**4.** *In vitro* **phytoremediation of** *L. camara* **for some heavy-metal-polluted media**

### **4.1 The aim of study**

The effect of different concentrations of some heavy metals (cadmium, cobalt, and lead) on the vegetative and root growth characteristics of big sage (*L. camara* L.) plants under *in vitro* conditions and their efficiency in accumulating these elements [27].

### **4.2 Materials and methods**

The study was conducted in the Plant Tissue Culture Laboratory, College of Agriculture, University of Basrah, Basrah, Iraq. The seeds of the local cultivar of the big sage (*L. camara* L.) plant obtained from Basrah nurseries were used. The fruits were soaked in sterile distilled water for 30 minutes to facilitate the removal of the fruit pulp. Then, the seeds were placed in a sterilizing solution of sodium hypochlorite at a concentration of 1.05% with the addition of three drops of Tween-20 for 20 minutes. Then, it was washed with distilled and sterile water thrice [27].

### *4.2.1 Preparation of nutrient medium*

The nutrient medium was prepared from ready-made MS salts [28] at a concentration of 4.43 g L−1 obtained from Cassion Lab, USA (**Table 1**). Other chemicals were added to the MS medium (**Table 2**). The pH was adjusted to 5.7–5.8 with a solution of sodium hydroxide (NaOH) or hydrochloric acid (HCl) 0.1 N. Then add the agar at a concentration of 6 g L−1. Then complete the MS to 1000 ml with distilled water. Then, the medium was heated to 90°C. After the medium became homogeneous and clear, the nutrient medium was poured into culture tubes of dimensions 2.5 × 18 cm (Pyrex) with a volume of 20 ml for each culture tube. Then, the tube nozzles were blocked with medical cotton, and the nozzles were wrapped with aluminum foil [27].

### *4.2.2 The proliferation of* L. camara *plantlets under* in vitro *culture conditions*

Sterilized seeds of the big sage plant were cultured in MS medium without the addition of hormones to obtain seedlings from which the shoot tips are taken as explants for subsequent experiments. The regenerated shoots of the *L. camara* were produced from branch proliferation on MS medium supplemented with 0.6 mg L−1 BA and 0.1 mg L−1 NAA after 8 weeks of culturing (**Figure 2A**). Then, these proliferated shoots were rooted by growing them on an MS medium supplemented with

*The Efficiency of Phytoremediation of the Big-Sage Plant in Accumulating Some Heavy Metals… DOI: http://dx.doi.org/10.5772/intechopen.109640*


### **Table 1.**

*Inorganic nutrient components of MS medium [28].*


**Table 2.**

*Inorganic nutrient components of MS medium [27].*

1.0 mg L−1 naphthalene acetic acid (NAA) and 0.1 mg L−1 benzyl adenine (BA) (**Figure 2B**). The plantlets having three pairs of leaves per plantlet were utilized in the accumulation of heavy metal investigations [27].

### *4.2.3 Heavy metal accumulation in root and vegetative part experiment*

Plantlets were cultivated on the MS media supplemented with 0.0, 0.2, 0.4, 0.6, and 0.8 mg L−1 of Co (CoCl2.6H2O), Cd (CdCl2.2H2O), or Pb (Pb (NO3)2) [27]. After 30 days of cultivating, the subsequent data were registered:


The investigations were designed by utilizing a randomized complete design. Each treatment included 10 replications (10 plantlets). The data were analyzed by utilizing analysis of variance with the statistical program SPSS Version 22. The treatments were compared between them utilizing the revised least significant difference test (R-LSD) at a probability level of 5% [31].
