Rice sample preparation: A total of 20 rice genotypes were collected; 4 (Binadhan-8 salt tolerant, Binadhan-10 salt tolerant, Iratom-24 and Binadhan-7) from Bangladesh Institute of Nuclear Agriculture, 10 (BRRIdhan76, BRRIdhan73 salt tolerant, BRRIdhan53 salt tolerant, BRRI dhan54 salt tolerant, BRRI dhan47 salt tolerant, BRRI dhan56 drought tolerant,BRRI dhan57 drought tolerant, BRRI dhan66 drought tolerant and BRRI dhan71 drought tolerant) from Bangladesh Rice Research Instituteand 7 land races (Basiraj, Pazam, Nazirshaildh,Gahinda, Jirakalani, Dulai) from different parts of Bangladesh. The research was conducted in Crop Physiology Division, Bangladesh Institute of Nuclear Agriculture, Mymensingh and Department of Agricultural Chemistry, Department of Biochemistry, Bangladesh Agricultural University, Mymensingh during 2018 to 2020. The samples were manually cleaned to remove cracks kernels and the husk of the paddy was removed to get rice. Rice grains were grinded for analyzing.
Determination of kernel length and breadth: Ten randomly selected whole kernels of rice in three sets were taken and length and breadth of each grain was measured by using a slide calipers. The average value for each observation was considered as final reading. The length and breadth of rice kernel were expressed in millimeter (mm).
Determination of kernel length/breadth (L/B) ratio: The L/B ratio was calculated by dividing the average length by the average breadth of kernel. L/B ratio= Average length of the rice (mm)/ average breadth of the rice (mm). The scores are recorded for brown rice to evaluate the traits as genetic characteristics avoiding the effect of milling on size and shape. The rice grains were classified by standard evaluation system (SES) for rice (IRRI 1996).
Estimation of protein: Micro-Kjeldahl method was used for the estimation of total nitrogen in rice grain. Then total nitrogen was multiplied by a conversion factor to obtained protein content.
Digestion: Powdered rice samples (0.2g) taken in a 75ml Kjeldahl flask and 5ml of concentrated H2SO4, 1 gm of digestion mixture was added. The flask was placed on digestion chamber and boiled until the mixture content becomes clear. The flask was cooled and the digested sample was diluted with distilled water.
Distillation: 25 ml of diluted digested samples was taken and 25 ml of 40% NaOH was poured into the flask slowly holding the flask about 45.angle and connected to the distillation set. The distillate was collected in a conical flask containing 10ml of 2% Boric acid solution and 2-3 drops of mixed indicator.
Titration: Total distillate was titrated with 0.1N HCL and titration value was recorded.
Percentage of N was calculated by the following × formula:
% of nitrogen= (Ts-Tb) × normality of acid×0.014×100/weight of samples (g),where Ts= Titre value of the sample, Tb=Titre value of the blank
0.014= Milli equivalent weight of nitrogen
% protein=% of nitrogen × C.F.
C.F.=Conversion factor(5.5 for plant sample)
Sample preparation and determination of Zn and Fe: Collected samples were dried in an oven at 65°C for 24 hours and ground by a grinding machine after cooling. The prepared samples were then kept into plastic bottles until extract preparation. The plant extract was prepared by wet oxidation method using di-acid mixture following Singh et al. 1999. Exactly, 1.0 g of finely ground plant material was taken into a 250 ml conical flask and 10 ml of di-acid mixture (HNO3: HClO4= 2:1) was added to it. Then, it was placed on the electric hot plate for heating at 180-200°C until white fumes were evolved and subsequently cooled at room temperature. The digest was washed with distilled water repeatedly and filtered into a 100 ml volumetric flask through filter paper (Whatman No. 42) and the volume was made up to the mark with distilled water. Amaranth extracts were preserved separately in plastic bottles for subsequent chemical analysis.
The concentrations of Fe and Zn ions in the extracts were analyzed by atomic absorption spectrophotometer (AAS) (Model: SHIMADZU AA-7000) at the wavelengths of 248.3 and 213.9 nm, respectively as described by APHA 2012.
Determination of amylose content: Amylose was determined following the method of Robyt and Whelan (1968). Accurately weighed 100mg of powdered sample was taken and 1mlk of 95% ethanol and 9ml of 1N NaOH were added and warmed for 5 min in water bath to gelatinize the starch. The content transferred in 100ml volume with water-cooled and brought to volume with water. 5ml solution was taken into a 100 ml volumetric flask, 1 ml of acetic acid and 2 ml of iodine solution were added and made up to the volume with water, stirred and allowed to stand for 20 min before taking optical density at by spectrophotometer at 590nm.
Preparation of standard curve: 100 mg of anhydrous potato amylose was dissolved in 100 ml of alcoholic NaOH (10 ml ethyl alcohol and 90 ml 1N NaOH). Portions containing 0.25, 0.50, 0.75, 1.00, 1.25, 1.50, 1.75 and 2 mg of amylose transferred to 100ml flask. The solution was acidified with 1N acetic acid by adding 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4 ml respectively and color was developed using iodine solution. Optical density was taken at 590nm in Spectrophotometer. The amylose content of the each sample was calculated from standard curve.
Calculation of amylopectin content: Amylopectin is a calculated value which is obtained from the value of total amylose. % of amylopectin= 100-% of amylose (Jane et al. 1999).
Determination of volume expansion ratio: Volume expansion ratio of raw milled rice and cooked rice was determined by water displacement method by using a measuring cylinder. A sample of 5 gm of rice grains poured into a measuring cylinder containing 15 ml of water and total volume was observed. The initial increase in volume after adding 5gm of rice was recorded (Y) and soaked for 10 min. Rice grain sample was cooked for 20 min in a water bath at 900C. All the 5 gm of cooked rice were placed in 50 ml water taken in 100 ml measuring cylinder and the increase in volume of water was measured (X). The volume raise was recorded (X-50). Where, (X-50) is the volume of cooked rice (ml) and (Y-15) is the volume of raw rice (ml).
Determination of kernel elongation ratio (KER): Kernel elongation after cooking and kernel elongation ratio (KER) was determined by Juliano 1971. In this method, 10 whole kernels after cooking (20 min in a water bath at 900C) was measured by using slide calipers and average kernel length was determined. Kernel elongation was calculated by dividing the average length of cooked kernel by the average length of the raw (uncooked) rice.