# Growth and Photosynthetic Response of _Capsicum annum_ L. in Biochar Amended Soil
## 2 Materials and Methods
### Experimental Design  
Four treatments, i.e., control (C), BC0.5, BC2.5, and BC5.0, were investigated in 7 replicates planted in the soil at the start of the experiment. BC0.5, BC2.5, and BC5.0 received 0.5%, 2.5%, and 5.0% biochar by weight, respectively. Control treatments received no biochar addition. The chosen amounts were determined based on earlier laboratory experiments and literature review [17; 32], indicating a small (0.5%), a medium (2.5%), and a relatively large (5.0%) amendment amount. Plants were grown from seeds for several weeks prior to the experiment. After the pepper plants reached the four leaf stage, seedlings subsequently were placed into pots (with dimensions of 14 cm in height and an 18 cm diameter with a drain outlet) to ensure minimizing plant growth differences prior to treatments. Each pots received two plants, where each treatments included the total plant numbers of 14, with ensuring that similar plant height and development phase were present in all treatments. All pots included 2 kg of soil (dry weight). Fertilizer (N:P:K of 20:20:20 in a concentration equal to 400 mg kg\({}^{-1}\) soil [33], manufacturer Elixir Zorka, Sabac, Serbia) was added to the soil during the third week of the experiment to eliminate nutrient limitations over the course of the study. At the beginning of the experiment, a PAR sensor (Apogee Instruments) was placed at 2 m above the ground (above investigated plant canopy) and the information was used as reference PAR data of radiation reaching the leaf surface. Within each treatments a PAR sensor was placed between plant replicates ensuring same distance from plant stems for all treatments (Figure 1). This setup resulted in continuous measurements of the response of four partial plants per treatment. From the measured PAR values we calculated the fraction of the absorbed PAR (fAPAR) as a difference of the hemispherical and below canopy radiation flux over the below canopy radiation flux [34]. The PAR and consequently the fAPAR values were used as supplementary data for the LAI measurements. Data, which might have been influenced by shadows from nearby trees were omitted and we only used sunshine hours when all below and above plant canopy sensors could receive the same amount of sunlight. Photon flux values are presented in \(\mu\)mol m\({}^{-2}\) s.  
Spectral reflectance sensor of PRI (Decagon Devices; measuring 532 and 570 nm spectral irradiance) were included in the experimental setup approximately four weeks after the beginning of the study. One hemispherical sensor was placed 2 m above ground, and three PRI sensors (field view) were placed at the same distance (45 cm, resulting in a 23 cm by 16 cm radii continuous leaf reflectance measurements with the total area of 0.11 m\({}^{2}\)) from two plant replicates' meeting point in terms to collect data on entering and reflecting the 532 and 570 nm wavelength of the sunlight (Figure 1). With the replicates, portions of four plants and their response to environmental changes were monitored continuously for the control, BC2.5, and BC5.0 treatments. For BC0.5 data was not obtainable. The experimental  
Figure 1: Schematics of the experimental setup at the beginning of the experiment. photochemical reflectance index (PRI) is the photochemical reflectance index and photosynthetically active radiation (PAR) is the photosynthetically active radiation. setup schematics of plants and sensors is shown in Figure 1, including the used instrumentation and treatment placements. The PRI values were calculated based on [PERSON] et al. [35] using the following equation:  
\[\text{PRI}=\frac{Pr/Pi_{532\text{ mm}}-Pr/Pi_{570\text{ nm}}}{Pr/Pi_{532\text{ nm}}+Pr/Pi_{570\text{ nm}}} \tag{1}\]  
where \(Pr\) represents the field stop lens sensor for reflected radiation from the canopy, while \(Pi\) the hemispherical sensor for incident radiation values. The uncorrected reflectance values (\(Pr\)/\(Pi\)) were calculated for each wavebands (532 and 570 nm) and used to calculate uncorrected PRI.