Effect of salicylic acid on morphological and biochemical parameters of Withania somnifera L.

Introduction

Withania somnifera (synonames: ashvagandha, suranjan, winter cherry, Indian ginseng) is a xerophytic plant that nurtures abundantly in Africa, Sri Lanka, Pakistan and India. Withania somnifera is a small shrub which belongs to family Solanaceae. This plant acclaimed as medicinally essential due to the high therapeutic and nutraceutical potential. It possessed a plethora of medicinal uses and pharmacological application. In Ayurvedic system of medicine, W. somnifera is regarded as an important medicinal herb (Singh et al. 2011). 'Ashwagandha' is commonly known as 'Indian ginseng', (Kulkarni and Dhir 2008). Ashwagandha has attributed immense pharmacological research interest. According to the researchers, the plant is reported to have adaptogenic, anticancerous, immunomodulatory, antioxidative properties and in treating neurological disorders International Journal of Advanced Biochemistry Research 2025; SP-9(11): 1233-1243 (Schliebs et al. 1997;Dhuley 2001;Naidu et al. 2003). The extract of Withania somnifera was reported to prevent the skin carcinomaas induced by DMBA (dimethyl benzanthracene) and croton oil (Davis and Kuttan 2001). It was also reported to treat arthritis and neurological behavioural problems (Kulkarni and Ninan 1997). Ashwagandha is one of the most commonly used sedatives in India, similar to ginseng in China (Bown 1995). It has also been reported to have rejuvenating effect on the reproductive and nervous systems and improves the vitality and helps in early recovery after an illness (Bown 1995; Chevallier 1996). Salicylic acid (SA) is a natural plant hormone that plays an important role in plant growth and development, as well as in the plant's response to biotic and abiotic stresses. SA is a defense-related plant hormone that plays a key role in resistance to different microbial pathogens, such as virus, bacteria, fungi, and oomycetes (Kunkel and Brooks, 2002; Vlot et al., 2009) [29] . SA has been shown to regulate plant growth by promoting cell division, elongation, and differentiation, as well as by modulating the balance between growth and defense pathways. SA has also been shown to increase the activity of photosynthetic enzymes and to enhance plant tolerance to various stresses, such as drought, salt, and heat. The mechanisms underlying the effects of SA on plant growth and stress tolerance are complex and involve the regulation of numerous genes and signaling pathways. SA interacts with other plant hormones, such as auxin, gibberellins, and cytokines, and modulates their biosynthesis, transport, and signaling. SA also regulates the expression of genes involved in the biosynthesis of secondary metabolites, such as flavonoids, alkaloids, and phenolics, which play important roles in plant defense against pests and diseases. The application of SA as a plant growth regulator has gained increasing attention in recent years, particularly in the field of horticulture and agriculture. SA has been shown to enhance the growth and yield of various crops, such as rice, wheat, soybean, and tomato, under both normal and stress conditions. SA has also been shown to improve the quality of fruits and vegetables by increasing their antioxidant content and delaying senescence. SA treatment increased the level of antioxidants in tomato plants which helped protect the plants from oxidative stress (Khan et al.2019). It also increased the growth and biomass of tobacco plants (Raskin, I., & Skubatz, H.1995). It can also enhance plant growth and stress tolerance by regulating various physiological and biochemical processes. The application of SA as a plant growth regulator can be done through different methods, such as foliar sprays, root drenches, and seed treatments. The optimal dosage and timing of SA application depend on the plant species, growth stage, and environmental conditions. Excessive or untimely application of SA can lead to negative effects, such as phytotoxicity and reduced yield. However, further research is needed to better understand the mechanisms underlying the effects of SA and to develop efficient and sustainable application strategies for different crops and environmental conditions. Withania somnifera is an adaptogenic herb that is commonly used in traditional Ayurvedic medicine to help the body cope with stress. Salicylic acid, on the other hand, is a type of plant hormone that plays a role in regulating plant growth and development, as well as in the plant's response to stress. There is limited research on the specific effect of salicylic acid on Withania somnifera. Some studies have investigated the effect of salicylic acid on other plants in the same family as Solanaceae, such as tomato and tobacco. So, based on these studies, it is possible that salicylic acid may have similar effect on Withania somnifera by increasing promoting growth. However, further research needed to determine the optimal dosage and application method of salicylic acid for Withania somnifera.

Material and Method

Seeds of Withania somnifera (L.) Dunal. Collected from shreeji beej company (Baroda) We took 108 Withania somnifera seeds and divided them into 4 parts. 27 seeds were put in Control, 27 seeds in 0.25mM, 0.5mM and 1mM concentration of SA respectively. After that seeds were socked into each concentration for 3-4 hrs and put it for the germination. Prepare black plastic nursery bags with soil for the further growth of plant. Keep triplicate for each concentration. Observed the growth of the plants for 9 weeks and performed qualitative and quantitative features at a particular time interval. Morphological characteristics like measurement of shoot and root length and number of leaves measured at the 5 th, 7 th and 9 th weeks intervals. Fresh weight of each plantlet was also recorded at harvested time and put them for drying for https://www.biochemjournal.com 4-5 days at 80 C, and then take dry weight for the same.

Biochemical estimation of chlorophyll, proline and phenol were estimated for all harvested plants (5 th, 7 th and 9 th week's interval).

Micromorphological analysis of leaf samples were done by simple scratch method and peel was prepared and observe the internal structures under a compound microscope (Karl zeiss). Chlorophyll Estimation done by Arnon et al. (1949) [4] , Proline estimation done by Folin -Ciocalteu method (Journal of Science of Food and Agriculture 95(1),204-209,2015) follow for the estimation and proline estimation done by Acid Ninhydrin method (Bates, L.S., Waldern, R.P. and Tare, I. D., et al.1973).

Result and Discussion: Plant growth is a complex phenomenon and determined by several endogenous and exogenous factors. Internal factor like hormones play a vital role in regulating growth and development. The role of SA in plant growth regulation was the first physiological process to be reported (Dekock et al., 1974). Thereafter, both exogenous and endogenous levels of SA have been shown to impart various effects (Raskin, 1992).

Effect of Salicylic acid on quantitative morphological characters

There are few morphological characteristics like number of leaves, root length, shoot length, biomass and leaf area of leaflets, considered to check for the growth of plant (Fig. 3, 4 and5).

Number of leaves

Salicylic acid is used to coat seeds, causing the number of leaf to increase. Number of leaves increased as the weeks are increasing. 0.25 mM showed negative result than the control, but 0.5 mM and 1 mM showed positive result. Among these two 0.5 mM concentrations of SA showed best results. After 9 weeks 0.5mM showed around 26 number of leaves, which was double than the control (Table 1; Graph 1(A)). Similarly, when it was applied to ornamental plants the biomass always increased. Table 2 showed that 0.5 mM and 1mmM showed more shoot length compared to lower concentrations. This result also represented in Graph 1 (B). Root length: Increasing the total soil volume explored by the root of the plant is one of the best ways to improve yield since more water and nutrients may be taken up by the plants. A second important aspect is to increase rooting density which occurs because of increased initiation of secondary roots. In experiments carried out with salicylic acid both aspects (root length and density) were found to be affected. Length of the plant's roots is increasing over time, but this growth is affected by the concentration of SA used to treat the plant. The plants treated with 0.25mM SA showed less root length compared to control. The plants treated with 0.5mM and 1mM showed more root length compared to control. Among these two 0.5mM concentration of SA showed more root length than the 1mM. The showed result of root length is represented in Table 3 which also showed that upto 7 th week length of root increasing but after that root length is decreasing. Graphical representation of results shown in Graph 1 (C).

Leaf Area

Exogenous is well known to be involved in the regulation of various physiological processes such as the activity of photosystem II, plant biomass and leaf area thereby improving plant growth under adverse environmental conditions (Miura and supplementation of salicylic acid in nutrient media may help to improve W. coagulans, maximum increase in leaf area was observed in SA 150 and 200 µM treated seedlings compared to their respective control. To calculate the leaf area, three different sizes of leaves are considered for the study: large, medium and small. Again 0.5mM concentration showed a positive effect on the leaf area of all sized leaves. The result of it is represented in Table 5 and Graph 2 (A). In our present study we also get same result in higher concentration of SA (0.5 mM) that's have more amount of chlorophyll a, chlorophyll b and total chlorophyll present.so that means the 0.5mM concentration prevents the improving photosynthesis in plant. Table 6 showed that 0.5mM concentration of SA showed maximum amount of chlorophyll a, chlorophyll b and total chlorophyll amount (Graph 2 (B)). ). In our present study the phenol content in those seedlings which are treated with 0.5mM concentration of SA showed higher amount compare to various concentration of SA (Table 7, Graph 2 (C)). If the concentrations are increased than amount of phenol is going to be reduced. That is more benefited to these plant because of phenol is defense such as defense against pathogens and UV radiation, and regulation of plant growth and development. Thus, the right amount of phenol present in plant depends on the specific physiological and environmental context. , 2020). Anomocytic type of stomata present in the plant, and are observed in all concentration of SA (Fig. 6 and7).

Stomatal index increasing as the number of weeks increased. 0.5mM and 1mM concentration of SA showed almost similar stomatal index and is 32.35 and 32.33 respectively. Multicellular, branched and unbrached type of trichome observed in plant on both the epidermises (Fig. 6 and7). 1mM concentration of SA showed almost highest trichome index and is 12.5.

Conclusion

Salicylic acid is a great plant hormone which performs a good defense reaction in plants. In the present study, based on the result, we can conclude that exogenous supplements of salicylic acid on seeds were effective in stimulation of morphological characters and secondary metabolites like chlorophyll, proline and phenol etc. production.

Salicylic acid influence leaf chloroplast structure and chlorophyll amount. It also affects the proline, leads to adjustments in osmotic pressure and improves the root system. The major role of proline as a scavenger and improving growth character. 0.5mM concentration of salicylic acid shows interesting results, which stimulate least effect on plant growth and production of secondary metabolites also in high amounts.

Number of leaves, root and shoot length, biomass, leaf area like morphological characters and chlorophyll, proline and phenol like secondary metabolites are increased at 0.5mM concentration of salicylic acid. So, can conclude that 0.5mM if salicylic acid is more suitable for the growth of the Withania somnifera.

References

1. Ahmad G, Khan AA, Mohamed HI. Impact of the low and high concentrations of fly ash amended soil on growth, physiological response, and yield of pumpkin (Cucurbita moschata Duch. ex Poiret L.). Environmental Science and Pollution Research. 2021;28:17068-17083.
2. Ahmad F, Singh A, Kamal A. Ameliorative effect of salicylic acid in salinity-stressed Pisum sativum by improving growth parameters, activating photosynthesis and enhancing antioxidant defense system. Biosci Biotechnol Res Commun. 2017;10:481-489.
3. Aminot A, Rey F. Determination of chlorophyll a by spectroscopic methods. ICES Tech Mar Environ Sci. 1999;1-15.
4. Arnon DI. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol. 1949;24(1):1-15.
5. Asif R, Yasmin R, Mustafa M, Ambreen A, Mazhar M, Rehman A, et al. Phytohormones as plant growth regulators and safe protectors against biotic and abiotic stress. Plant Hormones: Recent Advances, New Perspectives and Applications. 2022;115.
6. Bates LS, Waldren RP, Teare ID. Rapid determination of free proline for water-stress studies. Plant Soil. 1973;39(1):205-207.
7. Beck CB. An introduction to plant structure and development: Plant anatomy for the twenty-first century. Cambridge University Press; 2010.
8. D'Arcy WG, Keating RC, editors. The anther: Form, function and phylogeny. Cambridge University Press; 1996.
9. Dutta R, Khalil R, Green R, Mohapatra SS, Mohapatra S. Withania somnifera (Ashwagandha) and withaferin A: Potential in integrative oncology. Int J Mol Sci. 2019;20(21):5310.
10. Dzib-Ek G, Villanueva-Couoh E, Garruña-Hernández R, Vergara Yoisura S, Larqué-Saavedra A. Effect of salicylic acid on tomato germination and root growth. Rev Mex Cienc Agric. 2021;12(4):735-740.
11. Glazebrook J. Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens. Annu Rev Phytopathol. 2005;43:205-227.
12. Guilfoyle T, Hagen G, Liu X, Rockett KS, Kørner CJ, Pajerowska-Mukhtar KM. Salicylic acid signaling: New insights and prospects at a quarter-century milestone. Essays Biochem. 2015;58:101-113.
13. Hayat S, Hasan SA, Fariduddin Q, Ahmad A. Growth of tomato (Lycopersicon esculentum) in response to salicylic acid under water stress. J Plant Interact. 2008;3(4):297-304.
14. Hua T, Zhang R, Sun H, Liu C. Alleviation of boron toxicity in plants: Mechanisms and approaches. Crit Rev Environ Sci Technol. 2021;51(24):2975-3015.
15. Ibrahim FARAH, Jaber A, Ibrahim GHASSAN, Cheble EDMOND. Antioxidant activity and total phenol content of different plant parts of Lebanese Annona squamosa Linn. Int J Pharm Pharm Sci. 2020;100-105.
16. Jendoubi W, Harbaoui K, Hamada W. Salicylic acidinduced resistance against Fusarium oxysporum f. sp. radicis-lycopersici in hydroponically grown tomato plants. J New Sci. 2015;21:1-10.
17. Karami L, Hedayat M, Farahbakhsh S. Effect of salicylic acid priming on seed germination and morphophysiological and biochemical characteristics of tomato seedling (Lycopersicon esculentum). Iran J Seed Sci. 2020;[pages unavailable].
18. Khan M, et al. Salicylic acid as a plant growth hormone: Roles and application. Crit Rev Plant Sci. 2016;38(3):221-258.
19. Khan MIR, Fatma M, Per TS, Anjum NA, Khan NA. Salicylic acid-induced abiotic stress tolerance and underlying mechanisms in plants. Front Plant Sci. 2015;6:462.
20. Koo YM, Heo AY, Choi HW. Salicylic acid as a safe plant protector and growth regulator. Plant Pathol J. 2020;36(1):1-10.
21. Kumar S, Ahanger MA, Alshaya H, Jan BL, Yerramilli V. Salicylic acid mitigates salt-induced toxicity through modifications of biochemical attributes and key antioxidants in Capsicum annuum. Saudi J Biol Sci. 2022;29(3):1337-1347.
22. Mandal D, Hazarika TK. Salicylic acid maintained quality and enhanced shelf life of tomato 'Samrudhi' at refrigerated storage. Acta Hortic. 2017;1210:207-212.
23. Maurya B, Rai KK, Pandey N, Sharma L, Goswami NK, Rai SP. Influence of salicylic acid elicitation on secondary metabolites and biomass production in Withania coagulans (L.) Dunal. Plant Arch. 2019;19:1308-1316.
24. Movaghatian A, Khorsandi F. Salicylic acid effects on germination of mungbean (Vigna radiata L.) under salinity stress. Adv Environ Biol. 2014;566-571.
25. Richardson AD, Duigan SP, Berlyn GP. An evaluation of noninvasive methods to estimate foliar chlorophyll content. New Phytol. 2002;153(1):185-194.
26. Singh A, Sharma S, Gaurav SS, Kaur H. Salicylic acid: A plant hormone. J Pharmacogn Phytochem. 2019;8(2):32770-3281.
27. Singh M, Agrawal S, Afzal O, Altamimi AS, Redhwan A, Alshammari N, et al. Optimization of elicitation conditions to enhance production of potent metabolite withanolide from Withania somnifera (L.). Metabolites. 2022;12(9):854.
28. Siahmansour S, Nia AE, Nejad AR. Effect of salicylic acid application on morphophysiological traits of Physalis peruviana L. under deficit water stress. J Hortic Sci. 2022;[pages unavailable].
29. Vlot AC, Dempsey DMA, Klessig DF. Salicylic acid, a multifaceted hormone to combat disease. Annu Rev Phytopathol. 2009;47:177-206.
30. Singh N, Bhalla M, de Jager P, Gilca M. An overview on Ashwagandha: a Rasayana (rejuvenator) of Ayurveda. Afr J Tradit Complement Altern Med. 2011;8(Suppl):208-13. doi:10.4314/ajtcam.v8i5S.9.