Priming with Humic Acid to Reverse Ageing Damage in Soybean [Glycine max (L.) Merrill.] Seeds

Abstract

Soybean seed vigour declines with increase in storage duration, due to ageing, which can be alleviated through seed priming. The objective of this study was to investigate the effects of Humic acid (HA) priming on germination, vigour and seedling performance under laboratory and greenhouse conditions with two soil moisture level [50% and 80% field capacity (FC)]. Seeds stored for 12 months having 60% germination were primed either with 0.2 g/L HA solution or distilled water (hydro-primed) at 25 °C for 1, 3, 5 and 7 h. Non-primed dry seeds were used as control, giving nine treatment combinations. Various germination traits [mean germination time (MGT), final germination percentage (FG%), germination rate index (GRI), seedling emergence percentage (SEP)], mean emergence time (MET), seedling quality traits [seedling vigor index (SVI), shoot length, root length, root volume], antioxidant enzyme activities [catalase (CAT), peroxidase (POD)], lipid peroxidation [malondialdehyde (MDA)] and electrical conductivity (EC) were determined. A germination test in the laboratory was conducted as single factor (nine priming treatments), while the greenhouse experiment was conducted as two factors [2 soil moisture level (50 and 80% FC) and 9 priming treatments]. The results indicated that seeds primed with HA for 5 h was able to reinstate the CAT activities (25%), POD activities (50%) and reduced EC (51%) and MDA content (40%) compared with non-primed seeds, reduced the MET (from 4.3 to 3.5 days), increased FG% (from 62 to 71%), GRI (15.6–21.1) and SEP (from 35 to 54%) and (from 60 to 72%) at 50% FC level and 80% FC level, respectively. A strong negative correlation (r = −0.80 **) was found between MDA content and GRI, while CAT and POD activities had positive correlation with GRI r= 0.67 ** and r = 0.56 **, respectively. Thus, priming with 0.2 g/L HA for 5 h improved the vigour of minimally deteriorated soybean seeds resulting in increased emergence with more uniform field establishment.

1. Introduction

Seed ageing is considered as one of the primary consequences of seed deterioration during storage. De Vitis et al. [1] reported that seed deterioration is an inevitable process, even under proper storage conditions. Thus, all seeds undergo deterioration during storage, differing in rate depending on initial seed quality [2] and storage environment [3,4]. In third world countries, tropical in nature, the deterioration is enhanced when seeds are stored under ambient conditions. In general, decline in germination percentage occurs more quickly in soybean seeds during storage compared with other grain crops, mainly due to high oil content, physiological fragility and thin seed coat [5,6]. Several studies have found that lipid peroxidation [4], degradation of enzyme activities [7], disruption of cellular membrane integrity [8] and damage to genetic materials as the foremost roots of seed ageing [9]. In addition, seeds with low vigour may not perform well and result in delayed germination and low seedling quality especially under abiotic stress conditions. Thus, any reliable technique that can improve germination and uniformity of seedling stand of aged soybean seeds, especially under unfavourable field conditions such as water stress, will be beneficial at the time of field planting.

Therefore, pre-sowing seed enhancement treatments play a vital role in instating repair mechanism of aged seeds (up to some extent) by regulating metabolic processes in the early phases of germination [10,11]. Seed priming, a popular pre-sowing seed enhancement technique, has shown that primed seeds had increased germination percentage, uniformity and speed of germination in a wide range of crop species [12]. According to Mohamed et al. [13], priming leads to repair of aged seeds by resumption of metabolic activity to restore cellular integrity through improvement of enzyme activities and protein synthesis, repaired cell membrane, increased antioxidant defense mechanism which enhanced the seed and seedling performance compared with non-primed seeds.

During priming, seeds are hydrated to a certain point where the germination progresses to a stage prior to radical emergence. In other words, metabolic activities relating to germination are allowed to occur during seed priming due to the imbibition process while radical development is avoided. Priming success is affected by various factors such as priming duration, temperature, initial seed quality and species [14]. Several seed priming techniques are used currently, such as water (hydro-priming), osmotic solutions (osmo-priming), inorganic salts (halo-priming), matric materials (matric-priming) and various natural extracts (live or decomposition materials from plant or animal parts) to enhance seed and seedling vigour. In previous studies, various solutions were suggested as priming agents for soybean such as polyethylene glycol (PEG) [15], salicylic acid (SA) [16], potassium nitrate (KNO₃), potassium chloride (KCl) and ascorbic acid (ASA) [17]. Although the above-mentioned chemicals have shown a positive effect, concerns of its use on the environment and the search for user-friendly seed priming agent for sustainability is on-going. Thus, environmentally friendly additives for seed priming are becoming a trend.

Humic acid (HA) is one of the environmentally friendly compounds used in seed priming. The HA-primed seeds were reported to enhance germination rate in chilli [18], sesame [19], wheat [20,21] and pea [22], with varying concentrations of HA and soaking time requirement for different crops. Asgharipour and Rafiei [23] reported that barley seeds primed with 0.75 g/L HA for 12 h increased seedling vigour index (SVI) compared with non-primed seeds, while Souguri and Hannachi reported significant positive results in sesame seeds primed with 1 g/L HA for 7 h [19]. An hour of soaking with 2.5 g/L HA increased germination percentage, shoot and root growth compared with non-primed seeds of Raphanus sativus [24]. These results suggest that the success of priming with HA depend on the concentration and duration of soaking which is species specific. To date, there is no report on the use of HA as priming agent for soybean, particularly on the repair mechanisms based on the synthesis of enzymes and reduction of peroxidation activities.

Based on the above, we aimed to investigate the potential of HA to instate repair via enhancement of antioxidant defense mechanisms and reduction of peroxidation activities of stored soybean seeds, therefore providing a good stand establishment even under stress conditions, for minimally aged soybean seeds.

2. Materials and Methods

2.1. Site Description and Planting Material

The laboratory test was conducted at the seed technology laboratory while the soil tray studies were carried out at the greenhouse at Field 15, Faculty of Agriculture, Universiti Putra Malaysia. Soybean seed lot (variety PB-1), with an initial germination of 92%, moisture content of 9.0%, electrical conductivity of 27.5 µScm⁻¹ g⁻¹, catalase activity of 21.4 µmol/min/mg FW, peroxidase activity of 83.7 nmol/min/mg FW and malondialdehyde content of 3.4 µmol/g FW, was stored under ambient condition (average temperature and relative humidity, 28.8 °C and 70.6% respectively for 12 months (August 2019–August 2020). After 12 months of storage, the seed lot germination had declined to 60% and MC had increased to 9.4%. The study on priming with HA utilized the seed lot that was stored for 12 months as mentioned above to represent minimally aged seeds. The seeds were surface sterilised with 5% sodium hypochlorite for 1 min and later rinsed three times with sterile distilled water to minimize contaminations during the priming process.

2.2. Seed Priming

The priming treatments were selected based on a series of preliminary studies on imbibition with HA concentrations ranging from 0.05 g/L to 20 g/L) and priming duration from 1–10 h to obtain the effective range. Based on the preliminary results, 0.2 g/L was selected as the concentration of HA and priming time was narrowed down (1 h to 7 h) which was the maximum time prior to radicle protrusion. Accordingly, this priming study was conducted using autoclaved distilled water/hydro priming (HP) and using 0.2 g/L HA solution (priming with natural extract) at 25 °C for 1, 3, 5 and 7 h, under dark conditions. Seeds were immersed in distilled water/HA solution with the ratio of 1 g seeds per 5 mL priming solution. Treated seeds were dried to their original moisture content at room temperature (26 ± 2°C) for 72 h. Non primed (NP) seeds were used as the control. Upon priming followed by drying, a sample of seeds from each treatment were subjected to standard germination in laboratory and germination in greenhouse with two soil moisture level (50% FC and 80% FC). In addition, a sample of seeds were kept for biochemical analysis (antioxidant enzyme activity and malondialdehyde (MDA)) in deep freezer at −80 °C until the analysis was performed.

2.3. Experimental Design, Treatment and Germination Test

Nine treatments (

Table 1. Priming agent and the soaking duration for each treatment.

Treatments ID	Priming Agent/Duration
Control	non-primed seeds
HA-1 h	0.2 g/L HA (1 h)
HA-3 h	0.2 g/L HA (3 h)
HA-5 h	0.2 g/L HA (5 h)
HA-7 h	0.2 g/L HA (7 h)
HP-1 h	distill water (1 h)
HP-3 h	distill water (3 h)
HP-5 h	distill water (5 h)
HP-7 h	distill water (7 h)

) were arranged in a complete randomised design (CRD) for the germination test in the laboratory.

The germination test was conducted according to ISTA [25] with four replications of 100 seeds for each treatment at room temperature (26 ± 2 °C) using sterilised sand. Germination was counted daily for seven consecutive days. On the seventh day, the number of normal seedlings was counted and expressed as final germination percentage (FG%) and the daily germination count data were used to calculate the germination index (GI), the germination rate index (GRI), mean germination time (MGT), the coefficient velocity of germination (CVG) and seedling vigour index (SVI) using the formulas in

Table 2. Formulas for the calculation of germination related traits.

Parameters	Formula	References
Final germination % (FG%)	$=\frac{\mathrm{Number} \mathrm{of} \mathrm{normal} \mathrm{seedling}}{\mathrm{Number} \mathrm{of} \mathrm{seeds} \mathrm{used}}\times 100$	[25]
Germination index (GI)	$=\left(7\times \mathrm{n}1\right)+\left(6\times \mathrm{n}2\right)+\dots +\left(1\times \mathrm{n}7\right)$ Where n1, n2, and … are the number of germinated seeds on the first day, second day, and other day and numbers 7, 6, and … are respectively weight imposed on the number of seeds germinated on the first day, second day and other days.	[26]
Germination rate index (GRI)	$=\left(\frac{\mathrm{G}1}{1}+\frac{\mathrm{G}2}{2}+\dots .+\frac{\mathrm{G}7}{7}\right)$ Where G1 is the germination percentage on day 1, G2 is the germination percentage on day 2, and so on.	[27]
Mean germination time (MGT)	$=\frac{{\displaystyle \sum }\mathrm{f} \mathrm{x}}{{\displaystyle \sum }\mathrm{f}}$ Where “f” is the number of seeds germinated on day x.	[27]
Coefficient velocity of germination (CVG)	$=\left(\frac{\mathrm{N}1+\mathrm{N}2+\dots .+\mathrm{N}7}{\mathrm{N}1\mathrm{T}1+\mathrm{N}2\mathrm{T}2+\dots .+\mathrm{N}7\mathrm{T}7}\right)\times 100$ Where N is the number of seeds germinated every day and T is the number of days from seeding corresponding to N.	[27]
Seedling vigour index (SVI)	$=\mathrm{Seedling} \mathrm{length} \left(\mathrm{cm}\right) \times \mathrm{Germinaton} \%$	[28]

.

2.4. Experiment Design and Treatment for Greenhouse Seedling Emergence Test

The experiments were laid out in complete randomized design (CRD) with split-plot arrangement comprising two soil moisture level (Ideal (ID) = 80% field capacity and moderate drought (MD) = 50% field capacity) as main plots. The treatments as in Table 1 were assigned as sub-plots and randomized within the main plots. Each treatment was replicated four times.

About 7.5 kg of air-dried topsoil obtained from Field 10, Universiti Putra Malaysia (electrical conductivity of 221.0 µS cm⁻¹, pH 5.6) was added into 50 cm × 40 cm × 7 cm trays. The soil field capacity and dry soil moisture content were 39.3% and 7.3%, respectively. Thereafter, soil moisture content was adjusted to 50% FC (19.7%) and 80% FC (31.4%) and monitored daily using a digital soil moisture meter. The soil moisture content was maintained at the above-mentioned levels throughout the experiment.

2.5. Seedling Emergence in the Greenhouse

Seedling emergence test in the greenhouse was carried out in the moisture adjusted tray. Around 2.0 cm sowing depth with 1.5 cm and 5 cm row spacing within and between the seedlings were used, respectively. Seedling emergence percentage (SEP) was counted daily until the number remained constant and expressed as a percentage of emerged seedlings. The seeds were monitored for (10 days) to observe the emergence of new seedlings. The emerged seedlings (with the cotyledons completely above ground level) were counted daily to obtain the cumulative value. All the seedling emergence traits (SEP, MET, speed of emergence index (SEI) and speed of emergence coefficient (SEC) were calculated using formulas as described in

Table 3. Formulas for the calculation of seedling emergence related traits.

Parameters	Formula	References
Seedling emergence percentage (SEP)	$=\frac{\mathrm{Number} \mathrm{of} \mathrm{normal} \mathrm{seedling}}{\mathrm{Number} \mathrm{of} \mathrm{seeds} \mathrm{used}}\times 100$	[29]
Mean emergence time (MET)	$=\frac{{\displaystyle \sum }\mathrm{f}\mathrm{x}}{{\displaystyle \sum }\mathrm{f}}$ Where “f” is the number of seeds emerged on day x.	[30]
Speed of emergence index (SEI)	$=\left(\frac{\mathrm{E}1}{1}+\frac{\mathrm{E}2}{2}+\dots .+\frac{\mathrm{E}10}{10}\right)$ Where E1 is the number of emerged seeds on day 1, E2 is the number of emerged seeds on day 2, and so on.	[31]
Speed of emergence Coefficient velocity of germination (CVG)	$=\left(\frac{\mathrm{E}1+\mathrm{E}2+\dots .+\mathrm{E}10}{\mathrm{E}1\mathrm{T}1+\mathrm{E}2\mathrm{T}2+\dots .+\mathrm{E}10\mathrm{T}10}\right)\times 100$ Where E is the number of seeds emerged every day and T is the number of days from seeding corresponding to E.	[32]

.

2.6. Root Characteristics

Roots from three individual seedlings having consistent growth were taken from each experimental unit and examined using a root scanner (LA1600+- scanner Canada). The WinRHIZO root analytic program was used to analyse the scanned root system. Root length (length of the primary root of a seedling), total root length (sum of the length of primary and secondary roots of a seedling), average root diameter, total root volume and number of root tips were obtained.

2.7. Seedling Characteristics

Five normal seedlings were selected randomly from each treatment and replication to determine the shoot length at the final germination count, 7th day after sowing for laboratory level gemination test and 10 days after sowing for soil tray experiment under greenhouse. The seedlings were separated carefully from the sand germination boxes/soil tray. The shoot length (SHL) was measured from the base of the primary leaf to the base of hypocotyl with the help of a ruler and the average length of seedling was expressed in centimeters (cm). Seedling root length (RL) was measured from the tip of the primary root to the base of hypocotyl with the help of a ruler and average root length was expressed in centimeters (cm). The total seedling length (SL) was calculated as the sum of SHL and RL The seedling dry matter determination was done according to the method described by Ludwing et al. [9].

2.8. Electrical Conductivity

Electrical conductivity was measured with four replications of 25 seeds per treatment, weighed on a three decimal analytical balance and soaked in a beaker containing 75 mL distilled water for 24 h at 20 °C [33]. Reading was made using a digitised conductivity meter (labCHEM-CP Australia), and the results were expressed in µS cm⁻¹g⁻¹.

2.9. Antioxidant Enzyme Analysis

A spectrophotometer (Shimadzu UV-3150 UV-VIS Near IR, Japan) was used to detect the antioxidant enzyme activities [catalase (CAT) and peroxidase (POD)]. Dry seeds weighing 0.15 g were homogenised with 1.5 mL of 100 mM potassium phosphate buffer (pH 7) using cold mortar and pestle. The extracted sample was centrifuged at 13,500 rpm for 20 min at 4 °C. The supernatant was used to determine the CAT and POD activities. Four replications were tested for each treatment. Catalase (CAT) activity was calculated according to the method described by Aebi [34] and activity was expressed per milligram of extractable fresh weight (µmol/min/mg/FW), while Guaiacol peroxidase (POD) activity was measured as described by Maehly, [35] and expressed per milligram of extractable fresh tissue (nmol/min/mg/FW).

2.10. Malondialdehyde (MDA) Assay

The amount of lipid peroxidation was quantified by malondialdehyde content using method described by Stewart and Bewley [36]. A total of 0.15 g of seeds was homogenized in 1.5 mL of distilled water. The extracted samples were centrifuged 10,000 g for 15 min. The reaction mixtures were prepared by adding 1 mL of extracted sample and 2 mL of 0.5% thiobarbituric acid (TBA) in 20% trichloroacetic acid (TCA) solution and the samples were incubated at 95 °C in constant temperature water bath for 30 min. After incubation, the reaction tubes were immediately cooled in an ice tray. The solution with 1 mL of distilled water and 2 mL of TBA/TCA was used as a blank. Absorbance was read at 450, 532 and 600 nm using a micro-plate spectrophotometer (Thermo Scientific MULTISKAN GO Singapore). MDA content in treatments were recorded with four biological replications and two technical replications.

2.11. Statistical Analysis

The data from the laboratory trial were subjected to one-way analysis of variance (ANOVA). Soil tray experiment under greenhouse was subjected to two-way analysis of variance. Statistical analysis was carried out using SAS 9.4 software (SAS institute, Cary NC, USA). The least significant difference (LSD, 5% level) was used to measure statistical differences between treatment means. Furthermore, correlation analysis was performed among the studied parameters.

3. Results

3.1. Effects of Priming on Standard Germination under Laboratory Conditions

3.1.1. Germination Traits

The priming treatments had a significant effect on all the germination traits tested, final germination percentage (FG%), germination index (GI), germination rate index (GRI), mean germination time (MGT) and coefficient velocity of germination (CVG) at (p < 0.05 (Figure 1 and

Table 4. Effect of the priming treatments on germination related traits in soybean.

Treat.	FG%	MGT (day)	GI	GRI	CVG (%/day)
Control	62 ± 1.2 b	4.3 ± 0.2 a	232 ± 12.8 c	15.6 ± 0.7 b	23.6 ± 0.9 d
HA 1 h	58 ± 2.9 b	3.8 ± 0.2 bc	244 ± 11.2 bc	16.2 ± 0.6 b	26.6 ± 1.1 abc
HA 3 h	63 ± 1.0 b	3.7 ± 0.1 c	268± 2.8 b	17.8 ± 0.2 b	26.8 ± 0.6 abc
HA 5 h	71 ± 1.0 a	3.5 ± 0.1 c	320 ± 6.5 a	21.1 ± 0.4 a	28.7 ± 0.9 a
HA 7 h	60 ± 3.7 b	4.1 ± 0.1 ab	234 ± 12.5 bc	16.0 ± 0.9 b	24.6 ± 0.9 cd
HP 1 h	42 ± 2.6 c	4.1 ± 0.1 ab	164± 8.3 d	10.9 ± 0.6 c	24.5 ± 0.3 cd
HP 3 h	59 ± 3.0 b	3.5 ± 0.1 c	264 ± 16.1 bc	17.4 ± 1.0 b	28.3 ± 0.4 ab
HP 5 h	56 ± 3.7 b	3.8 ± 0.1 bc	234 ± 15.5 bc	15.5 ± 1.0 b	26.3 ± 0.8 bc
HP 7 h	41 ± 3.4 c	3.7 ± 0.1 c	175 ± 16.8 d	11.6 ± 1.1 c	26.8 ± 1.0 abc
LSD	7.7	0.3	35.5	2.3	2.4
CV %	9.4	6.1	10.3	10.0	6.2

FG% = final germination percentage, MGT = mean germination time, GI = germination index, GRI = germination rate index, CVG = coefficient velocity of germination. Means (±SE) values with different letters indicate significant difference at p < 0.05 level using LSD.

).

The highest FG% (71%) was recorded for treatment with 0.2 g/L HA for 5 h priming, 14.5% higher as compared to the control (62%). All other priming duration did not increase germination significantly (58 to 63%) compared with control. Both hydro priming (3 h, 5 h and 7 h) and HA priming (1 h, 3 h and 5 h) reduced the MGT and increased CVG compared to the non-primed seeds. Furthermore, seeds primed with 0.2 g/L HA for 5 h had increased the GI and GRI by 37.9% and 35.3%, respectively compared with the control (Table 4). Hence, an increase in vigour was clearly demonstrated despite the relatively low increase in final germination percentage.

3.1.2. Seedling Quality Traits

Seedling quality traits including shoot length (SHL), seedling length (SL), root dry weight (RODW), shoot root ratio on dry weight basis (SH: RO) and seedling vigour index (SVI) were affected significantly by priming treatments (

Table 5. Effect of priming treatments on seedling quality traits on the 7th day after sowing soybean seeds.

Treat.	SHL (cm)	SL (cm)	RODW (g Seedling−1)	SH:RO (Ratio)	SVI (Nos)
Control	10.1 ± 0.5 cd	15.6 ± 0.8 d	0.006 ± 0.000 e	11.6 ± 0.6 a	967.0 ± 44.5 de
HA1 h	9.1 ± 0.7 d	13.5 ± 0.7 d	0.009 ± 0.002 cde	8.0 ± 1.8 bcd	789.5 ± 73.5 ef
HA3 h	14.1 ± 0.8 ab	21.0 ± 0.8 b	0.01 ± 0.000 c	7.1 ± 0.6 cd	1323.9 ± 51.5 b
HA5 h	15.2 ± 0.6 a	23.6 ± 0.7 a	0.016 ± 0.001 a	5.2 ± 0.7 d	1677.4 ± 65.2 a
HA7 h	13.1 ± 0.5 ab	19.9 ± 0.5 bc	0.009 ± 0.000 cd	5.7 ± 0.4 cd	1197.5 ± 88.7 bc
HP1 h	9.5 ± 0.7 d	15.2 ± 0.5 d	0.007 ± 0.000 de	10.7 ± 1.6 ab	634.6 ± 34.0 f
HP3 h	12.4 ± 0.6 b	18.8 ± 0.7 bc	0.014 ± 0.000 ab	5.2 ± 0.6 d	1111.7 ± 82.0 cd
HP5 h	12.0 ± 1.1 bc	18.4 ± 1.0 c	0.011 ± 0.001 bc	6.7 ± 1.1 cd	1025.7 ± 72.9 cd
HP7 h	9.0 ± 1.0 d	15.7 ± 1.2 d	0.009 ± 0.002 cde	8.8 ± 1.7 abc	645.1 ± 67.4 f
LSD	2.2	2.3	0.003	3.3	193.2
CV	12.8	8.7	21	29.3	12.8

SHL—shoot length, SL—seedling length, RODW—root dry weight SH:RO—shoot root ratio (dry weight basis) SVI—seedling vigour index. Means (±SE) values marked with different letters indicate significant difference at p < 0.05 level of probability using LSD.

).

The maximum value (15.2 cm) seedling shoot length for recorded for soybean seeds primed for 5 h with HA (50% higher than control). There was a positive correlation between HA priming duration with seedling dry weight for the first 5 h, and the value declined thereafter. The SH:RO showed a decreasing trend with an increase in HA priming time from 1 h to 5 h; however, the value increased with further priming duration. In hydro-priming, SH: RO increased with increase in priming duration after 3 h. The significant increment of SVI was only recorded in HA priming with 3 h, 5 h and 7 h compared to control.

Furthermore, the results of analysis of variance revealed that the effect of priming on root length (RL), total root length (TRL), average diameter of root (ADI), total root volume (TRV) and number of root tips (NRT) at p < 5% was significant on the 7th day after sowing. The maximum root length (8.5 cm) of seedlings was obtained at HA-5 h priming, and it showed a 54.5% increment compared with control (5.5 cm) (

Table 6. Root morphological characteristics of the soybean seedlings on the 7th day after sowing.

Treat.	RL (cm)	TRL (cm)	ADI (mm)	TRV (cm3)	NRT (nos)
Control	5.5 ± 0.5 d	30.2 ± 1.9 f	0.62 ± 0.05 bc	0.08 ± 0.00 e	29.6 ± 3.6 f
HA1 h	4.4 ± 0.2 e	33.3 ± 5.2 ef	0.63 ± 0.02 bc	0.1 ± 0.02 de	41.3 ± 8.2 def
HA3 h	6.9 ± 0.5 b	51.8 ± 2.5 bc	0.65 ± 0.02 abc	0.17 ± 0.00 bc	63.0 ± 6.7 bc
HA5 h	8.5 ± 0.2 a	69.0 ± 3.5 a	0.71 ± 0.04 a	0.28 ± 0.03 a	83.3 ± 2.2 a
HA7 h	6.8 ± 0.3 b	57.7 ± 3.4 b	0.59 ± 0.02 bc	0.16 ± 0.02 bc	72.9 ± 6.6 ab
HP1 h	5.7 ± 0.3 cd	31.6 ± 1.0 f	0.57 ± 0.03 c	0.08 ± 0.01 e	36.3 ± 1.5 ef
HP3 h	6.4 ± 0.3 bcd	59.6 ± 3.1 ab	0.67 ± 0.03 ab	0.21 ± 0.02 b	75.2 ± 6.6 ab
HP5 h	6.4 ± 0.5 bcd	46.5 ± 4.2 cd	0.67 ± 0.01 ab	0.16 ± 0.02 c	56.0 ± 3.9 ab
HP7 h	6.7 ± 0.3 bc	41.7 ± 2.8 de	0.66 ± 0.01 ab	0.14 ± 0.01 cd	47.9 ± 1.2 de
LSD	1.0	9.6	0.08	0.04	14.9
CV	11.3	14.1	8.4	20.2	18.3

RL—Root length, TRL—Total root length, ADA—Average diameter of root, TRV—Total root volume, TRN—Total root number. Means (±SE) values marked with different letters indicate significant difference at p < 0.05 level of probability using LSD.

).

The maximum TRL (69.0 cm) was obtained by the HA-5 h priming. In contrast, minimum TRL was recorded in control, HA-1 h and HP-1 h treatments, ranging from 30.2 cm to 33.3 cm with no significant difference among these treatments. The TRL increased corresponding to increment in HA priming duration from 1 h to 5 h; however, further soaking reduced the values. In hydro priming, TRL decreases with a further increase in priming duration beyond 3 h. A similar trend was observed in both HA priming and HP in TRV and NRT. In terms of TRL, ADI and NRT, no significant difference was recorded for both HA-5 h priming and HP-3 h.

3.2. Effects of Priming and Soil Moisture Level on Seedling Emergence under Green House Conditions

3.2.1. Seedling Emergence Traits

According to the analysis of variance, a significant difference was recorded among the priming treatments, soil moisture level and its interaction on seedling emergence percentage (SEP), speed of emergence coefficient (SEC) and speed of emergence index (SEI), while there was no interaction observed for mean emergence time (MET). However, significant differences of main factor were detected in MET. All the tested quality in the green house experiment was significantly lower in drought conditions (50% FC) compared to the ideal condition (80% FC), across all the seed treatments. Among the priming treatments, HA 5 h priming recorded the highest performance in terms of SEP, SEC and SEI within the soil moisture level (Figure 2 and Figure 3).

Humic acid (0.2 g/L) priming for 5 h showed higher increment (54.4%) in SEP compared with control in water stress condition (50% FC level) while only 20% increment was observed under an ideal moisture content level (80% FC level), which clearly shows the impact of priming under stress conditions. Other priming treatments had decreased SEP under 80% FC conditions compared to the control. There was no significant difference in SEP among control, HP priming (3 h, 5 h and 7 h) under 50% FC condition. A similar trend was observed in SEI. Furthermore, the 0.2 g/L HA at 5 h of priming increased the SEC by 24.1% compared with the control under 80% FC condition. There was no significant difference on SEC among control, HA priming (3 h, 5 h and 7 h) and HP (1 h, 3 h) under 50% FC condition and ranged from 16.2–17.5. Irrespective of soil moisture condition, HA 5 h priming had reduced MET by 12.2% compared with the control.

3.2.2. Seedling Quality Traits

Significant differences were observed among the priming treatments, soil moisture level and its interaction on total seedling length (TSL), and root dry weight (RDW), while there were no interactions observed for shoot dry weight (SHDW) and seedling dry weight (SDW), only the main factors were significantly different at p < 0.05. In the water stressed condition (50% FC), 0.2 g/L HA with 5 h priming duration increased the TSL from 16.0 cm to 22.1 cm (38.1% increment), while it increased from 29 cm to 32.0 cm (9.5% increment) under 80% FC level. Similarly, HA with 5 h priming enhanced RDW by 70.0% and 15.8% compared to the control in 50% FC and 80% FC level, respectively. Irrespective of the field moisture condition, HA 5 h priming enhanced SHDW and SDW by 17.8% and 20.1% respectively compared with non-primed seeds (Figure 4).

Furthermore, there was a significant interaction of different priming treatments in different soil moisture conditions on root morphological traits [root length (RL), total root length (TRL), root surface area (RSA), root volume (RV), average root diameter (ARD) and number of roots tips (NRT)] at p < 0.05 (Figure 5). Priming with 0.2 g/L HA for 5 h significantly enhanced root length from 7.8 cm in control to 12.7 cm (an increase of 62.8%), and 12.3 cm to 14.6 cm (by 18.7%) for 50% FC level and 80% FC level, respectively. Priming of soybean seeds with 0.2 g/L HA for 5 h significantly increased TRL, RSA, RV, ARD and NRT by 64.3%, 67.9%, 109.4%, 13.3% and 91.7% compared to non-primed seed respectively at a 50% FC level. Similarly, root morphological traits were increased by 16.3%, 43.6%, 96.7%, 27.9% and 75.9% compared to non-primed seeds respectively at the 80% FC level at the same priming treatment. Interestingly, seeds primed with HA for 5 h showed improved root characteristics except for ARD, and values increased at a higher rate compared to the control in water stress conditions (50% FC) than ideal moisture conditions (80% FC) (Figure 5).

3.3. Effects of Priming on Electrical Conductivity

Analysis of variance showed that there was a significant difference on electrical conductivity (EC) value among the treatments. Furthermore, the lowest EC was attained from seeds primed for 3 h and 5 h with 0.2 g/L HA (103.4 and 109.4 µS cm⁻¹g⁻¹, respectively), it was reduced by 51.1% and 48.3%, respectively. No significant difference was recorded for 1 h HA priming and 1 h and 3 h hydro priming compared with control. However, EC value was reduced by 26.6% and 25.1% in HP-5 h and HP-7 h treatments, respectively, compared with the control (Figure 6).

3.4. Effects of Priming on Antioxidant Enzyme Activity

Catalase (CAT) and peroxidase (POD) activities were significantly affected by priming treatments. The highest amount of both CAT and POD activities was recorded in HA 5 h priming treatment. The CAT and POD activities upon 5 h of priming increased by 34.0% and 102.1%, respectively, compared with the control (Figure 7). There were no significant differences between hydro-priming for 1 h to 7 h, in both enzymes.

3.5. Effects of Priming on Malondialdehyde Content

The MDA content of freshly harvested seeds (3.4 µmol/g FW) increased to 6.8 µmol/g FW in non-primed (control) minimally aged soybean seeds after 12 months of storage period, an evidence of lipid peroxidation. Seed priming with 0.2 g/L HA for 1, 3 and 5 h significantly reduced the MDA content by 13.4%, 27.8% and 38.2%, respectively, compared with the control (6.8 µmol/g FW); however, prolonged priming (7 h HA) increased the MDA content compared with 5 h HA priming, while hydro priming for 3 h and 5 h significantly reduced MDA content by 16.0% and 9.6% compared to non-primed seed. The lowest MDA content was recorded at 0.2 g/L HA priming with 5 h. There was no significant difference between hydro-priming 1 h and the control. However, hydro priming for 7 h increased MDA content by 18.5% compared to the control (Figure 8).

3.6. Correlations

The correlation coefficients among the seed and seedling quality parameters are presented in

Table 7. Pearson correlations for seed and seedling quality parameters of sand germination test with different priming treatments.

	1	2	3	4	5	6	7	8	9
(1) FG	1.00
(2) MGT	−0.15 ns	1.00
(3) GI	0.92 **	−0.52 **	1.00
(4) GRI	0.94 **	−0.47 **	1.00 **	1.00
(5) CVG	0.17 ns	−1.00 **	0.53 **	0.48 **	1.00
(6) SVI	0.79 **	−0.30 ns	0.81 **	0.82 **	0.31 ns	1.00
(7) EC	−0.34 *	0.39 *	−0.45 **	−0.45 **	−0.38 *	−0.43 **	1.00
(8) MDA	−0.73 **	0.42 **	−0.80 **	−0.80 **	−0.42 **	−0.63 **	0.54 **	1.00
(9) POD	0.54 **	−0.18 ns	0.55 **	0.56 **	0.20 ns	0.54 **	−0.46 **	−0.60 **	1.00
(10) CAT	0.55 **	−0.48 **	0.68 **	0.67 **	0.50 **	0.61 **	−0.51 **	−0.69 **	0.48 **

*, **, ns Significant at 5% level, 1% level and not significant respectively n = 36.

.

According to the correlation analysis, seed vigour traits (GI and GRI) were strongly and positively correlated with MDA content of the seeds (r = 0.80 ** and r = 0.80 ** respectively). Meanwhile, positive correlation was observed in FG%, GI, GRI and SVI with catalase as r = 0.55 **, r = 0.68 **, r = 0.67 ** and r = 0.61 **, respectively. Strong correlations were also observed in seed vigour traits (GI, GRI and SVI) compared to germination traits (FG%) with EC, CAT, POD and MDA.

4. Discussion

Soybean seeds undergo ageing rapidly when stored under ambient conditions, particularly in the tropics with high humidity. Freshly harvested seeds with 92% germination declined to 62% within 12 months of storage (ambient condition). Besides the reduced viability, it is expected that the vigour of the seeds will be highly influenced, causing delayed germination as well as reduced seedling length and seedling dry weight. This scenario can result in poor field establishment and become more severe under water stress conditions. Thus, as a strategy, seed priming can be used to alleviate adverse effects of ageing on seed germination and seedling quality traits since priming is postulated to induce enzyme activation and protein synthesis, repair cell membrane and enhance antioxidant defense mechanism [13,37].

In the present study, the EC value of freshly harvested seed (27.5 µS cm⁻¹ g⁻¹) increased up to 211.5 µS cm⁻¹ g⁻¹ after 12 months of storage. This clearly shows that one of the reasons for reduced germination is the loss in plasma membrane integrity which enhanced electrolyte leakage under prolonged storage. Priming, both using HA (3 h, 5 h and 7 h) and HP (5 h and 7 h), showed a reduction in EC value compared to the control, suggesting repair of membrane damage, which was influenced by the exposure duration. Thant et. al. [15] reported that seed priming enhanced antioxidant defense mechanism of aged seed and consequently reduced cell membrane damage caused by the accumulation of ROS due to lipid peroxidation. This is similar to the findings of [38], in which different priming methods were able to reduce the rate of electrolyte leakage and thereby increased the membrane stability in rice. The results of this study showed that the lowest EC value was recorded in seeds primed for 3 to 5 h (103.4–109.4 µS cm⁻¹ g⁻¹), and it was 48–51% reduction compared with non-primed seeds. The significant negative correlations between EC value and seed and seedling qualities, FG% (−0.34 *), GI (−0.45 **), GRI (−0.45 **) and SVI (−0.43 **) further supports the effect of priming on membrane integrity.

Previous studies had suggested that the strength of the antioxidant enzymatic activity (CAT and POD) plays a vital role in seed and seedling quality traits since it scavenges ROS produced by various abiotic and biotic stress conditions. The amount of CAT and POD activities was highest (21.4 mmol/min/mg FW and 83.7 nmol/min/mg FW, respectively) at the time of harvest, which gradually declined with the increase in storage period, amounting to only 6.2 µmol/min/mg FW and 24.6 nmol/min/mg FW, respectively, for CAT and POD activities by the end of the 12 months storage. The weakening of antioxidant defense mechanism resulted in low seed and seedling quality performance [2,39]. Several studies suggested that priming triggers the ROS scavenging process by increasing antioxidant (SOD, CAT, POD and APX) activities and enhanced stress bearing ability of the seedling [11], [38,40]. In the present study, we observed that priming with HA increased the POD activities compared with non-primed seeds and the highest POD activity (49.6 nmol/min/mg FW) was recorded in HA priming for 5 h with 50% increment compared with non-primed seeds. Similarly, the highest CAT activity (8.3 µmol/min/mg) was recorded in the same treatment, and it was a 25% increment to non-primed seeds. Priming up to 3 h was not sufficient to increase the amount of CAT activity explaining the reason for non-enhancement of germination traits by these treatments (1 h and 3 h). The decline in germination at 7 h of priming is attributed to reduction in both CAT and POD activity. Enhancement of protective capacity against the ROS could be the main factor that improved the quality traits after appropriate seed priming treatments. On the other hand, hydro priming did not contribute to enhanced CAT and POD activities; this could be the reason for lower effectiveness of hydro priming on seed and seedling quality. Furthermore, correlation findings suggested that CAT activity was more correlated with germination traits compared to POD activity, suggesting that a critical amount of POD activity is required for effective ROS scavenging. These findings were further confirmed by the positive correlations between CAT activities with seed and seedling qualities (FG% (0.55 **), GI (0.68 **), GRI (0.67 **), CVG (0.5 **) and SVI (0.61 **)). Moreover, significant positive correlation was observed between POD activities with germination and related quality traits reported as FG% (0.54 **), GI (0.55 **), GRI (0.56 **) and SVI (0.054 **). Thus, these results confirmed that HA priming enhanced the activity of antioxidant defense mechanism of aged soybean seeds supporting the process of scavenging excessive ROS production and thereby minimizing lipid peroxidation.

In general, the amount of MDA signifies the level of lipid peroxidation. MDA is produced when polyunsaturated fatty acids in the cellular membrane suffers oxidative damage through the accumulation of ROS [41]. In the present study, it was observed that MDA content in freshly harvested seed increased from 3.4 µmol/g FW to 6.7 µmol/g FW after 12 months of storage under ambient conditions due to oxidative damage. This could have contributed to the lower seed and seedling quality values in non-primed seeds. The present study revealed that primed seeds (HA and HP primed for 3 h and 5 h) had reduced amount of MDA content compared with non-primed seeds believed to be attributed to minimization of oxidative damage by improved antioxidant mechanisms and repairing of the membrane damage. In agreement with [38,42], results of EC value with MDA content of this study also confirmed significant positive correlation (0.54 **).

The present study clearly showed that priming with HA influenced all tested biochemical (CAT, POD and MDA) and physiological (EC) traits, thus stimulating the process of seed germination and seedling growth, especially in minimally aged seeds, while priming with water did not show any positive effects on antioxidant enzyme activities but reduced MDA content (3 h and 5 h) and EC (5 h and 7 h). The lack of enhancement on both biochemical and physiological traits in minimally aged seeds using hydro priming suggests that reactivation of the antioxidant defense mechanism of aged soybean seeds plays a vital role in triggering seed quality upon priming. According to the results of HA priming, it is clearly observed that priming time greatly influenced germination and vigor traits by regulating the moisture content of the seeds to allow only initial resumption of metabolic activities that trigger repair mechanism of aged seeds, thus improving germination. Prolonged priming duration can cause damage due to progress of germination beyond the repair stage and therefore having a negative effect when dried back. According to our study, EC value and MDA content showed a reducing trend, while POD activity showed an increasing trend up to 5 h of priming; then, EC and MDA values increased concomitant to a decrease in POD value at 7 h of priming with HA, suggesting that prolonged priming negatively affected repair mechanisms. The highest antioxidant activity (CAT and POD) and the lowest EC and MDA content were recorded in HA (0.2 g/L) primed for 5 h, which consistently recorded positive consequences for all the tested germination related traits (FG%, MGT, GI GRI and CVG), in comparison to control and hydro priming treatments. Thus, our results suggest that 1 h and 3 h HA priming time is not adequate to provide the maximum pre-germinated metabolic activities and 7 h priming time exceed the maximum point of imbibition causing radicle emergence related events to occur. This is in accordance with the findings from Asgharipour and Rafiei [23] who reported that HA concentration and soaking duration have a direct effect on germination related traits. Several other studies also attributed the improvement due to HA priming on the metabolic repair processes, building up of germination metabolites or osmotic adjustments during the priming treatment [13,43,44].

Furthermore, seed priming modifies the seed’s physiological and metabolic state, therefore increasing the percentage, speed and uniformity of seedling emergence, particularly under unfavorable environmental conditions [38,45]. In terms of abiotic stress (soil moisture condition), the results indicated that HA-5 h primed seeds performed well compared to non-primed control in moderate drought conditions (50% FC) than 80% FC level. This could also be due to the controlled hydration process occurring in HA primed seeds, resulting in the increased water uptake potential and thereby exhibiting the smooth functioning of cellular activities. Our findings were similar to [46], who showed that seed priming will induce rapid emergence of seedlings through the regulation of metabolic processes in the early phases of germination. According to the seedling quality traits (RL, TRL, RSA, RV, ARD, NORT, TSL and RODW), the benefits of seed priming were found to be more apparent under unfavorable soil moisture environments compared to more optimal conditions. These findings were in agreement with [41] in sorghum where seed priming was able to enhance the enzymatic antioxidant activities in seedlings, which improved seedling growth under unfavorable soil moisture conditions. Vieira et. al. [47] reported that HA stimulated the appearance of root hairs, promoting a significant increase in the number of lateral roots emerging from the main axis resulting in greater capacity of the induction of lateral roots in the initial stage of development. Similarly, an increase in the number of lateral roots was observed in the present study with HA 5 h priming treatment irrespective of the soil moisture content.

Generally, drought stress is one of the major challenges in achieving good field stand establishment for soybean seed production in most of the tropical areas. The current study proved that priming with HA for 5 h increased germination percentage, seedling quality and field establishment especially in water shortage conditions particularly when minimally aged seeds (due to deterioration in storage) were used. Although literature has shown a positive effect of various other priming agents on the improvement of soybean seeds [15,16,17], to date, there is no record of improvement for seeds that has been stored for more than six months. Additionally, all the priming agents used are chemical in nature, hence the use of humic acid may serve as an environmental and user-friendly priming agent.

5. Conclusions

Based on this study, priming soybean seeds with HA (0.2 g/L) for 5 h significantly increased the antioxidant enzyme activities (CAT-25% and POD-50%), reduced the EC by 51% and MDA content by 40% compared with non-primed seeds. These biochemical and physiological changes due to priming with HA enhanced germination, seed and seedling vigour as well as field emergence traits especially under water stressed conditions (50 % FC). Thus, it is suggested that soybean seeds stored under ambient conditions, having undergone minimal deterioration (60% germination), are primed with (0.2 g/L) HA for 5 h to invigorate the seeds by initiating a repair mechanism as shown from the results of this study. This method can be adopted for priming seeds prior to field planting as an environmentally friendly method to improve stand establishment as well as seedling uniformity.