# Biochar-Improved Growth and Physiology of _Eltretia asperula_ under Water-Deficit Condition
## 2 Materials and Methods
### Data Collection  
_Growth Parameters_  
The growth rates of plant height were recorded from 4 May to 30 May (four times in pre-treating water stress period, five times in water stress period, six times in after re-watering period). The growth rates of plant height (cm/checking time) = PH\({}_{2}-\) PH\({}_{1}\)  
PH\({}_{2}\): Plant height this checking time; PH\({}_{1}\): Plant height last checking time  
The plant height (cm), the number of leaves, and biomasses of _Elretia asperula_ plants were measured after 10 days of re-watering. The dry biomasses of roots, shoots, and leaves were determined, and their contents were measured by drying the fresh roots, shoots, and leaves samples at 80 \({}^{\circ}\)C until a steady weight in an oven (MOV-212F, Sanyo Electric Co., Ltd., Osaka, Japan). _Physiological Attributes_  
The methods used to determine the physiological attributes of the plants were from our previous work [25]. The quantum efficiency of photosystem II (F\({}_{\rm v}\)/F\({}_{\rm m}\)) and chlorophyll content were also recorded from 4 May to 30 May (four times in pre-treating water stress period, six times in water stress period, and five times in after re-watering period). Chlorophyll contents were measured by using a chlorophyll meter (SPAD-502 Plus, Konica Minolta Sensing Inc., Osaka, Japan). The chlorophyll fluoresces (F\({}_{\rm v}\)/F\({}_{\rm m}\)) were measured by using a portable fluorometer (model OS-30p, Opti-Sciences Chlorophyll Fluorometer, Hudson, NY, USA). The initial fluorescence (F\({}_{0}\)), maximum fluorescence (F\({}_{\rm m}\)), and potential quantum efficiency of photosystem II (F\({}_{\rm v}\)/F\({}_{\rm m}\)) were measured. From these fluorescence data, the following parameters were calculated: variable fluorescence (F\({}_{\rm v}\) = F\({}_{\rm m}-\) F\({}_{0}\)) and the effective absorbed energy-conversion efficiency of photosystem II (F\({}_{\rm v}\)/F\({}_{0}\)). Fluorescence determinations were performed between 08:00 h and 11:00 h on the same leaves. The leaves were submitted to a 30 min dark adaptation period using leaf-clip holders, so that all the reaction centers in the foliar region under analysis acquired the 'open' configuration, indicating the complete oxidation of the photosynthetic electron transport system. The leaf relative water content was measured on the last day of withholding irrigation and 10 days after re-watering. To measure the leaf relative water content (RWC), 9 samples out of the 10 leaf discs, one per treatment, were obtained. Leaves were selected among the \begin{table}
\begin{tabular}{c c c c c} \hline
**Treating Conditions** & \multicolumn{4}{c}{**Biochar Rates (tons ha\({}^{-1}\))**} \\ \hline Non-WSC & 0 & 5 & 10 & 15 & 20 \\ \hline WSC & 0 & 5 & 10 & 15 & 20 \\ \hline \end{tabular}
\end{table} 
Table 2: Experimental design of different treatments. youngest fully expanded leaves. The leaf discs were immediately weighed (fresh weight; FW). The samples were bathed in distilled water (temperature range of 25-30 \({}^{\circ}\)C) inside a porous platform in order to obtain the turgid weight (TW). At the end of the imbibition period, the leaf samples were placed in a pre-heated oven at 80 \({}^{\circ}\)C for 48 h to obtain the dry weight (DW). Values of FW, TW, and DW were used to calculate RWC using the equation below. \[\text{RWC (\%)}=\left(\text{(FW - DW)/(TW - DW)}\right)\times 100\]  
The relative ion leakages in the leaves were measured on the last day of withholding irrigation. Relative ion leakage in the leaves was assessed by the leakage of electrolytes from the leaves of nine plants of similar size. Leakage of the electrolytes was determined with a conductivity meter (AG 8603, SevenEasy, Mettler Toledo, Switzerland). The leaf segments (disks of leaves with d = 1 cm\({}^{2}\)) were washed, blotted dry, weighted, and placed in stopped vials filled with an exact volume of deionized water. The vials were then incubated for 2 h in darkness with continuous shaking before the conduction (C1) was measured.