Tolerance Level of Butterfly Pea ( Clitoria ternatea L.) to Stress Acidity Through Tissue Culture Technique

Butterfly pea ( Clitoria ternatea L.) a high-quality legume that is rich in protein and grows on various soil types with a pH range of 5.5-8.9. This experiment was conducted to get the level of tolerance of butterfly pea plants to stress acidity at different levels through tissue culture technique. The study was designed using a complete randomized design with 6 treatments with the different levels of AlCl 3 addition using Murashige Skoog (MS) media with 20 replications (P0 (0 ppm AlCl 3 ), P1 (100 ppm AlCl 3 ), P2 (200 ppm AlCl 3 ), P3 (300 ppm AlCl 3 ), P4 (400 ppm AlCl 3 ), and P5 (500 ppm AlCl 3 )). Data were analyzed using analysis of variance (ANOVA), and if there was a significant difference, data were further analyzed using Duncan’s multiple range test. The variables observed were acidity media changes, plant height gain, number of leaves, number of branches, number of tillers, percentage of leaves withering, and leaf color. The results showed that the butterfly pea plant has mechanism of adaptation to acid stress on the parameters of plant height gain and number of leaves at the end of the observation. However, the level of plant tolerance on the parameters of the number of branches and the number of tillers was ≤ 300 ppm (pH 3.73).


Introduction
Feed is an important factor in the maintenance and productivity of ruminants.According to Prayitno et al. (2018), one alternative that can be done is using legumes as a source of natural protein because legume plants can grow anywhere and have not been utilized optimally.One type of legume with good nutritional content especially high protein is butterfly pea.
Butterfly pea flower is a plant originating from Central South America and has spread to the tropics since the 19 th century, especially to Southeast Asia, including Indonesia.It is one of the shrubs that commonly grows in Indonesia open spaces along roads and slopes (Cook et al., 2005).Butterfly pea has the characteristics of a perennial plant and has herbaceous habits, which has pinnate leaves, leaves with long petioles, podshaped fruits, and dark blue, purple, or white flower (Kosai et al., 2015).Butterfly pea is a source of leguminous forage, which has the potential as a source of forage for livestock because it has a high level of protein and digestibility (Sutedi, 2013).
Butterfly pea grows on various soil types, especially sandy soil and red clay with a pH range of 5.5-8.9(Cook et al., 2005).The area of acid-dry land in Indonesia is around 108.8 million/ha, and around 62.6 million/ha has the potential as agricultural land.Various land conditions are caused by acid stress, so the land experiences changes in soil physiochemical status and nutrient deficits, which cause plant growth to be not optimal.(Mulyani and Sarwani 2013).Acidic soils generally has pH characteristics of 4.6 -5.5, as well as fairly high clay content and relatively low potassium levels ranging from 0.1 -0.2 me/100 g of soil (Putra and Hanum, 2018).
According to Kasno (2020), the main limiting factors for production on dry land are soil acidity and high Al content and low C-organic.Intensive management of acid-dry land without considering nutrient balance can lead to low C-organic content, primary and secondary macronutrients such as phosphorus (P), calcium (Ca), and magnesium (Mg), as well as increase soil acidity, and Al can be exchanged so that can cause toxins and disrupted plant growth so that plant productivity decreases significantly.Butterfly pea is a plant with low diversity, so it has not developed in other areas (acid soil and saline soil).For this reason, it is necessary to determine the tolerance limit of the butterfly pea plant to acid stress with tissue culture.
Tissue culture is a method for isolating plant parts such as protoplasm, cells, tissues, and organs and growing them under aseptic conditions so that these parts can multiply and regenerate into whole plants again (Ziraluo, 2021).Butterfly pea has a relatively low diversity of production in Indonesia.Acid stress in the soil is caused by the high aluminium content, resulting in a decrease in the nutrients needed by plants, so their availability for plant growth and development is hampered.Butterfly pea has the potential to be developed as animal feed in dry and acidic areas, but the tolerance level to acid stress was unknown.This study aims to analyze the tolerance level to acid conditions in the butterfly pea through tissue culture techniques.

Research material
The tools used in this research are laminar air flow, autoclave, hot plate, magnetic stirrer, bunsen burner, calliper, 500 mL and 1 L beaker glass, 1 L plastic measuring cup, 100 mL capacity tissue culture bottles, pH meter, analytical scale, digital scale, pipette filler, mohr pipette, 200 mL glass bottle, scalpel, heat-resistant plastic, spatulas, blower, petri dishes, tweezers, Munsell color chart, and stationery.

Research procedures
Seed sterilization.The research was started by sterilizing the seeds using liquid detergent, Clorox solution, and sterile distilled water.The sterile seeds were placed on a petri dish lined with tissue sterilized in an autoclave, and then the sterile seeds were planted in bottles containing Murashige Skoog (MS) media.
Acid media creation.Preparation AlCl3 stock solution First weighed 1 g of AlCl3 crystals.Second, put AlCl3 into the crystal beaker and distilled water to a volume of 1 L or 1000 mL, then homogenized 1 g of AlCl3 and 1 L of distilled water using a magnetic stirrer.The stock solution was used according to the level of acid treatment (AlCl3 solution 100 mL/L for 100 ppm, 200 mL/L for 200 ppm, 300 mL/L for 300 ppm, 400 mL/L for 400 ppm, and 500 mL/L for 500 ppm).
The manufacture of acidic media weighed 2.215 g of Murashige Skoog (MS) media, 15 g of sugar, and 3.5 g of agar using an analytical scale.Next, the Murashige Skoog (MS) media, sugar, the stock solution of 0 mL, 50 mL, 100 mL, 150 mL, 200 mL, and 250 mL AlCl3 (according to treatment), and 0.5 mL of BAP stock solution were added for each treatment into a beaker glass.Then add distilled water so that the media reached a volume of 500 mL in each treatment.Homogenized the material that has been added.After that, the pH is checked using a pH meter.The finished media solution was removed and filled ±10 mL each into 35 bottles (each treatment), then closed the bottles using aluminium foil, and sterilized it using an autoclave at 121ºC, pressure 17.5 Psi for 15 min.The sterile media were arranged, stored in a tissue culture room at low temperature, and observed for one week.If there was contamination, they were not used as planting media.
Explant subculture on acid treatment media.Butterfly pea explants in the form of stems and shoots grown on germination media were transferred to the treatment medium through the subculture technique in laminar airflow.Each bottle contained one explant and there were 20 bottles in each treatment, so the total sample of explants was 120.The explants transferred to the treatment medium were then observed for 30 DAP (days after planting).

Research parameters
Acidity media changes.The measurement of changes in the acidity level of the media was carried out the difference between the acidity of the media last week (4 th week) and the acidity of the 0 th week of media using a pH meter.
Plant height gain.Measurement of plant height from the surface of the media to the highest point was measured using a caliper every 3 days from 0 to 30 DAP.Calculation of the increase in height was done by calculating the difference between the last observation of plant height and the previous observation.Data analysis was carried out for each result of the difference in days (increase in plant height on day 3 = plant height on day 3plant height on day 0).

Number of leaves.
The number of leaves was calculated at the end of the observation at 30 DAP, on each explant by counting the number of leaves formed in each treatment.
Number of branches.The number of branches was calculated at the end of the observation on 30 DAP in each explant, by counting the number branches formed in each treatment.
Number of tillers.The number of tillers was calculated at the end of the observation on 30 DAP, by counting the number of tillers formed in each treatment.
Percentage of leaves withering.Leaves withering was obtained at 30 DAP observations by counting withering leaves in each explant.Leaves withering is calculated by the following formula (Number of withered leaves for individual in each treatment / Number of leaves for each treatment x 100%).
Leaf color.Leaf was obtained by measuring the color change at the beginning and end of the observation using a leaf color application, namely the Munsell Color Chart for plants version 1.0.1.1.
The data were analyzed by analysis of variance (ANOVA) and if there was significantly different (p<0.05), then tested further by the Duncan's New Multiple Range Test using SPSS 22 software.

Acidity media changes
An increase in pH indicates the ability of plants to grow.Measurement of pH is done by measuring the initial pH and final pH.Changes in the pH of the media are presented in Table 1.Based on the results of the analysis of variance, it shows that different levels of AlCl3 gave significantly (p<0.05)different results on changes in the pH of the butterfly pea planting media.Further tests using Duncan's test showed that the 0 ppm treatment was not significantly different from the 100 ppm treatment but significantly higher in increasing the pH of the media than the 200 ppm, 300 ppm, 400 ppm, and 500 ppm treatments.This indicates that the butterfly pea plant could neutralize the pH at 0 ppm (pH 6.49) and increase the pH at 100 ppm (pH 5.39) so that the plant could absorb nutrients well for plant growth and metabolism compared to under conditions of acid stress.Treatments of 200 ppm (pH 3.79), 300 ppm (pH 3.49), 400 ppm (pH 3.21), and 500 ppm (pH 3.16) at the end of the observation experienced a decrease in the pH of the media indicating an adaptation mechanism to acid stress by increasing the use of cations which function to maintain plant growth and the availability of anions in large quantities which causes the pH of the media to decrease.
According to Firmansyah and Sumarni (2013), decreasing and increasing pH were related to the ability of roots to absorb cations under acid stress conditions of each different variety.If many cations were absorbed by the roots (NH 4+ ), then many H + ions would come out of the roots into the soil to become more acidic.If many anions were absorbed by the roots (NO 3-), then a lot of HCO 3 released by the roots goes into the soil to become more alkaline.An increase in pH affected the low solubility of Al 3+ so that P, which was previously bound to Al 3+ , became available in P form.The activity of Al in binding P occurred when the pH lowed because Al was in the form of Al 3+ .When the pH increased to ≥ 5.5, Al was in the form of oxide or hydroxide (precipitates), so it was not active in the P binding process (Fahrunsyah et al., 2023).

Plant height gain
Plant height, as the main parameter of plant morphology is visible when experiencing acid stress.The response of the butterfly pea plant height increase to acid stress for 30 days is presented in Table 2.
The analysis of variance showed that the application of different AlCl3 levels shows significant (p<0.05)different decreased the increase in the height of the butterfly pea plant on the 6 th and 9 th days.Further tests using Duncan's test showed that the treatment of 100 to 500 ppm significantly differed in reducing plant height gain compared to the control.This is caused by aluminium inhibiting the absorption of phosphorus by a plant, so the plant tissue becomes smaller and unable to grow properly.According to Zulputra et al. (2014), the high solubility of Al was toxic to plants and caused growth and development to be disrupted.In acid soils, symptoms of Ca, Mg, P, K, and N deficiency were often found and Al poisoning occurs.High Al content in acid soils has been shown to inhibit plant growth, such as plant height and stem diameter (Damayanti et al., 2017).pH also affected other factors, such as nutrient availability.Al and Fe solubility were also affected by soil pH.At an acidic pH, Al and Fe element's solubility was high.As a result, at very low pH, plant  growth would be stunted or abnormal (Karamina et al., 2017).
Plants undergo a homeostatic process or adaptation from the 12 th to the 30 th day to an acidstress environment, making the added value of plant height insignificant.This is because plants begin to tolerate the presence of Al by chelating the symplast (internal) in plant tissues to undergo cell division.According to Kochian et al. (2015), mechanism plant tolerance internally, where the plant could tolerate the presence of Al in in tissues by secreting organic acids that could binds to Al to form non-toxic complexes, compartmentalization of Al by vacuoles, and increased antioxidant capacity, which could play an important role in tolerating the presence of Al in plants, organic acids, or organic ligands.According to Hue et al. (1986), organic acids that have a strong influence in reducing the toxic power of aluminium are citric and oxalic acids, organic acids that have moderate ability to reduce the toxicity of aluminium are malic acid and salicylic acid and organic acids which have a weak ability to reduce the toxicity.Blum (1996), suggested that plants can adapt to high Al due to efficiency in the absorption, reduction, translocation, and redistribution of nutrients.

Number of leaves
The number of leaves, number of branches, number of tillers, withering leaves, showed the physiological and morphological response of the butterfly pea plant to acid stress.The results of measurements of the growth of the butterfly pea plant against acid stress at 30 DAP are presented in Table 3.
Based on the analysis of variance, different levels of AlCl3 gave results that were not significantly different on the growth of the number of leaves of the butterfly pea plant.This is thought to be caused by an internal tolerance mechanism to bind the accumulation of Al elements in the leaf organ tissues so that the leaves grow well and plants can reduce H + poisoning.This follows Santosa et al. (2016), the butterfly pea plant's mechanism in tolerating Al's presence is through an internal mechanism, namely tolerating the presence of Al in the tissue and then transporting and accumulating it in the leaf vacuole compartment.

Number of branches
The results of statistical analysis it was shown that giving AlCl3 levels of 200 ppm, 400 ppm, and 500 ppm to the butterfly pea plant was significantly different (p<0.05) in reducing the number of branches compared to the 0 ppm level indicating extreme acid stress.However, the levels of 200 ppm, 400 ppm, and 300 ppm gave results that were not significantly different compared to the 100 ppm and 300 ppm level.This showed that AlCl3 treatment at low levels could already cause inhibition of organ development in plants because acidic metals would bind nutrients essential for plant growth.The higher concentration of Al stress could affect cell division and enlargement, so growth in branches was inhibited.According to Silva et al. (2020), aluminum was a type of metal that has toxicity for plants which could adversely affect its biochemical and morphophysiological processes.High concentrations of aluminum in acidic soils caused disturbances in morphophysiological and biochemical processes so that plants experienced inhibition of the development of plant organs.

Number of tillers
Based on the analysis of variance, it was shown that the addition of AlCl3 levels of 400 ppm and 500 ppm was significantly different (p<0.05) in reducing the number of tillers of the butterfly pea plant compared to the 0 ppm treatment, but not significantly different from the 100 ppm, 200 ppm, and 300 ppm treatments.This was because the pH of the 400 ppm and 500 ppm treatments in Table 3 has a very acidic pH <4.00 and was also due to the inhibition of absorption of phosphorus elements because high aluminum concentrations will bind phosphorus nutrients.The binding of phosphorus nutrients would also occur if the acid metal AlCl3 is present, even at a low level.This was followed Zulputra et al. (2014) if the Al uptake was high, the P uptake of the plant was low so the number of tillers decreased.The increase in P uptake was due to the phosphorus plants need in cell division and as energy in every plant's metabolic process.

Withering leaves
The analysis of variance showed that giving different levels of AlCl3 gave significantly (p<0.05)different results on leaves withering of the butterfly pea plant.Further tests used Duncan's test showed that leaves withering in the treatment of 300 ppm and 500 ppm was significantly higher than in the treatment of 0 ppm and 200 ppm.This showed that plants experiencing acid stress will be experience changes due to physiological responses so that the  leaves will be turned yellow and die.This followed Zaeni et al. (2021), growth disturbances in plants were caused because plants have absorbed metals from the growing media and have translocated them to other parts of plant tissues.After Al enters plant tissue, Al could bind to nutrients that could cause disturbances in plant metabolism.This could cause the growth process in plants to be disrupted.This condition could be seen in the symptoms found in plants through the process of chlorosis on the leaves.The tips of the stems blacken, and the plants will wither and die.

Leaf color
Changed in the leaf color of the butterfly pea plant (Clitoria ternatea L.) to acid stress were presented in Table 4. Resulted from observations on the response of leaf color at the beginning and end of observations to acid stress showed that some plants were wilted so that the color changed from green to yellowish and even brownish.Based on the table, the different levels of AlCl3, 100 ppm, and 200 ppm, experienced a decrease in the green color of the leaves compared to the control becoming yellowish by 82% and 70%, respectively.In comparison, the treatment of 300 ppm, 400 ppm, and 500 ppm of the dominant leaf color experienced decreased green color and changed to brownish and yellowish color compared to control as much as 100%.
This showed that acid stress could cause the butterfly pea plant to experience leaf damage visually as a survival defense against toxicity by decreased leaf transpiration, stomata opening, and pigment degradation.This followed Santosa et al. (2016), which stated that the conspicuous symptoms seen were yellowing of the leaves, which indicated low chlorophyll content (N deficiency), and necrosis at the leaves' tips, indicating Mg deficiency.High Al stress on plants could cause the edges of the leaves to turn yellow and brown and then dry (Damayanti et al., 2017).The higher the concentration of aluminum stress treatment, were more yellow leaves were seen (Septiani et al., 2017).

Conclusions
Butterfly pea plant has mechanism of adaptation to acid stress on the parameters of plant height gain and number of leaves at the end of the observation.However, the level of plant tolerance on the parameters of the number of branches and the number of tillers was ≤ 300 ppm (pH 3.73).

Table 1 .
Changes in the pH of the butterfly pea plant (Clitoria

Table 2 .
Plant height gain of butterfly pea (Clitoria ternatea L.) acid treatment for 30 DAP