Induced syncyte formation via cytomixis in Trachyspermum ammi (L.) Sprague (Apiaceae)

ABSTRACT

Cytomixis is a remarkable cytological event which has been presumed to be a prime facilitator of the origin of infraspecific polyploidy. The phenomenon of intercellular chromatin transmigration through cytomictic channels was reported during meiotic analysis of gamma rays induced population of Trachyspermum ammi (L.) Sprague. Chromatin migration was observed at three administered doses (100, 200 and 300 Gy) of gamma rays in variety AA-1 of Trachyspermum ammi (L.) Sprague. The gamma ray treated sets exhibited the characteristic of intercellular migration of nuclear material by two means such as cytomictic channels or via complete fusion of the proximate pollen mother cells (PMCs). The whole phenomenon was more frequently observed at meiosis I and rarely in meiosis II. The occurrence of cytomixis was directly correlated with the doses of gamma irradiation. The study also documents the relative frequencies of syncyte formation at different doses of gamma rays. Owing to cytomixis, at the later stages of meiosis the abnormal post meiotic products such as dyads, triads, polyads and heterosized pollen grains were produced. Furthermore, along with cytomixis, other chromosomal aberrations were also observed which affected the pollen fertility. Syncytes may have evolutionary significances as they can produce the gametes of higher ploidy level. Moreover, the unreduced gametes have the potential application to create novelty in the genetic system of plants which can be applied for crop improvement programs.

KEYWORDS:

• Cytomixis
• gamma rays
• cytomictic channels
• syncytes
• infraspecific polyploidy
• heterosized pollen grains
• Trachyspermum ammi (L.) Sprague

Introduction

Cytomixis is a widespread, fascinating meiotic event marked by the migration of nuclear material and other cellular organelles between the proximate cells through intercellular cytomictic channels. It was discovered by Arnoldy (1900) in the reproductive organs of gymnosperms, followed by Koernicke (1901) and Miehe (1901) in microsporocytes of Crocus vernus and in epidermal tissues of Allium nutans, respectively. These researchers considered cytomixis as an artifact that arises either due to material fixation or traumatic intervention and were supported by other researchers in later studies (Takats 1959; Tarkowska 1965). However, Gates (1911) considered it as a spontaneous process and proposed the term “cytomixis” while working on Oenothera gigas. Baquar and Husain (1969) rebutted this conviction and described it as a nonspecific response to a direct mechanical or chemical impact on the plant cells. Since then, cytomixis has been reported in several flowering plants (Bellucci et al. 2003; Boldrini et al. 2006; Singhal and Kumar 2008; Kaur et al. 2013); even so, the origin and significance of this remarkable cytological event is still vague. It has been reported in diverse types of plant cells, such as apical meristem cells of woody plants (Guzicka and Wozny 2005), root meristematic cells (Sarvella 1958; Tarkowska 1960), vegetative tissues of anthers (Wang et al. 2004), ovary cells (Koul 1990), tapetal cells (Cooper 1952), proembryos of graminaceous plants (Klyuchareva 1983) and shoot apex of trees (Guzicka and Wozny 2005). However, intercellular migration of chromatin material has been most often studied in microsporogenesis of plants.

Cytomixis has been categorized into three different groups according to its intensity: slight (local), intensive, and destructive (Kravchenko, 1977). Kravets (2012) stated that local cytomixis does not cause destructive effects to meiosis, unlike the intensive and destructive type of cytomixis which causes several meiotic instabilities and autolysis of cells. Cytomixis occupies the specific status in biological studies because of the formation of syncytes which leads to the production of 2n gametes. Syncyte formation involves the fusion of two or more PMCs or nuclei, generally in early prophase of the first meiotic division, so that the syncyte produces 2n gametes after meiosis (Levan 1941; Sarbhoy 1980). It has been widely accepted that cytomixis is an attribute of hybrids, mutants and aneuploids (Premchandran et al. 1988; Peng et al. 2003; Zhou 2003). In some instances it has also been reported in polyploid plant species (Sheidai and Attaei 2005; Mursalimov et al. 2016).

An in-depth analysis of cytomixis is required to understand the rigorous mechanism of intercellular transmission of genetic material between the microsporocytes. Some researchers have anticipated that intercellular transfer of genetic material during microsporogenesis is involved in evolutionary pathways due to the formation of gametes with different ploidy levels or unreduced gametes (Falistocco et al. 1995; Ghaffari 2006; Lattoo et al. 2006; Negron-Ortiz 2007; Singhal et al. 2011). However to date, the evolutionary significance of cytomixis has not been proved experimentally.

Ajwain (Trachyspermum ammi (L.) Sprague, 2n = 2x = 18) is renowned for its medicinal and commercial value throughout the world. The essential oil of ajwain seeds is a rich source of various neutraceutical components, due to which it occupies a significant economic position in pharmaceutical industries. The major ajwain producing countries are India, Persia, Iran, Egypt, Afghanistan, Pakistan and North Africa. Despite the manifestation of cytomixis in several plant species, its occurrence in Trachyspermum ammi (L.) Sprague is still unmapped. The present investigation was carried out to assemble the comparative data on cytomixis induced by gamma rays and to analyze its outcome in the male reproductive system of this crop. Moreover, the study also documents the relative frequencies of syncyte formation and heterosized pollen grains. The present research work may contribute to understand the essence of infraspecific polyploidy in Trachyspermum ammi (L.) Sprague.

Materials and methods

Procurement of seeds

Fresh and healthy seeds of Trachyspermum ammi (L.) Sprague var. AA-1 were obtained from National Research Centre for Seed Spices (NRCSS), Ajmer, Rajasthan, India.

Gamma ray irradiation

The seed packets were exposed to gamma ray irradiation from a Co⁶⁰ source in gamma irradiation chamber-1200 Model at National Botanical Research Institute (NBRI), Lucknow. The gamma rays were administered in three different doses, 100, 200, and 300 Gy. The irradiated seeds were immediately sown in replicates of three in the field adopting a complete randomized block design (CRBD). Simultaneously, one set of untreated seeds was also sown as control.

Agro-climatic conditions of experimental site

The present experiment has been performed in the area of Roxburgh Botanical Garden, Department of Botany, University of Allahabad, Allahabad, U.P., India, during the rabi season. The exact experimental location is 25°27ʹ43.01ʺN, 81°51ʹ10.42ʺE. Allahabad is situated 98 m above mean sea level. Allahabad is in the sub-tropical climatic zone; the average rainfall is 1027 mm and relative humidity is 59%.

Meiotic studies

Of the 20 plants tested cytologically from each dose of gamma treatment, four plants in the case of 100 Gy and five plants in both the 200 Gy and 300 Gy gamma treatments showed cytomixis. For meiotic studies, approximately 20–25 buds from each dose were taken into consideration for cytological study of meiotic course. The young floral buds of suitable size were randomly selected and then fixed in freshly prepared Carnoy’s fixative (alcohol:glacial acetic acid in a 3:1 ratio) for 24 h and preserved in 70% alcohol at 4°C until use. Meiocyte preparations were made by squashing young anthers in 2% acetocarmine (Fürste 1962). Approximately 200–350 PMCs were analyzed for the replicates of each set. Slides were analyzed and meiocytes were photographed under a Nikon research photomicroscope (Nikon Eclipse, E200, Japan). Approximately 150 pollen particles were observed from each plant showing the phenomenon cytomixis. The pollen fertility was calculated using 1:1 glyceroacetocarmine mixture (Marks 1954). Pollen grains with stained cytoplasm were recorded as fertile whereas undersized and unstained pollen grains without nuclei were considered sterile.

Statistical analysis

All the statistical analyses of cytological observations have been done by using SPSS 16.0 software to measure mean values of variables.

Results

Meiotic study of the control population of Trachyspermum ammi (L.) Sprague showed the normal chromosomal arrangement (n = 9) at all stages of division without any irregularities. The control plant exhibited nine bivalents at metaphase I followed by symmetrical segregation at anaphase I which resulted in pollen grains of definite size and shape.

However, the gamma ray treated sets demonstrated the group of PMCs involved in intercellular transfer of chromatin material, resulting in abnormal post meiotic products (Figure 2(d–i)). The chromatin material was found to be migrated through different cytomictic channels which originated from the donor cell and transferred its nuclear content to the proximate recipient cell. In the present case, narrow cytomictic channels were observed (Figure 1(c–e)). In some instances, direct fusion of cell walls of PMCs was also observed (Figure 1(f–h)). However, the chromatin transfer through cytomictic channels was found to be more frequent as compared to direct fusion of cells. Usually, two to three PMCs were involved in chromatin transfer. Cytomixis between the PMCs was generally observed at the stages of meiosis I; however, it was also recorded in the later stages, i.e. meiosis II (Figure 1(l)) but its frequency of occurrence was found to be low. Cytomictic behavior was observed at all the three administered doses of gamma rays and their frequency at various meiotic stages was demonstrated in Table 1. The mean frequency of PMCs involved in cytomixis was 7.6%, 9.5%, and 13.3% for 100, 200 and 300 Gy, respectively (Table 1). The frequency of PMCs involved in cytomixis was reported to be directly proportional to the doses of gamma rays.

Table 1. Effect of gamma rays on the frequency of cytomixis and syncyte formation during microsporogenesis of Trachyspermum ammi (L.) Sprague.

		Cytomixis (%)				Frequency of cells showing cytomixis at various stages of meiosis (%)
			Types of cytomixis			Meiosis I						Meiosis II
						PI
Treatment Doses (Gy)	Total number of PMCs observed	Frequency of PMCs involved	CC	DF	Frequency of syncytes (%)	Pt	Dp	Dk	MI	AI	TI	MII	AII	TII	Other chromosomal abnormalities (%)
Control	348	-	-	-	-	-	-	-	-	-	-	-	-	-	-
100	332	7.6 ± 0.26	68.0	32.0	2.3 ± 0.33^a	2.3	1.4	1.2	1.7	0.3	0.3	-	-	0.7	9.9 ± 0.22^a
200	348	9.5 ± 0.21	66.7	33.3	4.7 ± 0.33^b	2.3	1.2	1.4	2.0	0.4	0.4	0.3	0.4	1.1	12.7 ± 0.17^b
300	325	13.3 ± 0.25	74.4	25.6	5.3 ± 0.33^b	2.9	1.5	1.9	2.7	0.6	0.9	0.7	0.6	1.3	16.4 ± 0.16^c

Abbreviations: CC, cytomictic channel; DF, direct fusion; PI, prophase I; Pt, pachytene; Dp, diplotene; Dk, diakinesis; MI, metaphase I; AI, anaphase I; TI, telophase I; MII, metaphase II; AII, anaphase II; TII, telophase II. Values followed by the different superscripts within same column differ at P < 0.05 between treatments by the DMRT.

Figure 1. (a) A PMC showing nine bivalents at diakinesis; (b) PMCs showing chromatin migration at early prophase-I (arrowed); (c) chromatin transfer through single and multiple cytoplasmic channels; (d) PMCs showing chromatin transfer through single cytoplasmic channel; (e) migration of a single chromosome (arrowed) through narrow cytoplasmic channels; (f) transfer of bivalents through broad cytoplasmic channel; (g) a group of PMCs showing chromatin migration between adjacent meiocytes; (h) unification of two meiocytes by direct fusion of walls; (i) PMCs showing resulting hypoploid (n = 1 + 3 + 2 small sticky fragments, n = 6 + 2 small fragments) and hyperploid (n = 9 sticky+ 2 small fragments, 9 + 2 small fragments) PMCs; (j) bidirectional transfer of bivalents from one meiocyte to two proximate meiocytes (two hypoploid PMCs and one hyperploid PMC); (k) two PMCs, one is donor and the adjacent is recipient; (l) channel formation at telophase II. Scale: 10 µm.

The direct fusion of two or three contiguous PMCs formed the syncytes (Figure 2(a) and (b)). After the fusion of two to three PMCs together, the chromosomes are transferred to a single cell (syncyte). The syncytes were giant sized and easy to distinguish from the normal PMCs. They were observed in low frequency and the mean percentage was 2.3%, 4.7% and 5.3% at 100, 200, and 300 Gy, respectively (Table 1). Figure 2(b) depicts a syncyte with 27 bivalents at diakinesis. Most of the syncytes were reported at the stage of meiosis I, i.e. at diakinesis. The partial chromatin migration resulted in the formation of hypoploid and hyperploid PMCs (Figure 1(i–k)). The resulting hypoploid (n = 1 + 3 + 2 small sticky fragments, n = 6 + 2 small fragments) and hyperploid (n = 9 sticky+ 2 small fragments, 9 + 2 small fragments) PMCs showed the variable chromosome numbers at M-I (Figure 1(i)). As the consequence of cytomixis, the production of abnormal post-meiotic products, i.e. abnormal tetrads such as dyads (Figure 2(g)), triads (Figure 2(f)) and polyads (Figure 2(d) and (e)). Owing to these aberrant post meiotic products, the process of microsporogenesis is greatly affected and consequently resulted into the formation of heterosized pollen grains. The mean size of pollen grains and the relative frequency of heterosized pollen grains along with pollen fertility and abnormal tetrad percentage were demonstrated in Table 2. The range of size of the large and medium pollen grains was recorded as 19.2–23.5 µm × 11.8–16.8 µm and 16.9–18.3 µm × 6.7–8.8 µm, respectively. The jumbo sized pollen grains (22.8 µm × 15.7 µm) were considered as the 2n pollen grains (Figure 2(i)). Other chromosomal aberrations such as precocious movement, stickiness, univalents, multivalents, laggards and bridges were also observed; these increased from 9.8% (at 100 Gy) to 16.4% (at 300 Gy) with the increase in the dose of gamma rays (Table 1). The pollen fertility was found to be 97.9% in the control, while it was recorded as 79.5%, 67.8%, and 58.6% for 100, 200 and 300 Gy, respectively.

Table 2. Effect of gamma rays on the post-meiotic products, fertility and size of pollen grains of Trachyspermum ammi (L.) Sprague.

		Sizes of heterosized pollen grains (µm)
Treatment doses (Gy)	Pollen fertility (%)	Large	Medium	Abnormal sporads (%)	Relative frequency of heterosized pollen grains (%)
Control	97.9 ± 0.22^d	18.3 × 8.3		-	100
100	79.5 ± 0.12^c	19.2 × 11.8	16.9 × 6.7	11.5 ± 0.15^a	3.6 ± 0.10^l 96.4 ± 0.10^m
200	67.8 ± 0.10^b	21.8 × 15.8	18.3 × 6.7	20.7 ± 0.13^b	5.5 ± 0.15^l 96.5 ± 0.15^m
300	58.6 ± 0.13^a	23.5 × 16.8	18.3 × 8.8	26.4 ± 0.15^c	6.8 ± 0.08^l 93.2 ± 0.15^m

Abbreviations: l, large; m, medium. Values followed by the different superscripts within same column differ at P < 0.05 between treatments by the DMRT.

Figure 2. (a) Syncyte (tetraploid) with 18 bivalents at diakinesis; (b) hyperploid PMC with 27 bivalents; (c) enucleated PMC; (d) pentad; (e) two sporads in which one is normal, i.e. tetrad and other is hexad; (f) triad; (g) dyad; (h) fertile heterosized pollen grains; (i) fertile jumbo sized pollen grain. Scale: 10 µm.

Discussion

Cytomictic mutants have the specific position among the mutagenic studies as it conferring the significant phenomenon of infraspecific polyploidy. Many hypotheses have been given for the reason of the occurrence of cytomixis. Cytomixis is an efficient means of cell-to-cell communication, as transportation of nutrient and signaling molecules among the cells occurs through cytomictic channels (McLean et al. 1997). However, another speculation about their occurrence is that their fundamental role is in the transfer of some genetic material from one cell to another, resulting in an increase in genetic diversity of the produced pollen or an adjusting and balancing in unbalanced genomes (Falistocco et al. 1995; Zhou 2003; Ghaffari 2006; Lattoo et al. 2006; Negron-Ortiz 2007; Singhal and Kumar 2008; Song and Li 2009; Singhal et al. 2011).

Cytomixis has been induced by various factors such as gamma rays (Saraswathy et al. 1990), ethyl methane sulphonate (EMS) and sodium azide (Dixit et al. 2013), methyl methane sulphonate (MMS) (Bhat et al. 2006), X-rays (Ghatnekar 1964), colchicine (Dwivedi et al. 1988), changes in the biochemical process that involve microsporogenesis modifying the microenvironment of affected anthers (Koul 1990) and environmental stress and pollution (Haroun et al. 2004). In the present study, gamma rays were found to be responsible for the induction of cytomixis and syncyte formation. From among the causes projected for cytomixis, gamma radiation is proposed to be most effective, resulting in the production of an imbalanced and sterile genetic system (Saraswathy et al. 1990).

The cytomictic channels are an important means by which the chromatin material of one cell migrates to cells of close proximity. Wang et al. (1998) and Mursalimov et al. (2013b) suggested that cytomictic channels were formed by the enlargement of single plasmodesma or fusion of several plasmodesmata and, on the other, de novo on the cell wall regions lacking any plasmodesmata however; Heslop-Harrison (1966) conflicting this statement and stated that cytomictic channels are formed de novo. The involvement of hydrolytic enzymes released by endoplasmic reticulum and Golgi apparatus in the formation of cytomictic channels was postulated by Wang et al. (1998) and Yu et al. (2004). This might be possible due to the substitution of pectin-cellulose wall by callose during prophase I and thus primary cytomictic channels disappear at the later stages (Wang et al. 2002; Mursalimov and Deineko 2012). The secondary cytomictic channels can also be formed by the specific organelles spherosome-like vesicles, containing the enzyme callase (Mursalimov et al. 2010, 2013a) which might be responsible for the formation of cytomictic channels at later stages of the meiotic course.

Typically, the nucleus resides in a specific place within a cell but in some cases nuclei do migrate to accomplish certain tasks, and in such cases the nuclear migration is provided by the interaction of cytoskeleton and motor proteins (Suelmann et al. 1997; Fischer 1999; Liu et al. 2003). The association of a migrating nucleus with a large number of filamentous structures in wheat endosperm (Zhang et al. 1990) and the occurrence of microtubule-like elements inside the cytomictic channels with migrating organelles in microsporocytes of lily (Wang et al. 2002) are some experimental examples which support the involvement of cytoskeletal elements in cytomixis. It is believed that actin rather than tubulin acts as a crucial agent in the execution of this phenomenon due to the fact that cytochalasin B halts migration of cell content through cytomictic channels (Zhang et al. 1990; Sidorchuk et al. 2007).

Cytomixis significantly influences the microsporogenesis of plants since the transmission of nuclear material of a cell to the contiguous microsporocytes resulted in the formation of gametes of different ploidy levels. Cytomictic transmigration occurring through cell wall dissolution among the neighboring PMCs generally leads to the formation of syncytes (Falistocco et al. 1995). Syncytes have been reported in several species such as in Chrysanthemum (Kim et al. 2009); Brachiaria (Mendes-Bonato et al. 2001); Anemone (Rana et al. 2013); Dianthus (Kumar et al. 2012); Phleum (Levan 1941); Achillea (Kaur et al. 2017), and Heracleum (Singhal et al. 2016). The syncyte PMCs are easily distinguishable, because of large size and their high genetic content leads to the production of giant pollen grains having higher ploidy levels. The syncyte PMCs in the plants are destined to produce heterosized or 2n pollen grains due to the non-reduction of gametes after meiosis (Sarbhoy 1980; Falistocco et al. 1995; Ghaffari 2006; Rana et al. 2013). Bretagnolle and Thompson (1995) stated that the presence of giant pollen grains has been used as an indication of the production of 2n pollen. Syncyte formation in diploid individuals has great significance in the initiation of low-level polyploidy and is very likely to play a major role in producing infraspecific polyploids (Kim et al. 2009). The occurrence of heterosized pollen grains might be the result of hypo- and hyperploid PMCs which were produced due to partial migration of chromatin material. Hypoploid PMCs in the present study may be responsible for the production of the aneuploid gametes resulting in the formation of sterile pollen grains. Simultaneously, several other chromosomal aberrations were also induced due to gamma irradiation, as a consequence of which some pollen sterility was observed.

To the best of our knowledge this is the first report on the intercellular migration of nuclear material between the PMCs of close proximity through cytomictic channels in Trachyspermum ammi (L.) Sprague, which subsequently produced syncytes and jumbo sized pollen grains. From the cytogenetical investigation, it can be elucidated that gamma rays can be used as an agent for raising the gametes of different ploidy levels mediated by syncyte formation. It can be concluded that gamma rays have the ability to induce variability by influencing the typical cytological events which govern this significant trait. There should be detailed studies based on syncytes in the future as they are conducive to the induction of infraspecific polyploidy.

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgments

The authors are very grateful to the National Botanical Research Institute (NBRI), Lucknow, India for providing the gamma irradiation facility. One of the authors (Harshita Dwivedi) thanks the University Grant Commission (UGC) for financial assistance and the Head of the Department of Botany, University of Allahabad, Allahabad, for providing the necessary facilities. Sincere thanks are also due to all the members of Plant Genetics Laboratory for their encouragement and support.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by the University Grants Commission.