Tolerance of Basil Genotypes to Salinity

Basil (Ocimum basilicum L.) is a medicinal species of Lamiaceae family, popularly known for its multiple benefits and high levels of volatile compounds. The species is considered to be one of the most essential oil producing plants. Also cultivated in Brazil as a condiment plant in home gardens. The objective of this study was to evaluate the effect of salinity on the growth of basil in nutrient solution of Furlani and to identify variables related to the salinity tolerance in this species. The first assay was performed with variation of five saline levels (0 control, 20, 40, 60 and 80 mM NaCl). In the second assay six genotypes were evaluated in two salinity levels 0 and 80 mM NaCl. The height, stem diameter, number of leaves, dry mass and inorganic solutes in different organs, photosynthetic pigments, absolute membrane integrity and relative water content were evaluated. All biometric variables in basil were significantly reduced by salinity. Dry matter yield and percentage of membrane integrity were the variables that best discriminated the characteristics of salinity tolerance among the studied basil genotypes. Basil genotypes showed a differentiated tolerance among the genotypes, the ‘Toscano folha de alface’ being considered as the most tolerant and ‘Gennaro de menta’ as the most sensitive, among the species studied.


Introduction
Basil (Ocimum basilicum L.) is a plant of the family Lamiaceae, a producer of essential oils of pharmaceutical importance, food, perfumery and cosmetics.It is also widely used in traditional medicine (Bharti, Barnawal, Wasnik, Tewari, & Kalra, 2016).The species is cultivated on a commercial scale in Asia, Africa, South America and the Mediterranean region, under natural conditions or in protected cultivation.The cultivation in greenhouses has the advantages of maximizing the yield and allowing a constant supply of material throughout the year (Sgherri, Cecconami, Pinzino, Navari-Izzo, & Izzo, 2010).
The use of brackish water in irrigation can result in soil salinization and compromise plant growth.The productivity of the crops in a salinized environment depends on the amount of soluble salts and the capacity of the plants to tolerate saline stress (Cova, Azevedo Neto, Ribas, Gheyi, & Menezes, 2016).The reduction of the water potential in the culture medium, due to the higher concentration of soluble salts, affects the water absorption and, consequently, the turgescence and the cellular expansion.Moreover, saline stress also leads to a reduction of photosynthesis by the closure of the stomata and, therefore, limits the absorption of carbon dioxide and, consequently, the reduction of growth and productivity occurs (Farooq, Wahid, Kobayashi, Fujita, & Basra, 2009).homeostasis between K + and Na + is fundamental for the regulation of the cellular osmotic potential (Zhu, 2003) and to avoid the deleterious effects of saline stress.
The integrity of cell membranes, enzymatic activity and photosynthesis are metabolic and physiological indicative functions of tolerance variation in the species as they are sensitive to salinity.In order to evaluate the tolerance of plants to salinity, growth is considered to be an effective measure as it integrates a set of physiological mechanisms that occur in the plant (Niknam & McComb, 2000).Tolerant plants may have ionic compartmentation mechanisms, while in sensitive plants this mechanism is not efficient.Plant responses to saline stress are complex and may vary between cultivars of the same species (Maas & Hoffman, 1977;Niknam & McComb, 2000).The genotypic variability may provide a differential tolerance to saline stress between plants from the same species, as evidenced by several authors (Azevedo Neto, Pereira, Costa, & Santos, 2011).
Knowing that basil is a very important crop, the objective of the present study was to evaluate the tolerance of six basil genotypes to saline stress and to classify them according to the degree of tolerance to stress.

Material and Methods
The study was conducted in a protected environment at the Federal Universidade do Recôncavo da Bahia, Centro de Ciências Exatas e Tecnológica, Cruz das Almas-BA (12º40′19″ S 39º6′23″ W), from January to March 2015.Two experiments were carried out: the first one was a completely randomized design with five saline levels and four replicates and the second one was a randomized block design with two saline levels and six genotypes with four replicates.

Assay I: Experimental Conditions and Treatments
Basil seeds var.Alfavaca basilicão, obtained at ISLA Sementes Ltda.were seeded in 150 mL plastic cups containing washed sand.At 21 days after emergence (DAE), when the seedlings presented a completely expanded pair of leaves, they were transferred to containers in a hydroponic "Floating" type system with aeration, containing 12 L of nutrient solution of Furlani (1998).After four days under these conditions, the seedlings received their respective saline treatments 0, 20, 40, 60 or 80 mM NaCl, corresponding to electrical conductivities of the nutrient solution of 2, 4, 6, 8 and 10 dS m -1 .NaCl was gradually added (20 mM day -1 ), to avoid osmotic shock.The volume of the solutions was completed daily with water and the pH maintained between 6.0 and 6.5 by the addition of HCl or NaOH.Plants were harvested 17 days after the treatments were applied.

Biometry and Dry Mass Production
The height of the plants, stem diameter (SD) and number of leaves (NL) were determined at the harvest.The height was measured with a graduated ruler, the main branch was measured from 0.5 cm from the insertion of the root to the apex of the main branch.The SD was measured with a digital caliper and counted the NL.Afterwards, the plants were collected and separated in leaves, stems and roots and the plant material was transferred to an oven with forced air circulation at 65 °C for 72 h.After this period, the determination of dry masses of leaf (LDM), stem (SDM) and roots (RDM) was performed on a precision scale.From the dry mass data of the plant parts, shoot dry mass (SHDM) and total dry mass (TDM) were calculated.

Analysis of Inorganic Solutes
For the determination of Na + , K + and Cl -contents in leaves, stems and roots, the extracts were prepared as described by Jones Júnior (2001), with minor modifications.In test tubes, 0.1 g of dried (in oven) and powdered material (in Willye-type knife mill) and 10 mL of deionized water were added.The test tubes were heated at 80 °C in a water bath for 1 h, being agitated every 15 min and then centrifuged at 5.000 × g.The supernatant was collected and stored at -20 °C for further analysis.The Na + and K + contents were determined by flame photometry (Faithfull, 2002) and the values of Cl -by spectrophotometry (Jones Júnior, 2001).

Assay II: Treatment and Execution
In the second assay, seeds of genotypes 'Gennaro de Menta', 'Alfavaca basilicão vermelho', 'Alfavaca basilicão', 'Toscano folha de alface', 'Limocino' and 'Grecco a palla', obtained from the company ISLA Sementes Ltd., were used.The seedlings production and the cultivation system were identical to Assay I.The seedlings of each genotype were submitted to 0 and 80 mM of NaCl in nutrient solution of Furlani (1998), with electrical conductivities of 2 and 10 dS m -1 , respectively.Addition of NaCl and control of nutrient solutions were also identical to those of Assay I. Plants were harvested after 20 days after the treatments were applied.

Dry Mass Production and Analysis of Inorganic Solutes
At the end of the experimental period, the plants were separated into leaves, stems and roots to evaluate dry mass and determination of Na + , K + and Cl -contents according to Experiment I.

Photosynthetic Pigments
To determine the levels of chlorophyll a, chlorophyll b and carotenoids (carotenes and xanthophyll), the samples were placed in 95% ethanol.Then, the spectrophotometric readings were performed at 649, 664 and 470 nm, according to the methodology described by Lichtenthaler and Buschmann (2001) and were calculated with Equations 1, 2 and 3, respectively: CHLa (µg ml -1 ) = (13.36× A 664 -5.19 × A 649 ) (1) CHLb (µg ml The AIP and RWC evaluations were performed on the third fully expanded leaf pair, for all genotypes except for 'Grecco a palla', where they were determined in the main branch, adapting the methodology to the morphology of this genotype.The determination of AIP was performed according to Pimentel, Sarr, Diouf, Aboud, and Roy-Macauley (2002), where 10 leaf discs with known area were placed in threaded tubes with 10 mL of deionized water.The tubes were placed for 24 h in a dark place and after that the electrical conductivity of the water (free conductivity-FC) was measured.Afterwards, the tubes were placed in a water bath at 100 °C for one hour and after returning to the ambient temperature the electrical conductivity of the water (total conductivity TC) was again measured.It was calculated using Equation 4: The RWC was determined according to Barr and Watherley (1962), in which 10 leaf discs with known area were removed and weighed to obtain the fresh mass (FM).The disks were then placed in Petri dishes, immersed in deionized water and left for 24 hours in a refrigerator.After this period, the discs were wiped with paper towel and weighed to obtain the turgid mass (TM).Afterwards, they were taken to dry in an oven until constant weight was obtained.The RWC was calculated as described in Equation 5:

Statistical Analysis
For the first assay, the results were submitted to analysis of variance (F test) and regression, using the Sisvar 4.6 statistical software (Ferreira, 2011).In the second assay, the results were submitted to analysis of variance (F-test) and the means compared by the Scott-Knott test at 0.05 probability.

Assay I
The variables plant height, stem diameter (SD) and number of leaves (NL) presented linear decreasing behavior with the increment of sodium chloride in the nutrient solution (Figure 1).Comparing the control treatment with the one using 80 mM NaCl, reductions of 37.31 and 27.5% were observed for height, SD and NL, respectively.

Note. Valu
The diame cultivation lodging.
For NL th 1C).eased n, the ely in K + to e ions ptake (Marschner, 2012).These results suggest a higher selectivity of K + ion transport in relation to Na + for the leaves, as well as a mechanism of Na + retention in stem and root tissues, avoiding damage due to toxicity in the leaves.
The osmotic adjustment, that is, the accumulation of solutes is an important mechanism, under conditions of saline stress, to obtain a gradient of favorable water potential and maintenance of cellular turgor.The accumulation of the inorganic solutes (K + , Na + and Cl -) has a lower energy cost for the cells when related to the accumulation of compatible organic solutes (Flowers, Munns, & Colmer, 2015).However, there must be a balance between these ions, because Cl -and Na + can be toxic when in excess in plant tissues.In this way, the accumulation of K + favors ionic homeostasis, reducing the toxic effects of Na + (Munns & Tester, 2008).Among all the variables studied in Assay I, the DM variable was the most accurate indicator of salinity effect, associated with the Cl -accumulation data.In this way, a larger reduction of DM occurred in the tissues with higher Cl -accumulation (leaves and stems) and, as the smallest DM reduction was in the root, where there was less accumulation of DM.The Cl -is a predominant anion under saline conditions (Tavakkoli, Rengasamy, & McDonald, 2010), when absorbed by the roots is easily translocated to the tissues of the aerial part (Li, Tester, & Gilliham, 2017), justifying its large accumulation in leaves and roots.
In this study, it is considered that 80 mM NaCl significantly decreased (50 to 60%) the dry mass production of basil organs in Assay I, this concentration was used for the subsequent salt stress experiments.

Assay II
Salinity reduced significantly the dry matter yield of leaf (LDM), stem (SDM), roots (RDM) and total (TDM) of basil genotypes, except for the 'Toscano folha de alface' (Figure 4).The highest reductions of LDM (54%), SDM (71%), RDM (55%) and TDM (61%) were observed in the 'Gennaro de menta' genotype.The reduction percentage in biomass production has been considered an effective indicator of tolerance to salt stress in plants (Munns, 2002).These data indicate that the 'Toscano folha de alface' genotype was more tolerant and the Gennaro de menta the most sensitive to salt stress when compared to each other (Figure 4).
The results of this study corroborate with the studies of Barbieri et al. (2012) and Prasad, Lal, Chattopadhyay, V. K. Yadav and A. Yadav (2007), who reported genotypic variability of basil in tolerance to saline stress.Barbieri et al. (2012) verified that the differentiated tolerance among the genotypes of this species was related to the morphological, physiological and metabolic adaptive characteristics to stress.
The Na + levels in the leaves (Figure 5A) of all basil genotypes were lower than in the stem (Figure 5B) and in the roots (Figure 5C).A significant variation might be observed between the mean values in the genotypes related to the rates of Na + in leaves (0.126 to 0.337 mmol g -1 DM), stem (1.814 to 3.414 mmol g -1 DM) and roots (2.338 to 3.720 mmol g -1 DM).It is interesting to note that the Na + content in leaves of the 'Toscano folha de alface' genotype (0.126 mmol g -1 DM) was 64% lower than the mean leaf content of the other genotypes (0.348 mmol g -1 DM).In this way, it can be inferred that there was a restriction in the transport of Na + to the leaves, with a retention of these ions in the roots of the genotype.This might explain, at least in part, the greater tolerance of this genotype to saline stress since such restriction inhibits Na + accumulation at toxic levels in leaves (Munns & Tester, 2008).

Figure g
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