**3.2 Germination and cultivation of chickpea seeds**

Twenty chickpea seeds were exposed to 4 ml Au NPs and C70 SWNTs and 15 ml deionized water mixture for 2 days, and they were grown in pots with perlite for 21 days in two groups. The control group of 20 seeds was also grown in other pots with perlite. All the plants in this group were watered every day in the morning only with water. Au NPs and C70 single-walled carbon nanotubes exposed to chickpea plants and control group were taken from the pots after 3 weeks, and herbarium materials were made. On the other hand, some of them were stored at 70% alcohol for microscopical investigations [48] (**Figure 9**).

## **3.3 Methods used to obtain anatomical data**

Cross sections were taken from taxa to determine and compare the characteristics of root and stem anatomy of runner bean and artichoke seedlings exposed to Cu and Pb heavy metal concentrations. While determining the samples from seedlings to take sections, 3–10 mm from the end of the roots and the middle part of the body above the ground were used. These fragments were used for sectioning with

### *Plant Phenology and An Assessment of the Effects Regarding Heavy Metals, Nanoparticles… DOI: http://dx.doi.org/10.5772/intechopen.91904*

microtome by the paraffin method. All plant samples were subjected to various treatments to make the sections suitable for microtome removal [47].

These operations were carried out on the samples taken from the abovementioned parts of the plants retained in 70% alcohol. These parts were passed through 80, 90, and 100% alcohol and 2 alcohol/1 xylol, 1 xylol/1 alcohol, 1 alcohol/2 xylol, and 100% xylol solutions, in this order. The paraffin was allowed to penetrate the interior of the samples which were kept in the laboratory drying oven at 60° for 48 hours. Sections of 20, 25, 30, 35, and 40 μm used in the investigations were obtained via samples placed in paraffin blocks. The sections were placed on slides properly by using a hot water bath set at 40 °, and they were fixed onto the slides with adhesive. Sections were cleared off paraffin using 100% xylol, 1 xylol/1 alcohol, absolute alcohol, 95% alcohol, 80% alcohol, 70% alcohol, and purified water, in this order, for 5 minutes each, and then they were stained with safranin and fast green. Samples were kept in pure water, 70% alcohol, 80% alcohol, 95% alcohol, absolute alcohol, 1 xylol: 1 alcohol, and 100% xylol, for 1 minute each, so that water removal from the tissues was completed [49–51]. After removal of all the water, the preparations which were made permanent using Entellan and allowed to dry for 4–5 days at room temperature were examined in general. In this way, the reaction of the samples (with different concentrations of heavy metal in the cells) to dyes and the possible staining status were determined.

During the examination of the sections, the treatment of plant tissues with dye has caused a problem since the samples contain heavy metals, such as copper and lead, and they affect the physiology of the plant. However, it is known that in permanent preparations, an artificial appearance is obtained by losing some of the chemical content of the plant material and pigments due to the fact that the plant materials pass through a considerable amount of chemical stages. Therefore, it has been stated in some studies that permanent preparation methods are not suitable for some plants [52, 53]. It was also tried to make hand cross sections on artichoke and runner bean samples. The anatomical features of the taxa were evaluated and interpreted according to Carlquist, Fahn, and Yentür [2, 54, 55].

Dyes were prepared using different ratios of safranin and fast green, and all of them were tested for staining of heavy metal-treated anatomical specimens in accordance with the literature [50, 51], and examinations were performed on the specimens deemed appropriate.

Furthermore, objectives of 4, 10, 20, and 40 were used for examination and photographing the anatomical structures of the root and stem of the taxa. Runner bean and artichoke root and stem photographs were taken with 4, 10, and 20 objectives, and the unit of measurement was determined as 100 (μm).

## **4. Results**

**3.2 Germination and cultivation of chickpea seeds**

**Figure 8.**

*20 ppm Pb.*

**Figure 9.**

**10**

*SWNTs and pots with late germination.*

*Plant Communities and Their Environment*

for microscopical investigations [48] (**Figure 9**).

**3.3 Methods used to obtain anatomical data**

Twenty chickpea seeds were exposed to 4 ml Au NPs and C70 SWNTs and 15 ml deionized water mixture for 2 days, and they were grown in pots with perlite for 21 days in two groups. The control group of 20 seeds was also grown in other pots with perlite. All the plants in this group were watered every day in the morning only with water. Au NPs and C70 single-walled carbon nanotubes exposed to chickpea plants and control group were taken from the pots after 3 weeks, and herbarium materials were made. On the other hand, some of them were stored at 70% alcohol

*General view of the control group grown in the climate cabinet, chickpea seedlings treated with Au NPs and C70*

*Artichoke seedlings treated with Pb grown in viol. (A) General view. (b) Close-up view of samples treated with*

Cross sections were taken from taxa to determine and compare the characteristics of root and stem anatomy of runner bean and artichoke seedlings exposed to Cu and Pb heavy metal concentrations. While determining the samples from seedlings to take sections, 3–10 mm from the end of the roots and the middle part of the body above the ground were used. These fragments were used for sectioning with

### **4.1 Morphological observations**

Runner bean seeds were germinated at all concentrations of Cu and Pb. However, there wasn't any germination observed in 640 and 1280 ppm concentrations of Cu in the artichoke seeds, but germination occurred in all concentrations of Pb [39, 47].

It was determined in the morphological observations of runner bean and artichoke plants subjected to heavy metals that significant differences occurred in different doses of phytotoxic effects of Cu and Pb [47].

The control group samples of the runner bean and artichoke plants were photographed in order to compare the heavy metal phytotoxic effects on the
