X-ray irradiation changes germination and biochemical analysis of two genotypes of okra (Hibiscus esculentus L.)

ABSTRACT The objective of this research was to examine the response of different X-ray doses to two okra genotypes, Hassawi okra and the commercial Clemenson genotype on germination and biochemical analysis. The two genotypes of the okra were subjected to various X-ray doses (0–100 Gy). The findings have been shown that the low doses of X-ray up to 5 Gy caused a significant increase in all morphological criteria, total pigment, enzymatic (ascorbic acid, glutathione, and anthocyanin) and nonenzymatic antioxidants (ascorbate peroxidase, catalase, and superoxide dismutase) in the two okra genotypes as compared with untreated plants. On the other side, the high doses of X-ray above 5 Gy considerably decreased all the above parameters and significantly increased lipid peroxidation and oxidative damage (hydrogen peroxide and superoxide anion) in the two okra genotypes as compared with untreated plants. In conclusion, the low doses of X-ray caused stimulation effect and may be used to improve plant growth and alleviate the stress effects. Okra Hassawi genotype is more sensitive to X-ray than the other genotype.


Introduction
Abiotic stress is a noteworthy worldwide issue restricting harvest profitability, as plants are being presented to these in an earthbound situation. Ionizing radiation, for example 'ultraviolet, gamma and X-rays' which happens normally, is the most unsafe worry for every living organism (Sidorov, 1994). Due to reactive oxygen species (ROS) production, crops suffer from oxidative stress when exposed to ionizing radiation. The activation of antioxidants can protect cells against oxidative stress, thereby improving resistance to plant stress Esnault, Legue, & Chenal, 2010). Exposure to X-and gamma-rays causes physicochemical effects hence influencing the growth and physiological changes in plants (Ahloowalia & Maluszynski, 2001). Effects of ionizing radiation at elevated doses are to a large level harmful and hindering. In this concern, some studies indicate that low ionizing radiation doses positively affect the growth of the plant and productivity (Afify, Rashed, Ebtesam, & El-Beltagi, 2013;Aly, Maraei, & Ayadi, 2018;Mohamed, 2011). Lower doses of radiation often cause germination and growth improvements of plants while higher doses result in growth abnormalities, germination retardation or even death of exposed wheat plants (Hong et al., 2018).
Okra (Hibiscus esculentus) is one of the most significant plant crops in Saudi Arabia which is one of the top 10 producers worldwide. The total production of okra in Saudi Arabia has been estimated to be 55.8 thousand tons, thus accounting for about 0.71% of the total world okra production. Okra is a member of the family Malvaceae and represents a significant vegetable plant in Africa and Asia (Kumar et al., 2010). It is developed economically in numerous nations, including 'India, Nigeria, Sudan, Pakistan, Ghana, Egypt, Benin, Saudi Arabia, Mexico, and Cameroon' (Benjawan, Chutichudet, & Kaewsit, 2007). Hassawi okra is a significant vegetable harvest in the Al Ahasa zone in the eastern district of Saudi Arabia (Azooz, Youssef, & AL-Omair, 2011;Youssef & Azooz, 2013). It is viewed as the best and most local cultivar. Okra principally spreads by seeds and has a cultivation cycle duration of 90-100 days. Okra contains protein, carbohydrate (Sharma & Prasad, 2010) and significant amounts of vitamins, nutrients, magnesium, calcium, potassium, and other mineral issues which regularly have a role in the eating routine of developing nations (Kumar et al., 2010). In addition, okra mucilage which is the slimy substance found in fresh as well as dried pods is used in many important medicinal applications such as protective food additives against irritating and inflammatory gastric diseases (Lengsfeld, Titgemeyer, Faller, & Hensel, 2004) and as a suspending agent in paracetamol suspension (Kumar, Patil, Patil, & Paschapur, 2009).
The objective of this research is to examine the response of two okra genotypes, Hassawi okra and the commercial Clemenson genotype to a range of X-ray irradiation (0-100 Gy) and to study the impact of X-ray doses on seedling growth, total chlorophylls, and antioxidants.

Plant materials
Seeds of the local landrace Hassawi okra genotype were collected from 'Hofuf Region, Agricultural Research Center, Ministry of Agriculture and Water, Saudi Arabia'. Clemson genotype seeds are accessible commercially. The seeds were cleaned with tap water before exposure to X-ray and then left to dry for 24 h in room temperature.

Irradiation treatments and experimental design
Using therapeutic X-ray devices 'Clinac 23EX Linear Accelerator, Varian Medical Systems, USA', the seeds of the two okra genotypes (Hassawi and Clemson) were irradiated with different X-ray doses (0, 0.25, 0.5, 1, 2.5, 5, 10, 25, 50, 75, and 100 Gy dose rates of 1.9 k Gy/min). The seeds of the two genotypes were seeded after irradiation in 20-cm plastic pots containing a mixture of soil, peat moss, and vermiculite (1:1:1) under natural conditions of temperature (30°C) and humidity (50%). To guarantee appropriate moisture, the pots were watered as required. There are 10 replicates for each treatment. The plants were harvested after 4 weeks of sowing to evaluate the morphological characteristics, physiological, and biochemical criteria.

Estimation of photosynthetic pigments
The two okra genotypes leaves (0.5 g) were extracted in 10 ml acetone 80% and then centrifuged (3000 × g for 5 min) to estimate the photosynthetic pigments according to Arnon (1949) method.

Estimation of total anthocyanine content
Fresh weight of okra genotype plants was homogenized in methanol containing 1% (v/v) HCl and then filtrated. The filtration was read at 530 and 657 nm using a spectrophotometer as described by Mancinelli et al. (1976).

Determination of ascorbic acid (AsA)
Levels of AsA followed the procedure described by Singh, Ma, Srivastava, and Rathinasabapathi (2006) with few modifications. Briefly, fresh samples (0.5 g) were extracted with 3 ml of 5% (w/v) trichloroacetic acid (TCA) and centrifuged at 18,000 × g for 15 min. AsA was determined in a reaction mixture consisting of 0.2 ml of the supernatant, 0.5 ml of 150 mM phosphate buffer (pH 7.4, containing 5 mm EDTA), and 0.2 ml of the deionized water. Color was developed in reaction mixtures with the addition of 0.4 ml of 10% (w/v) TCA, 0.4 ml of 44% (v/v) phosphoric acid, 0.4 ml of α,αdipyridyl in 70% (v/v) ethanol, and 0.2 ml of 3% (w/v) FeCl 3 . The reaction mixtures were incubated at 40°C for 40 min and the absorbance was recorded at 532 nm.

Enzyme extract preparation
Fresh samples of okra plants of 1.0 g were crushed into a fine powder using liquid nitrogen. Soluble protein was extracted by homogenizing the powder in 5 ml of 50 mM phosphate buffer (pH 7.8) containing 1 mM Ethylenediaminetetraacetic acid (EDTA) and 1% polyvinylpyrrolidone (PVP), with the addition of 1 mM ascorbate in the case of APX assay at 4°C. The homogenate was centrifuged at 15,000 × g for 20 min, and the supernatant was used for the following enzyme activity assay.

Determination of total soluble protein
Using the Bradford (1976) technique, crystalline bovine serum albumin is used as a standard to determine the protein in the supernatant of the enzyme extract of okra genotypes.
2.9. Determination of antioxidant enzymes 2.9.1. Assay of ascorbate peroxidase (APX) activity Ascorbate peroxidase activity (APX, EC 1.11.1.11) was determined spectrophotometrically by a decrease in the absorbance at 265 nm (e = 13.7 mM -1 cm -1 ) using the method reported by Nakano and Asada (1981). The reaction mixture contained 50 mM potassium phosphate buffer pH 7.0, 5 mM ascorbate, 0.5 mM H 2 O 2 , and enzyme extract. Addition of started the reaction. The rates were corrected for nonenzymatic oxidation of ascorbate by the inclusion of reaction mixture without enzyme extract.
2.9.2. Assay of catalase (CAT) activity Catalase activity (CAT; EC 1.11.1.6) was determined by the consumption of H 2 O 2 using the method of Aebi (1983). The reaction mixture (3 ml) contained 50 mM potassium phosphate buffer pH 7.0, 15 mM H 2 O 2 and 50 ml enzyme extract. The reaction was initiated by adding the H 2 O 2 . The consumption of H 2 O 2 was monitored spectrophotometrically at 240 nm (e = 39.4 mM -1 cm -1 ) for 3 min.
2.9.3. Assay of superoxide dismutase (SOD) activity Superoxide dismutase activity (SOD; EC 1.15.1.1) was assayed by measuring its ability to inhibit the photochemical reduction of NBT using the method of Beauchamp and Fridovich (1971). The 3 ml reaction mixture contained 50 mM phosphate buffer Ph 7.8, 13 mM methionine, 75 µM NBT, 2 µM riboflavin, 1.0 mM EDTA and 20 µl enzyme extract. Riboflavin was added last and the reaction was initiated by placing the tubes 30 cm below 15 W fluorescent lamps. The reaction was started by switching on the light and was allowed to run for 10 min. Switching off the light stopped the reaction and the tubes were covered with black cloth. Non-illuminated tubes served as control. The absorbance at 560 nm was recorded. One unit of SOD was defined as that being contained in the volume of extract that caused 50% inhibition of the SOD-inhibitable fraction of Nitro blue tetrazolium (NBT) reduction.

Determination of lipid peroxidation
The lipid peroxidation products were estimated by the formation of thiobarbituric acid reactive substances (TBARS) and quantified in terms of malondialdehyde (MDA) as described by Haraguchi, Saito, Okamura, and Yagi (1995). Okra plants (200 mg) were homogenized in 2 ml of 0.1% (w/v) trichloroacetic acid (TCA), followed by centrifugation at 12,000 × g for 20 min. The supernatant (1 ml) obtained was mixed with an equal volume of TCA (10%) containing 0.5% (w/v) TBARS or no TBARS as blank, and heated at 95°C for 30 min and then cooled in ice. The reaction product was centrifuged at 12,000 × g for 15 min and the supernatant absorbance was measured at 532 and 600 nm. After subtracting the nonspecific absorbance (600 nm), the MDA concentration was determined by its molar extinction coefficient of 155 mM −1 cm −1 , and the results are expressed as mmol/g FW.

Determination of hydrogen peroxide
Hydrogen peroxide was measured by the method described by Capaldi and Taylor (1983) with a slight modification. The ground okra plant tissues in 5% TCA (2.5 ml per 0.5 g tissues) with 50 mg active charcoal at 0 1C, and centrifuged for 10 min at 15,000 × g. The supernatant was collected, neutralized with 4 N KOH to pH 3.6 and used for H 2 O 2 assay. The reaction mixture contained 200 ml of leaf extract and 100 ml of 3.4 mM 3-methylbenzothiazoline hydrazone (MBTH). The reaction was initiated by adding 500 ml of horseradish peroxidase solution (90 U per 100 ml) in 0.2 M sodium acetate (pH 3.6). Two minutes later, 1400 ml of 1 N HCl was added. Absorbance was read at 630 nm after 15 min.
2.12. The production rate of O 2 ·T he production rate of O 2 _ was measured by the modified method as described by Elstner and Heupel (1976). Fresh mass (200 mg) from okra plant tissues was homogenized in 1 ml of 50 mM phosphate buffer (pH 7.8) and the homogenate was centrifuged at 10,000 × g for 10 min. Then, 0.5 ml of the supernatant was added to 0.5 ml 50 mM phosphate buffer (pH 7.8) and 0.1 ml of 10 mM hydroxylamine hydrochloride. After 1 h reaction at 25°C, 1 ml of 17 mM sulfanilamide and 1 ml 7 mM α-naphthylamine were added to the mixture at 25°C, and after 20 min, the specific absorbance at 530 nm was determined. Sodium nitrite was used as a standard solution to calculate the production rate of O 2

·2
.13. Statistical analysis SPSS 10 adaptation programming finished all statistical analyses. A mean and standard error was expressive proportions of quantitative data using variance sample evaluation (ANOVA) examination. P Values at 0.05 were regarded as important, with three replications in each parameter.

Effect of X-ray on morphological parameters
The data in Table 1 showed that treatment of the two okra genotypes (Clemenson and Hassawi) with the low doses of X-ray (0, 0.25, 0.5, 1.0, 2.5, and 5.0 Gy) caused significant increases in the morphological criteria 'shoot and root length, fresh and dry weights of shoot and roots' as compared to untreated plants.
The two okra genotypes irradiated with 5.0 Gy dose reported the most pronounced rise. On the other side, all of the above morphological criteria were significantly reduced when compared to untreated plants by increasing the X-ray doses (10, 25, 50, 75 and 100 Gy). The Clemenson genotype was more tolerant to X-ray than the genotype of Hassawi. Al-Enezi, Al-Bahrany, and Al-Khayri (2012) revealed similar outcomes, which discovered that low X-ray doses improved root growth and leaf length of date palm while the high doses of radiation caused a significant decrease in the same parameter. Exposure of the plants to ionizing radiation caused some physicochemical effects such as the decrease in plant growth, mineral content, and physiological criteria (Ahloowalia & Maluszynski, 2001;Mohamed, 2011). The elevated doses of radiation harm plants to a large extent (Hegazi & Hamideldin, 2010). In addition, there are some evidences from literature that the exposure of low ionizing radiation dose caused stimulation of growth (Aly, Eliwa, & Abd EL-Megid, 2019a;El-Beltagi, Mohamed, Mohammed, Zaki, & Mogazy, 2013).
The elevated doses of radiation that triggered the growth may be due to the effect on the permeability of the plasma membrane, transpiration, and the stomatal opening (Roy, 1974). Also, it induced the reduction in the plant growth hormones (Maherchandani, 1975) and a decrease in calcium absorption that plays an important role in plant growth and physiological process (Sanders, Pelloux, Brownlee, & Harper, 2002). Additionally, Preussa and Britta (2003) indicated that the high rate of gamma radiation during the G2/M stage contributed to cell cycle capture, resulting in a reduction in cell division growth rate and/or fluctuating harm to the entire genome. Reduction of fresh and dry shoot weights may be ascribed to the reduction of shoot moisture content owing to radiation stress (Majeed, Rehman, Ahmad, & Muhammad, 2010). In addition, small doses of radiation may stimulate plant growth and development owing to modifications in plasma membrane intake and safeguard the membrane from nutrient loss (Ashraf, Cheema, Rashid, & Qamar, 2004;Benderitter, Vincent-Genod, Pouget, & Voisin, 2003). Also, it stimulated cell proliferation, cell growth, and enzymatic antioxidants (El-Beltagi, Ahmed, & El-Desouky, 2011).

Effect of X-ray on total photosynthetic pigments
In the leaves of the two okra genotypes, total photosynthetic pigment contents were considerably improved by raising the X-ray doses to 5 Gy relative to untreated crops. The elevated doses of radiation, on the other side, induced a substantial reduction in complete photosynthetic pigments compared to control plants (Table 2). These findings are consistent with those of Al-Enezi et al. (2012) who noticed that total pigments decreased in date palm with increased X-ray doses. In addition, Dhawi and Al Khayri (2008) demonstrated that low levels of the magnetic field caused increment on the date palm photosynthetic pigments while the high levels caused a reduction in these contents. The effect of irradiation on increasing chlorophylls may be due to its effects on stimulation chlorophyll biosynthesis and/or delaying its degradation (Aly et al., 2018). The inhibition in the photosynthetic pigment contents may be due to the release of chlorophyll from its protein complex with ensuing dephytolization and potentially pheophytinization (Saha, Raychaudhuri, Chakraborty, & Sudarshan, 2010), Induction of free radicals (Foyer, 1994), the reduction in biosynthesis is due to a reduction in expression of respective genes (Kim et al., 2009) or an increase in their degradation (Kiong, Lai, Hussein, & Harun, 2008) or pigment decomposition owing to the creation of reactive oxygen species (Dartnell et al., 2011) and changes in the chloroplast such as "chloroplast swelling, thylakoid dilation, rupture of chloroplast outer membrane" (Latif & Mohamed, 2016). Table 2 showed that with increased X-ray doses of up to 5 Gy, ascorbic acid, reduced glutathione, and anthocyanin content was significantly improved, and maximally increased, but X-ray dose above 5 Gy induced a remarkable decrease in these antioxidant substances compared to untreated plants. The most marked increases in 5 Gy plants treated and the biggest decrease in 100 Gy plants treated. Similar outcomes are recorded by Abo-EI-Seoud, Hashim, and Farid (1994) who found that the 40 Gy of gamma ray had the capacity to enhance anthocyanin concentrations. Also, Štajner, Popovic, and Taški (2009) who reported that low irradiation doses caused increased glutathione content in soybean crops but reduced at the lowest dose of irradiation. Also, they were in coincidence with the same trend was obtained by Aly (2010) who found that ascorbic acid production was accelerated at low levels of radiation in-vitro propagated plantlets of Culantro (Eryngium foetidum L.). Ascorbate as an antioxidant was of great importance in preventing (or minimizing) the development of carcinogenic substances from nutritional material. Similarly, Hanafy and Akladious (2018) discovered that the treatment of fenugreek seeds with small doses of gamma rays improved the ascorbic acid content relative to control plants. In the same concern, Aly et al. (2019a) reported that it is possible to use small doses of gamma irradiation to increase crop productivity and increase the secondary metabolites which have commercial importance and are of high value in eggplant. The conceivable cause behind quickened decline of ascorbic acid at high doses of X-rays may be due to increased respiration levels resulting in improved action of enzymes causing rapid ascorbate degradation or due to incomplete conversion of AsA into dehydroascorbic (DHA), which could result in loss of AsA content in plants and may be due to its use by APX as an electron contributor to the cytosol H 2 O 2 equilibrium and in various cell compartments (Chaudhary & Agrawal, 2014). Radiation exposure triggers water ionization, an essential component of living tissues, to convey free radicals despite assistant receptive oxygen species that cause the growth of detoxifying substances (or antioxidants) to limit oxidative stress (Legue & Chanal, 2010). These radicals attack with macromolecules causing oxidative damage, including 'lipids, proteins, sugars, and nucleic acids'. (Pizzimenti, Toaldo, Pettazzoni, Dianzani, & Barrera, 2010).

Results in
Cell reinforcements by antioxidants are viewed as aggravates that can deferral, hinder, or counteract oxidation forms. They can meddle with oxidation by responding with free radicals, chelating metals, and furthermore by going about as oxygen scavengers, triplet just as singlet structure, and moving hydrogen atoms to the free radical structure (Kitazuru, Moreira, Mancini-Filho, Delincee, & Villavicencio, 2004). AsA plays a functional role in plants such as 'development, growth, differentiation, cell division' (Hanafy & Akladious, 2018). Glutathione (GSH) can play a defensive function in rummaging singlet oxygen, hydroxyl radicals, and peroxides. It is involved in the reuse of AsA in chloroplast ascorbate-glutathione (Foyer, 1993). The most significant nonenzymatic antioxidant in plants is GSH and ASA. By scavenging reactive oxygen species, GSH directly protects the plant tissues (Halliwell & Gutteridge, 1989). The increase in the content of anthocyanins, a pigment that plays an important role in radiation absorption. Anthocyanin is a protective pigment that depicts marked changes in response to different types of radiation (Ravindran, Indrajith, Balkrishnan, Venkatesan, & Kulanddaively, 2008). Anthocyanins are flavonoids, which are believed to be related to the overall antioxidant capacity of the plant. Anthocyanins function as major scavengers to eliminate free radicals (Radovanovic & Radovanovic, 2010).
The low doses of radiation stimulate the accumulation and synthesis of antioxidant molecules de novo may be due to increased gene expression (Gicquel, Esnault, Jorrin-Novo, & Cabello-Hurtado, 2011;Hong et al., 2014) and increased monomer degradation of some polyphenols (Ben Salem et al., 2013;Vardhan & Shukla, 2017). The elevated doses of radiation, on the other side, reduced the content of nonenzymatic and enzymatic antioxidants because the plant loses its capacity to overcome oxidative injury (De Micco et al., 2014).

Effect of X-ray on enzymatic antioxidants
Enzymatic antioxidants such as APX, CAT, and SOD were significantly increased at lower doses of X-ray in the two okra genotypes comparing to untreated plants. On the other side, higher X-ray doses above 5 Gy resulted in a significant reduction in antioxidant enzymes relative to nonirradiated plants (Table 3). Similar results are recorded by Chaomei and Yanlin (1993) who found that the high doses of irradiation '20, 40, 60, 80 Krad' caused enhancement in peroxidase and catalase activity of wheat plants. Also, the stimulation of APX activity was triggered by the exposure of two sugarcane varieties to gamma irradiation (Singh, Chandra, Singh, & Singh, 1993). Also, Štajner et al. (2009) found that exposure radish seeds to gamma irradiation (10 Gy) caused stimulation in POD, CAT, and SOD activities. In addition, garlic bulb irradiation (10-120 Gy) led to an enhancement in the activity of POD, CAT, and SOD (Kebeish, Deef, & El-Bialy, 2015). Several trials have imputed enzyme induction either to activate and current enzyme pools or to up-regulate encoding genes or by enzyme structure modulator effects (Foyer, López-Delgado, Dat, & Scott, 1997). Otherwise, Aly, Maraei, and Aldrussi (2019b) recorded an increase in POD and PPO operations resulting in plant protection against oxidative damage in wheat as a consequence of gamma irradiation. By accumulating enzymatic antioxidants that caused ROS scavenging, low doses of radiation-stimulated crop growth and development (Wi et al., 2007;Zaka, Vandecasteele, & Misset, 2002 (Chaitanya, Sundar, Masilamani, & Ramachandra, 2002). After treatment with low doses of radiation, the enzymatic antioxidants increased due to increased regulation of the corresponding genes to provide resistance to cells (Zaka et al., 2002).

Effect of X-ray on lipid peroxidation and oxidative damage
Of the two irradiated okra genotypes, contents of MDA, O 2 •̄, and H 2 O 2 were significantly higher than that of nonirradiated plants (Table 4). At the highest dose of X-ray (100 Gy), all of the above contents achieved its maximum rise compared to control plants. Most pronounced increases in the free radicals were detected in Okra Hassawi genotype than Okra Clemenson genotype. It means that Okra Hassawi genotype is more sensitive to X-ray than the other genotype. Hydroxyl radicals are generated either directly through water oxidation or indirectly through ROS formation (Apel & Hirt, 2004). It is thought that the fundamental impact of radiation on cell membranes by the manufacturing of free radicals stimulates lipid peroxidation. The largest dose of soybean seed irradiation caused MDA content to accumulate (Štajner et al., 2009). Gamma irradiation has resulted in increased content of hydrogen peroxide in various pumpkin tissues such as 'leaves, petioles and hypocotyls' (Wi et al., 2007). In addition, El-Beltagi et al. (2011)  and H 2 O 2 comparing with not treated plants. The rise in lipid peroxidation in okra crops following exposure to elevated doses of X-rays may be due to the breakdown of acylglycerol during radiation processing, leading to free fatty acid release (Niyas, Variyar, Gholap, & Sharma, 2003).

Conclusion
From the results, it is concluded that the two genotypes of okra were subjected to X-ray treatment and various important reactions emerged. The low doses up to 5 Gy were effective in improving plant growth and antioxidant defense system. On the other hand, higher doses (10 to 100 Gy) caused an adverse effect. The Clemson genotype is more tolerant to X-ray than the other genotype. The enzymatic and nonenzymatic antioxidants are  more pronouncedly increased in the Okra Hassawi genotype to help it to overcome the oxidative stress.