Effect of Humic acid, Cytokinin and Arginine on Growth and Yield Traits of Bean Plant Phaseolus vulgaris L. under salt stress

the


Introduction
The Leguminosae family is economically, environmentally, and, socially important, and it is known as the bean family.It is the third largest family in terms of the number of species after the 2024, 21(3): 0919-0936 https://dx.doi.org/10.21123/bsj.2023.8617P-ISSN: 2078-8665 -E-ISSN: 2411-7986 Baghdad Science Journal agriculture (about 10,000 years ago) 3 , as in common beans in Central America and chickpeas in the Middle East regions, and thus it is considered as one of the most important and richest plant families in terms of diversity because it has a high nutritional value, especially proteins that can reach 40% 4,5 .
Beans are a member of the legume family, which are considered the main food item in most countries of the world, as they have an important nutritional, economic, and industrial impact, and called green beans for their use of green pods bean or dry beans to use dry bean seeds.
Beans are considered one of the warmest season crops, as they are grown in Iraq in two seasons, spring season, starting from March to the end of June, the yield of green pods is harvested at the beginning of May, and the production of dry seeds at the end of June.Autumn is the second season, which begins in late August or early September for green pod production only, whereas, the yield is harvested in Octobers 6 .Harvesting the plant takes place in two stages: 1-The green beans harvesting stage, differs according to the variety, where the pods begin to be harvested 2-3 months after planting and collected 3 to 5 times.2-The dry beans harvesting stage, the dry beans are collected after 4-5 months until they are dried on the plant, then removed and placed to completely dry under the sunlight.
The common bean belongs to the leguminous family Leguminosae (Fabaceae), subfamily Papilionoidea, genus Phaseolus, and species P. vulgaris.
In a recent study, it was indicated that the accumulation of pharmaceuticals such as Diclofenac have anti-pain and inflammatory diseases such as gout, and mefenamic acid, which is resistant to inflammation and swelling, as well as metronidazole, which has antibacterial activity against anaerobic pathogens 15 .
Climate change-related stresses, such as salinity, drought, and heat, reduce the world's crop quantity and quality, leading to significant food, social, and economic insecurity.This is a reality in poor and developing countries, that more than two billion people do not have regular access to safe, nutritious, and sufficient food.According to the National Nutrition Surveys report, 36.9% of the world population suffers from food insecurity 16,17 , and with the world population reaching 10.4 billion in 2080, according to the United Nations estimation, 18 and increasing future expectations of food demand, 19 Therefore, effective measures to increase crop yields must be adopted to overcome the issues of the increase in world population and to mitigate the harmful effects of stresses associated with climate change.This is done through the optimal and effective use of sustainable agricultural practices, especially the addition of some materials that reduce the effect of salt stress resulting from the lack of fresh irrigation water, and the use of saline well water without compromising the quantity and quality of the crop, including leguminous family plants, especially the bean plant, it is very important to integrate into the diet that is important for human health.This is in line with an excellent strategy to achieve the United Nations Sustainable Development Goals for reducing malnutrition and achieving food security 20,21 .
According to the estimation of the Food and Agriculture Organization of the United Nations (FAO) for the year 2021, the cultivated area of the green bean crop in Iraq is 669 hectares, and the production reached 5.9013 tons/ hectare.The cultivated area of dry beans was 760 hectares and the productivity was 6.897 tons/ha. 22, while the cultivated areas of green beans in Iraq witnessed a Beans are considered one of the vegetable crops that are sensitive to salinity in the irrigation water, or in the soil.Salinity causes a severe reduction in the yield and quality, because of the rapid increase of saline soil in dry and semi-arid regions of the globe as a result of various climatic changes, insufficient irrigation water, or irrigation with saline-rich water, and widespread soil erosion caused by global warming 23,24 .
The phenomenon of salinity expanded, around 76 million hectares of irrigated lands have problems related to salt stress worldwide 25 .This accounts for 20% of the world's arable land, and an increase in the salinity problem is expected to lead to the loss of up to 50% of arable land by the middle of the 21 st century 26 .
Salt stress reduced growth factors, water content, and photosynthetic pigments of common bean plants 27 , also causing a lot of physiological, phenotypic, and biochemical changes in plants, and greatly affecting plant productivity.Salt-sensitive plants either die, or their productivity decreases after exposure to salt stress.Among the harmful effects of salinity on plants is root growth reduction, burns on the edges of leaves, inhibition of flowering, reduced germination, and seed and yield decline 28,29,30 .
Humic acid is a product that contains several elements to improve soil fertility and increase the availability of nutrients, thus affecting plant growth and yield, and mitigating the harmful effects of salt stress.It is a substance that is generally formed by the biodegradation of organic matter resulting in a mixture of acids containing carboxylic groups and phenols and has a low molecular weight and high oxygen content 31 .From previous available studies, it was found that the addition of humic acid led to a significant increase in plant length, stem diameter, average leaf area, number of leaves, and dry weight of shoot, as well as the yield of pods, the number of pods in the plant, and the length of the pod, compared to control plants 32 In addition, humic acid was used to mitigate the effect of stress of 8 types of salts (NaCl, Na2SO4, CaCl2, CaSO4, KCl, K2SO4, MgCl2, and MgSO4) on common beans.It was found that all types of salts led to a negative effect on the weights of the shoot and root, and Chlorine impact was more harmful to shoots, especially as NaCl salt, the addition of humic acid was also reported to have a mitigating effect on all types of salinity, leading to an increase in the internal levels of proline, and increase in the Nitrogen, Phosphorus and total chlorophyll content, and leaf area 33 .
Cytokinins influence many aspects of plant growth and development, as recent developments revealed complex physiological functions of cytokinins and discovery of the new function of cytokinins in plant defense systems and stress resistance.Studies have shown that cytokinin content and transmission are reduced due to salinity in different plant species.The decrease in cytokinin level in shoots under stress conditions may be due to suppression of IPT1,3,5 and/or activation of CKX1,3,6 in Arabidopsis, a decrease of transport of root cytokinins in xylem, and in the regulation of senescence through chlorophyll degradation, decreased photosynthetic activity, and ultimately cell death 34 .
Soil salinity leads to a severe decrease in the availability of water and essential minerals to plants, which hinders growth, but in a study of the role of benzyl adenine (BA) to mitigate the adverse effects of soil salinity in broad bean (Vicia faba L), plants were subjected to the stress of 150 mM NaCl, and were Sprayed with BA (0.9 mM), led to improving the growth characteristics of faba bean plants due to an increase in the uptake of K, Ca, and Mg ions, the accumulation of free amino acids, soluble sugars, and soluble proteins, and the activity of some antioxidant enzymes, which indicates that treatment with BA can reprogram metabolic processes to tolerate salinity 35 .In another study to prove the role of cytokinin in mitigating the effects of salt stress on rice plants under salt stress, it was found that the addition of cytokinin improved seed characteristics, including seed yield, the weight of 1000 seeds, and the percentage of seed filling 36 .Many studies on the effects of arginine have mainly focused on increasing abiotic stress tolerance in plants.It was reported that plants treated with arginine improved fruit yield and quality 37,38 .In another study, it was found that salinity stress causes a decrease of about 17.4% in the number of leaves, but the use of arginine at a rate of 10 and 20 mM showed the improvement of the fresh and dry weights of shoots (gm) recording 20.9%,15.8%,39.5%, and 31.6%,respectively in comparison to the control.It is interesting to note that the most pronounced effect of the growth parameters studied was found using the higher concentration of arginine 39 .In another study, the addition of arginine increase the vegetative growth length (cm), the weight of a thousand seeds (g), the dry weight of the root (g/plant), the total chlorophyll (µg/cm 2 ), the percentage of starch, and ash content, as well as the activity of catalase, peroxidase, and ascorbate peroxidase in different concentrations of salts 40 .
The present study aimed to evaluate the influence of the addition of humic acid and foliar spray of cytokinin and arginine on the growth and yield of the kidney bean plants under salinity in the local environment, taking into account the problems of deficiency of fresh water, increasing the saline areas and growing demand for this plant in the Iraqi market.

Materials and Methods
This experiment was carried out during the spring growing season in 2022 in one of the agricultural fields of Abu Ghraib district, 20 km West of Baghdad governorate, at longitude 44 East, latitude 33 North, and 34.1 m above sea level.A factorial design with three replicates was used.The first factor included three groups; control H0, H1 (6 Kg.h -1 Humic acid), and H2 (12 Kg.h -1 Humic acid).The second factor included two groups; control C0 (distill water spray), and C1 (100 mg.l -1 Cytokinins (benzyl adenine)), and the third factor included three groups; control A0 (distill water spray), A1 (100 mg.l -1 Arginine), and A2 (200 mg.l -1 Arginine) as a total of 18 treatments, identical to the combinations of humic acid, Cytokinins, and Arginine.Each treatment included 3 biological replicates, and contained 7 plants.The leaves were sprayed with solutions of distilled water containing 0.1 % Tween 20 (polysorbate 20, which is a polysorbate-type nonionic surfactant formed by the ethoxylation of sorbitan) as a surfactant agent.
The cultivation field was divided into three main panels (replicates), and then each panel was divided into 18 secondary panels to represent the experimental unit for each treatment, in a total of three transactions with 54 experimental units.The land was plowed and cleaned off all the growing bushes.Soil samples were taken from the field at a depth ofs 0-30 cms randomly and mixed well.The sample was analyzed in the Department of Soil Sciences and Water Resources of the College of Agriculture / University of Baghdad to conduct some chemical and physical analyzes of the soil, as in Table 1 and 2 The following parameters for growth and yield characteristics were recorded on randomly selected plants: plant height (cm), leaf area (dm 2 .plant - ), shoot dry weight (gm), root dry weight (gm), Number of pods (pod.plant -1 ), pods length (cm), Pod weight (g), Yield per plant (gm plant -1 ), and Total yield (ton hectare -1 ).For the statistical analysis, the analysis of variance (ANOVA) was performed, the analytical data were analyzed using the statistical program (Genstat Twelfth Edition, 2012), to examine the effects of humic acid, Cytokinin, Arginine, and their interactions on growth and yield of bean plant under salinity stress.

Results and Discussion
Plant height Table 3, demonstrated the results of the effect of the three treatments, the addition of humic acid, spraying of cytokinins (benzyl adenine), spraying of the amino acid (arginine), and their interactions in measuring the height of the bean plant, there was a significant difference in the treatments compared to untreated plants, and the highest value of height plant at the second concentration of humic acid (H2) was 45.3733 cm, the lowest value in the control treatment (H0) was 42.2822 cm.In addition, there was a significant difference in the treatment of cytokinin spraying, as the highest value at the first concentration of cytokinin (C1) was 44.7514 cm, compared to the control treatment, while the lowest value was (C0) 43.5935 cm, whereas, there were no significant differences for the treatment of spraying arginine in comparison to the control treatment.
Similarly, there were no significant differences in the interaction between cytokinin and arginine, whereas, there were clearly significant differences in the interaction between humic and cytokinin.The highest value of treatment with (H1C2) was 45.5000 cm, and the lowest value in the control (H0C0) was 41.1511 cm.Moreover, the interaction of humic and arginine gave the highest value (H2A2) recording 45.5540 cm, and the lowest height (41.8320 cm) was in the control treatment (H0A0).
In regards to the interaction of humic, cytokinin, and arginine, the highest value of (H2C1A2) treatment   For each parameter, treatments followed by the same letter are not significantly different at p ≤ 0.05.

Leaf Area
The results of Table 0 indicate the effect of treatments on the leaf area of the bean plant, where the highest value at the second concentration of humic acid (H2) and arginine (A2) was 10.11241 dm 2 and 9.50011 dm 2 respectively, and the lowest value for the control treatment (A0) was 9.22689 dm 2 .For cytokinin treatment, the highest value at the first concentration (C1) was 9.61546 dm 2 , compared to the control treatment (C0) at 9.18755 dm 2 .
On the other hand, the results revealed that there were no significant differences in the leaf area index, where a combination of cytokinin and arginine was used Whereas, there were obvious significant differences when the interaction between humic and cytokinin was used, as the highest value in the (H2C1) treatment was 10.2067 dm 2 and the lowest leaf area at (H0C0) treatment was 8.0443 dm 2 .Likely, the interaction between humic and arginine gave the highest value (10.1649 dm 2 ) in the (H2A2) treatment, and the lowest value was 8.1415 dm 2 in the control treatment (H0A0).
Moreover, the threesome interactions of humic, cytokinin, and arginine exert the highest leaf area value (10.2804 dm 2 ) when treated with (H2C1A2), and the lowest value with control (H0C0A0) was 7.6690 dm 2 .For each parameter, treatments followed by the same letter are not significantly different at p ≤ 0.05.

Dry weight of the shoot
The results of Table 1, showed the effect of treatments on the dry weight of the bean plant shoot, the highest rate was recorded at the second concentration of humic acid (H2) at 33.9444 gm, and the highest rate for the second concentration (A2) of the arginine was 29.2222 gm, and the lowest rate was in the control treatment (A0).It was found that there was a significant difference in the cytokinin treatment, as it reached the highest rate at the first concentration (C1) was 29.6667 gm, compared to the control treatment (C0).
The interaction between cytokinin and arginine revealed that there were no significant differences, while there were obvious significant differences in the interaction between humic and cytokinin, where the highest dry shoot weight in the (H2C1) treatment was 35,000 gm, and the lowest was in the (H0C0) treatment.
Similarly, the interaction between humic and arginine gave a 34,500 gm (H2A2) value, and the lowest was in the control treatment (H0A0).
For the triple interactions, there were significant differences when humic, cytokinin, and arginine were used in combination, as the highest value was 35,000 gm with (H2C1A2) treatment, and the lowest was in the control treatment (H0C0A0) For each parameter, treatments followed by the same letter are not significantly different at p ≤ 0.05.

Dry weight of the root
The results of Table 2, illustrated the effect of treatments on the dry weight of root, the highest value of the average at (H2) was 3.49222 gm compared to control (H0) 2.13889 gm, and there were significant differences.Likewise, the treatments of arginine, exhibits clear significant differences, with the highest rate for (A2) being 2.84056 gm, and the lowest rate for control (A0) being 2.64000 gm.In addition, there was a difference in the cytokinin treatment, as the highest rate at the (C1) was 2.91333 gm, compared to the control treatment (C0) at 2.58074 gm.
While the combination of two treatments, cytokinin, and arginine, there were no obvious significant differences, whereas, the highest value in the (H2C1) treatment was 3.6900 gm, and the lowest in the control treatment (H0C0) was 1.9478 gm, when the combination of humic and cytokinin was used, Similarly, the interaction between humic and arginine, gave higher value (3.6167 gm ) for the treatment (H2A2), and a lower value for the control treatment (H0A0) of 2.0033 gm for the dry root weight.
Moreover, there were significant differences in the interaction of humic acid, cytokinin, and arginine, as the highest value was 3.7133 gm with (H2C1A2) treatment, and the lowest value of the triple combination with no-treatment (H0C0A0) was 1.6733 gm.For each parameter, treatments followed by the same letter are not significantly different at p ≤ 0.05.

The number of pods
The results in Table 7, reflected the effect of the three treatments; addition of humic acid, spraying benzyl adenine, spraying of amino acid, and their interactions in measuring the number of pods of bean plants.The highest rate in the number of pods in the second concentration of humic acid was 27.241 and the lowest rate in the control treatment was 20.574., no differences were found in the interaction between cytokinin and arginine.
Unlikely, there were clear differences in the interaction between humic and cytokinin, as the highest number of pods in the (H2C1) treatment was 27,281, and the lowest number of pods in the (H0C0) treatment was 18,504.Furthermore, the results showed that there was no effect of the treatment of cytokinin and arginine on the average number of pods in the plant.However, the interaction has differed between humic and arginine, where the highest number of pods in the plant was 27.578 in the (H2A2) treatment, and the lowest number of pods with the control treatment (H0A0) was 18.933.
The triple interactions exhibit the highest number of pods in plants treated with (H2C1A2) 27.578, and the lowest number in the non-treated control (H0C0).For each parameter, treatments followed by the same letter are not significantly different at p ≤ 0.05.

Pods length
The findings of Table ., showed the effect of treatment on measuring the length of pods in bean plants, it was found that there were no significant differences in the average of humic acid, cytokinin, and arginine treatment on the length of pods.
Likely, no differences were observed in the interaction of arginine and cytokinin, and the interaction between humic and cytokinin.However, there was a significant effect of the interaction between humic and arginine, where the highest value in the (H2A0) treatment was 12.1167 cm, and the lowest value in the (H0A0) treatment was 11.1722 cm.
Interestingly, the highest value was found when a combination of all treatments was conducted (H2C1A2), and the lowest value was in no-treated control (H0C0A0).For each parameter, treatments followed by the same letter are not significantly different at p ≤ 0.05.

Pod weight average
The results of Table 9, revealed that there was a significant effect on plants treated with the treatments, where the highest rate of pod weight in the plants was 4.30557 gm at (2H), and the lowest average pod weight with the control treatment (H0) was 3.69496 gm.It was found that the cytokinin spraying treatment had a significant difference, as the highest rate with (C1) was 4.10010 gm in comparison to the control treatment, where the lowest rate at (C0) was 3.94772 gm.Whereas, there were no significant differences in arginine treatments compared to the control, Similar to the interaction between cytokinin and arginine.
Unlikely, the results showed that there were differences in the interaction between humic and cytokinin, was the highest value in the treated plant (H2C1) was 80.2594 gm, and the lowest value in the untreated plant (H0C0) was 78.3580 gm.The same with the interaction between humic and arginine, there were significant differences between the interactions, as the highest value in (H2A1) was 4.3459 gm, and the lowest value in the control (H0A0) was 3.6568 gm.
In regards to the threesome interactions of humic, cytokinin, and arginine, the highest value was 4.4343 gm when treated with (H2C1A2), and the lowest value was 3.4943gm when the control treatment (H0C0A0) was used.For each parameter, treatments followed by the same letter are not significantly different at p ≤ 0.05.

Yield per plant
The results of Table 14 indicated a crucial role of humic treatment in terms of the yield of the plant, as the highest mean of plant yield was 117.139 gm when the treatment (2H) is used.While the treatment of arginine didn't exert any significant differences in the yield per plant.It was observed that the cytokinin treatment excelled, as the highest rate of yield per plant was 103.739 g in treatment (C1), and the lowest rate of yield per plant was 93.051 gm in the control treatment (C0).
Whereas there were no differences in the interaction between the treatments of arginine and cytokinin in the experiment in terms of the yield of one plant.Otherwise, the interaction between humic and arginine, gave the best value of yield per plant in the treatment (H2A2), and the lowest value was in the control treatment (H0A0).
In the same context, with the interaction between humic and cytokinin, the highest value was obtained in the (H2C1) treatment, and the lowest value was in the control treatment (H0C0).
In consideration of using threesome interactions, there were very significant differences between the overlapping treatments for the yield per plant, the highest value was with (H2C1A2) treatment, and the lowest value was with no-treatment control (H0C0A0).For each parameter, treatments followed by the same letter are not significantly different at p ≤ 0.05.

Discussion
The outcomes of the present study can be initially discussed in the light of affecting the addition of humic acid, spraying of cytokinins (benzyl adenine), spraying of an amino acid (arginine), and their interactions on the bean plant sisPglolPsuloesahP L, to tolerate salinity stress via mitigating the salinity damage and reducing its toxicity for the plant by a significant increase in the vegetative growth traits, i.e. plant height, leaf area, fresh and dry weight for both shoot and root, and yield.Salt stress is one of the main environmental factors that weaken physiological processes in plants, including vegetative and flowering growth factors and water content, 27 by reducing the metabolism such as photosynthesis efficiency, decomposition of pigments, and vacuole function 41 .As well as deterioration of the chloroplast membrane, and other organelles such as mitochondria and endoplasmic reticulum 42 , disruption of plant hormones function, like auxin IAA, alteration of basic metabolic pathways, and manipulation of gene expression pattern 43 .Salinity stress leads to an ionic imbalance, which leads to the accumulation of harmful ions in plants, such as Na and Cl, 44 and reducing the absorption of macro elements such as N, P, K, Ca, and Mg, 45,46 and microelements such as B, Zn, Cu and Fe 47 .It leads to an increase in the production of reactive oxygen species (ROS), as it causes cellular imbalance, which mainly results in cell membrane damage, and deteriorates biomolecules such as lipids, DNA, and proteins 42,48 .
The significant reduction of salinity damage and stress tolerance resulting from the addition of humic acids to the plant leads to improve the vegetative growth characters 49 sby enhancing the absorption of important nutrients, and increasing their transfer and then their accumulation in the shoot system in a significant way, such as macronutrients N, P, K, Ca, Mg, S, and micronutrients such as Fe, Zn, Mn and Cus 50 , As well as reducing the accumulation of some toxic elements by reducing their uptake, such as Na 51, 52 , as well as elongating root cells, increasing oxygen uptake and respiration, and increasing chlorophyll pigments and thus the efficiency of photosynthesis, which leads to plant tolerance to high concentrations of salinity 53,54 .
It was found that spraying the growth regulator cytokinin mitigates the toxicity of saline water, perhaps through vegetation growth parameters, and also reduced lipid peroxidation, improved oxidative defense in leaves, and increases membrane permeability, as well as substitutes the oxidative damage by enhancing antioxidant defense mechanisms such as increasing the enzymatic activity of superoxide, catalase, peroxidase, ascorbate peroxidase, and scavenging ROS 55,56 Cytokinins also regulate the ability of plants to absorb many nutrients from the environment, including nitrogen, phosphorous, sulfur and iron 57 , which regulates mineral balance.The nutrient status of plants regulates plant growth 58 .Positive regulation of substances exerts protection against osmotic stress and ionic balance, antioxidant activity, and finally plant growth and yield 55 .
Amino acids can be used as neutralizing compounds against stress conditions, and as a useful strategy to mitigate salt stress, as the foliar spraying of arginine leads to protective effects on plants in alleviating salinity stress by both improving vegetative growth qualities, and improving the enzymatic activities of antioxidant enzymes.sCatalase, Peroxidase, Superoxide Dismutase, and Ascorbate Peroxidase 59,60 , and the increase in phenolic substances and osmotic modification, which lead to a better antioxidant defense system for the plant and osmolytes accumulation 44,61 .When using arginine, the pigments of photosynthesis increased significantly in plants treated with arginine under salinity stress, and thus the efficiency of the photosynthesis process increased, as a result, carbohydrates accumulated, which formed the basic framework for the plants 62,63 .In addition, free amino acids were increased, as well as a significant decrease in sodium, while a significant increase was found in potassium and phosphorus, and a nonsignificant increase in nitrogen, calcium, and magnesium, which leads to a significant increase in yield characteristics. 40,45 .Thus, the ratio of K + / Na + increased in the leaves, as increased K + absorption helps to maintain ionic balance, regulate osmotic balance, maintain swelling, and regulate membrane potential 64,65 .

Conclusion
In conclusion, the present study concluded the following: humic acid, cytokinin, arginine and their interactions enhance the growth and production of bean plants under salt stress.The optimum yield of bean plants under salinity was obtained when 12 Kg.h - humic acids was applied, in the overlapping treatment (H2C1A2) (12 Kg.h 1 Humic acid), (100 mg.l -1 Cytokinins (benzel adenine)) and (200 mg.l -1 Arginine) concentration for the local agricultural climate.

Authors' Declaration
-Conflicts of Interest: None.-We hereby confirm that all Tables in the manuscript are ours.
-Ethical Clearance: The project was approved by the local ethical committee in University of Baghdad.
2024, 21(3): 0919-0936 https://dx.doi.org/10.21123/bsj.2023.8617P-ISSN: 2078-8665 -E-ISSN: 2411-7986 Baghdad Science Journal clear fluctuation during the past years, and this fluctuation in the cultivated areas is due to many reasons, including the problems that agricultural lands suffer from, especially the issues of salinity, such as well as the lack of use practical techniques that lead to raising productivity through production per unit area.

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The experimental unit was represented by 1 m-apart and 3 m-long furrows, each containing 20 plants, and 45 cm apart.During the period of plant growth, furrow irrigation with drip pipe irrigation was used regularly with non-saline water EC=2.2, during the first growth stage until it reaches the stage of four true leaves then irrigated with well saline water EC=3.4 for the rest of the plant life cycle, and weeds were manually kept under control.The broad bean (sensitive to salinity) sisPglolPs uloesahP L. Var.Astraid (from MONARCH seeds, China) cultivars, was sterilized and circulated in local agriculture, were sowed in an open field on the first of March 2022.At curds collection (harvest), seven plants were randomly chosen for the experiment.The leaves of plants were sprayed with Cytokinin and Arginine, humic acid was added three times, the first time after the formation of the four leaves, the second time when the flowering began to form, and the third time after two weeks.

Table 4 .
The effect of humic acid, cytokinin, and arginine, and their interactions on the pod length (