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Full Research Article

Effect of Different Concentrations of Salicylic Acid on Increasing the Phenolic Content of Two in vitro-cultivated Prickly Pear Varieties

H
Hiba Ahmed Jawad1,*
0000-0002-6964-1071
M
Mohammed D. Abdulhadi1
L
Luma B. Hussein1
B
Bassim A. Essa1
1Faculty Members at Department of Horticulture and Landscape Engineering, Agriculture College, University of Diyala, Iraq.

ABSTRACT

Background: Salicylic acid (SA) is one of the most important simple phenolic compounds that has received considerable attention from scientists worldwide. Research aims to evaluate the response of two varieties of prickly pear fruit (Opuntia ficus indica) with desirable marketing characteristics that have recently been introduced to the country and to evaluate their vegetative cultivation using tissue culture technology to quickly obtain complete plants in large numbers and in a shorter period using different concentrations of benzyl adenine in combination with naphthalene acetic acid.

Methods: Several experiments were conducted, including sterilizing plant parts using sodium hypochlorite at a concentration of 10% and for different durations (10.5, 15 minutes).

Result: In the multiplication experiment using different concentrations of benzyl adenine (0, 2, 4 and 6 mg L-1) in combination with NAA at a concentration of 0.5 mg L-1, the Honey variety outperformed the others in average branch length, average number of branches and average number of roots, reaching 1.425 cm, 1.425 branches per plant and 1.300 roots per plant, respectively. However, the red variety outperformed the others in root length, achieving the highest root length of 1.661 cm. In the salicylic acid study, where 0, 50 and 100 µmol/L were used to increase the phenolic compound content using HPLC in prickly pear cactus branches grown in vitro, the Red variety was found to have the highest average branch length (3.000 cm), not significantly different from the Honey variety. The honey variety in branch content of the phenolic compound hydroxycinnamic acid. A concentration of 100 micromoles yielded the highest phenolic content at 28.061, compared to a concentration of 50 micromoles per liter, which yielded a phenolic content of 17.869.

INTRODUCTION

Prickly pear (Opuntia ficus indica) or prickly pear cactus is a flowering and fruiting crassulacean (CAM) metabolism plant belonging to the Cactaceae family. Cacti are widespread and cultivated throughout the world, especially in semi-arid and dry areas and the Mediterranean climate and constitute one of the most important and diverse groups of vascular plants. Prickly pear cultivated in marginal and sloping lands as a barrier against soil erosion. It is cultivated as a plant for the rehabilitation of ecosystems and land reclamation and it is also grown as a commercial crop, such as fruit for human consumption and livestock feed (Bouzroud, 2022) cacti are also known for their distinctive nutritional, medicinal and biological properties, which are attributed to the presence of highly valuable chemical compounds, such as phenolic compounds, carotenoids, vitamins, flavonoids and betalains (Al-Khayri et al., 2018).
       
In vitro tissue culture techniques are used to propagate commercial and endangered species (Giraldo-Silva, 2023) and tissue culture has been performed by other researchers. It has recently been suggested that the latter method is the most effective because it provides high propagation rates, reduces space requirements and produces healthy, pathogen-free plants. Recent evidence has revealed that micropropagation has been extensively studied and successfully developed in the cloning of several prickly pear cactus species (Ghaffari et al., 2013).
       
Salicylic acid (SA) is one of the most important simple phenolic compounds that has received considerable attention from scientists worldwide. Extensive research on salicylic acid in plants has revealed its role in diverse physiological and developmental responses, including seed germination, stomatal movement, pigment accumulation, photosynthesis, ethylene synthesis, heat production, enzyme activity, reversal of leaf fall, nutrient uptake, flowering stimulation, membrane function, root nodule formation in legumes and overall plant growth and development. Therefore, salicylic acid and its analogs are classified as plant hormones (Ali, 2021).
       
HPLC is the most widely used technique for separating and detecting phenolic compounds. It is a versatile and adaptable tool with numerous advantages, including high selectivity, sensitivity, accuracy and sample type. Several factors influence HPLC analysis of phenolic compounds, such as column type (the column in which the separation occurs is the core of the system), solvents used, mobile phase and the properties of the compounds used in the laboratory (Ali, 2022). Based on the above, the main objective of this study was to develop an effective protocol for the propagation of two prickly pear fruit varieties. The optimal duration for sodium hypochlorite sterilization was determined, along with the best concentrations of auxins and cytokinins for branch growth and rooting. The effect of different salicylic acid concentrations on increasing the plant’s content of hydroxybenzoic and hydroxycinnamic phenols was also evaluated.

MATERIALS AND METHODS

Experiments were conducted on prickly pear cactus at Plant Tissue Culture Laboratory, Department of Horticulture and Landscape Engineering, University of Diyala, during December 2024  up to September 2025.
       
Two newly introduced varieties of prickly pear cactus were used, first one, honey variety, second was, red variety. Mother plant selected from newly grown prickly pear cladodes and the seedlings were treated with a fungicide sprayed on the cladodes until they were wet. process was done two days before taking the plant part. After spraying them well, the cladodes were covered in transparent bags. This was the first method of surface sterilization. Then, after two days, cladode covered in the same bag was separated and taken to the laboratory for the purpose of carrying out the second stage of sterilization. cladodes cleaned with running water several times until the traces of the pesticide were removed. 100 mg L-1 of citric acid was prepared with 150 mg L-1 of PVP antioxidants and cladode was soaked in two solutions for 15 minutes for each solution separately. After that, the plant part was placed in sterile distilled water until it was submerged to remove the traces of the solutions.
       
Final stage of sterilization involved cutting the hand into small pieces, each containing a single eye. These small hand pieces were then placed in a 10% sodium hypochlorite bleach solution for 5, 10 and 15 minutes for both varieties, along with a few drops of surfactant, while stirring continuously to ensure thorough sterilization. Procedures performed inside a laminar air flow cabinet to guarantee contaminated pieces.
       
After sterilization of plant parts which containing sterilized developing buds, then lower limbs between the buds, affected by the sterilization solution, removed  by using a No. 11 surgical blade. Plant parts were ready for inoculation. For both varieties used, plant parts were cultured in MS culture medium without growth regulators to encourage new growth, process performed inside a laminar air flow cabinet.
       
Plant parts incubated in a growth chamber at 24±2°C and 1000 lux for 16 hours of light followed by 8 hours of darkness for four weeks. The percentage of contamination was calculated for the in vitro-cultured varieties. The plants resulting from the sterilization experiment, re-cultured to establish a tissue culture farm to maximize yield of Food Stuffs.
 
Effect of interacting different concentrations of benzyl adenine (BA) with NAA experiment  on vegetative propagation characteristics
 
Adding cytokinin BA with concentrations (0.2, 4, 6, mg L-1) in combination with 0.5 mg L-1 NAA was evaluated on stimulation vegetative propagation of branches for two prickly pear cultivars under incubation conditions. Plants incubated at a temperature of 25±2°C, with a light duration of 16 hours followed by 8 hours of darkness and a light intensity of 1000 lux.
1. Average number of formed branches.
2. Average branch height (cm).
3. Average number of roots.
4. Average root length (cm).
 
Effect of interfering with different kin concentrations and NAA on vegetative propagation experiment
 
Varieties, Red and Honey used for vegetative propagation to obtain the desired number of branches by tissue culture technique. Buds cut and re-cultured in MS medium containing different Kin concentrations (0, 1, 2 and 3 mg L-1) interfering with NAA at a concentration of 0.5 mg L-1, cultures were incubated at 25±2°C with 16 hours of light followed by 8 hours of darkness and a light intensity was 1000 lux.
1. Average number of formed branches.
2. Average branch height (cm).
3. Average number of roots.
4. Average root length (cm).
 
Effect of different salicylic acid concentrations on vegetative propagation and phenolic compound content experiment
 
Varieties, Red and Honey used for vegetative propagation to obtain the desired number of branches resulting from tissue culture technique. Buds cut and re-cultured in MS medium containing different concentrations of salicylic acid (SA) at concentrations of 0, 50 and 100 µmol/L. Cultures incubated at a temperature of 25±2°C, with a light duration of 16 hours followed by 8 hours of darkness and a light intensity of 1000 lux. measurements taken.
1. Average number of formed branches.
2. Average branch length (cm).
3. Extraction of phenolic compounds using HPLC from the cultures produced from the media containing the salicylic acid concentrations.
 
Extraction method
 
Extraction and quantification of the chemical compounds carried out at the research and technology station, environment and water department. 5 g of the ground and homogenized plant sample was taken and 12 ml of chloroform was added with continuous stirring for 8 hours at room temperature. The extract was then placed in an ultrasonic chamber for 15 minutes, followed by the addition of 100 ml of butanol. The mixture was then transferred to a separation funnel and the polar organic layer (butanol) was collected and transferred to a rotary evaporator to obtain a dry extract. The process was repeated three times to obtain a sufficient quantity before analysis (Ali, 2022).
 
Analysis procedures
 
Identification and quantification of chemical compounds (phenols) carried out at Science and Technology Laboratory, Department of Environment and Water using a high-performance liquid chromatography (HPLC) instrument manufactured by the Germany Sykam company. Instrument was equipped with a fixed phase column made of C18-DOS with dimensions of (4.6 mm × 25 cm) and a wavelength of 280 nm (Fig 1). The carrier phase consisted of methanol, water and formic acid at concentrations of 5, 25 and 70. The carrier phase flow rate was 1.0 ml/min. The instrument operated by determining the retention time and beam area of   the standard solution and the sample solution, according to the specific separation conditions for each type of chemical compound to be separated and quantified (Fig 2). The concentrations of the compounds were calculated according to the following equation:
 
Concentration of the model in the sample (Micrograms mL-1) = (Model package area × Standard model concentration)/(Standard model package area) × (Dilution factor)/(Model weight)

Fig 1: Standard solution curve of hydroxycinnamic compound measured by HPLC.



Fig 2: The standard solution curve of hydroxybenzioc compound measured by HPLC.

RESULTS AND DISCUSSION

Effect of different sterilization durations on the percentage of contaminated plant parts in in vitro, Table 1 shows differences in percentage of contamination in the studied varieties when different sterilization durations were used with a 10% bleach concentration. The red variety exhibited the lowest contamination rate at 26.6%, while the honey variety had the highest contamination rate at 36.6%. Regarding the durations, the same table indicates differences between 5, 10 and 15 minutes. The 10-minute duration resulted in the lowest contamination rate at 15%, while the 15-minute duration resulted in the highest contamination rate at 50%.

Table 1: Effect of different sterilization durations on the percentage of contaminated plant parts of two prickly pear varieties in vitro.


       
Effect of plant parts responding to the sterilizing agent (Sodium hypochlorite) may be due to the variation in response percentages among prickly pear varieties (Red and honey). Hypochlorite is characterized by its ability to kill microorganisms, viruses and fungi that contaminate plants, thus reducing biological risks and improving production quality. This acid is formed as a result of the dissolution of chlorine in water, as in the equation (2004): Cl2 + H2O = HCl + HOCl (Ramawat, 2004) and (Ibrahim et al., 2022).
 
Effect of BA concentrations on average branch length, number of branches, root length and number of roots in combination with NAA at a concentration of 0.5 mg/L after 4 weeks of culture on MS medium
 
Branch length
 
Table 2 indicate significant differences between the studied treatments. The honey variety outperformed the red variety, yielding the highest average plant length of 1.425 cm, while the Red variety exhibited the lowest average branch length of 1.286 cm. The BA treatment at a concentration of 6 mg/L resulted in the highest average branch length of 1.525 cm, while the lowest average branch length of 1.200 cm was observed when grown on a medium with a concentration of 2 mg/L. The honey variety grown in the control treatment and the Red variety grown on the 6 mg/L BA nutrient medium showed the highest average number of branches, reaching 1.600 and 1.650 cm, respectively.

Table 2: Effect of different concentrations of benzyl adenine (BA) on average plant trials in the presence of 0.5% auxin (NAA).


 
Number of branches
 
Results in the same table show that the honey variety significantly outperformed the red variety, with an average number of branches of 1,425 per plant, while the red variety yielded an average of 1,408 per plant. Branches grown on a medium treated with 4 mg/L of BA produced the highest average number of branches, 1,578 per plant and the lowest average number of branches was observed in the control treatment, at 1,450 per plant. The Honey variety grown on a medium treated with 4 mg/L also outperformed the red variety, yielding the highest average number of branches, 1,800 per plant.
 
Root length
 
Table 2 show significant differences between the treatments. Red variety had highest average root length, 1,661 cm, Honey variety had lowest average root length, 1,408 cm. Regarding the concentrations of benzyl adenine (BA) used in the nutrient medium, the 4 mg/L treatment resulted in the highest average root length of 1.474 cm, which was not significantly different from the 6 mg/L concentration. The control treatment, on the other hand, resulted in the lowest average root length of 1.275 cm. As for the interaction, the red variety grown on a medium prepared with a 6 mg/L concentration of BA exhibited an average root length of 1.600 cm.
 
Number of roots
 
Results of the same table indicate that no significant differences in the studied varieties in the trait of the average number of roots. As for the concentrations added to the nutrient medium, the control treatment was superior in the average number of roots, which did not differ significantly from the concentration of 2 mg L-1, as they gave an average number of roots of (1,400 and 1,372 plant-1 roots). As for the interaction between the variety and the concentration, the red variety was superior in the control treatment and the concentration of 2 mg L-1, as they gave the highest average number of roots of (1,400 and 1,444) plant-1 roots, respectively.
       
Table 2 shows that adding growth regulator BA at different concentrations led to an increase in the vegetative mass, the number of branches, the number of leaves and the number of nodes. This is perhaps attributed to the role this cytokinin plays in plant tissue development, cell division, breaking apical dominance, slowing senescence and increasing the plant’s ability to form aerial parts. Alternatively, these results could be due to the three double bonds present in this cytokinin’s molecular structure, as well as the presence of a benzyl ring, making it the most widely used cytokinin in plant propagation, results are consistent with Al-Sumaidi (2017) finding.
 
Effect of kin concentrations in combination with NAA at a concentration of 0.5 mg L-1, 4 weeks later of cultivation on MS medium
 
Branch length
 
Table 3 indicates a significant differences between the studied treatments. Honey variety outperformed the red variety, giving the highest average branch length of 1.912 cm, while the Red variety gave the lowest average branch length of 1.587 cm. The Kin treatment at a concentration of 3 mg L-1 resulted in the highest average branch length of 2.125 cm, while the lowest average branch length of 1.525 cm resulted from cultivation on medium prepared with a concentration of 1 mg L-1 of Kin. The honey variety cultivated with the 3 mg L-1 treatment showed the highest average branch length of 2.500 cm.

Table 3: Effect of different concentrations of Kin on average plant trials in interaction with 0.5% auxin (NAA).


 
Number of branches
 
Results show in the same table show that the red variety significantly outperformed the honey variety, with an average number of branches of 1,950 per plant, while the honey variety yielded an average of 1,855 per plant. Branches grown on a medium treated with 2 mg/L of Kin showed the highest average number of branches at 2,150 per plant and the lowest average number of branches in the control treatment at 1,611 per plant. The honey variety grown on a medium treated with 2 mg/L of Kin also showed the highest average number of branches at 2,400 per plant.
 
Root length
 
Table 3 shows significant differences between the treatments, honey variety had superiority of highest average root length of 2,033 cm at  control treatment, red variety had lowest average root length of 1,412 cm. Kin concentrations in nutrient medium, control treatment resulted in the highest average root length of 2.366 cm, while the 3 mg/L-1 treatment was lowest average root length of 1.325 cm. Interaction between varieties, honey variety result in highest root length (2.366 cm) when compared to control treatment, red variety showed the lowest average root length (1.500 cm) in comparison to control treatment.
 
Number of roots
 
Results in the same table indicate no significant differences between varieties in average number of roots. However, concentrations added to the nutrient medium, 3 mg/L-1 treatment resulted in highest average root number (1.850 roots per plant). Results of interaction between variety and concentration, red variety had highest average root number (2.100 roots per plant) at the 3 mg/L-1 treatment.
       
Table 3 shows that increasing the concentration of kinetin to a certain level led to an increase in the number of branches compared to the control treatment. This increase in the number of branches in the kinetin treatments may be attributed to kinetin’s role in breaking apical dominance, as it releases axillary buds by directing nutrients to them, thus playing a positive role in their growth. It also aids in RNA and protein synthesis in cultured tissues (Al-Sumaidi, 2017; Al-Ubaidi and Khairallah (2017).
 
Effect of salicylic acid concentrations on average branch length and number of branches, in interaction with Kin at a concentration of 3 mg/L, after 4 weeks of culture on MS medium
 
Branch length
 
Table 4 indicates no significant differences between the studied varieties in branch length. Regarding the salicylic acid treatment, the 50 mg concentration resulted in the highest average branch length of 3.750 cm, which was not significantly different from the 100 mg concentration. The red variety grown with the 50 mg concentration treatment showed the highest average branch length of 3.800 cm, which was not significantly different from the honey variety grown on the same medium, which produced 3.700 branches per plant.

Table 4: Effect of different concentrations of salicylic acid on average plant height and average number of branches of two varieties of prickly pear grown in vitro.


 
Number of branches
 
Results in the same table show that the red variety significantly outperformed the honey variety with an average number of branches of 2.733 per plant. Branches grown on a medium treated with salicylic acid at a concentration of 50 mg resulted in an average number of branches of 3.350 per plant, which was not significantly different from the 100 mg concentration, which produced the highest average number of branches. Branches amounted to 3,250 plant branches-1. As for the overlap of varieties with salicylic acid, the red variety was superior for both concentrations of 50 and 100, as it gave an average number of branches amounting to (3,350 and 3,250) plant branches-1.
 
Evaluation of active compounds
 
Effect of variety and salicylic acid concentration on the branch content of phenolic compounds in two prickly pear varieties grown in vitro
 
Phenolic compound hydroxy benzoic: Fig (3) shows superiority of the red variety over the honey variety in the branch content of hydroxy benzoic, as the concentration of 100 µmol L-1 gave the highest phenolic content of 54.445 compared to the concentration of 50 µmol L-1, which gave a phenolic content of 25.191.

Fig 3: The effect of salicylic acid on the amount of hydroxybenzoic phenolic compound produced from prickly pear tissue cultures after four weeks of cultivation on a nutrient medium.


 
Phenolic compound  hydroxycinnamic acid
 
Fig 4 shows red variety outperformed the honey variety in branch content of the phenolic compound  hydroxycinnamic acid. A concentration of 100 micromoles yielded the highest phenolic content at 28.061, compared to a concentration of 50 micromoles per liter, which yielded a phenolic content of 17.869.

Fig 4: Effect of salicylic acid on the amount of the phenolic compound hydroxy cinnamic acid from the tissue culture of prickly pear cactus branches after four weeks of cultivation on the nutrient medium.


       
Since correlation analysis revealed a strong or moderate positive correlation between the phenolic compound content and the biological activity of the extracts, it is possible to hypothesize that the increased biological activity is specifically related to the increased biosynthesis of secondary phenolic metabolites under the influence of salicylic acid. The positive effect of salicylic acid treatment on the total phenolic compound content is consistent with this finding (Skrypnik, 2022; Baqir et al., 2026). Salicylic acid is known to increase the activity of phenylalanine ammonia-lyase (PAL), the most important enzyme in the initial stages of phenolic compound synthesis (Xu et al., 2015, Ali et al., 2024; Afdal et al., 2026).

CONCLUSION

Research focuses on developing an efficient micropropagation protocol to meet the growing demand for these varieties for consumption as fruit, animal feed and as a drought- and water-scarce alternative crop, furthermore, it can be used in sustainable farming systems, also explores the potential of prickly pear cactus varieties in vitro as an anticancer agent due to their phenolic compound content. The red variety exhibited the best branch length and number of branches when cultured on a nutrient medium containing varying concentrations of salicylic acid. A concentration of 100 µmol/L of salicylic acid resulted in the highest content of the phenolic compounds hydroxyl benzoic and hydroxyl cinnamic acid.

CONFLICT OF INTEREST

The authors declare that there is no conflict of interest.

REFERENCES


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    Full Research Article

    Effect of Different Concentrations of Salicylic Acid on Increasing the Phenolic Content of Two in vitro-cultivated Prickly Pear Varieties

    H
    Hiba Ahmed Jawad1,*
    0000-0002-6964-1071
    M
    Mohammed D. Abdulhadi1
    L
    Luma B. Hussein1
    B
    Bassim A. Essa1
    1Faculty Members at Department of Horticulture and Landscape Engineering, Agriculture College, University of Diyala, Iraq.

    ABSTRACT

    Background: Salicylic acid (SA) is one of the most important simple phenolic compounds that has received considerable attention from scientists worldwide. Research aims to evaluate the response of two varieties of prickly pear fruit (Opuntia ficus indica) with desirable marketing characteristics that have recently been introduced to the country and to evaluate their vegetative cultivation using tissue culture technology to quickly obtain complete plants in large numbers and in a shorter period using different concentrations of benzyl adenine in combination with naphthalene acetic acid.

    Methods: Several experiments were conducted, including sterilizing plant parts using sodium hypochlorite at a concentration of 10% and for different durations (10.5, 15 minutes).

    Result: In the multiplication experiment using different concentrations of benzyl adenine (0, 2, 4 and 6 mg L-1) in combination with NAA at a concentration of 0.5 mg L-1, the Honey variety outperformed the others in average branch length, average number of branches and average number of roots, reaching 1.425 cm, 1.425 branches per plant and 1.300 roots per plant, respectively. However, the red variety outperformed the others in root length, achieving the highest root length of 1.661 cm. In the salicylic acid study, where 0, 50 and 100 µmol/L were used to increase the phenolic compound content using HPLC in prickly pear cactus branches grown in vitro, the Red variety was found to have the highest average branch length (3.000 cm), not significantly different from the Honey variety. The honey variety in branch content of the phenolic compound hydroxycinnamic acid. A concentration of 100 micromoles yielded the highest phenolic content at 28.061, compared to a concentration of 50 micromoles per liter, which yielded a phenolic content of 17.869.

    INTRODUCTION

    Prickly pear (Opuntia ficus indica) or prickly pear cactus is a flowering and fruiting crassulacean (CAM) metabolism plant belonging to the Cactaceae family. Cacti are widespread and cultivated throughout the world, especially in semi-arid and dry areas and the Mediterranean climate and constitute one of the most important and diverse groups of vascular plants. Prickly pear cultivated in marginal and sloping lands as a barrier against soil erosion. It is cultivated as a plant for the rehabilitation of ecosystems and land reclamation and it is also grown as a commercial crop, such as fruit for human consumption and livestock feed (Bouzroud, 2022) cacti are also known for their distinctive nutritional, medicinal and biological properties, which are attributed to the presence of highly valuable chemical compounds, such as phenolic compounds, carotenoids, vitamins, flavonoids and betalains (Al-Khayri et al., 2018).
           
    In vitro tissue culture techniques are used to propagate commercial and endangered species (Giraldo-Silva, 2023) and tissue culture has been performed by other researchers. It has recently been suggested that the latter method is the most effective because it provides high propagation rates, reduces space requirements and produces healthy, pathogen-free plants. Recent evidence has revealed that micropropagation has been extensively studied and successfully developed in the cloning of several prickly pear cactus species (Ghaffari et al., 2013).
           
    Salicylic acid (SA) is one of the most important simple phenolic compounds that has received considerable attention from scientists worldwide. Extensive research on salicylic acid in plants has revealed its role in diverse physiological and developmental responses, including seed germination, stomatal movement, pigment accumulation, photosynthesis, ethylene synthesis, heat production, enzyme activity, reversal of leaf fall, nutrient uptake, flowering stimulation, membrane function, root nodule formation in legumes and overall plant growth and development. Therefore, salicylic acid and its analogs are classified as plant hormones (Ali, 2021).
           
    HPLC is the most widely used technique for separating and detecting phenolic compounds. It is a versatile and adaptable tool with numerous advantages, including high selectivity, sensitivity, accuracy and sample type. Several factors influence HPLC analysis of phenolic compounds, such as column type (the column in which the separation occurs is the core of the system), solvents used, mobile phase and the properties of the compounds used in the laboratory (Ali, 2022). Based on the above, the main objective of this study was to develop an effective protocol for the propagation of two prickly pear fruit varieties. The optimal duration for sodium hypochlorite sterilization was determined, along with the best concentrations of auxins and cytokinins for branch growth and rooting. The effect of different salicylic acid concentrations on increasing the plant’s content of hydroxybenzoic and hydroxycinnamic phenols was also evaluated.

    MATERIALS AND METHODS

    Experiments were conducted on prickly pear cactus at Plant Tissue Culture Laboratory, Department of Horticulture and Landscape Engineering, University of Diyala, during December 2024  up to September 2025.
           
    Two newly introduced varieties of prickly pear cactus were used, first one, honey variety, second was, red variety. Mother plant selected from newly grown prickly pear cladodes and the seedlings were treated with a fungicide sprayed on the cladodes until they were wet. process was done two days before taking the plant part. After spraying them well, the cladodes were covered in transparent bags. This was the first method of surface sterilization. Then, after two days, cladode covered in the same bag was separated and taken to the laboratory for the purpose of carrying out the second stage of sterilization. cladodes cleaned with running water several times until the traces of the pesticide were removed. 100 mg L-1 of citric acid was prepared with 150 mg L-1 of PVP antioxidants and cladode was soaked in two solutions for 15 minutes for each solution separately. After that, the plant part was placed in sterile distilled water until it was submerged to remove the traces of the solutions.
           
    Final stage of sterilization involved cutting the hand into small pieces, each containing a single eye. These small hand pieces were then placed in a 10% sodium hypochlorite bleach solution for 5, 10 and 15 minutes for both varieties, along with a few drops of surfactant, while stirring continuously to ensure thorough sterilization. Procedures performed inside a laminar air flow cabinet to guarantee contaminated pieces.
           
    After sterilization of plant parts which containing sterilized developing buds, then lower limbs between the buds, affected by the sterilization solution, removed  by using a No. 11 surgical blade. Plant parts were ready for inoculation. For both varieties used, plant parts were cultured in MS culture medium without growth regulators to encourage new growth, process performed inside a laminar air flow cabinet.
           
    Plant parts incubated in a growth chamber at 24±2°C and 1000 lux for 16 hours of light followed by 8 hours of darkness for four weeks. The percentage of contamination was calculated for the in vitro-cultured varieties. The plants resulting from the sterilization experiment, re-cultured to establish a tissue culture farm to maximize yield of Food Stuffs.
     
    Effect of interacting different concentrations of benzyl adenine (BA) with NAA experiment  on vegetative propagation characteristics
     
    Adding cytokinin BA with concentrations (0.2, 4, 6, mg L-1) in combination with 0.5 mg L-1 NAA was evaluated on stimulation vegetative propagation of branches for two prickly pear cultivars under incubation conditions. Plants incubated at a temperature of 25±2°C, with a light duration of 16 hours followed by 8 hours of darkness and a light intensity of 1000 lux.
    1. Average number of formed branches.
    2. Average branch height (cm).
    3. Average number of roots.
    4. Average root length (cm).
     
    Effect of interfering with different kin concentrations and NAA on vegetative propagation experiment
     
    Varieties, Red and Honey used for vegetative propagation to obtain the desired number of branches by tissue culture technique. Buds cut and re-cultured in MS medium containing different Kin concentrations (0, 1, 2 and 3 mg L-1) interfering with NAA at a concentration of 0.5 mg L-1, cultures were incubated at 25±2°C with 16 hours of light followed by 8 hours of darkness and a light intensity was 1000 lux.
    1. Average number of formed branches.
    2. Average branch height (cm).
    3. Average number of roots.
    4. Average root length (cm).
     
    Effect of different salicylic acid concentrations on vegetative propagation and phenolic compound content experiment
     
    Varieties, Red and Honey used for vegetative propagation to obtain the desired number of branches resulting from tissue culture technique. Buds cut and re-cultured in MS medium containing different concentrations of salicylic acid (SA) at concentrations of 0, 50 and 100 µmol/L. Cultures incubated at a temperature of 25±2°C, with a light duration of 16 hours followed by 8 hours of darkness and a light intensity of 1000 lux. measurements taken.
    1. Average number of formed branches.
    2. Average branch length (cm).
    3. Extraction of phenolic compounds using HPLC from the cultures produced from the media containing the salicylic acid concentrations.
     
    Extraction method
     
    Extraction and quantification of the chemical compounds carried out at the research and technology station, environment and water department. 5 g of the ground and homogenized plant sample was taken and 12 ml of chloroform was added with continuous stirring for 8 hours at room temperature. The extract was then placed in an ultrasonic chamber for 15 minutes, followed by the addition of 100 ml of butanol. The mixture was then transferred to a separation funnel and the polar organic layer (butanol) was collected and transferred to a rotary evaporator to obtain a dry extract. The process was repeated three times to obtain a sufficient quantity before analysis (Ali, 2022).
     
    Analysis procedures
     
    Identification and quantification of chemical compounds (phenols) carried out at Science and Technology Laboratory, Department of Environment and Water using a high-performance liquid chromatography (HPLC) instrument manufactured by the Germany Sykam company. Instrument was equipped with a fixed phase column made of C18-DOS with dimensions of (4.6 mm × 25 cm) and a wavelength of 280 nm (Fig 1). The carrier phase consisted of methanol, water and formic acid at concentrations of 5, 25 and 70. The carrier phase flow rate was 1.0 ml/min. The instrument operated by determining the retention time and beam area of   the standard solution and the sample solution, according to the specific separation conditions for each type of chemical compound to be separated and quantified (Fig 2). The concentrations of the compounds were calculated according to the following equation:
     
    Concentration of the model in the sample (Micrograms mL-1) = (Model package area × Standard model concentration)/(Standard model package area) × (Dilution factor)/(Model weight)

    Fig 1: Standard solution curve of hydroxycinnamic compound measured by HPLC.



    Fig 2: The standard solution curve of hydroxybenzioc compound measured by HPLC.

    RESULTS AND DISCUSSION

    Effect of different sterilization durations on the percentage of contaminated plant parts in in vitro, Table 1 shows differences in percentage of contamination in the studied varieties when different sterilization durations were used with a 10% bleach concentration. The red variety exhibited the lowest contamination rate at 26.6%, while the honey variety had the highest contamination rate at 36.6%. Regarding the durations, the same table indicates differences between 5, 10 and 15 minutes. The 10-minute duration resulted in the lowest contamination rate at 15%, while the 15-minute duration resulted in the highest contamination rate at 50%.

    Table 1: Effect of different sterilization durations on the percentage of contaminated plant parts of two prickly pear varieties in vitro.


           
    Effect of plant parts responding to the sterilizing agent (Sodium hypochlorite) may be due to the variation in response percentages among prickly pear varieties (Red and honey). Hypochlorite is characterized by its ability to kill microorganisms, viruses and fungi that contaminate plants, thus reducing biological risks and improving production quality. This acid is formed as a result of the dissolution of chlorine in water, as in the equation (2004): Cl2 + H2O = HCl + HOCl (Ramawat, 2004) and (Ibrahim et al., 2022).
     
    Effect of BA concentrations on average branch length, number of branches, root length and number of roots in combination with NAA at a concentration of 0.5 mg/L after 4 weeks of culture on MS medium
     
    Branch length
     
    Table 2 indicate significant differences between the studied treatments. The honey variety outperformed the red variety, yielding the highest average plant length of 1.425 cm, while the Red variety exhibited the lowest average branch length of 1.286 cm. The BA treatment at a concentration of 6 mg/L resulted in the highest average branch length of 1.525 cm, while the lowest average branch length of 1.200 cm was observed when grown on a medium with a concentration of 2 mg/L. The honey variety grown in the control treatment and the Red variety grown on the 6 mg/L BA nutrient medium showed the highest average number of branches, reaching 1.600 and 1.650 cm, respectively.

    Table 2: Effect of different concentrations of benzyl adenine (BA) on average plant trials in the presence of 0.5% auxin (NAA).


     
    Number of branches
     
    Results in the same table show that the honey variety significantly outperformed the red variety, with an average number of branches of 1,425 per plant, while the red variety yielded an average of 1,408 per plant. Branches grown on a medium treated with 4 mg/L of BA produced the highest average number of branches, 1,578 per plant and the lowest average number of branches was observed in the control treatment, at 1,450 per plant. The Honey variety grown on a medium treated with 4 mg/L also outperformed the red variety, yielding the highest average number of branches, 1,800 per plant.
     
    Root length
     
    Table 2 show significant differences between the treatments. Red variety had highest average root length, 1,661 cm, Honey variety had lowest average root length, 1,408 cm. Regarding the concentrations of benzyl adenine (BA) used in the nutrient medium, the 4 mg/L treatment resulted in the highest average root length of 1.474 cm, which was not significantly different from the 6 mg/L concentration. The control treatment, on the other hand, resulted in the lowest average root length of 1.275 cm. As for the interaction, the red variety grown on a medium prepared with a 6 mg/L concentration of BA exhibited an average root length of 1.600 cm.
     
    Number of roots
     
    Results of the same table indicate that no significant differences in the studied varieties in the trait of the average number of roots. As for the concentrations added to the nutrient medium, the control treatment was superior in the average number of roots, which did not differ significantly from the concentration of 2 mg L-1, as they gave an average number of roots of (1,400 and 1,372 plant-1 roots). As for the interaction between the variety and the concentration, the red variety was superior in the control treatment and the concentration of 2 mg L-1, as they gave the highest average number of roots of (1,400 and 1,444) plant-1 roots, respectively.
           
    Table 2 shows that adding growth regulator BA at different concentrations led to an increase in the vegetative mass, the number of branches, the number of leaves and the number of nodes. This is perhaps attributed to the role this cytokinin plays in plant tissue development, cell division, breaking apical dominance, slowing senescence and increasing the plant’s ability to form aerial parts. Alternatively, these results could be due to the three double bonds present in this cytokinin’s molecular structure, as well as the presence of a benzyl ring, making it the most widely used cytokinin in plant propagation, results are consistent with Al-Sumaidi (2017) finding.
     
    Effect of kin concentrations in combination with NAA at a concentration of 0.5 mg L-1, 4 weeks later of cultivation on MS medium
     
    Branch length
     
    Table 3 indicates a significant differences between the studied treatments. Honey variety outperformed the red variety, giving the highest average branch length of 1.912 cm, while the Red variety gave the lowest average branch length of 1.587 cm. The Kin treatment at a concentration of 3 mg L-1 resulted in the highest average branch length of 2.125 cm, while the lowest average branch length of 1.525 cm resulted from cultivation on medium prepared with a concentration of 1 mg L-1 of Kin. The honey variety cultivated with the 3 mg L-1 treatment showed the highest average branch length of 2.500 cm.

    Table 3: Effect of different concentrations of Kin on average plant trials in interaction with 0.5% auxin (NAA).


     
    Number of branches
     
    Results show in the same table show that the red variety significantly outperformed the honey variety, with an average number of branches of 1,950 per plant, while the honey variety yielded an average of 1,855 per plant. Branches grown on a medium treated with 2 mg/L of Kin showed the highest average number of branches at 2,150 per plant and the lowest average number of branches in the control treatment at 1,611 per plant. The honey variety grown on a medium treated with 2 mg/L of Kin also showed the highest average number of branches at 2,400 per plant.
     
    Root length
     
    Table 3 shows significant differences between the treatments, honey variety had superiority of highest average root length of 2,033 cm at  control treatment, red variety had lowest average root length of 1,412 cm. Kin concentrations in nutrient medium, control treatment resulted in the highest average root length of 2.366 cm, while the 3 mg/L-1 treatment was lowest average root length of 1.325 cm. Interaction between varieties, honey variety result in highest root length (2.366 cm) when compared to control treatment, red variety showed the lowest average root length (1.500 cm) in comparison to control treatment.
     
    Number of roots
     
    Results in the same table indicate no significant differences between varieties in average number of roots. However, concentrations added to the nutrient medium, 3 mg/L-1 treatment resulted in highest average root number (1.850 roots per plant). Results of interaction between variety and concentration, red variety had highest average root number (2.100 roots per plant) at the 3 mg/L-1 treatment.
           
    Table 3 shows that increasing the concentration of kinetin to a certain level led to an increase in the number of branches compared to the control treatment. This increase in the number of branches in the kinetin treatments may be attributed to kinetin’s role in breaking apical dominance, as it releases axillary buds by directing nutrients to them, thus playing a positive role in their growth. It also aids in RNA and protein synthesis in cultured tissues (Al-Sumaidi, 2017; Al-Ubaidi and Khairallah (2017).
     
    Effect of salicylic acid concentrations on average branch length and number of branches, in interaction with Kin at a concentration of 3 mg/L, after 4 weeks of culture on MS medium
     
    Branch length
     
    Table 4 indicates no significant differences between the studied varieties in branch length. Regarding the salicylic acid treatment, the 50 mg concentration resulted in the highest average branch length of 3.750 cm, which was not significantly different from the 100 mg concentration. The red variety grown with the 50 mg concentration treatment showed the highest average branch length of 3.800 cm, which was not significantly different from the honey variety grown on the same medium, which produced 3.700 branches per plant.

    Table 4: Effect of different concentrations of salicylic acid on average plant height and average number of branches of two varieties of prickly pear grown in vitro.


     
    Number of branches
     
    Results in the same table show that the red variety significantly outperformed the honey variety with an average number of branches of 2.733 per plant. Branches grown on a medium treated with salicylic acid at a concentration of 50 mg resulted in an average number of branches of 3.350 per plant, which was not significantly different from the 100 mg concentration, which produced the highest average number of branches. Branches amounted to 3,250 plant branches-1. As for the overlap of varieties with salicylic acid, the red variety was superior for both concentrations of 50 and 100, as it gave an average number of branches amounting to (3,350 and 3,250) plant branches-1.
     
    Evaluation of active compounds
     
    Effect of variety and salicylic acid concentration on the branch content of phenolic compounds in two prickly pear varieties grown in vitro
     
    Phenolic compound hydroxy benzoic: Fig (3) shows superiority of the red variety over the honey variety in the branch content of hydroxy benzoic, as the concentration of 100 µmol L-1 gave the highest phenolic content of 54.445 compared to the concentration of 50 µmol L-1, which gave a phenolic content of 25.191.

    Fig 3: The effect of salicylic acid on the amount of hydroxybenzoic phenolic compound produced from prickly pear tissue cultures after four weeks of cultivation on a nutrient medium.


     
    Phenolic compound  hydroxycinnamic acid
     
    Fig 4 shows red variety outperformed the honey variety in branch content of the phenolic compound  hydroxycinnamic acid. A concentration of 100 micromoles yielded the highest phenolic content at 28.061, compared to a concentration of 50 micromoles per liter, which yielded a phenolic content of 17.869.

    Fig 4: Effect of salicylic acid on the amount of the phenolic compound hydroxy cinnamic acid from the tissue culture of prickly pear cactus branches after four weeks of cultivation on the nutrient medium.


           
    Since correlation analysis revealed a strong or moderate positive correlation between the phenolic compound content and the biological activity of the extracts, it is possible to hypothesize that the increased biological activity is specifically related to the increased biosynthesis of secondary phenolic metabolites under the influence of salicylic acid. The positive effect of salicylic acid treatment on the total phenolic compound content is consistent with this finding (Skrypnik, 2022; Baqir et al., 2026). Salicylic acid is known to increase the activity of phenylalanine ammonia-lyase (PAL), the most important enzyme in the initial stages of phenolic compound synthesis (Xu et al., 2015, Ali et al., 2024; Afdal et al., 2026).

    CONCLUSION

    Research focuses on developing an efficient micropropagation protocol to meet the growing demand for these varieties for consumption as fruit, animal feed and as a drought- and water-scarce alternative crop, furthermore, it can be used in sustainable farming systems, also explores the potential of prickly pear cactus varieties in vitro as an anticancer agent due to their phenolic compound content. The red variety exhibited the best branch length and number of branches when cultured on a nutrient medium containing varying concentrations of salicylic acid. A concentration of 100 µmol/L of salicylic acid resulted in the highest content of the phenolic compounds hydroxyl benzoic and hydroxyl cinnamic acid.

    CONFLICT OF INTEREST

    The authors declare that there is no conflict of interest.

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