Determination of Amylose Content in Mature Rice Seeds

Determination of Amylose Content (AC) in Mature Rice Seeds
1.1 Sample preparation

Collect some seeds in plant units. About 50 seeds were selected from mature seeds of each individual plant. Some of them were used to determine the gelatinization temperature, and some were ground to form polished rice flour. The method was applied to the amylose analyzer according to the method of the standard No. NY147-88. Amylose content was determined.
1.2 A large number of measurements After the seeds matured, they were harvested on a per plant basis. The determination of amylose content was carried out according to standard NY147-88 issued by the Ministry of Agriculture. Accurately weigh 50 mg of dried rice flour on a ten-thousandth scale and place in a 50 ml graduated test tube; then add 0.5 ml of absolute ethanol to the tube, gently shake the tube to wet the sample, and add 4.5 ml of 1.0 mol/l. NaOH solution, mix; put test tube in boiling water bath for 20min, cool to room temperature, dilute to volume with distilled water, mix thoroughly; absorb 5ml of digestive liquid, add in 100ml volumetric flask containing about 20ml distilled water; add 1.0ml 1.0mol/l acetic acid solution to acidify the sample; add 1.5ml of 0.02% iodine solution, dilute to volume with distilled water, shake for 15min; use spectrophotometer at a wavelength of 620nm with 1cm colorimetric The cup was measured for optical density; a standard curve was prepared using the optical density measured for the standard sample and its corresponding amylose content, and the amylose content of the sample to be tested was calculated accordingly. Make 2 repetitions for each sample.
1.3 Determination of single grains Take milled rice ground into powder and sieve through 100 mesh and dry. Accurately weigh 5~10mg dry rice powder and put it into a 50ml graduated test tube; then add 0.2ml absolute ethanol to the test tube, gently shake the sample to wet the sample, and add 0.9ml of 1.0mol/l NaOH solution. Evenly; put the test tube in a boiling water bath and cook for 20 minutes, cool to room temperature, dissolve with distilled water to the mark, and mix thoroughly. Pipette 25 ml of digestive juice into a 100 ml volumetric flask containing about 20 ml of distilled water; add 1.0 ml of 1.0 mol/ml. l Acidify the sample with acetic acid solution; add 1.5ml of 0.02% iodine solution, dilute to volume with distilled water, shake for 15min; measure the optical density with a 1cm cuvette at 620nm wavelength on the spectrophotometer Finally, a standard curve was prepared from the light-tight 22-degree value and the corresponding amylose content of the standard sample, and the amylose content of the sample to be tested was calculated based on this.
Determination of Gel Consistency and Gelatinization Temperature in Mature Rice Seeds. Some seeds were mixed in strain units. About 50 seeds were selected from the mature seeds of each individual plant. One part was used to determine the gelatinization temperature. A portion of the mixture was ground and then ground to a fine rice flour. The amylose content was determined according to the method shown in the ministerial standard NY147-88. And gel consistency.
2.1 Rice mature seed starch viscosity spectroscopy (RVA) analysis was performed using a 3-D RVA instrument manufactured by Newport Scientific Instruments, Australia, for rapid determination and analysis using TCW (thermal cycle for windows) software. According to the procedure, when the water content was 14.0%, the sample amount was 3.00 g and distilled water was 25.0 ml. During the stirring process, the temperature changes in the tank were as follows: 1 minute at 50°C; 12°C/min to 95°C (3.75 minutes); 95°C, 2.5 minutes; 12°C/min to 50°C ( 3.75 min); hold at 50°C for 1.4 min. The stirrer rotates at 960 r/min for the first 10 s and then remains at 160 r/min. Viscosity values ​​are expressed in RVU (RVA Viscosity Units). In addition to the peak viscosity, hot viscosity, and cool viscosity, the RVA profile also uses breakdown values ​​(peak viscosity, hot slurry viscosity), and subtractive values. (setback, cold-viscosity-peak viscosity) and return value (consistence, cold-adhesive-hot-slurry viscosity), etc. (AACC, 1995).
2.2 Scanning electron microscopy of rice grain During the maturation period, some transgenic lines and their controls were selected. The single ear of the same ripening period was selected and dried naturally. From each individual ear, strong grains of similar growth position were taken. The integrity of starch in the transverse direction of the grain was observed on a Philips XL 30-ESEM (circular scanning electron microscope) ring scan.
2.3 Field traits were investigated in a completely randomized block design. Transgenic homozygous and untransformed controls were planted and planted in 2 replicates in separate plots with 15 plants in each row with 8 plants in each row. Ten plants were randomly selected from each plot to investigate the main agronomic traits. The investigation included: plant height, panicle length, number of tillers per plant, seed setting rate and 1000-grain weight. The statistical analysis was conducted to compare the differences between the main agronomic traits of the transgenic homozygote and untransformed controls.

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