**2. Material and methods**

#### **2.1 Growth conditions**

The test was carried out in a greenhouse with natural light, forced ventilation, and controlled temperature in La Plata city (Argentina) (34°54<sup>0</sup> 45.5″ <sup>S</sup>–57°55<sup>0</sup> 51.5″ W) from April to July (2019).

*C. indica* L. (achira) seeds were superficially disinfected with NaClO (10%) for 5 min, flushed with sterilized water, and placed in Petri dishes with filter paper moistened with water for their germination. Previously, they were subjected to a mechanical scarification treatment to break their dormancy.

Once germination had occurred, the seedlings were transferred to 0.5 L pots and then to 5 L pots with a substrate composed of soil and sand (2:1 v/v). After 45 days, when the plants were approximately 50 cm tall, metal solutions were applied by immersion for 24 h. Cu(II) was added in the form of SO4Cu�5H2O in three concentrations (500, 1000, and 1500 ppm) and Zn(II) in the form of SO4Zn�7H2O in three concentrations (1000, 2000, and 3000 ppm).

After 21 days of the application, plants were harvested to perform the different physiological and biochemical determinations.

#### **2.2 Measurements performed**

#### *2.2.1 Biomass and leaf area*

At harvest, the dry weight per plant (DW) was determined for all treatments by oven-drying them at 80°C until constant weight, distinguishing the shoot from roots.

#### *2.2.2 Chlorophyll and carotene content*

For all treatments, the contents of chlorophyll and carotene were determined from a 1 cm diameter leaf disk. Pigment content calculation was performed using Wellburn *Phytoextraction of Zn(II) and Cu(II) by* Canna indica*: Related Physiological Effects DOI: http://dx.doi.org/10.5772/intechopen.102450*

technique [8] with a Shimadzu UV 160-A spectrophotometer (Kyoto, Japan). The results were expressed in μg of chlorophyll cm�<sup>2</sup> and μg of carotenoids cm�<sup>2</sup> .

$$\text{Chlorophyll } \left(\text{\(\mu\) cm}^{-2}\right) = \mathbf{12} \times A\_{643.8} - \mathbf{3.11} \times A\_{646} \tag{1}$$

$$\text{Chlorophyll} \left(\text{\(\mu g cm}^{-2}\text{)} = 20.78 \times A\_{646} - 488 \times A\_{663.8} \tag{2}$$

$$\text{Totalchlorophyll} (a+b) \left(\text{\(\mu g cm}^{-2}\text{)} = \text{17.67} \times A\_{646} + \text{7.12} \times A\_{6638} \tag{3}$$

$$\text{Carotenoids } \left(\text{\(\mu\)cm}^{-2}\right) = \frac{\left(1000 \times A\_{480} - 1.12 \text{ Ca} - 34.07 \text{ Cb}\right)}{245} \tag{4}$$

where A is absorbance, Ca is chlorophyll a content, and Cb is chlorophyll b concent.

#### *2.2.3 Soluble proteins content*

The soluble protein content was measured from 100 mg of fresh leaves and root material, employing the Bradford method [9]. The protein content calculation was carried out using a standard curve prepared with different concentrations of bovine serum albumin (BSA) (SiFMa Chemical Co.).

### *2.2.4 Proline content*

Proline determination was carried out taking 100 mg of fresh leaf and root material and homogenized with 2 ml of a 3% sulfosalicylic acid solution in water. The homogenate was centrifuged at 12,000g for 15 min, and 1 ml of the extract obtained was taken. Then 1 ml of the acidic ninhydrin reagent and 1 ml of glacial acetic acid were added to the extract in a 15 ml tube and put in a water bath at 100°C for an h. After this period, the reaction was stopped by rapidly cooling the tube. After, 2 mL of toluene was added to the above reaction mixture and vortexed for 15–20 s. The phases were allowed to separate and the aqueous phase containing the toluene-proline chromophore was taken. The absorbance at 520 nm was read using toluene as a blank. Proline content per unit of fresh weight was calculated according to:

$$\text{\(\mu mol\text{ poline} \cdot \text{g}^{-1}\text{ FW} = \frac{\frac{\text{pg poline} \cdot \text{ml}^{-1}}{\text{ml} \cdot \text{kg} \cdot \text{\mu mol}^{-1}}\text{}}{\frac{\text{g } FW}{5}}\tag{5}$$

where, FW is fresh weight.

#### *2.2.5 Malondialdehyde content (MDA)*

The amount of malondialdehyde (MDA) content in fresh tissues was determined by the reaction with thiobarbituric acid (TBA) described in the Heath and Packer method [10]. In total, 200 mg of fresh leaf tissue and 200 mg of fresh root tissue were ground with 1 ml of 0.1% trichloroacetic acid (TCA) and then centrifuged. The supernatant was reacted with 1 ml of the trichloroacetic acid (TCA), butylhydroxytoluene (BHT) and thiobarbituric acid (TBA) reagent (20% trichloroacetic acid (TCA), 0.37% thiobarbituric acid TBA and butylhydroxytoluene BHT 0.01 g), then the tubes were incubated for 30 min at 95°C. After this period, they were placed in an ice bath to rapidly stop the reaction, and then they were centrifuged at 10,000g for 10 min. Finally, the supernatant was separated, and the absorbance at 532 and 600 nm was read on a Shimadzu UV 160 UV/V spectrophotometer. The MDA concentration was calculated using an extinction coefficient of 155 mM�<sup>1</sup> cm�<sup>1</sup> :

$$\text{MDA equivalents (mmol ml}^{-1}) = \frac{A\_{532} - A\_{600}}{155,000} \tag{6}$$

where MDA is malondialdehyde content, *A* is the absorbance.

#### *2.2.6 Relative conductivity (RC) of cell membranes*

The determination of the relative conductivity (RC) of the cell membranes was made from 200 mg of fresh leaf material and 200 mg of fresh root material, from the different treatments, according to the Lutts method [11]. Immediately after sampling, the tissues were washed three times with redistilled water for 15 s, to remove the electrolytes adhering to the surface and those released by the wounds produced by the cut. Subsequently, each sample was immersed in a tube with 10 ml of double-distilled water where they remained for 4 h at room temperature. Following this, the electrical conductivity (dS m�<sup>1</sup> ) was determined using a Jenco model 3173 conductivity meter. Then, the tubes were capped and taken to an autoclave where they were kept for 20 min at a one-atmosphere pressure and 120°C, to affect the integrity of the membranes. Finally, the tubes were allowed to cool to room temperature, and the electrical conductivity of the medium was measured again. Based on the data obtained, the relative conductivity of cell membranes was estimated from the following formula:

$$RC\left(\%\right) = \left(\frac{L1}{L2}\right) \times 100\tag{7}$$

where *RC* is the relative conductivity; *L*1 and *L*2 are the electrical conductivity readings before and after autoclaving, respectively.

#### *2.2.7 Zn(II) and Cu(II) content in aerial part, root, and substrate*

Plant tissues were digested in triplicate with concentrated perchloric and nitric acids in a 1:4 ratio (Merck, analytical grade), for the analyses of Cu(II) and Zn(II) (FAO & SIDA, 1983). Luoma method [12] was used to analyze the Cu(II) and Zn(II) labile fraction of sediments, being mineralized with hydrochloric acid (1 N, Merck analytical grade) by shaking for 24 h. Then, the absorbance was read using an atomic absorption spectrophotometer (Shimadzu AA6650F Atomic Absorption Spectrophotometer, Japan). The data obtained were employed for calculating the bioavailability, accumulation, translocations, and bioaccumulation indexes. All values were expressed on the dry weight of the respective sample [13].

$$\text{BAI} = \frac{\text{mg Zn(II)} \cdot \text{kg}^{-1} \text{in roots}}{\text{mg Zn(II)} \cdot \text{kg}^{-1} \text{in the substrate}} \tag{8}$$

$$\text{AI} = \frac{\text{mg Zn(II)} \cdot \text{kg}^{-1} \text{in aerial part}}{\text{mg Zn(II)} \cdot \text{kg}^{-1} \text{in the substrate}} \tag{9}$$

*Phytoextraction of Zn(II) and Cu(II) by* Canna indica*: Related Physiological Effects DOI: http://dx.doi.org/10.5772/intechopen.102450*

$$\text{TI} = \frac{\text{mg Zn(II)} \cdot \text{kg}^{-1} \text{in aerial part}}{\text{mg Zn(II)} \cdot \text{kg}^{-1} \text{in roots}} \tag{10}$$

$$\text{BI} = \frac{\text{mg Zn(II)} \cdot \text{kg}^{-1} \text{in the biomass}}{\text{mg Zn(II)} \cdot \text{kg}^{-1} \text{in the substrate}} \tag{11}$$

where BAI is bioavailability index and indicates if the metal is extracted and accumulated in the root; AI is accumulation index and indicates if the metal is extracted and accumulated in the aerial part; TI is translocation index and indicates if the metal is translocated to the aerial part; BI is bioaccumulation index and indicates if the metal is accumulated in the biomass.

#### *2.2.8 Statistical analysis*

The experimental design was fully randomized with a control (without addition of heavy metals solutions), two metals, and three concentrations for each one. The number of repetitions per treatment was *n* = 5. The data were subjected to analysis of variance (ANOVA) and the means compared by the 5% least significant difference test (LSD test) and the Pearson correlations using the software InfoStat version 2019.
