*2.4. Biometric and Biochemical Analysis*

To determine the impact of different light conditions and fertilizer treatment, careful selection and preparation of plant samples were made. Leaves of the selected samples (for plants that have not reached the stage of forming flower stalks) were fresh, healthy, and undamaged. Their shape, color, and smell corresponded to their botanical type and variety. The leaf surface was not damaged by pests or their waste products. The assimilating leaf surface showed no sign of excessive external moisture.

The lettuce was sampled by taking one plant from each container and then cutting a rosette at the base of the plant. All samples were placed in hermetically sealed bags for further study. The total number of samples was 25 plants for each type of experiment (variety, lighting, fertilizer). Twelve plants were used to determine morphological parameters and 13 plants to determine their photosynthetic parameters. Morphological parameters (weight, number, and area of leaves) were determined for each selected plant. The biometric parameters of the morphological organs of the plants by the phases of their development were assessed 15, 30, and 45 days after the lettuce shoots appeared.

To determine the mass of dry matter in the plants, a sample was crushed manually using a hand cutting tool until the fragment size of no more than 1 cm. After grinding, the sample was mixed to avoid the inhomogeneity of fragments. Then, two weighed portions with mass of at least 5 g were isolated in two replicates using an analytical balance. The samples were dried in an oven for either 3 h at 60–70 ◦C or 1 h at 105 ◦C.

To estimate the moisture in fresh green leafy mass, the test sample was crushed and mixed, and two portions of 25–50 g were weighed on a balance with an accuracy of 0.001 g. Then the portions were placed in weighing bottles pre-dried to a constant weight. The containers with weighed portions were placed for 20–30 minutes in an oven heated to 120–130 ◦C to inactivate enzymes and then dried at 105 ◦C to constant weight.

The quantitative analysis of pigments (chlorophylls, carotenoids) included their extraction from the plant tissues using acetone, separation of the mixture into individual components, and spectrophotometry.

The concentrations of pigments in 100% acetone were calculated according to Holm– Wettstein as follows:

$$\mathcal{C}\_{\mathcal{C}lh\_{\mathfrak{a}}} = 9.784 \times D\_{662} - 0.990 \times D\_{644}$$

$$\mathcal{C}\_{\mathcal{C}lh\_{\mathfrak{b}}} = 21.426 \times D\_{644} - 4.650 \times D\_{662}$$

$$\mathcal{C}\_{\mathcal{C}lh\_{\mathfrak{a}} + \mathcal{C}lh\_{\mathfrak{b}}} = 5.134 \times D\_{662} + 20.436 \times D\_{644}$$

$$\mathcal{C}\_{car} = 4.695 \times D\_{440.5} - 0.268 \times \mathcal{C}\_{\mathcal{C}lh\_{\mathfrak{a}} + \mathcal{C}lh\_{\mathfrak{b}}}$$

Here *CChl<sup>a</sup>* , *CChl<sup>b</sup>* , and *Ccar* are the concentrations of chlorophylls *a* and *b* and the carotenoids. *D<sup>ω</sup>* is the optical density of the extract at the corresponding wavelength *ω* in nm.

To determine nitrate ion concentration, a 1% solution of aluminum-potassium alum was used as the extraction solution. The concentration of nitrate ions was measured using an NO3-selective electrode connected to an Ekspert-001 ionometric station (Econiks, Russia). The station was calibrated using solutions containing a known concentration of KNO<sup>3</sup> (Sigma-Aldrich, SL, USA).

Vitamin C in the leaves of lettuce plants was determined by a spectrophotometric method with a dye solution of 2,6-dichlorophenol indophenol [43]. The sucrose concentration was determined by refractometry [44].
