The impact of irrigation water salinity levels and alpha-tocopherol on some vegetative and chemical characteristics of 'Wonderful' pomegranate seedlings

Introduction

The pomegranate is one of the oldest fruits known to humankind. Its name derives from the Old French words "pome," meaning apple, and "granate," referring to numerous seeds, thus meaning "apple with many seeds" (Abdul Hamid et al., 2017) [7] . The region extending from Iran to the Himalayas in northern India is considered the pomegranate's original habitat. From Iran, it spread to Syria, Jordan, Palestine, and Lebanon, and then to North Africa, Egypt, and the Mediterranean basin (Abdul Hamid et al., 2008). Syria holds a prominent position in pomegranate cultivation, with an area of 6,845 hectares in 2020, and a domestic production of 106,506 tons, according to the Statistical Abstract of Agriculture (Ministry of Agriculture, 2020). Pomegranates have been used in traditional medicine in many cultures and are revered in the Abrahamic religions, being mentioned in the Holy Quran. The pomegranate is characterized by the presence of numerous bioactive compounds beneficial to the human body, concentrated in parts of the fruit such as the juice and peel. The importance of these compounds stems from their therapeutic properties, such as their ability to prevent oxidation and combat free radicals, which play a significant role in the development of many serious diseases, including cancer and atherosclerosis. These compounds include phenols, anthocyanins, tannins, and others, all of which contribute to antioxidant activity (Abdul-Barr & Sadiq, 2020) [6] . Furthermore, these bioactive compounds provide the body with energy and immunity, as they contain a good proportion of simple sugars like glucose and fructose, making them a good source of nutrition and energy. They are also rich in potassium, ascorbic acid (vitamin C), niacin (vitamin B), and several lipid-active compounds such as sterols and plant steroids (Hassan, 2020). Pomegranates are also rich in minerals such as calcium, magnesium, potassium, and zinc, giving their fruit nutritional value (Gil M: Thomas, 2000). These compounds are also rich in minerals such as calcium, magnesium, potassium, and zinc. The juice is extracted from pomegranate fruits, which is also used to produce pomegranate molasses, a method of preserving the fruit for consumption out of season by condensing the juice to remove moisture using techniques such as boiling or vacuum to preserve it for a long time. The results of a study conducted by Abdel-Barr and Sadiq (2020) [6] on the Syrian pomegranate variety (Laffan) confirmed that the optimal temperature for preparing pomegranate molasses under vacuum was 60°C at a vacuum pressure of 30 mmHg. In a study involving six varieties of the Molar group of pomegranates in Spain (Paul et al., 2012) [26] , the highest soluble solids content was found in the sixth, sweet variety (15.81%) The results of studies conducted by Paul et al. (2012) [26] indicated that glucose is the main sugar in all pomegranate varieties, comprising 60-64% of the total sugars, followed by fructose, maltose, and sucrose. Citric acid was the predominant acid. Researchers (Anahita et al., 2015) [8] studied the vitamin content of Spanish pomegranate varieties and found that the concentration of vitamin A in pomegranate juice was 22.8 µg/100 g, the concentration of vitamin C was 57.8 mg/100 g, and the amount of phenols in the juice was 2502 mg. Soil salinity is a harmful environmental factor that hinders the growth and production of horticultural plants worldwide. It is estimated that approximately 20% of agricultural land and half of irrigated land are affected by salinity. Among the most prominent factors contributing to soil salinization are the excessive use of chemical fertilizers, the use of saline irrigation water, and rising groundwater levels (Mastrogiannidou et al., 2016) [22] . This has led to significant degradation in pomegranate orchards, necessitating the adoption of methods to halt this decline and mitigate the damage caused by salt stress in lands irrigated with highly saline water. One such method is the use of certain types of Nano-fertilizer treatments to enhance water and nutrient uptake by plants and reduce salt stress (El-khawaga, 2013) [14] Alpha-tocopherol is an antioxidant chemical compound belonging to the vitamin E family and is generally produced in photosynthetic plants. It is considered a vitamin used to reduce the effects of salinity and is a fat-soluble isoprenoid from the tocoromanol family, primarily synthesized in photosynthetic organisms (Fritsche et al., 2017) [15] . Tocopherols are of great importance, working synergistically with tocotrienols, and are the only natural compounds exhibiting vitamin E activity. They are considered essential nutrients (Traber et al., 1996) [30] . Plants treated with alpha-tocopherol exhibit stress tolerance and protect themselves from oxidative stress caused by various factors (Kumar et al., 2012) [20] .

Objectives of the study 1. To investigate the effect of spraying alpha-tocopherol on pomegranate seedlings irrigated with saline water. 2. Studying the response of pomegranate seedlings to irrigation with saline water. 3. Studying the interaction effect of experimental factors on pomegranate seedlings.

Materials and Methods

Study Location

The study was conducted in the fields belonging to the Department of Horticulture and Landscape Engineering at the College of Agriculture, Al-Qasim Green University, during the 2025 growing season, at the University of Babylon site.

Seedling Preparation

The research was conducted on one-year-old pomegranate seedlings of the Wonderful variety, as uniform in size as possible. The seedlings were obtained from the General Company for Horticulture and Forestry/Horticulture Station in Al-Hindiya, Karbala Governorate, totaling 150 seedlings planted in 2 kg plastic bags. The seedlings were then transferred to 10 kg black plastic (polyethylene) pots, which were filled with soil taken from the ground adjacent to the wooden shade structure at the College of Agriculture after being cleaned of impurities and debris. From the available seedlings, 135 seedlings were selected and distributed equally among the three replicates. Thus, the number of treatments is 9 with three replicates, making the total number of treatments 27. Each treatment is randomly applied to an experimental unit containing five seedlings, for a total of 135 seedlings, with each replicate containing 45 seedlings.

Soil Analysis

Random samples were taken from different areas of the pots' soil before the experiment. This procedure was carried out prior to the seedling treatment to study some chemical and physical properties. The soil analysis was performed at the Ghaya Al-Maarefa Private Laboratory.

Experimental Design

The experiment was designed as a factorial experiment according to the design of the completely randomized block (R.C.B.D), where the experiment includes two factors: the first factor is the salinity levels of irrigation water (river water comparison, 6.3 dS/L), and the second factor is the concentration of alpha-tocopherol (0, 150, 300 mg per liter) produced by the Indian company HIMEDIA, with three replicates (Al-Rawi and Khalaf Allah, 2000) [2]

Date and Number of Sprays

The treatments were sprayed four times, 15 days apart, on the following dates:

Preparation of Study Factors 1. Preparation of Irrigation Water Concentrations

A 2000-liter tank filled with drainage water from the Ibrahimia area was used as the base solution. 500 liters were prepared, and the salinity was fixed at 3 decimeters. Another 500 liters were prepared, and the salinity was fixed at 6 decimeters after adding sodium chloride to the water. This was done using the equation for converting salinity measurements from decimeters to grams. The amount of irrigation water was estimated at one liter per seedling.

Preparation of alpha-tocopherol concentrations

Dissolve each concentration of alpha-tocopherol separately in 100 ml of water in a 1-liter volumetric flask, then complete the volume to 1 liter according to the required concentration.

The seedling height was measured using a tape measure from the soil surface to the top of the main stem. Five seedlings were used per experimental unit, with three replicates.

The diameter of the main stem of the seedlings growing above the point where the main stem meets the soil was measured using a caliper. Five seedlings were used per experimental unit, with three replicates.

(Average Leaf Area (cm²)

The leaf area was measured by taking one leaf from each plant in each experimental unit and using a scanner and the Images software. The average was then multiplied by the number of leaves per plant to calculate the area, according to the method used by Al-Zaidy (2016) [3] . 6-2 Chemical Properties:

Estimation of NPK content in leaves Digestion of samples

A sample of mature leaves and flower heads was taken, then cut and air-dried in a well-ventilated room. The sample was placed in perforated paper bags inside an electric oven at 68°C until weight stabilized. Afterward The sample was transferred to airtight plastic containers and stored in a dry place until its constituent elements could be determined and analyzed (Al-Sahaf, 1989) [4] .

A 0.2 g sample of the ground sample was then dissolved by adding 4 mL of concentrated sulfuric acid and 1.75 mL of concentrated perchloric acid. This was then placed in digestion flasks on a heated plate. Upon completion of digestion, a colorless solution was obtained. The liquid was then transferred to 100 mL plastic containers, and the solution was topped up with distilled water to the mark. It was then transferred to plastic containers according to the method described (Jones and Steyr, 1973) [16] , and the constituent elements were determined as follows:

The percentage of total nitrogen was determined using a Kjeldahl micrometer according to the method described by Page et al. (1982) [24] at the Plant Analysis Laboratory, Faculty of Agriculture, Al-Qasim Green University. Five milliliters of the digested sample were taken, and then five milliliters of 2% boric acid and an indicator of Methyl Red Bromocresal Green were added, along with 10 milliliters of 40% sodium hydroxide (NaOH). The mixture was then distilled, and the released ammonia was collected in a glass flask. The collected ammonia was then rectified using 0.005 M hydrochloric acid (HCl).

The percentage of phosphorus in the plant was estimated using the ammonium molybdate-phenolate method (Al-Sahaf, 1989) [4] . 22.5 g of ammonium molybdate was dissolved in 400 mL of distilled water (Solution 8), and then 1.25 g of ammonium phenolate was dissolved in 300 mL of hot distilled water (Solution 8). Next, solution 8 was added to the other solution, and the mixture was allowed to cool. Then, 250 mL of concentrated nitric acid (3HNO₃) was carefully added and allowed to cool to room temperature. The volume was then brought up to 1 liter of distilled water.

Five mL of the digested sample was then taken, and 5 mL of ammonium molybdate and phenolate solution were added.

The volume was then brought up to 50 mL with water.

The distillate was left to stand for 30 minutes. Afterward, the optical absorption reading was taken using a spectrophotometer at a wavelength of 410 nm. The control sample (Bank) was prepared in the same way, except that the standard curve was prepared using potassium dihydrogen phosphate (4KH2PO). Potassium Concentration (%) Measured using a Flame Photometer according to Page's method (1982) [24] Results and Discussion

Vegetative Growth Characteristics Seedling Height cm

The results in As for the interaction between the two experimental factors, it was observed from the data in the same table that there were significant differences for most of the interactions with the comparison treatment, as the interaction treatment of irrigating the seedlings with ordinary river water (1.5 dS/m-1) with spraying with alpha-tocopherol at a concentration of (300 mg/L-1) achieved the highest rate of seedling height, which reached (82.27 cm), compared to the seedlings that were sprayed with water with a salinity level of (6 dS/m-1) with spraying with alpha-tocopherol at a concentration of (0 mg/L-1), as the rate of seedling height reached the lowest level, which reached (62.33 cm) The same table also shows significant differences in the average stem diameter of pomegranate seedlings based on alpha-tocopherol treatment. A concentration of 300 mg L⁻¹ significantly resulted in the highest average stem diameter of 7.66 mm, compared to seedlings that were not sprayed, which recorded the lowest average stem diameter of 6.91 mm.

Regarding the interaction of experimental factors, the data showed that irrigating seedlings with water at a salinity level of (6 dS/m-1) while spraying with alpha-tocopherol at a concentration of (300 mg/L-1) resulted in a significant increase in the average stem diameter of pomegranate seedlings, reaching (7.92 mm plant-1). Conversely, the lowest average stem diameter was recorded at a salinity level of (3 dS/m-1) while spraying with alpha-tocopherol at a concentration of (0 mg/L-1), reaching (6.49 mm)).

The results in Table ( 4) indicate significant differences in the effect of irrigation water salinity levels on the nitrogen percentage (%). Normal river water significantly outperformed other sources, achieving the highest nitrogen percentage at (2.707%), while the (6 dS•m-1) level recorded the lowest percentage at (2.031%).

The same results also showed significant differences in the percentage of nitrogen achieved when spraying alphatocopherol (mg/L), with the concentration (150 mg/L) achieving the highest percentage (2.260%). Regarding the interaction between the two experimental factors, the data in the same table showed significant differences for most interactions compared to the control treatment. The interaction treatment of irrigating seedlings with normal river water (1.5 dS/m) combined with spraying alphatocopherol at a concentration of (300 mg/L) achieved the highest percentage of nitrogen (3.064%), compared to seedlings sprayed with water at a salinity level of (6 dS/m) combined with spraying alpha-tocopherol at a concentration of (0 mg/L), where the average seedling height reached the lowest level (1.416%).

The results in Table (5) indicate significant differences in the effect of irrigation water salinity levels on the average phosphorus percentage (%). Normal river water level (1.5 dS/m⁻¹) showed the highest phosphorus percentage (0.410%), while level (6 dS/m⁻¹) recorded the lowest (0.295%).

The same results showed no significant differences in the average phosphorus percentage (%) when alpha-tocopherol (mg/L⁻¹) was sprayed. Regarding the interaction of experimental factors, the data in the table indicated no significant differences in the average phosphorus percentage (%). The treatment of irrigating the seedlings with ordinary river water (1.5 dS/m-1) with spraying with alpha-tocopherol at a concentration of (300 mg/L-1) gave the highest percentage of (0.468%), compared to the seedlings treated with water with a salinity of (6 dS/m-1) with spraying with alpha-tocopherol at a concentration of (0 mg/L-1), which achieved the lowest percentage of (0.267%)

Table (6) results showed no significant differences between irrigation water salinity levels in the average percentage of potassium (%), as the normal river water level (1.5 dS.m-1) significantly outperformed, achieving the highest percentage of potassium at 1.091% compared to the level (6 dS.m-1)

The same table also showed no significant differences in the percentage of potassium when alpha-tocopherol was used. Salinity also affects photosynthesis, respiration, and electron transport pathways. This decrease may also be due to indirect effects of salinity on soil properties, which negatively impact seedling growth. The increased osmotic potential of the soil solution at high irrigation water salinity levels leads to a water absorption deficit, reducing cell swelling pressure. This, in turn, affects cell wall flexibility, cell dilation, and elongation, ultimately reducing plant height and vegetative branch growth (Orcutt and Nielsen, 2000) [23] . These results are consistent with those reported by Tozlu et al. (2000) [29] in their study of trifoliate oranges regarding plant height and stem diameter, as well as with those reported by Khalil (2004) [5] in his study of sour orange seedlings regarding the average number of branches, the average number of leaves, and the average leaf area. These findings also align with those reported by Ahmed (1984) [1] .

Effect of alpha-tocopherol concentrations on vegetative characteristics

Tables 1,2, and 3 illustrate the effect of alpha-tocopherol spraying. Spraying at a concentration of 300 mg/L improved the studied vegetative growth indicators. This is due to its effect on increasing cell division and the activity of a number of enzymes such as glucosidase, amylase, βamylase, and phosphatase, in addition to its assistance in the synthesis of other enzymes such as protase, α-amylase, and Smirnoff lipase. It also increased the uptake of macronutrients that accumulate within the plant, leading to increased plant cell division and differentiation processes, and the maintenance of chloroplasts and their effect on carbon synthesis and its products (Dowdle et al., 2007) [12] .

Effect of Salinity Levels on Chemical Properties

Tables (4, 5, 6) indicate a decrease in leaf NPK content. The significant decrease in leaf chlorophyll content at high salinity concentrations may be due to inhibition of chlorophyll biosynthesis or degradation (Kumar, 2021), or dysregulation of chloroplasts (Camejo et al., 2006) [11] . Madan et al. (2004) [21] reported that salinity stress led to a decrease in the rate of photosynthesis and chlorophyll content in salt-tolerant plants, while the decrease was greater in salt-tolerant plants.

Effect of Alpha-Tocopherol Concentrations on Chemical Properties

Tables (4, 5, 6) show a decrease in leaf NPK content. The chlorophyll content in leaves increases significantly when sprayed with alpha-tocopherol, and this increase is associated with an increase in the amount of spray applied. This can be attributed to the effect of alpha-tocopherol, an antioxidant that protects green membranes and chloroplasts from photo-oxidation, creating favorable environmental conditions for photosynthesis. It acts as an internal signal transmitter, increasing cell and electron transport in the PSII photocatalytic system. The results also indicate that spray treatment with a concentration of 300 mg/L of alphatocopherol is superior in increasing leaf content of nitrogen, phosphorus, and potassium (N, P, K). This is because vitamins enhance plant efficiency in terms of carbon content and growth, leading to increased synthesis and translocation of substances to the roots. This, in turn, promotes growth and nutrient uptake, contributing to nutrient accumulation, particularly of NPK. Furthermore, it improves membrane permeability and increases the activity of membraneassociated enzymes, protecting plants by maintaining the structural integrity of the plasma membrane and enhancing its role in mineral nutrient uptake. This aligns with the findings of Semida et al. (2014) [28] and Rady et al. (2015) [27] ).

References

1. Ahmed, Latif RA. Water in Plant Life. Directorate of the House of Books. University of Mosul 1984.
2. Al-Rawi, Mahmoud K and Muhammad KAAA. Design and Analysis of Agricultural Experiments. University of Baghdad. Ministry of Higher Education and Scientific Research. Republic of Iraq 2000.
3. Al-Zidi, Nahir KA. The Effect of Adding Wheat Grafting and Spraying with its Extract on the Growth and Production of Red Cabbage. Master's Thesis. College of Agriculture, University of Baghdad, Republic of Iraq 2016.
4. Al-Sahaf, Hussein F. Applied Plant Nutrition. University of Baghdad. Ministry of Higher Education and Scientific Research. Republic of Iraq 1989.
5. Khalil, Haqqi N. The Effect of Irrigation Water Salinity, Soil Moisture Level, and Soil Texture on the Growth of Bitter Orange Seedlings. (Citrus aurntium L.) Master's Thesis. College of Agriculture. University of Baghdad 2004.
6. Abdulbar, Rahaf and Sadiq S. A study of the effect of applying the concentration under vacuum method on some physical and chemical properties of pomegranate molasses. Arab Journal of Food and Nutrition, 47, 9985 (2020).
7. Abdulhamid, Qatma RG, Majd Al-Ayoubi AD and Anton A. A study of the biodiversity of pomegranate varieties (Punica granatum L.) in the Darkoush region of northern Syria. Jordanian Journal of Agricultural Sciences 2017;(131): 219-22.
8. Anahita A, Asmah R, and Fauziah O (2015).
9. Barkat MR and Sayed TA. Response of Newhall Naval orange to a-Tocophrol under newly reclaimed area conditions I: Vegetative growth and nutritional status. J. Hort. Sci. and Ornamen. Plants, 2012;4(1): 1825.
10. Camejo D, Jime´nez A, Alarco´n JJ, Torres W, Go´mez, JM and Sevilla F. Changes in photosynthetic parameters and antioxidant activities following heatshock treatment in tomato plants. Funct. Plant Biol., 2006;33: 177-187.
11. Dowdle J, Ishikawa T, Gatzek S, Rolinski S and Smirnoff N. Two Genes In Arabidopsis Thaliana Encoding GDP-L-Galactose Phosphorylase Are Required For Ascorbate Biosynthesis And Seedling Viability. The Plant Journal, 2007;52: 673-689.
12. Dutt SK, Bal AR and Bandyopadhyay AK. Salinityinduced chemical changes in Casuarina equisetifolia Forst. Egyptian Journal of Soil Science, 1991;31, 57-63.
13. El-Khawaga AS, Zaeneldeen EM, and Youssef ΜΑ. Comparative Responses of Three Pomegranates (Punica Granatum L.) Varieties to Salinity. Proceedings of Basic and Applied Sciences. Vol 2013;1(1):454-469. Egypt.
14. Fritsche S, Wang X, and Jung C. Recent advances in our understanding of tocopherol biosynthesis in plants: an overview of key genes. Functions, and Breeding of Vitamin E Improved Crops. Antioxidants 2017;6:99. doi: 10.3390/ antiox6040099.
15. Jones JB and steyn WJA. Sampling, Handling and Analyzing plant tissue sampels. Joslyn pp: 1973;248268.
16. Joslyn, MA. Methods in Food Analysis, Physical, Chemical and Instrumental Methods of Analysis 2nd ed. Academic press, New York and London (1970).
17. Kafkafi U, Siddiqi MY, Ritchie RJ, Glass ADM, and Ruth TJ. Reduction of nitrate (13NO3) influx and nitrogen (13N) translocation by tomato and melon varieties after short exposure to calcium and potassium chloride salts. Journal of Plant Nutrition, 1992;15(67):959-975.
18. Kafkafi U, Siddiqi MY, Ritchie RJ, Glass ADM, and Ruth TJ. Reduction of nitrate (13NO3) influx and nitrogen (13N) translocation by tomato and melon varieties after short exposure to calcium and potassium chloride salts. Journal of Plant Nutrition 1992;15(67):959-975.
19. Kumar S. Singh R and Nayyar H. αTocopherol application modulates the response of wheat (Triticum aestivum L.) seedlings to elevated temperatures by mitigation of stress injury and enhancement of antioxidants. J. Plant Growth Regul. 2012;32(2):307314.
20. Madan-Pal, Singh DK, Rao LS and Singh KP. Photosynthetic characteristics and activity of antioxidant enzymes in salinity tolerant and sensitive rice cultivars. Indian J. of Plant Sci., 2004;9(4):407412.
21. Mastrogiannidou E, Chatzissavvidis C, Antonopoulou C, Tsabardoukas V, Giannakoula A and Therios I. Response of pomegranate cv. wonderful plants to salinity. Journal of Soil Science and Plant Nutrition, 2016;16(3), 621-636
22. Orcutt DM and ET Nilsen. The Physiology of Plants under Stress: Soil and Biotic Factors. John Wiley & Sons, Inc, USA 2000.
23. Page AL, Miller RH, & Keeney DR. Methods of Soil Analysis, Part 2, Agron. Monogr, ASA and SSSA, Madison 1982.
24. Pessarakli M. Hand Book of Plant of Crop Stress. Second Edition, University of Arizona. U.S.A 1999.
25. Pual L. Melgarejo J and Martines J. Evaluation of Spanish Pomegranate Juices: Organic Acid, Sugars, and Anthocyanins. International Journal of Food Properties (2012);15(3):481-490.
26. Semida WM, Taha RS, Abdelhamid MT, Rady MM. Foliarapplied α-tocopherol enhances salt-tolerance in Vicia faba L. plants grown under saline conditions. S Afr J Bot 2014;95:24-31.
27. Tozlu I. Moore GA and Guy CL. Effect increasing NaCl concentration on stem elongation, dry mass production, and macro and micro nutrient accumulation in Poncirus trifolita. Australian Journal of Plant Physiology, 2000;27(1):35-42.
28. Traber MG, and Sies H. Vitamin E in Humans: Demand and Delivery. Annu. Rev. Nutr. 1996;16:321-347.