ABSTRACT
III. MATERIALS AND METHODS
Experiments were conducted in the lath-house under the jurisdiction of Plant Breeding and Genetics (PBG) department. Whereas all other experimental work was carried out in the department of Agricultural Chemistry, The University of Agriculture Peshawar. The experiments mainly consisted of two parts. The first part consisted of treating wood coal with dilute acids and base solutions and the other part consisted of pots arrangement and transplanting tomato plants of Rio Grande cultivar.
Pots were kept in triplicates, where one plant per pot was transplanted. These pots created a set of 37 rows, where acid base treated wood coal (32) and different controls (4) were applied along with one untreated soil pots row.
Wood coal was obtained from local shops of Peshawar city and then grinded (powdered) using lab mill, which was then treated with dilute acids and base solutions. The acids and base solutions were prepared in the agricultural chemistry laboratory from commercial grade acids and bases. The amount of acids and bases for solution was calculated. The solid bases were weighted and dissolved in distilled water as required.
Acids used for the solutions were HCl, HNO3 and H2SO4. Concentration of the acids and bases was made by studying some literature related to the present experiment. HCl and H2SO4were made as 4% and HNO3 was made 15%. Keeping in mind the acids solution and their neutrality, bases i.e. KOH and NaOH solution were made 5% each in concentration. Fifty gram wood coal in powder form was first treated with 200mL of each acids and bases solution individually. Then the same amount of wood coal was treated with the all possible mixtures of the acids and bases solutions mentioned above (abstract) in 1:1 ratio. The mixture treatment was done in sequential manner i.e. after completion of the reaction of wood coal with one chemical (acids or bases) in 1-2 days; wood coal was treated with second solution for similar time and so on. These treatments resulted in 32 different treatment products of wood coal. Each individual product of wood coal along with controls was applied to the same number of sets of pots. Each set contained 3 medium sized pots with 2.5 Kg of sandy loam soil. The products were applied in diluted form (10mL of product solution/100mL of distilled water) at regular interval of time (about 15 days). Along with 32 sets of products, 5 sets of pots were used as control (NPK, Humic Acid,
Wood Coal, Farm Yard Manure and Untreated Soil). Tomato seedlings of cultivar Rio Grande were transferred (2-3 per pots) to each set of pots. The plants number per pot was then reduced to one plant pots-1 after successful growth in day or two. These products were expected to be good fertilizers which would improve soil properties, growth and yield parameters of the tomato plants. The data regarding physical and chemical parameters of the products, soil and tomato plants and fruits were recorded and presented in result discussion chapter.
3.1 Wood Coal Product Quality Parameters
Wood coal treated with different acid and base solutions was analyzed for pH, Acidity, carbon, nitrogen, phosphorous, sodium, potassium, calcium and magnesium.
All the parameters were determined by standard procedures of AOAC (2000) 3.2 Soil Parameters
Soil was analyzed for different chemical parameters like carbon, nitrogen, phosphorous, potassium, sodium calcium, magnesium, and physical parameters like pH, Acidity, Electrical conductivity (EC) using standard methods of AOAC (2000).
3.2.1 Soil Organic Carbon (SOC):
Soil organic carbon was determined by the modified method of Nelson and Sommers, (1982). In this method one gram of air dried soil sample was treated with 10ml of 1N K2Cr2O7 solution and 20ml of concentrated sulphuric acid for 1 minute.
Then the sample was cooled for 10-15 minutes. After cooling, 200ml distilled water were added and filtered. The filtrate was titrated against 0.5N FeSO4.7H2O solution after adding 5-6 drops of Ortro- Phenolphthalein indicator, the change in color from greenish to dark brown showed the end point. A blank reading was also taken at the same time. Soil organic C was determined by the following formula;
Organic C %=(meqK2Cr2O7 - meq FeSO4)× meq of C Weight of dry sample ×(0. 75) ×100
Whereas 0.75 is derived from the assumption that only 75% of organic matter is oxidized in this method and miliequilant weight (meq.) of Carbon is 0.003.
3.2.2 Soil Total Nitrogen
Soil total N for each treatment was determined calorimetrically, following the Kjeldahl procedure (Bremmer and Mulvaney, 1982). In this method, 0.2g of sampled soil was digested with 3ml of concentrated H2SO4 in the presence of digestion mixture containing K2Cr2O7 and Se on block digest for about 4-5 hours. The digestion was initially started at 50ºC and then the temperature was raised gradually to 100, 150.
200, 250, 300 and finally to 350ºC , which was maintained at least for 1 hour to turn the sample colour to light greenish or colourless. After cooling, the digest was transferred to a 100ml volumetric flask and the volume was made up with distilled water. 20ml of the digest was distilled in the presence of 5ml of 40% NaOH solution and 5ml boric acid mixed indicator. The distillate was titrated against standard 0.005M HCl, and N was calculated as 1ml of 0.005M HCl is equivalent to 70μg. A blank reading was also taken at the same time.
(S – B) x N x 0.0014 x Dilution
% Nitrogen = ---x 100 Weight of sample x volume
3.2.3 Soil pH, EC and Acidity
Soil water suspension of 1:5 was used to measure pH of the soil with the help of pH meter (Mc-Clean, 1984). In this method, 10g soil sample and 50mL water was shaked for 30 minutes on a reciprocal shaker. The suspensions were then analyzed for pH and EC using pH meter and EC meter after calibration the pH for different buffers.
Acidity of this solution was measured by titration method. Where 0.1 N NaOH solution was used, while Phenolphthalein was used as indicator.
3.3 PHYSICAL CHARACTERISTICS OF PLANTS
Physical characteristics of plants like Plant height, leaf area, number of leavesPlant-1, number of leaflets plant-1, number of branches plant-1, number of flowers Plant-1, number of fruits plant-1were recorded.
3.3.1 Plant Height (cm)
Plant height was recorded with the help of measuring tape in each treatment in each replication from soil level to the top of the plant and their average was calculated.
3.3.2 Leaf Area (cm2)
Leaf area was calculated before and after with the help of leaf area meter (LICOR, Li-3100) by randomly selected six leaves plant-1 in each treatment.
3.3.3 Number of Leaves Plant-1 and Number of Leaflets Plant-1
Number of leaves plant-1and number of Leaflets plant-1was recorded (at 15 days interval started from transplantation till crop harvesting).
3.3.4 Number of Branches Plant-1
Number of branches plant-1was recorded (at 15 days interval started from transplantation till crop harvesting).
3.3.5 Number of Flowers Plant-1
Number of flowers plant-1was recorded (weekly counted).
3.3.6 Number of Fruits Plant-1
Number of fruits plant-1was recorded (weekly counted).
3.4 CHEMICAL CHARACTERISTICS OF PLANTS 3.4.1 Proximate Composition
The recommended methods of the association of official analytical chemists (AOAC) were employed for determining the level of Moisture, Ash, Crude Fat, Crude Protein and Crude Fiber. Moisture content was determined by oven drying method.
Ash was determined by direct ashing method using muffle furnace maintained at 550
°C. Crude protein (% nitrogen x 6.25) was determined by kjeldhal method. Crude fat was obtained by exhaustively extracting in a soxhlet apparatus using petroleum ether
as the extractant. Crude fiber was determined by acid and alkali digestion. Nitrogen Free Extract (NFE) representing the total digestible carbohydrates was calculated by difference.
NFE = 100 - % (Ash + Crude Fat + Crude Protein + Crude Fiber) 3.4.2 Mineral Analysis.
The samples were first digested with nitric acid and perchloric acid and then the aliquot for determination of minerals. (AOAC, 2000).
After the acid digest preparation Na and K was determined by using Flame photometer. As in Phosphorus determination was made the colour assay so the Spectrophotometer was used. The other micro nutrients were determined by using Atomic absorption spectrophotometer.
3.5 Chlorophyll Analysis
Chlorophyll was determined using At-leaf chlorophyll meter where reading was note in unit µg/g. Determined by the method described by Sandoval-villa et al., (2002).
3.6 Lycopene Analysis
Lycopene was determined using spectrophotometer sp 3000. The reagents used in the procedure were of HPLC grade and 2:1:1 hexane, acetone and ethanol were mixed for the extraction of Lycopene from tomato fruits juice. Tomato fruits were washed and made into a juice. Then 100 µl of juice was pipette into 20mL screw cap tube. Eight mL of the mix reagent was added and vertexed then incubate in dark.
After 2-3 hours incubation 1 mL distilled water was added and vertexed again. The sample was rested for 10 to 20 minutes for phase separation. For blank 100µl of water was used with same procedure. The cuvette was washed and filled with the upper layer of the two phase sample. The absorption was noted at 503nm. The spectrophotometer was made zero with water blank.
Lycopene
(
mgKgfreshwt .)
=(A503×537×8×0.55)/(0.10×172)where 537 g/mole is the molecular weight of Lycopene, 8 mL is the volume of mixed solvent, 0.55 is the volume ration of the upper layer to the mixed solvents, 0.10 g is the weight of tomato added, and 172mM-1 is the extinction coefficient for Lycopene in hexane
3.7 Statistical Analysis
The data was analyzed using Statistix 8.1. Analysis of variance was carried out using simple CRD and or factorial design with 2 and 3 way interaction where necessary. Data means were compared using LSD test option of the software.