iqbal Sofi et al. mic channels (Figs. 1 H, I) were observed. At anaphase-I no PMC with normal segregation was observed out of 20 cells scanned (Table 1). Chiasmata frequency per PMC calculated at diplotene in this population is 10.9 while RI calculated is 17.9. In Karpokhar population, a total of 114 cells were scanned at different stages of meiosis with 24 cells (21.05%) at diplotene (Fig. 2 A), 17 cells (14.91%) at diak- inesis, 54 cells (47.36%) at metaphase, 13 cells (11.40%) at anaphase I and 6 cells at telophase-I (5.26%) (Table 1). At diakinesis stage perfect 7IIs and 4 cells with 1IV+5IIs (Fig. 2 B) were found. At metaphase-I, 5IIs (Fig. 2 C), 6IIs (Fig. 2 D), cells with perfect 7IIs (Fig. 2 E), clump- ing, satellite chromosomes (Fig. 2 D) and cytoplasmic channels (Figs. 2 H, I) were observed. At anaphase-I no PMC with normal segregation was observed, PMC’s with chromatin bridges (Fig. 2 F) and clumping (Fig. 2 F) were recorded (Table 1). Chiasmata frequency per PMC calculated at diplotene in this population is 11.3 while RI calculated is 18.3. In Changoyal, a total of 92 cells were scanned at dif- ferent stages of meiosis with 34 cells (36.95%) at diplo- tene (Fig. 3 A), 42 cells (45.65%) at metaphase and 16 cells (17.39%) at anaphase I (Table 1). At metaphase- I, perfect 7IIs (Fig. 3 B), 5IIs (Fig. 3 C), 14 cells with Is (Fig. 3 D), clumping (Fig. 3 E) and laggards (Fig. 3 F) were observed. At anaphase-I no PMC with normal seg- regation was observed out of 16 cells scanned, all the cells with huge clumping (Fig. 3 G) were recorded. Chi- asmata frequency per PMC calculated at diplotene in this population is 12.4 while RI calculated is 19.4. Similarly, in Rungdum population, a total of 81 cells were scanned at different stages of meiosis with 19 cells (23.45%) at diplotene (Fig. 4 A), 29 cells (35.80%) at met- aphase, 21 cells (25.92%) at anaphase I, 7 cells (8.64%) at telophase-I and 5 cells (6.17%) at telophase-II (Table 1). At metaphase-I, perfect 7IIs (Figs. 5 B-E) and clumping (Figs. 4 F, G) containing 7IIs were observed. At ana- phase-I a PMC with abnormal segregation of 6:3 (Fig. 4 G) with a chromatin bridge was observed. However, Figure 2. (A) A PMC at diplotene, (B) A PMC at diakinesis, (C) A PMC at metaphase with 5 IIs, (D) A PMC at metaphase with 6 IIs and 1 satellite, (E) A PMC at metaphase with 7 IIs, (F) A PMC at anaphase with chromatin bridge formation, (G) A PMC at telophase, (H & I) PMCs showing migration of chromosomes through cytoplasmic channels. Scale bars =10 µm. 33Meiotic behavior and its implications on the reproductive success of Arnebia euchroma in rest of cells clumping were observed. Chiasmata fre- quency per PMC calculated at diplotene in this popula- tion is 12 while RI calculated is 19. The average proportion of meiotic irregularity, pol- len fertility, and seed set observed in the four studied populations of the Arnebia euchroma is shown in Table 2. It was evident from results that the percentage of pol- len fertility and seed set declined with increase in per- centage of meiotic anomalies in the four populations under study. The linear regression between seed set (%) and mei- otic abnormality (%) revealed a significant (p<0.001) decline of seed set with the increase in meiotic abnor- mality in the studied sites of target plant species (Fig. 5). The correlation analysis also depicted negative relation- ship between meiotic abnormality and pollen viability (r = -0.96), meiotic abnormality and seed set (r = -0.99) and positive correlation between pollen viability and seed set (r = 0.98), (Fig. 6). DISCUSSION The present study has documented chromosome number and meiotic behaviour of Arnebia euchroma from the four natural populations. The study con- firms chromosome number 2n = 2x = 14 in accordance with previous studies (Sharma et al. 2013). The pres- ence of chromosomal stickiness, cytomixis, laggard formation and other chromosomal abnormalities have been observed in all the studied populations. The most prevalent chromosome abnormality observed was chro- mosomal stickiness and chromosomal clumping in all studied sites. During the current study, cytomixis which involves the transfer of chromatin material primar- ily between proximal PMCs (Guan et al. 2012) was also observed. As a result of the chromatin material being transferred between PMCs, the irregularities associated with this transfer including chromosomes stickiness, sterility of pollen grains (Páez et al. 2021) was observed. This phenomenon functions as an additional potential Figure 3. (A) A PMC at diplotene, (B) A PMC at metaphase with 7 IIs, (C) A PMC at metaphase with 5 II s, (D) A PMC at metaphase with 14 Is, (E) A PMC at metaphase showing clumping of chromosomes, (F) A PMC at metaphase showing laggards, (G) A PMC at anaphase. Scale bars =10 µm. 34 Irfan Iqbal Sofi et al. genetic recombination mechanism (Mursalimov and Deineko 2017; Rashid et al. 2022b) and is a natural mei- otic aberration that may have evolutionary importance (Singhal et al. 2018). The cytoplasmic channels and chro- matin migration has also been reported in meiocytes of Arnebia hispidissima (Baquar and Husain., 1969). The phenomenon of cytomixis and its effects on meiotic developments and pollen fertility has been reported in various taxa of Himalayan region (Tantary et al. 2021). Cytomixis causes various meiotic abnormalities which include interbivalent connections, chromosome sticki- ness, laggards, bridges, late disjunction, pyknotic chro- matin and unorganized chromatin threads (Singhal and Kumar 2008) as observed in present study also. Unre- duced gametes or aneuploids and polyploids plants with certain morphological traits can both result from cyto- mixis (Falistocco et al. 1995; Arabi et al. 2022) leading to increase or decrease of basic chromosome count of the species (Tantary et al. 2021). Chromosome clustering in A. euchroma was associ- ated with both the intense (entire genome affected) and mild (few chromosomes affected) chromosomal sticki- ness that was observed in some PMCs. The presence of clumping distorts the chromosome shape making it difficult to determine the chromosome count. In the majority of cases stickiness was observed at Metaphase- I, and Anaphase-I in the present study. The cause of chromosome stickiness in many plant species has been attributed to environmental and genetic causes, as well as the interplay between the two (Pessim et al. 2015; Arabi et al. 2022). The sticky chromatin in various flow- ering plants have been attributed to gene mutation that disrupts proteins which in normal circumstances helps the chromosomes stay apart and prevents adherence (Tantary et al. 2021). However, the low temperature and high UV exposure in the alpine habitats (Rashid et al. 2022b) may be responsible for the observed chromo- somal stickiness in the Arnebia euchroma. Chromo- somal stickiness and the ensuing lack of chromosomal segregation at anaphase I can be suspected as the cause of meiotic abnormalities in the current investigation as seen in case of other studies (Masoud et al. 2010; Sin- Figure 4. (A) A PMC at diplotene, (B-E) PMCs at metaphase with 7 IIs, (F) A PMC at metaphase showing clumping, (G) A PMC at ana- phase showing chromatin bridge, (H) A PMC at telophase I, (I) A PMC at telophase II. Scale bars =10 µm. 35Meiotic behavior and its implications on the reproductive success of Arnebia euchroma gh et al. 2022). The chromosome bridges may occur as a result of chiasma interlocking in bivalents, and lag- gards may develop as a result of delayed terminalisation of stickiness at the ends of chromosomes (Chaudhari and Chaudhary 2012). Pollen fertility may be totally or partially impacted by chromatin stickiness, depend- ing on degree of presence (Rana et al. 2013). The pres- ence of five and six bivalents and few quadrivalents in some cells of the studied populations as against the nor- mal seven bivalent formation can lead to development of aneuploids in the target plant species. The rarity of quadrivalents points to translocation instead of segmen- tal allopolyploidy as the cause (Dawson et al. 1993; Lat- too et al. 2006). Normal segregation of chromosomes occurs as a result of optimal spindle orientation and chiasma development, while any deviation can lead to laggard formation (Arabi et al. 2022). The frequency of abnormal PMCs decreased as the cells progressed dur- ing meiosis, as seen by a comparison of the stages dur- ing the course of meiosis. A recovery mechanism that may successfully combat the anomalous behaviour of PMCs and restores fidelity during division with cell cycle advancement (Grewal and Rani 2022). The mei- otic aberrations present in the target species can lead to abnormal microsporogenesis without micronuclei. This type of atypical meiotic behaviour results in sterile pol- len grains, which lowers pollen viability, seed and fruit set as seen during the present study. The intrinsic factors (meiotic abnormalities) asso- ciated with the species is a constrain in its reproduc- tive process. Therefore, these factors can lead to loss of reproductive efficiency with low seed and fruit forma- tion and reduction in the pollen fertility of the species (Rashid et al. 2022a; Rashid et al. 2022a). It is evident from the current study that designing of effective future conservation programs of the target species should also consider the intrinsic factors that hold capacity to reduce the population of the species in nature. Figure 5. Relationship between seed set and meiotic abnormality as shown by linear model of regression with 95% confidence interval highlighted in grey shade. Figure 6. Relationship between meiotic abnormalities, seed set, and pollen viability as depicted by correlation plot.Table 2. Coordinates of sites and mean meiotic irregularity, pollen fertility, and seed set observed in the 4 studied populations of the Arnebia euchroma Population Latitude Longitude Mean Meiotic abnormality Pollen viability Seed set Matayen 34.37 75.59 26.88 85 42.5 Karpokhar 34.24 75.97 50.00 67 37.5 Changoyal 34.35 76.13 45.65 75 38.75 Rungdum 34.05 76.20 35.80 78 40 36 Irfan Iqbal Sofi et al. AUTHOR CONTRIBUTIONS MAS envisioned the idea of the present work. 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