Characterizing Maize Genotypes for Salt Tolerance Using Morphological and Ionic Traits at Seedling Stage

bstract: Maize crop is the third most important cereal crop, mostly grown for food, feed and fodder purpose. In spite of the fact the crop is susceptible to salt stress but exhibits a considerable genotypic variability for salt tolerance. The present study was carried out to determine to which extent the maize genotypes with contrasting morphological traits respond to salt stress. Seven days old seedlings of thirty maize genotypes were exposed to NaCl stress of EC less than 4 (control), equal to 8 and 12 dS m -1 for further 28 days in a temperature controlled greenhouse. The salt stress imposition was completed in two increments every other day from the date of planting. At the age of 35 days, the seedlings were evaluated for contrasting morphological traits. The genotypes exhibited considerable variations for each of the 10 measured morphological and ionic traits. PCA was employed to identify the most suitable morphological trait to be used as selection criterion for salt tolerance. Based on the PCA results, dry shoot weight (DSW) was used to classify thirty maize genotypes into salt-tolerant-T, moderately tolerant-MT and salt sensitive-S groups. Two (2) out of thirty genotypes i.e. SB-9617 and FH-949 had the highest average of percent dry shoot weight (PDSW) values > 70% were classified as salt tolerant (T). The fifteen (15) genotypes showed average of PDSW values in the range of 55-69.9 % were classified as moderately tolerant (MT) by indicating the sequence order of salt tolerance as YH-1898 > MMRI-yellow > S-2002 > FH-988 > FH-1292 > HC-12 > MS-2018 > NCEV-1270-7 > Pahari > Pearl White > FH-1114 > NCEV-1270-3 > Iqbal > NCEV-1297 > DK-6724. However, thirteen (13) out of total thirty (30) genotypes attained the average PDSW values <55% were classified as salt sensitive (S) following the sequence order as NCEV-1530-9 > Composite > FRI-22 > Azam > MS-2015 > P-1543 > Neelum > Afgoi > Malka-2016 > MS-1501 > HNG > NCEV-1270-5 > NCEV-7004. It was also noticed that the declared salt tolerance was positively correlated with dry shoot weight (DSW), shoot length (SL), stem diameter (SD), fresh shoot weight (FSW), fresh root weight (FRW), dry roots weight (DRW), root length (RL), leaf area per plant (LA), number of leaves per plant (NL) and K + /Na + ratio. The highest positive Pearson correlation coefficients were determined in LA vs SD (r=0.900), DSW vs SD (r=0.899), SL vs DSW (r=0.891), SL vs FSW (r=0.890) and DSW vs FSW (r=0.887). Additionally, the salt tolerant (T) group of genotypes maintained higher K + /Na + ratios compared to moderately tolerant (MT) and salt sensitive (S) groups. The results clearly showed that dry shoot weight (DSW) could be viable option to classify maize genotypes into different salt tolerance groups and to identify the most suited and best adapted salt tolerant genotype for cultivation in saline soils. Furthermore, this scientific information could help the plant scientists to improve and develop the new salt tolerant cultivar.


Introduction
Maize (Zea mays L.) crop is on rank 3 rd after wheat and rice. In Pakistan it is grown on area of 1,418 thousand hectares with the total production of 8.47 million tons in 2020-21 [18] contributed 0.6 percent to GDP. The crop is moderately sensitive to salt stress and mostly grown for multiple purposes such as food, feed and fodder.
Salinity impacts crop growth and development through its osmotic and ionic effects via restricting the water and nutrients supply to plants [54,34,59,47]. Moreover, the influence of salt stress on plant growth occurs in two phases: limits water supply to plants in the first phase i.e. osmotic phase [48,49,31] and disrupts nutrients supply through higher accumulation of toxic ions specifically Na + in the second phase i.e. ionic phase [49]. Furthermore, the reactive oxygen species (ROS) when produced in mitochondria; chloroplast and peroxisomes in response to saline stress, are more reactive than molecular O2 and have the greater tendency to react with cellular membrane causing more leakiness of membranes [44,2,24]. Additionally, the ROS also cause significant damage to plant genomics and disrupts various physiological, biochemical, and metabolic processes, more specifically the bio-synthesis of proteins and chlorophylls [46,43,41,66,64,36,25,12].
However, different breeding procedures such as introduction, selection and hybridization are reported in the literature for the development of improved varieties. Thus, the existence of sufficient genetic variability among the genotypes for various attributes is perquisite for a successful breeding programme. Among the traits, morphological and ionic attributes can effectively be used to determine genotypic variability for salt tolerance among the genotypes [23]. The genetic variability based on these traits determines salt tolerance among the genotypes [6].
As maize crop is categorized as moderately sensitive to salt stress but exhibits considerable genetic variability for morphological and ionic traits among genotypes [40,15,26,33,7]. The morphological and ionic traits which experience negative impacts of salt stress greater are shoot length (SL), root length (RL), stem diameter (SD), number of leaves (NL) & leaf area (LA), fresh & dry root weights (FRW & DRW), fresh & dry shoot weight (FSW & DSW) and K + /Na + ratios [13,20]. Such type of variations may serve as mile stone for developing new/advance breeding lines and varieties with improved characters. Therefore, the acquisition of knowledge about the genotypic variability in the available germplasm resources, especially regarding the morphological and ionic attributes, is the foremost step before starting any breeding programme (3,55). It enables the plant breeder to select the most suitable/appropriate genetic material for developing salt tolerant cultivars by considering the morphological traits as selection criteria [57,69]. To ease the selection of desirable maize genotypes, the maize breeders must have information regarding the nature and extent of relationships among the morphological and ionic attributes [37]. Some powerful methods and strategies to analyze the degree of functional genetic divergence in the available genotypes include multivariate analysis [27,68]. Various statistical techniques such as correlation, PCA, regression, cluster analysis, and factor analysis are used to assess the genotypic variability [6,37,68].
In view of the above perspective, the present study was conducted with the objectives (i) to find out the strength of relationships among studied attributes of the experimental genotypes (ii) to explore the existence of genetic variability among the tested genotypes (iii) to identify the most suitable trait to use as selection criterion among the studied morphological and ionic attributes. PCA and then Murillo et al. [5,4] classification were employed on the data to categorize maize genotypes into different salt tolerance groups i.e. tolerant (T), moderately tolerant (MT) and sensitive (S) genotypic groups.

Materials and Methods
The study was planned to categorize promising maize genotypes into different salt tolerance groups based on genotypic variability using morphological and bio-chemical traits as a selection criterion. Sargodha. The seed of each genotype was surface sterilized with hypochlorite (1%) solution. After sterilization the seed was washed with distil water 2-3 times and allowed to dry under the shade. Thereafter, sand was purchased from the local market and sieved properly to remove all the inert material and gravels. The sieved sand was then washed with water three time and spread on the polythene sheet for sun drying. After that, the sand was filled in seedling trays. One tray was maintained for each genotype by planting one healthy seed per hole of the seedling tray. These seed planted trays were placed in the rain protected glass house with regulated temperature (30 0 C) at Institute of Soil Science, PMAS-Arid Agriculture University Rawalpindi (33.6518 0 N and 73.0806 0 E). The trays were moistened daily to avoid drying. On the day 7 th from planting of seed, five healthy and uniform seedlings from each genotype were uprooted carefully and wrapped separately with foam at the root shoot junction. After wrapping, each seedling was fixed into the holes of polystyrene thermo pole sheet floating on the water in a plastic tub (60×40×30 cm 3 ). Moreover, five seedlings per replica were maintained. Furthermore, a weighed quantity of sodium chloride (NaCl) of analytical grade was added into each tub to develop the requisite salt stress levels viz. <4 (control), 8 and 12 dS m -1 . It was noteworthy that salinity stress was imposed in two equal splits on each alternate day after the shifting of seedlings. Half-strength of the Hoagland's [14] nutrient solution was also added in each tub on the very next day to meet the nutrients requirement of the seedlings. Additionally, the pH of the solution was maintained at 6.5±0.5 with 1M NaOH or HCl when required. Nutrient solution was changed accordingly after one week interval. An aeration pump (ACO-208, Guangdong Hailea Group Co., Ltd, China) was installed in each tub with all its piping arrangements to ensure the continuous supply of air.
At the age of 35 days, the seedlings of each genotype from each salt stress treatment were taken and separated into root and shoot parts with the help of a sharp paper cutter blade. Accordingly, the morphological and bio-chemical traits were recorded by employing the standard procedures as under: Fresh weights of the shoots and roots were recorded with digital balance (Model: AND-GF 3000). Shoot and root lengths of the seedlings were measured with the help of an ordinary foot scale. Stem diameter of the seedlings measured using vernier calliper. Leaves of the five seedlings from each genotype were counted and then averaged to compute number of leaves per seedling. Leaf area was measured by multiplying the maximum length and width of the leaves with 0.75. Dry weights of the shoots and roots were recorded after oven drying at 70 0 C for 72 h. However, the Na + and K + contents were determined in the dried shoots were determined by digesting the plant material with perchloric acid and concentrated H2SO4. The digested samples were diluted and run at flame photometer (Digiflame, DV-710) for the quantification of K + and Na + .
After the quantification of all the morphological and bio-chemical attributes, PCA was performed to identify the most suitable trait to be used as selection criterion. By relying on the PCA results, dry shoot weight (DSW) and fresh root weight (FRW) were appeared as the major contributing traits to be used as selection criterion for salt tolerance. Moreover, both the variables contributed maximally and equally to explain the overall variations. Furthermore, it has been proven previously that shoots experiences more salts stress than roots. In this study, dry shoot weight (DSW) was therefore used as a sole criterion to categorize maize genotypes for salt tolerance using Murillo et al., [5,4] classification. Accordingly, dry shoot weights (DSW) were used to compute PDSW8 (A) and PDSW12 (B) and finally to work out average of PDSW (C) for each genotype by using the formulae given as under: Where PDSW8 and PDSW12 are the percent dry shoot weights at EC 8 and 12dSm -1 , respetively.

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According to this classification, the genotypes which showed average of PDSW ≥ 70 % were designated as tolerant (T). However the genotypes whose average of PDSW values falls between 55-69.9 % were categorized as moderately tolerant (MT). Moreover, the genotypes that exhibited the average of PDSW ˂ 55 were placed under sensitive (S) category.
Statistical Analysis: Analysis of variance (ANOVA) was performed by using a statistical package, SPSS version 16.0 and the means were compared by standard error.

Shoot length (cm):
The thirty (30) maize genotypes responded differently in response to salt stress and considerable variations between the control and salt treatments were observed in almost all traits. Data presented in the  Table 2 indicated the deleterious effects of incremented salt stress levels on fresh shoot weight (FSW) of thirty maize genotypes. The maximum fresh shoot weights viz., 5.78±0.09 3.82±0.16 2.33±0.07 g per plant were obtained in SB-9617 at EC <4, 8 and 12 dS m -1 , respectively followed by FH-949 produced fresh shoot weights of 5.56±0.24 3.81±0.14 2.02±0.07 g per plant at the above salt stress levels respectively. The minimum fresh shoot weights viz., 2.74±0.06 1.35±0.03 0.54±0.06 g per plant were attained with NCEV-7004 with the increasing levels of salt stress. Furthermore, SB-9617 showed the lowest reductions of fresh shoot weight 33.9 & 59.7 % at 8 and 12dSm -1 with respect to control treatment, respectively. Mean ± Standard error (Mean ± S.E)

Dry shoot weight (g) per plant:
The increasing salt stress also caused drastic effects on the dry shoot weight (DSW) in all the maize genotypes as indicated in the Table-3. The maximum dry shoot weights viz., 1.45 ± 0.09, 1.19 ± 0.05 and 0.90 ± 0.03 were determined in SB-9617 where EC less than 4 and equal to 8 and 12dSm -1 were maintained in the nutrients medium solution. The minimum dry shoot weights viz., 0.68 ± 0.10, 0.37 ± 0.03 and 0.11 ± 0.01 were recorded in NCEV-7004 at same levels of salt stress respectively. The other tested maize genotypes were noticed between the two genotypes with respect to dry shoot weight. International Letters of Natural Sciences Vol. 86

Number of leaves and Leaf area (cm 2 ) per plant:
The reductions of numbers of leaves (NL) and leaf area (LA) per plant in all the genotypes were observed with incrementing the salt concentrations in the nutrient medium (Table-4 & 5). In the present study the highest numbers of leaves (6.00 ± 0.00, 5.67 ± 0. 33

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Root length (cm) per plant:
Root length (RL) was also seriously affected by higher levels of salt stress in all the studied genotypes (Table-7). In the present study, root lengths in all the genotypes were drastically influenced by incrementing the salt stress levels. However, the maximum reductions in the root lengths (10.93 ± 0.35, 8.20 ± 0.42 & 7.03 ± 0.37 cm) were recorded in NCEV-7004 at <4, 8 and 12dSm -1 respectively. Additionally, these reductions were recorded 33.1 % greater than control at 8dSm -1 and 55.4 % higher than control at 12dSm -1 in the same maize genotype. Among the maize genotypes, SB-9617 proved the best performing genotype having more root lengths (17.60 ± 0.91, 15.10 ± 1.10 & 13.57 ± 0.62 cm) at all the salt stress levels. On overall basis, SB-9617 attained 20.3 % higher more root length than NCEV-7004.

Fresh root weight (g) per plant:
Fresh root weights (FRW) in the tested genotypes were also affected by the negative impacts of increasing salinity (Table-8). In comparison, almost all the maize genotypes experienced the reduction of fresh root weight with the higher levels of salt stress but SB-9617 was found least affected by the increasing salt concentrations i.e. at EC=8 dSm -1 the reduction of fresh shoot weight was 12.8 % and in the similar way the reduction in the FRW was 45 % at EC equal to 12dSm -1 compared to control. The highest reductions of FRWs i.e. 36.4 & 74.4 % were recorded in NCEV-7004 at 8 and 12dSm -1 respectively. On an average, SB-9617 produced 212.3% higher fresh root weight (FRW) than NCEV-7004 under the influence of salt stress. The remaining genotypes were present in between them with respect to FRW (Table-8).

Dry root weight (g) per plant:
Dry root weight (DRW) presented in the Table-9 showed the deleterious effects of salt stress in thirty (30) maize genotypes. The results depicted that SB-9617 performed better among the other tested genotypes with respect to DRW i.e. 0.39 ± 0.01, 0.35 ± 0.01 and 0.30 ± 0.01 g per plant at EC less than 4 (non-saline), equal to 8 and 12dSm -1 respectively. It is furthermore added that SB-9617 produced 10.3 and 23.1 % higher DRW at 8 and 12dSm -1 respectively, compared to control treatment. However, NCEV-7004 was noticed as the least performer yielding DRWs of 0.17 ± 0.01, 0.11 ± 0.01 and 0.08 ± 0.01 g per plant where EC was less than 4, equal to 8 & 12dSm -1 respectively. On overall basis, SB-9617 (showing highest mean DRW) produced 192 % higher dry root weight than NCEV-7004 (showing least mean DRW).

K + /Na + ratios
All the 30 maize genotypes were also evaluated for K + /Na + ratios against the graded levels of salt stress. Data presented in the Table-10 clearly indicated that all the genotypes responded differently to incrementing salt concentrations. The maize genotype SB-9617 comparatively exhibited the highest K + /Na + ratios i.e. 7.24 ± 0.836, 2.76 ± 0.112 and 1.13 ± 0.019 at EC less than 4, equal to 8 & 12dSm -1 respectively. However, the maize genotype NCEV-7004 attained the lowest K + /Na + ratios i.e. 0.64 ± 0.101, 0.35 ± 0.032 and 0.09 ± 0.003 at the above levels of salt stress. The other maize genotypes were positioned between these two genotypes with respect to K + /Na + ratios on overall basis.   fresh shoot weight (32.04%) contributed maximally to PC2 (Figure-2). However the ion accumulation traits such as K + : Na + ratio showed their maximum influence on PC3 i.e. 70.63 % (Figure-2).

Principle components (PCs)
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ii. Moderately tolerant group (MT)
Based on the average of PSDW values (Table-11     FSW=Fresh shoot weight, DSW=Dry shoot weight, SL=Shoot length, LA=Leaf area, FRW=Fresh root weight, DRW=Dry root weight, RL=Root length, NL=Number of leaves per plant, K + /Na + =Potassium to Sodium ratio **Correlation is significant at 0.01 levels of significance

Discussion
Salt stress adversely affects almost all the developmental stage in maize but causes severe damage to early seedling establishment [49]. In spite of the fact that maize crop is sensitive to salt stress but contains excellent genotypic variability for salt tolerance [10]. To cope with salinity, the crop undergoes some necessary alterations in the shoot and roots morphology [53,67]. Therefore, quantification of the morphological traits associated with shoot and roots at seedling stage are of great significance to be used as selection criterion for salt tolerance in maize. In the present study, different morphological traits such as lengths of shoot and root, fresh and dry weights of shoot and roots, stem diameter, number of leaves per plant and leaf area per plant were studied.
Among all the morphological traits, shoot growth characters in maize exhibits higher vulnerability to salt stress [22]. In the current study, shoot morphological traits such as shoot length (SL), fresh shoot weight (FSW) and dry shoot weight (DSW) were severely affected by incrementing the salt stress level. The decreased shoot length with increasing salt stress levels might be attributed to the stunted growth with reduced intermodal development. The results of the present study were in accordance with the findings as reported in the previous studies held by Akram et al. [29]; Qu et al. [11]; Batool et al. [32]; Chen et al. [63] and Shafique et al. [16] wherein it was investigated that leaf abscission and /or senescence disrupts the production of photosynthates process and their translocation to growing tissues, thereby leading to the production of seedlings with stunted growth and shorter length. As far as the fresh and dry shoot weights are concerned, a significant number of research reports are available which clearly indicate the dual aspect of salt stress either positive or negative. Furthermore, the response of crop to salt stress changes with salt type, its concentration and the plant type [35,9,61,56]. In our findings, the gradual reductions of fresh and dry shoot weights were also noticed in all the maize genotypes with higher levels of salt stress. However, the reduced SFW and DSW were recorded in SB-9817, FH-949 and YH-1898 might be due to having more inherited potential to prevent the accumulation of lethal concentration of salts in the biological tissues. Similar findings were also reported by Maqbool et al. [38]; Huqe et al. [37]; Shafique et al. [16]; Zahra et al. [42] that salt lenient maize genotypes attained higher shoot biomass than sensitive genotypes under salt stressed conditions.

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Stem diameter (SD), number of leaves (NL) and leaf area (LA) are the other morphological stress indicators whose estimation also determines the intensity of salt damage to crop. In the present results, it was observed that the increasing levels of salt stress caused significant reduction of the stem diameter (SD). Tas and Basar [8]; Farhana et al. [53]; Gao et al. [62] and Nassar et al. [51] also concluded the similar findings that salinity stress alters the stem anatomy which eventually reduces the stem thickness, thereby affects the stem diameter (SD).
Salt stress through its ionic and osmotic effects accelerates the leaf senescence which resultantly cause wilting and then death of the leaves [49,42,60]. These effects significantly reduced the number of leaves and leaf area per plant in our current study. The results of the presents study are further supported by the findings of Szalai and Janda [17], Qu et al. [11] and Agami et al. [50] where it was investigated that salts when applied in higher concentrations suppress the leaf growth and leaf initiation by reducing the number as well as the rate of elongating cells. Similar reports were also presented in various studies that increasing concentration of salt stress deleteriously impacts the leaf area by reducing the number of leaves per plant [21,28,42,16].
Saltiness also had drastic effects on the root morphological traits such as length of the roots and fresh and dry weights. The reduction of these parameters might be associated with inhibited roots growth due to excessive accumulation of Na + in the roots (Munns and Tester, 2008). Similar reports were also published by Farhana et al. [53]; Hasan and Miyake [45] and Shafique et al. [16] wherein the excessive accumulation Na + in the roots tissues was held responsible for altered morphology and anatomy of the roots. Furthermore, the sodium ions displace the calcium ions from the cellular membranes causing them more perforated, thereby leading to the loss of vital electrolytes [19], which resultantly reduced the cell division and cell elongation [17].The results of the present study were further supported by the findings as reported by Maqbool et al. [38], Dikobe et al. [58] and Sandhu et al. [13] that salt stress causes significant reduction of roots length and fresh & dry weights of roots.
Based on the morphological traits, all the genotypes were found superior at least in one character and inferior in other characters. Therefore, describing maize genotypes for each character separately was very difficult. Hence, PCA-Biplot multivariate analysis was employed using nine morphological traits across 30 maize genotypes against the graded levels of salt stress (EC <4, 8 & 12dSm -1 ) to identify the most suitable characters to be used as selection criterion for salt tolerance. PCA is a type of that combines characters and objects in two dimensions together and minimizes overlapping variations, facilitating the determination of main characters for selection [1]. The first and second principle components (PC1& PC2) were used which contributed maximally (90.7 & 3.3 %, respectively) to explain the overall variation (94.0%) as shown in the Figure-1. Two (DSW & FRW) out of nine studied morphological characters were found of great significance which exhibited the highest and equal contribution (10.55%) to explain the variations in PC1. Thus, they are the fair estimates of performance of the maize genotypes under salinity stress.
After determining the most suitable morphological character i.e. DSW (Based on PCA results) was used as selection criterion for salt tolerance in maize genotypes using Murrillo et al. [5,4] classification to categorize genotypes into tolerant (T), moderately tolerant (MT) and salt sensitive (S) groups.
Based on morphological features, the tolerant (T) group was least affected by the adverse effects of salinity stress than moderately tolerant (MT) and sensitive (S) genotypic groups. Varsha et al., [23] and Huqe et al. [37] also used the similar statistical procedure of multivariate analysis i.e. PCA to identify the salt tolerant maize genotypes on the basis of morphological traits. The differences among the genotypes for morphological traits as observed in the current study might be associated with the differential strategic behaviour of genotypes to cope with harmful effects salinity. The sensitivity of maize genotypes to increased levels of salt stress depends on their inability to retain higher K + contents in the plant tissues that eventually reduced the K + /Na + ratio. Under the influence of salt stress, plants tend to accumulate more Na + than K + in the shoot and roots tissues. Whereas, the salt tolerant genotypes retain lower Na + and maintained higher levels of K + in the plant body thereby leading to higher K + /Na + ratio (65,37,30), which is very much consistent with our findings (Table-10). In short summary, the current study provided scientific information to the breeders to boost the development process of new, more productive and better adapted maize cultivars.

Conflicts of Interest
Author declares not any conflict of interest regarding the publication of this manuscript.