Experimental Description

Five trials were executed at the Microbiology Lab of the Agronomy Department, University of Agriculture Faisalabad, Pakistan, during 2019-2020. Seeds of guar (cv. BR-17) were placed in each Petri plate (9 cm diameter) having three layers of Whatman No. 1 filter paper and replicated thrice. Before each trial, seeds were soaked for 5 minutes in 0.5% NaClO (sodium hypochlorite) solution and then washed with distilled water thoroughly (by repeating the procedure thrice to minimize the residual chlorine) to avoid fungal attack and allowed to dry at room temperature for 5 hrs, before placing those at the filter papers. According to the requirement, the filter papers were moistened regularly with deionized water using a pipette. For sowing depth treatments, plastic pots were filled with soil, and germination was noted for three weeks. The humidity (40%) and constant fluorescent light (400-700 nm and 100 µmol m^-2 s^-1) conditions were kept similar for all treatments in five trials of cluster bean.

Imposition of Abiotic Stresses and pH Level Adjustments

For imposing HS, different levels of constant temperature (T, 10, 20, and 30°C) were maintained to examine its effect on seed germination in a growth chamber (ICO105, Memmert GmbH+ Co. KG, Schwabach, Germany). Data from each trial were recorded daily for two weeks except for sowing depth treatments. Seeds were considered to have germinated when radical protrusion reached up to 2 mm. For seed size categorization, guar seeds were classified by visual observation and then based on 1000-grain weight. In each Petri plate (9 cm), 9 guar seeds were placed according to their sizes (small, medium, and large) with three replicates and incubated in a growth chamber at all the above levels separately.

For imposing salinity stress treatments, salt solutions were prepared by adding 1 molar solution of NaCl (58.5 g molecular mass of NaCl) in 1000 ml of distilled water in accordance with [26].

So, in this way, 2.92, 5.85, 8.77, and 11.7 grams of NaCl were dissolved in 1000 ml of distilled water to obtain 50, 100, 150, and 200 mM salt solutions. Different-sized seeds of guar (9 per Petri plate) were treated with all these above solutions. Initially, Petri plates were treated with 5 ml of each solution (50, 100, 150, and 200 mM) separately, but further concentrations of these solutions were applied according to requirement. Petri plates were kept in the incubator at 27 ^⁰C temperature.

For imposing the osmotic stress treatments, different osmotic solutions were prepared as suggested by [27].

Where C = concentration of PEG in g/kg of distilled water and T = temperature (°C)

These solutions were prepared by using PEG 6000, and the experiment was conducted in a growth chamber. In the control treatment, distilled water was used, whereas osmotic stress was imposed using different concentrations of PEG (0.028, 0.045, 0.058, 0.069, and 0.079 g dissolved in 1000 ml of water) for imposing stress levels of -0.2, -0.4, -0.6, -0.8, and -1.0 Mpa, respectively.

To assess the impact of different pH levels, these were adjusted by using different solutions, such as pH₅ and pH₆ solutions of 2 mM MES [2-(N- morpholino) ethanesulfonic acid], which was adjusted by using a 0.1 N solution of sodium hydroxide. For making pH₇ and pH₈ solutions, HEPES [N-(2-hydroxymethyl) piperazine–N– (2-ethane sulfonic acid) solution was adjusted by a 0.1 N of sodium hydroxide solution. Tricine was adjusted by 0.1N sodium hydroxide solution for pH levels of 9 and 10 [24].

Sowing Depth Treatments

Sowing depth treatments were comprised of 0, 2, 4, 6, and 8 cm depth, wherein control treatment (0 cm) was implied by placing seeds on the soil surface. Twenty guar seeds were placed in plastic pots (15 cm), which were filled with soil (40% sand, 30% silt, and 30% clay), and seeds were sown according to the treatments with the help of a wooden scale. Distilled water was provided (by maintaining sufficient soil moisture level at different depths by pulverizing the soil with an iron rod and ensuring water application until it started to leak out from the pot’s bottom hole) to pots according to their requirements and seeds were considered to have germinated by the visibility of plumule (2 mm).

Response Variable Recordings

Germination (G, %) was assessed by using the formula described by Iqbal [28].

Seed germination was recorded daily to determine the germination index (GI) as suggested by Iqbal [7].

Mean germination time was also estimated by following the equation reported by Ali et al. [29].

Where D = number of days from the start of germination/emergence and n = number of seeds germinated/ emerged on day D.

Moreover, T₅₀ was calculated according to the formula given by Farooq et al. [30].

t_j and t_i represent the adjacent times. N denotes the final germinated seed, while n_i and n_j represent the cumulative number of germinated seeds by contiguous counts at time t_j and ti, where n_i<N/2 < n_j.

Statistical Analyses

The collected data of all response variables of guar were arranged and subjected to a one-way Fisher’s analysis of variance (ANOVA) technique for estimating the overall significance of employed treatments with the help of the computer-run statistical package Statistix (8.1 version). Thereafter, the least significant difference (LSD) test at the 5% probability level was employed to sort out the significance among the treatment means [31].

Temperature and Seed Sizes Interactive Effects

The recorded findings revealed that different sizes of cluster bean seeds and different temperature regimes interacted significantly to influence germination and seedling growth (Table 1). The maximum germination (100%) was recorded at 20-30°C for all sized seeds, but at 10°C, small-sized seeds showed a significant reduction in final germination (70%). Likewise, the germination index and time to 50% germination were also significantly affected under different temperatures and with seed sizes, but at 20°C, the final germination and germination index were increased. Thus, these findings showed that any increase and decrease in temperature from 20°C and decrease in seed size led to a significant increase in the germination time and time taken to 50% germination. For all seedling parameters under investigation, the maximum root and shoot length (4.7 and 7.3 cm, respectively) were recorded by the large seeds when incubated at 20°C, and the minimum (1.2, 2.3 cm) corresponding values were demonstrated by small-sized seeds at 10°C. Moreover, the maximum root, shoot fresh, and dry weights were also observed for large seeds incubated at 20°C. These findings were also correlated with previous studies whereby a significant influence of temperature was noted on seed germination and seedling growth [32, 33]. Gresta et al. [34] concluded the same results, as the mean germination time was pronouncedly increased with fluctuations in the optimum temperature. Likewise, these results were in accordance with EL-Abady [35], who noted the significant impact of temperature alterations on the seedling growth traits of maize. Chaturvedi et al. [13] observed that temperature regulated the germination process by influencing many metabolic processes (enzymatic activity). Moreover, it was also demonstrated that many catabolic activities were inhibited by chilling stress, whereas elevated temperature led to the inactivation of many enzymes and the denaturation of many vital proteins [36]. Similar to our findings, Al Khteeb [26] inferred that germination rate and seedling parameters of panicum turgidum were significantly enhanced at the optimum temperature of 20-30°C, whereas higher temperatures caused a significant reduction in germination indices and seedling growth traits.

Salinity Levels and Seed Sizes Interactive Effects

Germination and seedling growth of different-sized seeds of guar were evaluated under various salinity levels (SS, control, 50, 100, 150, and 200 mM), and the results exhibited that an increase in SS and a decrease in seed size led to a pronounced decrease in the germination of guar (Table 2). Small seeds showed tolerance to SS of 50 mM. Mean germination time and time to 50% germination were increased with the increase in SS coupled with a decrease in seed size. Small-sized seeds showed the lowest germination index when exposed to the 200 mM salt solution, and the highest germination index was observed in large-sized seeds when treated with distilled water (control) up to 100 mM salt solution. In terms of seedling growth traits under investigation, large-sized seeds performed better even when exposed to the 100 mM salt solution. Likewise, the large-sized seeds depicted the highest root and shoot length (8.9, 7.6 cm) as compared with small and medium-sized seeds. Under high SS levels, seedling fresh and dry weights were greatly reduced, particularly in small-sized seeds. These results were contradictory to Kaya et al. [37], who reported that larger seeds of chickpea acquired the highest mean germination time under all SS levels; however, Teolis et al. [38] noted that guar seeds showed variations in germination under various SS levels. Chauhan and Johnson [39] also recorded similar results in African mustard and inferred that the reduction in germination under high SS levels might be due to the toxic effects of accumulated salts, which reduced the uptake of water and nutrients. Like our findings, higher levels of salt ions caused a reduction in the germination of okra and suppressed cell division, causing inhibition in growth [40]. The same findings were reported by [41] in shallot, whereby a saline environment restricted moisture and nutrient uptake, and growth was pronouncedly restricted. Moreover, SS seriously reduced plant traits, while hydrolyzed gellan gum reduced the deleterious effects of a saline environment [42].

Osmotic Stress and Seed Sizes Interactive Effects

Germination and seedling growth of different-sized seeds of guar were evaluated under different osmotic stresses (OS, control, -0.2, -0.4, 0.6, -0.8, and -1.0 Mpa), and the results exhibited that large-sized seeds performed much better under all osmotic stresses as compared to medium and small-sized seeds (Table 3). The maximum germination was recorded in all-sized seeds in the control treatment that supplied the distilled water, while the lowest germination (25%) was recorded by small seeds when exposed to the -1.0 Mpa solution. Likewise, the GI was also reduced in small-sized seeds exposed to a higher osmotic stress level (-1.0 Mpa). Likewise, the mean germination time and time taken to 50% germination were significantly increased with higher levels of OS, whereas these parameters were also reduced with a decrease in seed size (large to small). Seedling parameters were also reduced under high OS, particularly in small-sized seeds. The maximum root and shoot length were recorded in large-sized seeds in all osmotic stress levels as compared with medium and small-sized seeds. Moreover, seedling fresh and dry weights recorded a significant reduction with the increase in OS, especially the small-sized seeds that were not able to develop their seedling at -1.0 Mpa solution. These findings agreed with those of Mut and Akay [43], who reported that large-size seeds effectively offset the deleterious effects of osmotic stress owing to being more vigorous in nature and resulting in a pronouncedly greater germination percentage. Likewise, in line with our findings, previous results have been reported by Pratiwi et al. [44] in shallot (Allium ascalonicum L.) and triticale [45]. Muscolo et al. [46] described that root length and germination percentage recorded a pronounced reduction under OS in all lentil genotypes because the germination process involved several enzymes, while osmotic stress disrupted the activities of enzymes like glucosidase and amylase. Contrastingly, the OS inhibited germination by restricting the seed imbibition process due to a significant reduction in water uptake [18]. Ghorbani et al. [47] also observed that different morphological traits of guar were reduced under osmotic stress. Mujtaba et al. [48] and Khayatnezhad et al. [49] inferred that no wheat seedling showed survival at the OS level of -1.0 Mpa, and it also caused a significant reduction in seedling growth of maize. In agreement with our findings, several other studies have also reported the deleterious effects of OS on germination and seedling growth in many field crops like maize, rice, and camelina [50-52]. The underlying reason was the osmotic stress-driven injury in mitochondria and chloroplast that resulted in the overproduction of reactive oxygen species (ROS), and growth was hampered in Melilotus albus [53]. Moreover, PEG-induced osmotic stress pronouncedly reduced germination and seedling growth by triggering the biosynthesis of ROS in Sophora viciifolia, whereas exogenous abscisic acid mitigated the adverse effects of OS [54].

pH Solutions and Seed Sizes Interactive Effects

The results pertaining to the different-sized seeds of guar sown in a wide range of pH solutions (5, 6, 7, 8, 9, and 10) revealed that the maximum germination percentage was recorded at 7-8 pH (Table 4). Large-sized seeds performed superiorly under all pH levels as compared to medium and small-sized seeds. The highest germination (98%) was recorded by large seeds at 7 pH, whereas germination was reduced significantly with increasing pH levels, particularly for small-size seeds (20%) when exposed to 10 pH. Following the trend, the highest mean germination time (8.6 days) was exhibited by small seeds at 10 pH. Interestingly, it was also observed that the time to 50% germination was also increased with an increase or decrease of optimum pH (7), especially in small-sized seeds. Moreover, the large seeds demonstrated the highest germination index (GI) at 7 pH (13.8), whereas the lowest corresponding value was recorded by small seeds at 10 pH. Furthermore, seedling growth was significantly affected by the pH levels and seed sizes (Table 4). The minimum development in root and shoot traits (length, fresh and dry weights) was recorded by small seeds at pH 5 and 10 levels. Small-sized seeds even did not develop their seedling when exposed to a 10-pH solution. Like these results, Dorner et al. [55] demonstrated that any increase or decrease in optimum pH caused a reduction in canary grass, and it was attributed to a disruption in many enzyme (lipase, protease, and diastase) activities. Contrastingly, it was inferred that soil pH influenced the availability of essential plant nutrients and altered the correlations between the uptake of macro and micronutrients [56- 58]. Moreover, biochar-induced increment in pH caused osmotic stress along with nutrient imbalance owing to the insolubilization of minerals, and thus reduced availability of vital nutrients suppressed the seedling growth in tomatoes. Moreover, Yang et al. [59] stated that high alkalinity caused an ionic imbalance, and inhibition of ion uptakes led to the reduction of seedling growth. However, these findings were in contradiction with those of Gentili et al. [60], who recorded minimum growth of Ambrosia artemisiifolia at neutral pH, which might be interpreted in terms of different species and plant materials used in the study.

Sowing Depth and Seed Sizes Interactive Effects

The recorded findings regarding the emergence and various seedling parameters of different-sized seeds of guar sown under various sowing depths (SD, 0, 2, 4, 6, and 8 cm) revealed that the maximum emergence (80%) was recorded by large seeds, and it was significantly decreased with an increase in the SD (Table 5). The highest mean emergence (9.8 days) and time to 50% emergence (7.5 days) were recorded in small seeds at 8 cm depth. The maximum emergence index was recorded in large-sized seeds at 4 cm and the minimum emergence index (1.96 seed day^-1). In terms of all seedling parameters, large-size seeds performed much better under all sowing depths as compared to medium and small-sized seeds. Large seeds had better root length (7.6 cm) and shoot length (8.6 cm) under 2 cm depth. Seedling fresh and dry weights were highly reduced in small-sized seeds when these seeds were sown at 8 cm SD. It might be interpreted that seed germination tends to depend on various ecological conditions, particularly moisture, light, and temperature, and these conditions vary at different soil depths, which led to different germination indices in this study. In previous studies, it was inferred that seeds showed the minimum emergence when buried too shallow due to insufficient moisture at the uppermost layer of soil [61, 62]. Likewise, Heckman et al. [63] reported that seeds sown at 2 cm depth recorded greater germination and developed a better root system to uptake water and nutrient contents, which resulted in better seedling vigor. The same conclusions have been given by Emenky and Khalaf [64] in chickpeas. Deep planting depth caused a negative effect on seedling emergence, as reported by Nabi et al. [65] in cotton. Boyd and Van Acker [61] reported that seed emergence was reduced by the increase in planting depth in some annual and perennial weed species. Similar results were reported for Galium tricornutum seeds, and seeds sown at 8 cm of depth showed no germination, while seeds sown at depths of 0.5-2 cm recorded the highest seedling emergence [62]. Moreover, it was revealed that seeds buried in different soil depths tend to experience atypical environmental conditions, particularly; oxygen availability, CO₂ exchange between soil layers, temperature, moisture, and nutrient deficiency, which affect seed germination, time taken to 100% germination, and seedling growth traits (root and shoot length and their fresh and dry weights). Interestingly, deeply buried seeds need to consume extra food reserves in pushing their shoot apices at the soil surface in comparison to the seeds sown at shallower depths. Contrastingly, seeds present on the soil surface were exposed to air and litter, which reduced their viability owing to rapid loss of moisture [25]. Thus, the findings of our trial put forward the concept of optimization of seed size and SD reconciliation for ensuring maximum emergence and seedling growth under normal and suboptimal growth conditions.