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


Research Articles | Published:

Print ISSN : 0970-4078.
Online ISSN : 2229-4473.
Pub Email: contact@vegetosindia.org
Doi: 10.1007/s42535-022-00545-6
First Page: 1427
Last Page: 1436
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Keywords: Salt stress, Ultrasonic waves, Fenugreek, Biochemical constituents, Seedling growth, RAPD-PCR


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.

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Ahmad A, Rafatullah M, Sulaiman O, Ibrahim MH, Hashim R (2009) Scavenging behaviour of meranti sawdust in the removal of methylene blue from aqueous solution. J Hazard Mater 170:357–365

Aladjadjiyan A (2002) Study of the influence of magnetic field on some biological characteristics of Zea mays. J Central Euro Agric 3(2):89–94

Aladjadjiyan A (2007) The use of physical methods for plant growing stimulation in Bulgaria. J Cent Eur Agric 3:369–380

Aladjadjiyan A (2012) Physical factors for plant growth stimulation improve food quality. In: Aladjadjiyan A (ed) Food Production: Approaches, Challenges and Tasks, Intech Open 145–168

Aniat-ul-Haq RV, Agnihotri RK (2010) Effect of osmotic stress (PEG) on germination and seedling survival of lentil (Lens culinaris Medik.). Res. J. Agric. Sci 1(3):201–204

Anjum SA, Wang LC, Farooq M, Hussain M, Xue LL, Zou CM (2011) Brassinolide application improves the drought tolerance in maize through modulation of enzymatic antioxidants and leaf gas exchange. J Agron Crop Sci 197(3):177–185. https://doi.org/10.1111/j.1439-037X.2010.00459.x

Ashraf M, Foolad MR (2007) Roles of glycine betaine and proline in improving plant abiotic stress resistance. Environ Exp Bot 59(2):206–216

Bates LS, Waldren RP, Teare ID (1973) Rapid determination of free proline for water-stress studies. Plant Soil 39(1):205–207

Begum F, Ahmed IM, Nessa A, Sultana W (2010) The effect of salinity on seed quality of wheat. J Bangladesh Agric Univ 8:19–22

Behairy RT, El-Hamamsy SMA, El-khamissi HAZ (2017) Alleviation of salinity stress on Fenugreek seedling growth using salicylic acid, citric acid and proline. Middle East J Agric 474–483

Bochu W, Jiping S, Biao L, Jie L, Chuanren D (2004) Soundwave stimulation triggers the content change of the endogenous hormone of the Chrysanthemum mature callus. Colloids Surf B Biointerfaces 37(3–4):107–112. https://doi.org/10.1016/j.colsurfb.2004.03.004

Carrillo-Lopez LM, Garcia-Galicia IA, Tiradogallegos JM (2021) Recent advances in the application of ultrasound in dairy products: effect on functional, physical, chemical, microbiological and sensory properties. J Ultrasonics Sonochemistry 73:105467

Chartzoulakis KS, Klapaki G (2000) Response of two greenhouse pepper hybrids to NaCl salinity during different growth stages. Sci Hortic 86:247–260. https://doi.org/10.1016/S0304-4238(00)00151-5

Chauhan A, AtulKumar BA, Verma JS, Ghramh HA, AliKhan K, Ansari MJ (2016) Influence of gibberellic acid and different salt concentrations on germination percentage and physiological parameters of oat cultivars. Saudi J Biol Sci 26(6):1298–1304

Chaves MM, Flexas J, Pinheiro C (2009) Photosynthesis under drought and salt stress: regulation mechanisms from whole plant to cell. Ann Bot 103:551–560. https://doi.org/10.1093/aob/mcn125

Chelli-Chaabouni A, Ben Mosbah A, Maalej M, Gargouri K, GargouriBouzid R, Drira N (2010) In vitro salinity tolerance of two pistachio rootstocks: Pistacia vera L. and P. atlantica Desf. Environ Exp Bot 69:302–312

Chen JY, Wen PF, Kong WF, Pan QH, Zhan JC, Li JM, Wan SB, Huang WD (2006) Effect of salicylic acid on phenylpropanoids and phenylalanine ammonialyase in harvested grape berries. Postharvest Biol Technol 40:64–72

Cuartero J, Bolarin MC, Asins MJ, Moreno V (2006) Increasing salt tolerance in the tomato. J Exp Bot 57(5):1045–1058. https://doi.org/10.1093/jxb/erj102

Delachiave MEA, De Pinho SZ (2003) Germination of Senna occidentalis link: seed at different osmotic potential levels. Brazlian Arch Tech 46:163–166

Dhingra HR, Varghese TM (1986) Effect of NaCl salinity on the activities of amylase and invertase in Zea mays L. Pollen Ann Bot 57:101–104

Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15

Fateh E, Sohrabi SS, Gerami F (2012) Evaluation the allelopathic effect of bindweed (Convolvulus arvensis L.) on germination and seedling growth of millet and basil. Adv Environ Biol 6(3):940–950

Florez M, Carbonell V, Martínez E (2007) Exposure of maize seeds to stationary magnetic fields: Effects of germination and early growth. Environ Exp Bot 59:68–75. https://doi.org/10.1016/j.envexpbot.2005.10.006

Goda SM, Ahmed SA, El Sherif F, Ibrahim HHA, AK, (2017) Genetically stable plants with boosted flavonoids content after in vitro regeneration of the endangered Capparis spinosa L. Global Drugs Ther 2(4):1–7

Goussous SJ, Samarah NH, Alqudah AM, Othman MO (2010) Enhancing seed germination of four crops species using an ultrasonic technique. Exp Agric 46:231–242

Hanafy RS, Akladious SA (2018) Physiological and molecular studies on the effect of gamma radiation in fenugreek (Trigonella foenum-graecum L.) plants. Journal of Genetic Eng Biotechnol 16(2):683–692. https://doi.org/10.1016/j.jgeb.2018.02.012

Heuer B (2003) Influence of exogenous application of proline and glycinebetaine on growth of salt-stressed tomato plants. Plant Sci 165(4):693–699

Iqbal MZ, Rahmati K (1992) Tolerance of Albizia lebbeck to Cu and Fe application. Ekologia (CSFR) 11:427–430

Kala S, Godarara AK (2011) Effect of moisture stress on leaf total proteins, proline and free amino acid content in commercial cultivars of Ziziphus mauritiana. J Sci Res 55:65–69

Kapoor K, Srivastava A (2010) Assessment of Salinity Tolerance of Vigna mungo Var. Pu-19 using ex vitro and in vitro methods. Asian J Biotechnol 2:73–85

Kapoor N, Pande V (2015) Effect of salt stress on growth parameters, moisture content, relative water content and photosynthetic pigments of fenugreek variety RMt-1. J Plant Sci 10(6):210–221

Keshavarzi B, Rafsanjani MSO, Moussavinik M, Lak AP (2011) Effect of salt (NaCl) stress on germination and early seedling growth of Spinach (Spinacia oleracea). Ann Biol Res 2:490–497

Kökten K, Karaköy T, Bakoğlu A, Akçura M (2009) Determination of salinity tolerance of some lentil (Lens culinaris M.) varieties. J Food Agric Environ 81:140–143

Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurements with Folin phenol reagent. J Biol Chem 193:25–275

Machikowa T, Kulrattanarak T, Wonprasaid S (2013) Effects of ultrasonic treatment on germination of synthetic sunflower seeds. Int J Agr Biol Eng 7(1):1–3

Mamatha NC, Tehlan SK, Srikanth M, Shivaprasad MK, Karthik RP (2017) Molecular characterization of fenugreek (Trigonella foenum-graecum L.) genotypes using rapd markers. Int J Curr Microbiol Appl Sci 6(6):2573–2581. https://doi.org/10.20546/ijcmas.2017.606.306

Moosavi SA, Siadat SA, Poshtdar A, Direkvand F (2018) Ultrasonic assisted seed priming to alleviate aging damages to milk thistle (Silybum marianum) Seeds. Not Sci Biol 10(2):275–281

Morsi MM, Abdelmigid HM, Aljoudi NGS (2018) Exogenous salicylic acid ameliorates the adverse effects of salt stress on antioxidant system in Rosmarinus officinalis L. Egypt J Bot 58:249–263. https://doi.org/10.21608/ejbo.2018.1772.1124

Munns R, Tester M (2008) Mechanisms of salinity tolerance. Ann Rev Plant Biol 59:651–681. https://doi.org/10.1146/annurev.arplant.59.032607.092911

Nazarli H, Faraji F, Zardashti MR (2011) Effect of drought stress and polymer on osmotic adjustment and photosynthetic pigments of sunflower. Cercetari Agronomice in Moldova 44(1):35–41. https://doi.org/10.2478/v10298-012-0022-9

Nounjan N, Nghia PT, Theerakulpisut P (2012) Exogenous proline and trehalose promote recovery of rice seedlings from salt-stress and differentially modulate antioxidant enzymes and expression of related genes. J Plant Physiol 169(6):596–604. https://doi.org/10.1016/j.jplph.2012.01.004

Oktem HA, Eyidogan F, Selcuk F, Silva JATD, Yucel M (2006) Osmotic stress tolerance in plants: transgenic strategies. Floricult Ornament Plant Biotechnol 194–208

Okuma E, Murakami Y, Shimoishi Y, Tada M, Murata Y (2004) Effects of exogenous application of proline and betaine on the growth of tobacco cultured cells under saline conditions. Soil Sci Plant Nutr 50(8):1301–1305. https://doi.org/10.1080/00380768.2004.10408608

Olfa B, Maha Z, Nada B, Zeineb OA (2018) Effects of Nacl on plant growth and antioxidant activities in Fenugreek (Trigonella foenum graecum L.). Biosci J Uberlândia 34(3):683–696

Pinheiro C, Chaves MM (2011) Photosynthesis and drought: can we make metabolic connections from available data? J Exp Bot 62:869–882. https://doi.org/10.1093/jxb/erq340

Qureshi MI, Israr M, Abdin MZ, Iqbal M (2005) Responses of Artemisia annua L. to lead and salt-induced oxidative stress. Environ Exp Bot 53:185–193

Rahneshan Z, Nasibi F, Lakehal A, Bellini C (2018) Unravelling salt stress responses in two pistachio (Pistacia vera L.) genotypes. Acta Physiologiae Plantarum 40:172. https://doi.org/10.1007/s11738-018-2745-1

Rao G, Ashraf U, Huang S, Cheng S, Abrar M, Mo Z, Pan S, Tang X (2018) Ultrasonic seed treatment improved physiological and yield traits of rice under lead toxicity. Environ Sci Pollut Res 25:33637–33644

Ratnakar A, Raib A (2013) Effect of sodium chloride salinity on seed germination and early seedling growth of Trigonella foenum-graecum L. Var PEB Octa J Environ Res 1:304–309

Saberali SF, Moradi M (2019) Effect of salinity on germination and seedling growth of Trigonella foenum-graecum, Dracocephalum moldavica, Satureja hortensis and Anethum graveolens. J Saudi Soc Agric Sci 18:316–323. https://doi.org/10.1016/j.jssas.2017.09.004

Santos C, Andrade S (2005) Comparison of carbohydrate metabolism in Na2SO4-and naturally-induced senescent sunflower leaves. Agrochimica Pisa 49(5):252–259

Shamshiri MH, Fattahi M (2014) Evaluation of two biochemical markers for salt stress in three pistachio rootstocks inoculated with arbuscular mycorrhiza (Glomus mosseae). J Stress Physiol Biochem 10(1):335–346

Shekari F, Mustafavi SH, Abbasi A (2015) Sonication of seeds increase germination performance of sesame under low temperature stress. Acta Agric Slovenica 105:203–212

Snedecor GW, Cochran WG (1980) Statistical methods, 6th edn. Iowa State University Press, Ames

Srivastava D, Rajiv J, Naidu MM, Puranaik J, Srinivas P (2012) Effect of fenugreek seed husk on the rheology and quality characteristics of muffins. Food Nutr Sci 3(11):1473–1479. https://doi.org/10.4236/fns.2012.311191

Sundaram S, Purwar S (2011) Assessment of genetic diversity among fenugreek (Trigonella foenum-graecum L.), using RAPD molecular markers. J Med Plants Res 5(9):1543–1548

Tahany AAA, Abdullateef TM, Lei Z, Xiaojie Y, Abu Elgasim AY, Haile M, Li C, Cunshan Z (2021) Interaction effects of salinity and ultrasound pretreatment on the phytochemical compounds of clover sprouts. Acta Sci Nutr Health 5(3):90–101. https://doi.org/10.31080/ASNH.2020.05.0838

Tort N, Turkyilmaz BA (2004) physiological investigation on the mechanisms of salinity tolerance in some barley culture forms. J.F.S 27:1–16

Umbreit WW, Burris RH, Stauffer JF, Cohen PP, Johanse WJ, Lee PGA, Potter VR, Schneider WC (1959) Manometric Techniques. Minnieap. Burgess Publ. Co. a manual description method, applicable to study of desiring metabolism. Burgess Publishing Company. c.f. Razak, A.A. 1979. 239

Verbruggen N, Hermans C (2008) Proline accumulation in plants: a review. Amino Acids 35:753–759

Wang J, Wang J, Vanga SK, Raghavan V (2020) High-intensity ultrasound processing of kiwifruit juice: effects on the microstructure, pectin, carbohydrates and rheological properties. Food Chem 313:126121. https://doi.org/10.1016/j.foodchem.2019.126121

Wang S, Liu P, Chen D, Yin L, Li H, Deng X (2015a) Silicon enhanced salt tolerance by improving the root water uptake and decreasing the ion toxicity in cucumber. Front Plant Sci 17(6):759. https://doi.org/10.3389/fpls.2015.00759

Wang WW, Meng TT, Guo DZ, Hai-Le MA, Cao Y, Wang WX (2015b) Research progress on ultrasonic biological effect of food processing, Sci. Technol. Food Ind.

Wani SA, Kumar P (2016) Fenugreek enriched extruded product: optimization of ingredients using response surface methodology. Int Food Res J 23(1):18–25

Xiujuan W, Bochu W, Yi J, Chuanren D, Sakanishi A (2003) Effect of sound wave on the synthesis of nucleic acid and protein in chrysanthemum. Colloids Surf, B 29(2003):99–102

Yaldagard M, Mortazavi SA, Tabatabaie F (2008) Influence of ultrasonic stimulation on the germination of barley seed and its alpha-amylase activity. Afr J Biotech 7:2465–2471

Yasseen BT, Al-Thani RF, Alhadi FA, Abbas RAA (2018) Soluble sugars in plants under stress at the Arabian gulf region: possible roles of microorganisms. J Plant Biochem Physiol 6:224. https://doi.org/10.4172/2329-9029.1000224

Yi J, Bochu W, Xiujuan W, Daohong W, Chuanren D, Toyama Y, Sakanishi A (2003) Effect of sound wave on the metabolism of chrysanthemum roots. Colloids Surfaces b: Biointerfaces 29(2):115–118. https://doi.org/10.1016/S0927-7765(02)00155-8

Yiyao L, Wang B, Xuefeng L, Chuanren D, Sakanishi A (2002) Effects of sound field on the growth of Chrysanthemum callus. Colloid Surface B 24:321–326

Zayed MA, Zeid IM (1998) Effect of water and salt stresses on growth, chlorophyll, mineral ions and organic solutes contents, and enzymes activity in mung bean seedlings. Biol Plant 40:351–356

Zhang YQ, Fu EH, Liang JH (2008) Effect of ultrasonic waves on the saccharification processes of lignocellulose. Chem Eng Technol 31(10):1510–1515

Zhao H, Wu J, Zheng L, Zhu T, Xi B, Wang B, Cai S, Younian W (2003) Effect of sound stimulation on Dendranthema morifolium callus growth. Colloids Surf B Biointerfaces 29(2–3):143–147. https://doi.org/10.1016/S0927-7765(02)00184-4

Ziwei S, Keli S, Jun Y, Guoyuo C, Baoshu X (1999) The secondary structure changes of plant cell-wall proteins aroused by strong sound waves using FT-IR. Acta Photon Sin 28(7):600–602



The authors would like to thank Botany and Microbiology Department, Faculty of Science, Helwan University, Egypt for support with chemicals and instruments required for this work.

Author Information

El-Sattar Amira Mohamed Abd
Botany and Microbiology Department, Faculty of Science, Helwan University, Cairo, Egypt
Tawfik Eman
Botany and Microbiology Department, Faculty of Science, Helwan University, Cairo, Egypt