Effect of plant biostimulants on red and green romaine lettuce (Lactuca sativa) growth in indoor farming

By controlling environmental conditions, indoor farming can increase plant growth, productivity, and harvest quality. An additional element that can enhance lettuce yield is the utilization of plant biostimulants. These substances can boost nutrient absorption efficiency, enhance resistance to environmental stresses, and foster positive impacts on plant growth and maturation. This study examined the impact of red and green romaine lettuce with different plant biostimulants (Viva®, Megafol®, Radifarm®) added to AB mix on lettuce growth under white LED lighting on 30 days of cultivation. The MANOVA tests showed that different varieties of romaine lettuce and plant biostimulants substantially affected chlorophyll b, total chlorophyll, leaf number, leaf area, leaf weight, and root weight. A polynomial growth model was also obtained from height data collected during 30 days of planting, with an optimal R2, RMSE, MAE, and MAPE reaching the optimal value of 0.9926; 0.50; 0.46; 1.72, respectively, for red lettuce with Viva®, and 0.9930; 0.11; 0.29; 2.37, respectively, for green lettuce with Viva®. In addition, Pearson Correlation analysis between each treatment indicated a positive and substantial correlation in various growth indices, up to 0.901.


Introduction
Indoor cultivation is currently a viable option for maintaining food security in Indonesia's major cities.As a nation with large cities with a high population density, indoor farming can be developed since it requires limited space, and it is possible to farm year-round.The tropical climate in Indonesia poses difficulties for certain plant species.Indoor farming lets users control temperature, humidity, and lighting to create optimal growth conditions for various plant species.Indoor farming is a technique for cultivating plants in a controlled environment, such as a building or greenhouse, using a light-emitting diode (LED).The indoor farming method employing LEDs is advantageous because, in addition to being more effective in land use, indoor farming can rapidly increase plant growth and quality [1].The yield quality of romaine lettuce grown indoors using a hydroponic system revealed an average fresh weight of 112 grams per m 2 per day, which is superior to lettuce grown in open fields using the sun as a light source, producing 10 g of fresh weight per m 2 daily [2].Lettuce cultivated indoors under white LED lighting exhibited a greater leaf area and fresh weight than lettuce grown using the control approach of red and blue LED lighting [3].Other reports discovered that the white LED light increased the lettuce's height and chlorophyll content by 13% [4].Utilizing biostimulants is a further action that can be taken to support crop quality and maintain crop productivity.Plant biostimulants are complex compounds that, when administered to plants, trigger innate mechanisms that encourage the advancement and maturation of plant growth [5].Protein hydrolysate (PH) is a widely embraced plant biostimulant favored due to its many beneficial impacts on enhancing crop productivity [6].The plant biostimulant classification includes PH, distinguished by its composition of peptides and amino acids acquired through chemical or enzymatic hydrolysis.In addition to its primary constituents, PH can contain carbohydrates, trace quantities of mineral elements, phytohormones, and phenols [7].Biostimulants for plants can enhance plant quality by enhancing nutrient assimilation and preventing abiotic stress.Utilizing PH as a biostimulant can increase nutrient assimilation and stimulate plant growth, increasing yield.In addition, using biostimulants can enhance the quality of damaged soil while reducing the need for fertilizer [8], [9].
This study aims to evaluate the effects of utilizing diverse biostimulants, particularly protein hydrolysate, on the growth attributes of both red and green romaine lettuce.This study encompasses parameters such as plant height, shoot and root fresh weight, leaf fresh weight, leaf count, leaf area, and chlorophyll content.Furthermore, this study's outcomes can inform the advancement of lettuce cultivation within indoor plant facilities that utilize artificial lighting (PFAL) utilizing plant biostimulants.

Plant material and growth condition
Red and green romaine lettuce (Lactuca sativa) of Known You Seed® was cultivated using a planting medium in the form of a mixture of cocopeat, peat moss, and perlite (70:20:10 w:w:w) in a tray measuring 24 × 19 cm [10].Each tray contains 300 grams of planting medium; 30 holes with a 2 cm distance were used by planting 4 seeds on each hole with 0.5 grams of total seed placed.Every tray is positioned within a planting chamber with dimensions of 75 × 60 × 28 cm at a temperature of 24-26℃ and RH 68-70%.The planting room is made of an iron frame with 95% para net covered.Plants were watered daily using AB Mix (Nutrisiip) as plant nutrient with 700 ppm until each tray had 700 g of weight [10].AB mix nutrition is a mixture of fertilizer A, calcium nitrate-based (N, P2O5, K2O, CaO, and Fe-EDTA), and fertilizer B, magnesium sulfate-based (Mg, S, N, P2O5, K2O).
After 24 hours of dark germination time, for 30 days of cultivation, artificial white LED was used for 12 hours of photoperiod illumination from 7:00 a.m. to 7:00 p.m., which was controlled using a time switcher, 95.92 mol•m -2 •s -1 the Photosynthetic Photon Flux Density (PPFD), it was set at 1000 lux, with a wavelength of 550 nm.The measurements for PPFD, lux, and wavelength were conducted utilizing distinct instruments, including a light meter of Lutron LX-107, a PAR-quantum sensor of LI-250 Light Meter and LI-190R Quantum Sensor (LICOR LI-250Q PAR), and a Visible Near Infrared Spectroscopy (VIS-NIR Ocean Optics spectrometer), with series of fiber-optic probes and tungsten halogen lamp (HL-2000-HP-FHSA Ocean Optics) for measuring the lamp's wavelength.

Treatment
Four applications of plant biostimulants started on days 15, 20, 25, and 30 after planting.Plant biostimulants, including Viva®, Radifarm®, and Megafol®, were applied as substrate drenches by mixing in nutrient solutions on the seed tray based on product recommendations dose.The experimental study utilized a biostimulant classified as a plant-derived protein hydrolysate (PH) purchased from Valagro, Indonesia.The PH was obtained using an advanced enzymatic hydrolysis process of plantbased protein, resulting in the extraction of various bioactive components, as shown in Table 1.

Plant growth analysis
The plant height (cm) was randomly measured for red and green lettuce on each tray at 0, 5, 10, 15, 20, 25, and 30 days, using 10 replications per tray.Twenty-four replications of red and green lettuce from each tray were performed to harvest random samples 30 days after planting.The leaf area (cm 2 ) was assessed using the ImageJ software, and leaf samples and ruler were gathered and positioned on black fabric to prevent any overlap, ensuring accurate measurements of the leaf area as a uniform reference for the image processing procedures.The 8-bit image was extracted in the subsequent steps, and thresholding techniques were applied.The fresh weight (g) of the shoot, root, and leaf weights were established employing an analytical balance.In contrast, the leaf count was carried out by visually examining the physical foliage of mature plants.
Table 1.The main active ingredient and doses of plant biostimulant used in the research [11], [12].

Biostimulant
Active ingredients Dose on treatment Viva ®  Polysaccharides, proteins, polypeptides, amino acids, vitamin complexes, folic acid, vitamins (B6 and PP), polysaccharides, and humic acid Megafol ® Auxin, amino acids (proline, tryptophan, glycin, glutamic acid), cytokines, gibberellins, betaines, proteins, and vitamins (B5, PP, B1, B6) Radifarm ® Amino acids (arginine and asparagine), betaines, glycosides, microelements, organic acids, polysaccharides, saponins, vitamins Red and green lettuce samples were weighed for 0.5 g and macerated for 24 hours with a 2:1 (v/v) mixture of acetone and ethanol until the samples turned completely white, using eight replicate lettuce samples on each tray.The chlorophyll content was quantified by utilizing an Ultraviolet-Visible Spectrophotometer (UV-Vis, LW Scientific UV-200-RS) with specific wavelengths of 645 nm and 663 nm, which align with the peak absorption points of chlorophyll a and b.Optical Density (OD) values were obtained from the spectrophotometer readings of the filtrate.The chlorophyll content was then determined using an equivalency calculation, as shown in Equations ( 1)-(3) [13].

Statistical and model performance analysis
For every five days of plant height, ANOVA was used to compare the significance means between treatments.The study employed multivariate analysis of variance (MANOVA) to investigate the influence of different romaine lettuce varieties and plant biostimulants; the study focused on evaluating parameters including shoot, root, and leaf fresh weight, leaf area, leaf count, as well as chlorophyll a, chlorophyll b, and total chlorophyll levels.Other statistical techniques, including ANOVA and further test, Duncan's multiple range test (DMRT), were applied to assess the statistical significance of each plant biostimulant based on the available data.Furthermore, the t-test has been used to identify any statistically significant variances among the different types of lettuce.Pearson correlation analysis assessed the magnitude of the association between the treatments and the parameters under investigation.Regression polynomials were used to estimate the plant height of 40 samples of red and green romaine lettuce cultivated under different plant biostimulants for 30 days, beginning on days zero, five, ten, fifteen, twenty, twenty-five, and thirty days after transplanting.Ten replicates' plant height data were divided into five sets of calibration data and five sets of prediction data.The performance accuracy of the model was assessed by analyzing the predictive error values of the calibration and prediction sets using metrics such as root mean square error (RMSE), mean absolute error (MAE), mean absolute percentage error (MAPE), and coefficient of determination (R 2 ).Similar analysis structures are utilized when multiplying independent variables by a polynomial degree in linear regression.Equations ( 4)- (8) list the polynomial equation and model evaluation; n is the number of samples, ý is the predicted value of the plant height at the i-point, and y is the actual plant height at the i-point [14].

Plant height of red and green romaine lettuce on biostimulant application
The t-test showed that red and green lettuce had significantly different average plant heights on days 5, 10, and 15.The ANOVA test showed no significant difference between nutrient solution treatments throughout cultivation.However, the addition of Radifarm ® resulted in the highest average final yield (19.55 cm) among other nutrient solution treatments (Table 2).The application of Radifarm ® has been demonstrated to enhance the growth and development of plant roots [15], although there is currently no literature that explicitly discusses the effect of Radifarm ® on plant height.Based on other studies, adding hydrolyzed protein to several plants resulted in favorable plant growth, including increased plant height [16]- [18].Protein hydrolysate (PH) enables plants to assimilate more nutrients.pH encompasses a variety of amino acids and peptides that plants swiftly absorb and employ to facilitate the comprehensive growth and development of the entire plant structure [19].Moreover, the utilization of PH also improves the uptake of essential nutrients crucial for plant growth, such as nitrogen and nitrate [20].Therefore, adding PH contributes to plants' overall growth and height by absorbing plant nutrients [6] [21].A robust and well-developed root system will support the growth and development of the stems and leaves.Moreover, a healthy root system will increase the plant's assimilation of water and nutrients, influencing the development of all plant parts [22], [23].

Plant Height Growth Model of red and green lettuce on biostimulants application
The growth and development of red and green lettuce plants respond differently to each biostimulant.Concerning plant height, all treatments showed no significant difference between nutrient solution treatments on each red and green lettuce (Figure 1).The polynomial model equation representing the plant growth of red and green romaine lettuce plants using plant height of 30 days of cultivation, including one control and three treatments of biostimulant application, is presented in Table 3.
Similar to red lettuce, green romaine lettuce has the optimal high growth model in the combination of AB Mix + Viva®, based on the calibration set's R 2 value of 0.9981, 0.10 of RMSE, 0.20 of MAE, and 2.43 of MAPE, and the validation set's R 2 value of 0.9930, 0.16 of RMSE, 0.29 of MAE, and 2.37 of MAPE (Table 4).However, the four developed growth models can predict plant height since their R 2 values are near 1, and other model evaluation values also show a minimal difference between the growth model's predicted height and the actual height.The developed polynomial growth model is suitable for predicting the height development of romaine lettuce plants due to its low coefficient of determination.The polynomial regression model is also suitable for predicting plant growth because it considers nonlinear relationships between variables [25].

Plant growth and chlorophyll content of red and green romaine lettuce on biostimulant application
The t-test indicated that green lettuce exhibited notably greater leaf area, shoot fresh weight, and chlorophyll content (both a, b, and total) in comparison to red lettuce (Table 5).In the previous study, Son et al. [26] found that green lettuce's shoot fresh weight and leaf area differed from red lettuce's, but generally, the two cultivars had similar growth trends.In addition, Table 5 indicates that ANOVA analysis and Duncan's test, based on nutrient solution, revealed that the AB Mix + Radifarm ® treatment resulted in a greater leaf area than the other treatments.The MANOVA examination indicated a notable correlation between the types of romaine lettuce and the nutrient solution concerning factors like leaf count, leaf size, weight of fresh leaves and roots, as well as levels of chlorophyll b and overall chlorophyll content.to lettuce plants, except for the leaf area, led to a 9.0% increase in shoot fresh weight compared to the control group [28].The biostimulants Viva ® and Radifarm ® comprise various components, including amino acids, polysaccharides, glycosides, and organic acids.These chemicals are crucial in activating phytohormones and growth substances within plants.Additionally, the presence of humic acid in Viva ® has been found to promote the processes of photosynthesis and root respiration [29].This study also demonstrates that the application of biostimulants Viva® and Radifarm® resulted in greater leaf weight than the other treatments.
In contrast to the control plants, the elevated chlorophyll levels in the biostimulant-treated plants imply that these biostimulants can expedite the process of photosynthesis by enhancing the uptake of crucial nutrients like nitrogen and magnesium.These nutrients are vital components that facilitate plant chlorophyll production [30].In several other investigations, using biostimulants to increase chlorophyll content in plants was also successful.The chlorophyll a, b, and total chlorophyll levels exhibited a 1.25fold increase in lettuce cultivated with biostimulants [31].In another study on lettuce plants, adding the initial quantity of biostimulant from 0 to 10 ml/L resulted in a 1.43-fold rise in both the SPAD value and nitrogen content observed in the leaves [20].

Pearson correlation of red and green romaine lettuce on biostimulant application
A heatmap shows Pearson's correlation between romaine lettuce variety, nutrient solution, and growth parameters (Figure 2).A Pearson correlation coefficient greater than 0.50 implies multi-collinearity, which refers to significant intercorrelations, which are particularly common in lettuce varieties.The variety of lettuce correlated positively with leaf area (0.375), shoot fresh weight (0.414), and chlorophyll content (0.786-0.901) but negatively with final plant height (-0.419).The only positive correlation in plant nutrition was leaf area (0.546).Regarding their physical attributes, the red and green lettuce varieties exhibit similarities; the sole distinction lies in anthocyanin pigments, causing the red lettuce to display a reddish hue [26], [32].The chlorophyll concentration of lettuce varieties also varies.An earlier investigation revealed significant differences in chlorophyll a and b concentrations overall chlorophyll content between romaine lettuce and oak leaf lettuce [31].Pearson's correlation analysis between each growth parameter indicates a strong interconnection among these parameters, highlighting that alterations within one category can impact the others (Figure 2).Positive relationships were also discovered between growth parameters, particularly chlorophyll a, b, and total chlorophyll (0.811-0.983).This phenomenon indicates that if one form of chlorophyll increases in response to the same treatment, the other varieties will increase proportionally.For instance, if chlorophyll a increases, chlorophyll b will also increase since chlorophyll b is synthesized via numerous enzymatic reactions in the chlorophyll cycle, including chlorophyllide and oxygenase [33].In addition, chlorophyll b and total chlorophyll were significantly correlated with shoot fresh weight (0.332-0.333), shoot fresh weight with leaf number (0.459), and plant height with leaf fresh weight (0.326) and leaf areas (0.383).A suitable light photoperiod can increase chlorophyll content, which improves plant production, which is linked to leaf number, plant weight, and plant height [34].

Conclusions
The combination of lettuce type and nutrient treatment resulted in a non-significantly impact on the height of the plants.However, the supplementing of biostimulants to nutrient solution had a simultaneous effect on different varieties of lettuce, successively within the range of 5.65-6.50number of leaves, 12.99-20.58cm 2 of leaf area; 0.204-0.466g of leaf fresh weight; 0.085-0.168g of root fresh weight; 20.082-45.706mg/g of chlorophyll b content; and 42,854-69,930 of mg/g total chlorophyll content, compared to AB mix as a control.Modeling with polynomials can predict the height of romaine lettuce plants, with the most appropriate plant height prediction model for red lettuce obtained from the AB Mix + Viva ® treatment, with 0.9926 of R 2 , 0.50 of RMSE, 0.46 of MAE, and 1.72 of MAPE, for the prediction set.In comparison, the optimal predictive model for green lettuce is also from the AB Mix + Viva ® combination, with 0.9930 of R 2 , 0.11 of RMSE, 0.24 of MAE, and 2.32 of MAPE for the prediction set.The Pearson correlation analysis showed that lettuce variety was positively connected with leaf area (0.375), fresh shoot weight (0.414), and chlorophyll content (0.786-0.901) but negatively correlated with final plant height (-0.419).The sole instance of a positive correlation within plant nutrition was evident in the leaf area (0.546).

Figure 1 .
The plant height of red (a) and green (b) romaine lettuce after biostimulant treatment.

Figure 2 .
Figure 2. Pearson correlation heatmap of red and green romaine lettuce growth on different nutrients.

Table 2 .
Effect of biostimulant application and lettuce variety on plant height.The MANOVA analysis determined the significance level at P≤0.05 to distinguish between significant and nonsignificant results; in the RL column, various letters show statistically significant differences based on the ttest; in the NS column, various letters represent significant differences determined by ANOVA and Duncan's test.

Table 4 .
Performance model of calibration and prediction of plant height on red and green lettuce.

Table 5 .
Effect of biostimulant application and lettuce variety on growth and yields mean.