Cytogenetic impact of sodium chloride stress on root cells of Vigna radiata L. seedlings

Introduction

The soil salinization is the most important abiotic stress in the barren zone of land [1]. Due to which there is an excess accumulation of salts in the rooting region ensured complete or limited loss of soil structure and fertility [2]. Salty soils consist of carbonate, magnesium, potassium, sodium, calcium, sulfate, chloride, nitrate and borate. In which sodium chloride will be standout amongst the salts in nature and has a dangerous impact due to its superior solubility [1]. It badly devastates seedlings biochemistry and inducing ionic stress [3], [4] by storing Na⁺ & Clˉ ions in the cytosol. These ionic toxicities distort the double helix structure of DNA and orientation of chromosome at metaphase that caused chromosome abnormalities like stickiness in chromosomes, C-mitosis, lagged and disturbed anaphase [3], [5]. The decline in mitotic index usually down-regulated the development of seedlings [2] which hampered agrarian productivity [5]. Over fact, no hazardous compound hinders the development of crops besides salt stress on the earth scale [6].

As a matter of fact, the presence of excessive amount of salts in the soil is hazardous to most of the comestible species like Vigna radiata. The Vigna genus is comprised of about 150 species mainly belong to Asia and Africa [7]. Vigna radiata (2n=2×=22) is one of its cultivated species grown in the tropic locale of Pakistan, India, Bangladesh, Central Asia, Sri Lanka, and South China [7], [8]. It is generally known as mungbean or green or black gram [9]. In Pakistan, beans are cultivated around 950 hectares of lands that yield 0.715 tons per hectare [10]. Its seedling has high nutritive value but very sensitive for saline stress [11]. It was reported that salt alters double helix of DNA and become a root cause of the chromosomal aberration in onion [12], barley [13], and wheat [5]. However, no precise research on high salt (350 mM NaCl) induced Vigna radiata seedlings roots damage, and chromosomal aberrations have been reported during last few decades. Therefore, the aim of this research is to evaluate the impact of salt stress over root cells through the study of mitotic index (MI), and distinct chromosomal anomalies, utilizing root tip cells from NM-92 and NM19-19 Vigna radiata varieties. As suggested by Çavuşoğlu et al. [14] and Singh and Roy [15], it is conceivable to assess the cytotoxicity of tested chemical utilizing above-mentioned criterion. Thus, the cytogenetic investigation of Vigna radiata may provide valuable information about the impact of high salinization of NaCl at seedlings stage.

Materials and methods

Results and discussion

Table 1 of ANOVA showed that two Vigna varieties NM-92 and NM19-19 performed differently for mitotic index (MI), stickiness, spiralization, lagged, bridge and disturbed anaphase. However, NM-92 and NM19-19 differed non-significantly for total chromosomal aberrations and C-mitosis. The effect of five levels of NaCl stress were statistically different for all inspected cytogenetic parameters. The interaction of Vigna varieties with five levels of NaCl stress on lagged anaphase, spiralization, bridge and disturbed anaphase was highly significant at 1% probability level. In contrast, the impact of interaction between five concentrations of NaCl on two Vigna varieties showed non-significant difference for mitotic index, total chromosomal aberration, C-mitosis, and stickiness of chromosomes. Moreover, the coefficient of variation found maximum in C-mitosis and minimum in mitotic index that were 73% and 9%, respectively (Table 1).

Salts generated ionic stress on seedlings morpho-physiology and biochemistry have been studied [20], [21] While, the impact of NaCl stress at cytological level is not well examined [5]. According to Lubini et al. [22], cytotoxicity of any chemical could be determined through promotion or inhibition in mitotic index. The MI revealed cell division and proliferation frequency and rate of meristem growth [23]. Here, it is utilized to investigate the impact of NaCl stress over Vigna seedling root cells. It was seen that varying levels of NaCl imposed the deleterious effect on root cells. The lower level of NaCl stress inhibited 7% cell division while 350 mM manifested 20% inhibition (Figure 1A). Further, the severe NaCl stress caused the highest level of mitotic aberrations that was 75% than H₂O control cells (Figure 1B). Moreover, various NaCl levels induced number of chromatin (C-mitosis, and stickiness of chromosomes) and spindle fiber associated abnormalities (lagged anaphase, spiralization, bridge anaphase, and disturbed anaphase) in Vigna root cells.

Figure 1:

Cytological impact of 0, 50, 150, 250, and 350 millimolar NaCl concentration on seedlings root tip cells of Vigna radiata varieties.

Above examination was the outcome of four replicates. Post hoc test for means comparison was conducted at p≤0.01 level. (A) mitotic index; (B) total chromosomal aberrations; (C) C-mitosis; (D) chromosome stickiness; and (E) lagged-anaphase. The data expressed in percentage while, trendline showed promotion and inhibition pattern in the graphs.

The least inhibition in mitotic index at 50 mM NaCl concentration indicated an active division of root meristem cells. In contrast, maximum decrease in MI at 350 mM salt exhibited extreme stress over biochemical mechanism which caused decline in cell division that retard Vigna seedling growth. However, cell division in apical meristem became the cause of root growth that is directly related to the mitosis duration. The greater mitotic index in NM-92 under each concentration showed salt stress tolerance whereas lower MI in NM19-19 indicated genotype sensitivity against salinity stress. These findings are well in conformity with Kielkowska [12] and Çavuşoğlu et al. [2] in onion. Marakli et al. [13] tested 150 and 250 mM NaCl impact over barley seedlings. Both concentrations caused the decrease in mitotic index and promoted disorganized prophase, metaphase, and anaphase and bridge in anaphase. However, the total absence of mitotic activity was seen from 300 to 600 mM of NaCl stress [5], [12]. In wheat, 250 mM NaCl reduced mitotic activity and induced chromosomal anomalies, for instance, irregular prophase, stickiness, C-mitosis, bridges, and multipolarity [5]. In another study 300, 350, 400 and 450 mM saline stress inhibited mitotic index of barley (cv. ‘Bulbul 89’) and the higher number of chromosomal aberrations as salt concentration increase [1]. These findings support our results (Figure 2).

Figure 2:

Cytological impact of 0, 50, 150, 250, and 350 millimolar NaCl stress for 24 h on seedlings root tip cells of two Vigna radiata varieties.

Above examination was the outcome of four replicates. (A) normal interphase; (B) normal prophase; (C) normal metaphase; (D) normal anaphase; (E) normal telophase; (F) sticky metaphase; (G) spiralization at metaphase; (H) c-mitosis; (I) lagged anaphase; (J) disturbed anaphase; (K) spiralization at anaphase; and (L) bridge anaphase.

The server level of NaCl (350 mM) stress showed 99% C-mitosis in both examine varieties (Figure 1C). However, 250 mM NaCl stress exhibited highest (68%) stickiness of chromosome at metaphase in variety NM19-19 and lower (40%) in NM-92 (Figure 1D). Similarly, lagged anaphase was found higher (69%) in NM19-19 and lesser (44%) in NM-92 at 150 mM salt concentration (Figure 1E). The reason for the occurrence of C-mitosis and lagged anaphase may result in spindle formation failure due to sever salt stress [24], [25]. These observed chromosome fragmentation and breaks showed clastogenic action [26] while, stickiness in chromosomes may be due to inter-chromosomal linkages coupled with the excessive formation of nucleoproteins that caused stickiness in chromosomes [24]. These nucleoproteins production and inter-chromosomal linkage may be because of unwanted Na⁺ and Clˉ ions accumulation in the cytosol and vacuole of the cell of Vigna seedlings.

In addition, various other aberrations like spiralization, bridge-anaphase, and disturbed anaphase were also noticed under salt stress (Figure 3). The maximum 12% spiralization, and 15% disturbed anaphase in variety NM19-19 while, 1.5% spiralization and 6% disturbed anaphase were observed in NM-92 at 250 mM salt stress (Figure 3A,B). Whereas, 7% bridges at anaphase was noted in NM19-19 and 3.5% in NM-92 under 150 mM salt level (Figure 3C). The spiralization, disorder, and bridges at anaphase could be as a result of inversions or unequal allocation of nuclear chromatin and spindle dysfunction. Spindle dysfunction may induce aberrant segregation of chromosomes because of faulty kinetochore and microtubule synergy which provokes bridges creation [1] at anaphase. In short, nuclear chromatin degradation is the consequence of NaCl ions toxicity [27], that was responsible for root and shoot growth retardation in Vigna seedlings. Also, it was observed that the maximum types of aberrations found at 150 and 250 mM concentration of sodium chloride though, 350 mM salinity level was lethal for both varieties seedling’s cell division and because of this single type of aberration found. However, the higher percentage of stickiness, spiralization, lagged, bridge and disturbed anaphase aberrations in NM19-19 at each level of NaCl stress showed genetic inability to resist salt stress. While in the current examination, NM-92 proved as salt tolerant variety due to exhibiting less percentage of chromosomal aberrations at all concentration of salt.

Figure 3:

Cytological impact of 0, 50, 150, 250, and 350 millimolar NaCl concentration on seedlings root tip cells of Vigna radiata varieties.

Above examination was the outcome of four replicates. Post hoc test for means comparison was conducted at p≤0.01 level. (A) spiralization at meta and anaphase; (B) bridge-anaphase; and (C) disturbed-anaphase. The data expressed in percentage while, trendline showed promotion and inhibition pattern in the graphs.

In short, the salinization has chromo toxic effect over seedling cells that inhibits DNA, nuclear proteins synthesis, compatible osmolytes, antioxidant defense enzymes, polyamines and restrict water availability [28]. Other than these, it originates responsive oxygen species, ion toxicity, enhance alkalinity and osmotic pressure, disturb metabolic mechanism, cellular physiology, and ion transport that notable raises altogether types of chromatin anomalies in radical cells [12], [14], [24] which leads to fewer cells production cause reduction in meristem size [12]. While concentrations greater than 300 mM cause severe damage at the cellular level and lead to cell death [1].

The impact of stress over seedlings depends on the chemical and physiological nature of stress-inducing compound such as NaCl. Sodium chloride is an ionic compound induce ion toxicity in the cytosol of the cell. It could be concluded that chloride and sodium ions toxicity induced various cytological anomalies in seedlings roots that adversely affect the growth of Vigna radiata. Hence, cytological examination of Vigna species may play a key role in understanding the impact of salinization over seedling growth.