The Effect of Different Doses of Salt Stress on Germination and Emergence in Cannabis (Cannabis sativa L.) Seed Treated with Pre-Salicylic Acid

Abstract

It is known that salinity stress, one of the abiotic stress factors, significantly limits the germination, growth, development, and crop yield of plants. Therefore, this study was conducted to evaluate the effects of salinity (control, 50, 100 ve 150 mM) on seed germination and the effect of salicylic acid (control, 0.25, 0.50, 0.75 ve 1.00 mM) on seed germination to improve salt tolerance in cannabis seeds. The physiological characteristics of the plant, such as germination percentage, germination duration, germination index, radicle length, plumule length, wet radicle weight, dry radicle weight, wet plumule weight, and dry plumule weight, were analyzed. Depending on the salinity application dose, it was observed that there was a significant decrease in the germination parameters of cannabis seeds compared to the control. It was observed that salicylic acid treatments under salinity stress positively affected all the characters examined and reduced germination arrest due to increasing concentration levels. Although it is recommended to prepare seeds with 0.5 mM salicylic acid pre-application dose against salt stress of the cannabis plant,it is essential to expand the studies on its transfer to practice. As a result, salicylic acid will provide positive results that can be transferred to practice by increasing the resistance of cannabis plants against salinity, especially in agricultural soils with salt problems, as it will be more sensitive to pests and diseases.

Keywords:
Cannabis sativa L; germination percentage; hemp seed; salt stress; salicylic acid

HIGHLIGHTS

Cannabis sativa is a plant with psychoactive properties.

There is limited information on the adaptability of cannabis to different salinity-alkalinity stresses.

Salicylic Acid affects seed germination, cell structure, and cell function.

Salicylic Acid has a high potential for use in agricultural production.

Salicylic Acid affects plant germination, growth, and regeneration

GRAPHICAL ABSTRACT

INTRODUCTION

Plants are exposed to constantly changing environmental conditions due to global warming and climate change. In particular, they must resist salinity stress due to drought and high temperatures. Soil salinity is one of the most important problems limiting production worldwide. Salt stress, which limits plant growth and impairs grain yield and quality, is a major global threat to agriculture [¹, ², ³]. The average amount of saline and alkaline soils worldwide is estimated at 830 million hectares [⁴]. Although the timing, duration, and severity of stress are highly effective in determining crop losses, it is clear that total yield and profit losses are significant [⁵]. Since salt stress affects 20% of land worldwide and more than 50% of irrigated agricultural land, it is predicted that 30% of agricultural land may be lost in the next 25 years, and this loss percentage may reach 50% by 2050 [⁶].

For germination, there must be appropriate moisture in the environment and water penetration into the seed. Germination, the most sensitive stage of seeds, is affected by many adverse environmental conditions, including salinity [⁷]. Although salt stress has a negative effect on all developmental stages of the plant, germination is the most sensitive stage to salinity. Regardless of the environment in which the seed is germinating, the amount of salt reduces the germination percentage and delays germination [⁸]. The increase in the amount of salinity in the environment in which the plants germinate negatively affects the germination percentage both because it creates a toxic effect on the seed and because it prevents the enzymatic activities from being activated by preventing water entry into the seed [⁹]. In a study conducted on two cannabis cultivars (YM5 and BM), it was reported that cannabis seed was sensitive to salt. The tolerance of cannabis seed to salt stress varied according to salt type and concentration, but both cultivars showed resistance to salt at mode percentage doses [¹⁰]. In a study conducted to test the germination responses of cannabis seed varieties to salt stress, the inhibition of salt stress on cannabis seed germination varied with salt type, salt concentration, the varieties were found to be more sensitive to alkaline salt stress and 50 mM salt treatment stimulated seed germination of Cannabis sativa [²]. Salt-resistant seeds can be identified by creating NaCl-induced ionic stress during seed germination [¹¹].

Pretreatment (priming technique) is very effective on germination; it is the process in which germination processes start by partially moistening in a controlled environment by treating with a substance that will be effective before germination for a certain duration [¹²]. This technique promotes germination by causing a wide range of biochemical changes in the seed [¹³]. To achieve this, growth regulators and chemicals are used to increase the germination percentage of many crops' seeds, rooting, vegetative growth, and productivity to grow, improve, and protect against stress factors [¹⁴]. “Seed priming is also called hydro-priming or osmo-priming, and this method induces germination, improves plant growth, and increases stress tolerance in plants [¹⁵]”. In recent years, many studies have determined that hormones, vitamins, nanoparticles, or signal molecules are useful for this method. H₂O₂-primed and H₂O₂+NO-primed plants had significantly higher shoot dry weight than the non-primed plants [¹⁶]. [¹⁷], showed that priming with salicylic acid (SA), gibberellin (GA₃), CaCl₂, and abscisic acid (ABA) improves the germination potential and germination rate in rapeseed seeds under low temperatures and drought. [¹⁸], showed that NO treatment was more beneficial for plant growth. Besides this, [¹⁹], reported that seed-primed SiO₂ NPs reduce Cr-accumulation and raise plant growth and biomass. Salicylic acid, a highly effective natural compound, plays a central role in specific physiological processes and defense responses in seed germination and seedling development in plants [²⁰]. TiO₂ nanoparticles are a useful tool to increase seed permeability by improving morphological and physiological properties of the medicinal-industrial plant Cannabis sativa [²¹]. [²²], suggested that low-dose gamma irradiation pretreatment can enhance the tolerance of common vetch seedlings against stress due to salt and drought.

Plants have different mechanisms to perceive and enhance stress factors [²³] so that growth and development, as well as interactions with the environment, are regulated by plant hormones [²⁴, ²⁵]. External application of growth regulators to plants under stress conditions is a critical and essential method that effectively increases yield, improves crop quality, and regulates plant mineral nutrient uptake [²⁶]. Salicylic acid (SA), one of the plant growth regulators, is a phenolic compound with a biochemical structure of ortho hydroxybenzoic acid [C₆H₄ (OH)CO₂H] and is classified as a plant growth regulator [²⁷]. Salicylic acid has positive effects as a signaling molecule in inducing plant tolerance by external application under stress conditions [²⁸]. SA, one of the intrinsic growth regulators with the potential to ameliopercentage the adverse effects of salt stress, is effective against changing adverse climatic conditions [²⁹]. SA has been observed to prevent the accumulation of Na⁺ ions in plant tissues and improve protection by providing the activity of the antioxidant enzyme system [³⁰]. [³¹], reported that SA application reduced salinity's harmful effects in stimulating plant vegetative growth. SA application has been found to help plants cope with stress under saline conditions by regulating photosynthesis percentage, stomatal conductance, and plant water balance transpiration [³²]. External application of SA is efficacious in improving seed germination, seedling growth, and photosynthesis percentage; it has been reported to be highly effective in antioxidant biosynthesis, activation of many enzymes, regulation of stomatal opening and development of chloroplasts [³³].

Cannabis (Cannabis sativa L.) is widely distributed in China, Europe, and Canada, among the countries with the highest production. Cannabis, one of the few plant species with a long history of cultivation in China, is rich in fiber, oil, and medicinal use [³⁴]. Although cannabis's THC (tetrahydrocannabinol) content is <0.3%, not above the European Union standard of 0.2%, it is internationally recognized in the industrial field. Cannabis has been reported to be very important in many agricultural areas and for producing sustainable bioenergy [³⁵]. Studies on cannabis are mainly in the form of fiber, seed, and the use of chemical analysis. There is limited information on salt and alkali stresses on cannabis and insufficient data on its ability to adapt to different salinity-alkalinity stresses. This study aimed to determine the effects of salicylic acid pretreatment doses (0.25, 0.50, 0.75 and 1.00 mM) on germination and seed characteristics of cannabis seeds exposed to salt stress (0, 50, 100 and 150 mM).

MATERIAL AND METHODS

Material procurement

This study was conducted in 2023 at Sinop University, Faculty of Arts and Sciences, Department of Biology, Plant Physiology laboratory. The seed of the Vezir55 cannabis (Cannabis sativa L.) variety used in the study was obtained from the Black Sea Agricultural Research Institute of the Ministry of Agriculture and Forestry. Salicylic acid (CAS Number: 69-72-7/Sigma-Aldrich) at 5 different doses (0.25, 0.50, 0.75, and 1.00 mM) was kept for 24 hours. NaCl (CAS Number: 7647-14-5 Sigma-Aldrich) solutions at different doses (0, 50, 100, and 150 mM) were applied to the designated petri dishes.

Experimental setup

Seed experiments were carried out in the acclimatization room. Firstly, cannabis seeds with a plump appearance, robust, and similar size were selected for the test. Before use, the seeds were surface sterilized (treated with 1% sodium hydrochloride solution for 10 minutes, washed with distilled water, and dried on filter paper at room temperature). The experiment was conducted in three replications according to the "Randomized Plots Experimental Design" using different doses of salicylic acid and salinity concentrations. In salicylic acid germination experiments, cannabis seeds were kept in 5 different doses of salicylic acid (control, 0.25, 0.50, 0.75, and 1.00 mM) and distilled water (H₂O) for 24 hours at 20±1°C in dark conditions at appropriate concentrations for pretreatment of salicylic acid. After the application, the seeds were dried and kept at +4 ^oC until use. For germination of cannabis seeds, 51 seeds for 1 dose were placed in 3 Petri dishes with 17 seeds in each petri dish in which two layers of blotting paper were placed. Salinity solutions at different doses (control, 50, 100, and 150 mM) were applied to the petri dishes. 3 mL of pure water was added to each petri dish for the control and salicylic acid groups. 3 mL of salinity was added at the appropriate concentration for the salinity groups. The germination process was left to germinate for 7 days in the dark at 22±1⁰C, according to [³⁶]. During the germination duration, the seeds in the glass Petri dishes were checked every day, and the seeds with a rootlet length of 2 mm were considered germinated and recorded. During the control, contaminated seeds were removed from the environment and recorded. Calculations were made over 50 seeds in each group.

Physiological measurements

Germination percentage (%)

The germinated seeds were counted at the same time every day, and the seeds were considered as germinated when the radicle reached 2 mm.

$Ger\min ationpercentage\left(\%\right)=\frac{Numberofger\min atedseeds}{Totalnumberofseeds}x100$

Germination duration (day)

Mean germination duration was determined as per the method of [³⁷]:

$Meanger\min ationduration=\frac{n_{1}x{d}_1+n_{2}x{d}_2+n_{3}x{d}_3+\dots +n_{n}x{d}_n}{Totalnumberofdays}$

Where, n = the number of germinated seeds, d = day.

Germination index

The germination index was calculated according to [³⁸]:

$Ger\min ationindex=\frac{n_1}{d_1}+\frac{n_2}{d_2}+\frac{n_3}{d_3}+\dots +\frac{n_n}{d_n}$

Where, n = number of germinated seeds, d = day.

Wet and dry weights of radicle and plumule of germinated plants (g)

Six plants with similar characteristics were taken from each group, and their wet weights were first recorded. Then, they were kept at 60 ^oC for 24 hours, and their dry weights were measured [³⁹].

Radicle and plumule lengths of germinated plants (mm)

The lengths of the radicle and plumule were measured by taking 6 plants of similar size from each group.

Statistical analysis

The two-year data obtained from the research were subjected to analysis of variance with the help of the JMP 5.0.1, package according to the Random Full Blocks Trial plan. According to the Duncan Multiple Comparison Test, significant differences between the applications were compared and grouped at the 5% probability level.

RESULTS

Germination percentage

The effect of salinity doses and salinity x salicylic acid interaction on germination percentage was statistically significant (p<0.01). Salt stress significantly reduced germination without salicylic acid (Table 1). While increasing salinity caused a decrease in germination, salicylic acid promoted germination at all salt levels. The highest germination percentage was obtained from control and 0.5 salicylic acid pretreatment, while the lowest was obtained from the interaction of 150 mM salinity and 1 mM salicylic acid pretreatment. The 0.5 mM dose of salicylic acid pretreatment reduced the adverse effects of salinity stress and showed significant differences in germination percentage. In addition, a 1 mM dose of salicylic acid pretreatment showed an inhibitory effect on germination percentage in all treatments (Table 1). Salicylic acid 0.5 mM dose positively affected germination in cannabis seeds and showed that they tolepercentaged the damages of salinity stress. When we look at the salinity x salicylic acid interaction, it is seen that as salicylic acid doses increase, it prevents the negative effect of salt doses on germination percentage.

Germination duration

While the effect of salinity doses on germination time was insignificant, the effect of salicylic acid application and salinity x salicylic acid interaction was statistically significant (p<0.01) (Table 1). Despite the adverse effects of salinity stress on germination time, salicylic acid application doses contributed significantly to the shortening of germination time. The shortest germination time (1.17 days) was obtained from 0.5 mM salicylic acid pretreatment dose, while the lowest germination time (2.73 days) was obtained from 50 mM salinity treatment dose. Although 0.75 and 1 mM salicylic acid pretreatment doses decreased the germination time, 0.5 mM salicylic acid pretreatment dose significantly shortened the germination time (Table 1).

Table 1
Germination percentage, germination duration, germination index averages and variance analysis results of hempseed varieties germinated in different salinity and salicylic acid doze

S/SA	Germination Percentage (%)
	0	0.25	0.5	0.75	1.00	Mean
0	96.7±1.87a	75.9±2.68ce	91.2±0.89ab	84.12±2.65bc	75.3±3.24ce	84.63±2.42a
50	70.6±0.41dg	70.8±0.07dg	78.4±6.04cd	75.3±3.82ce	68.5±1.14dg	72.89±3.17b
100	64.5±2.18ac	71.6±0.85df	73.0±1.17cf	70.8±0.16dg	65.7±1.91eg	72.94±1.76b
150	65.9±1.48eg	64.0±2.82eg	62.5±1.07fg	61.70±0.82fg	58.8±0.36g	62.58±0.86c
Mean	79.42±3.71	70.54±1.54	76.30±4.64	73.00±3.01	67.10±1.97	73.26
Germination Duration (day)
0	2.70±0.115ab	2.03±0.007df	2.30±0.011c	1.83±0.046hı	1.90±0.046fh	2.15±0.087
50	2.73±0.106a	1.50±0.029k	2.07±0.040de	2.00±0.029dg	1.67±0.005j	1.99±0.115
100	1.97±0.011eg	1.68±0.027j	1.73±0.017ıj	2.57±0.043b	1.87±0.023gh	1.96±0.086
150	2.11±0.063d	2.13±0.028d	1.17±0.040l	1.70±0.017ıj	1.70±0.029ıj	1.76±0.094
Mean	2.38±0.109a	1.84±0.078b	1.82±0.129b	2.03±0.101ab	1.78±0.033b	1.9670
Germination Index
0	17.47±0.271c	13.08±0.132h	19.92±0.467a	16.17±0.098d	14.75±0.115ef	16.28±0.63a
50	20.61±0.438a	16.25±0.086d	15.50±0.175de	14.50±0.204fg	12.00±0.275ı	15.77±0.76a
100	13.09±0.610h	18.70±0.294b	15.42±0.215de	13.75±0.511gh	12.08±0.138ı	14.61±0.64a
150	11.06±0.611j	13.83±0.051gh	15.25±0.075ef	12.08±0.046ı	10.92±0.400j	12.63±0.46b
Mean	15.56±1.14ab	15.50±0.67ab	16.52±0.60a	14.13±0.46bc	12.44±0.44c	14.82
Analysis of Variance		d.f	Germination Percentage		Germination Duration	Germination Index
Salinity		3	16.44**		2.749ns	6.591**
Salicylic Acid		4	2.38ns		6.658**	4.962**
Salinity x Salicylic Acid		20	5.25**		69.547**	76.889**
CV			7.70		4.10	3.70

• **Statistically significant at 1% (P<0.01), ns: nonsignificant, S: salinity, SA: Salicylic acid, The difference between the averages denoted by different letters is significant. The difference between the averages denoted by different letters is significant.

Germination index

Salinity, salicylic acid, and salinity x salicylic acid interactions on germination index were statistically significant (p<0.01) (Table 1). The increase in salinity doses applied to cannabis seeds had a negative effect on the germination index, and the highest (16.28) and lowest (12.63) average germination indexes were obtained from control and 150 mM salinity doses, respectively. According to salicylic acid pretreatment doses, the highest (16.52) and lowest (12.44) average germination index was obtained from 0.5 and 1 mM salicylic acid doses. In terms of salinity x salicylic acid interaction, the highest germination index was obtained from control (20.61) and 0.5 mM salicylic acid (19.92) application dose, while the lowest germination index was obtained from 150 mM salinity (11.06) and 1.0 mM salicylic acid (10.92) application dose (Table 1). While 0.5 mM salicylic acid application dose increased the germination index against salinity stress, 1 mM salicylic acid dose decreased the germination index. According to these results, we can say that the best germination index was obtained from 0.5 mM salicylic acid application dose.

Radicle length

Salinity and salicylic acid application doses on radicle length were statistically significant at p<0.05, and the interaction of salinity x salicylic acid was statistically significant at p<0.01 (Table 2). The highest mean radicle length value (1.405 mm) was obtained from the control treatment in salinity application doses. It was observed that radicle length was negatively affected with increasing salt concentration. In the interaction of salicylic acid and salinity x salicylic acid, the highest mean radicle length (1.565 and 2.672 mm, respectively) was obtained from the 0.25 mM salicylic acid application dose. In comparison, the lowest mean radicle length (0.756 and 0.513 mm, respectively) was obtained from the 0.5 mM salicylic acid application dose (Table 2).

Table 2
Radicle length, plumule length averages and variance analysis results of hempseed varieties germinated in different salinity and salicylic acid doze

S/SA	Radicle Length (mm)
	0	0.25	0.5	0.75	1.00	Mean
0	1.025±0.4888cd	2.672±0.2419a	0.513±0.0505d	1.348±0.4096bd	1.468±0.3279bc	1.405±0.230a
50	1.361±0.6110bc	2.092±0.2083ab	0.947±0.0971cd	1.086±0.4602cd	1.237±0.3520cd	1.345±0.182ab
100	1.002±0.3190cd	0.754±0.2500cd	0.909±0.2940cd	0.972±0.1402cd	0.808±0.1502cd	0.889±0.094b
150	1.1389±0.1195cd	0.742±0.1950cd	0.656±0.0944cd	0.731±0.1158cd	1.430±0.1367bc	0.940±0.094b
Mean	1.1317±0.0775ab	1.565±0.271a	0.756±0.088b	1.034±0.152b	1.236±0.137ab	1.1440
Plumule Length (mm)
0	2.215±0.295ae	2.653±0.604ac	2.038±0.194af	2.789±0.380ab	2.883±0.428bf	2.516±0.177a
50	2.567±0.581ad	2.758±0.651ab	2.133±0.500af	2.164±0.626ae	1.733±0.069cf	2.271±0.222a
100	1.776±0.155bf	1.288±0.098ef	1.806±0.0580bf	1.433±0.292ef	1.614±0.270df	1.583±0.091b
150	1.117±0.044f	1.389±0.200ef	1.347±0.0764ef	1.262±0.120ef	1.522±0.248ef	1.327±0.070b
Mean	1.919±0.217	2.022±0.284	1.831±0.148	1.912±0.250	1.938±0.205	1.924
Analysis of Variance		d.f	Radicle Length		Plumule Length
Salinity		3	2.7560*		13.374**
Salicylic Acid		4	2.7050*		0.091ns
Salinity x Salicylic Acid		20	3.0007**		2.557**
CV			6.7		12.4

• *, ** Statistically significant at 5 % (P<0.05) and 1% (P<0.01), ns: nonsignificant, S: salinity, SA: Salicylic acid, The difference between the averages denoted by different letters is significant. The difference between the averages denoted by different letters is significant.

Plumule length

In this study, Salinity and Salinity x salicylic acid interactions were statistically significant (p<0.01) except for salicylic acid application dose on plumule length (Table 2). The effect of different salinity doses on plumule length was negative. Salinity doses (50, 100, and 150 mM) shortened plumule length by 9.7%, 37.1%, and 47.3%, respectively, and the highest shortening was observed in 150 mM Salinity treatment (47.3%). Although the effect of salicylic acid pretreatment doses on plumule length was insignificant, the best results were obtained from 0.25 mM salicylic acid treatment dose (Table 2). According to Salinity x salicylic acid interaction, the highest plumule length (2.88 cm) was obtained from salinity (control) x salicylic acid (1.00 mM) interaction. In comparison, the lowest plumule length (1.12 cm) was obtained from salinity (150 mM) x salicylic acid (control) interaction. According to the salinity x salicylic acid interaction, salinity application was in the control group, while salicylic acid application had a positive effect on plumule length except 0.5 mM.

Wet radicle weight

While the effect of salinity and salicylic acid concentrations on wet radicle weight was insignificant, the effect of salinity x salicylic acid interaction was statistically significant (P<0.05) (Table 3). When salinity application doses were analyzed, the highest average wet radicle weight (0.0078 g) was obtained from a 50 mM salinity dose, and the lowest average wet radicle weight (0.0055 g) was obtained from a 150 mM salinity dose. According to the averages of salicylic acid concentrations, the highest wet radicle weight (0.0080 g) was obtained from 1.00 mM salicylic acid treatment, and the lowest wet radicle weight (0.0050 g) was obtained from 0.25 mM salicylic acid treatment. Regarding salinity and salicylic acid interaction, the highest wet radicle weight (0.0098 g) was obtained from 150 mM salinity x 1.00 mM. In comparison, the lowest wet radicle weight (0.0028 g) was obtained from 0 mM salinity x 0.5 mM salicylic acid interaction (Table 3).

Table 3
Wet radicle weight, dry radicle weight, wet plumule weight, dry plumule weight averages and variance analysis results of hempseed varieties germinated in different salinity and salicylic acid doze

S/ SA	Wet Radicle Weight (g)
	0	0.25	0.5	0.75	1.00	Mean
0	0.0073±0.0031ae	0.0092±0.0011ab	0.0028±0.0006h	0.0078±0.0018ae	0.0080±0.0010ae	0.0070±0.0008
50	0.0085±0.0027ad	0.0089±0.0013ac	0.0065±0.0005ah	0.0072±0.0014af	0.0077±0.0005ae	0.0078±0.0006
100	0.0050±0.0013dh	0.0046±0.0013eh	0.0048±0.0007dh	0.0071±0.0009ag	0.0066±0.0004ah	0.0056±0.0005
150	0.0053±0.0004ch	0.0035±0.0003fh	0.0057±0.0008bh	0.0033±0.0005gh	0.0098±0.0016a	0.0055±0.0007
Mean	0.0065±0.001	0.0066±0.0009	0.0050±0.0005	0.0064±0.0008	0.0080±0.0006	0.0065
Dry Radicle Weight (g)
0	0.0007±0.0003be	0.0013±0.0001ab	0.0008±0.0002ad	0.0010±0.0003be	0.0012±0.0003ce	0.0010±.00012
50	0.0011±0.0002ad	0.0012±0.0003ac	0.0007±0.0001be	0.0011±0.0004ac	0.0013±0.0002ab	0.0011±0.0001
100	0.0005±0.0002ab	0.0009±0.0004be	0.0004±0.0002de	0.0012±0.0003ac	0.0010±0.0002be	0.0008±0.0001
150	0.0009±0.0001be	0.0009±0.0004be	0.0008±0.0003be	0.0003±0.0002e	0.0018±0.0001a	0.0009±0.0007
Mean	0.0008±0.0001bc	0.0011±0.0002ab	0.0007±0.0003c	0.0009±0.0007bc	0.0013±0.00011a	0.0010
Wet Plumule Weight (g)
0	0.036±0.0061	0.050±0.0028	0.038±0.0058	0.048±0.0071	0.048±0.0038	0.0440±0.0038ab
50	0.052±0.0062	0.053±0.0059	0.042±0.0022	0.049±0.0106	0.039±0.0017	0.0470±0.0027a
100	0.040±0.0015	0.034±0.0011	0.039±0.0036	0.043±0.0044	0.043±0.011	0.0398±0.035bc
150	0.038±0.0071	0.033±0.0025	0.034±0.0026	0.032±0.0019	0.041±0.0103	0.0356±0.031c
Mean	0.0415±0.0027	0.0422±0.032	0.0383±0.0024	0.043±0.0018	0.0430±0.0030	0.0416
Dry Plumule Weight (g)
0	0.0083±0.0011	0.0126±0.0016	0.0143±0.0015	0.0125±0.0012	0.0119±.0012	0.0119±0.007b
50	0.015±0.0021	0.0133±0.0010	0.0134±0.0004	0.0134±0.0007	0.0128±0.0007	0.0136±0.0005a
100	0.0155±0.0014	0.0138±0.0013	0.0142±0.0003	0.0134±0.0006	0.0139±0.0009	0.0142±0.0004a
150	0.0123±0.0008	0.0132±0.0020	0.0113±0.0023	0.0141±0.0010	0.013±0.0004	0.0128±0.006ab
Mean	0.0128±0.001	0.0132±0.0006	0.0133±0.0007	0.0134±0.0004	0.0129±0.0005	0.0131
Analysis of Variance		d.f	Wet Radicle Weight	Dry Radicle Weight	Wet Plumule Weight	Dry Plumule Weight
Salinity		3	2.547ns	0.874ns	4.456**	2.8250*
Salicylic Acid		4	1.967ns	4.170**	0.460ns	0.1232ns
Salinity x Salicylic Acid		20	76.880**	2.320*	1.560ns	1.3910ns
CV			3.70	3.51	8.2	9.5

• *, ** Statistically significant at 5 % (P<0.05) and 1% (P<0.01), ns: nonsignificant, S: salinity, SA: Salicylic acid, The difference between the averages denoted by different letters is significant. The difference between the averages denoted by different letters is significant.

Dry radicle weight

While c treatments (P<0.01) and salinity x salicylic acid interaction (P<0.05) were statistically significant on dry radicle weight, salinity treatments were not significant (Table 3). The lowest dry radicle weight (0.0008 mg) was obtained from a 100 mM salinity dose, while the highest dry radicle weight (0.0011 mg) was obtained from a 50 mM salinity treatment. These results showed a 27.3% difference between the highest and lowest dry radicle weight doses. There was a significant difference between salicylic acid doses regarding dry radicle weight. The highest dry radicle weight of salicylic acid application was obtained from a 1.00 mM salicylic acid application dose. The root dry weight obtained from this dose was 62.5% higher than the control plant.

Wet plumule weight

While the effect of salinity application on wet plumule weight was significant (p<0.01), salicylic acid and salinity x salicylic acid interactions were not significant. The highest wet plumule weight (0.0470 g) was obtained from a 50 mM salinity application dose, increasing by 6.8% more than the control. While the total wet plumule weight value decreased with increasing salinity doses, the decrease in wet plumule weight decreased because salicylic acid application doses inhibited the negative effect of salt (Table 3).

Dry plumule weight

According to the variance analysis results, salinity application's effect on dry plumule weight was statistically significant (P<0.05). In contrast, the effect of salicylic acid and salinity x salicylic acid interaction was insignificant (Table 3). The lowest dry plumule weight was obtained from the control and 150 mM salinity dose (0.0119 and 0.0128 g, respectively). In contrast, the highest dry plumule weight was obtained from 50 and 100 mM salinity doses (0.0136 and 0.0142 g, respectively).

DISCUSSION

It is seen that cannabis plants are sensitive to salinity stress during the germination duration, as in many plants. Similarly, [³²] and [⁴⁰] reported that salicylic acid applications prevented the adverse effects of salt on germination percentage. Indeed, germination percentage values generally decreased with increasing salicylic acid concentrations [⁴¹]. According to these results, it was determined that the germination percentage varied depending on the salinity level.

[⁴²], reported that salicylic acid inhibits the activity of catalase enzyme in plants, and with the decrease in the activity of catalase enzyme, the percentage of hydrogen peroxide increases and plays an active role in many physiological events such as seed germination, ion uptake, photosynthesis, growth, and development. In addition, it was determined that salicylic acid applications eliminated the effect of salt [⁴³]. [³²], reported that salicylic acid prevents the effects of salinity stress in barley depending on plant species and applied concentration. Similar situations emerged in our study, and the germination duration of cannabis seeds was shortened with salicylic acid application.

According to these results, we can say that the best germination index was obtained from 0.5 mM salicylic acid application dose. Salicylic acid increase protects plants against environmental stresses by increasing enzyme activity in the biosynthetic pathway. Therefore, salicylic acid improves salinity stress tolerance mechanisms in many plants [⁴⁴]. Indeed, the authors clearly state that salicylic acid increases seed germination by reducing oxidative damage in the face of a certain amount of salt stress [⁴⁵, ⁴⁶]. In addition, the inhibition of seed germination by increasing salicylic acid concentration is thought to be due to increased abscisic acid synthesis [⁴⁷]. These sources support that specific salicylic acid application doses increase the germination index while high doses decrease it.

It was found that high salinity stress and increasing salicylic acid concentration negatively affected radicle length. It has been reported that the decrease in radicle length with increasing salinity concentration may be due to osmotic differences, Na+ accumulation in leaves, and inhibition [⁴⁸, ⁴⁹]. Salicylic acid has been shown to cause the progression of cell division and increase productivity in the roots of plants exposed to salinity stress [⁵⁰]. [⁴⁰] and [⁵¹], also reported that root elongation was inhibited in parallel with the increase in salicylic acid concentration under salinity stress conditions. Similarly, [⁵²], reported that the radicle length development of barley seeds was negatively affected by the increase in salinity. Our results are supported by the literature mentioned by the researchers.

As a result of the study, we can say that Salinity suppresses plumule length. As a matter of fact, [⁴⁰], reported that Salinity suppressed plumule length and showed a 30% reduction. Salicylic acid application doses positively affected plumule length, which decreased due to salinity stress. According to the results of many studies, salinity stress caused a significant decrease in plumule length development because it slowed down cell division [⁵³, ⁵⁴, ⁵⁵]. Many researchers are trying to develop powerful treatments, such as salicylic acid, to reduce the harmful effects of salinity stress. As a matter of fact, it has been reported that seeds pretreated with salicylic acid showed tolerance to salt stress and improved the development process positively by increasing the water content in the material, especially in some plants, low doses of salicylic acid pretreatment increased plumule length to a certain extent [⁵⁶].

While wet radicle weight decreased due to salinity stress, salicylic acid pretreatment increased wet radicle weight. As salinity concentrations increased, wet radicle weight was negatively affected [⁵⁷], reported that salinity stress decreased wet radicle weight from 7.4 g to 3.7 g. Similar results were also reported by [⁵⁸]. The salicylic acid application was reported to have a positive effect on the wet radicle weight of the plant in a dose-dependent manner [⁵⁹]. It was determined that increasing salicylic acid concentrations positively affected plant wet radicle weight and increased plant wet radicle weight while increasing salinity concentrations decreased wet radicle weight. The results we obtained support the literature.

As a result, while increasing salt concentrations decreased the dry root weight, increasing salicylic acid doses increased the dry root weight. Thus, under stress conditions, compounds such as abscisic acid, ethylene, and brassinosteroids are synthesized in plants, and these compounds have been reported to promote root elongation at low doses and to reduce root growth at low doses [⁶⁰]. [⁶¹], reported that increasing salinity concentrations reduced root growth, and dry radicle weight decreased by about 20% at the highest salinity concentration compared to the control. These results parallel previous studies showing that salinity stress reduces dry radicle weight. This was attributed to the fact that salicylic acid stabilizes IAA (indole acetic acid), suppressing proteins and causing inhibition of auxins, resulting in a decrease in the amount of free IAA [⁶²].

While the total wet plumule weight value decreased with increasing salinity doses, the decrease in wet plumule weight decreased because salicylic acid application doses inhibited the negative effect of salt. Although salinity is toxic, it acts as a nutrient element in low-dose seeds [⁶³,⁶⁴]. Many researchers have reported that plumule weight decreases in parallel with the increase in salinity concentration [³]. Salicylic acid application has been reported to help plants cope with salinity stress by increasing photosynthesis percentage, stomatal conductance, vegetative growth of plants, and regulating transpiration for plant water balance [³¹]. [⁴³], reported that wet plumule weight decreased with salinity applications, and salicylic acid applications slowed down the decrease.

These results showed that salinity application positively affected dry plumule weight up to 100 mM. Table 3 shows salicylic acid increased dry plumule weight up to 0.75 mM. Indeed, [⁶¹], found that salicylic acid increased dry plumule weight compared to the control. Similarly, [³⁷] investigated the effect of different salicylic acid applications on dry plumule weight under salinity stress. They reported that 1.5 mg L^-1 salicylic acid applications obtained the highest dry plumule weight.

CONCLUSION

According to the general results of this study, salinity stress at different doses decreased all seed germination parameters of cannabis except dry plumule weight. In addition, it was determined that salicylic acid doses applied to cannabis seeds improved the germination characteristics by reducing some negative characteristics of salinity stress. The most significant damage on germination of cannabis seeds under salinity stress was obtained at 150 mM application dose. It can be said that low pretreatment doses of salicylic acid give better results on the germination of cannabis seeds than high doses. Indeed, 0.5 mM salicylic acid pretreatment dose significantly improved germination percentage and germination. According to this result, treating cannabis seeds with a modepercentage salicylic acid pretreatment dose will positively affect seedling formation by increasing the germination time. There are positive results that salicylic acid applications can be transferred to practice in cannabis production, especially since there will be more sensitivity to pests and diseases in agricultural soils with salt problems. Although it is recommended to prepare seeds with 0.5 mM salicylic acid pre-application dose against salt stress of the cannabis plant, it is essential to expand the studies on its transfer to practice and to evaluate it by making economic analyses.

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• Funding:
  This research was not funded by any organization.

Edited by

• Editor-in-Chief:
  Bill Jorge Costa

• Associate Editor:
  Adriel Ferreira da Fonseca

Publication Dates

• Publication in this collection
  28 Oct 2024
• Date of issue
  2024

History

• Received
  16 Jan 2024
• Accepted
  11 June 2024