Determination of Seed Size Properties of Soybean Cultivars and Their Response under Salinity during Early Growth

Four soybean cultivars (Kocatürk, Batem Erensoy, Göksoy and Ataem) were obtained from the Batem, Antalya, Türkiye. The experiment was conducted in the seed technology laboratory of Field Crop Department of Ankara University Faculty of Agriculture in 2023. The seeds were graded individually into two categories depending on their genotypes as small (6-7 mm for Ataem, 4-6 mm for Kocatürk, 4-7 mm for Batem Erensoy and 4-5 mm for Göksoy) and large (>8 mm for Ataem and Batem Erensoy, >7 mm for Kocatürk and >6 mm for Göksoy.  The one thousand seed weight and water uptake of four cultivars is shown in Table 1.
 
Germination test

The electrical conductivity of NaCl concentrations was adjusted to 10 and 20 dS m^-1 before the start of the experiment by using YSI 3200 conductivity meter (Day and Aasim, 2017). Distilled water was used as control (0 dS m^-1).

The experiment made use of 200 seeds per treatment for each category of seeds. These were equally divided into four replications containing 50 seeds in each replication. The seeds were germinated in between 3 rolled filter papers with 10 ml of respective test solutions and put into sealed plastic bags. The seeds were incubated at 25±1°C in the dark for 10 days. The papers were replaced to prevent the accumulation of salts after every two days. When the emerging radicles elongated to 2 mm, the seeds were considered germinated. Mean germination time (MGT) was calculated using the formula given below to evaluate germination speed (ISTA, 2017).
   
n= Number of germinated seeds on day t.
t= Number of days from the beginning of the germination test.
Ʃn= Total number of germinated seeds.

The germination percentage calculation was conducted following Day and Koçak-Şahin, (2023a).
   
Where:
GP= Germination percentage.
NG= Number of germinated seeds.
NT= Total number of seeds.

Root length (RL), shoot length (SL), seedling fresh weight (FW) and dry weight (DW) of 10 seedlings randomly selected from each replicate were measured on the 15^th day.

Dry weight was measured after drying samples at 70°C for 48 hours in an oven (Kaya et al., 2013). Dry matter was calculated with the given formula:  
  One thousand seed weight of each genotype was determined with the formula given below:
  Water uptake
 
Ten grams of seeds from each of both seed sizes of soybean cultivars were placed in petri dishes containing distilled water to observe the water uptake of respective genotype to germinate. The percentage increase of moisture contents was calculated individually for each size of each genotype. The seeds in each replicate were weighed before and after germination and water uptake was calculated as given below:
             
Electrical conductivity (EC) test
 
The electrical conductivity (EC) of three replicates of 25 seeds each of soaked soybean seeds with distilled water was measured using a YSI 3200 Conductivity Instrument (Day and Koçak-Şahin, 2023b). The results were expressed in µS cm^-1 to take account of variability in different seed cultivars and sizes.
 
Seed pigment content
 
The seed samples of cultivars (500 mg) for each seed size of each variety were ground to powder with a mortar. The grounded seeds were mixed with 2 ml of petroleum ether (PE 60-80°C, pure) and tetrahydrofuran (THF) in a ratio of 1:1 with continuous grinding in a mortar with 3 ml of PE added. The samples were rinsed twice with 3 ml of PE: THF (4:1) mixture. The homogenate was then filtered through micro filters with a pore size of 0.45 µm. The filtrate of each sample was used to measure chlorophyll a (Chl a) and chlorophyll b (Chl b) using a spectrophotometer at wavelengths of 645 and 655 nm. The pigment contents were calculated with the equation reported by Bulda et al. (2008).
 
Emergence test
 
To determine the impact of seed size on different cultivars of soybean, four replications of 50 seeds were sown in pots filled with sterilized peat and perlite mixture to a depth of 3 cm. The trays were placed in greenhouse at 25±1°C 45 µM photons m^-2 s^-1 light for 16 h. The peat used for sowing had 40 mS m^-1 EC and 6.5 pH. The trays were irrigated with tap water every two days. Emerged seedlings (unfolding cotyledons on the surface) were counted daily. The emergence percentage (EP) and the mean emergence time (MET) were calculated with the formula given in the germination test. After 25 days in greenhouse, shoot length, root length, seedling fresh weight and dry weight were measured from randomly selected ten plants. The seedling weights were recorded soon after the end of the experiment to avoid any loss in weight (Day, 2016 a; b). The seedling dry weight determined after sample drying in an oven at 105°C for 2 h (Yildiz et al., 2010).
 
Pigment analysis
 
The fresh leaf samples (0.1 g) were extracted in 10 mL of 100% acetone to determine the content of chlorophyll (a+b) and total carotenoids (x+c). Thereafter, the absorbance of extracts was measured at 470, 644.8 and 661.6 nm using a Hettich universal 30 RF spectrophotometer. The contents of chlorophyll (a+b) and carotenoids (x+c) were determined using adjusted extinction coefficients (Lichtenthaler, 1987; Sharma et al., 2021).
 
Electrolyte leakage (EL) test
 
Electrolyte leakage (EL) measurement was done as described by Lutts et al., (1996); Hnilièková et al.  (2019). The leaves were divided into 1 cm diameter (0.2 g) and washed with deionized water, placed in tubes with 10 ml of deionized water and incubated for 24 h at 25°C. Later, the electrical conductivity of the solution (L1) was determined. The samples were autoclaved at 120°C for 20 min and the final conductivity (L2) was measured after equilibration at 25°C. The EL was calculated as follows:
    
Experimental design and statistical analysis
 
The experiment was arranged in a completely randomized design with four replications. The data given in percentages were subjected to arcsine transformation before statistical analysis. The differences among the means were compared with Duncans multiple range test (p<0.01 or p<0.05). For all investigated parameters, analysis of variance was performed using the MSTAT-C computer software program (Michigan State University, version 2.10).
 

Seed characteristics tests
 
Diversities in one thousand seed weight, water uptake and electrical conductivity of the investigated soybean cultivars are given in Table 1. One thousand seed weight of cultivars and seed sizes showed significant variation among them. Large and small-sized seeds of cv. Ataem gave heavier seeds compared to both sizes of other cultivars. Water uptake was higher in large seeds of all cultivars (Table 1). EC among cultivars and sizes did not show variety except for large seeds of cv. Batem Erensoy (Table 1). The maximum and the minimum EC of the seeds were noted for large seeds of Batem Erensoy with 701.10 µS cm-1 and for the small seeds of Göksoy with 114.50 µS cm^-1.

Seed Chl a and Chl b content of cultivars represented a statistical difference (Table 2). The maximum chl a and chl b content of 7.04 mg g dw^-1 and 10.58 mg g dw^-1 was noted in cv. Göksoy. The seed size impact on seed chl a and chl b showed significant differences. The large seeds had less chl a and chl b with the values of 3.96 mg
g dw^-1 and 6.99 mg g dw^-1 and the small seeds had the higher chl a and chl b with the values of 6.76 mg g dw^-1 and 8. 94 mg g dw^-1.

Seed size impact on one thousand seed weight, water uptake and seed EC along with the seedling growth parameters of soybean cultivars was significantly clear. The EC test is a type of seed vigor test. Usually, high levels of leakage are considered a sign of low vigor (Araújo et al.,  2022). However, it was found that larger soybean seeds with higher germination rate and vigor tend to leak more (Table 1).



It was found that small seeds contain higher Chl a and Chl b compared to large seeds (Table 2). A decrease in GP and EP in small seeds could be attributed to less degradation of chlorophyll of small seeds during seed maturation (Nakajima et al., 2012). Similar reports related to the inhibiting effect of seed high chlorophyll content on seed germination have been particularly noted by Costa et al., (2014) in rice.


 
Germination test
 
The impacts of seed size and NaCl levels on MGT, GP, RL, SL and DM are given in Table 3. MGT represented a statistical difference over seed size and cultivars. Large sized seeds of cultivars germinated earlier and a higher GP was obtained (3.11 days; 93.50%) compared to small sized seeds (3.34 days; 68.33%). NaCl level increase from 10 dS m^-1 to 20 dS m^-1 caused increased MGT. The minimum and the maximum time to germination were noted in control (2.50 days) and 20 dS m^-1 (4.46 days). A decrease in GP was observed in 20 dS m^-1 (79.12%) and was significantly less compared to the control (84.00%). However, the GP recorded was similar at 10 and 20 dS m^-1. The results recorded significant impact of NaCl levels on the shoot length and root length. A comparison of the mean root length values showed that the maximum root length (6.78 cm) was recorded in control. Minimum root length was noted (1.24 cm) on 20 dS m-1 NaCl. Increase in dry matter was observed with the increase in NaCl levels. The minimum dry matter of seedlings was observed in control treatment (14.10%) and the maximum dry matter was detected at 20 dS m^-1 NaCl level (29.89%).



A three-way interaction was observed for the MGT, root length and shoot length, (Table 4). MGT showed variation for all the cultivars, both the sizes studied which delayed time to germination with increasing NaCl levels. GP varied between 46% to 100%. 



At a NaCl level of 10 dS m^-1, there was a small increase in the root length, but at 20 dS m^-1, there was a significant decrease in the root length. However, at 20 dS m^-1 NaCl level, large and small seeds recorded similar root length in all the cultivars studied.

Analysis of variance showed significant differences among treatments for shoot length. The maximum shoot length value of 14.51 cm was observed in the control treatment for small seeds of Kocatürk. The shoot length of small seeds was superior or gave statistically similar values for large seeds especially in control treatments and 10 dS m^-1.

The harmful impact of NaCl levels applied to soybean cultivars was especially observed in MGT and EP parameters of seedlings of small-sized seeds. The larger seeds have a higher germination rate (Table 3). The MGT of the soybean varied and NaCl applied small seeds recorded delay in germination compared to large seeds. The early germination of large seeds could elevate competing capacity with the surrounding weeds and efficient uptake of the resources. The detrimental impacts of high levels of NaCl are in line with earlier observations in soybean by Putri et al., (2017) and Yin et al., (2022).

Under NaCl stress, dry matter increased significantly, indicating that increasing NaCl decreased tissue water contents (Table 3). De Oliveira et al., (2020) in sorghum determined lower tissue water content and higher dry matter contents under stress conditions. The big seeds’ superiority particularly in MGT and GP along with dry matter could be attributed to larger embryos and more carbon-based reserves in seeds (Pandey et al., 2017). The findings of the study are in line with Okonwu et al., (2022). The results of the salinity experiment revealed that 20 dS m^-1 NaCl level showed its detrimental effect on germination and seedling growth of all the cultivars studied in both the sizes and is in line with Day et al., (2008).
 
Emergence test
 
The seedling emergence performance of the cultivars in relation to seed size was tested by emergence test. The EP of the cultivars differed from GP (Table 5). The seed size of the cultivars under the study significantly affected the root length. Large seeds recorded longer root lengths with 9.47 cm compared to small seeds. In emergence test, shoot length of soybean seedlings showed significant differences between seed sizes. A comparison showed that the maximum (31.30 cm) shoot length was noted in large seeds. Fresh weight and dry weight of seedlings also showed significant differences and seedlings of large seeds had the maximum values with 2.01 g in fresh weight and 0.216 g in dry weight.



Results about chlorophyll contents measurements clearly showed the impact of seed size on chl a and carotenoid pigments (Table 6). The maximum chl a and carotenoids were observed in seedlings from large seeds with 30.28 and 5.65 mg g fw^-1.

Based on the emergence test, it was found that seedlings from larger seeds had longer roots and shoots, leading to higher fresh and dry matter compared to seedlings from smaller seeds (Table 5). Earlier research by Bianchi et al., (2022) also supports the idea that larger seed growth parameters lead to better performance.

Chl a and carotenoids in seedlings of large seeds were more compared to small seeds (Table 6). Carotenoids are essential in absorbing light energy and transferring to chlorophylls in the spectrum 450-550 nm, a range that chlorophylls do not absorb (Hashimoto et al., 2016). The vital role of carotenoids in photoprotection is to sustain adaptation to changing light (Simkin et al., 2022). Emphasizing the significance of chlorophyll a and carotenoids also brings attention to the significance of seed size and its vitality in soybean. The large seeds contain a significant amount of Mg and N reserves, which can aid in the rapid synthesis of chlorophyll pigments.



Leaf electrolite leakage is a test used under stress (Demidchik et al., 2014). However, developing cells and organs in plants are under the stress of mechanical internally apart from the stress caused by the environment. During germination and initial growth of seedlings, seeds experience mechanical stress because of fluctuations in osmotic and turgor pressure and this stress could lead to diversity in leaf electrolyte leakage. The principal mechanical stress that is experienced by all living plant cells is turgor pressure. Turgor is important for the structural stability of the plant cell. Moreover, turgor pressure is the driving force behind cell expansion and it works together with tightly regulated cell-wall extensibility to determine the size and shape of a plant cell. In the context of seed mechano-responses due to expanding cells, this creates stresses which could change depending on the species, cultivars and seed size (Monshausen and Haswell, 2013). In this study, it is found that leaves obtained from small-sized seeds leaked more or equal over large seeds. Leaf electrolyte leakage diversity among cultivars and different sizes showed that in mechanostress perception their intrinsic mechanical stresses are changing depending on cultivars and seed size (Table 7).