Chapter 3

MATERIALS AND METHODS

3.1 Preliminary Experimental Details

These studies were carried out in Faisalabad (31.4180° N, 73.0790° E) conditions and
comprised of pots experiment in wire house and in field conditions during winter (two successive
years 2016-2017 and 2017-2018) in the Department of Botany, University of Agriculture
Faisalabad, Pakistan to study the mitigating effect of fresh moringa leaf extract (MLE 3%),
Sorgaab (3%), hydrogen peroxide (100 µM) and Ascorbic acid (500 µM) in Quinoa
(Chenopodium quinoa Willd.) under high temperature stress. Quinoa genotype UAF-Q7 was
used in all the experiments were kindly provided by Alternate Crops Lab, Department of
Agronomy, University of Agriculture Faisalabad, Pakistan. Part of studies was also conducted at
University of Copenhagen, Denmark. All the experiments were conducted related to mitigation
of high temperature effects in quinoa for two years. The details of these studies are as under;

3.2 Mitigation of Heat Tolerance in Quinoa under Glass Canopy Conditions

3.2.1 Experimental location and growth conditions

Sowing of seeds: The experiment was conducted in plexiglass fitted canopies (Fig. 3.1), located
in the wire-house of the Old Botanical Garden, Department of Botany, University of Agriculture
Faisalabad. Ten seeds of quinoa genotype UAF-Q7 were sown in clay pots (diameter = 23 cm
and depth = 29 cm) containing 10 kg loamy soil and after two week of germination plant were
thinned up to five plants of equal size. The design of the experiment was Completely
Randomized Design (CRD) Factorial with three replicates. The plants were grown for about 68
days to reach the anthesis stage.
Fig. 3.1: Plexiglass-fitted canopies used to develop high temperature environment for quinoa.
Light transmission index of the canopy was 75‒80%.

High temperature treatments: At anthesis stage (after 68 days of sowing) experimental plants
were divided into two sets. One set was kept under wire-house and considered as control, while
other set was shifted to open door plexiglass fitted canopies. Daily maximum and minimum
temperatures in wire house and in canopy were recorded on daily basis (Fig. 3.1). The canopies
had 7-10ºC high temperature as compared to ambient temperature.

3.2.2 Selection of optimum level and application of treatments

The optimum concentration of fresh moringa leaf extract (3%), Sorgaab (3%), hydrogen
peroxide (100 µM) and ascorbic acid (500 µM) used here has already been established in
different studies as optimum growth enhancers (Basra et al., 2011; Jahangeer, 2011; Yasmeen et
al., 2013b). The foliar spray treatments were: 1) control (no spray), 2) water control (plants
sprayed with water), 3) 3% fresh moringa leaf extract (3% MLE) spray, 4) 3% Sorgaab spray, 5)
100 µM hydrogen peroxide (H2O2) and 6) 500 µM ascorbic acid (AsA).
 60 Maximum ambient temperature Minimum ambient temperature 2016
Canopy maximum temperature Canopy minimum temperature
50
Temperature (oC)

40

30

20

10

0
2-Mar

8-Mar
4-Mar
6-Mar

10-Mar
12-Mar
14-Mar
16-Mar
18-Mar
20-Mar
22-Mar
24-Mar
26-Mar
28-Mar
30-Mar
1-Apr

7-Apr

13-Apr
15-Apr
16-Feb
18-Feb
20-Feb
22-Feb
24-Feb
26-Feb
28-Feb

3-Apr
5-Apr

9-Apr
11-Apr
60 2017

50
Date (in 2016)
Temperature (oC)

40

30

20

10

0
2-Mar

8-Mar
4-Mar
6-Mar

10-Mar
12-Mar
14-Mar
16-Mar
18-Mar
20-Mar
22-Mar
24-Mar
26-Mar
28-Mar
30-Mar
1-Apr

7-Apr

13-Apr
15-Apr
16-Feb
18-Feb
20-Feb
22-Feb
24-Feb
26-Feb
28-Feb

3-Apr
5-Apr

9-Apr
11-Apr
Date (months/years)

Fig. 3.2: Changes in the minimum and maximum temperature inside and outside the glass
canopies. The light transmission spectrum was about 0.8.

Sixty eight days old plants were shifted to glass canopies and kept for one week to
acclimate to heat stress condition. After a week, the plants were foliar sprayed with the
abovementioned selected levels of growth enhancers. Since all the growth enhancers were
prepared in water, the foliar spray of water was done to find out the specific effect of water (if
any) on the plant attributes. The plants were harvested two weeks after foliar spray. Data were
recorded for growth, photosynthetic pigments and gas exchange properties, oxidative stress and
antioxidants, secondary metabolites, nutritional relationships of leaf and seed yield and
nutritional attributes were determined using standard protocols as given under various sections
below. For different biochemical analyses, a part of the plant material was preserved in a freezer
at 40oC until analyzed.

3.3 Mitigation of Terminal Heat Stress at Different Sowing Dates

The proposed study was conducted at Research Area of Directorate of Farms, University
of Agriculture Faisalabad, Pakistan. Seeds of genotype UAF-Q7 were sown during the
experiments. There was 30 cm row to row and 15 cm plant to plant distance. The design of
experiment was randomized complete block design (RCBD) with three replicates. Standard crop
husbandry practices including, fertilization, irrigation and weed removal were carried out. The
meteorological conditions under both the conditions by transforming on weekly basis have been
presented in Fig. 3.3. Foliar spray of abovementioned growth enhancers and their levels were
carried out at anthesis stage as per following schedule during both the years:.

Year Sowing date Foliar spray date Growth and physiological Maturity harvest date
data recording date
2016 20 Nov 2016 5 Mar 2017 12 Mar 2017 18 Apr 2017
16 Dec 2016 -do- -do- 2 May 2017
27 Dec 2016 -do- -do- 30 May 2017
2017 17 Nov 2017 1 Mar 2018 10 Mar 2018 20 Apr 2018
13 Dec 2017 -do- -do- 30 April 2018
29 Dec 2017 -do- -do- 12 May 2018

3.4 Recording of Data

The data were recorded for growth, physiological, biochemical, nutritional and seed yield
attributes as detailed below for both experiments.
100 Total rainfall (mm) 2016-17
90 Maximum temperature (oC)
Minimum temperature (oC)
80 Relative humidity (%)
70
60
50
40
30
20
10
0
100
19–25 Jan
29 Dec–4 Jan
5–11 Jan
12–18 Jan

9–15 Feb
16–22 Feb

9–15 Mar
16–22 Mar
23–29 Mar

27 Apr–3 May
4–10 May
11–17 May
18–24 May
25–31 May
1–7 Dec
16–23 Nov
24–30 Nov

2–8 Feb

23 Feb–1 Mar
2–8 Mar

30 Mar–5 Apr

13–19 Apr
20–26 Apr
8–14 Dec
15–21 Dec
22–28 Dec

26 Jan–1 Feb

6–12 Apr
2017-18
90
80
70
Date (weekly interval)
60
50
40
30
20
10
0
19–25 Jan
29 Dec–4 Jan
5–11 Jan
12–18 Jan

9–15 Feb
16–22 Feb

27 Apr–3 May
4–10 May
11–17 May
18–24 May
25–31 May
16–23 Nov
24–30 Nov

2–8 Feb

23 Feb–1 Mar
2–8 Mar
9–15 Mar
16–22 Mar
23–29 Mar
30 Mar–5 Apr
1–7 Dec
8–14 Dec
15–21 Dec
22–28 Dec

26 Jan–1 Feb

6–12 Apr
13–19 Apr
20–26 Apr

Date (weekly interval)

Fig. 3.3: Changes in the meteorological conditions during the course of experiment.

3.4.1 Growth and yield components

The height was measured of the intact plants growing in the pots or field. For shoot fresh
weight, the plants were cut and immediately weighed by using a top-loading balance. For dry
weight, the plants were transferred to paper bags and dried in an oven at 70 oC for one week and
then dry weights were taken using the top-loading balance. The seeds were carefully extracted
from the panicles and weighed to express seed yield on per plant basis. For 1000 seed weight, the
seeds were counted and weighed. Total aboveground dry mass per plant was weighed and used
for deriving the harvest index (HI) using the following equation:

Seed yield per plant

HI (%) = ----------------------------------- × 100
Straw yield per plant

3.4.2 Physiological and biochemical analysis

a. Estimation of photosynthetic pigments:

Arnon (1949) protocol was used for the estimation of chlorophylls while the carotenoids
were measured by using Davies (1976) method. For these measurements, 0.1 g freshly harvested
leaves were grinded in 80% acetone solution; filtered the material and made final volume up to
10 mL. Absorbance of the samples was taken on spectrophotometer at 480, 645 and 663 nm.

The following formulas were used for the determination of chlorophylls and carotenoids:

Chlorophyll a (mg/g fresh wt.) = (1.27 (OD663)-2.69(OD645) ×V/1000×W

Chlorophyll b (mg/g fresh wt.) = (22.9 (OD645)-4.68(OD663) ×V/1000×W

Total chlorophyll (mg/g fresh wt.) = (20.2 (OD645) + 8.02 (OD663) ×V/1000×W

Carotenoids (mg/g fresh wt.) = (OD480 + 0.114 (OD663)-0.638 (OD645)/2500) × 1000

b. Leaf gas exchange parameters

Third fully expanded leaf was selected for this purpose. Infrared gas analyzer (Analytical
Development Company, Hoddesdon, England) was used for the determination of following gas
exchange characters:

1. Net CO2 assimilation rate (A) (µmol m-2 s-1)

2. Transpiration rate (E) (mmol m-2 s-1)
3. Water use efficiency (A/E)
4. Stomatal conductance (gs) (mmol m-2 s-1)
5. Sub-stomatal CO2 concentration (Ci)
 These measurements were made between 10 am and 2.00 pm with the following
specifications/adjustments: leaf surface area 6.25 cm2, ambient CO2 concentration (Cref) 342.12
µmol mol-1, temperature of leaf chamber (Tch) varied from 36.2 to 42.9 oC, leaf chamber volume
gas flow rate (v) 396 mL min-1, leaf chamber molar gas flow rate (U) 251 µmol s -1, ambient
pressure (P) 99.95 kPa, molar flow of air per unit leaf area (Us) 221.06 mol m -2 s-1, PAR (Q leaf)
at leaf surface was maximum up to 918 µmol m-2 s-1.

c. Oxidative stress parameters

Hydrogen peroxide (H2O2): The hydrogen peroxide was measured by following the protocol of
Velikova et al. (2000). Fresh leaves were (0.1 g) were grinded in 5 mL of 0.1% (w/v)
trichloroacetic acid (TCA), in a pre-chilled pestle and mortar. Then homogenized leaf samples
were centrifuged for 15 minutes. After that 0.5 mL supernatant was thoroughly mixed with 0.5
mL potassium phosphate buffer (pH 7.0) and 1 mL of potassium iodide solution. The absorbance
was noted at 390 nm by using spectrophotometer. Distilled water was used as blank. The
concentration of H2O2 was calculated by constructing a standard curve.

Malondialdehyde (MDA): The concentration of MDA was determined with the procedure of
Heath and Packer (1968). Fresh leaf samples were crushed in 1 mL (1%) trichloroacetic acid
(TCA) and centrifuged for 15 minutes. Supernatant (1 mL) was mixed in 1 mL (0.5%) of
thiobarbaturic acid (prepared in 20% TCA) and heated at 100 oC. Then the sample mixture was
centrifuged for 5 minutes at 7500 rpm, incubated at 95oC for 50 minutes and then cooled on ice.
The absorbance was taken on spectrophotometer at 532 nm, 600 nm, and 1% TCA was used as
blank. By subtracting the A600 from of A532, non-specific turbidity was corrected. To calculate the
concentration of MDA, an extinction coefficient 155 nmol/L was done with the equation:

MDA equivalents (nmol mL-1) = [(A532-A600) / 155000] × 106

d. Antioxidant activities determination:

Enzyme extraction: A 0.5 g fresh leaves were grinded in 10 mL phosphate buffer of pH = 7.8.
The leaf extract was centrifuged at 15000 rpm for 20 minutes. The supernatant of enzyme extract
was stored in Eppendorf tubes at -20oC and further used to determine the amount of soluble
proteins and activities of antioxidants.
Soluble proteins: The amount of soluble proteins was measured using coommassie brilliant blue
(CBB) dye binding method of Bradford (1976).

Superoxide dismutase (SOD) activity: The activity of SOD was checked by using the protocol
of Giannopolitis and Ries (1977). In cuvettes, 50 µL enzyme extract, 50 µL NBT (nitroblue
tetrazolium), 50 µL riboflavin, 100 µL methionine, 250 µL phosphate buffer, 40 µL distilled
water and 100 µL Triton X was added and kept it under light for 15 min. After that, the
absorbance was noted at 560 nm by using spectrophotometer.

Catalase (CAT) activity: This was measured following the method of Beers et al. (1952). In
cuvette 100 µL enzyme extract, 1.9 mL phosphate buffer and and 1 mL H2O2 (5.9 mM) was
added. By using spectrophotometer absorbance was taken at 240 nm.

Peroxidase (POD) activity: The POD activity was measured by using the procedure of Chance
and Maehly (1955). Enzyme extract (15 µL) was added in cuvette containing reaction mixture of
750 µL phosphate buffer, 100 µL guaiacol (20 mM) and 100 µL hydrogen peroxide (40 mM).
Absorbance was recorded at 470 nm on spectrophotometer.

Ascorbic acid (AsA): AsA was estimated by following the procedure of Mukherjee and
Choudhuri (1983). Fresh leaves (0.1 g) were crushed in 2 mL of 6% TCA solution. Added 1 mL
of extract in 1 mL of 2% dinitrophenyl hydrazine (in acidic medium) and one drop of 10%
thiourea (prepared in 70% ethanol). Reaction mixture was heated in a water bath for 15 minutes
and chilled on ice. Then added 5 mL H2SO4 (80%) to the cooled reaction mixture. The
absorbance was noted at 530 nm using spectrophotometer. The concentration of AsA in each
sample was analyzed from a standard curve plotted with known concentration of AsA.

e. Osmoprotectants determination

Total free amino acids (TFAAs): TFAAs were measured with the method of Hamilton and Van
Slyke (1943). A 0.1 g fresh sample was grinded in phosphate buffer (pH 7), 1 mL of this extract
was mixed with 1 mL ninhydrin solution (2%) and I mL pyridine solution (10%). Heated the
reaction mixture for 30 minutes. Then the mixture was diluted up to 25 mL. The absorbance was
taken at 535 nm, and phosphate buffer was run as blank.

Free proline: The free proline was determined by the method of Bates et al. (1973). A 0.1 g
fresh plant material was extracted in 20 mL aqueous sulfosalicylic acid (3%). The 1 mL of
extract was mixed with 1 mL of acid ninhydrin and 1 mL of glacial acetic acid in a test tube
followed by heating for 60 min at 100 oC. After cooling the reaction mixture on ice bath, 2 mL of
the toluene was added, and vortexed for 15-20 sec. The colored complex containing free proline
was aspirated in a test tube and warmed to room temperature. The absorbance of the colored
complex was taken at 520 nm. Aqueous sulfosalicylic acid (3%) was used as a blank. For the
calculation of free proline, the following equation was used.

µg proline/mL × mL of toluene
µmoles proline/g fresh weight = -------------------------------------------------
(115.5 µg/mole)/g sample/5

Glycinebetaine (GB): The gycinebetaine was determined by using the method given by Grieve
and Grattan (1983). Fresh plant material (0.1 g) was homogenized in 5 mL distilled water. Then
1 mL of the extract was mixed with 1 mL of 2N H 2SO4 in test tube. From this solution 0.5 mL
was taken and mixed with 0.2 mL potassium periodide (prepared by dissolving excess of iodine
in potassium iodide solution). Test tubes were kept at 4 oC for 16 hours. Then this mixture was
centrifuged at 10,000 × g for 15 minutes at 4 oC and discarded the supernatant. The periodide
crystals settled in the bottom of the centrifuge tube were dissolved in 10 mL of 1, 2-
dichloromethane, left at room temperature and after 15-20 minutes, the absorbance was taken at
365 nm by using spectrophotometer. The 1, 2- dichloromethane was used as blank.

f. Secondary metabolites determination

Soluble phenolics: Soluble phenolics were determined by Julkunen-Titto (1985) method. A 0.1
g plant sample was extracted in 1 mL acetone (80%). After centrifugation for 15 min at 12000
rpm, supernatant was collected in microfuge tubes and stored at -30 oC until used. In a test tube,
100 µL supernatant (diluted it up to 1 mL) 0.5 mL of folin phenol reagent (Fluka) and 2.5 mL of
20% sodium carbonate (Na2CO3) and made the volume up to 5 mL. Then vortexed the reaction
mixture for 5-10 sec and kept them for 20 min at room temperature. The absorbance of the
colored complex was noted at 750 nm, and 80% acetone was used as a blank.

Anthocyanins: Anthocyanins were measured by following the procedure of Stark and Wray
(1989). A 0.1 g fresh plant leaves were ground in 2.5 mL of acidified methanol (1% HCl, v/v)
and warmed at 50oC for 60 min. Absorbance noted at 535 nm using acidified methanol as blank.
g. Nutrient estimation

Seed and shoot dried material was digested in digestion flasks using 2 mL digestion
mixture. The digestion mixture (50 mL) was prepared by dissolving 0.42 g Se and 14 g
LiSO4.2H2O in 350 ml distilled water. Conc. H2SO4 was added by keeping mixture in an ice bath
[Se (0.42g) + LiSO4.2H2O (14g) + H2O2 (350 mL) + slowly added conc. H2SO4 (420 mL) by
keeping it digestion flasks were heated up to 250ºC on hot plate until fumes were produced.
Removed the flasks from hot plate, cooled the samples and slowly added 1 mL H 2O2 (35% A.R.
Grade extra pure) and placed the flasks back to hot plate. Repeated the above step until material
was colorless. The volume of extract was maintained up to 50 mL, filtered and used for the
determination of the following mineral elements.

Ca2+ and K+ determination: Ca2+ and K+ ions were estimated by using flame photometer
(Sherwood PFP 410). A graded series of standards (10 ppm, 20 ppm, 30 ppm and 40 ppm) of
Ca2+ and K+ were run and standard curves were drawn. The values of Ca 2+ and K+ from flame
photometer were compared to standard curves and final amounts were calculated.

Mg2+, Zn2+ and Fe2+ determination: For the estimation of Mg2+, Zn2+ and Fe3+ from the shoot
and/or seed extract, the samples were run on Atomic Absorption Spectrometer (A100, Perkin
Elmer, Waltham Massachusetts, USA). The exact quantities of all these elements were
determined from the standard solutions supplied by the manufacturer.

Nitrate-N estimation: Kowalenko and Lowe (1973) method was used for this purpose. Dried
plant (0.5 g) were boiled in 5 mL distilled water for 60 minutes and made the volume of filtrate
up to 50 mL. Took 3 mL extract in test tube and add 7 mL working solution of chromotropic acid
(CTA) and briefly vortexed. Yellow colored complex intensity was noted at 430 nm.

Phosphate-P estimation: Phosphate-P was determined by using the procedure of Yoshida et al.
(1976), 0.5 g dried plant material was heated in 5 mL of distilled water for one hour; made the
volume of filtrate up to 50 mL. Took 1 mL extract in test tube; added 2 mL of 2N nitric acid and
1 mL of molybdate-vandate reagent, and made the volume up to 10 mL. Vortexed the mixture
and after 20 minutes, absorbance was taken at 420 nm. Distilled water was run as blank.

Sulfate-S estimation: The method of Tendon (1993) was used for this purpose. Ten mL of the
extract in a conical flask was added with 1 mL each of 6 N HCl and 0.5% gum Arabic solutions
and swirled for 10-15 seconds. Then 0.5 g BaCl 3 crystals were added and swirled the flasks until
the crystals were dissolved and solution was clear. The sample transmittance was taken at 340
nm. To prepare Sulfate-S standard curve, 4-20 mg/L graded series were made from 100 mg/L
K2SO4 stock solution. To each flask of standard 25 mL of the buffer solution (prepared as 50 g
MgCl2.6H2O, 4.1 g KNO3 and 28 mL ethanol) was added while rest of the protocol was same.

3.5 Experiments at University of Copenhagen, Denmark

3.5.1 Experimental setup

A pot experiment was conducted in a growth chamber at the experimental station of

Taastrup, at the Faculty of Science, University of Copenhagen, Denmark. Purpose was to
investigate heat tolerance level of quinoa (Chenopodium quinoa) cv. Titicaca at anthesis stage,
and the mitigation effect of ascorbic acid (AsA). Pots (with 25 cm height x 16 cm diameter) were
filled with peat moss. Five seeds per pot were sown and thinned to one plant per pot at the two-
leaf stage. Growth conditions were set as: day/night air temperature 24/18oC, relative humidity
75% from sowing day to till anthesis stage (40 days). Light intensity of 580–680 µmol m-2s-1 for
13 h. At anthesis stage, plants were sprayed with 500 µM AsA. One set of plants was kept at
same climatic conditions and considered as control group, while other group shifted to another
growth chamber for heat stress, which had day/night air temperature 33/24oC (day/night), relative
humidity 65%. After one week of heat stress, pots were again shifted to ambient condition
growth chamber to check for recovery from heat stress.

3.5.2 Measurements

Fresh and dry biomass: To measure fresh weight plants were harvested at anthesis stage by
cutting the stems at the soil surface. After measuring fresh weight, plants were kept in oven at
70°C for 72 hours to measure plant dry weight.

Relative water content (RWC): Fresh leaves (FW) of equal weight were floated in water for
four hours under light to get turgid weight TW), then dried for 24 hours at 80°C to measure dry
weight (DW). The RWC was determined as: RWC (%) = (FW-DW)/(TW-DW)
Abscisic Acid (ABA): ABA concentration was measured following the protocol of Asch et al.
(2001). Fresh leaves were grinded in liquid nitrogen; took 0.1 g of crushed material in eppendorf
tubes, added 1 mL of distilled water. For homogenation samples were kept on rotatory shaker for
over-night at 4°C. Then extracts were centrifuged at 14000 rpm for 5 minutes at 4°C and
supernatant was collected. ABA concentration was determined by using an enzyme-linked
immunosorbent assay (ELISA) using a monoclonal antibody for ABA (AFRC MAC252)
(Philosophhadas, Hadas and Aharoni, 1993).

Gas Exchange measurements: Net photosynthetic rate (A), stomatal conductance (gs) and
transpiration rate (E) of fully grown leaf was recorded with the help of LI-COR Li-6400 (USA)
portable photosynthetic system. Readings were noted during 10:30 am to 12:00 noon. Chamber
temperature was 25°C with 400 µmols/mol CO2 flow level. The quantum flux for IRGA was
adjusted according to clear sunny conditions, using sunlight as light source.

Enzymatic antioxidants activity: Activity of SOD, CAT and POX were determined as
mentioned above. Dehydroascorbate reductase (DHAR) and monodehydroascorbate reductase
(MDHAR) activities were measured by the protocol of Murshed et al. (2008).

3.6 Statistical Analysis

Main and interactive effects of sowing date and foliar spray in quinoa crop was
evaluated on various response variables by analysis of variance (ANOVA) technique at 5%
probability level. Standard deviations of means were calculated. MS Excel (v.2010) was used
for the graphical presentation of data.

3.7 Chemicals

All the chemicals used for foliar application and plant analyses were of high purity
grade and purchased from Merch, Sigma, Fluka, or Aldrich.