Biofortification of calcium on mustard (Brassica juncea L.) and lettuce (Lactuca sativa) cultivated in floating hydroponic system

*Corresponding author munandar.unsri@gmail.com Calcium (Ca) is one of the essential macrominerals needed by the human body as a major component in the formation of bones and teeth. Calcium is fulfilled by eating calcium-rich foods, both animal and vegetable. Mustard and lettuce are vegetables that can be a source of Ca. Efforts to increase the Ca content in plants can be done through increasing the concentration of Ca given through fertilizer or in nutrient solution. However, excessive Ca application is not recommended because it will be toxic to plants. This study aims to determine the effect of various Ca concentrations in hydroponic nutrient solutions on the growth and yield of mustard vegetables (Brassica juncea L) and lettuce (Lactuca sativa L.). This study used a floating hydroponic system with a completely randomized design. Treatment of Ca concentrations of hydroponic nutrient solutions that were tried were 0, 100, 200, 300, and 400 ppm. The parameters observed were plant height, number of leaves, level of greenness of leaves, fresh and dry weight, and concentration of calcium in the leaves. The results showed that Ca treatment significantly affected the number of leaves and the level of leaf greenness. Application of 400 ppm Ca in hydroponic nutrient solution is the highest Ca concentration that could increase Ca content in mustard and lettuce plants. Application of Ca 300 ppm is the highest concentration of hydroponic nutrient solution that can increase the Ca content of plants without causing a decrease in plant biomass, and therefore the treatment of Ca 300 ppm can be used for biofortification of Ca by hydroponic in mustard and lettuce plants.


INTRODUCTION
The human body needs enough minerals as a raw material for enzyme activity for metabolic processes. Calcium is one of the essential macro minerals as a major component for the formation of bones and teeth, and maintains the rigidity of the body's skeleton. Needs per person every day of Ca is 535 mg, depending on age category (Wrzosek, Woźniak, Kozioł-Kaczorek, & Włodarek, 2019). Pregnant women need a higher calcium intake, at least 1300 mg every day, therefore calcium supplementation in pregnant women is important. Hidden hunger is a condition of the human body that lacks of nutrients and minerals. This deficiency can occur when the quality of food consumed cannot meet needs adequately, so that micronutrients are lacking to support body growth and development (Hofmeyr, Lawrie, Atallah, & Torloni, 2018). Consuming calcium-rich foods is an alternative to meeting calcium needs. Sources of calcium are divided into two, namely animal and vegetable. Calcium content in vegetables is not as much as in animals, but the ability of vegetables to provide Ca can be increased through biofortification in plant tissue (Galera, et al 2010).
Mustard and lettuce are green vegetables which have high mineral content and high economic value. Natesh, Abbey, and Asiedu (2017) reported that horticultural crops, especially leafy vegetables play an important role because they contain more vitamins and minerals than other types of vegetables. Conventionally cultivated vegetables and lettuce leaves each contain 103 mg and 35 mg of calcium per 100 g (Zand, LaValle, & Spreen, 1999).
Biofortification of plant Ca is an effort to increase the concentration of Ca in plants by increasing the application of Ca through fertilizer or plant nutrients. Thus, the amount of Ca absorbed by plants will increase. This method is difficult to do in conventional cultivation systems that utilize soil as a growing medium. The availability of calcium in the soil is influenced by soil reactions, interactions with other elements, and the activity of microorganisms. Therefore, biofortification is more friendly by implementing a hydroponic system (Lenni, Suhardiyanto, Seminar, & Setiawan, 2020). The advantages of a hydroponic system include efficient land use, nutrient in growing solution media could be controlled, free of pesticide poisons, more efficient use of fertilizers and water, and easier to control pests and diseases (Sharma, Acharya, Kumar, Singh, & Chaurasia, 2019). The floating hydroponic system is currently popular as a floating hydroponic farming technique. The floating hydroponic technique emphasizes the method of cultivating plants in styrofoam tray that float on the surface of containers containing nutrient solutions. This technique allows plant roots to be submerged in nutrient solution.
Increasing Ca concentration of nutrient solution up to 300 ppm results in an increase in calcium content in fresh lettuce leaves from an average of 179 mg (100 g) -1 to 229 mg (100 g) -1 (Neeser, Savidov, & Driedger, 2007). High Ca absorption efficiency followed by an increase in Ca content in plant tissue (vegetables) can be implemented by increasing the concentration of Ca in nutrient solution. However, excessive application of Ca in plants is not recommended because it will be toxic and dangerous for plant growth. Therefore, this study aimed to determine the highest concentration of Ca in hydroponic nutrient solution that can increase the Ca content of plants but not to inhibit the growth and yield of mustard and lettuce plants.

Research Design
The research was carried out at the Hydroponic House of the Agronomy Study Program of the Faculty of Agriculture, Sriwijaya University, Indralaya, Ogan Ilir, and South Sumatra, Indonesia in June -November 2019. The study used a completely randomized design method with 2 types of plants and 5 levels of Ca concentration in the AB Mix nutrient solution. Two type of crops were mustard greens and lettuce. The Ca concentration of culture solution consisted of 0 ppm as a control plant, 100, 200, 300, and 400 ppm. Each treatment was repeated four times, so there were a total of 20 experimental units. Each unit experiment consisted of a container containing 10 liters of AB Mix nutrient culture solution on which was placed the styrofoam panel planted with 2 mustard plants and 2 lettuce plants.

Plant Preparation
The mustard and lettuce seed were sown on rockwool media for 3 weeks until the seedling had 3-4 leaves. The seedlings were then put into a netpot that functions as a planting medium. The net pot was then transplanted into a floating panel on a container filled with 10 of AB mix nutrient solutions. The concentration of Ca in the solution was modified according to treatment by adding stock solution of CaCl2. Every 5 days, the nutrient solution for all treatment concentrations was replaced with a new nutrient solution. The pH and electronic conductivity (EC) of the solution were measured at the beginning and end before the solution was replaced. The observed parameters were plant height (cm), number of leaves, the level of leaf greenness, plant fresh weight (g), plant dry weight (g) and calcium (Ca) concentration in leaves.

Analysis of calcium content
Two grams of leaves and stems of mustard and lettuce samples were dried and finely ground, then burned in a furnace at 500 O C for 4 hours. After becoming ash, the sample was cooled in a dexicator. Then it was put into the beaker and add 20 ml of HCl (1+1). After that, the glass was covered using a watch glass and heated on a heater until it boiled for 10 minutes. The glass was taken from the heater, allowed to cool and added 20 ml of distilled water. The solution was put into a 50 ml glass beaker, then filtered using filter paper. The residue left in the glass was rinsed 2 times using distilled water. Then the remaining residue in filter paper was washed using distilled water and diluted to 50 ml using distilled water (Mardiah, 2017). The dilution solution was used to be analyzed for Ca solution using Atomic Absorption Spectrophotometry.

Results
Analysis of variance on observed growth and yield parameters showed that the concentration of calcium (Ca) in nutrient culture solutions in the cultivation of mustard and lettuce plants had a very significant effect on the parameters of the number of leaves and the level of greening of the leaves, but had no significant effect on the parameters of plant height, weight fresh and dry plant weights (Table 1).  Table 5% 3.06 F Table 1% 4.89 Notes: ** is highly significantly difference, * is significantly difference, ns is not significant diffrence

Plant Height
Plant height showed that increasing Ca concentration did not significantly influence the height of mustard and lettuce ( Figure 1), but plant height tended to decrease with higher Ca concentration of nutrient solution. The highest plant height was shown in the control treatment of 32.3 cm in mustard plants and 19.15 cm in lettuce. The lowest plant height was shown in the concentration of Ca 400 ppm, which was 29.55 cm in mustard and 19.15 cm in lettuce. The results of the analysis of variance showed that differences in the concentration of calcium (Ca) significantly affected the number of mustard and lettuce leaves (Table 2). Increased Ca concentration in nutrient culture solution increases the number of leaves in both mustard and lettuce plants. The highest number of leaves was produced in the treatment of 400 ppm Ca and the lowest was in the control treatment for both plants. The highest number of leaves was 15 in mustard plants and 12.5 in lettuce, while the lowest number of leaves is 9.75 for both mustard and lettuce plants.

Level of greenness of leaves
Increasing the concentration of Ca in culture solution significantly increases the level of greenness of leaves in plants and lettuce. The highest level of greenness of leaves was indicated by mustard plants obtained in the treatment of 300 ppm (39.1) and was significantly different with the treatment of 400 ppm Ca (37.05), whereas in lettuce plants, application of Ca 400 ppm resulted in the highest level of greenness of leaves 36, 85, not significantly different with the treatment of 400 ppm Ca (36.85). The lowest level of greenness of the leaves is shown in the control treatment that is 33.6 in mustard greens and 32 in lettuce.

Plant fresh weight
Although statistically different concentrations of calcium (Ca) in hydroponic nutrient solutions did not significantly affect the fresh weight of mustard and lettuce plants, but the higher Ca concentrations tends to reduce the fresh weight of plants. The control treatment of 0 ppm Ca produced the highest fresh weight in mustard and lettuce plants respectively 32.82 g and 38.72 g. The fresh weight of the plant slowly decreased from in control treatment to a Ca concentration of 400 ppm, each producing a fresh weight of 38.2 g in mustard greens and 37.07 g in lettuce. Fresh weight reduction due to addition of Ca nutrient solution is sharper in mustard compared to lettuce.

Plant dry weight,
Although the analysis of variance did not show the significanteffect of Ca increasing concentrations in nutrient solutions on plant dry weight, it appeared that the dry weight tended to decrease slightly with the addition of higher Ca concentrations. Fresh weight reduction due to the addition of Ca was sharper in mustard plants compared with lettuce. In the lettuce, a reduction occurred in the plants treated with Ca 400 ppm, whereas in the reduction of dry weight in mustard plants had occurred in plants treated with Ca 100 ppm (Figure 3). The highest dry weight of 1.94 g respectively in mustard greens and lettuce in the control treatment and decreased to 1.91 g in mustard greens and to 1.85 g in lettuce plants as Ca concentraation increase up to 400 ppm.

Leaf Ca content
Ca content at all levels of Ca aplication was higher in mustard than in lettuce. The response pattern of leaf Ca content to the increase in Ca aplication in nutrient solutions was relatively same in both plants. The increase in Ca in culture solution from 0 ppm to 300 ppm caused the mustard leaf Ca content to increase from 3.43% to 4.99%, or increase by 31.3%, and after that it only increased to 3.03% in Ca 400 ppm. In lettuce, leaf Ca content increased from 1.77% to 2.97% or increased to 40.4%, as Ca culture solution increased from 0 ppm to 300 ppm. After that, addition of Ca to 400 ppm only increased leaf Ca content to 3.03%. The concentration of Ca in nutrient solution for both plants may have reached saturation level at a concentration of 400 ppm.

DISCUSSION
The results of this study indicated that increasing Ca concentrations in hydroponic nutrient solutions increased the Ca nutrient content in plants. The leaf Ca content increased to 4.99% in mustard plants and up to 2.97% in lettuce plants as 300 ppm when CaCl2 was added. Increased leaf Ca content affected several growth components of the two plants. The corelation and effect of increasing leaf Ca content with observed growth parameters are shown in Table 4. Leaf Ca was positively correlated with the number and degree of greenness of the leaves, and negatively correlated with plant height, and did not correlate with wet weight and dry weight.  Level of significant at 1% = 0.444; 5% = 0.561; ** is highly signifiacant; * is Significant; and ns is not significant High Ca uptake caused a higher level of greenness and number of leaves, lower plant height and no effect on plant weight and plant weight. One of the roles of calcium is to control the development of chloroplasts. The application of calcium can increase the efficiency of the use of nitrogen which is utilized to help increase photosynthesis (Ayyub et al., 2012). The same research results have been reported by Hernández et al., (2003) and Lenni et al., (2020) that calcium ions play a role in gene expression by controlling light (phytochromes), and therefore calcium application increases photosyn-thetic pigments, nitrogen use efficiency and increase photosynthetic rate This study also showed that the addition of calcium concentrations up to 400 ppm did not increase the height of mustard and lettuce plants, instead the growth of plant height was hampered by the increase in plant Ca content. This is in line with research conducted by Rachmah, Nawawi, and Koesriharti (2017) which showed that the application of calcium and gibberellins fertilizer had no significant effect on plant height, fresh fruit weight per plant, fruit diameter, and fruit quality of tomato plants. These results are different from the research reported by Parvin, Ahamed, Islam, & Haque (2015) in tomato plants that an increase in Ca nutrient concentrations causes stunted plant growth was inhibited.
The number of leaves is a very important character for plant growth and development because the leaves are the main photosynthetic organ. In this study the number of leaves increased with the addition of Ca nutrient solution. Similar research results reported by Hernández et al., (2003), that Ca concentration has a significant effect on the number of leaves and number of branches of tomato plants by increasing calcium to 5 mM. Likewise, research conducted by Ayyub et al., (2012) who reported that the application of calcium fertilizer sourced from CaCl2 at different growth stages could increase the number of compound leaves in tomato plants.
In both mustard and lettuce plants, leaf Ca content increased to reach 4.99% in mustard plants and up to 2.97% in lettuce plants at the addition of 300 ppm CaCl2, and relatively did not increase again despite the addition of CaCl2 concentration to 400 ppm (Figure 4.) Furthermore, although the wet weight and dry weight of plants decreased with increasing concentrations of Ca in nutrient solution up to 300 ppm, but the decrease in wet weight and dry weight was not significantly different than at the addition 0 ppm concentrations (Figures 2 and 3).
From this fact it can be concluded that the treatment of 300 ppm CaCl2 concentration is a concentration level of nutrient Ca in a hydroponic growing media solution that can increase the Ca content of plants several times the normal, and does not cause inhibition of growth of mustard plants and lettuce. Increasing the concentration of 300 ppm CaCl2 nutrient solution in the cultivation of floating raft hydroponic systems can be recommended as a biofortification technique for increasing Ca mineral mustard and lettuce plants. This result is in line with the results of Rohmaniyah et al., (2015) research which stated that the calcium content in plant leaf tissue will be higher if the concentration of Ca applied to the nutrient solution is higher. Calcium is an important and essential macro nutrient that is absorbed in the form of Ca ++ . Calcium in plants cannot move or cannot be translocated to other parts of the plant (Marschner, 2012). Most calcium found in lamella of cell wall as calcium pectate. Calcium acts as a structural component of cell membranes, maintains membrane stability and cell integrity (regulating ion absorption selectivity, regulating membrane permeability and preventing leakage of solution in cells). Calcium stimulates the formation of root hairs, harden plant stems, neutralize organic acids produced in metabolism, and acts as calmodulin (CaM) enzyme regulation and control gene expression (second messenger). The increase in gene expression activity and membrane stability will increase the ability of plants to deal with environmental stress (Marschner, 2012).
The results of this study also showed that the range in which the plant was deficient, sufficient and toxic or poisoning by Ca nutrients differed between mustard and lettuce plants. Deficient levels Ca nutrient in mustard and lettuce respectively were below 450 mg (100 g) -1 FW and 250 mg (100 g) -1 FW, sufficient range in mustard was between 450-480 mg (100 g) -1 FW, in lettuce between 250-280 and the level of toxic or toxicity occurs if the content Ca more than 480 mg (100 g) -1 FW for mustard and more than 280 mg (100 g) -1 FW for lettuce ( Figure  5).
The absorption of Ca follows apoplastic pathway mechanism which allows Ca to enter xylem without affecting in cell activities and the plants were able to absorb Ca with the concentration up to 400 ppm and increase their Ca content (White & Broadley, 2003). The 0 ppm Ca control treatment is a treatment with optimal nutrient concentration, so various growth parameters will show the best value. In the sufficient zone, an increase in Ca increases dry weight, then increasing the concentration of Ca nutrients to 400 ppm causing several growth parameters to decrease. This shows that at concentration of 400 ppm Ca uptake of Ca by plants has reached the toxic zone. These results are in line with research conducted by Domingues et al., (2016) which stated that increasing the concentration of calcium can increase plant dry weight because with the addition of calcium the ability of plants to absorb and use photosynthate results becomes more efficient. The addition of calcium in plants below the level of toxic zone will affect the structure of plant cells for improving the strength and thickness by forming cross-links in the polysaccharide pectin matrix (Easterwood, 2002). These results were in line with the study of Domingues et al., (2016) which revealed that increasing concentration of calcium can increase plant biomass as calcium addition improving the ability to absorb and utilizing photosynthate more efficiently. Calcium is one of the important mineral elements and is needed by the human body. The average calcium intake of Indonesian people is only around 254 mg day -1 person -1 , which is only 21.2% of the international standard requirement for calcium adequacy, the human body that requires calcium as much as 1200 mg day -1 person -1 (Centeno et al., 2009).
Consumption of 100 g of mustard plants cultivated at 0 ppm Ca hydroponics, will be able to meet 28.58% of the daily requirement of Ca, whereas if consuming mustard plants resulting from biofortification of 300 ppm Ca will be able to meet the daily calcium requirements up to 41.6%. In lettuce, consumption of 100 g of Ca 0 ppm hydroponic plants can only meet 14.75% of the daily calcium requirement, whereas if consuming 100 g of lettuce produced in Ca 300 ppm biofortification will be able to meet the daily calcium needs up to 24.8% (Table 3). Hydroponically biofortification of mustard and lettuce with 300 ppm Ca can be an alternative way to meet the daily needs of Ca. In order to meet the recommended daily Ca requirements of 1200 mg, people need to consume ± 240.5 g of mustard or ± 404 g of lettuce per day.

CONCLUSION AND RECOMMENDATIONS
Concentration 300 ppm of Ca is an opimum concentration for hydroponically biofortification of Ca in mustard and lettuce. In mustard plants, increasing Ca concentration of hydroponic nutrient solution increases Ca content from 343 mg (100 g) -1 FW at 0 ppm Ca to 499 mg (100 g) -1 FW at 300 ppm Ca; in lettuce plants Ca content increasing from 177 mg (100 g) -1 FW at 0 ppm Ca to 297 mg (100 g) -1 FW at Ca 300 ppm. Consumption of 100 grams mustard and lettuce produced hydroponically biofortificated in 300 ppm Ca, respectively can fulfill 41.6% and 24.8% of the daily Ca mineral needs. Increased absorption of Ca nutrients by hydroponically cultivated mustard and lettuce plants increases the number of leaves, the level of greenness of leaves, and reduces the height. The range of deficiency, sufficient and toxic zones of Ca nutrient in mustard plants are higher compared with in lettuce plants.