Effects of ultrasonic waves on seedling growth, biochemical constituents, genetic stability of fenugreek (Trigonella foenum-graecum) under salinity stress

Fenugreek is a globally important legume that is widely cultivated for its therapeutic benefits in most parts of the world. Seeds on the other hand have a poor germination and growth rate when exposed to salinity. The effect of ultrasonic exposure period on germination and early seedling behaviors of fenugreek seeds under salt stress was investigated in a laboratory experiment. During germination and early seedling stages, all tests were conducted at 40 kHz in a water bath ultrasonic device with two durations (10 and 20 min) under salinity stress using different concentrations of NaCl (0, 1000, 3000, and 5000 mg/l). The results revealed a substantial decrease in germination percentage, all growth criteria, with increasing NaCl concentration and a significant increase in biomass produced by the Fenugreek (total soluble protein, total soluble carbohydrate, and proline), all of which are thought to be mechanisms for salinity resistance. Ultrasonication of fenugreek seeds for 10 and 20 min has a significant impact on seed germination, early seedling development and biochemical constituents under normal and stress conditions. The genetic stability of fenugreek DNA content was affected by these different treatments. This variation was estimated by RAPD-PCR molecular marker, and resulted in a total polymorphism percentage of 49.72% from all the primers. All these different treatments caused variation in the physiological responses and DNA content. This variation enhanced with more ultrasonic and salt treatments. Hence, these stresses can be used for enhancing the variable metabolic processes in fenugreek plant and stimulate its medicinal properties.


Introduction
Fenugreek (Trigonella foenum-graecum L.) is an annual herb in the Fabaceae family whose leaves and seeds are used as spices and condiments due to their strong flavor and scent.Fenugreek has also gained popularity as an ingredient in traditional remedies (Shrivastava 2012).Fenugreek is well-known for its therapeutic properties, including anti-diabetic, anti-carcinogenic, hypocholesterolemia, antioxidant, and immunological effects.Besides its therapeutic use, it is also utilized as a food stabilizer, glue, and emulsifying agent in the manufacture of numerous food products (Wani and Kumar 2016).Fenugreek is a spice, vegetable, and significant medicinal crop that is grown all over the world (Olfa et al. 2018).Many medicinal plants must be professionally farmed in order to meet rising demand for indigenous systems of medicine as well as the pharmaceutical industry, but soil salinity poses major challenges to plant cultivation (Qureshi et al. 2005).Most plant species' seed germination and seedling growth phases are known to be particularly sensitive to salt stress (Cuartero et al. 2006).Drought stress, or salinity, has a devastating secondary effect on cells.The impacts can be direct, such as decreased CO 2 availability (Chaves et al. 2009), or indirect, such as changes in photosynthetic metabolism (Pinheiro and Chaves 2011), or secondary, such as oxidative stress.Saberali and Moradi (2019) investigated the effect of salinity on germination and seedling growth of fenugreek, dragonhead, savory, and dill, and found that as the NaCl concentration increased, germination and seedling growth decreased, with the greatest reduction occurring at the highest salt level.All of the species studied experienced a linear reduction in germination, germination velocity, and seedling growth as a result of salinity stress, but the losses differed.The effects of salinity on seedling germination and growth are mainly linked to osmotic effects, which can cause water uptake inhibition, and ion toxicity, which can prevent cell growth and division (Munns and Tester 2008;Morais et al. 2018).Olfa et al. (2018) investigated the effect of different NaCl concentrations (0, 50, 100, 150, and 200 mM) on Fenugreek plant growth and determined that the salt effect reduced the biomass generated by the Fenugreek, as measured by leaf, stem, and root dry weight, at 150 and 200 mM.
Utilizing inaudible sound frequencies, ultrasonic interaction with materials is a ground-breaking physical technique (20-100 kHz).It has been shown that treating substances with ultrasound can change their state and even expedite processes (Aladjadjiyan 2007).This approach is unique among seed preparation techniques because it is straightforward, affordable, environmentally friendly, and multifunctional (Goussous et al. 2010).Many seed varieties have been treated with ultrasound to encourage germination, including maize, barley, rice, and sunflower (Aladjadjiyan 2002;Florez et al. 2007;Yaldagard et al. 2008).Ultrasound has a significant impact on plant growth and development when used as a kind of stress.The growth of various plant organs can be affected by ultrasonic treatment.When seeds are subjected to ultrasonic process, researchers discovered that a small amount of ultrasound can encourage cell division, a medium dose of ultrasound can inhibit cell division, and a higher amount of ultrasound can kill cells.Ultrasonic processing boosted the rate of germination of spinach and cabbage seeds, according to the study.Mild ultrasonic can cause plant root cells to actively proliferate, improve plant development ability, and encourage the plant to strike roots, which can reduce yearly plant respiration intensity while increasing plant respiration intensity for two years (Zhang et al. 2008).Seeds from two rice cultivars, Guangyan1 and Huahang 31, were treated to ultrasonic vibration for 30 min before being transplanted into lead contaminated soil, according to Rao et al. (2018) the Pb content in roots, stems, leaves, panicles (at heading), and brown rice (at maturity) was lower in the ultrasonic treatment than in the control, indicating that seed treatment with ultrasonic waves could improve rice performance and minimize brown rice lead accumulation in lead polluted soils.Previous research has ascribed the improvement to a variety of factors, including: Ultrasound improves water uptake and oxygen availability by increasing the porosity of the seed through acoustic cavitation.Ultrasound speeds up mass transfer by allowing extra-absorbed water to react freely and readily with the cell embryo, releasing gibberellic acid and speeding up metabolic activities in aleurone cells.By disrupting cell membranes, ultrasound aids in the mobilization of endosperm nutrients.The same results were also showed by Wang et al. (2020) and Carrillo-Lopez et al. (2021).
With salt stress brought on by NaCl, this study sought to ascertain the effects of ultrasonic wave duration on specific seedling growth, metabolic components, and genetic stability in fenugreek.

Material and methods
The present study was conducted at the Laboratory of Plant Physiology of Helwan University, Faculty of Science, Ain Helwan, Cairo.Healthy and uniform seeds of fenugreek (Trigonella foenum-graecum L.) were selected and used for the ultrasonication that were carried out at 40 kHz on the digital ultrasonic cleaner (MFUC-80A).Ultrasound treatments were performed in a water bath at constant temperature (25 °C) and for two time periods (10 and 20 min).After exposure to ultrasonic waves equal number (25 seeds) of fenugreek seeds were transferred to sterile Petri dishes with one filter paper Whatman No. 1. Four replicates were prepared for each treatment.The seeds were germinated under different osmotic stress values of NaCl (0.0, 1000, 3000, 5000 mg/l).A seed was considered to be germinated at the emergence of both radicle and plumule up to 2 mm length (Chartzoulakis and Klapaki 2000).

Germination percentage and growth parameters
The experiment was terminated on at the 7th day, data on germination percentage, plumule and radicle length (cm), fresh and dry weight of plumule, radicle (mg), plumule to radicle length ratio and seedling vigour index were recorded.The data on plumule and radicle dry weight was recorded after drying in hot air oven at 65 °C for 48 h.
The germination percentage was determined by using the following formula (Aniat-ul-Haq and Agnihotri 2010).
The plumule to radicle length ratio of seedling was calculated by the following formula (Kökten et al. 2009): The seedling vigour index was determined by multiplying the sum total of mean length of plumule and radicle of a seedling with germination percentage of the respective seedling by the following formula (Iqbal and Rahmati 1992): The germinating seeds were used for measuring the total soluble sugars, total soluble proteins and proline.

Chemical analysis
A known weight of germinated seeds was extracted in 70% ethanol and completed to a known volume with distilled water and used for estimation of total soluble sugars using anthrone reagent (Umbreit et al. 1959) and the total soluble proteins by the procedure of Lowry et al. (1951).Approximately 0.5 g leaves were homogenized in 10 ml of 3% aqueous sulfosalicylic acid, and then the homogenate filtered through Whatman filter paper no.2.and used for estimation of proline according to Bates et al. (1973).

DNA isolation and RAPD-PCR bioassay
The total genomic DNA was extracted by CTAB method, according to Doyle and Doyle (1990).Half gram of leaves was mixed with 800 µl of 2% CTAB buffer, then incubated 45 min at 65 °C (vortex each 10 min).The tubes were centrifuged at 12,000 rpm for 12 min, then the supernatant transferred into new tubes with addition of equal volume of chloroform: isoamyl alcohol (24:1) and set for 3 min at room temperature.After that, tubes were centrifuged (12,000 rpm/10 min/4 °C).Then the upper aqueous layer was transferred to new Eppendorf tubes with addition of 800 µl of absolute ice-cold ethanol and left overnight at − 20 °C.Tubes were centrifuged to precipitate DNA pellets then washed them with ice-cold 70% ethanol.Finally, resuspend the pellets in 50 µl of TE buffer and kept at − 20 °C till applying RAPD-PCR.
Seven decamers were applied in this study, however only four of them yielded scorable and reproducible bands (Table 2).The RAPD-PCR reaction was carried out in Biometra thermocycler with a total volume of 25 µl of 12.5 µl Taq master mix (COSMO PCR RED M. Mix, W1020300x), 3 µl of genomic DNA, 1.5 µl for each primer (Willowfort) and 8 µl ddH2O.The reaction program was designed for 40 cycles as follow: Denaturation for 30 s at 94 °C, annealing 30 s at different degrees and extension for 1 min at 72 °C; followed by one step of final extension at 72 °C for 10 min then cooling at 4 °C.The amplified PCR product was run on 1.4% agarose gel compared to (New England Biolab, #N3232S) ladder.

Statistical analysis
The significance of the results was determined using Least Significant Difference (LSD) values at p = 0.05 according to Snedecor and Cochran's method of complete randomized blocks design (CCRBSE.Bas) utilizing analysis of variance (1980).The analysis of gel electrophoresis resulted in images that were analyzed by band scoring (1, 0).These computations were carried out using Bio-Rad Quantity one (4.6.2).

Effect of salt stress on germination and growth parameters
According to the findings, there was a general drop in germination percentage with increasing salt content, with a maximum loss of 74% at concentration 5000 mg/l NaCl (Fig. 1).The use of the seed's stored food supply is necessary for seed germination.Water absorption by seedlings is hampered by salinity.As a result, the hydrolysis of seed reserves is inhibited, causing seed germination to be delayed and reduced Error bars are standard errors of the mean (LSD least significant difference) (Begum et al. 2010).Furthermore, due to the concentration of soluble solutes around the seeds, which increases the osmatic pressure, the seeds require more water intake during germination when exposed to salt.As a result of the excessive uptake of ions, the plant becomes poisonous.Furthermore, due to the concentration of soluble solutes around the seeds, which increases the osmatic pressure, the seeds require more water intake during germination when exposed to salt.As a result, the uptake is enormous.
Toxicity in the plant is caused by a buildup of ions.Furthermore, there is a difference in water potential between the external environment and the seeds, (lower water availability) suppresses the development of main roots (Delachiave and DePinho 2003).
Many authors, including Saberali and Moradi (2019), Olfa et al. (2018); and Behairy et al. (2017), have documented salt damage on the seed germination of fenugreek (Trigonella foenum-graecum).However, after exposing seedlings to ultrasonic waves for 10 or 20 min, the detrimental effects of salt stress were significantly decreased, and the germination percentage in both stressed and unstressed circumstances improved.There was a notable increase in germination percentage of unstressed seeds from 84 to 96% with exposure of seeds to ultrasonic waves for 20 min, while under salinity stress it was observed that the germination values at 20 min were consistently greater than those at 10 min.Goussous et al. (2010) on wheat, Yaldagard et al. (2008) on barley, and Aladjadjiyan (2002) on Zea mays all observed Improved germination performance following ultrasonic application.The main effects of ultrasonic are revealed to be mechanical effects (acoustic cavitation) and rupture of plant cell walls, resulting in increased water uptake.Cavitation during the sonication process was hypothesized as another possible reason that induced micro fissures on the seed coat, which may have improved the imbibition of moisture for enhanced germination speed (Yaldagard et al. 2008).
As shown in Table 1, there was a negative relationship between salinity stress and all morphological traits of growth parameters such as (plumule and radicle length (cm), fresh and dry weight of plumule, radicle (mg), seedling vigor index, and plumule to radicle length ratio of seedling) that were optimal in the absence of salt and reduced by increasing salt concentrations.The lowest values for all growth parameters were obtained at 5000 mg/l of salt.Our findings corroborate previously published findings.Many authors (Ratnakar and Raib 2013) showed that salt stress hindered the growth of fenugreek seedlings.Olfa et al. (2018) investigated the growth responses of fenugreek to salt stress and found that dry weights of leaves, stems, and roots were reduced.These alterations were linked to a decrease in water content, K + , and Ca 2+ concentrations, as well as a significant increase in Na + and Cl − concentrations in many organs.Salinity stress Inhibited seedling development by reducing assimilate supply and turgor pressure (Munns and Tester 2008).Salt's direct impacts on plant growth include (1) a drop in the osmotic potential of the soil solution, which limits plant accessible water, and (2) toxicity of excessive sodium or chloride towards the plasma membrane (Oktem et al. 2006).Seeds' ability to keep harmful substances out for salt tolerance in Suaeda salsa, a medicinal halophyte (Wang et al. 2015a), was dependent on the ability of seeds to remove harmful Na + from the developing embryo.
Although the plumule to radicle length ratio is a derived property, it was also discovered that the general trend of salinity stress indicated a steady decrease in plumule to radicle length ratio of seedlings as salt concentration rises, when compared to control.Such observation was reported earlier in fenugreek (Kapoor and Pande 2015;Ratnakar and Raib 2013), in spinach (Keshavarzi et al. 2011) and in oat (Chauhan et al. 2016).
The seed vigor Index, also known as seed power or seed viability, is a measurement of a seed's capacity to emerge under stressful condition and seeds with a higher seed vigor index is more vigorous.Table 1 shows that all growth metrics had a negative and substantial relationship with seedling vigor index that dropped as salinity concentrations rose.Despite the deleterious effects of salt stress on seedling growth, ultrasonic treatment had a significant impact on seedling growth, with all growth parameters after 10 or 20 min of exposure.According to research, a small amount of ultrasound can accelerate cell division in some plant organs whereas a substantial dose of ultrasound can inhibit cell division (Zhang 2008).According to Machikowa et al. (2013) germination, Seed vigor index, and the root and shoot lengths of seedlings treated with ultrasonic were different depending on the ultrasound's intensity, from the control the highest level at 10 min of ultrasonic treatment, the seedling's Seed vigor index was discovered therapy at intensities of 40-60% and the treatments, which lasted between 5 and 20 min, yielded the best results.
The effects of ultrasonic assisted seeds priming (20, 40, 80, and 160 s) with a frequency of 24 kHz on seed enhancement of aged Milk thistle seeds were investigated by (Moosavi 2018) who showed that root growth was significantly improved by using ultrasonic energy for 20 s, and root length was 10.39 cm, compared to 5.48 cm without ultrasonic energy.The effects of salinity and ultrasonic pretreatment on the development of clover sprouts were investigated by Tahany et al. (2021) after being processed with ultrasound at 20, 28, and 40 kHz for 30 min at 30 °C, the clover seeds were steeped in deionized water with 1000 and 2000 ppm of sodium chloride for 9 h, and then sprouted for 3 days in the dark.Clover sprout length dramatically decreased as salinity concentration was increased to 2000 ppm NaCl and increased for samples that had previously had ultrasonic treatment, indicating that the growth of clover sprouts was less negatively impacted by salinity.Aladjadjiyan (2012) found comparable findings with lentils, Fateh et al. (2012) with fennel, and Shekari (2015) with sesame.

Effect of salt stress on various physiological parameters
Fenugreek had a higher concentration of total soluble proteins and total soluble carbohydrate and the greatest value was reported at 5000 mg/L NaCl concentration as compared to the control value as shown from (Figs. 2, 3) This coincides with the findings of Tort and Turkyilmaz (2004), who found a significant increase in protein content in barley plants (Hordeum vulgare L.) after treating them with 120 mM sodium chloride.The findings of a recent study on Vigna mungo (L.) by Kapoor and Srivastava (2010) back up the earlier findings.When the salt concentration was increased, they saw a rise in protein content.Under salinity stress, amylase and protease enzymes become more active, which could explain this response.
As a process of osmoregulation under salt stress, an increase in cellular content of organic solutes, such as soluble carbohydrates and soluble proteins, reduces the cellular water potential.The increased activity of the amylase enzyme, according to Dhingra and Varghese (1986) was also responsible for the reduction in polysaccharide levels that was linked to a rise in total soluble sugars.Furthermore, Yasseen et al. (2018) speculated that soluble sugars in polysaccharide formation might be underutilized.Zayed and Zeid (1998) on mung bean and Santos and Andrade (2005) on sunflower both came up with similar results.Plants accumulate osmo-protective solutes like proline and soluble carbohydrates in response to salt stress (Chelli-Chaabouni et al. 2010).One of the most prevalent responses of plants to changes in the external osmotic potential is the formation of metabolites that act as compatible solutes (Munns and Tester 2008).Plants' defensive mechanisms against ROS-induced damage frequently entail the accumulation of soluble protein, which aids osmoregulation and cell turgor maintenance (Anjum et al. 2011).The soluble protein in fenugreek seedlings was significantly increased after treatment with ultrasonic waves when comparing untreated controls to 20 min of ultrasonic treatment, the greatest values for soluble protein and soluble carbohydrates of fenugreek seedlings were reported as showed from (Figs. 2, 3).Sound waves of particular intensities (100 dB) and frequencies (1000 Hz) raised the quantities of soluble sugar, protein, and amylase activity in Chrysanthemum, indicating that sound stimulation could boost the metabolism of roots and the growth of Chrysanthemum (Yi 2003).Sound waves of various frequencies and intensities have been found to change the secondary structure of tobacco cell wall proteins by altering Amide I and Amide II links (Ziwei et al. 1999).Sound at specific frequencies and intensities also increased the content of soluble proteins and carbohydrates in the cytoplasm of Dendranthema morifolium callus (Zhao et al. 2003).The ideal intensity and frequency sound field stimulation at the right strength and frequency significantly increased soluble protein content and chrysanthemum growth (Bochu et al. 2004;Yiyao et al. 2002;Yi et al. 2003).
Salt stress led to accumulation of proline while it was reduced by ultrasonication for short periods especially at exposure time 10 min (Fig. 4).In pistachios, proline accumulation under salinity stress can be used as a biochemical indicator of salt stress levels (Shamshiri and Fattahi 2014).Proline has been shown to have several functions during stress, including osmoregulation, osmo-protection, free radical scavenger and antioxidant, protection of macromolecules from denaturation, regulation of cytosolic acidity, carbon and nitrogen reserve after stress relief, reducing lipid membrane oxidation, and stabilization and protection of membranes, proteins, and enzymes from salt damage (Verbruggen and Hermans 2008).Proline accomplishes these goals by safeguarding the photosynthetic apparatus (Ashraf and Foolad 2007).Exhibiting antioxidant activity (Okuma et al. 2004) and acting as an oxygen radical scavenger (Heuer The quantities of proline and soluble protein in the seeds of two rice cultivars, Guangyan1 and Huahang31, were significantly raised after ultrasonic wave treatment for both 10 and 20 min, according to Rao et al. (2018).

Molecular analysis
RAPD is a PCR-based molecular technique, simple and require only small quantities of DNA samples.In this study, the reproducible 4 RAPD primers were: OPA-05, OPA-12, OPA-13 and OPD-15.These primers gave a total number of 26 bands for fenugreek.The total polymorphism percentage was 49.72%.These primers' data in detail were illustrated in the (Table 2; Fig. 5).
The polymorphism percentage and variation in the propagated plants' genetic stability could be explained for many reasons.Such reasons are explained by Sundaram   and Purwar (2011) and Mamatha et al. (2017) who applied RAPD-PCR to characterize the different fenugreek genotypes and accessions.Also, Hanafy and Akladious (2018) applied RAPD-PCR to estimate the effect of gamma radiation on fenugreek plant.In general, it is essential to confirm that the regenerants in genetic fidelity are genetically true-to-type of their donor plants.The RAPD marker system has been used for this target to know where there is an aberration in the regenerated plants.This scheme has been shown to be a potential marker for the distinction between genetic variation and genetic fidelity of different plants (Goda et al. 2017).
In this study, there were variation reflected in the morphological and physiological behavior of fenugreek plant.This could be due to variation in DNA content which is translated into altered proteins.This was confirmed by other studies as follow: it was claimed that certain sound waves could speed up plant growth and alter the transition temperature of cell walls, which was closely related to plant cell division.It was discovered that sound waves sped up (Xiujuan et al. 2003).At the molecular level, ultrasound can have positive or negative effects on the environment, enzymes, substrates, and interactions between them.Ultrasound produces the effects of cavitation, magnetostrictive force, and mechanical oscillation under the right frequency and low intensity conditions.To hasten the contact between the enzyme and substrate, it modifies the conformation of the enzyme.Enzyme biological activity is thereby encouraged (Wang et al. 2015b).

Conclusion
Osmotic and ionic effects on fenugreek seeds cause salinity to suppress germination and plant growth.At 5000 mg/l NaCl, the largest decrease in germination percentage was observed.At the early seedling stage, salinity stress also decreased all growth characteristics.As sodium chloride concentrations rose, levels of total soluble carbohydrates, total soluble protein, and proline rose sharply, indicating that this accumulation was necessary for osmotic correction as salinity levels rose.Fenugreek germination and seedling growth were increased and improved by ultrasonically treating seeds at 40 kHz for 10 and 20 min under control and salinity conditions.By increasing the seed's porosity, ultrasound increases its ability to absorb water and oxygen.It also speeds up metabolic processes, which helps the endosperm's ability to mobilize nutrients through the cell membrane.Due to modifications in the ultrasonic and salt stress treatments, the polymorphism percentage is high and could reach 49.72%.This demonstrates how sensitive plants are to changes in salt and ultrasonic.

Fig. 1
Fig. 1 Effect of ultrasonic exposure time (10 and 20 min) and salt stress on germination percentage of fenugreek at early seedlings stage.Values represent the mean of three replicates.Different letters (a, b, c, d and e) indicate statistical differences at 5% probability according to Duncan's test.Error bars are standard errors of the mean (LSD least significant difference)

Fig. 2 Fig. 3
Fig. 2 Effect of ultrasonic exposure time (10 and 20 min) and salt stress on soluble proteins (mg g −1 d.m) content of fenugreek at early seedlings stage.Values represent the mean of three replicates.Different letters (a, b, c, d, e, f, g, h, i and j) indicate statistical differences at 5% probability according to Duncan's test.Error bars are standard errors of the mean, dm-dry matter (LSD least significant difference)

Fig. 4
Fig.4Effect of ultrasonic exposure time (10 and 20 min.)and salt stress on proline (mg g −1 f.m) (milli gram/gram of fresh matter) content of fenugreek at early seedlings stage.Values represent the mean of three replicates.Different letters (a, b and c) indicate statistical differences at 5% probability according to Duncan's test.Error bars are standard errors of the mean, dm-dry matter (LSD least significant difference)

Table 1
Effect of ultrasonic exposure time and salt stress on growth characteristics of fenugreek at early seedlings stage Data shown in the table represent the mean ± standard error, followed by a small letter; similar letters indicate that means were not different significantly at 5%, probability based on Duncan's test