*2.1. Experimental Site and Plant Material*

The experimental trial, aimed at evaluating the effects of NaCl salt stress in basil (*Ocimum basilicum* L.), was carried out in spring–summer 2021 in the greenhouses of the Federico II University of Naples Department of Agriculture (DIA) (Portici, Italy; 40◦48 N, 14◦20 E, 29 m.s.l.). On 5 May 2021, basil plants 'Anise' (*Ocimum basilicum* L. var thyrsiflora; Blumen, Milan, Italy), 'Cinnamon' (*Ocimum basilicum* L. cv Cinnamon; Blumen, Milan, Italy) and 'Lemon' (*Ocimum* × *citriodorum*; Pagano Domenico & Figli Sementi, Scafati, Italy) were seeded in 54-hole polystyrene trays (52 × 32 × 6 cm, L × W × D; volume: 0.06 L) until two true leaves appeared. At 14 days after sowing (29 May 2021; 1 day after transplanting), seedlings were transplanted into round anti-spiraling pots (0.1 m × 0.1 m × 0.15 m) filled with a mixture (*v/v*) of 1/3 perlite and 2/3 peat (Vigorplant, Fombio, Italy) (Figure 1). The pots were placed in rows with a spacing of 0.27 m × 0.15 m with a density of 25 plants m<sup>−</sup>2. Nutrient solution (NS) was distributed through drippers with a flow rate of2Lh−<sup>1</sup> (1 dripper/plant).

**Figure 1.** Illustrative pictures of the different types of basil at transplant. From left to right: *Ocimum basilicum* L. var thyrsiflora (Anise), *Ocimum basilicum* L. cv Cinnamon, and *Ocimum* × *citriodorum* (Lemon).

#### *2.2. Experimental Design*

Basil seedlings were arranged in the greenhouse according to a bifactorial design in which three basil cultivars ('Anise', 'Cinnamon', and 'Lemon') and two NSs (salt and a non-salt control) were considered as factors. The control NS was a modified Hoagland with the following nutrient element composition: 13.0 mM NO3-N, 1.0 mM NH4-N, 1.5 mM P, 5.0 mM K, 1.75 mM S, 4.5 mM Ca, 2 mM Mg, 9 μM Mn, 20 μM Fe, 0.3 μM Cu, 20 μM B, 1.6 μM Zn, and 0.3 μM Mo. Each experimental unit was replicated three times and included 15 plants (45 plants per treatment). Saline NS was prepared by adding 60 mM NaCl to the control NS.

#### *2.3. Plant Collection*

At harvest (26 days), eight plants per replicate were cut at root collar and sampled to determine biometric parameters and yield. Freshly sampled plants were weighed for total fresh and leaf weight measurements (g plant<sup>−</sup>1). The height (cm) and number of leaves per plant were then determined. The collected samples were dried in a ventilated oven at 60 ◦C until constant weight (about three days) for the measurement of the dry weight (g plant<sup>−</sup>1) and the percentage of dry matter. The dried plant material was then finely ground using an MF10.1 cutting head mill (IKA®, Staufen im Breisgau, Baden-Württemberg, Germany) for the measurement of the mineral concentration. Four plants per replicate were sampled and immersed in liquid nitrogen, stored at −80 ◦C, and subjected to a freeze-drying cycle (Christ, Alpha 1–4 (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany) for the measurement of antioxidant activity, carotenoids, and phenolic acids, while another part was stored at −20 ◦C for the measurement of chlorophyll concentration.

#### *2.4. Digital Quantification of the Leaf Area*

Digital quantification of the leaf area was performed using photos of the leaves of each plant sampled. Specifically, at 26 days, the leaves of each plant were placed on a white panel perpendicular to the camera lens (Nikon D80, Nikon AF S DX 18-135/3.5-5.6G IF-ED lens; Nikon Corporation, Tokyo, Japan). The captured photos were processed with Adobe® Lightroom Classic and Adobe® Photoshop 2022 software (Adobe Inc., San Jose, CA, USA) for distortion correction and brightness and contrast adjustment. Leaf area was quantified using ImageJ v1.52a software (U.S. National Institutes of Health of the United

States, Bethesda, Rockville, MD, USA). The analyses were performed in triplicate and the leaf area was expressed in cm2.

#### *2.5. Color Measurement*

Just before harvest, colorimetric indices were determined on ten healthy and fully expanded leaves per replicate using a Minolta CR-400 portable colorimeter (Minolta Camera Co. Ltd., Osaka, Japan). Color was converted according to the CIE 1976 L, *a*\*, *b*\* (CIELAB) color space, where L indicates brightness, a and b chromaticity (greenness and yellowness, respectively). Chroma and Hue angle (◦) were calculated according to the formulas proposed by Kheng [18].

$$\text{Clroma } = \sqrt{a^{\*2} + b^{\*2}} \tag{1}$$

$$Hue = \ tan^{-1} \left(\frac{a^\*}{b^\*}\right) \tag{2}$$

where,

$$0^{\circ} < Hue < 90^{\circ} \text{ if } a^{\*}, b^{\*} > 0$$

$$90^{\circ} < Hue < 180^{\circ} \text{ if } a^{\*} < 0, b^{\*} > 0$$

$$180^{\circ} < Hue < 270^{\circ} \text{ if } a^{\*}, b^{\*} < 0$$

$$270^{\circ} < Hue < 360^{\circ} \text{ if } a^{\*} > 0, b^{\*} < 0$$

#### *2.6. Leaf Gas Exchange and Leaf Fluorescence*

At 26 days, six fully expanded and well illuminated leaves per replicate were labeled and used to determine gas exchange and leaf fluorescence. Measurements were taken in the middle of the day (11:00 a.m.–3:00 p.m. solar time).

The net CO2 assimilation rate (ACO2; μmol CO2 m−<sup>2</sup> s−1), transpiration (E; mmol H2O m−<sup>2</sup> s−1), and stomatal conductance (gs; mol H2O m−<sup>2</sup> s−1) were measured with the LI-6400 portable gas exchange system (LI-COR Biosciences, Lincoln, NE, USA). The CO2 in the measurement chamber was set at ambient values (about 400 ppm) and the photosynthetically active radiation at 1000 μmol m−<sup>2</sup> s−1. The leaves were closed in the measurement chamber until equilibrium was reached (about 15 min).

Chlorophyll fluorescence measurement was performed using a portable Fv/Fm Meter fluorometer (Plant Stress Kit, Opti-Sciences, Hudson, NH, USA) on dark-adapted leaves for 10 min using leaf clips. Maximum chlorophyll fluorescence (Fm) was induced with a saturating light pulse of 3000 μmol photons m−<sup>2</sup> s−<sup>1</sup> for 1s, while initial fluorescence (Fo) was recorded with an internal blue LED light of 1–2 μmol photons m−<sup>2</sup> s−1. Fv/Fm was estimated as (Fm − Fo)/Fm.

#### *2.7. Pigment Measurement*

The concentrations of leaf chlorophyll and carotenoid (lutein and *β*-carotene) concentrations were determined by spectrophotometry and high-performance liquid chromatography with diode array detection (HPLC-DAD), respectively. All reagents were purchased from Sigma-Aldrich (Milan, Italy). Analyses were performed in triplicate.

To determine chlorophyll according to Wellburn [19]'s protocol, 0.5 g of fresh frozen sample was extracted in 3 mL of 90% ammonia acetone (*v*/*v*) and homogenized by IKA® T10 basic Ultra Turrax® homogenizer (Staufen im Breisgau, Baden-Württemberg, Germany). Chlorophyll a and b concentrations were determined using a UV-Vis spectrophotometer (DR 4000, Hach Co., Loveland, CO, USA) with an absorbance of 647 and 664 nm, respectively. Total chlorophyll was calculated as chlorophyll a + chlorophyll b and expressed as mg g−<sup>1</sup> of fresh weight (fw).

Lutein and *β*-carotene concentrations were determined in freeze-dried basil leaves according to the protocol of Salomon et al. [20]. A 0.1 g freeze-dried sample was extracted in a mixture of ultra-pure water (1 mL) and ethanol/n-hexane (5 mL; 60:50, *v*/*v*) and subjected to a vacuum centrifugation cycle to separate the pellet from the solvent. The pellet was mixed with methanol and MTBE (methyl-t-butyl ether) in a 1:1 (*v*/*v*) ratio and analyzed by HPLC-DAD. The results were expressed as mg kg−<sup>1</sup> of dry weight (dw).

#### *2.8. Antioxidant Activity*

To determine the antioxidant activity, three spectrophotometric methods were compared: DPPH (1,1-diphenyl-2-picrylhydrazyl), ABTS (2,2 -azinobis-(3-ethylbenzothiazoline-6-sulfonate)), and FRAP (ferric ion reducing antioxidant power), according to protocols of Brand-Williams et al. [21], Re et al. [22], and Rajurkar and Hande [23], respectively.

To determine the antioxidant activity of DPPH, 1 mL of DPPH solution (0.1 mM) and 96% methanol were added to 200 μL of the aqueous extract, mixed and incubated at room temperature for 30 min in the dark. The absorbance was recorded against the blank at 517 nm.

To determine ABTS antioxidant activity, a stock solution of ABTS+ was prepared by reacting 7 mM aqueous solution of ABTS<sup>+</sup> with 2.45 mM aqueous solution of potassium persulfate in equal parts. After incubation in the dark (16 h at 23 ◦C), the stock solution was diluted with ethanol in a ratio of 1:88 until an absorbance of 0.700 ± 0.020 at 734 nm was reached. A 0.1 mL aliquot of each sample, previously filtered and diluted (1:10) with 70% methanol, was mixed with 1 mL of ABTS<sup>+</sup> solution and stored at room temperature for 2.5 min. The absorbance was read at 734 nm.

For measurement of FRAP antioxidant activity, a FRAP solution was prepared containing 1.25 mL of Fe2+/2,4,6-tris (2-pyridyl)-s-triazine (10 mM) in HCl (40 mM) + 1.25 mL of FeCl3 (20 mM) in water + 12.5 mL of acetate buffer (0.3 M, pH 3.6). An aliquot of 150 μL of the sample was mixed with 2.850 mL of FRAP solution and incubated for 4 min in the dark. The absorbance was read at 593 nm against a blank containing all the reagents.

The absorbances of the DPPH, ABTS, and FRAP essays were recorded with a UV-vis spectrophotometer (Shimadzu, Japan). The results were expressed as Trolox equivalent mmol kg−<sup>1</sup> dw. All analyses were performed in triplicate.
