This research was conducted in a date orchard in Jahrom city in Fars province for two consecutive years (2016-2017). In this experiment, pollens of eight pollinating cultivars including ‘Sheikhali’, ‘Faryab’, ‘Beraem’, ‘Zahedi’, and ‘Kabkab’ cultivars as local pollinators, and ‘Jarvis’ and ‘Fard’ cultivars as international pollinators were used for pollination of 32 (four replications; in each replication, one tree) twelve-year-old ‘Piarom’ date tree. Pollens were obtained from a research station located in Saadabad city of Bushehr province during ripening of male spots in March. To prevent unwanted pollination, ‘Piarom’ date inflorescences were covered with kraft paper before pollination. During pollination, three strands of male inflorescences were placed inside each female inflorescence and covered again with a paper bag for 4 weeks.
Measurement of pollen grain composition.
Carbohydrate and starch. To measure the amount of carbohydrates in pollens of different cultivars, 0.1 g of pollen was homogenized in 80% ethanol. The supernatant was then dried in an oven at 40°C and dissolved by adding water. The amount of soluble sugars was determined by injecting it into reverse-phase high-performance liquid chromatography (HPLC) (Portuguese Unicam model) with a carbohydrate column.
To measure the amount of starch, 2 ml of hydrochloric acid was added to the remaining solution from the ethanol extract prepared for measuring carbohydrates and boiled for 30 min. After cooling, the pH was reduced to 4.5 by adding potassium hydroxide to its solution. To digest the starch, 36 units of amyloglucosidase were added to the solution and kept at 50°C for 60 min. Finally, the solution was kept at 25°C for 30 min, and the absorption rate was read at 340 nm16.
Protein. To measure the protein, 0.05 g of pollen were homogenized with 2 ml of 0.1 M phosphate buffer with an acidity of 6.8 and centrifuged for 15 min. Then, 50 µl of the supernatant was removed and the volume was increased to 100 µl. Finally, 5 ml of Bradford solution was added, and the absorbance of the solution was read at 595 nm17.
Total phenol. The amount of total phenol in pollens of different cultivars, was measured by preparing an ethanol extract of pollen grains and using the Folin-Ciocalteu reagent colorimetric method. In this method, granulated ethanol extract (0.5 ml) was mixed with 2.5 ml of Folin-Ciocalteu reagent (diluted 1: 10 with distilled water) and 2 ml of 4% sodium carbonate. The solution was placed in the dark for two hours then the absorbance was measured at 740 nm. The amount of total phenol in terms of mg of gallic acid per gram of plant dry weight was expressed18.
Flavonoids. To measure flavonoids in pollens of different cultivars, 0.5 ml of ethanol extract (4.3 mol of 80% ethanol) was mixed with one tenth of a milliliter of aluminum nitrate and 0.1 ml of 1M potassium acetate. After 40 min, the absorbance at 430 nm was measured using a spectrophotometer and the amount of total flavonoids in mg of quercetin per gram of dry weight of plant matter was expressed19.
Pectin methylesterase (PME). To measure the activity of PME enzyme in pollen grains of different cultivars, 50 mg of pollens were added to 250 µl of PME extraction buffer containing 0.1 M citrate and 0.2 M sodium phosphate buffer and 0.1 M sodium chloride with pH 5 and centrifuged for 10 minutes at 14000 rpm. The supernatant was collected and one ml of the extract was removed, to which 0.2 M sodium phosphate was added (pH 6.3) and incubated at 37°C for 24 h. Then 0.2 ml of 0.05% red ruthenium and 0.5 ml of 0.6 M calcium chloride were added to the solution and centrifuged for 15 minutes at 14000 rpm. Finally, the supernatant was collected, and the absorption rate was measured at 534 nm20.
Amylase. To measure the activity of amylase enzyme in pollen of different cultivars, pollen grains (1 g) were homogenized with 15 ml of sodium citrate buffer at pH 5. The solution was then centrifuged at 10,000 rpm for 10 min. The 0.5 ml of the resulting extract was added to 3.5 ml of hydrochloric acid solution, and the absorbance was read at 580 nm21.
Measurement of ‘Piarom’ date fruit compositions.
Fruit chlorophyll and carotenoid. To measure the amount of chlorophyll and carotenoids in the fruit at four stages of development (kimri, khalal, rutab, and tamar), one gram of the outer layer of fruit flesh was removed and extracted with 80% acetone and then centrifuged for 10 min. The absorbance of the supernatant at 663 nm (chlorophyll a), 646 nm (chlorophyll b), and 470 nm (carotenoid) was measured using a spectrophotometer. Finally, using the following formulas, the amounts of chlorophyll and carotenoids in terms of mg/g wet weight of the sample were calculated22.
Chlorophyll a = (19.3 × A663 – 0.86 × A646) V/W
Chlorophyll b = (19.3 × A646 – 3.6 × A663) V/W
Carotenoids = 100(A470) – 3.27(mg chl. a) – 104(mg chl. b)/227
V = Volume of supernatant
A = Light absorption at 663, 646, and 470 nm
W = Wet weight of sample in term of gr
Fruit reducing and non-reducing sugars. To measure reducing and non-reducing sugars (glucose, fructose, and sucrose), date fruit flesh texture in tamar stage was divided into small pieces and homogenized with 100 ml of distilled water and kept at room temperature for one hour. The resulting extract was centrifuged at 10,000 rpm for 15 min. Then, 500 µl of the supernatant was removed and diluted with 500 µl of distilled water and was increased to 4 ml with 3 ml of acetonitrile. The prepared solution was passed through a 0.2 µm membrane filter. Glucose, fructose, and sucrose standards at concentrations of 2.5, 5, 10, 15, and 20 mg/l were also prepared. Then, 25 µl of standards and extracts of date fruit were injected into the HPLC (Portuguese Unicam model) with a C18 column. Finally, the glucose, fructose, and sucrose levels were calculated23.
Fruit soluble tannin. To measure the soluble tannins, 5 g of fruit skin and flesh tissue in four stages of development (kimri, khalal, rutab, and tamar) were homogenized with 25 ml of 80% methanol in a porcelain mortar and then centrifuged at 360 rpm for 5 min. The supernatant was separated, and the precipitate was extracted with 80% methanol and centrifuged. The supernatants were added together and made up to 100 ml with deionized water. One milliliter of the resulting solution was mixed with 6 ml of deionized water and half milliliter of Folin-Ciocalteu reagent (previously diluted 1: 10 with distilled water). After 3 min, 1 ml of saturated sodium carbonate and 1.5 ml of deionized water were added and after one hour, the amount of light absorption of the solution at 750 nm was read using a spectrophotometer. The concentration of soluble tannin was calculated based on the standard curve of different concentrations of pure gallic acid prepared at the same time as the samples24.
Fruit total phenol. To measure total phenol, 200 mg of fruit skin and flesh tissue in four stages of development (kimri, khalal, rutab, and tamar) were mixed with 2 ml of 50% ethanol in a porcelain mortar and kept for one hour. The resulting solution was centrifuged for 10 min. Then, 200 µl of the solution was mixed with 1.5 ml of Folin-Ciocalteu reagent (diluted 1: 10 with distilled water) and kept under the same conditions for 5 min, followed by the addition of 20% sodium carbonate (1.5 ml). After 90 min, the absorbance of the solution at 750 nm was measured using a spectrophotometer. The concentration of total phenol was calculated based on a standard curve of different concentrations of pure gallic acid25.
Fruit anthocyanin. To measure anthocyanin 0.5 g, fruit peel in four stages of fruit development (kimri, khalal, rutab, and tamar) were homogenized with 25 ml ethanol hydrochloric acid solution (980 ml 95% ethanol + 20 ml concentrated hydrochloric acid). Finally, the optical density of the solution at 530 nm was measured using a spectrophotometer. The amount of anthocyanin was measured in mg per 100 g of fruit peel according to the following equation26.
Anthocyanin in 100 gr of fruit peel =\(\frac{\text{o}\text{p}\text{t}\text{i}\text{c}\text{a}\text{l} \text{d}\text{e}\text{n}\text{s}\text{i}\text{t}\text{y} \times \text{v}\text{o}\text{l}\text{u}\text{m}\text{e} \text{o}\text{f} \text{e}\text{x}\text{t}\text{r}\text{a}\text{c}\text{t}\text{e}\text{d} \text{s}\text{o}\text{l}\text{u}\text{t}\text{i}\text{o}\text{n} \times 100}{\text{w}\text{e}\text{i}\text{g}\text{h}\text{t} \text{o}\text{f} \text{t}\text{h}\text{e} \text{s}\text{a}\text{m}\text{p}\text{l}\text{e} \times 98.2}\)
Fruit cellulase enzyme. To measure cellulase activity, 50 g of fruit mesocarp tissue in four stages of fruit development (kimri, khalal, rutab, and tamar) were mixed in 85 ml of 12% polyethylene glycol and 2% sodium bisulfite for one minute. After centrifugation for 10 min at 10,000 rpm, the resulting material was divided into two parts to measure the enzyme activity. The resulting material was mixed with 50 mM sodium acetate at pH 5 and 0.5 M sodium chloride at 4°C in a shaker for one hour and was centrifuged. The supernatant was separated and diluted by 5 mM sodium acetate at pH 5. By measuring the amount of reductant groups isolated from carboxymethylcellulose, cellulase activity was determined. The reaction mixture contains 0.25 ml of pure enzyme, 0.5 ml of 1% carboxymethylcellulose, 0.25 ml of 100 mM sodium acetate buffer with pH 5 and incubated at 37°C for 1, 2, 3, 4, and 5 h. The precursor was added to the control tubes after heat treatment, and the samples were incubated in a hot water bath for 5 min, the amount of absorption was measured at 550 nm. For determining the concentrations of reducing groups, the D-glucose standard was used. Cellulase activity as a unit of enzymatic activity was defined as the amount of enzyme isolated under standard conditions from one micromole of glucose per hour27.
Fruit invertase enzyme. To measure the amount of invertase enzyme in fruit at four stages of growth and development (kimri, khalal, rutab, and tamar), 5 g of fruit samples were homogenized in a mortar using sodium acetate extraction buffer. After two centrifuges, a dinitrosalicylic acid solution was added to form a color solution. Color samples were recorded using a spectrophotometer at 540 nm, and the data were expressed in micromoles per gram of fresh material. The extraction of invertase enzyme was similar to that of cellulase. The assay mixture consists of 1.5 M sucrose, 1 ml enzyme extract and 0.5 M acetate buffer (pH 4.5), in a total volume of 5 ml. This mixture was incubated at 37°C for one hour and 1 ml samples were taken at 10 min intervals during this period, and the reducing sugar was determined using dinitrosalicylic acid. The amount of reduced sugar isolated was calculated using a calibration chart, and one unit of invertase enzyme was defined as the amount of enzyme required for the hydrolysis of sucrose (0.5 µM) per minute28.
Fruit polygalacturonase enzyme. To measure the polygalacturonase enzyme, fruit tissue (2.5 g) was homogenized in four stages of fruit development (kimri, khalal, rutab, and tamar) with a pH of 3. The mixture was centrifuged for 15 min at 8000 rpm, and the supernatant was removed. The precipitate was washed with distilled water, and after centrifugation, the supernatant was removed. The resulting solution was mixed with 1.2 mol of sodium chloride in a ratio of 1:1 and its pH reached 6, after which the supernatant was removed after centrifugation for 20 min at 10,000 rpm. Then, 50 µl of the extract was added to 350 µl of buffer containing 0.2% polygalacturonic acid and 0.4 mol of sodium acetate with pH 4.4. After 10 min of incubation at 37°C, the reaction was stopped with the buffer at a concentration of 0.1 mol and the pH equal to 9. The amount of light absorption was measured at 276 nm, and the enzyme activity in the samples was evaluated using a standard curve29.
Statistical analysis. This study was performed on a total of 32 twelve-years-old trees (eight treatments, four replications and one tree in each replication). A randomized complete block design was used to analyze the data. Duncan’s multiple range test was used to determine the statistical significance of the means (p < 0.05). SPSS software was used for the data analysis.
Statement on guidelines. All experimental procedures on date palm plants comply with relevant institutional, national, and international guidelines and legislation.