Chapter 3: Materials and Methods
3.4 Modes of cultivation
3.4.2 Hydroponics system experiment
The aphids were controlled by spraying Mospilan at a rate of 4 g/20 L water, and Confidor at a rate of 20 cm3/20 L water. Weeds were controlled manually.
The two varieties of the wheat seeds were germinated in perlite in seed planting tray (Figure 3.8). On 9th December 2018 with intervals of twenty days, each plant was transferred from the tray to the hydroponic pot containing approximately 2 kg of perlite and peat moss mixture at a rate of 1:2 (Figure 3.9). The reservoir tank had to be filled with the hydroponic solution and maintain the irrigated water treatment (Treated wastewater and well water supplemented with nutrients) by adding the hydroponic solution regularly.
Figure 3.8: Seed germination Figure 3.9: Wheat cultivation using hydroponic system
There are countless numbers of and Hoagland updated versions of nutrient solutions that have been released in the past 50 years. Hoagland and Arnon's (1950) agricultural experimental research station bulletin from the University of California (Berkeley) is they are known as the basis for many formulations currently being used by researchers and commercial companies.
The growth solution was prepared according to the hydroponic solution method described by Hoagland and Arnon (1950) using chemicals from Sigma-Aldreich (Table 3.1).
The temperature of the atmosphere ranged from 21.1◦C to 38.4◦C with a natural light rotating between 12 hours of light and 12 hours darkness during the experimental timing which was reported during the time of the experiment by the National Center of Meteorology (Al Foah Automatic Weather Station).
The temperature, electrical conductivity (EC) and pH were recorded for each growth solution every 2 days. The measurements were recorded using a combination portable pH/TDS/EC/temperature meter (manufactured by Hanna Instruments, USA).
The plant sampling date 5th of March 2019 after 12 weeks to study the differences of agronomy/physiological growth parameters such as Plant and root length, Plant weight, and dry weight, Flag length, and area, number of the head by plant and head length. The plants were harvested on the 20th of April 2019. The duration of the experiment was 133 days.
86 Table 3.1: The growth solution composition for the hydroponic experiment
No. Chemical Stock solution
(g/1L)
Stock solution (M)
Growth solution (M)
mL in 2L
mL in 50L
mL in 75L
1 Potassiumnitrate (M.wt=101.1) 121.3 g 1.2 0.0025 4.2 104.2 156.3
2 Calcium nitrate (M.wt=236.15) 188.9 g 0.8 0.0025 6.3 156.3 234.4
3 Magnesium sulfate heptatahydrate (M.wt=246.48) 98.6 0.4 0.001 5.0 125.0 187.5
4 Potassium hydrogen phosphate 23.816 g 0.175 0.0005 5.7 142.9 214.3
5 Fe-EDTA (M.wt Total=571.04) 30.836 g 0.054 0.00005 1.9 46.3 69.4
No. Chemical Stock solution
(g/1L)
Stock solution (µM)
Growth solution (µM)
mL in 2L
mL in 50L
mL in 75L
1 Boric acid (M.wt=61.83) 2.860 g 46200 7.1 0.3 7.7 11.5
2 Manganese (II) chloride (M.wt=198) 1.801 9000 7.3 1.6 40.6 60.8
3 Zinc sulfate (M.wt=287.54) 0.22 760 0.9 2.4 59.2 88.8
4 Copper (II) sulfate (M.wt=159.61) 0.0495 g 310 0.5 3.2 80.6 121.0
5 Ammonium molybdate (M.wt=1236) 0.037 g 29.9 0.0016 0.1 2.7 4.0
6 Nickel (II) Nitrate-6-Hydrate (M.wt=290.79) 0.29079 1000 0.5 1.0 25.0 37.5
3.4.2.1 Estimation of chlorophyll and carotenoids
Complete phytochemical profiling of a representative plant sample is required before it is used for yield characteristics. Usually, the first step in the research with any crop is the profiling of their phytoconstituents. Chlorophyll, carotenoids, and pigments, which are generally secondary metabolites in a plant, affect the yield characteristics.
Chlorophyll extraction is achieved using 80% acetone, and the absorbances were measured in a spectrophotometer at 663 and 645 nm. Thus, it is mandatory to determine the phytoconstituents in the plant quantitatively. A standard method for the assay of chlorophyll (both A & B) and carotene contents are given by Rajput and Patil (2017) was used.
Reagents Acetone (85%) Procedure
Briefly, 0.5 g of freshly picked representative leaf sample was taken after 12 weeks from cultivation and placed into mortar and pestle to be grinded with view drops of acetone. The green color was released from the sample then the green solution is poured onto a 100 mL volumetric flask with filter paper and the volume was completed with the acetone. Dilution was done to the samples with higher intensity. The intensity of the color was read at 663, 645 nm and 440 nm for chlorophyll (both A & B) and carotene contents respectively using Spectrophotometer (Model Helios Alpha). The final result was in mg/g by the below equation:
Total Chlorophyll (mg/g) = 20.2 (OD645) + 8.02(OD663) x (V/(1000 x wt)) Chlorophyll A (mg/g) = (12.7 (OD663) – 2.69(OD645)) x (V/(1000* wt))
Chlorophyll B (mg/g) = (22.9 (OD645) – 4.68(OD663)) x (V/(1000* wt)) Carotene mg/g = 4.695 (O.D 440) (V/wt)
Where;
OD: optical density at certain wavelength (645 or 663 or 440 nm) V: final volume (mL)
wt: weight of sample (g)
3.4.2.2 Estimation of fiber and crude protein content
Proximate chemical composition of representative wheat grain samples was carried out bestowing to the procedure described by EU commission regulation No 691/2013 of 19 July 2013 amending Regulation (EC) No 152/2009 (Kashlan et al., 1991). To estimate the crude fibre content, it was necessary to digest the fiber by treating the sample overnight at 1.25% H2SO4 and then subjecting it to the hot NaOH treatment. After digestion, wheat samples were aspirated into ANKOM 220 Fiber Analyzer. Acid detergent fiber (ADF) was analyzed using 1 N sulphuric acid and cetyl trimethyl ammonium bromide and Neutral Detergent Fiber (NDF) was analyzed using NaSO2 and sodium lauryl sulphate (AOAC, 1984).
Principle
The wheat plant materials were divided into less digestible cell walls and more digestible cell contents viz. It contains lignin, cellulose, and hemicellulose, and includes sugars and starch, respectively. Van Soest et al. (1991) reported neutral and acid detergents to separate fiber contents such as Na-lauryl sulfate, EDTA, pH =7.0, and acetyl trimethyl ammonium bromide in 1 N H2SO4, respectively. In animals' digestive systems, the plant cell walls of consumed plant cells are classified as either less
digestible or more digestible content. The cellulose, lignin, and hemicellulose containing cell walls are less digestible, whereas sugars and starch are more digestible cell contents. The less digestible cell wall contents of cellulose, lignin, and hemicellulose are not digestible in non-ruminants. Cellulose and hemicelluloses are digestible to a certain extent in ruminants.
Neutral Detergent Fiber = Lignin +Cellulose + Hemicellulose. This is an indicator of “feed intake”. As it is described to be indigestible, therefore NDF is indicated as “bulk”. Acid detergent Fiber = Lignin +Cellulose. This is an indicator of energy intake because it is recognized to be digestible.
Reagents for NDF determination (Neutral detergent fiber) 1. Neutral detergent solution:
✓ Disodium ethylenediaminetetraacetate (EDTA, C10H14N2Na2O8)
✓ Disodium phosphate anhydrous (Na2HPO4) 4.56 g – 10 mL
✓ 1 L distilled water in a beaker, combine sodium borate and disodium EDTA and dissolve with H2O. Then heat with 2 ethoxyethanol and lauryl sulfate dissolve the disodium phosphate in a small volume of distilled water and heat it until it dissolves completely. Mix the two solutions together with the remaining distilled water and change the pH to between 6.9 and 7.1
✓ Sodium borate decahydrate (Na2B4O7 - 10 H2O) 6.81 g
✓ 2-ethoxyethanol (Ethylene glycol monoethyl ether, Cellosolve, C4H10O2) 30 g
✓ Sodium sulfite anhydrous (Na2SO3)
✓ 18.61 g Sodium lauryl sulfate neutral (C12H25NaO4S)
✓ n-octanol (C8H18O) octilic alcohol
✓ Acetone
Reagents for ADF determination Acetone 1. Acid detergent solution:
✓ C19H42BrN - 20 g of Cetyltrimethylammonium bromide technical grade
✓ Dissolve tensioactive into acid while stirring to promote dissolution.
✓ H2SO4, 49.04 – (g/l) 1 l - Sulfuric acid 1 N 2. n-octanol (C8H18O) octilic alcohol.
3.4.2.3 Estimation of NDF determination
The neutral detergent fiber content was determined by the method of Waldern (1971). Briefly, fresh wheat samples were collected, and the sample was washed under running water to remove foreign and dirty materials. The sample was oven-dried at 60◦C and the dried sample was made into a fine powder using a kitchen blender. The oven-dried powder sample was collected from a sieve with a size of 1 mm.
A crucible was filled with precisely 1 gm of fine powder, 100 mL of neutral detergent solution, 0.5 gm sodium sulfite, and a few drops of n-octanol. In boiling conditions, the reaction mixture was kept for 60 minutes. The reaction mixture was filtered, and the extract was washed three times with hot water and twice with acetone. The resulting mixture was allowed to dry for 8 hours before being incubated at 105°C and eventually put in a desiccator. The content's weight was expressed in gm.
(weight of crucible + weight of residue) - weight of the crucible
NDF % = ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯×100
Weight of sample
3.4.2.4 Estimation of ADF
The neutral detergent fiber content was determined by the method of Dong and Rasco (1987). Briefly, the sample was washed under running water to remove foreign and dirty materials. The sample was oven-dried at 60◦C, and the dried sample was made into a fine powder using a kitchen blender. The oven-dried powder sample was collected from 1 mm sieve. A crucible was packed with precisely 1 gm of fine powder, 100 mL of acid detergent solution, and a few drops of n-octanol. In boiling conditions, the reaction mixture was allowed to stand for 60 minutes. The reaction mixture was filtered, and the extract was washed three times with hot water and twice with acetone.
The resulting mixture was allowed to dry for 8 hours before being incubated at 105°C and then put in a desiccator. The weight of the content was articulated in gms. ADFwas calculated as
(Weight of crucible + Weight of residue) - Weight of crucible ADF % =⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯×100 Weight of sample
The mixture was left in a muffle furnace at 550 C for 2 hours and then cooled in a desiccator. The resulting ash was weighed.
The NDF of ash was calculated as NDF = loss on ashing/weight of sample x 100.
3.4.2.5 Estimation of crude fiber
The crude fiber content in various samples was assessed using AACC (1969) method.
Reagents
1. 200 mL of 1.25% of sulphuric acid in water – 2.5 mL of sulphuric acid mixed with 197.5 mL distilled water
2. NaOH – 200 mL of 1.25% NaOH – 5 gms of sodium hydroxide made up to 200 mL distilled water
3. 95% C2H5OH
Procedure for crude fiber estimation
Exactly two gms of the defatted sample were weighed into a 500 mL beaker, and 200 mL of 1.25% of sulphuric acid in water was added and permitted to digest for 30 minutes. The residue/filtrate was later added with boiled water to remove excess acid. The acid-free residue/filtrate was extracted repeatedly with 200 mL of 1.25%
NaOH at room temperature for 30 min. The entire contents were filtered hot through cheesecloth. The residue/filtrate was concentrated in a gooch crucible until the residue/filtrate was NaOH free; the residue/filtrate was washed with 15 mL of 95%
C2H5OH. The contents of the crucible were concentrated at 100°C. The mixture was ignited at 550°C ± 15°C in a muffle furnace for 30 minutes.
3.4.2.6 Elemental analysis
Analysis of plant, seeds and water samples were done by using Inductively Coupled Plasma Optical Emission Spectrometer (ICP) to determine levels of, Cadmium, Calcium, Copper, Iron, Lead, Magnesium, Phosphorus, Potassium, Sodium, Tin and Zinc for food commodities. Also, that the scope of analysis covered Calcium, Iron, Magnesium, Potassium, Sodium, Copper, Zinc and Phosphorus for water samples.
Acid treated samples were aspirated into ICP-OES (VISTA-MPX, USA).
Microelements, secure elements, and trace element content were assessed using the procedure described by Zhang et al. (2014).
Plant, seeds and soil samples
The plant and wheat seed varieties collected for proximate element analysis must be subjected to pre and post cultivation assessment. Moreover, soil samples were analyzed before and after cultivation. 1 g of the ground sample was digested using a mixture of (HNO3 + HSO4) at a rate of 2:1 overnight in the hot plate with temperature of 400℃. The final concentration was calculated:
Sample concentration (mg/Kg) = C (mg/L) * V * D Where;
C = Analyte concentration direct from instrument reading, in mg/L V= Final Volume (50 mL) of solution
W = initial weight (0.5 g) of sample
D = Dilution factor (Diluted volume/aliquot volume), if secondary dilution was made.
Water samples
Water samples were collected and analyzed using (ICP-OES). Samples were filtered before measurement with the instrument and were diluted so that TDS does not exceed 0.1% (w/v). It has been employed for the investigation of 18 mineral elements.
SAR can be calculated using the equation:
SAR ( Sodium Absorption Ratio) = 𝑁𝑎+ (√(𝐶𝑎2++𝑀𝑔2+)
2 )
Where; Concentration of calcium, sodium and magnesium must be in meq/L
3.4.2.7 Estimation of nitrogen Principle
Nitrogen content in the wheat sample was determined by Cavell (1954) method using a flame photometer. When inorganic or organic samples are digested with concentrated H2SO4, it results in ammonium sulphate from nitrogen. The resultant solution is alkaline treated with 40% NaOH, and the escaped ammonia gas is collected in H3BO3 solution. The formation of ammonia was evaluated by titration with standard acid.
Wheat sample + H2SO4→(NH4) 2SO4+ H2O + CO2 + by-products (NH4) 2SO4 + 2NaOH → 2NH3 + Na2SO4 +2H2O
NH3 + H3BO3 → NH4+ + H2BO3
H2BO3 + HCl → H3BO3 +Cl- Reagents
1. 0.1 N HCl
2. NaOH – (40% for distillation and 15% for digestion) 3. K2SO4 and CuSO4 (10:1)
4. Boric acid (H3BO3) in 100 mL distilled water a. 480 mL of 2 percent boric acid
b. 20 mL of 0.1% bromocresol green (in 95% ethyl alcohol) c. 4 mL of 0.1 percent methyl red solution.
Procedure
The total Nitrogen (N) content was studied conferring to the Kjeldahl method.
All the plant parts viz., root, shoot, spike biomass. The protein content of samples was assessed according to the Kjeldahl method defined in AOAC (1984). A digestion mixture containing 10 parts of K2SO4 and 1 part of CuSO4 was prepared. About 20 mL of concentrated sulphuric acid was mixed with 5 gms of digestion mixture and 2 gms of the sample. The Kjeldahl flask containing this reaction mixture was digested until the digested sample was carbon-free. This was transported into a 100 mL volumetric long-necked flat bottom flask, and distilled water was added to a final volume100 mL.
About 10 mL of digested sample was mixed with 40% sodium hydroxide. The solution was shaken robustly and distilled to liberate ammonia. This was then collected in 25 mL beaker containing 20 mL of 2% H3BO3 and 2-3 drops of indicator. It was titrated against 0.1 N HCl. The following equation was then used to calculate the nitrogen content in g/Kg of the sample.
1 mL of 0.1 M HCl or 0.05 M H2SO4 = 0.0014 g N
𝑁𝑖𝑡𝑟𝑜𝑔𝑒𝑛 % =𝑅 ∗ 𝑁𝑜𝑟𝑚𝑎𝑙𝑖𝑡𝑦𝑜𝑓𝐻𝐶𝑙 ∗ 0.014 𝑊𝑡𝑜𝑓𝑡ℎ𝑒𝑠𝑎𝑚𝑝𝑙𝑒 (𝑘𝑔)
Where; R= Volume of std. HCl used – volume of std NaOH
% Protein = % Nitrogen X Conversion Factor
The value of 5.71 was used as a conversion factor for the wheat.
3.4.3 Open Top Chambers (OTC) experimental site and growing conditions