Acta agriculturae Slovenica, 118/2, 1–13, Ljubljana 2022 doi:10.14720/aas.2022.118.2.1557 Original research article / izvirni znanstveni članek Morphological, biochemical, and nutritional value of prickly and smooth fruit spinach Reza ABOLGHASEMI 1, 2, Maryam HAGHIGHI 3, Nematollah ETEMADI 3 Received March 11, 2020; accepted May 10, 2022. Delo je prispelo 11. marca 2020, sprejeto 10. maja 2022 1 Ph.D. student, Department of Horticulture, College of Agriculture, Isfahan University of Technology, Isfahan, Iran 2 Corresponding author, e-mail: r.abolghasemi@ag.iut.ac.ir 3 Associate Professor, Department of Horticulture, College of Agriculture, Isfahan University of Technology, Isfahan, Iran Morphological, biochemical, and nutritional value of prick- ly and smooth fruit spinach Abstract: This study aimed to investigate the morpho- logical (qualitative and quantitative traits) and biochemical characteristics (such as leaf pigments and total antioxidant capacity, vitamin E and C content, total soluble carbohydrate, total amino acid content, nitrate concentration, nitrate reduc- tase assay, oxalic acid content, Ca and Fe content) in spinach. The selected accessions in this study were prickly (’Varamin Prickly’) and smooth (’Monatol’) fruits of spinach selected among 44 accessions. This experiment was carried out in spring, arranged as a complete randomized block with three replicates and 18 observations. Results showed no significant differences between the two accessions for most qualitative and quantitative morphological traits. In contrast, biochemi- cal characteristics showed significant differences between the two accessions. Both accessions had high yields, but the dry biomass of ’Varamin Prickly’ accession was more than ’Monatol’ (smooth fruit). The results indicated that the fruit type does not appear to cause variations in morphological traits, and differences in accessions could be due to genetic sources and environmental distribution. The prickly fruit accession showed a significant superiority for most qualita- tive nutraceutical traits, including DPPH, flavonoid, phenol, carbohydrate, amino acid, fiber, and Fe content compared to smooth fruit accession. Finally, it was found that prickly fruit accession is very suitable for mechanized harvesting and human diet due to its appropriate plant, leaf, petiole, and qualitative nutraceutical traits and can be used for breeding purposes and cultivation fields. Key words: Iranian spinach accession; prickly fruit; smooth fruit; nutraceutical traits; Spinacia oleracea Morfološka, biokemična in hranilna vrednost špinače z gladkimi in bodečimi plodovi Izvleček: Namen raziskave je bil preučiti morfološke (kakovostne in količinske) in biokemijske lastnosti (listna barvila, celokupno antioksidacijsko sposobnost, vsebnost vitaminov E in C, celokupnih topnih ogljikovih hidratov, ce- lokupnih amino kislin, nitrata, oksalne kisline, Ca in Fe, prei- skus nitrate reduktaze) v špinači. Iz 44 akcesij so bile izbrane sorte z bodečimi (’Varamin Prickly’) im gladkimi (’Monatol’) plodovi. Poskus je bil izveden spomladi kot popolni naključni bločni poskus s tremi ponovitvami in 18 opazovanji. Rezultati so pokazali, da v večini kakovostnih in količinskih morfolo- ških lastnostih obeh tipov ni bilo značilnih razlik. Nasprotno so se vbiokemičnih lastnostih obeh tipov pokazale značilne razlike. Oba tipa akcesij sta imela velike pridelke, a je bila bio- masa ’Varamin Prickly’ večja kot pri akcesijah sorte Monatol. Rezultati so pokazali, da tip plodov ne povzroča raznolikosti v morfoloških lastnostih in, da bi razlike med akcesijami lah- ko bile genetskega ali okoljskega izvora. Akcesije z bodečimi plodovi so bile značilno superiorne v večini kakovostnih hranilnih lastnosti kot so DPPH, vsebnost flavonoidov, feno- lov, ogljikovih hidratov, amino kislin, vlaknin in Fe v primer- javi z akcesijami z gladkimi plodovi. Povzamemo lahko, da bi bile akcesije z bodečimi plodovi zelo primerne v bodočih pro- gramih žlahtnenja in pridelave na polju zaradi primernosti za strojno spravilo in prehrano ljudi zaradi primernih lastnosti habitusa, listov, listnih pecljev rastlin in ugodnih hranilnih lastnosti. Ključne besede: akcesije iranske špinače; bodeči plodo- vi; gladki plodovi; hranilne lastnosti; Spinacia oleracea Acta agriculturae Slovenica, 118/2 – 20222 R. ABOLGHASEMI et al. 1 INTRODUCTION Spinach (Spinacia oleracea L.) is one of the criti- cal and commercial vegetables planted worldwide for fresh and processed consumption (Morelock & Correll, 2008). As one of the origins of spinach, Iran has ranked the seventh producer globally (Shi et al., 2016). This vegetable is a fast-growing, cold season, and nutritious that is highly recommended in the human diet. Spinach has high levels of iron, dietary fiber, vitamins, antioxi- dants, and several phytochemical components. On the other hand, some compounds, such as nitrate and oxa- late, can be high accumulated in leaves that have poten- tial health hazards to humans (Koh et al., 2012). There- fore, selecting and introducing low-capacity nitrate and oxalate varieties is an essential aim in spinach breeding programs (Koh et al., 2012). Pprevious studies were devoted to the genetic structure associated with the morphological traits of spinach (Arif et al., 2013; Sabaghnia et al., 2014). High variation yield was reported among 54 Iranian spinach accessions (Sabaghnia et al., 2014). Nevertheless, vari- ation in the phenotypic characteristics of plants such as leaf type, fruit type (seed type), and even leaf color can affect plant growth and biochemical traits. Like, leaf color affects morphological and biochemical char- acteristics of Sassafras tsumu (Hemsl.) Hemsl. Jiang et al., 2016). Phenotypic differences were also reported in Vachellia nilotica subsp. indica (Benth.) Kyal. & Boatwr. due to having two types of smooth and prickly seeds. (Gorain et al., 2014). Sunflowers from different seeds differ in biochemical properties such as oil and protein content (Balalic et al., 2012). Fruit types, leaf morphology, leaf color (green vs. purple), and day to flowering are spinach’s most critical phenotypic characteristics for breeding classification and commercial purposes (Shi et al., 2016). Wrinkled leaves are marketable in the USA, whereas flat leaves are favorable in Iran (Avsar, 2011). Moreover, fruit forms used in mechanization and handy cultivation are differ- ent (Wu et al., 2015). There are two types of taxonomic varieties of spinach (S. oleracea var. spinosa Moench.) with prickly and (S. oleracea var. inermis Peterm.) with smooth fruits (Mei et al., 2010). Spinach cultivars, with prickly fruits are not suitable for mechanized cultiva- tion. So, it has been suggested that round and smooth fruits, is suited for mechanized spinach cultivation (Morelock & Correll, 2008). Some countries have solved the prickly problem of spinach fruit by using fruit coat- ing (Shi et al., 2016). It is reported that most European varieties of spinach fruits are smooth (Meng et al., 2017). Conversely, most prickly spinach fruits belong to the Asian region consisting of Korea, Japan, Iraq, and Iran. There is a remarkable diversity among the fruits of Asian spinach cultivars due to the vast territory and different climates (Meng et al., 2017; Shi et al., 2016). The researchers reported that prickly fruit spinach varieties had narrow and small leaves with long peti- oles that are generally resistant to low temperatures but sensitive to high temperatures (Meng et al., 2017; Mei et al., 2010). On the other hand, thick and wrinkled leaves with short petioles are generally observed in smooth fruit varieties (Mei et al., 2010). It is reported that smooth fruit varieties of spinach are tolerant to high temperatures and are sensitive to low temperatures (Mei et al., 2010). So, the appearance of spinach fruit is also an essential feature for spinach classification (Wu et al., 2015; Liu et al., 2004). Besides, the type of fruit may be affected by spin- ach quality. Some relationships were observed between the morphological and biochemical characteristics of spinach (Mei et al., 2010). Wu et al. (2015) investigated a wide variety of morphological traits of two smooth and prickly fruits spinach varieties. He reported signifi- cant differences in the growth habits of two fruits types, and morphological and biochemical classification of spinach fruits are critical to distinguish among them (Wu et al., 2015). Therefore, the qualitative assessment of prickly and smooth fruits of spinach, such as antioxi- dants, fiber, iron content, and accumulation of nitrate and oxalate, provided helpful information on cultivar selection (Wu et al., 2015). Recently, due to the extensive cultivation of spin- ach, there has been a vast demand for high-quality seed material for farmers (Jafari & Jalali, 2015). There is a lack of information on the differences between smooth and prickly fruit spinach for the future breeding program. Therefore, this study seems necessary as primitive in- formation for broadcasting future research. The present experiment was conducted to investigate and compare the morphological and biochemical characteristics of the prickly and smooth fruit accessions and the relation of fruit morphology with spinach’s biochemical and nutraceutical characteristics. 2 MATERIALS AND METHODS 2.1 PLANT MATERIAL AND FIELD EXPERI- MENT DESIGN Iranian spinach accession ’Varamin Prickly’ (prickly fruit) and ’Monatol’ accession (smooth fruit), in text later indicated as seeds, were selected from 44 spinach accessions provided by the Seed and Plant Im- provement Institute and the Gene Bank of Iran (SPII) Acta agriculturae Slovenica, 118/2 – 2022 3 Morphological, biochemical, and nutritional value of prickly and smooth fruit spinach and the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) gene bank (IPK, 2018) based on the previous author’s study (Abolghasemi et al., 2019). The selection was based on spinach’s yield and mecha- nized planting characteristics, such as leaf features, plant height, petiole length, fresh mass and yield, dry mass, days to flowering, and percentage of the female plant. In addition, these two accessions had different seed type that was selected for this study. The experiment was designed based on a randomized complete block design (RCBD) with 3 replications and 18 observations. The seeds were sown in a field located at the Isfahan University of Technology, Isfahan, Iran, in March 2018 (spring sowing). The conditions of daylight and tem- perature during spring are presented in Fig 1. The soil was sandy-clay, and manure fertilizer mixed with soil 40 t ha-1 pre-planting. The plot size was 2 m2. After seed germination and plant growth, 18 observations (plant bush) in each plot were selected. It should be noted that this study was carried out in the growth chamber for a more precise evaluation and better comparison of mor- phological and biochemical characteristics of these two accessions (smooth and prickly seed). Finally, the results of our research in controlled conditions confirmed our field cultivation results (data was not presented). There- fore, due to the similarity of the two studies, the field study results are presented in this manuscript. 2.2 MORPHOLOGICAL CHARACTERIZATION To investigate morphological features, 35-50 days after planting (due to non-homogeneous growth of spinach), 18 plants were selected from each plot. Then four leaves were selected from four directions of plants to measure the desired parameters. In the first step, the qualitative and visual characteristics of spinach acces- sions were evaluated based on the descriptor of the In- ternational Plant Genetic Resources Institute (IPGRI) presented in Table 1. (Arif et al., 2013; Jafari & Jalali 2017). Quantitative and morphological characteristics studied in this study included: 1000-grain mass, ger- mination percentage was calculated using the formula when 50 percent of the seeds were germinated (Ikic et al., 2012): Germination percentage = (number of germinated seeds / total number of seeds) × 100 when 50 percent of the seeds were germinated, leaf length (cm), leaf width (cm), petiole length (cm), petiole diameter (mm), leaf area (mm2), plant height (cm), fresh and dry mass (g), yield (kg ha-1), leaf numbers, male and female plants percent, days to flowering. The dry mass of the shoot was measured after putting the bush in an oven at 70 °C for 48 hours (Arif et al., 2013; Jafari & Jalali, 2017). 2.3 BIOCHEMICAL ANALYSIS Leaf pigments: Some fresh leaf tissue (5 g) was mixed with 80 % acetone, then filtered and balanced to 10 ml, and its absorption was measured in 663, 647, and 470 nm with a spectrophotometer (U-2100, JASCO, Japan) (Lichtenthaler, 1987). Chlorophyll a = (19.3 × A663 ‐ 0.86 × A647) Vol- ume / 100 Mass Chlorophyll b = (19.3 × A647 ‐ 3.6 × A663) Vol- ume /100 Mass Total chlorophyll = Chlorophyll a + Chlorophyll b Carotenoids = 100(A470) ‐ 3.27(mg g-1 Chl. a) ‐ 104 (mg g-1 Chl. b)/227 Total antioxidant capacity: The total antioxidant capacity of spinach was measured by the 2, 2-diphenyl- 1-picrylhydrazyl hydrate (DPPH) method (Prasad et al., 2008). Fresh tissue (0.1 g) was mixed in 1.0 ml MeOH and shacked for 2 hours, then centrifuged at 12000 g for 30 min. The supernatant (0.5 ml) was added to the 2.8 ml DPPH solution (0.1 mM) and incubated for 30 mat room temperature. Absorbance was read at 517 nm. The control sample was 2.8 ml DPPH, with the addition of 0.5 ml MeOH. The scavenging activity was determined by the following equation (Stojichevich et al., 2008): Inhibition % = (A control – A sample / A control) × 100 A: Absorption at 517 nm Total flavonoid content: 0.30 ml of the extract was mixed with 0.50 ml of NaNO2 (5 %). After that, Fig 1: Changes in temperature and day length in spinach spring cultivation Acta agriculturae Slovenica, 118/2 – 20224 R. ABOLGHASEMI et al. AlCl3.6H2O (10 %), 0.4 ml NaOH, and 0.20 ml were added. Then, its absorption was performed at 510 nm with a spectrophotometer (U-2100, JASCO, Japan) (Krizek et al., 1998). Total phenolic content: The Folin–Ciocalteu meth- od measured total phenolic content. We were using a spectrophotometer (U-2100, JASCO, Japan) at 765 nm. The standard curve was plotted with the Gallic acid so- lution (Raven, 2003). Vitamin E content: 0.2 ml of alcoholic extract mixed with 5.0 ml toluene, 1.0 ml of ferric chloride, and 3.5 ml of 2, 2-Bipyridine. The mixture was diluted with 10 ml ethanol (95 %), and after 2 min, absorbance was recorded at 525 nm. The standard curve was plotted with vitamin E (Wang & Galletta, 2002). Vitamin C content: Fresh leaf (1.0 g) mixed with 50 ml meta-phosphoric acid (6 %), keeps in the dark for 45 min then centrifuged at 6000 g for 15 min, then 1.0 ml of extract was added to 9.0 ml dichlorophenol indophenol solution (DCPIP) (0.025 %). The next ab- sorbance was read at 515 nm. The standard curve was plotted with different vitamin C concentrations (Djioua et al., 2009). Total soluble carbohydrate content: Total soluble carbohydrate content is measured by the anthrone method (Mc-Cready et al., 1950). For this purpose, 2.0 ml of anthrone was added to the alcoholic extract of the samples. Then, samples were boiled for 2 minutes in the water bath. After cooling and creating the color phase, the absorbance was read at 625 nm. The standard curve was plotted with the glucose standard (Mc-Cready et al., 1950). Total amino acid content: The ninhydrin method was used to measure free amino acids. The ethanol ex- tract of powdered samples was mixed with 1.0 ml of ninhydrin; after boiling and cooling, absorption was read in 575 nm wavelength by spectrophotometer (U- 2100, JASCO, Japan). Calibrated curves were plotted with alanine: y = 0.0198x - 0.0025(r2 = 0.997) (Shih- wen et al., 2006). Crude fiber content: Dried leaves (1.0 g) boiled with 200 ml of H2SO4 for 30 min. The extract was fil- tered, washed with boiling water, the residue boiled with 200 ml NaOH for 30 min then filtered, the solids solid part removed from the filter and placed in a tarred crucible for drying and ashing. It has placed tarred cru- cible with residue in an oven set at 100 ˚C for 8 hours. Then weighted the crucible with fiber residue and cal- culated the residue mass by subtracting the empty cru- cible mass from the crucible and sample mass. Then, the crucible was placed with the dried residue in the oven (550 ˚C) for 4 hours for ashing. Finally, we weighted the crucible with the ash residue in the analytical balance and calculated the ash mass by subtracting the empty crucible mass from the crucible and ash mass. The per- centage of crude fiber was calculated using the follow- ing equation: % Crude fiber content = ((dried residue mass – ash mass) / sample mass) × 100 Nitrate concentration: Nitrate concentration was evaluated by Narayana & Sunil (2009). Briefly, water ex- traction (0.1 g fresh leaf mixed in deionized water) was placed in a boiling water bath for 30 min and centri- fuged at 4000 g for 30 min. Then, extraction was mixed with 0.8 ml salicylic acid (5 %) in concentrated sulfuric acid (95 %) and cooled at room temperature. Then, 19 ml of sodium hydroxide (NaOH, 2N) was added, and the absorbance at 410 nm was determined with a spec- trophotometer (U-2100, JASCO, Japan). The standard curve was plotted with different KNO3 concentration using following standard formula get from standard curve; y = 0.053x + 0.035 (r2 = 0.997). Nitrate reductase assay: The leaf sample (100 mg) was suspended in 5.0 ml of phosphate buffer (0.1 M), KNO3 (0.02M), and propanol (5 %). The solution was kept in the dark water bath (37 °C) for 30 min. The solution was treated with 1.0 ml of sulfanilamide (1 %) and N-1-naphthyl-ethylenediamine (0.02 %). After 15 min, the absorbance was measured at 540 nm with a spectrophotometer (U-2100, JASCO, Japan). The stand- ard curve was plotted with the KNO2 solution: y = 0.0049x + 0.0092 (r2 = 0.988) (Narayana & Sunil, 2009). Oxalic acid content: The dry sample (0.5 g) was mixed with 30 ml HCl (0.25N) and put in a water bath for 15 min. Then, the supernatant was filtered and mixed with 5.0 ml sulfuric acid (2 N) and 2.0 ml potassium permanganate (0.003 M). After 10 minutes, the absorb- ance was detected at 528 nm. y = 0.9126x - 0.0705 (r2 = 0.9327) and standard oxalic acid solution (1 mg ml-1) was prepared with distilled water. (AOAC, 1970). Ca and Fe content: The concentration of Ca and Fe shoot was measured by atomic absorption (Perkin Elmer, 3030, Netherland) after digesting for 12 h with 2.5 ml HCl (36 %) (Nolte, 2003). 2.4 ANALYSIS OF DATA The experiment was arranged in RCBD with three replicates. The morphological and biochemical ob- tained data were analyzed using analysis of variance (ANOVA) by SAS 9.4 comparison of means was per- formed using the least significant difference (LSD) test at a 0.05 level of probability. The correlation between morphological and biochemical traits was also tested Acta agriculturae Slovenica, 118/2 – 2022 5 Morphological, biochemical, and nutritional value of prickly and smooth fruit spinach using the least significant difference (LSD) test at p ≤ 0.05. 3 RESULTS AND DISCUSSION 3.1 QUALITATIVE TRAITS The qualitative characteristics of two accessions based on the spinach descriptor are shown in Table 1. Usually, smooth seeds of spinach are generally more favorable in the world. The ’Monatol’ accession has a smooth seed (presented in Fig 2, C). On the other hand, prickly seed accession (Fig 2, A) is less desirable; they are challenging to plant (Asadi & Hasandokht, 2007). It has been reported that the best seed type for mecha- nized planting of spinach was a smooth and round seed (Morelock & Correll, 2008). In this study, both acces- sions had a gray background in seed color (Table 1). It has been reported that accessions with petiole standing, leaf sheath standing, wrinkled, or slightly wrinkled, are suitable for mechanical harvesting (Mei et al., 2010). Accordingly, ’Varamin Prickly’ (prickly seed) accession is more suitable for mechanical har- vesting than ’Monatol’ (smooth seed) accession because of petiole and leaf form (Table 1). Prickly seed acces- sion has a feature of leaf standing, petiole standing, and low wrinkle, green leaf color appropriate, which can be used in breeding programs, mechanical harvesting, and genetic modification (Table 1). In the USA and other western countries, wrinkled leaves of spinach are more marketable than flat leaves (Kuwahara et al., 2014). In confirmation of this report, smooth seed accession had wrinkle leaves (Fig 2, C, and D). The smooth seed accession has wrinkled dark green leaves and is more suitable for storage because the ventilation is better but hardly washable (Fig 2, D). Usually, spinach cultivars with lower leaf wrinkles have less nitrate content, and there is a direct correlation between leaf wrinkle and nitrate content (Arshi, 2000). So, no-wrinkle leaves as a desirable attribute are inter- ested in researchers. While in Iran, the most favorable spinach has a large, fleshy, thick, low leaf wrinkle and juicy leaf (Fig 2, B), and according to Table 1, the en- demic accession with prickly seed has these features. In Iran, the optimal form of spinach leaf is round, and overseas desirable triangular shapes have been reported (Kunicki et al., 2010). The seed type did not affect leaf shape (Table 1). Leaf color is critical in leafy vegeta- bles since green pigments are desirable and marketable for fresh and frozen spinach (Eftekhari et al., 2010). In this study, both accessions had a complimentary green color, although the foreign accession with smooth seed Se ed ty pe Se ed co lo r Pe tio le at tit ud e W ri nk le s of le af Le af th ic kn es s Le af Sh ea th at tit ud e Le af sh ap e Le af co lo r Le af Sh ea th Lo ba tio n of le af ti p Th e sh ap e of le af ti p W av e m ar gi n of le af Pr ic kl y se ed G ra y- ye llo w Er ec t Ve ry Lo w Th ic k Er ec t Br oa d ov al D ar k gr ee n C on ca ve Be nd C ir cu la r Ye s Sm oo th se ed G ra y- ye llo w H or iz on ta l H ig h Ve ry th ic k H or iz on ta l Br oa d ov al Ve ry da rk g re en C on ca ve U pw ar d C ir cu la r N o Ta bl e 1: Q ua lit at iv e m or ph ol og ic al fe at ur es o f p ri ck ly a nd sm oo th sp in ac h ac ce ss io ns (V ar am in P ri ck ly a nd M on at ol ) a cc or di ng to sp in ac h de sc ri pt or (A ri f e t a l., 2 01 3; Ja fa ri an d Ja la li 20 17 ) Acta agriculturae Slovenica, 118/2 – 20226 R. ABOLGHASEMI et al. was darker in color (Table 1, Fig 2). The leaf shape, leaf sheath, and leaf tip shape were similar in both acces- sions. In general, although the type of seed did not af- fect most of the quality characteristics of spinach in this study, the prickly seed type of spinach was much more desirable for mechanical harvesting than the smooth seed due to the attitude of the petiole and leaf sheath (Table 1). Researchers have reported that prickly seed spinach has better growth in appearance characteristics (Wu et al., 2015; Amoli, 2012) that can be observed in mechanical harvesting for the prickly seed (Table 1). 3.2 QUANTITATIVE TRAITS Analysis of variance showed a significant dif- ference between the two accessions in dry mass, 1000-grain mass, germination percentage, male and fe- male plants at 1 % probability level, and 5 % for petiole length (data was not presented). Mean comparison in Table 2 showed no significant difference in leaf num- ber, plant height, leaf length and width, petiole diam- eter, yield, fresh mass, leaf area, and day to flowering between prickly and smooth seed accessions. There is not much similarity between the investigations that can be reported in each region according to different spin- ach weather and growth conditions. In this study, peti- ole length was more than 100 % higher in the prickly seed accession (Iranian accession) than in the smooth accession, indicating that the plant’s form was more sta- ble in the prickly seed than the smooth seed accession and more suitable for mechanical harvesting (Table 2). Confirming this, it has been reported that plant shape and petiole length are crucial for mechanical harvesting (Shi et al., 2016). Dry mass was the highest in prickly seed accession (Table 2). So, the highest amount of dry mass (14.77 g) was observed in ’Varamin Prickly’ ac- cession (Table 2). yield, fresh and dry mass of ’Varamin Prickly’ was more suitable (Asadi & Hasandokht, 2007; Eftekhari et al., 2010; Jafari & Jalali, 2015). One of the desirable traits for spinach processing and packaging is dry mass, which directly relates to the smoothness of the leaf spinach (Arshi, 2000; Eftekhari et al., 2010). It can also be stated that the different water content in the tissues of these two accessions leads to the dif- ference in dry mass. In Asadi & Hasandokht’s (2007) study, the highest dry mass in ’Qom’ accession was re- ported with prickly seed. The mass of 1000-grains of prickly seed accession was 16.81 g, which had bigger seeds than the smooth seed accession 48.3 % more than the 1000-grain mass of smooth seed accession (Table 2). According to our results, Eftekhari et al. (2010) re- ported that the 1000-grain mass of prickly seed acces- sions was higher than that of smooth seed accession. Prickly seeds appear larger in appearance than smooth ones. The percentage of seed germination of smooth seed accession was higher than the prickly seed acces- sion (showed a 52.3 % increase in germination). There was a significant difference between the two accessions (Table 2), which may be due to more water absorption of smaller seeds during germination. Although prickly seeds are high in mass, it is hard to say that prickly seeds have a much larger surface area than smooth seeds. It is believed that the small seed varieties have higher wa- ter absorption capacity and better establishment due to the higher surface area to volume ratio (Zaferaniye, 2015). Observations on the germination process of spinach seeds have shown that genotypes with better germination had better vegetative growth and yield. (Jaliliyan, 2009; Zaferaniye, 2015). However, this study’s performance was not statistically significant (Table 2). Spinach is a leafy vegetable, so longer vegetative growth is desirable. It is reported that the late-flowering varie- ties are a priority to increase the spinach production period (Jaliliyan, 2009). In this regard, this study classi- fied prickly and smooth seed accessions as spring late flowering (near to 70 days) (Table 2). The researchers reported that the late-flowering accessions were mostly economically desirable (Asadi & Hasandokht, 2007). Therefore, spinach with better vegetative growth, yield, and appearance is more favorable to farmers. It should Fig 2: Comparison of ’Varamin Prickly’ spinach seed (A); leaf (B) and ’Monatol’ spinach seed (C); leaf (D) Acta agriculturae Slovenica, 118/2 – 2022 7 Morphological, biochemical, and nutritional value of prickly and smooth fruit spinach be noted that the result of two traits of yield and day to the flowering of spinach in two different seasons (spring and autumn) can be different because these two traits are affected by the type of growing season (Asadi & Hasandokht, 2007). In this study, both Iranian and foreign accessions (the prickly and smooth seed) were similar in day to flowering and yield (Table 2)). In con- firmation of various reports of spinach being dioecious , both of the accessions were male or female in this study as well (Morelock & Corell, 2008). As can be seen in table 2, the smooth seed accession had significantly more female plants than the Iranian prickly seed acces- sion. So smooth seed accession showed 93.5 % more female plants (Table 2). According to studies, spring spinach cultivation is valuable if the plant can flower later with female flowers (Zaferaniye, 2015). Jaliliyan (2009) recommends planting female varieties for eco- nomic yield. As a result, the smooth seed accession in this study confirms this statement. It has been reported that prickly seeds in winter and spring conditions usu- ally have better growth than smooth seeds (Wu et al., 2015). It can be concluded that prickly seed accession was similar to the smooth seed accession in many traits in spring cultivation. According to the spinach descriptor coding (Table 5), prickly seed accession showed higher valuable traits (a score of 33) in yield. Smooth seed accession has more scores in the mechanical harvesting traits (rated 31), indicating the superiority of mechanical harvesting. According to qualitative characteristics (Table 1), these results are related to more standing leaves and petiole in the Iranian accession, than in the foreign accession. Our results confirm previous reports on favorable veg- etative and mechanical harvesting characteristics for prickly seed accessions (Asadi & Hasandokht, 2007; Eftekhari et al., 2010; Jafari & Jalali, 2015). In the cat- egory of breeding traits, smooth seed accession (with a score of 13) was better than the Iranian accession (with a score of 12), which indicates that the foreign accession is better for breeding traits (Table 5). However, it seems that prickly seed accession should also be considered in terms of breeding characteristics due to near score according to the descriptor scale to smooth seed acces- sion (Table 5). 3.3 BIOCHEMICAL TRAITS Currently, many domestic and wild spinach acces- sions in the country may have more favorable biochem- ical characteristics than foreign accessions. Identifying these genotypes and crossing them improved cultivars with good biochemical characteristics (Sabaghnia et Fe m al e pl an t (% ) M al e pl an t (% ) D ay s t o flo w er in g G er m i- na tio n pe rc en t- ag e (% ) 10 00 -g ra in m as s (g ) Le af a re a (m m 2 ) D ry m as s (g ) Fr es h m as s (g ) Yi el d (k g h- 1 ) Pe tio le di am et er -( m m ) Pe tio le le ng th (c m ) Le af w id th (c m ) Le af le ng th (c m ) Pl an t he ig ht (c m ) Le af nu m be r Se ed ty pe 49 .5 9b 51 .8 6a 69 .6 6a 64 .7 1b 16 .8 1a 48 01 5a 14 .7 7a 60 .0 2a 30 86 7a 4. 35 a 12 .6 6a 7. 33 a 13 .7 3a 25 .0 0a 15 .6 6a Pr ic kl y se ed 95 .9 6a 3. 82 b 69 .0 0a 98 .5 8a 11 .3 3b 60 08 5a 8. 63 b 81 .7 7a 42 05 6a 4. 75 a 6. 20 b 9. 06 a 12 .0 0a 19 .6 6a 20 .2 0a Sm oo th s ee d 11 .2 0. 44 3. 16 13 .2 0. 04 57 66 8 1. 14 25 9. 2 68 59 5 0. 26 2. 84 1. 28 5. 24 8. 48 28 .4 0 St an da rd E rr or Ta bl e 2: M or ph ol og ic al tr ai ts o f p ri ck ly a nd sm oo th s ee d sp in ac h ac ce ss io ns D iff er en t l et te rs w ith in th e sa m e co lu m n in di ca te si gn ifi ca nt d iff er en ce s o f e ac h ty pe a t p ≤ 0 .0 5 by th e LS D te st Acta agriculturae Slovenica, 118/2 – 20228 R. ABOLGHASEMI et al. al., 2014). Results of the analysis of variance showed that there were differences between DPPH, flavonoids, amino acid, and iron content in a 1 % probability level and 5 % probability level in phenol, carbohydrate, and fiber content. No significant differences were observed in some biochemical traits such as photosynthetic pigments, vitamins E, C, nitrate and nitrate reductase activity, and the amount of oxalic acid. (data was not presented). There were significant differences between DPPH, flavonoids, and phenolic compounds they are increased in prickly Iranian accession (69.38 %, 0.76 %, and 79.55 mg 100 g-1 fresh mass, respectively, Table 3). Phenol and antioxidant properties of every region depend on many parameters such as climate, soil, altitude, and different species of plants (Mirzaei et al., 2010). Phenolic and an- tioxidant compounds of Iranian accessions ’Saleh-abad’ and ’Langrood’ had the highest amount of antioxidant compounds, and both of them had prickly seeds (Yosefi et al., 2010). Accordingly, the antioxidant capacity of prickly seed accession was 6.5 % higher than smooth seed accession (Table 3). Plants are one of the im- portant sources of antioxidants compounds that can protect cells from oxidative damage. Secondary plant metabolites such as total phenols and flavonoids de- rived from plants have strong potential for free radical sweeping in different parts of the plant, such as spinach leaves. Iranian spinach is rich in phenolic compounds with antioxidant properties (Chan et al., 2009). Spinach leaves contain active components of fla- vonoids, high antioxidants, and wide pharmacological and biochemical applications, including anti-allergic, anti-viral, and anti-cancer (Lamnitski et al., 2003; Berg- man et al., 2001). In the present study, the content of flavonoids in prickly seed accession was 67.1 % higher than in smooth seed varieties (Table 3). Flavonoids are one of the most widespread and diverse natural com- pounds that can absorb free radicals like other phenolic compounds. In oxidative stresses, phenolic compounds, especially flavonoids, can interact with membrane phospholipids through hydrogen bonding to the polar heads of phospholipids, thereby contributing to mem- brane integrity (Mirzaei et al., 2010). Under non-stress conditions, there were differences in antioxidant, fla- vonoid, and total phenolic activity in different spinach varieties (Barbarin et al., 2005). Soluble carbohydrates are nutrient chemicals valu- able to humans and a source of plant energy. Carbohy- drates of spinach are very important (Bavec et al., 2010). The highest amount of carbohydrate was observed in prickly seed accession with 0.64 mg g-1 fresh mass of spinach (Table 3). Hagen et al. (2009) reported that the carbohydrate content of leafy vegetables varied among the different accessions under the same growth condi- tions. The content of soluble sugars has been affected by pre-harvest growth temperature (Steindal et al., 2015). Amino acids are involved in the structure of spin- ach protein, and amino acids make up about 30 % of all spinach dry matter (Lisiewska et al., 2011). Few arti- cles have been published investigating factors affecting spinach amino acid content (Trejo-Tellez et al., 2005). The level of amino acids in spinach has been correlated with the amount of nitrogen content (Trejo-Tellez et al., 2005). Although nitrogen was not used as fertilizer in the present study, amino acid content was higher in Iranian prickly seed accession (Table 3) (66.6 %) than in other cultivars (Table 3). The studied accessions had significant differences in total fiber content. Prickly Iranian accession showed the highest fiber content (2.03 %) (Table 3). One of the causes of vegetable consumption is fiber. In this opin- ion, the studied Iranian spinach has superiority over the foreign accession in fiber content was more suggested for consumption as a diet food (Erfani et al., 2006). In confirming Iranian spinach accession showed 27.6 % more crude fiber content (Table 3). Spinach is one of the most significant nitrate accumulators because it has a very efficient absorp- tion system and an inefficient nitrate recovery system (Cantliffe, 2005). According to the results of the analysis of variance, nitrate accumulation and nitrate reductase activity in prickly and smooth seed accessions were not significant (data was not presented). It has been report- ed that there is a difference in nitrate accumulation only in smooth and wrinkled leaves (Arshi, 2000). Although the difference in leaf wrinkling between the two acces- sions was obvious, their leaves were not sufficiently dif- ferent in this trait. Probably, nitrate accumulation was not statistically significant (Table 3). Nitrate accumu- lation in vegetables is also reported to be affected by cultivars and even specific genotypic differences such as ploidy levels (Alamian et al., 2014). The studied accessions had significant differences in Fe content (Fig 3). Zaferaniye’s (2015) study reported that the Iranian accessions have higher iron content than the foreign accessions. In the present study, the Iranian spinach accession had higher Fe content, which was 62.1 % more than the foreign accession (Fig 3). Also, the concentration of iron in the Iranian and for- eign accessions of spinach varied from 30 to 50 mg g-1, which was consistent with the amount of iron in this study (Fig 3). Prickly accession was superior in mor- phological traits in most studies (Asadi & Hasandokht, 2007), and the present study also showed the superi- ority of nutraceutical traits in prickly seed accession. In the nutraceutical category, the superiority of the Acta agriculturae Slovenica, 118/2 – 2022 9 Morphological, biochemical, and nutritional value of prickly and smooth fruit spinach prickly seed accession over the smooth seed accession was quite evident (Table 5). Therefore, it is concluded that prickly seed accession had significant antioxidants, flavonoids, total phenols, carbohydrates, amino acids, fibers, and iron content, which was higher than the for- eign accession of smooth seed. Following the results of other researchers, Iranian accessions, including ’Vara- min Prickly’ have a significant superiority in terms of nutritional and functional traits compared to imported foreign accessions, which can be emphasized in the se- lection and improvement of Iranian spinach accessions (Erfani et al., 2006; Zaferaniye, 2015). 3.4 CORRELATION AMONG THE MORPHOLOG- ICAL AND BIOCHEMICAL TRAITS Morphological characteristics of leaves in spring conditions were impressed, as a positive correlation was observed between leaf length and leaf width (r = 0.79*) (Table 4). Similar to our results, Eftekhari et al. (2010) reported a significant correlation between leaf length and width in spinach accessions. Plant height was positively but not significantly correlated with petiole length (r = 0.74), indicating that increasing petiole length was desirable for mechanized harvesting (Table 4). Leaf number is one of the most important yield components in spinach (Jafari & Jalali, 2015), and as shown in table 4, this trait had a significant correla- tion with yield (r = 0.85*). Also, a significant positive relationship was observed between fresh and dry mass (r = 0.86*). There was also a positive relationship be- tween iron content, leaf area (r = 0.79*) and dry mass (r = 0.89**). A good correlation was observed between female plants and the amount of antioxidants’ activity (r = 0.81*), flavonoids (r = 0.96**) and total phenols con- tent (r = 0.61) (Table 4). Also, there was a significant correlation between the percentage of the female plant Se ed ty pe C hl or op hy ll (m g g F M -1 ) C ar ot en oi d (m g g F M -1 ) D PP H (% ) Fl av on oi d (% ) Ph en ol (m g/ 10 0 g F M -1 ) V it E (m g 10 0 g F M -1 ) V it C (m g 10 0 g F M -1 ) C ar bo hy dr at e (m g g F M -1 ) A m in o ac id (μ g g F M -1 ) Fi be r (% ) N itr at e (m g N O 3 g- 1 F M ) N itr at e R ed uc ta se (μ gr N O 2- g F M -1 h ) O xa lic A ci d (m g g- 1 F M ) Pr ic kl y se ed 7. 27 a 1. 65 a 69 .3 8a 0. 76 a 79 .5 5a 1. 04 a 20 .5 4a 0. 64 a 0. 05 a 2. 03 a 10 6. 2a 29 .1 1a 2. 09 a Sm oo th s ee d 6. 1a 1. 43 a 65 .1 1b 0. 25 b 78 .7 5b 0. 76 a 19 .4 9a 0. 36 b 0. 03 b 1. 59 b 10 7. 2a 38 .0 3a 3. 02 a St an da rd E rr or 0. 63 0. 02 5 2. 25 0. 00 3 29 .8 0. 02 5 1. 52 0. 00 7 0. 00 5 0. 04 1 46 5. 3 24 0. 8 0. 31 Ta bl e 3: B io ch em ic al tr ai ts o f p ri ck ly a nd sm oo th s ee d sp in ac h ac ce ss io ns D iff er en t l et te rs w ith in th e sa m e co lu m n in di ca te si gn ifi ca nt d iff er en ce s o f e ac h ty pe a t p ≤ 0 .0 5 by th e LS D te st Fig 3: The mineral concentration of prickly and smooth seed spinach accessions Acta agriculturae Slovenica, 118/2 – 202210 R. ABOLGHASEMI et al. Ta bl e 4: C or re la tio n co effi ci en ts 3 0 m or ph ol og ic al a nd b io ch em ic al tr ai ts st ud ie d on p ri ck ly a nd sm oo th s ee d sp in ac h ac ce ss io ns ** in di ca te s s ig ni fic an t c or re la tio ns a t t he le ve l o f p < 0 .0 1; * in di ca te s s ig ni fic an t c or re la tio n at th e le ve l o f p < 0. 05 . L .N : L ea f n um be r; P. H : P la nt h ei gh t;L .L : L ea f l en gt h; L .W : L ea f w id th : P .L : P et io le le ng th ; P .D : P et io le d ia m et er , Y : Y ie ld ; F .M : F re sh m as s; D .M : D ry m as s; L. A : L ea f a re a; S .W : 1 00 0- gr ai n m as s; G .P : G er m in at io n pe rc en ta ge ; D .F : D ay to fl ow er in g; M : M al e; F : F em al e; C h: C hl or o- ph yl l; C ar : C ar ot en oi ds ; F la v: F la vo no id s; Ph : P he no ls ; C ar b: C ar bo hy dr at es ; A .A : A m in o ac id s; Fi : F ib er ; N it: N itr at e; N itR D : N itr at e re du ct as e; O A : O xa lic a ci d Se ed ty pe N ut ra ce ut ic al a tt ri bu te s To ta l gr ad e C hl or op hy lls C ar ot en oi ds D PP H Fl av on oi ds Ph en ol s V it E V it C C ar bo hy dr at es A m in o ac id s Fi be r N itr at e N itr at e re du ct as e O xa lic ac id C a Fe Pr ic kl y se ed 3 2 4 4 3 3 4 4 4 5 2 1 3 3 4 48 Sm oo th s ee d 2 1 3 2 3 2 3 3 2 2 1 2 2 4 2 34 Yi el d an d m ec ha ni ca l h ar ve st in g at tr ib ut es Le af nu m be r Pl an t he ig ht Le af le ng th Le af w id th Pe tio le le ng th Pe tio le d ia m et er Yi el d Fr es h m as s D ry m as s Le af a re a G er m in at io n Pr ic kl y se ed 3 4 4 1 3 2 3 3 4 3 3 33 Sm oo th s ee d 4 2 2 2 1 2 4 4 1 4 5 31 Br ee di ng fe at ur es Se ed ty pe Se ed w ei gh t D ay to fl ow er in g M al e Fe m al e Pr ic kl y se ed 2 4 2 2 2 12 Sm oo th s ee d 1 2 2 4 4 13 Ta bl e 5: D es cr ip tiv e st at is tic s o f t he m ea su re d tr ai ts in sm oo th a nd p ri ck ly s ee d of sp in ac h ac ce ss io ns (S ab ag hn ia e t a l., 2 01 4; Ja fa ri a nd Ja la li, 2 01 5) † (1 = L ow p oi nt ; 2 = M ed iu m p oi nt ; 3 = H ig h po in t; 4 = ve ry h ig h po in t) Acta agriculturae Slovenica, 118/2 – 2022 11 Morphological, biochemical, and nutritional value of prickly and smooth fruit spinach with antioxidant properties and leaf iron content (Table 4). In confirmation of our results, Asadi & Hasandokht (2007) reported that female plants of spinach had more leafy, yield, and antioxidant properties. A significant correlation was observed between chlorophyll and carotenoid (r = 0.90*). Carotenoid has been reported to have a protective effect on chlorophyll (Macfarland & Burchett, 2001). A positive correlation was observed between ni- trate and petiole length (r = 0.63). However, this rela- tionship was not significant but consistent with other researchers’ reports about the direct relationship be- tween nitrate accumulation and petiole length (Asadi & Hasandokht, 2007; Jafari & Jalali, 2015). The correlation in Table 4 shows that vitamin C has a negative relationship with nitrate (r = -0.41) and a positive relationship with oxalic acid (r = 0.85*). In- creasing nitrate reduces vitamin C in spinach leaves. No significant negative relationship was observed between oxalic acid and nitrate (r = -0.50). According to studies, different results have been reported on nitrate and oxa- late relation; (Kaminishi & Kita, 2006) reported a nega- tive relationship between nitrate and oxalate in spin- ach. Koh et al. (2012) and Zhang et al. (2005) reported a positive correlation between these traits. These reports may indicate a complex pathway involved in nitrate and oxalate metabolism. Fiber content was positively correlated with female plant percentage (r = 0.90*) and negatively with male plant percentage (r = -0.78). It seems that in addition to photosynthetic pigments (Asadi & Hasandokht, 2007), the fiber content will also increase in the female bushes. 4 CONCLUSION It can be concluded that although the foreign smooth seed accession was better in breeding charac- teristics such as 1000-grain mass, germination percent- age, leaf thickness, and female plant %, ’Varamin Prick- ly’ (prickly seed) accession was significantly superior to nutraceutical traits such as antioxidant activity, total phenols, fibers, and iron content. Therefore, to enhance the quality traits in new spinach varieties, ’Varamin Prickly’ (prickly seed accession) is suitable for transfer- ring these traits and breeding purposes. These features may help obtain more information on the qualitative characteristics of Iranian spinach accessions or clarify the factors that influence the nutraceutical properties of Iranian accessions. Therefore, desirable Iranian ac- cessions for nutraceutical and morphological traits can be used in breeding programs for spinach cultivar pro- duction. 5 AUTHOR CONTRIBUTION STATE- MENTS Each named author has contributed to conducting the underlying research and drafting this manuscropt. 6 ACKNOWLEDGMENTS We thank Isfahan University of technology and the Iran National Science Foundation for supporting this work. 7 COMPLIANCE WITH ETHICAL STAND- ARDS (E.G., CONFLICT OF INTEREST) To the best of our knowledge, the named authors have no conflict of interest, financial or otherwise. 8 REFERENCE Abolghasemi, R., Haghighi, M., Etemadi, N., Soorni, A., & Jafari, P. (2019). Screening of some native and foreign ac- cessions of spinach for spring culture in Isfahan. Iran Ag- ricultural Research, 38(1), 87-99. Alamian, M., Eftekhari, S. A., Heidari, M., & Alamzadeh An- sari, N. (2014). 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