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Full Research Article

Effect of Foliar Spraying with Lysine on Growth and Yield Performance and Assessment of Genetic Diversity using ISSR Markers in Faba Bean (Vicia faba L.)

G
Ghuffran Ali Hussien1
E
Eman Noaman Ismail2
D
Dania Salman Qahraman1
E
Enas Abdul-Raheem AL-Wagaa1
D
Duha M. Majeed2
A
Ahmed Flayyih Hasan2,3,*
1Department of Field Crop Sciences Agriculture College, Diyala University, Diyala, Iraq.
2Biotechnology Research Center, Al-Nahrain University, Jadriya, Baghdad. Iraq.
3Department of Medical Laboratory Techniques, College of Health and Medical Technology, Al-Farabi University, Baghdad, Iraq.

ABSTRACT

Background: A field experiment was conducted at the experimental farm of the Department of Field Crop Sciences, College of Agriculture, University of Diyala, during the winter growing season of 2024-2025. The aim of the study was to investigate the effect of spraying the amino acid lysine on growth characteristics and yield of Vicia faba L. and to determine genetic variation by using ISSR-PCR DNA markers.

Methods: The study was conducted during the 2023-2024 growing season in the fields of the College of Agriculture, University of Diyala. The genetic analysis was carried out at the Institute of Genetic Engineering, Al-Nahrain University. The experiment was conducted using a randomized complete block design (RCBD) with a split-plot system and three replications The experiment included two factors: The first factor, placed in the main plots, consisted of three concentrations of lysine: T1 (control treatment - no spray), T2 (100 ml L-1 lysine spray) and T3 (200 ml L-1 lysine spray), The second factor, placed in the subplots, consisted of three genotypes: G1 (Spanish genotype), G2 (Veto genotype) and G3 (local Iraqi genotype).

Result: Lysine spraying was carried out twice, on January 14, 2024 and the second one on January 20, 2024. The Studied Traits were plant height, Number of branches,  number of pods per plant, pod length, pod fresh weight, plant yield and total yield. The application of Lysine at a concentration of 200 mg L-1 resulted in the highest performance for the Vito genotype, which excelled in most studied parameters, including pod length, fresh weight per pod and total yield per Plant. The UPGMA tree results, based on ISSR data and the Dice similarity coefficient, indicated clear genetic variation among the studied samples, which were clustered into two major groups with closely related internal subgroups.

INTRODUCTION

The faba bean (Vicia faba L.) is one of the most important legume crops worldwide. It plays a significant part enhancing agricultural sustainability, it represents a great source of protein and it is classified as one of the most protein-rich crops (Burstin et al., 2011). Also it’s proved its importance in nitrogen fixation and controlling the regulation of crops rotation (Abbas, 2012). Recent research focused on the use of various stimulants to enhance plant growth and development. Amino acids play a significant role in agriculture, as they are the building blocks of proteins and are important in plant biochemical processes. And they participate in plant defense against pests and diseases as a part of biological control, Furthermore they are important in improving plant tolerance to both biotic and abiotic stresses (Dellero, 2020). Amino acids have several beneficial effects on plants. Amino acids are structural units of proteins and play a role in biosynthesis of glutamine which is a precursor of nucleoids 1995 (Abdelhameed et al., 2024). There are many amino acids, lysine considered one of the most important (Ibraheim et al., 2021), in spite of its significance, the concentration of lysine is low in cereal and legume crops (Wenefrida et al., 2009), in turn its reduces their nutritional value to 50-75% (Galili and Amir, 2013). Therefore, it necessary to enhance lysine levels in field crops through genetic engineering approaches (Galili and Amir, 2013). It is also possible to determine genetic diversity by using molecular techniques, which lead to  genetic improvement of plants. Scientists focused on several molecular technology, including ISSR-PCR, this technology characterized by not requiring prior knowledge of DNA sequences and the ability  to amplify multiple regions across the genome, thus revealing differences between cultivars.
       
The aim of the study is to investigate the effect of spraying the amino acid lysine on growth characteristics and yield of Vicia faba L. and to determine genetic variation by using ISSR-PCR DNA markers.

MATERIALS AND METHODS

The study was conducted during the 2023-2024 growing season in the fields of the College of Agriculture, University of Diyala. The genetic analysis was carried out at the Institute of Genetic Engineering, Al-Nahrain University.
       
The experiment was conducted using a randomized complete block design (RCBD) with a split-plot system and three replications.
       
The experiment included two factors: The first factor, placed in the main plots, consisted of three concentrations of lysine: T1 (control treatment - no spray), T2 (100 ml L-1 lysine spray) and T3 (200 ml L-1 lysine spray), The second factor, placed in the subplots, consisted of three genotypes: G1 (Spanish genotype), G2 (Veto genotype) and G3 (local Iraqi genotype). Lysine spraying was carried out twice, on January 14, 2024 and the second one on January 20, 2024.

The data were analyzed according to a randomized complete block design (RCBD) arranged in a split-plot system, Duncan’s multiple range test (DMRT) was used to compare treatment means at the 0.05 probability level.
 
DNA extraction from a plant
 
Genomic DNA was extracted by using a DNA extraction kit supplied by Bioneer/Iraq.The isolating of DNA was from faba bean leaves and its quality was verified by using agarose gel electrophoresis (0.1%) and visualized under ultraviolet (UV), then the purity and concentration of the DNA were subsequently evaluated using a NanoDrop spectrophotometer.
 
Polymerase chain reaction (PCR)
 
In this study we used Six primers based on the ISSR-PCR technique. The ISSR analysis was performed in a 50 µL reaction volume as described by Khalifa et al., (2025).
       
The reaction mixture contained 0.2 mM of deoxynucleotide triphosphates (dNTPs), 1.5 mM MgCl2  0.5 µL of reaction buffer, 0.2 µL of primer (50 µM), 0.3 µL of template DNA (50 µg µL-1) and 0.3 µL of Taq DNA polymerase (5 U µL-1). The final volume was adjusted to 50 µL with sterile distilled water.
       
The polymerase chain reaction (PCR) was performed for 40 cycles, following an initial denaturation step of 5 minutes at 95°C, Each cycle consisted of three steps: denaturation at 95°C for 1 minute, annealing at 36°C for 2 minutes and extension at 72°C for 1 minute, The final extension step was performed at 72°C for 10 minutes.
       
The products of ISSR-PCR were separated by electrophoresis on 1.2% agarose gel stained with ethidium bromide in 1.5× TBE buffer. The gels were visualized under ultraviolet (UV) light and photographed. Fragment sizes were estimated using a 100-3000 bp DNA ladder as a molecular size standard.


 
The unweighted pair group method of cluster Analysis (UPGMA) dendrogram based on the Dice similarity coefficient was constructed to assess the genetic diversity and relationships among three broad bean species (Vicia faba). Only clear and reproducible bands were scored and converted into a binary data matrix, where the presence of a band was recorded as (1) and the absence of  a band was recorded as (0).

RESULTS AND DISCUSSION

(Table 1) showed the effect of genotype, spraying lysine  and their interaction on plant height (cm) in faba bean no significant differences between the studied genotypes, the Iraqi genotype recorded the highest plant height (70.66 cm), while the Vito genotype recorded the lowest plant height (60.2 cm) and there were no significant differences observed between  lysine levels. This is possibly due to differences in the concentration of the amino acid (lysine), or may be variation in crop species and environmental conditions. Or It’s because the effects of individual components are less understood Henderson et al., (2025); Rafie et al., (2017) showed no significant difference in onion by using free amino acids.

Table 1: Physical and chemical properties of the soil for the 2025 growing season.


       
For the interaction effect, the result of statistical analysis was consistent with the results of the principal factors, showing that no significant differences were observed with the interaction treatments. However, the interaction treatment (Spanish genotype without spraying) recorded the highest plant value, reaching 74.67 cm (Table 1).
       
The results in (Table 2) showed significant differences between the genotypes. The Iraqi genotype exhibited the highest number of branches per plant, reaching 9.6, compared to the lowest number of branches in the Vito genotype, which was 6.95. The number of branches was not affected by lysine spraying, as no significant differences were observed between its levels.

Table 2: Effect of genotype, lysine spraying and their interaction on plant height (cm) in faba bean.


       
The number of branches was not affected by lysine spraying, as no significant differences were observed between its levels. The results also indicate a significant interaction effect, with a significant increase in the number of branches per plant observed in the interaction treatment between the Iraqi variety and under a 200 mg/plant-1 spray, compared to the other interaction treatments. This increase reached 10.73 branches, compared to the lowest number of branches in the Vito variety under a 200 mg/plant-1 spray level, which was 6.86 branches. This may be attributed to the effect of amino acid spraying on vegetative growth characteristics (Hussein et al., 2026). This result is consistent with the findings of (Abdelkhalek et al., 2022), who also reported an increase in the number of branches in maize plants after lysine spraying, this is alsoagreed with the results of EL-Bassiouny (2005) who showed that the foliar application of amino acids increased number of spikes, that due to the amino acids effect in stimulating the physiological and biochemical processes such as protein biosynthesis,carbohydrate metabolism by stimulating photosynthesis and stimulate Enzymes and coenzymes Baqir and Al-Naqeeb (2019); Al-Said and Kamal (2008); Francessco and Michele (2009) (Table 2).
       
The number of branches was not affected by lysine spraying, as no significant differences were observed between its levels.
       
The results in (Table 3) regarding the effect of genotype, lysine spraying and their interaction on the number of pods per plant in broad beans indicate significant differences between the genotypes. The Iraqi genotype outperformed the others, achieving the highest number of pods per plant (14), while the Spanish genotype achieved the lowest (7.73).

Table 3: Effect of genotype, lysine spraying and their interaction on number of branches in faba bean.


       
The same table also shows that lysine spraying had no significant effect on the number of pods per plant. However, the interaction between the genotypes and lysine spraying significantly affected the number of pods per plant. The Iraqi genotype, under a spray level of 100 mg/L, produced the highest number of branches (14.6), compared to the lowest number of branches (5.8) in the Spanish genotype under a spray level of 200 mg/L. This result is consistent with the findings of) on maize. Ismail and Fayed (2020) showed the same result, also Zeboon et al. (2017) showed the significant differences between the two wheat cultivarsin the number of tillers,its because of amino acids stimulates the plant to produce auxins and proteins and enhancing cell division which lead to increases plant’s growth traits of the oat plant (Al-Badrawi and Alabdulla, 2021).
       
Table 4, which shows the effect of genotype, lysine spraying and their interaction on the number of pods per plant in broad beans, reveals significant differences between the genotypes. The Vito and Spanish genotypes outperformed the Iraqi genotype, achieving the highest pod lengths of 20.46 cm and 19 cm respectively, compared to 11.43 cm for the Iraqi genotype.

Table 4: Effect of genotype, lysine spraying and their interaction on number of pods per plant trail in faba bean.


       
The same table indicates a significant effect of lysine spraying, as spraying at a concentration of 200 mg/L resulted in the longest pod, reaching 19.32 cm, compared to the shortest pod length of 16.32 cm in the non-sprayed variety. The interaction between genotypes and lysine spraying also significantly influenced pod length. The Phyto and Spanish genotypes showed a significant advantage at all spray levels, with the highest pod length (20.86 cm) observed in the Phyto genotype at 100 mg/L spraying. This contrasts with the shortest pod length (9.5 cm) in the Iraqi genotype under the 100 mg/L spray level. This is attributed to the positive effect of amino acid spraying on broad beans, as amino acids play a role in stimulating physiological and biochemical processes, including protein synthesis and carbohydrate synthesis through chlorophyll production and photosynthesis. (Sabry et al., 2022).
       
The results in (Table 5) show significant differences between the genotypes. The phyto genotype achieved the highest fresh pod weight of 20.13 g, compared to the lowest fresh pod weight of 11.73 g in the Iraqi genotype.

Table 5: Effect of genotype, lysine spraying and their interaction on pod length (cm) in faba bean.


       
The same table also indicates a significant effect of lysine spraying. Spraying at a concentration of 200 mg/L resulted in a higher fresh pod weight of 18.09 g than other concentrations, while the lowest fresh pod weight was 13.2 g in the non-sprayed variety. Furthermore, the results show a significant interaction effect, with a significant increase in fresh pod weight (22.24 g) observed in the Phyto genotype treatment under 200 mg/L spray, compared to the lowest fresh pod weight (11.05 g) in the Iraqi variety under 100 mg/L spray, this increase due to the role ofamino acids which play an important role in plant metabolism and protein assimilation this is necessary for cell formation and consequently increase in fresh and dry matter  (Sadak et al., 2014), (Anwar et al., 2025; Rasheed et al., 2025) also showed that amino acids are regulating and improving themetabolic pathways and osmotic effortsincreases tiller number and enhance growthvariables.
       
The results in (Table 6) regarding the effect of genotype, lysine spraying and their interaction on plant yield in broad beans indicate significant differences between the genotypes. The Vito genotype outperformed the others, achieving the highest yield per plant at 198.2 g, while the Spanish genotype achieved the lowest yield per plant at 111.18 g.

Table 6: Effect of genotype, lysine spraying and their interaction on pod fresh weight (g) in faba bean.


       
The results in the same table indicate a significant effect of lysine spraying on plant yield. The highest yield was 165.5 g at a concentration of 200 mg L-1, while the lowest yield was 143.92 g without spraying. The interaction between genotypes and lysine spraying also had a significant effect on plant yield. The genotype “Veto” under a spray level of 200 mg L-1 produced the highest yield of 213.45 g, compared to the lowest yield of 106.06 g in the genotype “Spanish” under the same spray level. This is attributed to the role of amino acids in stimulating physiological and biochemical processes (Baqir and Al-Naqeeb, 2019), contributing to increased cell capacity for water and nutrient absorption from the soil and promoting vegetative growth (Ghiath et al., 2025).
       
From the (Table 7) showing the effect of genotype, lysine spraying and their interaction on the total yield (kg/dunum-1) in broad beans, we observe significant differences between the genotypes. The vito genotype outperformed the others, achieving the highest total yield of 4955 kg/dunum-1, compared to the lowest total yield of 2779.4 kg/dunum-1 in the Spanish genotype.

Table 7: Effect of genotype, lysine spraying and their interaction on plant yield (g) in faba bean.




       
The total yield of the Spanish genotype was 2779.4 kg/dunum-1. The results in the same table indicate a significant effect of lysine spraying, as spraying at concentrations of 100 and 200 mg L-1 outperformed no spraying, achieving the highest total yield of 4067.6 and 4137.5 kg dunam-1, respectively, while the lowest total yield was 3598 kg dunam-1 in the no-spray situation. The interaction between genotypes and lysine spraying also resulted in a significant effect on total yield, with the Vito genotype and spraying at a concentration of 200 mg L-1 producing the highest total yield of 5336.2 kg dunam-1, compared to the lowest total yield of 2651.4 kg dunam-1 in the Spanish genotype under the 200 mg L-1 spray level, its because the positive effect of Amino acid which led to the highest degree of photosynthesis and increased grain productivity (Ashoori et al., 2013; Rasheed et al., 2026), this result agreed with (Jari et al., 2014; Al-Mashhadani et al., 2026) showed that amines play a role in many physiological processes, including cell division and growth, flowering and development and an increase in the level of internal hormones.
       
From the (Table 8) (Fig 1 and Fig 2) The UPGMA tree based on Dice similarity coefficient and ISSR data showed that the nine samples were clustered into two main groups. The first group included the samples V1, V2 and VC with VC and V2 being morecloselyrelated,followed by V1. The second group included T1, SC, S2, IC, S1 and I2 and was further divided into two subgroups. The first subgroup comprised T1, SC and S2 with T1 and SC showingthehighest degree of similarity, whereas the second subgroup comprised IC, S1 and I2 with IC and S1 being more closest pairs. Overall, the results indicated clear genetic variation among the studied samples, with higher genetic similarity observed between certain pairs of samples within each cluster than among the remaining samples.

Table 8: Effect of genotype, lysine foliar application and their interaction on the total yield (kg.donum-1) of faba bean.



Fig 1: A) The amplified result of ISSR 1 was subjected to agarose gel electrophoresis. B) The amplified result of ISSR 2 was subjected to agarose gel electrophoresis. C) The amplified result of ISSR 3 was subjected to agarose gel electrophoresis. D) The amplified result of ISSR 4 was subjected to agarose gel electrophoresis.



Fig 2: A) The amplified result of ISSR 5 was subjected to agarose gel electrophoresis. B) The amplified result of ISSR 6 was subjected to agarose gel electrophoresis.


       
The UPGMA tree results, based on ISSR data and the Dice similarity coefficient, indicated clear genetic variation among the studied samples, which were clustered into two major groups with closely related internal subgroups. The clustering pattern reflects a degree of genetic similarity among some samples, whereas the divergence of others suggests underlying genetic differences. Furthermore, the apparent closeness between certain pairs, such as VC and V2 andIC and S1, may indicate that they share similar genetic traits or a more closely related ancestry than to the other samples. Overall, these results confirm the effectiveness of ISSR markers in detecting genetic variation and revealing genetic relationships among the studied samples.

CONCLUSION

The study results indicate that foliar spraying with lysine, particularly at a concentration of 200 mg/L, improved the growth and yield characteristics of okra, with the Phyto genotype exhibiting the best performance across most of the studied traits. Furthermore, ISSR analysis using the UPGMA tree and Dice similarity coefficient revealed significant genetic variation among the genotypes.
 
Funding
 
None.

CONFLICT OF INTEREST

The authors declare that there is no conflict of interest among them.

REFERENCES


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    Full Research Article

    Effect of Foliar Spraying with Lysine on Growth and Yield Performance and Assessment of Genetic Diversity using ISSR Markers in Faba Bean (Vicia faba L.)

    G
    Ghuffran Ali Hussien1
    E
    Eman Noaman Ismail2
    D
    Dania Salman Qahraman1
    E
    Enas Abdul-Raheem AL-Wagaa1
    D
    Duha M. Majeed2
    A
    Ahmed Flayyih Hasan2,3,*
    1Department of Field Crop Sciences Agriculture College, Diyala University, Diyala, Iraq.
    2Biotechnology Research Center, Al-Nahrain University, Jadriya, Baghdad. Iraq.
    3Department of Medical Laboratory Techniques, College of Health and Medical Technology, Al-Farabi University, Baghdad, Iraq.

    ABSTRACT

    Background: A field experiment was conducted at the experimental farm of the Department of Field Crop Sciences, College of Agriculture, University of Diyala, during the winter growing season of 2024-2025. The aim of the study was to investigate the effect of spraying the amino acid lysine on growth characteristics and yield of Vicia faba L. and to determine genetic variation by using ISSR-PCR DNA markers.

    Methods: The study was conducted during the 2023-2024 growing season in the fields of the College of Agriculture, University of Diyala. The genetic analysis was carried out at the Institute of Genetic Engineering, Al-Nahrain University. The experiment was conducted using a randomized complete block design (RCBD) with a split-plot system and three replications The experiment included two factors: The first factor, placed in the main plots, consisted of three concentrations of lysine: T1 (control treatment - no spray), T2 (100 ml L-1 lysine spray) and T3 (200 ml L-1 lysine spray), The second factor, placed in the subplots, consisted of three genotypes: G1 (Spanish genotype), G2 (Veto genotype) and G3 (local Iraqi genotype).

    Result: Lysine spraying was carried out twice, on January 14, 2024 and the second one on January 20, 2024. The Studied Traits were plant height, Number of branches,  number of pods per plant, pod length, pod fresh weight, plant yield and total yield. The application of Lysine at a concentration of 200 mg L-1 resulted in the highest performance for the Vito genotype, which excelled in most studied parameters, including pod length, fresh weight per pod and total yield per Plant. The UPGMA tree results, based on ISSR data and the Dice similarity coefficient, indicated clear genetic variation among the studied samples, which were clustered into two major groups with closely related internal subgroups.

    INTRODUCTION

    The faba bean (Vicia faba L.) is one of the most important legume crops worldwide. It plays a significant part enhancing agricultural sustainability, it represents a great source of protein and it is classified as one of the most protein-rich crops (Burstin et al., 2011). Also it’s proved its importance in nitrogen fixation and controlling the regulation of crops rotation (Abbas, 2012). Recent research focused on the use of various stimulants to enhance plant growth and development. Amino acids play a significant role in agriculture, as they are the building blocks of proteins and are important in plant biochemical processes. And they participate in plant defense against pests and diseases as a part of biological control, Furthermore they are important in improving plant tolerance to both biotic and abiotic stresses (Dellero, 2020). Amino acids have several beneficial effects on plants. Amino acids are structural units of proteins and play a role in biosynthesis of glutamine which is a precursor of nucleoids 1995 (Abdelhameed et al., 2024). There are many amino acids, lysine considered one of the most important (Ibraheim et al., 2021), in spite of its significance, the concentration of lysine is low in cereal and legume crops (Wenefrida et al., 2009), in turn its reduces their nutritional value to 50-75% (Galili and Amir, 2013). Therefore, it necessary to enhance lysine levels in field crops through genetic engineering approaches (Galili and Amir, 2013). It is also possible to determine genetic diversity by using molecular techniques, which lead to  genetic improvement of plants. Scientists focused on several molecular technology, including ISSR-PCR, this technology characterized by not requiring prior knowledge of DNA sequences and the ability  to amplify multiple regions across the genome, thus revealing differences between cultivars.
           
    The aim of the study is to investigate the effect of spraying the amino acid lysine on growth characteristics and yield of Vicia faba L. and to determine genetic variation by using ISSR-PCR DNA markers.

    MATERIALS AND METHODS

    The study was conducted during the 2023-2024 growing season in the fields of the College of Agriculture, University of Diyala. The genetic analysis was carried out at the Institute of Genetic Engineering, Al-Nahrain University.
           
    The experiment was conducted using a randomized complete block design (RCBD) with a split-plot system and three replications.
           
    The experiment included two factors: The first factor, placed in the main plots, consisted of three concentrations of lysine: T1 (control treatment - no spray), T2 (100 ml L-1 lysine spray) and T3 (200 ml L-1 lysine spray), The second factor, placed in the subplots, consisted of three genotypes: G1 (Spanish genotype), G2 (Veto genotype) and G3 (local Iraqi genotype). Lysine spraying was carried out twice, on January 14, 2024 and the second one on January 20, 2024.

    The data were analyzed according to a randomized complete block design (RCBD) arranged in a split-plot system, Duncan’s multiple range test (DMRT) was used to compare treatment means at the 0.05 probability level.
     
    DNA extraction from a plant
     
    Genomic DNA was extracted by using a DNA extraction kit supplied by Bioneer/Iraq.The isolating of DNA was from faba bean leaves and its quality was verified by using agarose gel electrophoresis (0.1%) and visualized under ultraviolet (UV), then the purity and concentration of the DNA were subsequently evaluated using a NanoDrop spectrophotometer.
     
    Polymerase chain reaction (PCR)
     
    In this study we used Six primers based on the ISSR-PCR technique. The ISSR analysis was performed in a 50 µL reaction volume as described by Khalifa et al., (2025).
           
    The reaction mixture contained 0.2 mM of deoxynucleotide triphosphates (dNTPs), 1.5 mM MgCl2  0.5 µL of reaction buffer, 0.2 µL of primer (50 µM), 0.3 µL of template DNA (50 µg µL-1) and 0.3 µL of Taq DNA polymerase (5 U µL-1). The final volume was adjusted to 50 µL with sterile distilled water.
           
    The polymerase chain reaction (PCR) was performed for 40 cycles, following an initial denaturation step of 5 minutes at 95°C, Each cycle consisted of three steps: denaturation at 95°C for 1 minute, annealing at 36°C for 2 minutes and extension at 72°C for 1 minute, The final extension step was performed at 72°C for 10 minutes.
           
    The products of ISSR-PCR were separated by electrophoresis on 1.2% agarose gel stained with ethidium bromide in 1.5× TBE buffer. The gels were visualized under ultraviolet (UV) light and photographed. Fragment sizes were estimated using a 100-3000 bp DNA ladder as a molecular size standard.


     
    The unweighted pair group method of cluster Analysis (UPGMA) dendrogram based on the Dice similarity coefficient was constructed to assess the genetic diversity and relationships among three broad bean species (Vicia faba). Only clear and reproducible bands were scored and converted into a binary data matrix, where the presence of a band was recorded as (1) and the absence of  a band was recorded as (0).

    RESULTS AND DISCUSSION

    (Table 1) showed the effect of genotype, spraying lysine  and their interaction on plant height (cm) in faba bean no significant differences between the studied genotypes, the Iraqi genotype recorded the highest plant height (70.66 cm), while the Vito genotype recorded the lowest plant height (60.2 cm) and there were no significant differences observed between  lysine levels. This is possibly due to differences in the concentration of the amino acid (lysine), or may be variation in crop species and environmental conditions. Or It’s because the effects of individual components are less understood Henderson et al., (2025); Rafie et al., (2017) showed no significant difference in onion by using free amino acids.

    Table 1: Physical and chemical properties of the soil for the 2025 growing season.


           
    For the interaction effect, the result of statistical analysis was consistent with the results of the principal factors, showing that no significant differences were observed with the interaction treatments. However, the interaction treatment (Spanish genotype without spraying) recorded the highest plant value, reaching 74.67 cm (Table 1).
           
    The results in (Table 2) showed significant differences between the genotypes. The Iraqi genotype exhibited the highest number of branches per plant, reaching 9.6, compared to the lowest number of branches in the Vito genotype, which was 6.95. The number of branches was not affected by lysine spraying, as no significant differences were observed between its levels.

    Table 2: Effect of genotype, lysine spraying and their interaction on plant height (cm) in faba bean.


           
    The number of branches was not affected by lysine spraying, as no significant differences were observed between its levels. The results also indicate a significant interaction effect, with a significant increase in the number of branches per plant observed in the interaction treatment between the Iraqi variety and under a 200 mg/plant-1 spray, compared to the other interaction treatments. This increase reached 10.73 branches, compared to the lowest number of branches in the Vito variety under a 200 mg/plant-1 spray level, which was 6.86 branches. This may be attributed to the effect of amino acid spraying on vegetative growth characteristics (Hussein et al., 2026). This result is consistent with the findings of (Abdelkhalek et al., 2022), who also reported an increase in the number of branches in maize plants after lysine spraying, this is alsoagreed with the results of EL-Bassiouny (2005) who showed that the foliar application of amino acids increased number of spikes, that due to the amino acids effect in stimulating the physiological and biochemical processes such as protein biosynthesis,carbohydrate metabolism by stimulating photosynthesis and stimulate Enzymes and coenzymes Baqir and Al-Naqeeb (2019); Al-Said and Kamal (2008); Francessco and Michele (2009) (Table 2).
           
    The number of branches was not affected by lysine spraying, as no significant differences were observed between its levels.
           
    The results in (Table 3) regarding the effect of genotype, lysine spraying and their interaction on the number of pods per plant in broad beans indicate significant differences between the genotypes. The Iraqi genotype outperformed the others, achieving the highest number of pods per plant (14), while the Spanish genotype achieved the lowest (7.73).

    Table 3: Effect of genotype, lysine spraying and their interaction on number of branches in faba bean.


           
    The same table also shows that lysine spraying had no significant effect on the number of pods per plant. However, the interaction between the genotypes and lysine spraying significantly affected the number of pods per plant. The Iraqi genotype, under a spray level of 100 mg/L, produced the highest number of branches (14.6), compared to the lowest number of branches (5.8) in the Spanish genotype under a spray level of 200 mg/L. This result is consistent with the findings of) on maize. Ismail and Fayed (2020) showed the same result, also Zeboon et al. (2017) showed the significant differences between the two wheat cultivarsin the number of tillers,its because of amino acids stimulates the plant to produce auxins and proteins and enhancing cell division which lead to increases plant’s growth traits of the oat plant (Al-Badrawi and Alabdulla, 2021).
           
    Table 4, which shows the effect of genotype, lysine spraying and their interaction on the number of pods per plant in broad beans, reveals significant differences between the genotypes. The Vito and Spanish genotypes outperformed the Iraqi genotype, achieving the highest pod lengths of 20.46 cm and 19 cm respectively, compared to 11.43 cm for the Iraqi genotype.

    Table 4: Effect of genotype, lysine spraying and their interaction on number of pods per plant trail in faba bean.


           
    The same table indicates a significant effect of lysine spraying, as spraying at a concentration of 200 mg/L resulted in the longest pod, reaching 19.32 cm, compared to the shortest pod length of 16.32 cm in the non-sprayed variety. The interaction between genotypes and lysine spraying also significantly influenced pod length. The Phyto and Spanish genotypes showed a significant advantage at all spray levels, with the highest pod length (20.86 cm) observed in the Phyto genotype at 100 mg/L spraying. This contrasts with the shortest pod length (9.5 cm) in the Iraqi genotype under the 100 mg/L spray level. This is attributed to the positive effect of amino acid spraying on broad beans, as amino acids play a role in stimulating physiological and biochemical processes, including protein synthesis and carbohydrate synthesis through chlorophyll production and photosynthesis. (Sabry et al., 2022).
           
    The results in (Table 5) show significant differences between the genotypes. The phyto genotype achieved the highest fresh pod weight of 20.13 g, compared to the lowest fresh pod weight of 11.73 g in the Iraqi genotype.

    Table 5: Effect of genotype, lysine spraying and their interaction on pod length (cm) in faba bean.


           
    The same table also indicates a significant effect of lysine spraying. Spraying at a concentration of 200 mg/L resulted in a higher fresh pod weight of 18.09 g than other concentrations, while the lowest fresh pod weight was 13.2 g in the non-sprayed variety. Furthermore, the results show a significant interaction effect, with a significant increase in fresh pod weight (22.24 g) observed in the Phyto genotype treatment under 200 mg/L spray, compared to the lowest fresh pod weight (11.05 g) in the Iraqi variety under 100 mg/L spray, this increase due to the role ofamino acids which play an important role in plant metabolism and protein assimilation this is necessary for cell formation and consequently increase in fresh and dry matter  (Sadak et al., 2014), (Anwar et al., 2025; Rasheed et al., 2025) also showed that amino acids are regulating and improving themetabolic pathways and osmotic effortsincreases tiller number and enhance growthvariables.
           
    The results in (Table 6) regarding the effect of genotype, lysine spraying and their interaction on plant yield in broad beans indicate significant differences between the genotypes. The Vito genotype outperformed the others, achieving the highest yield per plant at 198.2 g, while the Spanish genotype achieved the lowest yield per plant at 111.18 g.

    Table 6: Effect of genotype, lysine spraying and their interaction on pod fresh weight (g) in faba bean.


           
    The results in the same table indicate a significant effect of lysine spraying on plant yield. The highest yield was 165.5 g at a concentration of 200 mg L-1, while the lowest yield was 143.92 g without spraying. The interaction between genotypes and lysine spraying also had a significant effect on plant yield. The genotype “Veto” under a spray level of 200 mg L-1 produced the highest yield of 213.45 g, compared to the lowest yield of 106.06 g in the genotype “Spanish” under the same spray level. This is attributed to the role of amino acids in stimulating physiological and biochemical processes (Baqir and Al-Naqeeb, 2019), contributing to increased cell capacity for water and nutrient absorption from the soil and promoting vegetative growth (Ghiath et al., 2025).
           
    From the (Table 7) showing the effect of genotype, lysine spraying and their interaction on the total yield (kg/dunum-1) in broad beans, we observe significant differences between the genotypes. The vito genotype outperformed the others, achieving the highest total yield of 4955 kg/dunum-1, compared to the lowest total yield of 2779.4 kg/dunum-1 in the Spanish genotype.

    Table 7: Effect of genotype, lysine spraying and their interaction on plant yield (g) in faba bean.




           
    The total yield of the Spanish genotype was 2779.4 kg/dunum-1. The results in the same table indicate a significant effect of lysine spraying, as spraying at concentrations of 100 and 200 mg L-1 outperformed no spraying, achieving the highest total yield of 4067.6 and 4137.5 kg dunam-1, respectively, while the lowest total yield was 3598 kg dunam-1 in the no-spray situation. The interaction between genotypes and lysine spraying also resulted in a significant effect on total yield, with the Vito genotype and spraying at a concentration of 200 mg L-1 producing the highest total yield of 5336.2 kg dunam-1, compared to the lowest total yield of 2651.4 kg dunam-1 in the Spanish genotype under the 200 mg L-1 spray level, its because the positive effect of Amino acid which led to the highest degree of photosynthesis and increased grain productivity (Ashoori et al., 2013; Rasheed et al., 2026), this result agreed with (Jari et al., 2014; Al-Mashhadani et al., 2026) showed that amines play a role in many physiological processes, including cell division and growth, flowering and development and an increase in the level of internal hormones.
           
    From the (Table 8) (Fig 1 and Fig 2) The UPGMA tree based on Dice similarity coefficient and ISSR data showed that the nine samples were clustered into two main groups. The first group included the samples V1, V2 and VC with VC and V2 being morecloselyrelated,followed by V1. The second group included T1, SC, S2, IC, S1 and I2 and was further divided into two subgroups. The first subgroup comprised T1, SC and S2 with T1 and SC showingthehighest degree of similarity, whereas the second subgroup comprised IC, S1 and I2 with IC and S1 being more closest pairs. Overall, the results indicated clear genetic variation among the studied samples, with higher genetic similarity observed between certain pairs of samples within each cluster than among the remaining samples.

    Table 8: Effect of genotype, lysine foliar application and their interaction on the total yield (kg.donum-1) of faba bean.



    Fig 1: A) The amplified result of ISSR 1 was subjected to agarose gel electrophoresis. B) The amplified result of ISSR 2 was subjected to agarose gel electrophoresis. C) The amplified result of ISSR 3 was subjected to agarose gel electrophoresis. D) The amplified result of ISSR 4 was subjected to agarose gel electrophoresis.



    Fig 2: A) The amplified result of ISSR 5 was subjected to agarose gel electrophoresis. B) The amplified result of ISSR 6 was subjected to agarose gel electrophoresis.


           
    The UPGMA tree results, based on ISSR data and the Dice similarity coefficient, indicated clear genetic variation among the studied samples, which were clustered into two major groups with closely related internal subgroups. The clustering pattern reflects a degree of genetic similarity among some samples, whereas the divergence of others suggests underlying genetic differences. Furthermore, the apparent closeness between certain pairs, such as VC and V2 andIC and S1, may indicate that they share similar genetic traits or a more closely related ancestry than to the other samples. Overall, these results confirm the effectiveness of ISSR markers in detecting genetic variation and revealing genetic relationships among the studied samples.

    CONCLUSION

    The study results indicate that foliar spraying with lysine, particularly at a concentration of 200 mg/L, improved the growth and yield characteristics of okra, with the Phyto genotype exhibiting the best performance across most of the studied traits. Furthermore, ISSR analysis using the UPGMA tree and Dice similarity coefficient revealed significant genetic variation among the genotypes.
     
    Funding
     
    None.

    CONFLICT OF INTEREST

    The authors declare that there is no conflict of interest among them.

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