Combination Effect of Temperature and Salinity Stress on Germination of Different Maize (Zea mays L.) Varieties

Abstract

Temperature and salinity stress are currently spreading widely across the globe and have been proven to have a negative impact on maize (Zea mays L.) crops as early as the germination stage. However, more research must be conducted on the interactive or combined effects of salinity and temperature stress on maize germination. This study aims to determine the impact of combined temperature and salinity stress on 16 different maize varieties. The maize seeds were incubated at three different temperatures (15 °C, 20 °C, and 35 °C) and two sodium chloride (NaCl) levels (0 mM and 100 mM) simultaneously. Germination percentage, root and shoot growth, root:shoot length ratio, and seed vigor index (SVI) were recorded and analyzed. The presence of salinity reduced maize germination qualities at all three temperatures tested. However, at high 35 °C temperatures, significant reductions in germination performances were observed compared to lower temperatures with salt stress. Three varieties (V1, V10, and V16) had the best overall germination performance in all three temperatures under saline stress, while V4, V5, V12, and V14 showed higher salt tolerance at 35 °C than at lower temperatures. In conclusion, increased temperature amplifies the salt stress in maize germination, and the varietal effect influences the seed tolerance towards a combination of salt and temperature stress.

1. Introduction

Zea mays L. (corn) is a versatile crop. It can be used for human and animal consumption or generating energy as biofuel for daily use. Historically, maize was first cultivated around 8700 years ago in Mexico [1]. As maize breeding knowledge and technology has advanced, the area where this crop is grown has widened across the globe, from southern latitude 40° S to northern latitude 50° N and up to 3500 m above sea level [1]. In 2020, it was estimated that around 1.16 billion tonnes of maize were harvested from about 202 million hectares of land worldwide. In comparison, 760 million tonnes of wheat were produced from 219 million hectares of land in the same year [2]. Without proper agriculture practices, it was estimated that 50% of global croplands would be salinized by 2050. The global temperature is predicted to increase by at least 1.5 °C, with more frequent and intense heatwaves, by the same year [3,4]. Producing and cultivating crop varieties tolerant to multiple abiotic stresses may become a priority for researchers, breeders, and farmers to ensure worldwide food security in the future.

Maize was believed to be domesticated around 8000–10,000 years ago from a wild grain of teosinte (Z. mays ssp. parviglumis) [5]. Over the millennia, maize has evolved through selective breeding and hybridization, which improves the chemical compounds, yield quality, and physiological adaptability and tolerance to biotic and abiotic stress [1]. Cross-pollination between varieties is still currently one of the methods in producing improved maize hybrids, including to achieve better abiotic stress tolerance, other than through the genetic modification method [6]. However, cross-pollination may produce progeny with undesirable traits, such as low tolerance towards abiotic stresses [7]. Therefore, the new progeny must be evaluated for the desired traits before varieties are released and cultivated on the field.

It was discovered that maize plants are more susceptible to salt stress at germination and early vegetative growth stages [8,9]. High salinity in soil solutions results in high osmotic pressure that restricts the seed imbibition by preventing water absorption and entry into the seed [10]. The inability to absorb water because of high salt concentration will also prevent the mobilization of essential nutrients needed for germination. Besides that, a high sodium chloride (NaCl) concentration during the early growth stage also causes Na⁺ and Cl⁻ toxicity to the embryo and young seedlings, resulting in stunted plant development [10,11]. Salt stress caused by NaCl also decreases the content of essential hormones for germination, such as gibberellins, while increasing the abscisic acid (ABA) levels [12]. Soil with a high saline content, especially from NaCl, will prevent the roots from absorbing macronutrients and micronutrients such as nitrogen, potassium, calcium, and iron [8]. Salinity stress during the growth and reproduction stages will affect both green fodder and kernel yield of sensitive maize hybrid by decreasing the grain weight and count [8,13,14]. Research shows that salt stress significantly inhibits maize acid invertase activity by up to 50% in soil experiments and can cause kernel reduction of about 50% in maize ears. Acid invertase activity is responsible for sucrose hydrolysis in the phloem to be transferred into the maize kernel [15]. In a salt-tolerant maize variety, it was observed that high phenolic compounds such as anthocyanins and polyphenols are produced by the plant, which exhibits high antioxidant activities that limit the oxidative damage by the Reactive Oxygen Species (ROS) [16].

Each plant seed requires a specific temperature range to initiate germination [17,18,19,20,21]. Temperature regulates the activity of phytohormones such as ABA and gibberellin (GA), which are responsible for cascades of biochemical reactions in breaking the seed’s dormancy and initiating germination [22,23,24]. The optimum germination temperature is defined as that at which the highest seed germination rate with the least number of days is achieved. Under the optimum temperature, seed germination and plant growth rates increase as the temperature increases and gradually decrease as the temperature passes the optimum level [23,24]. In maize, several studies have reported that the optimum germination temperature is between 25 °C and 30 °C [25,26,27,28]. A recent survey of a temperate maize hybrid shows germination and seedling growth occur at the highest rate at 20 °C, and the best growing range for the seedling in vitro is between 20 °C and 35 °C [17]. Temperature outside this optimal range will cause a lower germination rate, and no germination at extreme temperatures, such as outside the range between 5 °C and 40 °C [17,29].

Several recent studies have shown that an interaction between abiotic stresses can inflate the negative impact of one stressor [30,31,32]. High temperature simultaneous with other abiotic stresses, including salt stress, can cause irreversible damage to the plant photosynthesis apparatus and reproductive plant development [33]. However, studies have found that plants develop different acclimation mechanisms, including osmoprotectant accumulation and synthesis of ROS-scavenging enzymes, in response to combined abiotic stresses, and that these mechanisms differ for each stress condition [34,35,36,37]. In wheat and sorghum, studies observed that high temperatures can cause more detrimental effects on germination under salinity stress than in saline conditions under optimum temperatures. Simultaneous stresses also cause more negative impacts on the germination rate, plant growth, photosynthetic rate, chlorophyll fluorescence, and seedlings’ dry weight compared to under one stress alone [30,38]. Meanwhile, in maize, a combination of drought and heat stress affects the growth and yield of the crop compared to a single stress [32,39].

However, to the best of the authors’ knowledge, studies about the combination of temperature and salinity stress in maize germination are still lacking, as most of the past research has concentrated on the independent effect of individual factors. Therefore, this research aims to study the combined effect of temperature and salinity stress on the germination of 16 maize varieties. Seeds from 16 maize varieties were chosen for this germination trial as studies showed varieties in a plant species could produce seeds with different tolerance to abiotic stresses [14,40,41]. Therefore, the null hypotheses to be tested in this trial was “1. combination of temperature and salinity stress does not influence the germination quality in maize” and “2. all 16 maize varieties have the same tolerance against each abiotic factor”. The findings of this study will serve as a basis for future research on maize against these two stresses. At the end of this study, the best variety against each combined stress will be determined.

2. Materials and Methods

2.1. Seeds Materials

Sixteen maize varieties were used to study the interaction of salinity and temperature on maize seed germination quality, as summarized in

Table 1. Maize varieties tested for germination performances.

Source	Genotype		Hybrid/Parent
Martonvásár	V1	B1026/17	Parent
	V2	TK222/17	TC hybrid
	V3	TK15/DV	Parent
	V4	TK1083/18	Parent
	V5	TK623/18	SC hybrid
	V6	MCS901/19	Parent
	V7	TK256/17	DC hybrid
Szeged University	V8	GK155	Parent
	V9	GK131	Parent
	V10	GK 154 x155	SC hybrid
	V11	Szegedi 521	SC hybrid
	V12	GK 154	Parent
	V13	GK 150	Parent
	V14	GK 140	Parent
	V15	GK 144 x150	SC hybrid
Producer	V16	Margitta	Hybrid

. The seeds were obtained from a local producer, Szeged University, Hungary, and the Center of Agricultural Research, Martonvasar, Hungary. The varieties studied in this trial consist of single cross (SC), double cross (DC), and three-way cross hybrids (TC), parental lines, and one commercially available variety (V16). Variety 16 (V16) is a Hungarian dent maize variety called Margitta and is widely grown regionally. Margitta lies in the FAO 280 group, is considered a cold tolerance variety, and has a short growing cycle. However, the salinity and temperature tolerance were not published by the producer. The other 15 varieties were chosen to evaluate the different tolerance towards salt and temperature stress between the parental lines and the progeny regarding germination activities.

2.2. Growing Conditions

The trials were conducted in the laboratory of the Crop Production Institute of the Hungarian University of Agriculture and Life Sciences, Gödöllő, Hungary. The seeds were germinated at three different temperatures (15 °C, 20 °C, and 35 °C), with 20 °C chosen as the optimal temperature for maize germination based on a recent study on temperate maize hybrid, which used the same hybrid of V16 [19]. Based on other studies, temperatures below 10 °C delay seedling emergence [42,43], while a study showed temperate maize hybrids are more vulnerable to high-temperature stress (>35 °C) compared to tropical hybrids [44]. Therefore, temperatures of 15 °C and 35 °C were used to introduce the temperature stress conditions. Temperatures of 15 °C and 35 °C were considered the lowest temperature (LT) and highest temperature (HT), respectively, to initiate germination within nine days [17]. Two sodium chloride (NaCl) concentrations, 0 mM (control) and 100 mM (8.6 dS/m), were used to test the simultaneous effect of temperature and salt stress on germination. Normally, soil with electrical conductivity (EC) higher than 4 dS/m is classified as saline soil [45]. Several maize varieties were reported to be unable to germinate at EC of more than 8 dS/m, while several other varieties successfully germinated at EC of 15 dS/m [46,47,48]. Therefore, the experiment consisted of 6 different treatments: control (20 °C + 0 mM NaCl), LT (15 °C + 0 mM), HT (35 °C + 0 mM), salinity (20 °C + 100 mM), LT + salinity (15 °C + 100 mM), and HT + salinity (35 °C + 100 mM).

Before use, all of the seeds used in this trial were surface sterilized with 5% sodium hypochlorite for five minutes, rinsed with distilled water five times, and filtered to remove excess water. Each treatment was repeated four (4) times for all 16 maize varieties. In each repetition, six seeds from each variety were placed on a 9 cm petri dish containing single-layer filter paper, allowing enough space for the seedling growth within 9 days. As the seed size varied between varieties, the solution volume was fixed at 10 mL for every Petri dish. The Petri dishes were sealed with Parafilm sealing film to avoid moisture loss and were incubated in a Memmert climate chamber with a 70–80% humidity level. The germination percentage (GP) and the length of the radicle (RL) and plumule (PL) were recorded on days 3, 5, 7, and 9 of the experiment. Germination was considered to have started when the radicle length was more than 0.1 cm. On the last day of the incubation, the seed vigor index (SVI) and the root:shoot length ratio (R:S) were calculated based on the radicle and plumule and percentage. The GP and SVI were measured using the following equations [49]:

$$\mathrm{Germination}\mathrm{percentage}=\frac{\mathit{T}\mathit{o}\mathit{t}\mathit{a}\mathit{l}\mathit{n}\mathit{u}\mathit{m}\mathit{b}\mathit{e}\mathit{r}\mathit{o}\mathit{f}\mathit{s}\mathit{e}\mathit{e}\mathit{d}\mathit{s}\mathit{g}\mathit{e}\mathit{r}\mathit{m}\mathit{i}\mathit{n}\mathit{a}\mathit{t}\mathit{e}\mathit{d}}{\mathit{T}\mathit{o}\mathit{t}\mathit{a}\mathit{l}\mathit{n}\mathit{u}\mathit{m}\mathit{b}\mathit{e}\mathit{r}\mathit{o}\mathit{f}\mathit{s}\mathit{e}\mathit{e}\mathit{d}\mathit{s}\mathit{i}\mathit{n}\mathit{p}\mathit{e}\mathit{t}\mathit{r}\mathit{i}\mathit{d}\mathit{i}\mathit{s}\mathit{h}}\times \mathbf{100}$$

SVI = (shoot length + root length) × GP

2.3. Statistical Analysis

Multivariate analysis of variance (MANOVA) and two-way analysis of variance (ANOVA) at the 0.05 probability level were used to analyze the interaction between the independent variables and the dependent variables using IBM SPSS (Version 27, Armonk, NY, USA). Assumption tests, including for normality, homogeneity, correlation, and data outliers, were also conducted before the parametric test. Before statistical analysis, GP and seedling growth data were transformed into arcsine and ln(x + 1) to ensure a normal distribution. However, untransformed data are presented in the figures. Besides that, the mean value of each treatment was compared with the Games−Howell post hoc test at the 0.05 probability level. Microsoft Excel 2010 was also used for data management and to produce figures.

3. Results

3.1. Germination Duration and Seedling Growth

Based on the results, exposure of the seeds to salt stress (SS) influenced the germination percentage (GP), radicle length (RL), and plumule length (PL) at all three temperatures tested. Figure 1 shows that SS reduced the GP as the temperature increased from 15 °C to 20 °C and 35 °C, with the highest GP achieved at 15 °C after 9 days of incubation. Although the seeds germinated rapidly for the first 3 days at 35 °C, compared to 15 °C and 20 °C, the percentage reached a constant level after 5 days of incubation. In the 35 °C saline condition, the rate of seed germination did not increase after 5 days of incubation and produced the lowest GP compared to the other two temperatures. Therefore, the data show that at higher temperatures, the GP is more susceptible to salt stress than at lower temperatures.

Meanwhile, the radicle and plumule growth results also show a growing trend as the incubation days increase, especially for the control treatment (Figure 2a,b). The results show that the control condition at 20 °C was the optimum condition for the maize radicle and plumule growth compared to 15 °C and 35 °C. The results show that the radicle and plumule growth had the shortest length at 15 °C and only started growing at day 5, while the longest was at 20 °C. However, at 35 °C, the seedling growth, especially the radicle, did not improve as the incubation day increased for the SS seeds. Therefore, it can be concluded that SS inhibits the growth of the maize radicle and plumule, and a temperature below and above the optimum level aggravates the stress responses. The significant interaction between SS and temperature is further discussed in Section 3.2.

3.2. Interaction between Salinity and Temperature on Maize Germination

Figure 3 shows no significant difference in GP between the control and SS seeds at 15 °C and 20 °C, whereas, at 35 °C, the control seeds show a significantly higher percentage than the SS seeds. Figure 3 also indicates that GP gradually increased as the temperature increased for the control seeds, whereas the salinity stress caused the GP to drop at 35 °C. Besides that, the presence of SS significantly reduced the length of both the radicle and plumule as the temperature increased to 20 °C and 35 °C, as shown in Figure 4a,b. The results also show that the RL and PL gradually increase as the temperature increases from 15 °C to 20 °C but reach a plateau as the temperature rises to 35 °C. As the temperature increased to 20 °C and 35 °C, significant reductions in RL and PL were observed with the presence of SS. A two-way MANOVA was conducted to test the important interaction between salt treatment and temperature on the seeds’ GP, RL, and PL (

Table 2. Pillai’s trace MANOVA of interaction between salinity and temperature on germination performance of Z. mays L.

Pillai’s Trace	Value	F	Hypothesis df	Error df	Sig. *	Partial η2
Salinity	0.382	307.947	3.000	1492.000	<0.001	0.382
Temperature	0.436	138.753	6.000	2986.000	<0.001	0.218
Salinity × Temperature	0.151	40.575	6.000	2986.000	<0.001	0.075

df degrees of freedom, Sig. significance, * Statistically significant difference: p < 0.05.

). Several assumption tests were run before the MANOVA, as explained in Appendix A.

Results of the MANOVA in Table 2 present a significant effect of the independent variables (salinity and temperature) individually and of the interaction between them on the germination quality parameters. Based on this result, it can be concluded that the interaction between the salt treatment and the temperature significantly affects maize seeds’ germination qualities. The partial η² also shows that the interaction between the factors has a medium effect on the dependent variables compared to the large effect size of the factors individually. Therefore, the results made it sufficient to reject our first null hypothesis. Thus, follow-up ANOVA was carried out to determine the effect of the factors on the dependent variables individually (

Table 3. ANOVA of germination percentage, radicle length, and plumule length of Z. mays L. at different salinity treatments, temperatures, and combinations of the factors.

		Type III Sum of Squares	df	Mean Square	F	Sig. *	Partial η2
Salinity	Germination %	8568.480	1	8568.480	7.968	0.004	0.005
	Radicle length	5879.823	1	5879.823	745.233	<0.001	0.333
	Plumule length	271.305	1	271.305	212.022	<0.001	0.124
Temperature	Germination %	70,884.300	2	35,442.150	32.959	<0.001	0.042
	Radicle length	3347.884	2	1673.942	212.162	<0.001	0.221
	Plumule length	808.970	2	404.485	316.100	<0.001	0.297
Salinity * Temperature	Germination %	12,240.479	2	6120.239	5.691	0.003	0.008
	Radicle length	1161.793	2	580.896	73.625	<0.001	0.090
	Plumule length	16.119	2	8.060	6.299	0.002	0.008
Error	Germination %	1,606,547.641	1494	1075.333
	Radicle length	11,787.522	1494	7.890
	Plumule length	1911.736	1494	1.280

df degrees of freedom, Sig. significance, * Statistically significant difference: p < 0.05.

).

Table 3 shows a significant difference between the independent variable and their combination on each dependent variable, i.e., GP, RL, and PL. The combined effect between salinity and temperature shows statistically significant results for all three dependent variables, with probability values ranging between <0.01 and <0.001. The partial η² values also show temperature and the salt treatment greatly affected RL and PL. Meanwhile, the interaction between salinity and temperature had a larger effect on RL than on PL and GP. Therefore, based on this result, the interaction between salinity and temperature significantly affects maize seeds’ germination and the elongation of the radicle and plumule.

Games−Howell post hoc comparisons were made to evaluate pairwise differences between the temperatures and are summarized in

Table 4. Mean data ± standard deviation of germination and seedling growth traits of Z. mays L. for different treatments at different temperatures.

	Treatment	Temperature	Mean ± SD
Germination %	Control	15 °C	47.46 ± 39.7 Ba
		20 °C	54.29 ± 28.65 Ba
		35 °C	68.52 ± 22.63 Bb
	100 mM NaCl	15 °C	43.81 ± 39.93 Ba
		20 °C	55.86 ± 33.08 Bb
		35 °C	56.25 ± 29.19 Ab
Radicle length	Control	15 °C	1.99 ± 2.36 Ba
		20 °C	7.41 ± 5.21 Bb
		35 °C	6.52 ± 3.21 Bb
	100 mM NaCl	15 °C	0.44 ± 0.54 Aa
		20 °C	1.68 ± 1.87 Ab
		35 °C	1.91 ± 1.39 Ab
Plumule length	Control	15 °C	0.59 ± 0.69 Ba
		20 °C	3.06 ± 2.82 Bb
		35 °C	2.93 ± 1.75 Bb
	100 mM NaCl	15 °C	0.2 ± 0.31 Aa
		20 °C	1.07 ± 1.32 Ab
		35 °C	1.21 ± 0.94 Ab

Different letters indicate significant differences with p < 0.05, starting with a letter (A) or (a) showing the lowest mean value. Capital letters compare the differences between treatments, while lowercase letters compare the differences between temperatures at specific treatment conditions.

. The test revealed a significant pairwise difference between control and saline-treated seeds with p < 0.05 and p < 0.001. However, the test shows no significant difference in the GP at 15 °C and 20 °C between the control and the SS seeds, with p = 0.307 and p = 0.575, respectively, as illustrated in Figure 3. The result also shows that the GP, RL, and PL were not significantly different at 20 °C and 35 °C, whereas the dependent variables differed between 15 °C and the other two temperatures.

Furthermore, the root:shoot length ratio (R:S) and seed vigor index (SVI) were also measured and tested with two-way ANOVA. The data were assumed to be normal; the skewness and kurtosis of the ratio were 0.991 and 0.705, respectively, and those of the SVI were 1.251 and 0.926 [50]. However, the homogeneity was violated, influencing the decision to use the Games−Howell post hoc test. The results in Figure 5 show that the combination of SS and temperature also affects the R:S ratio and the SVI. Based on the results, the highest R:S ratio at 15 °C produced the highest ratio, while there was no significant difference in the ratio at 20 °C and 35 °C. The control had a higher ratio than the SS seeds, but both conditions produced a similar decreasing pattern as the temperature increased. Furthermore, Figure 5b shows SS significantly reduced maize SVI, especially at a temperature of 20 °C. The ANOVA results (

Table 5. ANOVA of root:shoot ratio and seed vigor index of Z. mays L. at different salinity treatments, temperatures, and combinations of the factors.

		Type III Sum of Squares	df	Mean Square	F	Sig. *	Partial η2
Salinity	Root: shoot	103.041	1	103.041	206.724	<0.001	0.358
	SVI	60,624,256.825	1	60,624,256.825	427.145	<0.001	0.535
Temperature	Root: shoot	76.610	2	38.305	76.849	<0.001	0.293
	SVI	46,702,743.493	2	23,351,371.747	164.529	<0.001	0.470
Salinity * × Temperature	Root: shoot	8.534	2	4.267	8.561	<0.001	0.044
	SVI	14,239,692.975	2	7,119,846.487	50.165	<0.001	0.213
Error	Root: shoot	184.923	371	0.498
	SVI	52,655,647.016	371	141,928.968

df degrees of freedom, Sig. significance, * Statistically significant difference: p < 0.05.

) indicate a significant effect between the combination of salt treatment and temperature on the R:S and the SVI with p < 0.001.

3.3. Comparison of Germination Performance between Varieties

To study the impact of different temperatures on the germination performance of 16 maize varieties exposed to salt stress, a two-way multivariate analysis of variance (MANOVA) was conducted. The significant differences in the GP, RL, and PL between 16 maize varieties at each temperature and the relation between the two independent variables, i.e., maize varieties and temperature, in influencing the dependent variables were tested. Several assumption tests were run before the MANOVA, as explained in Appendix A. Results of the MANOVA presented in

Table 6. Pillai’s trace MANOVA of interaction between variety and temperature on germination performance of Z. mays L.

	Value	F	Hypothesis df	Error df	Sig. *	Partial η2
Variety	0.467	72.904	6.000	1436.000	<0.001	0.233
Temperature	0.272	4.785	45.000	2157.000	<0.001	0.091
Temperature × Variety	0.211	1.817	90.000	2157.000	<0.001	0.070

df degrees of freedom, Sig. significance, * Statistically significant difference: p < 0.05.

show a significant main effect of the factors and the combined factors on all three dependent variables (GP, RL, and PL) with p values < 0.001. The variety factor yielded the largest effect size with a partial η² of 0.233. Based on these results, evidence was sufficient to reject the second null hypothesis. Thus, follow-up ANOVA was carried out (

Table 7. ANOVA of germination percentage, radicle and plumule growth of different Z. mays L. varieties, temperatures, and combination of the factors.

Source		Type III Sum of Squares	df	Mean Square	F	Sig. *	Partial η2
Variety	Germination %	31.938	15	2.129	11.301	<0.001	0.191
	Radicle length	117.213	15	7.814	7.405	<0.001	0.134
	Plumule length	78.116	15	5.208	5.209	<0.001	0.098
Temperature	Germination %	6.597	2	3.298	17.507	<0.001	0.046
	Radicle length	316.017	2	158.008	149.729	<0.001	0.294
	Germination %	304.026	2	152.013	152.063	<0.001	0.297
Temperature × Variety	Germination %	17.461	30	0.582	3.089	<0.001	0.114
	Radicle length	75.253	30	2.508	2.377	<0.001	0.090
	Plumule length	56.578	30	1.886	1.887	0.003	0.073
Error	Germination %	135.459	719	0.188
	Radicle length	758.756	719	1.055
	Plumule length	718.764	719	1.000

df degrees of freedom, Sig. significance, * Statistically significant difference: p < 0.05.

).

As shown in Table 7, the independent variables, i.e., variety, temperature, and interaction, significantly affect the GP, RL, and PL of maize seeds growing in saline solution. Based on the partial η², the maize variety showed a larger effect size on GP and RL than PL, whereas temperature had a larger effect size on RL and PL than GP. Finally, the interaction between temperature and variety had the largest effect size on GP and a medium effect size on RL and PL [51].

Finally, the Games−Howell post hoc test results revealed that all post hoc mean comparisons were statistically significant with p < 0.005 and p < 0.001. The post hoc test results and the impact of different temperatures on the germination performance of 16 maize varieties exposed to salinity stress are summarized in

Table 8. Mean data of germination and seedling growth traits for different Z. mays L. varieties in 100 mM NaCl solution at different temperatures.

	Germination Percentage			Radicle Length			Plumule Length
Var	15 °C	20 °C	35 °C	15 °C	20 °C	35 °C	15 °C	20 °C	35 °C
1	50.00 ± 42.60 Aa	87.50 ± 17.74 Bfg	65.63 ± 31.31 Abde	0.62 ± 0.67 Aa	2.94 ± 2.08 Bd	2.55 ± 1.91 Bbcde	0.28 ± 0.35 Aa	2.03 ± 1.79 Bc	1.44 ± 1.22 Bbcd
2	40.63 ± 43.87 Aa	75.00 ± 40.82 Bdefg	59.38 ± 29.17 ABbcde	0.44 ± 0.57 Aa	2.22 ± 1.92 Babcde	1.97 ± 1.26 Bbcde	0.22 ± 0.32 Aa	1.49 ± 1.68 Babc	1.10 ± 0.85 Babcd
3	45.83 ± 42.82 Aa	67.71 ± 31.90 Acdefg	55.21 ± 23.35 Abde	0.54 ± 0.60 Aa	2.40 ± 2.00 Bbcde	1.21 ± 0.54 Bbcde	0.24 ± 0.34 Aa	1.76 ± 1.88 Bbc	0.86 ± 0.54 Bbcd
4	34.38 ± 37.75 Aa	48.96 ± 26.85 Abcd	54.17 ± 20.64 Aabcd	0.29 ± 0.37 Aa	0.81 ± 0.48 Babcde	1.90 ± 1.30 Cbcde	0.15 ± 0.22 Aa	0.81 ± 0.97 Babc	1.45 ± 0.77 Cbcd
5	34.38 ± 36.75 Aa	48.96 ± 23.94 ABcde	72.92 ± 13.44 Bbde	0.29 ± 0.36 Aa	1.02 ± 1.12 Babcde	2.56 ± 1.36 Ccde	0.17 ± 0.27 Aa	0.64 ± 0.71 Aabc	1.76 ± 1.13 Bbcd
6	36.46 ± 36.12 Aa	47.92 ± 23.47 Acde	47.92 ± 29.74 Aabc	0.36 ± 0.44 Aa	1.17 ± 0.94 Babcde	1.62 ± 1.23 Babcde	0.11 ± 0.22 Aa	0.73 ± 0.80 Babc	1.13 ± 0.95 Babcd
7	35.42 ± 39.85 Aa	62.50 ± 31.91 Bdefg	53.12 ± 23.74 ABbd	0.37 ± 0.58 Aa	1.86 ± 2.10 Babcde	1.46 ± 0.63 Bbcd	0.13 ± 0.22 Aa	1.08 ± 1.34 Babc	1.06 ± 0.63 Bbcd
8	40.63 ± 44.71 Aa	59.38 ± 29.79 Acdef	61.46 ± 14.55 Abd	0.40 ± 0.51 Aa	1.43 ± 1.46 Babcde	1.85 ± 0.84 Bbcde	0.13 ± 0.21 Aa	1.13 ± 1.53 Babc	1.14 ± 0.68 Bbcd
9	42.71 ± 37.99 Ba	13.54 ± 16.35 Aa	30.21 ± 13.90 ABa	0.49 ± 0.56 Aa	0.33 ± 0.55 Aa	1.15 ± 0.89 Aab	0.22 ± 0.34 Aa	0.24 ± 0.42 Aa	0.78 ± 0.62 Aabc
10	54.17 ± 46.55 Aa	82.29 ± 23.94 Bfg	58.33 ± 36.00 ABefg	0.61 ± 0.71 Aa	3.30 ± 2.83 Bcd	2.53 ± 2.05 Bde	0.28 ± 0.39 Aa	1.52 ± 1.50 Babc	1.19 ± 1.12 Bbcd
11	29.17 ± 33.05 Aa	16.67 ± 18.26 Aab	34.37 ± 32.47 Aac	0.24 ± 0.40 Aa	0.54 ± 0.90 Aabe	1.38 ± 1.59 Aabc	0.08 ± 0.16 Aa	0.34 ± 0.68 ABab	0.86 ± 0.96 Bab
12	48.96 ± 36.24 ABa	26.04 ± 18.23 Aabc	64.58 ± 23.47 Bdef	0.41 ± 0.49 Aa	0.62 ± 0.47 Aabce	2.00 ± 0.88 ABcde	0.23 ± 0.35 Aa	0.49 ± 0.48 Aabc	1.54 ± 1.05 Bcd
13	46.87 ± 40.92 ABa	59.37 ± 27.87 Bcdefg	28.13 ± 23.35 ABa	0.47 ± 0.52 Aa	1.70 ± 1.59 Bbcde	1.02 ± 1.16 ABa	0.18 ± 0.26 Aa	1.02 ± 1.11 Babc	0.53 ± 0.68 ABa
14	54.17 ± 40.60 Aa	60.42 ± 27.81 Acdefg	68.75 ± 36.45 Bfg	0.60 ± 0.67 Aa	1.85 ± 1.77 Bbcde	2.77 ± 1.88 Cde	0.25 ± 0.35 Aa	1.11 ± 1.28 Babc	1.57 ± 1.25 Cd
15	46.87 ± 39.07 Aa	55.56 ± 26.48 Acdef	64.58 ± 29.11 Abde	0.37 ± 0.41 Aa	1.62 ± 1.79 Bbcde	1.95 ± 1.19 Bbcd	0.23 ± 0.34 Aa	0.97 ± 1.06 Babc	1.25 ± 0.80 Bbcd
16	60.42 ± 42.55 Aa	80.21 ± 18.48 ABfg	80.21 ± 20.38 Bfg	0.60 ± 0.67 Aa	2.77 ± 2.47 Bcd	2.81 ± 1.41 Be	0.32 ± 0.44 Aa	1.5 ± 1.50 Bbc	1.79 ± 1.03 Bd

Different letters indicate significant difference with p < 0.05, starting with a letter (A) or (a) showing the lowest mean value, and bold values are the highest values. Capital letters compare the difference between a variety at different temperatures, while the lowercase letters compare the performance between varieties at a particular temperature.

. Under 100 mM NaCl conditions, each variety showed the highest germination performance at different incubation temperatures. Variety 16 showed the highest GP at 15 °C compared to the other 15 varieties, but there was no significant difference with other varieties. V16 also showed the highest GP at 35 °C, which was significantly different from other varieties except V10 and V14. The highest GP was that of V1, of 87.50% at 20 °C, and it was not significantly different from V2, V3, V7, V10, V13, V14, and V16. Six varieties (V3, V4, V6, V8, V11, V15) were unaffected by temperature changes as there was no significant difference in GP at all three temperatures.

Furthermore, at 15 °C, there was no significant difference in RL and PL between varieties. V10 produced the longest radicle at 20 °C and was only significantly different from V9, V11, and V12. On the other hand, V16 produced the longest radicle at 35 °C and was significantly different from V7, V9, V11, V13, and V15. Besides that, 13 out of 16 varieties showed a significant difference in RL between 15 °C and 20 °C. Meanwhile, V4, V5, and V14 showed significantly higher RL at 35 °C compared to 20 °C, while RL of the other varieties was not significantly different at 20 °C and 35 °C. In addition, V1 showed the longest plumule length at 20 °C, with an average of 2.03 cm, but it was not significantly different from the 13 other varieties. Meanwhile, V16 produced the longest plumule at 35 °C and was only significantly different from V9, V11, and V13. Finally, all varieties showed no significant difference in PL between 20 °C and 35 °C, except V4, V5, V12, and V14.

Meanwhile, the differences in root:shoot ratio (R:S) and seed vigor index (SVI) between maize varieties were also analyzed at different temperatures. The MANOVA results show a significant difference between the combination of variety and temperature on the R:S ratio and the SVI; Pillai’s trace = 0.275, F(4, 742) = 29.56, p < 0.001, partial η² = 0.137. The result shows that the interaction between the two factors significantly affects the dependent variables. Thus, follow-up ANOVA was conducted after testing the normality and homogeneity assumptions. The data were assumed to be normal; skewness and kurtosis of the ratio were 0.819 and 0.545, respectively, and those of the SVI were 1.204 and 0.405 [50]. However, the homogeneity was violated, influencing the decision to use the Games−Howell post hoc test.

Table 9. ANOVA of germination percentage and radicle and plumule growth of different Z. mays L. varieties, temperatures, and combination of the factors.

		Type III Sum of Squares	df	Mean Square	F	Sig. *	Partial η2
Variety	Root: shoot	9.450	15	0.630	2.841	<0.001	0.237
	SVI	5,374,528.096	15	358,301.873	10.170	<0.001	0.527
Temperature	Root: shoot	13.092	2	6.546	29.515	<0.001	0.301
	SVI	4,075,822.817	2	2,037,911.409	57.845	<0.001	0.458
Variety × Temperature	Root: shoot	12.247	30	0.408	1.841	0.010	0.287
	SVI	4,381,965.942	30	146,065.531	4.146	<0.001	0.476
Error	Root: shoot	30.384	137	0.222
	SVI	4,826,616.448	137	35,230.777

df degrees of freedom, Sig. significance, * Statistically significant difference: p < 0.05.

shows the interaction between the factors affecting the R:S ratio and SVI with p values < 0.005 and <0.001, respectively. Partial η² shows that the interaction affects SVI more than the R:S ratio, while temperature had a larger effect than variety on the dependent variables.

The Games−Howell post hoc test was carried out to determine the significant difference in R:S ratio and SVI between the varieties and temperatures under 100 mM NaCl (

Table 10. Mean data of germination and seedling growth traits for different Z. mays L. varieties in 100 mM NaCl solution at different temperatures.

Var.	Root: Shoot			Seeds Vigor Index (SVI)
	15 °C	20 °C	35 °C	15 °C	20 °C	35 °C
1	1.86 ± 0.46 Aa	1.24 ± 0.10 Aabcd	1.28 ± 0.24 Aa	215.97 ± 33.77 Aa	1029.17 ± 114.48 Bc	350.07 ± 277.58 Aab
2	1.76 ± 0.26 Aa	1.26 ± 0.32 Aabcd	1.84 ± 0.53 Aa	181.39 ± 91.75 Aa	895 ± 122.34 Bbc	302.29 ± 275.58 Aab
3	2.01 ± 0.82 Aa	1.18 ± 0.22 Aabcd	1.22 ± 0.44 Aa	188.61 ± 51.09 Aa	863.82 ± 298.45 Babc	302.01 ± 207.52 ABab
4	1.71 ± 0.34 Ba	0.74 ± 0.35 Aab	1.04 ± 0.44 ABa	107.85 ± 46.93 Aa	231.53 ± 69.31 Aa	329.24 ± 254.28 Aab
5	1.52 ± 0.61 Aa	1.48 ± 0.71 Aabcd	1.19 ± 0.48 Aa	118.54 ± 34.89 Aa	259.86 ± 209.74 Aab	387.08 ± 137.33 Aab
6	2.42 ± 0.95 Aa	1.37 ± 0.28 Aabcd	1.37 ± 0.20 Aa	118.70 ± 39.26 Aa	258.68 ± 136.23 Aa	208.54 ± 275.03 Aab
7	2.35 ± 0.58 Aa	1.68 ± 0.31 Aabcd	1.37 ± 0.22 Aa	136.39 ± 104.22 Aa	714.31 ± 443.59 Aabc	218.82 ± 106.88 Aa
8	2.64 ± 0.61 Aa	1.06 ± 0.31 Aabcd	1.43 ± 0.28 Aa	169.44 ± 45.10 Aa	487.08 ± 322.77 Aabc	290.97 ± 100.12 Aa
9	1.96 ± 0.53 Aa	1.89 ± 0.83 Aabcd	0.91 ± 0.37 Aa	165.56 ± 110.04 Aa	83.06 ± 43.60 Aa	84.63 ± 68.35 Aa
10	1.93 ± 0.49 Aa	2.01 ± 0.33 Acd	1.70 ± 0.18 Aa	249.17 ± 62.22 Aa	1096.11 ± 175.8 Cc	629.17 ± 44.55 Bb
11	2.76 ± 0.54 Aa	1.27 ± 0.45 Babcd	1.73 ± 0.94 ABa	77.78 ± 84.91 Aa	119.81 ± 157.19 Aa	112.04 ± 53.87 Aa
12	1.51 ± 0.23 Aa	0.96 ± 0.11 Bac	1.08 ± 0.38 ABa	154.93 ± 80.42 Aa	91.11 ± 56.02 Aa	426.11 ± 136.49 Bb
13	2.33 ± 0.76 Aa	1.40 ± 0.09 Abd	2.06 ± 1.12 Aa	159.79 ± 19.89 Aa	471.53 ± 349.26 Aabc	16.18 ± 6.23 Ba
14	1.99 ± 0.33 Aa	1.39 ± 0.24 Aabcd	1.43 ± 0.37 Aa	223.82 ± 68.26 Aa	585.42 ± 393.14 Aabc	687.22 ± 249.97 Ab
15	1.28 ± 0.18 Aa	1.61 ± 0.34 Aabcd	1.63 ± 0.76 Aa	148.82 ± 66.31 Aa	333.26 ± 395 Aabc	229.93 ± 153.49 Aab
16	1.54 ± 0.27 Aa	1.65 ± 0.23 Aabcd	1.52 ± 0.31 Aa	257.08 ± 57.37 Aa	1029.24 ± 196.49 Bc	672.64 ± 203.61 Bb

Different letters indicate significant differences at p < 0.05, started with a letter (A) or (a), showing the lowest mean value, and highest values are bolded. Capital letters compare the difference of a variety at different temperatures while the lowercase letters compare the performance between varieties at a particular temperature.

). Table 10 shows that V10 produced the highest SVI at 20 °C, whereas V13 produced the lowest SVI at 35 °C. The comparison between varieties shows that at 15 °C there were no significant differences in the R:S ratio and the SVI between all 16 varieties and no significant differences in the R:S ratio between varieties at 35 °C. At 20 °C, the ratio of V10 was significantly different from that of V4, while V13 was significantly different from V12. V10 also produced the highest SVI at 20 °C, of 1096.11, and was significantly different from V4, V5, V6, V9, V11, and V12. Besides that, SVI of V10 was also significantly different from that of V7, V8, V9, V11, and V13 at 35 °C. V14 had the highest SVI at 35 °C but was not significantly different from V16 and V10. Furthermore, increases in temperature from 15 °C to 20 °C significantly decreased the R:S ratio of V4, V11, and V12, while significantly increasing the SVI of V1, V2, V3, V10, and V16. V10 was the only variety that had significantly different SVI between each temperature. At the same time, SVI of V12 was significantly improved at 35 °C, but SVI of V13 was negatively affected at 35 °C compared to the lower temperatures of 20 °C and 15 °C.

Finally, based on the five germination parameters studied in this trial, V1 (B1026/17), V10 (GK 154 x155), and V16 (Margitta) showed the highest tolerance to a combination of two abiotic stresses. In contrast, V9 (GK 131) and V13 (GK 150) were the most vulnerable to salt stress, especially at 20 °C and 35 °C. Meanwhile, all varieties showed significantly similar sensitivity to the low temperature of 15 °C, and while most varieties showed the highest growth at 20 °C, V4 (TK1083/18), V5 (TK 623/18), V12 (GK 154), and V14 (GK 144) showed the highest seedling growth at 35 °C.

4. Discussion

Abiotic stress tolerance is achieved by expressing multiple genes responsible for producing stress tolerance metabolites and regulatory proteins such as osmolytes, antioxidants, and ABA in the case of exposure to triggering stress levels. The level of activation of the responsible genes may be different between varieties, causing different degrees of stress tolerance [52]. It was discovered in barley that two varieties with higher tolerance towards combined drought and salinity stress accumulate a lower Na⁺:K⁺ ratio and lipid peroxidation, and higher Ca²⁺Mg²⁺ATPase activities and proline and water use efficiency, due to higher antioxidant activity compared to the susceptible variety [53]. A study of Bromus inermis showed that the optimum temperature of 20 °C alleviated the salt stress and improved the tolerance as the salt level increased during germination compared to lower and higher temperatures [54]. Meanwhile, a study of Arabidopsis thaliana proved that a combination of salt, heat, and mannitol stress-activated unique biosynthesis pathways is not a simple combination of individual stress responses. It was suggested that under combined stress, the plant might only activate the most effective gene as a defensive mechanism in a limited resource situation instead of triggering all genes responsible for individual stresses [55].

Based on our results, the temperature changes affect the germination qualities of maize seeds incubated in saline solution, indicating the role of temperature in maize salinity stress response. Combining 100 mM NaCl and a temperature of 35 °C decreased the GP and seedlings’ growth more severely than at high temperature stress alone. At a lower temperature of 15 °C, the difference in germination performances was insignificant with or without salt stress. The combination of the two factors also shows similar results in the germination of other plant species, including Sorghum bicolor, wheat, several medicinal plants, and three salt-resistant halophytes [30,38,56,57]. Temperature plays an important role in cell elongation and plant division, including during germination. High temperature disrupts the cell production, thus affecting the elongation of radicles and plumules [58,59,60]. It was stated that salinity could cause problems for crops in two ways: salt in soil solution decreases the availability of water to roots or seeds due to osmotic stress, while accumulated salt in plant cells can reach toxic levels in plant tissue [61,62]. A study showed maize germination speed reduced as salt concentration increased; NaCl concentration below 80 mM did not affect maize germination and seedling growth, while a concentration more than 320 mM caused root deformation [63].

In this study, we observed that salinity decreases seedling growth, especially at 20 °C and 35 °C. However, we also observed similar seedling growing patterns in both saline and non-saline conditions as the temperature changed (Figure 4). In a recent study, similar findings were observed in desert grass Lasiurus scindicus exposed to high temperature and salinity, which also decreased the level of chlorophyll and increased the level of proline in the seedlings [49]. A similar study of Sorghum bicolor showed the combination of both abiotic stresses was more damaging in germination and early seedling stages than at later stages, while also affecting the chlorophyll and proline content [38]. In rice crops, the combination of high temperature and salt stress immediately reduced the shoot’s fresh weight and dry matter 5 days after exposure, followed by irreversible leaf damage and desiccation as the exposure continued [37]. Besides that, similar to our results, several studies showed that a temperature of 15 °C delays the emergence of maize seeds in temperate and tropical maize varieties [17,27]. In another recent study, it was observed that young maize seedlings’ growth was more affected by a combination of salinity and 14 °C than at a higher temperature of 24 °C [31]. However, in our study, the combination of cold temperature and high salinity only significantly affected the R:S ratio but did not affect the GP and seedling growth.

Based on our results, salt stress also reduced the root:shoot (R:S) length ratio compared to the control condition. The significant reduction in radicle and plumule growth in saline conditions may have influenced the ratio generated. However, the R:S ratio was higher at 15 °C compared to 20 °C and 35 °C (Figure 5a). A higher R:S ratio shows that the seedling produced a higher proportion of root than shoot structures [64]. A study showed that environmental factors and seed size may regulate the R:S ratio in the early seedling stage [64]. Previous studies in wheat show salinity stress increases the R:S length ratio of 19 wheat genotypes tested [65]. The shorter plumule formation at 15 °C compared to the other temperatures may influence the higher R:S ratio at 15 °C. Studies in maize, sorghum, and elephant grass revealed that cold temperature affects plant shoot growth more than that of the roots [31,38]. Besides that, the high temperature can also reduce the R:S ratio by decreasing the root length, as observed in several crop plants such as wheat, sorghum, and lupin [59,60,66,67]. Furthermore, based on our analysis, salinity and non-favorable temperatures also decreased the maize seed vigor index (SVI) (Figure 5b). SVI illustrates seeds’ activity level, germination performance, and seedling development in a particular condition [68]. Our results show that the highest SVI was observed at 20 °C for both saline and non-saline conditions. As the SVI is influenced by the germination percentage and the seedling length, reducing these parameters during the stress period caused the number to plummet. In maize, it was discovered that the presence of 150 mM salt stress could reduce the SVI by 63% [69]. A similar observation was found in various main crops, including sorghum and rice [70,71]. Besides that, in maize and cotton, below-optimum temperatures also reduced the seed vigor, resulting in poor germination activities [72,73].

Lastly, maize varietal performance under various abiotic stresses at different growth stages has been unveiled by many previous studies [27,45,74,75]. Our previous study discovered that grain maize is more susceptible to salt stress and a combination of salt and temperature stress than sweet maize [76]. In this study, it was observed that salt tolerance was present in parent and single cross hybrids. It was also observed that several hybrids did not perform well under exposure to a combination of salt and heat stress. Genetic variability within the same species allows multiple reactions to stress factors due to different signal-transmitting mechanisms that would enable appropriate physiological and biochemical responses to tolerate stresses [77,78]. Stress tolerance between varieties may be triggered by genetic variation within species caused by various factors, including germplasm variety, induced mutation, genetic engineering, or intraspecific hybridization [79]. A study of 18 maize varieties shows that variation in genotypic component was the biggest contributor to the total variation in reaction against salt stress between varieties [45]. For example, some maize varieties tolerate high soil temperatures, while others tolerate low temperatures to allow the germination process to start [27]. This study can be further improved through soil pot trials with a wider range of temperature differences. A pot trial allows the seeds to fully come into contact with soil particles and allows better seedling growth observations. Using a Petri dish for a temperature above 35 °C increases the evaporation rate of the solution to the top cover of the dish, which may prevent complete seed imbibition.

5. Conclusions

More comprehensive studies on how combined abiotic stresses affect crop growth and development should be conducted by researchers, as simultaneous stresses are more likely to happen in the field than individual stresses. Our study shows that a combination of temperature and salinity stresses was more detrimental to the germination performance of maize seeds than individual stresses alone. The combination of 35 °C and 100 mM salinity reduced the germination performance and maize seeds’ vigor more than at 35 °C and 100 mM individually. The germination percentage was the most affected by the temperature increase from 20 °C to 35 °C compared to the radicle and plumule growth. This study also identified the varieties that were more tolerant and susceptible to combined salt and temperature stresses regarding germination performance. Even though we found all varieties performed significantly similarly at 15 °C with the presence of salt stress, V1 (B1026/17), V10 (GK 154x 155), and V16 (Margitta) showed the highest germination activities at both 20 °C and 35 °C. More interestingly, V4 (TK1083/18), V5 (TK 623/18), V12 (GK 154), and V14 (GK 144) showed higher germination activities at 35 °C than at 20 °C, showing the highest tolerance towards combined stresses compared to other varieties. This information is important for further breeding programs and for farmers to choose the best planting material depending on their land conditions. The genetic activities of the tolerance varieties should be compared with the susceptible varieties, such as V9 (GK131) and V13 (GK150), to determine the responsible genes controlling the response of the seed to the combination of temperature and salinity stresses. Researchers should also treat combined abiotic stress as unique, not just a simple fusion of two or more stressors. This research should be further conducted in pot or field trials to determine the effect of this stress on the further development and reproductive yield of the plant.