# Hyperspectral Imaging to Evaluate the Effect of Irrigation Water Salinity in Lettuce
## 2 Materials and Methods  
### Materials and Analytical Measurements  
The lettuce plants (_Lactuca sativa_ L. var _capitata_ variety \"Tempo neutrino\") were grown from seeds. The seeds were inserted into polystyrene cylinders that were positioned through holes in the plastic lid of containers with Hoagland's nutrient solution inside, thus enabling the roots to be in contact with the nutrient solution. The composition of the nutrient solution was 7 mM K\({}^{+}\), 4 mM Ca\({}^{2+}\), 14 mM NO\({}_{3}\)\({}^{-}\), 1 mM Mg\({}^{2+}\), 1 mM PO\({}_{4}\)\({}^{-}\), 1 mM SO\({}_{4}\)\({}^{-}\), 20 \(\upmu\)M Fe\({}^{2+}\), 2.5 \(\upmu\)M B\({}^{3+}\), 2 uM Mn\({}^{2+}\), 2 uM Zn\({}^{2+}\), 0.5 uM Cu\({}^{2+}\) and 0.5 uM Mo\({}^{6+}\); it was maintained between pH levels of 5.5 and 6.5 through routine replacement of the hydroponic solution. After 14 days, plants with similar development were selected and placed in containers (five plants per container) using 15 plants per treatment. Four salt treatments were then applied--a control treatment without NaCl (Ct) and three levels of salt through the addition of different concentrations of NaCl to the nutrient solution: S1 (50 mM NaCl), S2 (100 mM NaCl) and S3 (150 mM NaCl). The application of salt treatments was conducted for a total of 10 days. The average EC of the nutrient solution during the experiments were 1.23 dS/m, 6.09 dS/m, 10.77 dS/m and 14.95 dS/m, for Ct, S1, S2 and S3, respectively. The experiments were performed in a controlled growth chamber with a temperature (T) of 23 \({}^{\circ}\)C/18 \({}^{\circ}\)C (day/night), a photoperiod of 16 h/8 h (light/darkness) and a photosynthetically active radiation of 400 \(\upmu\)mol\(\cdot\)m\({}^{2}\)/s. The leaves were harvested at the end of the experiments and a sample of 10 leaves per treatment, with similar size, was selected for the acquisition of hyperspectral images. Figure 1 shows, as an example, Red-Green-Blue (RGB) images of one leaf per treatment.  
After image acquisition, the water content and osmotic potential were determined for the harvested leaves. The leaf water contents were estimated following the method described by [23] with modifications. Twenty grams of leaf pieces per treatment were taken, homogenized with a commercial grinder, and divided into three samples of five grams per treatment. The samples were dried in an oven (Thermocenter T C40/100, Salvis Lab, Rotkreuz, Switzerland), at 65 \({}^{\circ}\)C until achieving a constant weight. The water content (WC) was calculated as:  
\[\%\ WC=\frac{\text{Fresh weight}-\text{Dry weight}}{\text{Fresh weight}}\times 100 \tag{1}\]  
For the calculation of the osmotic potential (V\(s\)), three 5-g samples of leaf pieces per treatment were collected and frozen at \(-20\)\({}^{\circ}\)C. The samples were centrifuged at 2800\(\times\)\(g\) (9.8 m/s\({}^{2}\)) for 15 min (Centronic centrifuge, J.P. Selecta, Barcelona, Spain). The supernatant was analysed with a micro-osmometer (Roebling 13 DR, Loser Mestechnik, Berlin, Germany) to determine osmolarity. The osmotic potential was calculated with the Van't Hoff equation [24]:  
\[\Psi s=-R\cdot x\cdot T\cdot x\cdot cs \tag{2}\]  
where \(R\) is the ideal gas constant (m\({}^{3}\cdot\)Pa\(\cdot\)mol\({}^{-1}\cdot\)K\({}^{-1}\)), \(T\) is the temperature (K), and \(cs\) is the osmolarity (Osm\(\cdot\)m\({}^{-3}\)).