Biostimulants Based on Humic Acids , Amino Acids and Vitamins Increase Growth and Quality of Lettuce Seedlings

Humic acids (HA), amino acids (AA) and vitamins can increase plant growth; however, there are still few studies that determine the ideal concentration for use in lettuce (Lactuca sativa L.) production. Research was conducted to evaluate the effects of HA enriched with sources and levels of AA and vitamins on lettuce seedlings morphology and quality. Two experiments were conducted in a completely randomized design with four replicates. The first experiment had 16 treatments, 3 × 5 + 1 factorial design, the first factor was betaine, glycine and proline used in the HA enrichment, and second factor five concentrations of AA (0, 0.5, 1.0, 1.5, and 2.0 g L), plus the control, without HA and AA. The second experiment had 21 treatments, 4 × 5 + 1 factorial design, the first factor was the use of four vitamins (A, E, B1 and B2) in the enrichment of HA, while the second factor was composed of five concentrations of each vitamin (0, 0.5; 1.0, 1.5, and 2.0 g L) plus the control, without HA and vitamins. The application of HA and HA+AA promote the improvement in the seedling’s morphology and quality, while the application of the HA+vitamins negatively affected the morphology, reducing seedling quality.


Introduction
Biostumulants are substances and/or microorganisms that promote plant growth and development.The global market of these products is projected to reach $ 2.24 billion by 2018 with an annual growth rate of 12.5% between 2013 and 2018 (Calvo et al., 2014).These products are considered low-cost and high value-added, as well as a promising technology for plant cultivation, since they increase productivity with low investment and in a sustainable manner (Meirelles et al., 2017).
Humic acids (HA) are the most stable reactive fraction of humic substances (HS), containing plant hormones such as auxins, which promote cell stretching, root growth and nutrient absorption, leading to plant growth and development (Calvo et al., 2014).Due to these characteristics, they can be used as biostimulants in increasing lettuce (Lactuca sativa L.) yields (Façanha et al., 2002).The application of HA induces the formation of lateral roots and at the cellular level promotes an increase in the activity and quantity of proton pumps in the biological membranes, making them more efficient in nutrient transport (Baldotto et al., 2011;Canellas & Olivares, 2014).The influence of HA on the production and quality of lettuce seedlings is still poorly understood, however, studies by Bezerra et al. (2007), Rodrigues (2013), andSantos et al. (2018) have demonstrated positive effects of HA as a biostimulant on the development and quality of lettuce seedlings.
Associated to HA an alternative that can promote improvement in the quality of lettuce seedlings is the exogenous application of amino acids and vitamins.Amino acids act as activators of physiological metabolism, which can increase the percentage of germination, yield, produce stronger roots and more vigorous plants (Ludwig et al., 2011).Among the amino acids, glycine, in addition to forming proteins, also participates directly in nitrogen metabolism (Stitt et al., 2002).Proline acts as an important cofactor for the maintenance of water balance within the cell, leading to cell elongation as the turgor of cells increases (Goulart et al., 2011).
Unlike amino acids, the main action of vitamins is as an enzyme cofactor (vitamin B1 or thiamine), oxidation-reduction reactions through FAD coenzymes (vitamin B2 or riboflavin), amino acids synthesis (vitamin B1, B2), and germination of pollen grains (vitamin A, B1, B2) (Sinha, 2004).Vitamins are involved in increasing the biochemical reactions efficiency, nutrient assimilation and efficacy of agricultural pesticides (Floss & Floss, 2007).However, the effect of exogenous application of vitamins is still poorly understood, especially in lettuce cultivation.
In other crops, as common bean, vitamin applications in the plant development did not affected the percentage of germination and productivity (Castro & Boaretto, 2001).In contrast, positive results were described by Aires et al. (2007), who observed an increase in castor bean, Ricinus communis L., germination as a result of vitamins and carbon sources.
Lettuce is the most abundant and consumed salad crop in Brazil and in the world, corresponding to 11% of the Brazilian vegetable production, with about 4.9 million metric tons (Blind & Silva Filho, 2015).However, the success in the cultivation of this vegetable depends mainly on the use of quality seedlings, corresponding to about 60% of the success of the crop, due to the greater adaptive capacity after transplanting.
Research has been conducted with olive plants to develop biostimulant techniques that will maximize crop production (Izidório et al., 2015).And, although there is a growing interest and use of biostimulants by growers, research documenting biosimulants impact on lettuce development remain insufficient (Vavrina, 2014).Although there are indications that combining the use of humic acids, amino acids, and vitamins has a potential to benefit plant growth, there is limited research documenting their impact on seedling lettuce development and the ideal concentrations.Therefore, the aim of this research was to evaluate the effect of humic acids, amino acids, and vitamins on the morphology and quality of lettuce seedlings.

Seedling Production, Growth Conditions, and Treatments
Two experiments (A and B) were conducted simultaneously under greenhouse conditions at Experimental Field of Universidade Federal do Tocantins (UFT), Campus Gurupi (11°43′45″ S and 49°04′07″ W), located in the southern region of the Tocantins state at an altitude of 280 m.According to the Köppen climate classification, the regional climate is of the B1wA'a' moist type with moderate water deficiency (Alvares et al., 2013).The average annual temperature is 29.5 °C, with an average annual precipitation of 1804 mm.
The lettuce seedlings were produced in a greenhouse with plastic cover, 50% shade side panels, in multicellular trays of 128 cells each, filled with commercial substrate (Tropstrato®) mixed with 1:1 carbonized rice husk.Five seeds were placed in the center of each cell at a depth of 5 mm.Seedling thinning was performed seven days after germination (DAG), maintaining one plant per cell.The experiment consisted of production system of lettuce seedlings, cv.Elba, with the application of humic acids (HA) supplemented with sources, and levels of vitamins and amino acids.The seedling trays were irrigated water daily until the final stage of seedling formation.
The HA source for this research was extracted from alternative organic compound, of cattle rumen residue from municipal slaughterhouse, and was quantitatively characterized as: 10 g kg -1 nitrogen (N) and 25 g kg -1 organic carbon (C).C and N levels of HA were determined according to Mendonça and Matos (2005).
Experiment A: was conducted in a completely randomized design with four replicates and constituted by 16 treatments in factorial design 3 × 5 + 1, factor 1 included three amino acids (betaine, glycine, and proline), combined with HA (22 ml L -1 ), and factor 2 included five concentrations of amino acids (0, 0.5, 1.0, 1.5, and 2.0 g L -1 ), plus an additional treatment which did not have either HA or amino acid applications.
Experiment B: was conducted in a completely randomized design with four replicates, consisted of 21 treatments, in a 4 × 5 + 1 factorial design.The first factor was the HA (22 ml L -1 ) enriched with four different vitamins (A, E, B1, and B2).The second factor was composed of five vitamin concentrations (0, 0.5, 1.0, 1.5, and 2.0 g L -1 ).And the last treatment, the control, did not receive HA or vitamin applications.
The experiment treatments were applied to the seedling leaves at 7 and 14 days after emergence (DAE) with a manual sprayer, applying approximately 3.0 mL plant -1 .The HA concentration used in this research was based on research results described by Rodrigues (2013) and Santos (2013).Biometrics parameters of lettuce seedlings were evaluated at 21 days after sowing.

Biometric Parameters, Nitrogen Determination, and Quality of Seedlings
To determine the shoot dry mass the material was oven dried at 60 ºC for 72 hours.After drying, the shoot was weighed on an electronic analytical balance (0.0001 g), The number of leaves was determined by counting, excluding cotyledons.Plant height was measured from base to the end of the seedling using a ruler graduated in mm.Stem diameter was determined by measuring the medial part of the stem with a digital caliper (mm).Root dry mass was determined after the roots were oven dried at 60 ºC for 72 hours.After drying, the roots were weighed in on electronic analytical balance (0.0001 g).Root length was measured from the base of the seedling to the tip of the longest root, using a ruler graduated in mm.
The total N content in leaves and roots were extracted through acid digestion and determined according to the Kjeldahl method (Bremner & Mulvaney, 1982).

Statistical Analysis
The data were subjected to analysis of variance and regression through the Sigma Plot 10® program.The regression models were chosen based on the significance of the coefficients of the regression equation and the coefficient of determination, using 1 and 5% of probability.

Results
The addition of increasing levels of amino acids or vitamins associated with HA resulted in differences in respect to lettuce seedling development.For dry mass of shoot and number of leaves, the increase in concentrations promoted a linear increase, showing that the variables would still respond to the application of concentrations higher than 2 g L -1 , regardless of the amino acid source (Figures 1a and 1c).According to the regression equations, for each 0.5 g L -1 of amino acids added to the HA there was an increase of 0.0056, 0.0068 and 0.0044 g per plant in the dry mass of shoot and 0.2, 0.1 and 0.05 in number of leaves with betaine, glycine and proline, respectively.For the dry mass of shoot, the concentration of 2 g L -1 of glycine was greater when compared to the control (Figure 1a), while for leaf numbers the best result was obtained by the application of betaine, in which the maximum level of 2.0 g L -1 produced approximately 3.5 leaves (Figure 1c), doubling the number of leaves in relation to the control.
In relation to the addition of vitamins levels, and the impacts to the parameter dry mass of shoot, was observed the quadratic effect for the vitamins E and B, exponential effect for application of vitamin B1 and linear reduction for vitamin A application (Figure 1b).For the number of leaves, there was a quadratic response due to the addition of vitamins E, B1 and B2, while the addition of vitamin A promoted a linear reduction in the number of leaves ( this param      The development of a better root system provides seedlings with more ideal conditions for nutrient and water absorption (Reis et al., 2012).If a short period of water deficiency occurs, there is a greater probability that a plant will survice when it poccesses an enhanced root system.In this sense, it can be inferred that the seedlings produced with application of humic acids would have greater ability to establishment in the field, due to the greater root development.
Positive results in the DQI of lettuce seedlings with the use of amino acids were also found by Santos (2013), who reports that the application of products based on HA promote better quality indexes then the control.According to Fonseca et al. (2002), andBatista et al. (2014), DQI considers the robustness and the balance of biomass distribution in the seedlings, considering the results of several important parameters used for quality evaluation, and, thereby, being a good indicator of seedling quality.Thus, it can be said that the seedlings produced under the application of fertilizer composed of HA would produce more robust seedlings with a greater chance of survival after transplantation to the final location.

Conclusion
The use of HA in the presence or absence of other compounds, such as amino acids and vitamins, demonstrate positive changes in the development of lettuce seedlings, improving aspects such as nutrient absorption and morphology.In this way the use of HA, vitamins, and amino acids can contribute to the production of more robust lettuce seedlings, increasing the survival chances after transplanting.However, high concentrations of these compounds can cause negative effects on plant development; thus, further research is needed to clarify the effects of these substances on plants, as well as to calibrate the ideal level for each species in the condition in which it is grown.
Figure con Fig c

Figure
Figure

Figure
Figure 5. D