Ocimum basilicum L. Seeds Quality as Submitted to Saline Stress and Salicylic Acid

The quality of the seeds is of great importance for the cultivation of plants, however, the quality can be altered by the abiotic effects, amongst such, saline stress. Salinity is one of the great obstacles faced by modern agriculture, however, alternatives have been researched to mitigate its effects, where it is possible to mention the use of salicylic acid. The objective of this study was to evaluate the quality of basil (Ocimum basilicum L.) seeds cultivated using applications of salicylic acid to attenuate the irrigation water salinity. The experimental design was a completely randomized complete block design in incomplete factor scheme, with five conductivities for irrigation water (0.5, 1.3, 3.25, 5.2, 6.0 dS m) and five salicylic acid doses (0.0, 0.29, 1.0, 1.71 and 2.0 mM), with five and four replicates, respectively. The electrical conductivity presented an effect on germination, while the salicylic acid presented an effect on the germination speed index, emergence speed index, shoot length, root and seedling length, and seedlings dry mass. The highest correlations were obtained amongst the germination variables. Therefore, it is possible to affirm that salicylic acid can be used up to the dose of 1.0 mM with no further damage to basil seeds’ quality.


Introduction
The seed's physiological quality can be influenced by biotic and abiotic factors, amongst such saline stress.With this regard, excess salt in addition to reducing the water absorption by the seeds also causes toxicity, which can lead to the death of the embryo (Santos et al., 2016).Under these conditions, basil (Ocimum basilicum L.), a species belonging to the botanical family Lamiaceae, involved in ornamental trade and spices (mainly due to the high linalool content present in the essential oil) presents germination and viability of seeds (McMahon et al., 2004).
Currently, alternatives are researched to attenuate the effects of saline stress on plants, amongst such alternatives, the salicylic acid (SA) attenuators are highlighted.This acid is a phenolic nature hormone, acting as a regulator of the plant development, which participates in several physiological plant processes and has been studied as attenuating to biotic and abiotic stresses (Taiz et al., 2017).Salicylic acid plays an important role in the response to abiotic stresses, including water deficit, low temperature, and salt stress (Miura & Tada, 2014).Several studies have been carried out using salicylic acid with a salt stress attenuator (Ashraf et al., 2010;Bahini et al., 2012;Jini & Joseph, 2017).
Knowing that seed quality is a parameter of great importance in improving medicinal herbs yields and that salicylic acid can be used to mitigate the deleterious effects caused by saline stress.Thus, the objective of this study was to evaluate the quality of basil seeds (Ocimum basilicum L.) cultivated under the application of salicylic acid as an attenuation of the irrigation water salinity.

Obtaining Basil Seeds
The basil plants were cultivated in a under greenhouse conditions at the Agricultural Sciences Center of the Federal University of Paraíba located in the city of Areia, PB (6°51′47″S, 35°34′13″O; 575 m), during the months of May to August 2017.
Irrigation was performed by drainage lisimetry as needed.The water with the desired electrical conductivities (ECw) were prepared by adding a mixture of NaCl, CaCl 2 •2H 2 O and MgCl 2 •6H 2 O salts (in the equivalent ratio of 7:2:1, respectively) in water (0.5 dS m -1 ) from the experiment site.The treatments with saline waters started after transplanting the seedlings until the end of the experiment.For the doses preparation of salicylic acid, distilled water was used, adding 0.05% Tween 80 as surfactant, to improve plants absorption.The control treatment for this factor was prepared with distilled water and Tween 80.The experimental design was in randomized blocks, with five electrical conductivities of irrigation water (ECw: 0.50,1.30,3.25,5.20 and 6.00 dS m -1 ) and five doses of salicylic acid (SA: 0.00, 0.29, 1.00, 1.71 and 2.00 mM L -1 ), with five replicates and two plants per replicate, totaling in 9 combinations generated through the Central Composite Box (CCB) matrix (Table 1).The plants were sprayed (about 10 mL per plant) until thoroughly wetted with the salicylic acid solutions weekly for 21 days.At 120 days after planting the seeds were collected and stored for further quality analysis.The experiment was carried out at the Laboratory of Seed Analysis (LAS), at the Agricultural Sciences Center, in the Federal University of Paraíba (CCA-UFPB), Areia, PB.The seeds water content was determined using the oven method at 105±3 °C for 24 hours, using four replicates of five grams of seeds, according to Brasil (2009).
The germination test was performed using a completely randomized design, with four 50 seeds replicates, and the seeds were sowed in blotting paper inside germination acrylic boxes, moistened with distilled water (2.5 times the weight of the dried paper) and placed in a BOD (Biochemical Oxygen Demand) type germination chamber regulated at a constant temperature of 25 °C and 12 hours photoperiod.The germination was evaluated from the 4 th to the 15 th day after the test, considering the seeds germination that presented radicle protrusion and the two primordia leaves, with the results expressed as percentage of normal seedlings (Brasil, 2009).
The first germination count was performed on the 4 th day after the germination test, and the results were expressed as a percentage of normal seedlings.The germination rate index was obtained through daily counts of the number of seeds that produced normal seedlings, calculating results using the formula proposed by Maguire (1962).The average germination time was established through daily counts of normal seedlings on each day of the germination test, calculated using the formula proposed by Labouriau (1983).
The seedling emergence assay was performed in a greenhouse, and four 50 seeds replicates were used, where such were seeded in plastic trays with a seven liters capacity, containing as substrate washed and sterilized sand.The percentage of emergent seedlings was evaluated daily by counting the seedlings that presented visible cotyledons above the substrate, performed until the 15 th day after sowing, with results expressed as a percentage.The emergence speed index was obtained via daily counts of the number of seedlings emerged, calculated using the formula proposed by Maguire (1962).
At the end of the emergence test, the length of root, shoot and seedling length was evaluated, taking into consideration the hypocotyl and epicotyl.The seedling length was obtained based on the sum of the root and shoot length values, using a millimeter ruler, and the results were expressed in centimeters (cm).
The dry mass was created by packing the seedlings in kraft-type paper bags and drying them in an oven equipped with air circulation at 65 °C until reaching constant weight.The weighing was performed in an analytical scale with a 0.001 grams accuracy, and results were expressed as grams (g) seedling -1 .

Statistical Analysis
Data was further submitted to variance analysis and when significant, a regression analysis was performed.We also performed a correlation analysis between the factors and variables analyzed, all of such using the statistical software R (R Core Team, 2017).Only the data that presented significant results were submitted to regression analysis and presented in the discussion.

Results and Discussion
The initial water content for basil (Ocimum basilicum L.) seeds varied by 0.97 percentage points (8.9 to 9.95%), cultivated according to different saline levels and salicylic acid doses, whereas such values are within tolerable limits between samples (Table 2).According to Marcos Filho (2015), the maximum variation limit is 2.5%, indispensable to the uniformity in the water content of the seeds, allowing obtaining results that are more coherent.Note.ECw = Electrical conductivity for irrigation water; SA = salicylic acid.
A significant interaction was observed between the factors electrical conductivity for irrigation water (ECw) and salicylic acid (SA) in relation to the percentage of germination.However, ECw demonstrated no effects on the other variables.
Germination is one of the most crucial and decisive phases in the plant growth cycle, since it determines the establishment and final yield of the plant (Bahrai & Pourreza, 2012).Germination can be affected by several biotic and abiotic factors, such as saline stress, however, the application of phytohormones may attenuate such effects.In the current study herein presented, the application of salicylic acid in the basil growth phase favored the germination of seeds produced by these plants (Figure 1).hormone, allowing for greater seedlings development.Salicylic acid is a phenolic compound that plays important roles in the plant, regulating plant growth, developing and defending the plant against stress conditions (Miura & Tada, 2014).This stimulating behavior in the growth until the tolerated levels is verified in other vegetal species, as in cucumber -Cucumis sativus L. (Hao et al., 2012), wheat -Triticum aestivum L. (Kang et al., 2013), mustard -Sinapis alba L. (Nazar et a., 2015) and rice -Oryza sativa L. (Jini & Joseph, 2017).
The seedlings dry mass (Figure 3C) presented the same behavior for seedlings length (Figure 3C), with an increase in biomass as a function of salicylic acid doses, being the highest values obtained in seedlings grown under 1.0 mM doses, thereafter a decrease in dry seed biomass content for the seedlings occurred.In addition, the biomass accumulation as a function of the salicylic acid application allows the plant to sustain growth even under adverse conditions, promoting greater proteins expression involved in the photosynthesis and plant defense metabolism (Sharma et al., 2017).
According to Mazzuchelli (2014), these increases in biomass production may be related to the direct salicylic acid action on a specific tissues differentiation and also the growth of plant organs.It is noted that salicylic acid promoted an increase in biomass production to a certain extent, followed by a decrease with increasing doses.This may be due because such acts as a hormone antagonistic to auxin, that is, when a high concentration in the cellular tissue tends to inhibit plant growth.
When correlation was performed between the variables (Figure 4), it was observed a high positive correlation between the germination variables and seedlings vigor expressed by their growth.The germination rate index was highly correlated with the first germination count (0.96).Regarding the emergence, it was observed it had a higher positive correlation with the emergence rate (0.87).The highest positive correlations were determined between the first germination count and germination rate index (0.96) and between root length and seedling length (0.96); and a negative correlation between the first germination count and the mean germination time (0.40). Figu

Table 1 .
Matrix of Central Composite Box (CCB)

Table 2 .
Initial water content for basil (Ocimum basilicum) seeds, produced under different saline levels and salicylic acid doses