Morpho-Physiological Phenotyping of Fifteen Farmers Rice Varieties and Assessment of Grain Quality

The present study high-lighted on the yet-explored extensive analyses of eating and cooking quality parameters in fifteen indigenous rice varieties. As rice is a vital staple crop globally that can feed one-third world’s population solely, it is an imperious task to develop research for enhancement of its nutritional as well as eating and cooking quality (ECQ). The exploitation of identified nutrient-enriched rice germplasm for breeding application is the easiest way to get better quality rice. Satabdi IET-4786 (SAT) appeared to be very demanding by virtue of having sufficient level of amylose which is considered as key determinant of grain quality. The importance of Sabita (SAB), with regards to higher content of amylose and maltose with higher α-amylase activity and of Dudheshwar (DUD) with respect to protein and total free amino acid content in rice grain was also significant. Here we analyzed fifteen different local rice germplasms including aromatic and some high yielding cultivars to identify some qualitative as well as some quantitative traits related to nutrition and consumers’ acceptance. The knowledge gained from this study will largely provide a path direction for the making a proper decision of considering germplasms for large-scale cultivation and dietary consumption.


Introduction
Rice is a main cereal crop contributing to the world's most caloric consumption and instant nutrition. ¹ Besides the yielding quantities, the determination of rice grain quality is also important because it can meet up the consumer's demand. Rice grain quality (RGQ) includes milling, storage, cooking, eating, nutritional and market quality of rice grain RGQ can be judged based on physical properties (size, shape, color), biochemical composition (amylose content, gelatinization temperature, viscosity, and aroma), and nutritional properties (micronutrients, protein, amino acid content). Rice is consumed in milled or polished form, subtracting most of the nutritional components. This milled rice contains only starchy endosperm having low sugar, low fat, low cholesterol, trace amount of gluten and enough portion of protein containing all eight essential amino acids which make a perfect diet food for hypertension patients advised to take salt-limited and low fiber diet. ² Among the carbohydrates within the rice grain, starch is most abundant and important component due to its smallsized granules and hypoallergic effects. ³ The most prominent endo-hydrolase enzyme within the grain is α-amylase, which helps in the rapid degradation of starch into a soluble substrate, the available form for utilization of other enzymes. Another salient source of carbohydrate is sucrose which enzymatically broken down to produce hexoses (mainly glucose) which are considered to power and support the growth of the sink tissues. ⁵ Starch is considered a vital reserve food in plants.
Carbohydrates produced by photosynthetic tissues during the different growth stages are either transported to other organs in form of soluble sugars or accumulated in leaf tissues. It has been reported that the ability of plants to recover from most abiotic stresses normally increases with increasing concentrations of photosynthetic assimilates in plant tissues during or after stress. ⁴ Rice grain has a great biological value for providing energy and calories in high amount. As major constituent, carbohydrate in form of complex polysaccharides present in rice grain, and these are broken down into glucose providing the most vital energy source for human brain. Besides carbohydrates, there are some minor constituents within the starch present either at the surface or inside the granule itself. Rice starch granule contains approximately 1% of lipids and ~0.25-0.30% of proteins. ⁵ The present investigation was taken into account for qualitative assessment of some local rice cultivars grown under natural conditions. This precised us to investigate the eating and cooking qualities viz. amylose content, total soluble sugar, protein, free amino acid content, α-amylase activities etc in the selected local rice varieties. Statistical analysis was performed to reveal significant variance of data from biochemical analyses. The collected mature seeds were categorized into two subtypes, viz., aromatic and high-yield cultivars.

Growth Condition
For biochemical assessment at seedling stage, seeds were surface sterilized with 1% sodium hypochlorite solution and imbibed in distilled water for 24 h. ⁶ Then germinated seedlings were raised in soil up to 14 days. Seedlings were maintained in natural conditions (Rice experimental field, University of North Bengal, attitude 26˚84' North, longitude 88˚44' East)⁷ at 28 ± 1˚C, 70-80% relative humidity and 12h day/night photoperiod in july 2022.

Seed Sample Preparation
For biochemical assessment of rice grain, first dehusking the grains from hull of both types, followed by grounding the grains into fine powder using ceramic morter-pestle.⁸ Resulting fine powder stored at -20˚C till further use.

A l k a l i S p r e a d i n g Va l u e / G e l a t i n i z a t i o n Temperature
The gelatinization temperature is estimated based on alkali spreading value (ASV). The alkali spreading value was determined as described. ⁹ In short, six rice kernels of each cultivar were placed in small-sized glass Petri plates (50×42×22 mm) containing 1.7% Potassium Hydroxide (KOH) solution. Kept kernels undisturbed for 24h and then take a reading of kernels disintegration visually. The disintegration of kernels was scored as follows-Score 1: unaffected kernels, score 2: swollen kernel, score 3: swollen kernel with incomplete-narrow collar, score 4: swollen kernel with complete-wide collar, score 5:splitted kernel with complete-wide collar, score 6: dispersed kernel with merged collar, score 7: complete, intermingled dispersed kernel. Gelatinization temperature is inversely related to ASV i.e., low ASV value has corresponded to a high gelatinization temperature.

Gel Consistency
Gel consistency was measured according to the method.¹⁰ Briefly, take 100 mg rice flour into a culture tube (13×100 mm),then add 95% ethyl alcohol containing 0.02% thymol blue and kept them for boiling by adding 2ml of 1(N) KOH. After 8 minutes of boiling, test tubes were left at room temperature for 5 minutes and then placed in the ice bath for a few minutes. Finally, placed the tube horizontally on graph paper for measuring the spreading. GC was scored as follows-Spreading of gel <40 mm (hard), 41-60 mm. (medium), >60 mm. (soft).

Estimation of Amylose by Spectrophotometer
Amylose content is estimated as described by. ¹¹ Briefly, 100 mg grain flour was taken into a volumetric flask and add allow to digest using 9 ml of 1(N) NaOH by heating for 10 minutes in a water bath. After the gelatinization of starch kept the flask at room temperature and made up the volume of up to 100 ml of distilled water. To the 500µl of a test sample, add 5 ml distilled water, 100µl of acetic acid, and 200µl of iodine solution. A dilution was made by adding 4.2 ml of distilled water again. The absorbance of reaction mixture was taken at 620 nm. by using a spectrophotometer (Microprocessor UV-VIS Double Beam Spectrophotometer-2700). The amylose content within the test sample was estimated using pure amylose (Sigma-Aldrich, USA) standard curve.

Estimation of Maltose Content
The content of maltose is estimated according to the method described by. ¹² Breifly,100mg sample was extracted with 80% warm alcohol and taken supernatant after centrifugation at 8000 rpm. Then evaporated the supernatant and made the volume up to 2ml. Add an equal volume of dinitro salicylic acid reagent (containing Na-K-tartrate and NaOH) to the sample extract and leave it in the water bath for 10 minutes. After cooling add 10 ml of distilled water and absorbance was taken at 520 nm. against the reagent blank. The concentration of maltose is calculated from a prepared standard curve of pure maltose (1mg/ml) purchased from Himedia.

Estimation of Total Soluble Sugar Content
The total soluble sugar content is estimated according to the procedure described ¹³ with some modifications. Briefly, a 100 mg sample was boiled with 5ml 0f 2.5 (N) HCl. Then cool down at room temperature, neutralized with the solid form of sodium carbonate until effervescence ceases. Then made volume up to 50 ml and centrifuged at 10000 rpm for 10 minutes. With 1 ml of supernatant add 4 ml of ice cold anthrone reagent. Then absorbance of the reaction mixture was taken at 630 nm. against reagent blank. The concentration of total soluble sugar was estimated from a standard curve using glucose as standard soluble sugar (1mg/ml). The concentration of sugar is calculated as follows-

Estimation of Reducing Sugar Content
The reducing sugar content was estimated using method described by. ¹⁴ Briefly, 100mg sugar sample was extracted using 80% ethanol. Then centrifuged the sample and evaporated the supernatant. Volume made up to 10 ml with distilled water. Finally, the reaction mixture contained 3 ml of sugar sample and 3 ml of dinitrosalicylic acid reagent. After 5 minutes of boiling in a water bath, a reddishorange color developed, lastly add 1 ml of sodiumpotassium-tartrate to the mixture. Then absorbance of the solution was taken at 510 nm. using UV-Vis spectrophotometer. The concentration of reducing sugar is estimated from a standard curve of glucose (Himedia). Calculated as follow.

Estimation of Protein Content
The protein content was estimated using method described by ¹⁵ with some minor modifications. In brief, 1g powder sample was subjected to extraction by 0.1 (M) Phosphate buffer (pH-7.4) for subsequent soluble protein estimation. Then centrifuged at 10000 rpm for 10 minutes and collected the supernatant. The final reaction mixture contained 1 ml of supernatant, 5 ml of solution C (mixture of sodium carbonate and copper sulphate-sodium potassium tartrate), and 500 µl of Folin-ciocalteu reagent. After 30 minutes of dark incubation, absorbance was taken at 660 nm. against blank. The concentration of protein was calculated from the standard BSA (bovine serum albumin) curve having a stock concentration 1mg/ml.

Estimation of Total Free Amino Acids
Total free amino acid content was estimated using the method described by. ¹⁶ Briefly, 1g of seeds were homogenized in 80% ethanol. Centrifuged the sample and supernatant was collected. The final reaction volume contained 300 µl of sample extract and 1 ml of ethanolic ninhydrin reagent. After 30 minutes of boiling water bath incubation, a purple color was developed. The absorbance of sample was taken at 570 nm. using UV-Vis spectrophotometer.

Qualitative Assay for α-Amylase Activity
For qualitative assessment of α-amylase activity, we used starch agar plate assay using embryo-less half seeds as described by ¹⁴. Briefly, rice seeds are surface sterilized according to method. ¹⁵ And then incubated with distilled water at 28±2˚ C for 48h. after that seeds were cut transversely and placed embryo-less halfseed on 2% agar plate. The agar plate contained soluble potato starch 0.2%, and 20 mM of CaCl 2 , and 20 mM of sodium succinate (pH-5.0). Petri plates were incubated at 28˚C for 48h. After the incubation period plates were flooded with KI solution, and after 5 minutes, due to the reaction between iodine starch the agar-plate turns blueishpurple except for the region of hydrolysis of starch by α-amylase producing halo-zones. The diameter of halo zones was measured using millimeter scales which corresponds to α-amylase activity.

Result and Discussion Amylose Content
Amongst the samples of examined cultivars, the highest amylose content is registered in SAT (figure 1), while the lowest in HAR (10-folds lower than SAT, following the trend.

Maltose Content
The maltose content is registered to be highest in GIT (figure 1), and lowest in DUD and AMA (1.8-fold lower than AMA). The trend followed is. Considering the grain quality of above selected cultivars, the free amino acid content is an important anti-oxidant molecule. As in cultivar Amalmona (AMA), Sabita (SAB), and Dudheshwar (DUD) higher free amino acid content is directly correlated with protein content. ⁸ Alkali spreading value (ASV) is another important determinant in ECQ supported by. ¹⁸ ASV is the measure of tendency to alkali digestion. This ASV is inversely related to gelatinization temperature (GT). ASV is directly related to the presence of short chain amylopectin in grain starch that means short chain amylopectin easily digests the grain under an alkali solution (figure 4), which make its gelatinization temperature high (i.e., cooking time high) as shown by cultivar Dudheshwar (DUD). ²⁰ In cultivar Gitanjali (GIT), a lower gelatinization temperature (GT) value can be seen (figure 4), due to low amylose content, which helps to gelatinize the starch comparatively at lower temperature.³ A similar result can also be seen for cultivars Kartick khas (KAR), Khas dhan (KHA), and Amalmona (AMA).  Considering the eating and cooking quality (ECQ) of rice, gel consistency is an important indicator of ECQ. Gel consistency (GC) measures the coldpaste viscosity of cooked milled rice flour, an index used in distinguishing cooked rice texture of high amylose genotype²¹ ( figure 5). GC is inversely proportional to the amylose content and directly proportional to the amylopectin short chain as shown by cultivar Sabita (SAB) and Sukumar C (SUK). As amylose is known to restrict the gelatinization property of starch molecules, the high amylose containing rice viz. SAT, SUK, DUD, KAL, HAR were shown to have high GT value as well as high gel consistency. The nature of gel consistency is also get affected by amylose content. 22 Upon heating, water molecules entered within the amorphous layer of a starch molecules and double-helical crystalline structures get disrupted. After that, the starch granules undergo hydration, resulting in granule swelling and subsequent solubilization.³ The amylopectin molecules are known to catalyse the starch swelling process or gelatinization whereas amylose restricts it as shown by cultivar GIT and AMA with soft gel consistency i.e., more spreading due to less amylose content.³

GIT > KAN > SAT > SUK > KHA > KAR > MTU > HAR > GOB > KAL > SAB > CHI > RAD > DUD > AMA
If we focused on the α-amylase activity of cultivars it is directly correlated with the conversion of stored carbohydrates²³ into soluble and reducing sugar which subsequently help in seedling growth and better adaptability. ⁴٫¹⁹ On other hand, maltose content is also directly related to the α-amylase activity (figure 6), as α-amylase hydrolyses the polysaccharide starch.⁴

Fig. 6: Measurement of α-amylase activity on agar plate
Currently, the above-mentioned varieties are cultivated only in some specific regions within west Bengal. The knowledge achieved from our above studies with help the farmers as well as the consumers to be aware of taking nutritionally improved rice for their proper nourishment.

Conclusion
Amylose content was high in Satabdi IET-4786 (28%) and lowest observed in Harinakhuri. Alpha amylase activity was high in Sabita (2.63 cm) and lowest in Kartickkhas (0.89 cm). In our study, we provided some vital information regarding the morpho-physiological depictions of local farmer's varieties. This will help plant breeders and rice researchers in near future for developing better, quality varieties.