Study site and experimental materials
The research work was conducted at Plant Breeding and Genetics Division, Nuclear Institute of Agriculture (NIA), Tando Jam, Pakistan during the Kharif season, 2018–2019. The experimental material consisted of 12 diverse cotton advanced lines and cultivars (NIAB-878, NIA-M30, NIA-H24, NIA-HM48, NIA-Bt.30, NIA-H32, NIA-H67, NIAB-112, Sadori, NIAB-Kiran, and NIA-82) including one glandless genotype (Glandless-1) as the susceptible check.
Evaluation of cotton genotypes against sucking insects under field conditions
Field experiment design
The experimental materials were sown in Randomized Complete Block Design (RCBD) blocks during May, 2018. The plot size was 6.1 m × 3.0 m with four rows. Plant × plant and row × row distance was 30 cm and 75 cm, respectively. Three replicates, each of 12 genotypes, produced a total of 36 plots. Each plot comprised four rows of 20 cotton plants for a total of 80 plants. Standard agronomic practices were performed throughout the growing season. Plant protection measures were not adopted in order to expose the genotypes to insect attack. The experimental materials were harvested during October, 2018.
Estimation of sucking insect pest infestation
The population of adults per leaf of cotton whitefly (Bemesia tabaci), thrips (Thrips tabaci) and jassid (Amrasca devastans) were recorded at weekly intervals, early in the morning, from 20 days after sowing up to the picking of seed cotton. Five plants were tagged randomly in each replication of the treatment. From each plant, data were recorded from three leaves, one from the upper leaf, one from the middle and one from the lower portion of the plant and then converted into per leaf basis (Ahmad et al. 2011). A hand magnifying lens was used for counting the sucking insect population. A total of 11 observations were recorded for about 3 months during the study.
Estimation of morphological and biochemical HPR traits
Leaf area and glanding
The above randomly selected five plants were also used to measure morphological HPR traits (leaf area and leaf glanding). Five leaves per plant from different positions (upper, middle and lower) of each plant were cut, bagged, and carried to the laboratory. The leaf area (cm2) of 25 randomly selected leaves was measured with a leaf area meter and means were computed. The leaf glanding was also estimated from the lower side of these leaves and scored by using the following scale given by Romano and Scheffler (2008).
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0
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= Glandless
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2
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= Very few gland on the interveinal region
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4
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= Few number of interveinal glands (between very few glands = 2 and normal glanding = 6)
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6
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= Normal glanding on throughout the interveinal area and on the margins and veins
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Note = If categories 0, 2 or 4 had normal glanding on leaf margins or veins, 1.0 was added to score.
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Total soluble sugars (TSS)
The amount of TSS (mg·g−1) in fully expanded youngest leaves randomly collected from different plants of each genotype was estimated by phenol sulphuric acid reagent method given by Dubois et al. (1951). Leaf samples (0.05 g) were extracted in 95% methanol solution (2 mL) for 48 h at room temperature. Assay solution was prepared by using 96% sulphuric acid, 5% phenol and sample extract. A 1 mL of leaf extract, 0.5 mL phenol solution, and 2.5 mL sulphuric acid were added in glass tubes. Each tube was gently agitated during acid addition and then kept in a water bath at 26–30 °C for 20 min. Then absorbance of the colored solution was measured at 490 nm using spectrophotometer VIS-1100 (BMS, Canada). Different concentrations (0.1–3 mg with 0.5 mg interval and 3–6 mg with 1 mg interval) of glucose were prepared in 4 mL distilled water. After mixing the standards and placing them in the water bath at 26–30 °C for 20 min, absorbance was measured at 490 nm. A standard curve was drawn by plotting glucose concentration on the x-axis against their absorbance at 490 nm on the y-axis.
Total soluble proteins (TSP)
The TSP was measured using Bradford protein estimation assay (Bradford 1976). Briefly, 0.5 g fully expanded youngest leaves randomly collected from different plants were ground and homogenized in 10 mL of ice–cold potassium phosphate buffer. Then samples were centrifuged for 100 min at 15000×g. The supernatant (5 μL) was then mixed with a 1 mL reaction solution containing Coomassie Brilliant Blue G-250 (0.02 g), which was dissolved in 10 mL of 95% ethanol. To this solution, 20 mL of 85% phosphoric acid was added. The resulting solution was diluted to a final volume of 200 mL, and bovine serum albumin was used as a standard.
Total phenolics (TP)
The TP was measured using the Folin-Ciocalteu reagent following a method adapted from Singleton and Rossi (1965). The fully expanded youngest leaves of 0.05 g randomly collected from different plants were extracted in a 95% methanol solution (500 μL) for 48 h at room temperature. The extract was centrifuged at 14 000 r.min− 1 for 10 min. The supernatant (100 μL) was added to a 100 μL of 10% Folin-Ciocalteu reagent, after 2 min, 800 μL of 7.5% Na2CO3 solution was added. After shaking, the solutions were maintained at room temperature for 1 h. Absorption was measured at 765 nm using the spectrophotometer VIS-1100. Gallic acid standard solutions were used to calibrate the method and the results were expressed as gallic acid equivalents in milligram per gram in fresh weight.
Tannins
Fresh leaf samples (0.05 g) were extracted in a 95% methanol solution (500 μL) for 48 h at room temperature with occasional stirring. The extract was centrifuged at 14 000 r.min− 1 for 10 min. The supernatant was used to estimate tannins following the method described by Burns (1971). Tannin in the sample was measured at 765 nm using tannic acid as standard. The total tannins were expressed as milligram per gram fresh weight.
Total flavonoids (TF)
The TF were determined following the method described by Chang et al. (2002). The fully expanded youngest leaves of 0.05 g randomly collected from different plants were extracted in a 95% methanol solution (500 μL) for 48 h at room temperature. The TF assay was performed using an extract (200 μL) dissolved in 800 μL distilled water, 10% aluminium chloride (50 μL), 1 mol·L− 1 potassium acetate (50 μL), and 1.4 mL distilled water. After shaking, the mixtures were incubated at room temperature for 40 min and the absorbance was then measured at 415 nm using the spectrophotometer VIS-1100. The TFs were expressed as rutin equivalents in milligram per gram in fresh weight.
Statistical analysis
The data collected were analyzed separately for each parameter and subjected to analysis of variance following Steel et al. (1997). The means were compared using least significant differences (LSD) test (alpha = 0.05). Correlation matrix was computed using XLSTAT 2012. The values presented are mean of three replicates ± standard error (SE), respectively.
