CHAPTER THREE

MATERIALS AND METHODS

3.1 study site

The study sites for this study are Mayfair motor park (7029’ 24.916”N, 4031’ 54.893”E),

Oshogbo/Ede motor park (7029’ 53.105”N,4031’ 11.461”E), OAU campus gate motor park(7029’

50.821”N,4031’ 21.576”E), toll gate trailer garage (7029’ 38.825”N,4029’ 32.319”E), OAU

central market car park(7031’ 0.190”N,4030’ 45.711”E), New buka car park(7 030’

59.718”N,4030’ 51.048”E), first bus-stop on OAU campus(7031’ 2.247”N,4031’ 23.428”E), .

These locations are ideal because: (1) there is a lot of traffic; (2) there are no large-scale

agricultural activities nearby that need the use of agricultural chemicals containing heavy metals.

Three samples would be taken from each location. Samples to be used for this research will be

collected from these sites (0-3cm deep and 0.5-1meter from the road) and taken to the

Department of Botany Obafemi Awolowo University, Ile-Ife (07 0 30’N, 040 40’E), where the

planting would be done. Top soil samples from a site that is unaffected by vehicular emissions

(about 50m from the road) will also be collected for control experiment. Experimental plants

(Pteris vitata) would be collected from the walls of computer building (geo location) and

identified at IFE herbarium. The plants would be cultivated in the department of botany OAU in

pots containing soil samples from the study sites.

3.2 Soil preparation

The sites of interest are places with high automobile traffic. Seven sites are of interest in this

study, all located in Ile-Ife. The elemental concentration of the soil and the parent plant would be

carried out so as to know the heavy metal concentrations of the soil especially that of lead(Pb)
using atomic absorption spectrophotometry. The factor that affect the metal availability to plant

is the physical properties of the soil, this will be determined using Gee and Bauder’s method

1986.

The soil samples were each grinded to a fine powder in an agate mortar. 1.0g each of the grinded

samples was weighed into a clean Teflon beaker. 5ml concentrated HF and 20ml HNO 3 were

carefully added. The beaker was gently shaken to allow the sample dissolve in the acid mixture

and then heated in the fume hood until the sample was digested. After about an hour, heating was

removed and the beaker allowed to cool, this was filtered and the filtrate made up to mark in a

25ml std flask and kept refrigerated till AAS analysis. When it has been ascertained that the lead

concentration of the soil from these sites are higher than the WHO and/or FEPA’s permissible

limit. Soil samples from these sites of interest will be collected and put in planting pots and

labeled accordingly. Each site would have three replicates. Soil will be adequately watered prior

to transplanting.

3.3 Plantlets transplanting

Fernlets having 2 to 4 healthy fronds of about 4 weeks old will be transplanted into the already

prepared planting buckets containing soil from the study sites and. The pot containing the control

experiment is label CT. There will be 24 planting buckets in all. The fernlets are generated by

vegetative propagation, they will be of uniform height and size. The plants will be grown in the

soils for a period of 12weeks after transplanting for further study and will be watered when

necessary.

3.4 Growth measurement and analysis

Growth and development of each of the plants will be closely monitored weekly throughout the

duration of the study. The frond length will be carefully measured, the length and breadth of

chosen leaflets will be measured. These measurements will be done with a metric ruler. The

number of leaflets per frond and the number of fronds per plants will be determined weekly. At

the end of the period of the experiment the plants will be harvested and the shoot and root will be

separated. The soil on the roots will be thoroughly washed off with water. The dry weights of the

root and the shoot biomass will be determined after drying in the oven at 60 0C for 48 hours. The

leaflet area will be calculated using the length and breadth data of the leaflet.

Leaflet Area (LA) = L x W x 2.325 (Osei-Yeboah et al., 1983).

The unit is cm2 W and W are leaf length and with respectively while 2.325 is the correction

factor.

3.5 Anatomical Studies

A rotary microtome will be used to cut transverse sections of the leaflets and stipes of the fronds

in each treatment of the two plants at a thickness of 8µm. After 15 minutes, these will be

submerged in a few drops of 1% Safranin O and washed three times in distilled water. They will

then be dehydrated using ethanol concentrations of 50%, 70%, 80%, 90%, and 100% before

being counterstained in an Alcian blue solution of 1% for 3 to 5 minutes. These were mounted

for microscopic inspection in 25% glycerine.

By measuring the number of cells in precisely defined locations in the cross sections, the mean

cell area (in m2) of the parenchyma (PCA) in the stipe ground tissue and the mesophyll of the

leaflet midrib will be determined. The Picasa 3.9.139 version program will be used to measure

mean cell areas from additional randomly selected portions of the parenchyma tissues from
photographs of cross sections that would be imported into the computer. Using this formula, the

percentage reduction in the PCA of experimenrs will be calculated: Percentage reduction is equal

to the difference between the control and the treatment divided by the control multiplied by 100.

That is, % reduction = (control- experiment) ÷ control.

3.6 Spore viability test

Each of the pots will yield fresh fertile fronds with fully developed sori. In order for the sori on

these fronds to burst apart and release the spores, they will be dried at room temperature for two

weeks while wrapped in paper envelopes. To prevent contamination, the spores' surface was

sterilized in 20% sodium hypochlorite for a few minutes. Then, it was rinsed three times with

distilled water using sterilized whatman filter paper. Each of the sterile, labeled petri dishes will

receive 20 ml of 0.8% nutritional agar, which will be poured into each plate and allowed to set.

Each petri dish will receive the sterilized spores, which will be maintained at a pH of 5.8.

Throughout the experiment, 4 ml of Parker's nutrition will be poured into each Petri dish housed

in the microflow chamber at regular intervals. The spores were cultured at 23 ± 2°C under 1000

lux of cool fluorescent white light for 16 hours per day. In the microflow chamber, the

germination in each of the petri dishes will be observed under a dissecting microscope. For each

petri dish, the average spore germination % will be computed.

3.7 Chemical analysis

By oven-drying at 60 °C for 48 hours and grinding the biomass into a powdered state, the dry

weight of the shoot and root biomass was calculated. For each of the study sites, the soil and
ground plant materials will be analyzed using atomic absorption spectrophotometry to determine

the amount of lead and other heavy metals. The bioconcentration factor-the ratio of lead

concentrations in the shoot and root to those in the soil—and the transfer factor—the ratio of the

shoot's concentration to that of the root will be calculated.

3.8 Data analysis

The average of three replicates was used to express all the results. The significant difference

between the lead contents in the shoot and root biomass will be tested using one-way analysis of

variance (ANOVA). Two-way analysis of variance will be used to analyze the significant effect

of time and study site soil on the leaflets area, frond length and mean number of the plant.

REFERENCES

Gee G.W. and Bauder J.W. (1986). Particle size analysis In: methods of soil analysis part

1, Klute A. (Ed.). Soil Science Society of America, Madison, Wisconson. Pp. 1-67.