The experiment was done in Dept. of Agricultural Chemistry experimental plots of the Hajee Mohammad Danesh Science and Technology University, Dinajpur, Bangladesh. Duration of the study was from months October 2011 to July 2012. Experimental fields are located between 25.13ºN latitude and 88.23ºE longitude. Elevation of the area was 37.5 m. Study site belongs to the Old Himalayan Piedmont Plain. The site belongs to Agro-Ecological Region (AEZ) One13. Experiment field was medium high land and texture was sandy loam. Soil pH was 5.6. The experimental area possesses sub-tropical climate. Usually there is no or less rainfall during winter season. The air temperature ranges were low to moderate and low during the winter season and increases as the season proceeds towards Kharif season (monsoon crops during July-October) with occasional gusty winds. Seeds of nine selected rice land races and or cultivars and or variety were used for this study. Seven cultivars were local and rest two of them was HYV varieties. The cultivars were Bashful (V1), Pasursail (V2), Gochi (V3), Taipi (V4), Pariza (V5), Lafayaa (V6), Jirashail (V7), BRRI dhan 28 (V8) and Banglamoti (V9). Design of experiment was randomized complete block design. Seedlings that were of fourty days used for transplanting in experimental plots. Fertilization, intercultural operations such as weeding by hand picking and applications of pesticides by hand sprayer at the vegetative growth stages were done as and when necessary. Data collections of the following parameters from the experiment on different growth stages were done. Leaves from nine cultivars were collected for determination of proline content. Grains were used for determination of minerals Ca, Fe and Zn and determination of physical properties, i.e., length/breadth ratio, shape and size, moisture content etc. Plants leaves samples were collected at 35 DAG (days after germination), 25 DAT and 50 DAT (days after transplanting) from the seed bed and from main field. Grains were collected after crop harvest.
Determination of L/B ratio of selected rice grains: Slide caliper was used to measure length and breadth (L / B). L/B proportion was obtained by dividing length with breadth. Long (>6mm) size and slender shape, Medium (5-6) size and bold shape; and Short (<5) size and round shape categories were of rice was found. While, L / B proportions were >3, 2-3 and <2 for size category long, medium and short respectively.
Procedure for proline determination from plant leaves sample: Sample holder was marked by labeling, small test tube considered as micro fuse tube1.5 ml-2.0 ml. Micro fuse tube (sample holder) was weighed. The micro fuse tube + leaf sample → (leaf → about 60-80 mg) → (freshly in prepared) was weighed, dipped in a liquid N2 solution for 3 minutes, 500 µl (micro liter) 3% sulfosalicylic acid with micro-pipette was added. Then, samples were ground with a stick/plastic rod to mix well. Test tube was placed with a vortex mixer for about 30 sec ~1 min. On another micro test tube, the mixer was placed for 1 ~ 15 min in a thermomixure. 500 µl more suftosalicylic acid 3% was added to micro fuse tube. It was centrifuged for 20 ~ 30 min at 250C and with 15000 rpm. Ten ml test tube with a glass tube pipette was used to collect supernatant. One ml sulfosalicylic acid was further added to the small test tube + sample. Place again in the test tube mixer for 10 min. Again, mixer was placed in the centrifuge machine for 20 min at 250C and with 15000 rpm. Supernatant was collected again and mixed with previously collected supernatant. Ninhydrin solution was prepared. Standard proline solution 0, 1, 2, 5, 20, 50, 100, 150, 200 and 300 µg/2ml, 3% sulfosalicylic acid was taken in a test tube for standard curve. 2 ml acetic acid was added into each sample + proline standard solution. 2 ml Ninehydrin acid was added into each sample + proline standard solution → mix well by end to end. Test tube was heated for exact 15 minutes in thermo aluminum bath/water bath 96-1000C. It was cooled in an ice box. The optical density was measured at 520 nm wave length with a spectrophotometer for standard curve and for sample solutions.
Moisture content: Working steps that were involved for moisture content determination; i. 5g ground samples (rice grain) were taken in a petridis, ii. oven dry samples prepared at 600C for 72 hrs, iii. dried samples were taken in desiccators, iv. oven dried sample were weighed, and v. Moisture % was calculated.
Estimation of minerals (Ca, Fe and Zn) content in rice grain: Digestion was done with 0.5 g sample. 25 ml acid mixture (H2SO4, HClO4 and HNO3–proportion of 0.5:1.0:0.5) by volume was used. Approx. 2000C temperatures was used during digestion and high neck volumetric flask was used which was thermal proof. Digestion was continued to get clear and or greenish solution. Solution was taken into test tube after cooling. Concentration of Fe2+, Ca2+ and Zn2+ were determined after dilution and Chemito atomic absorption spectrophotometer was used for this purpose. Wavelength used for Fe2+, Ca2+ and Zn2+ were 248.3, 422.7 and 213.9 nm respectively. A calibration curve was used for all analyzed samples. Glassware contamination was avoided with caution. Reagents and deionized water were used during mineral analysis.
Data were statistically analyzed using MSTAT-C program. Then, analysis followed by least significance difference test and duncan’s multiple range test.