Acta agriculturae Slovenica, 121/3, 1–12, Ljubljana 2025 doi:10.14720/aas.2025.121.3.18392 Original research article / izvirni znanstveni članek Sodium chloride, colchicine, and 6-benzylaminopurine can change an- tioxidant property and phenols content in Hypericum perforatum L.: An in vitro study Ara MANTEGHI TAFRESHI 1, Reza MOHAMMADHASSAN 1, 2 Received March 12, 2024, accepted August 07, 2025 Delo je prispelo 12 marec 2024, sprejeto 7. avgust 2025 1 Plant Sciences Department, Amino Techno Gene Private Virtual Laboratory (NGO), Tehran, Iran. 2 corresponding author: rezarmhreza22@gmail.com Sodium chloride, colchicine, and 6-benzylaminopurine can change antioxidant property and phenols content in Hyperi- cum perforatum L.: An in vitro study Abstract: Hypericum perforatum, a medicinal plant from the Hypericaceae family, is known for its wide range of bioac- tive properties, including antidepressant, antiviral, antibacte- rial, and anti-cancer activity. These effects are attributed mainly to its antioxidants and among them phenolic secondary me- tabolites. This in vitro study aimed to investigate the impact of various concentrations of colchicine (0.05, 0.1, 0.2 mg l-1), sodium chloride (NaCl; 0.5, 1. 2 mg l-1), and 6-benzylaminopu- rine (BAP; 0.25, 0.5, 1 mg l-1) on antioxidant activity and total phenol content in H. perforatum over three weeks. A factorial experiment was conducted in a completely randomized design with three replications. The results revealed that all treatments significantly enhanced (p < 0.01) antioxidant capacity, phenol content, and morphological characteristics. Notably, the high- est levels of antioxidant activity and total phenol content were observed in the third week following treatment with 0.25 mg l-1 BAP, 2 mg l-1 NaCl, and 2 mg l-1 colchicine, separately. These findings suggest that the higher concentrations of NaCl and colchicine, along with a lower concentration of BAP, can ef- fectively enhance the biosynthesis of phenolic compounds and antioxidant activity in H. perforatum. Key words: Hypericum perforatum, antioxidant property, phenol content, colchicine, sodium chloride, 6-benzylamino- purine, plant tissue culture. Natrijev klorid, kolhicin in 6-benzilaminopurin lahko spre- menijo antioksidacijske lastnosti in vsebnost fenolov v šen- tjanževki (Hypericum perforatum L.): in vitro raziskava Izvleček: Šentjanževka (Hypericum perforatum L.), zdra- vilna rastlina iz družine krčničevk (Hypericaceae), je zelo po- znana zaradi svojih bioaktivnih lastnosti kot so antidepresiv- na, protivirusna, protibakterijska in protirakovna aktivnost. Ti učinki so v glavnem posledica vsebnosti antioksidantov in med njimi fenolnih sekundarnih metabolitov. Namen te in vitro raziskave je bil preučiti vpliv različnih koncentracij kolhicina (0,05; 0,1; 0,2 mg l-1), natrijevega klorida (NaCl; 0,5; 1, 2 mg l-1) in 6-benzilaminopurina (BAP; 0,25; 0,5; 1 mg l-1) na antioksi- dacijsko aktivnost in vsebnost celokupnih fenolov v šentjanžev- ki v obdobju treh tednov. Popolni naključni faktorski poskus je bil zasnovan v treh ponovitvah. Rezultati so pokazali, da so vsa obravnavanja značilno povečala (p < 0.01) antioksidacijsko sposobnost, vsebnost phenolov in morfološke lastnosti. Najve- čji antioksidacijska aktivnost in vsebnost celokupnih fenolov sta bili ugotovljeni tretji teden po obravnavanjih z 0,25 mg l-1 BAP, 2 mg l-1 NaCl in ločeno po obravnavanju z 2 mg l-1 kolhici- na. Te ugotovitve nakazujejo, da lahko večje koncentracije NaCl in kolhicina hkrati z manjšimi koncentracijami BAP učinkovito pospešijo biosintezo fenolnih spojin in povečajo antioksidativ- no aktivnost šentjanževke. Ključne besede: Hypericum perforatum, antioksidacijke lastnosti, vsebnost fenolov, kolhicin, natrijev klorid, 6-benzila- minopurin, rastlinske tkivne kulture Acta agriculturae Slovenica, 121/3 – 20252 MANTEGHI TAFRESHI and MOHAMMADHASSAN 1 INTRODUCTION Hypericum perforatum L., belonging to Hyperica- ceae, includes approximately 400 species, with 10 mor- phologically and chemically essential species world- wide. H. perforatum is a perennial, herbaceous, erect and rarely glabrous plant with a height of 90-100 cm. The shoot contains many branches covered by red or black, amber glands. The leaves are on smaller and nar- rower branches, ovate to almost linear, with 5.35×2-14 mm dimensions (Saleh, 2023; Shamilov et al., 2019). This plant has a long history of medicinal use, initially applied for treating burns and skin injuries. In recent decades, it has gained substantial attention as an effective treatment for depression. Clinical and pharmacological investigations have indicated that H. perforatum exhibits antidepressant efficacy superior to several conventional antidepressants, with a notably lower incidence of side effects (Leandro et al., 2017). In addition to the neuropharmacological potential, H. perforatum has shown broad-spectrum antiviral activi- ties, particularly against hepatitis C (HCV), human im- munodeficiency virus (HIV), and neurotropic viruses (Tariq et al., 2019), contributing to neuroinflammation and neurodegenerative diseases (Ramedani et al., 2015; Marawne et al., 2022). The antibacterial properties of H. perforatum extract have also been observed against My- cobacterium tuberculosis Zopf 1883 and various wound- associated bacterial strains (Imreova et al., 2017). Mo- reover, the plant has been traditionally utilized for a wide range of therapeutic purposes, including tonic and digestive support, choleretic and urinary antiseptic ac- tions, anti-influenza and anxiolytic effects, astringency, enhancement of respiratory and uterine function, and alleviation of arterial inflammation (Budantsev et al., 2021; Velingkar et al., 2017). Secondary metabolites cause therapeutic effects. The hydroalcoholic extract from the upper parts of the plant contains 6 major groups of natural products, including naphthodianthrone, phloroglucinol (par- ticularly hypericin and pseudohypericin), flavonoid, biflavone, phenylpropane, and proanthocyanidin. In addition, there are lower amounts of tannin, xanthone, and amino acids, which have been found in all parts of Hypericum spp (Suryawanshi et al., 2024; Saleh, 2023). Phenol is a simple organic compound formed by bonding a hydroxyl group to a benzene ring. The compound with the formula C6H5OH is known as hy- droxybenzene or, more commonly, phenol. It is also historically referred to as carbolic acid. Pure phenol is a white solid, usually water-soluble, with a melting point of 42  °C and low acidity (Albuquerque et al., 2021). Almost all phenolic compounds show antimicrobial activity, which is not very specific. The antimicrobial activity of phenols maight be caused by damaging the structure and changing the permeability of the cell wall and membrane in microorganisms and lysosomes (Ou- lahal and Degraeve, 2022). Although this antimicrobial activity is specific to some antibiotics, the general anti- microbial effects of many phenols are irreversible when diluted with water. In addition, bacteria cannot acquire immunity against the initial inhibitory concentration of a phenolic compound. Consequently, phenols are eco- nomically valuable antimicrobial agents (Lima et al., 2023). Phenol has a specific anthelmintic activity that increases with the presence of alkyl groups. The most effective anthelmintic phenolic compounds need to be low water soluble, so they are not absorbed from the intestine (Manjusa and Pradeep, 2022). Antioxidants are chemical compounds that neu- tralize free radicals, an active, harmful ingredient for human health, to preserve or delay oxidative damage to biomolecules. Antioxidants are used as food addi- tives to increase the shelf life of oils and fatty foods and maintain their quality during storage and consumption (Gulcin, 2020). Many studies indicate that some syn- thetic antioxidants may harm the body. Antioxidants reduce the risk of cardiovascular diseases and stroke by neutralizing free radicals. On the other hand, antioxi- dants can suppress the cancer process and protect cell membranes (Parcheta et al., 2021). Natural antioxidants have recently been used in food, medicine, and phar- maceutical industries. Consequently, the provision of herbal essential oils and plant-derived secondary me- tabolites as natural antioxidant alternatives plays a cru- cial role in mitigating the adverse effects of oxidative stress. Many scientists extract these compounds from medicinal plants, with fewer side effects and high effi- ciency (Zeb, 2020). Salinity is a vital factor in reducing crop yield; the effects of salinity are not limited to a specific stage of plant growth but are effective throughout the entire growth period of the plant and ultimately lead to a decrease in yield (Hameed et al., 2021). Salt stress can damage plants in two ways; first, the high concentration of salts, especially the high concentration of sodium, destroys the soil structure. As soil porosity decreases, both soil ventilation and hydraulic conductivity are de- bilitated in such soils. Second, high salt concentrations are closely related to water stress. High concentrations of solutes reduce the water potential of the soil and make it more difficult for plants to absorb water and nu- trients. In other words, some physiological drought oc- curs. Because both drought and salinity cause osmotic stress. Hence, the reactions of the plant are similar to Acta agriculturae Slovenica, 121/3 – 2025 3 Sodium chloride, colchicine, and 6-benzylaminopurine ... property and phenols content in Hypericum perforatum L.: An in vitro study the osmotic stress (Giordano et al., 2021; Etesami et al., 2021; Khan et al., 2020). Colchicine (Col) has been used as the most effec- tive chemical compound in studies to induce polyploidy in plants. Chol is an alkaloid extracted from the seeds and pods of Colchicum autumnale L.. The synthesized form of the compound is known as clomid. Col, as a natural inhibitor of mitosis, leads to the creation of a cell with a doubled number of chromosomes by creat- ing an inhibitory state in the formation of spindle fi- bres and, as a result, preventing the polar migration of chromosomes and, eventually, cell division (Natt and Sattely, 2021; Gracheva et al., 2020). Technically, this compound prevents the formation and polymerization of microtubules by linking with the protein subunit of microtubules called tubulin. Finally, the chromosomes simultaneously enter the cell at the metaphase stage. Consequently, the ability has turned into a polyploid inducer. Thus, Col is the most critical chemical agent for chromosomal doubling that is widely used (Cui et al., 2023; Yuling et al., 2022). In the present study, we aimed to analyze the ef- fects of Col, sodium chloride (NaCl), and 6-benzylami- nopurine (BAP), as the common synthetic form of cyto- kinin widely used in plant tissue culture, on antioxidant property (AP) and total phenol content (TPC) of H. perforatum, in vitro conditions. 2 MATERIALS AND METHODS 2.1 PLANT MATERIALS AND CHEMICAL REA- GENTS Seeds of H. perforatum were provided from Pakan Bazr, Isfahan, Iran. All Media, chemical compounds and synthetic phytohormones, particularly NaCl, Col, and BAP, were from Sigma-Aldrich, St. Louis, Missouri, United States. All experiment stages were conducted in Amino Techno Gene Private Laboratory (NGO), Tehran, Iran. 2.2 PREPARING AND CULTURING EXPLANTS The seeds were sanitized and then cultured into the basal MS agar media to germinate and grow for 2 months at 25 ºC and 16/8 light and darkness. After that, the ex- plants were regenerated in different hormone-free MS (Murashige and Skoog, 1962) agar media containing dif- ferent dosages of BAP (0.25, 0.5, 1 mg l-1), Col (0.05, 0.1, 0.2 mg l-1), and NaCl (0.5, 1, 2 mg l-1). 2.3 MORPHOLOGICAL ANALYSIS For 3 weeks, the explants were weekly taken out from the treated media, and then different plant parts, including aerial parts, were quickly weighed to meas- ure the fresh mass (FM; g) of the aerial parts (leaves and shoots) and other morphological characteristics, includ- ing length of shoots (cm) and the number of leaves and shoots. 2.4 EXTRACTION OF THE ESSENTIAL OIL The essential oil of the treated H. perforatum was extracted with methanol solvent for measuring thymol production, elicited by different treatments. 20 ml of methanol was added to a laboratory mortar containing a sample from the aerial parts of each treatment, and then well ground with a pestle. After that, the well-ground samples were moved into a flask and covered with alu- minium foil and then incubated and shaken in a shak- er incubator (TSHE 53, Nour Sanat Tajhiz Co., Karaj, Alborz, Iran) at 140 rpm for 48 hours, without any set temperature. In the next step, the solutions were centri- fuged (Sahand Azma Tajhiz, Tehran, Iran) at 20 °C for 15 minutes at 3000 g. The above phase was transferred into a beaker and then allowed to evaporate in the open air and darkness for 24 hours. Consequently, the solvent was removed from the concentrated extract and maintained in cool and dark conditions. 2.5 TPC ANALYSIS First, 10 ml of methanolic extract and 490 ml meth- anol 80 % were poured into a 15 ml Falcon. Next, 500 ml of Folin-Ciocalteu was added to the content. After 2 min, 1 ml of sodium carbonate 7 % was added to the mixture. The final volume was made up to 6 ml using distilled wa- ter. The falcon, containing the mixture, was incubated in a bain-marie bath at 30 ºC in the dark for 90 min; then, the absorption spectrum was measured using a spectro- photometer (S2100, Unico, USA) at 725 nm. Gallic acid was used as a standard solution. 2.6 2,2-DIPHENYL-1-PICRYLHYDRAZYL (DPPH) ANALYSIS Different concentrations of methanolic extraction were used to analyze antioxidant potential (AP). After that, 1 ml of DPPH 0.4 mM was added to each concen- tration. The final volume was made up to 5 ml using pure Acta agriculturae Slovenica, 121/3 – 20254 MANTEGHI TAFRESHI and MOHAMMADHASSAN methanol. The reaction mixture was vigorously mixed and then incubated at room temperature in the dark for 30 min. The positive control sample contained 1 ml DPPH 0.4 mM and 4 ml pure methanol. The spectropho- tometer was calibrated with methanol. The absorption spectrum was measured using a spectrophotometer at 517 nm. 2.7 STATISTICAL ANALYSIS This experiment was a factorial, completely rand- omized design with three replications. Duncan’s multi- ple-range test (p < 0.01) was used to analyze variance and means comparison. Also, the relationship between traits was analyzed by the correlation coefficient. SAS software (Version 9.2) and Microsoft Excel (2016) were employed for statistical analysis and graph drawing, respectively. 3 RESULTS AND DISCUSSION 3.1 BAP According to the results, BAP, duration, and BAP×duration significantly influenced all traits, includ- ing leaf number, shoot number and length, FM, AP, and TPC (p < 0.01; Table 1). The means of the BAP effect on the traits were compared by Duncan’s multiple-range test at p < 0.05. The maximum and minimum numbers of leaves were observed in the highest BAP concentra- tion, the most prolonged duration (13), and the lowest BAP × time interaction (3). Besides, the highest number of shoots was in 1 mg l-1 BAP in the 3rd week (7), and the lowest number of shoots was measured in 0.25 mg l-1 in the 1st and 2nd weeks, and 0.5 mg l-1 in the 2nd week (1 cm). Also, the highest shoot length was observed in 1 mg l-1 BAP in the 3rd week (29 cm), and the lowest length of shoot was in 0.25 mg l-1 in the 1st week (6 cm). In case of FM, the highest and the lowest amounts were measured in 1 mg l-1 BAP in the 3rd week (g) and 0.25 mg l-1 in the 1st week (g), respectively. However, AP and TPC were higher in the lowest BAP concentration and increased over time (233 and 8.01, respectively; Figure 1). More- over, there are significant correlations (p < 0.01) among leaf number, shoot number and length, FM, AP, and TPC affected by BAP; however, the correlation between shoot length and TPC was significant at p < 0.05 (Table 2). Nazir et al. (2022) studied the BAP effect on in vitro micropropagation of mg –l Valeriana jatamansi Jones ex Roxb. They found that shoot length and leaf number can be enhanced when BAP concentration increases in the MS culturing medium. In another study, growth in BAP level caused higher shoot length and number rates in Plectranthus amboinicus Lour. (Arumugam et al., 2020). Also, Kharel et al. (2022) found that higher BAP concentrations can enhance shoot numbers in highbush blueberry explants (Vaccinium corymbosum L.). In addi- tion, a study demonstrated that the highest FM level of Lamprocapnos spectabilis (L.) Fukuhara was significantly induced by higher BAP concentrations (Kulus, 2020). Kozak et al. (2021) reported the same results in Mandev- illa sanderi (Hemsl.) Woodson micropropagation. In an in vitro study on Lycium barbarum L. (goji berry), MS medium containing various hormones was used to in- vestigate plant regeneration and somatic embryogenesis. Among cytokinins, BAP had a lower effect on shoot pro- duction compared to TDZ and other compounds, while the combination of TDZ and 2,4-D induced the highest number of shoots and embryos. This study showed that the selection of the appropriate type and concentration of cytokinin has a significant effect on the efficiency of regeneration and production of secondary metabolites (Verma et al., 2022). In another in vitro study on L. barbarum, the highest number of branches (23.33) and percentage of regeneration (100 %) were obtained from nodal explants in MS medium containing only 0.5 mg l-1 benzyladenine (BA). Also, the combination of BA/NAA significantly increased chlorogenic acid and caffeic acid in callus. The results showed that the type of plant growth regulator has a direct effect on regeneration and produc- tion of phenolic metabolites (Karakas, 2020). Chatoui et al (2020) found that Lepidium sativum L. seeds are rich in fatty acids (especially linolenic acid and oleic acid), phytosterols (β-sitosterol) and γ-tocopherol. The metha- nol extract showed the highest antioxidant activity and phenolic content compared to the ethanol extract. The results indicate a high potential for these seeds to be used in food supplements or additives as antioxidants. In an in vitro study on Pyrus spinosa Forssk., two genotypes rich in phenolic compounds and antioxidant capac- ity were identified from the Agia Anastasia region and used for micropropagation. Pear Medium 1, containing 5 μM BAP, provided the highest shoot regeneration (22.7 shoots per explant). This concentration of BAP was con- sidered suitable for the efficient propagation of the rich and native germplasm of this wild species (Alexandri et al., 2023). In the in vitro culture of Isatis indigotica Fort. leaves, the combination of 2 mg l-1 BAP, 0.1 mg l-1 NAA, 1.5 mg l-1 activated carbon and 0.2 % phytagel produced the highest callus regeneration and the lowest brown- ing. Mature leaf callus had higher total phenolic and POD activity than seedling leaves, despite the addition of browning inhibitors. Browning during the dedifferentia- tion process is related to phenol accumulation and PPO Acta agriculturae Slovenica, 121/3 – 2025 5 Sodium chloride, colchicine, and 6-benzylaminopurine ... property and phenols content in Hypericum perforatum L.: An in vitro study enzyme activity (Su et al., 2023). Also, the highest Salvia tebesana Bunge callus induction (100 %) was obtained in the apical meristem on MS medium containing 2,4-D and BAP. The highest accumulation of phenolic com- pounds, flavonoids and antioxidant activity was also observed in treatments containing 2, 4-D + BAP. The strong correlation between total phenolic content and antioxidant activities (DPPH and FRAP) indicates the efficiency of this method for mass production of me- dicinal metabolites (Hemmati et al., 2020). In H. perforatum, BAP at higher concentrations increased vegetative growth (number and length of shoots, fresh mass), but the highest antioxidant activity and total phenolics were obtained at lower concentra- tions (0.25 mg l-1). This pattern is consistent with results reported in plants such as V. jatamansi, P. amboinicus, and P. spinosa, where high concentrations of BAP in- creased shoot growth, but contrasts with studies such as S. tebesana and I. indigotica, where the combination of BAP with auxins produced the highest phenolics and regeneration. Therefore, the effect of BAP varies depending on the species, the type of explant, and the purpose of culture (growth or metabolite production). BAP is a widely used synthetic cytokinin as a phy- tohormone with several physiological effects. Cytokinin plays critical roles in plant cell division and enlarge- ment, consequently in plant growth and development. The phytohormone can deactivate apical dominance, on the contrary, enhance lateral buds, and then sub- branches can be germinated more. Consequently, cyto- kinin can positively induce shoot germination, develop- ment and growth (Hallmark & Rashotte, 2019; Wybouw and De Rybel, 2019; Delche et al., 2014). Also, the phy- tohormone is effective in nutrient transportation into the leaves. It has been proven that cytokinin is involved in processing metabolism and preventing senescence in leaves (Mock, 2019). Cytokinin induces leaf formation by producing plant stem cells in shoot apical meristems to generate leaf primordia. Also, the phytohormone can determine the final morphology of the leaf and phyllo- taxis pattern. Cytokinin decreases sugar accumulation but enhances chlorophyll levels and the period of pho- tosynthesis in the leaf, particularly during senescence (Wu et al., 2021). There is much evidence suggesting the role of cy- tokinin in responding to abiotic stresses, including drought, heat, salinity, and low temperature, as well as resisting parasites and pathogens, as the agents causing biotic stress (Li et al., 2021; Cortleven et al., 2019). Cy- tokinin can induce the biosynthesis and accumulation of metabolites, particularly phenolic and antioxidant compounds, to tolerate stress (Liu et al., 2020). Besides, the phytohormone increases the expression of the gene encoding an antioxidant enzyme to promote antioxi- dant systems. Generally, TPC, AP, and flavonoid con- tent can be enhanced when cytokinin levels rise (Aremu et al., 2020). 3.2 NACL The results indicated that salinity, duration, and the interaction of both treatments could significantly (p < 0.01) influence the number of leaves, the number of shoots and length, FM, AP, and TPC (Table 3). Moreover, the means comparison showed that 0.5 mg l-1 NaCl observed the highest number of leaves (19.08) during the first week, and the lowest amount of leaves (6) was in 2 mg l-1 NaCl during the 2nd and 3rd weeks. Changes in shoot numbers were in the range from 1 to 2; most numbers were measured in 0.5 mg l-1 NaCl during the first and second weeks; however, fewer numbers resulted from 1 mg l-1 NaCl in the exact dura- tions and also 2 mg l-1 for the first and second weeks. In addition, the longest shoots (16 cm) were caused by 0.5 mg l-1 NaCl in the first week; in contrast, the smallest shoots (7 cm) were observed with 1 mg l-1 NaCl in the third week. 0.5 mg l-1 NaCl × 1st week and 1 and 2 mg l-1 × 3rd week could also cause the highest and lowest FM (1.15 and 0.7 g), respectively. Moreover, the highest AP Source of Variation df Mean Square Leaves Num Shoot Num. Shoot Length (cm) FM (g) AP (mg ml-1) TPC (mg g-1) Week 2 859** 285** 3678** 428** 69436** 124** BAP 2 327** 69** 553** 34** 39652** 86.03** BAP×Week 4 52** 12** 14** 2** 2913** 36** Error 124 139.096 85 391 55 13048 211.07 Total 134 16.60 5.81 5.446 6.06 7.8452 6.41 Table 1: Variance analysis of BAP effect on the traits. *, ** significance in 5 % and 1 %, respectively Acta agriculturae Slovenica, 121/3 – 20256 MANTEGHI TAFRESHI and MOHAMMADHASSAN (150 mg ml-1) was observed by 2 mg l-1 NaCl for 3 weeks, but the 0.5 mg l-1 NaCl caused the lowest AP (20 mg ml- 1) in the first week. Besides, the highest TPC (56 mg g-1) was measured under a high range of salinity, caused by 2 mg l-1 NaCl, for 3 weeks; on the contrary, 0.5 mg l-1 NaCl could lead to the lowest TPC in the 1st week (6 mg g-1). Generally, when NaCl concentration increased, morpho- logical traits, including leaf number, shoot number and length, and FM, were raised, but AP and TPC decreased (Figure 2). Interestingly, there was a significant correla- tion among all traits, including leaf number, shoot num- ber and length, FM, AP, and TPC, under NaCl treatment (p < 0.01; Table 4). Salinity can decrease plant growth rates, shoot num- bers, plant and shoot height, seed germination, and leaf numbers. For instance, an in vitro study indicated that high NaCl concentrations can significantly decrease morphological traits, including shoot length and num- ber, FM, and dried mass, in Paronychia argentea Lam.. High salt concentration causes osmotic stress, leading to low water adsorption by plants. The osmotic stress eventually inhibits photosynthesis, impairs homeostasis, and peroxidises membrane lipids, causing ion stress to damage membrane permeability and accumulate reac- tive oxygen species (ROS), leading to oxidative stress (Hao et al., 2021; Osman et al., 2021; Li et al., 2021). In in vitro Paulownia culture, the addition of NaCl by reduc- ing the water potential of the culture medium increased genetic diversity and selected lines resistant to salinity. The presence of NaCl led to the expression of specific molecular markers in resistant lines that were not seen in control plants. These results indicate the effect of NaCl in stimulating genetic pathways associated with resist- ance under laboratory culture conditions (Salem et al., 2022). In a study, the effect of different concentrations of NaCl (0 to 250 mM) on the in vitro growth of pep- per (Capsicum annuum L.) was investigated. The results showed that increasing salinity significantly reduced germination, shoot and root growth, and physiological indices and increased visual damage. The high sensitivity of pepper to salinity stress was confirmed in vitro (Kara et al., 2025). Ghasemi-omran et al. (2021) reported that melatonin at concentrations of 5 and 10 μM improved growth, ionic balance, photosynthetic pigments, antioxi- dants, and increased steviol glycoside production in Ste- via rebaudiana Bertoni. This effect was associated with a decrease in ROS and upregulation of kaurenoic acid hydroxylase and uridine diphosphate glycosyltransferase genes expression. In contrast, a higher concentration of melatonin (20 μM) had a negative effect on growth under salinity. In another in vitro study, Red pitaya (Selenicereus polyrhizus (F.A.C. Web.) Britton & Rose ) showed greater tolerance to salinity stress (up to 150 mM NaCl) and less reduction in shoot and root length than white and hybrid pitaya. Increased salinity increased electro- lyte leakage and decreased chlorophyll a and membrane Leaves Num. Shoot Num. Shoot Length (cm) FM (g) AP (mg ml-1) TPC (mg g-1) Leaves Num. 1 Shoot Num. 0.001** 1 Shoot Length (cm) 0.001** 0.001** 1 FM (g) 0.001** 0.001** 0.001** 1 AP 0.001 0.001 0.001** 0.001** 1 TPC -0.023 -0.005 0.001* 0.001** 0.001** 1 Table 2: Correlation among traits effected by BAP *, ** significance in 5 % and 1 %, respectively Figure 1: The means comparison of BAP effect on the traits Acta agriculturae Slovenica, 121/3 – 2025 7 Sodium chloride, colchicine, and 6-benzylaminopurine ... property and phenols content in Hypericum perforatum L.: An in vitro study integrity in all species. The results introduce red pitaya as a resistant option for cultivation in saline areas and emphasize the efficiency of in vitro culture for screening of salinity tolerance (de Vasconcelos Dias et al., 2025). In H. perforatum, low NaCl concentration increased growth, but high concentration reduced growth traits and increased AP and TPC. This pattern was similar to the results of plants such as C. annuum L. and P. argentea L., in which salinity reduced growth. In contrast, plants such as red pitaya and paulownia showed greater toler- ance to salinity. During oxidative stress, plants use enzymatic and non-enzymatic antioxidant mechanisms to cope with stress. The key antioxidant enzymes include peroxidase, superoxide dismutase, and catalase. Also, the non-enzy- matic antioxidant system can provide several secondary metabolites containing many phenolic compounds (Ku- mar et al., 2023; Hassanuzzaman et al., 2021; Moham- madhassan et al., 2021; Barzin et al., 2016). 3.3 COLCHICINE The findings showed that duration significantly af- fected leaf number, length, FM, AP, TPC (p < 0.01), and shoot number (p < 0.05). All traits could be significantly influenced by Col × duration (p < 0.05). Also, Col sig- nificantly changes all traits (p < 0.01), but TPC (Table 5). Furthermore, the mean comparison indicated that Source of Variation df Mean Square Leaves Num Shoot Num. Shoot Length (cm) FM (g) AP (mg ml-1) TPC (mg g-1) Week 2 454** 3** 189** 7** 148957** 19831** NaCl 2 422** 18** 380** 1** 28851** 4794** NaCl × Week 4 58** 4** 6** 0.0001** 38220** 6353** Error 124 315 12 182 0.0001 53756 5298.045 Total 135 12.407 2.56 1.8507 1.9 12.98920 13.5801 Leaves Num. Shoot Num. Shoot Length (cm) FM (g) AP (mg ml-1) TPC (mg g-1) Leaves Num. 1 Shoot Num. 0.001** 1 Shoot Length (cm) 0.001** 0.001** 1 FM (g) 0.001** 0.001** 0.001** 1 AP 0.001** 0.001** 0.001** 0.001** 1 TPC 0.001** 0.001** 0.001* 0.001** 0.001** 1 *, ** significance in 5 % and 1 %, respectively Table 4: Correlation among traits influenced by NaCl *, ** significance in 5 % and 1 %, respectively Table 3: Variance analysis of NaCl influence on the traits Figure 2. The means comparison of the traits effected by NaCl. Acta agriculturae Slovenica, 121/3 – 20258 MANTEGHI TAFRESHI and MOHAMMADHASSAN increasing Col concentrations could raise leaf number and shoot length, then decrease in higher concentra- tions. The highest and lowest leaf numbers and shoot length were 5 and 11, respectively, caused by 0.05 mg l-1 Col × 1st week and 0.1 mg l-1 Col × 3rd week, also 7 and 16 cm resulted from 0.05 and 0.2 mg l-1 Col ×1st week and 0.1 mg l-1 Col ×3rd week. Besides, the lowest shoot number (0.1) was observed in 0.2 mg l-1 Col in the 2nd and 3rd weeks. Also, the highest number (1.083) of shoots was measured in 0.05 mg l-1 Col×3rd week. There was no change for 0.1 mg l-1 Col for 3 weeks. In other measured traits, increasing colchicine concentration and treatment duration were also associated with an increase in FM; However, duration increased and then decreased FM in samples treated by 0.1 mg l-1 Col. Consequently, the lowest and highest FM was 0.03 and 0.12 g caused by 0.1 mg l-1 × Col 1st week, and 0.1 mg l-1 Col ×2nd week and 0.2 mg l-1 Col × 3rd week, respectively. At least, Col concentration can enhance AP and TPC over time and individually. Thus, the highest AP (134 mg l-1) and TPC (160 mg g-1) were observed in samples treated with 0.2 mg l-1 Col in the 3rd week. Also, the lowest AP (87 mg ml-1) and TPC (36 mg g-1) were caused by 0.5 mg l-1 Col × 1st week, as well as 0.1 mg l-1 Col in the same duration for TPC (Figure 3). According to the results, there was a significant cor- relation between the number of leaves and shoots and other traits. Also, FM was correlated with AP and TPC. The correlations were observed between AP and TPC, as Source of Variation df Mean Square Leaves Num Shoot Num. Shoot Length (cm) FM (g) AP (mg ml-1) TPC (mg g-1) Week 2 130** 1.08* 248** 0.013** 10034** 218999.024** Col 2 116** 2** 546** 0.027** 20140** 1783ns Col × Week 4 30** 2** 155.06** 0.023** 1347** 1910** Error 124 10414 200.063 18592 1 1704305 212843 Total 134 6.56 6.095 16.97 0.0001 5.7671 3.9272 Leaves Num Shoot Num. Shoot Length (cm) FM (g) AP (mg ml-1) TPC (mg g-1) Leaves Num. 1 Shoot Num. 0.001** 1 Shoot Length (cm) 0.001** 0.001** 1 FM (g) 0.001** 0.001** -0.051 1 AP 0.001** 0.001** -0.021 0.001** 1 TPC 0.001** 0.001** 0.001* 0.001** 0.001** 1 Figure 3: The means comparison of the traits treated by Col. *, ** significance in 5 % and 1 %, respectively *, ** significance in 5 % and 1 %, respectively Table 6: Correlation among Col-treated traits Table 5: Variance analysis of Col effect on the traits Acta agriculturae Slovenica, 121/3 – 2025 9 Sodium chloride, colchicine, and 6-benzylaminopurine ... property and phenols content in Hypericum perforatum L.: An in vitro study well as shoot length and TPC (p < 0.01); however, shoot length was not correlated with FM and AP (Table 6). Eng et al. (2021) demonstrated that Col can signifi- cantly increase shoot number and length, and leaf num- ber in Neolamarckia cadamba (Roxb.) Bosser, although higher Col concentrations showed negative effects on these morphological traits. The same Col effects on FM, shoot length and number were observed in Dracaena sanderiana Mast. (Mujib et al., 2023) and Glycin max (L.) Merr. (Mangena, 2020). Çömlekçioğlu and Özden (2020) studied the effects of the different Col concentrations on AP and TPC in Physalis peruviana L.. They found that TPC and AP can be enhanced when Col concentration increases. The same results were reported from in vitro research study- ing the effects of Col on AP and TPC in Lavandula stricta Delile (Nouri Dashlibroon et al., 2020) and Nigella sativa L. (Gupta et al., 2021). There are many studies reporting growth regulatory function for Col, as well as phytohormones. Moreover, other studies indicated that low Col concentration could improve morphological traits (Abd El-Latif et al., 2018); however, it has not revealed how Col can influence AP, TPC, and secondary metabolites production (Mangena and Mushadu, 2023), although it might be related to the cytotoxicity, mutation, and tubulin-binding ability of high Col concentrations (Eng and Ho, 2019; Manzoor et al., 2019). 4 CONCLUSIONS It could be concluded that 1 mg/l BAP can increase the studied morphological traits of H. perforatum, for 21 days, whilst 0.25 mg l-1 BAP can be more beneficial for higher AP and TPC levels in the same duration. Addi- tionally, shoot length and leaves number, shoot number, and FM were respectively enhanced by 0.1, 0.05, and 0.2 mg l-1 Col. The production of the highest AP and TPC levels were induced by 0.2 mg l-1 Col. 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