MORPHO-PHYSIOCHEMICAL AND GENETIC CHARACTERIZATION OF RICE VARIETIES (ORYZA SATIVA L.) IN DIFFERENT GEOGRAPHICAL AREAS OF NORTHERN INDIA.

Sonu Kumar Chaudhary 1 , Diksha Varma 2 , Dr. Krishan Pal 1 * and Dr. Manisha Tiwari 2 *. 1. Department of Agriculture, Shri Venkateshwara University, Gajraula, Uttar Pradesh 244236, India. 2. Bio-Organic Chemistry Laboratory, Dr. B. R. Ambedkar Centre for Biomedical Research, University of Delhi, Delhi 110007, India. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

In the present study, prominently evolved varieties of Indica rice (Oryza sativa L.) were studied and characterized. Effect of temperature and humidity on the grain yield and grain quality in different geographical areas of Northern India was studied. This would ensure the selection of rice varieties with respect to different geographical climatic regions. Seven varieties of rice were studied and characterized by the effect on their morpho-physiochemical characters in different climatic conditions of Uttar Pradesh and Haryana, India. All the seven varieties of rice except Sambha Sub-1 showed presence of fgr gene whose protein product are responsible for the peculiar fragrance of Basmati rice. This was also confirmed by the chemical assay for aroma. Morho-physiological characters of the crop namely; plant height, plant biomass, root biomass, leaf area, panicle length, spikelets per panicle, grain yield per plant and days of maturity were found to significantly correlate with each other with (P > 0.5). Biochemical studies performed showed the effect of temperature in different climatic conditions on total chlorophyll, chlorophyll a and chlorophyll b, total sugar and total protein. A significant correlation (p > 0.05) was observed between the higher and lower temperature and humidity respectively and different morpho-physiochemical characters. Four varieties of Basmati namely VB-21, VB-24, PB-1509, PB-1121 has been found to show higher morphophysiochemical characters and grain yield per plant. VB-21 and VB-24 has been observed to resist the high temperature and low humidity in the region of Haryana as they have shown to produce higher grain yield, seed weight and grain quality.

…………………………………………………………………………………………………….... Introduction:-
Rice is one of the most important field crops of the world providing staple food to millions. It is grown in 114 countries across the world and the population of 150 million has continuing cultivation in near 11% of the world's cultivated land. More than 90% of the world rice is produced and consumed in Asia as it is an indispensable source of calories for almost half of the world population within the continent. Rice belongs to the genus Oryza and has two cultivated and 22 wild species. The two cultivated species are Oryza.sativa L and Oryza.glaberrima. Oryza.sativa L In this study an attempt has been made to study the effect of temperature and humidity on the morphological, physiochemical, genetic characters, grain yield and grain quality of seven rice varieties namely; Vallabh Basmati-21, Vallabh Basmati-22, Vallabh Basmati-23, Vallabh Basmati-24, Pusa Basmati 1509, Pusa Basmati 1121, Sambha Sub-1 in different geographical areas of Northern India. This will ensure the selection of rice varieties with respect to different geographical climatic regions and will further help to improve their yield and grain quality to fulfill the substantial food requirement of India and the other counties of the world.

Materials and Methods:-
Collection and sowing of the paddy Crop:-In the present study seven varieties of paddy Oryza sativa L were collected from National Seeds Corporation, New Delhi, Indian Agriculture Research Institute, New Delhi and Basmati Import and Export Foundation, Meerut. Seven varieties of paddy were selected for the study namely, Vallabh Basmati-21 (VB-21), Vallabh Basmati-22 (VB-22), Vallabh Basmati-23 (VB-23), Vallabh Basmati-24 (VB-24), Pusa Basmati 1509 (PB-1509), Pusa Basmati 1121 (PB-1121) and Sambha Sub-1 (SS-1). The seeds were collected and sown on the agricultural field of Hastinapur, Uttar Pradesh and Asaudha, Haryana, India as given in Table 1. The experiment was laid out in Randomized Block Design (RBD) with three replications for each genotype with size of each plot -8m × 1m, plot distance 45 cm 2 and row distance 20 cm 2 in sandy loamy soil in kharif season in 2016. Healthy seeds were selected by putting them in water and stirring well and then the floated seeds were rejected. The germinated seeds were then sown in raised seedbed. The seedlings were transplanted on the main field after 28 days. Only one seedling was transplanted per hill. N: P: K -20: 10: 10 Kg/Ha (in two split doses) of the fertilizer was used in the field during maturation stage while Chloroform 3g @ Kg/Ha was used against Hispa. The temperature and amount of humidity in the environment during various stages of rice development are listed in the Table 1.  ), root length (cm), dry and fresh root biomass (g), dry and fresh plant biomass (g), panicle length (cm), spikelets per panicle, Grain yield per Hill, Grain yield per plant, days taken by plants to mature and 1000 seeds weight were studied and recorded. The plant height was measured from the base of the shoot above the soil up to the edge of the leaf. Panicle length was measured from the upper topmost node up to the tip of the panicle. Grain yield was measured after harvesting the seeds when the moisture content of the seeds was approximately 14%. Leaf area was measured by length-width measurement method given by Yoshida et al (1981) [20]. It uses a correction factor (k). Leaf area was measured by the following equation: Leaf Area (cm 2 ) = k x length (cm) x breadth (cm) The correction factor (k) used for the rice leaves ranges from 0.67 to 0.80, depending on the variety and the growth stage. The value of 0.75, however, can be used for the all growth stage except the seedling and maturity stage.  [7]. Leaf tissue from each sample of the rice variety was collected in the vegetative stage from the fields and stored in -80°C freezer. The frozen leaf tissue was cut into pieces and grinded in liquid nitrogen. The grounded fine powder was then dissolved in 500 µl DNA extraction buffer (0.038 g sodium bisulphite added to pre-warmed buffer at 60°C) and thoroughly vortexed. The solution was incubated at 65°C for 40 minutes and then kept at room temperature for 5 minutes. 200 µl of 5M chilled potassium acetate was added to the sample solution, vortexed and kept at 20°C for 20 minutes. Solution was then centrifuged at 1000xg for 15 minutes at 4°C and to 100µl of the supernatant 100µl of chilled isopropanol added. After keeping the supernatant at room temperature for 10 minutes, it was centrifuged at 1000xg for 5 minutes at 4°C. The pellet obtained was then washed thrice with absolute and 70% alcohol respectively at 100xg for 10 minutes at 4°C. The final pellet of DNA obtained was dried in the lyophilizer and dissolved in DEPC water and stored at 20°C. The DNA samples were quantified by A 260/280 method.

Selection of Markers:-
The fgr gene responsible for the rice fragrance was studied and four markers that have been shown to be present in most of the fragrant variety of rice were selected. [2,6,10,15]. The primer sequence of the gene markers is given in Table 2. Primers ESP + IFAP + INSP + EAP were used for performing Nested PCR for gene specific amplification. Biochemical Study:-Isolation of Chloroplast and Estimation of Chlorophylls:-2 g of rice sample leaves harvested in the flowering stage and was washed thoroughly under the tap water, cut into small pieces and grinded in liquid nitrogen. To the powder 1ml of 1xCIB buffer (0.33M Sorbitol, 0.1M Tris HCl 824 PH-7.8, 5mM MgCl2, 10 mM NaCl, 2mM EDTA) with bovine serum albumin (BSA) and centrifuged at 200xg for 2 minutes. The supernatant was transferred leaving white pellet behind, into the chilled 50 ml centrifuge tubes and centrifuged at 1000xg for 7 minutes. A green pellet obtained was secured. The green pellet was broken gently by finger tapping and resuspended in 2 ml of 1x CIB buffer with BSA and mixed gently by pipetting up and down. The suspended pellet was then centrifuged at 300xg for 5 minutes and pooled into one centrifuge tube. The pellet was finally mixed with 500 µl of 1x CIB buffer without BSA. Absorbance of the solution was read at 645, 663 & 652 nm against the solvent (80% acetone) as blank. The amount of the chlorophyll present in the extract (mg chlorophyll/ g tissue) was calculated using the following equation Assay of rice Fragrance:-Twenty six freshly harvested milled rice grains from each of the rice varieties were crushed into fine powder and taken in the conical flask. About 10 ml 1.7% KOH solution was added to each of the conical flask and covered immediately with aluminum foil and left at room temperature for about 1 hour. The samples were scored on 1-4 scale with 1, 2, 3 and 4 corresponding to absence of aroma, slight aroma, moderate aroma and strong aroma, respectively. The score for each sample was recorded by a panel of five experts who have experience in aromatic rice breeding and quality evaluation [3,6,12].

Estimation of total Soluble sugar:-
The total soluble sugar present in the different varieties of rice seeds was measured by Anthrone method as described by Hedge and Hofreiter et al (1962) [5]. 100 mg of the powdered grains was taken in a test tube and 80 % of 10 ml ethanol was added, vortexed and hydrolysed by keeping it in a boiling water bath for 30 minutes. After cooling to the room temperature the sample was centrifuged at 1000xg for 10 minutes and collected in a beaker. To the remaining residue 10 ml of 80% ethanol was added and the method was repeated and the supernatant was pooled together. The total collected supernatant was evaporated in the vacuum evaporator and 50 ml of water added and vortexed to solubilize the sugar to use it for total sugar estimation. For estimation 0, 0.2, 0.4, 0.6, 0.8 and 1.0 ml of the working standard (1mg/ml) of D-glucose was used while water served as blank. After making the volume to 1 ml in all the test tubes including the sample tubes with distilled water, 4ml of anthrone reagent (200 mg anthrone dissolved in 100 ml ice cold 95% H2SO4 just before use) was added to all the tubes. The tubes were heated in a boiling water bath for 8 minutes. Rapidly the tubes were cooled in an ice bath and the change in color was read from green to dark green at 630 nm in a spectrophotometer. Standard curve was plotted by drawing the graph for the concentration of the standard on the X-axis versus the absorbance on the Y-axis. The amount of total sugar present in the sample tube was measured.
Amount of carbohydrate = Sugar value from graph (mg) X Total Vol of extract (ml) X100 in sample (%mg) Aliquot sample used (0.5 ml) X Wt. of sample (mg)

Estimation of Total Protein:-
To the finely powdered seed samples of 0.02g, 400 µl of the extraction buffer was added containing 0.5 M Tris-HCl (pH 8.0), 0.2% SDS, 5 M urea and 1% 2-mercaptoethanol. The mixture was than vortexed and centrifuged at 1000xg for 5 minutes at room temperature. After centrifuging samples, the crude proteins were recovered as clear supernatant on the top of the tube. Then the supernatant was transferred into new 1.5 ml eppendorf tubes and were stored at -20°C [18]. Bradford's method for protein quantification, modified by Nicholas J. Kruger was used for estimation of total protein in the seeds of different rice varieties by Standard Assay Method [13]. The supernatant containing the protein was diluted to (1, 1:10, 1:100, 1:1000) in duplicates. Protein BSA standard was prepared in volumes of 10, 20, 40, 60, 80, and 100 μL of 1 mg/ml and made each up to 100 μL with distilled water for the preparation of the calibration curve. 100 μL of distilled water was used as a blank. To these 5 mL of protein reagent was added and mixed well by inversion or gentle vortex mixing. Absorbance was measured at A595 between 2 min and 1 hour after mixing. The 100 μg standards should give an A595 value of about 0.4.

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Statistical Analysis:-The data of the morpho-physiochemical characters was statistically analyzed using Microsoft Excel 2010. To find the correlation dependence of morphological and biochemical parameters on temperature and humidity Student's t-test was applied. Correlation coefficient for each correlation between the morphological and biochemical parameters with each other was also found.

Results and Discussion:-
Morphological and Physiological:-Different morphological characters of each rice variety from each region of Uttar Pradesh and Haryana were measured and standard deviation from the mean and standard error for each observation has been recorded as listed in Table 3

*Standard Error (SE) < 1, **SE < 2, SD-Standard deviation
Evaluation of the fragrance marker fgr gene:-The fragrance of Basmati rice varieties is a peculiar phenotypic trait which increases its demand and cultivation. The presence of fgr gene in rice is a single recessive allele at a locus on chromosome 8. The product of this gene is 2acetyl-l-pyrroline (2AP) that gives Basmati rice its typical mild desirable aroma [3,9]. Rice genotypic study was carried out to evaluate the presence of fgr gene in seven varieties of rice selected in this study. Using the markers specific to the fgr gene PCR was carried out as per the protocol. The marker specific gene amplification of the fgr gene was observed in all the rice varieties taken in the study. Exception was observed with Sambha Sub -1 variety which showed minimal or null amplification with the Nested PCR primers ESP + IFAP + INSP + EAP and RM 515 respectively. Lower or Higher DNA bands from PCR of few of the varieties were also observed. This may be due to difference of few base pairs in the fgr gene length under environmental adaptability. Thus, Fragrance genotyping helped us to confirm the presence of the fragrance gene in the varieties of rice considered in this study.

Assay of Rice Fragrance:-
Apart from quality of rice seed such as grain size, seed luster and weight of the seeds; aroma of the rice, especially among the varieties of Basmati makes them a grain of choice in the market. To assess the fragrance in the seven varieties selected in the present study, the rice seeds were assayed for fragrance by KOH method. For the assessment 1-4 scoring scale with 1, 2, 3 and 4 corresponding to absence of aroma, slight aroma, moderate aroma and strong aroma, respectively was considered. The score for each sample was recorded by a panel of five experts who have experience in aromatic rice breeding and quality evaluation is listed in Table 6  Estimation of total sugar and total protein:-Rice is the staple food of people among the Asian countries because it fulfills the requirement of carbohydrates. The amount of starch content in rice grain determines its quality and gives the estimate of calories it can produce. The protein content of the rice is however found to be low as the amount of starch stored in the seeds is higher. Thus, to evaluate the amount of starch protein content in the rice grain seeds of different varieties were collected after harvesting and were used for estimation of total sugar and total protein by Anthrone method and Bradford's method respectively. The results are listed in the Table 6. As observed in the table, the total protein content in the rice seeds is low as compared to the total sugar content. However, it was also observed that the amount of total protein was higher in the rice varieties cultivated in the region of Haryana. This may be because of effect of high temperature and less humidity at the time of grain maturity and development. As evident the total sugar content in each rice variety in the region of Uttar Pradesh was found to be higher as the humidity and temperature required for plant maturity and grain development were optimally favorable.

Association of morphological parameters:-
Knowledge of the co-dependence of plant characters like grain yield with other morphological parameters paramount an important knowledge to the breeders for making improvement in the quantitative as well as qualitative characters in paddy. Hence, association of these characters was analyzed with respect to each rice variety as given in Table 7. The morpho-physiochemical characters of each rice variety in its area of cultivation and respective deviation from the mean value are listed in Table 3and 4.The correlation of the parameters with their respective correlation coefficient that are associated with each other is listed in Table 7. Plant height, Plant Biomass, Root Biomass, Leaf area, area, Length of the panicle, Spikelets per panicle, Days of Maturity and Grain yield per plant are found to be significantly correlated (P-value > 0.5) with each other. The plant height was significantly correlated (P=0.560), panicle length (0.868), Spikelets per panicle (0.561), plant biomass (0.651) and days of maturity (0.746). Tillers per hill were found to be significantly correlated to root biomass (0.612), panicle length (0.54) and days of maturity (0.655). On the other hand, Plant biomass was significantly correlated to root biomass (0.66), leaf area (0.605), panicle length (0.611), spikelets per panicle (0.574), grain yield per plant (0.603) and days of maturity (0.566). Similar correlation between the morpho-physiochemical characters was found in a study conducted by Touhiduzzaman et al (2016) [19]. These parameters are significantly dependent upon each other such as if plants will be tall they will acquire proportionate amount of biomass in the shoot as well as the root. While, when leaf area is large plants can absorb more amount of sunlight that will help them to increase plant biomass and plant height. This requires more number of days to attain maturity and thus, this in turn will increase the yield of the grains per plant.

Effect of Temperature and Humidity on the morphological characters:-
Paddy is the plant of the Kharif season that requires humidity and optimum temperature as it attains maturity. Student's t test was done to find the co-dependence of the important morphological parameters upon temperature and humidity. The P value respective to each codependence of each parameters on temperature and humidity is listed in the Table 8. In this study, paddy was grown in two regions which have considerable difference in the temperature and humidity. The Maximum and minimum temperatures and humidity in the environment at the different stages of rice plant development are listed in Table 1. Uttar Pradesh has pleasant temperature and high humidity ideal for paddy cultivation. Haryana is comparatively dry with temperature higher to that in the region of Uttar Pradesh. In accordance to this, morphological parameters such as plant height, plant biomass, root biomass, root length were significantly correlated with the temperature and humidity that plants are subjected to. However, parameters such as leaf area, panicle length, spikelets per panicle, grain yield and weight and days taken by the plant to attain the maturity were found to be negatively correlated with the temperature and humidity. As the temperature rises and humidity is less the leaf area reduces so as to minimize the rate of water evaporation from the surface. Also, the chlorophyll content in the leaf decreases that affects the rate of photosynthesis. As described in the previous section reduced leaf area will affect the length of the panicle, spikelets per panicle and thus grain yield and weight that are found to be reduced in the region of Haryana with higher temperature and lower humidity compared to Uttar Pradesh region.
Effects of temperature and humidity on biochemical parameters and their association of with the morphological parameters:-As found in the Table 9, the biochemical parameters such as total protein and total sugar content in the seeds was found to be positively correlated with chlorophyll content in the leaves, plant biomass, root biomass, spikelets per panicle, panicle length and grain yield per plant. However, with the increase in the temperature and decrease in humidity the total protein in the grains increases as the plants increase in size while the total sugar content decreases as amount of food stored in the grain is reduced. This result positively correlates the fact that as the grain yield per plant deceases, the total sugar in the seeds decreases that reduce the weight of the seeds.

Conclusion:-
The statistical studies clearly explain a significant correlation (P > 0.05) between the morpho-physiochemical characters of rice. Student's t test showed that the climatic temperature and the humidity have direct effects on the rice plant growth, gain yield and quality. The six out of seven rice varieties namely; VB-21, Vb-22, VB-23, VB-24, PB-1509, PB-1121 have aroma of Basmati and presence of fgr gene in the genome while Sambha Sub-1 is a nonbasmati variety. VB-21, VB-24, PB-1509, PB-1121 are among the rice varieties that show higher grain yield per plant and grain quality in both the regions of Northern India despite of climatic variations observed. While VB-21 and VB-24 shown higher grain yield as these can withstand higher temperature and lower humidity. These varieties can thus be used by the farmers in differential climatic regions of Northern India for Basmati production.