WO2018092155A1

WO2018092155A1 - A non-gmo rice variety with high resistance starch and dietary fibre

Info

Publication number: WO2018092155A1
Application number: PCT/IN2017/050535
Authority: WO
Inventors: Selvakumar GURUNADHAN; Venkataramanana MUDILI
Original assignee: Lorven Biologics Pvt. Ltd.
Priority date: 2016-11-16
Filing date: 2017-11-16
Publication date: 2018-05-24

Abstract

The present invention provides a rice variety - LRO-23 which is a non-GMO giving better yields; having high dietary fibre (DF), High resistance starch, and Less rapidly digestible starch (LRDS); and having good cooking quality with appealing aroma and taste. The present rice variety - LRO-23 is developed, using forward genetics approach. Identification of the mutant line with desired qualities through Bio-chemical analysis was carried out. The present rice variety owing to its high dietary fibre (DF), Resistant starch (RS) and less rapidly digestible starch (LRDS) content could be really useful for the people suffering with diabetes in low and middle income countries in meeting their dietary needs.

Description

FIELD OF INVENTION:

This present invention relates to bio-fortification of the cereals to manage the health risks associated with diabetes. More particularly, it relates to a rice variety with high resistance starch and dietary fibre and process of developing the same.

BACKGROUND OF INVENTION: Cereals are staple food source in human life and it provides half the calories in human diet. Among the cereals, rice {Oryza sativa L.) is the most notable crop and staple food of over half the world's population. 'Rice is life' for human beings especially in the Asian subcontinent, where 90% of world's rice is grown and consumed by 60 % of population (Khush and Virk, 2000).

Rice grain releases energy rapidly and thus has a relatively high glycemic response compared with other starch based foods (Chassy et al., 2008) and very limited efforts have been made to improve the health benefits of rice by reducing its glycemic index (Gl) which is interlinked with Resistance starch (RS) and Dietary Fibre available in the cereal grains. The level of RS in available cooked rice grain is 3-3.5% and this level is insufficient to meet the demand of 20g per capita intake per day. It is essential to increase the RS level to 8-8.5% so that the demand will be met by consuming 250-300g cooked rice per day and fibre level 1 .25% to 8% for sustained energy release. Hence, this can be noted as a major drawback in the case of diabetic people who needs the slowly digestible starch thus to control the blood glucose levels and to maintain the body homeostasis. According to the World Health Organization (WHO), diabetes is currently one of the biggest health concerns globally. India ranks among top 3 countries with diabetic population. The current national diabetes prevalence is 8.6% in India, with more than 1 million annual diabetes-related deaths in the 20 to 79 age group. Burden of diabetes has shown a progressively increasing trend in India as evidenced by the upsurge of the diagnosed burden from approximately 62.4 million in 201 1 to 66.8 million in 2014. Moreover, the projected prevalence for the year 2030 was measured to be 79.4 million. Recent studies have shown that the prevalence of diabetes among adults has reached approximately 20% in urban populations and 10% in rural populations. Because of the considerable disparity in the availability and affordability of diabetes care, as well as improper diet and nutrition practices, the glycemic outcome in treated patients is far from ideal.

One of the major reasons for the high prevalence of the Type 2 diabetes is the consumption of carbohydrate rich foods capable of raising the blood sugar concentration. The concept of Gl was introduced by Jenkins et al., 1981 as a quantitative indicator of the ability of carbohydrates to raise blood glucose in relation to time. Foods that raise blood sugar content quickly have high Gl values. Conversely, foods that raise blood sugar content slowly have low Gl values (Gelencser, 2009). The Gl consists of a scale from 1 to 100, indicating the rate at which 50 grams of carbohydrate in a particular food is absorbed into the bloodstream as blood-sugar (Jenkins et al., 1981 ).

Nevertheless, planning and achieving a proper diet for diabetic patients is the mainstay in clinical strategy in the diabetic management. Hence, the global trend in increasing levels of diabetes along with obesity warrants an alteration in the starch fractions of grains to make them into a medically acceptable food (Morrell et al., 2010). Even though the carbohydrate portion measured is available or digestible, the Gl measures the impact of all carbohydrates on blood glucose levels without correcting for available vs. non-digestible carbohydrates (e.g. resistant starch (RS), dietary fibre). The Gl values of foods are grouped into low Gl (<55), medium (55-70), and high (>70) (Miller et al, 1992). At this junction, the global need of value added cereals to manage the profound risk of health due to low availability of dietary fibre, resistant starch, micronutrients can be addressed alternatively by the way of external fortification of micronutrients and other value added ingredients like dietary fibre etc., to cereals based food products. However, these products may not be accessible to the people who situated at low and middle income countries due to the high prices and low availability. Other alternatives also reported by increasing the soil fertility in terms of increasing the micronutrient availability of the plants. However, these methods are not viable for large-scale applications where the agriculture practices were diverse among the agro-climatic regions of the country.

Hence, to address the problem of high Gl of rice, the viable solution will be to increase the fraction of resistant starch (RS) in the grains (Rahman et a/., 2007). RS is similar to dietary fibre with respect to its three major effects when included in the diet: dilution of dietary metabolizable energy, a bulking effect similar to non-fermentable fibre, and fermentation to short-chain fatty acids and increase in expression of Peptide YY (PYY) and glucagon-like peptide (GLP)-1 in the gut (Englyst et al., 1992; Higgins, 2004; Nugent, 2005). RS that has physiologic effects similar to fibre is of utmost importance in rice based diet. Understanding the genetic control of Gl and RS in rice is of utmost importance for enhancing the nutritional quality of rice. Research on RS content in rice assumes considerable significance given the dramatic increase in the incidence of type II diabetes and colo- rectal cancer in South East Asian countries that are increasing through the adoption of "western" diets.

Starch may be divided into three categories based on digestibility and absorbability of the breakdown products - rapidly digestible starch (RDS), slowly digestible starch (SDS) and resistant starch (RS) (Englyst and Hudson, 1996; Englyst and Hudson, 1997). The amylose:amylopectin ratio of the starch source is a determining factor in the formation of RS. High amylose maize is more resistant to enzymatic hydrolysis compared to its counterpart amylopectin (Leeman et al, 2006; Lehman and Robin, 2007) and has been used commercially to increase the RS content of processed foods. Physiological factors such as mastication influence the particle size of the starchy food. Large particles travel faster whereas increased chewing results in increased digestibility (Topping etal., 2003). Rice goes through a variety of processes before it is ready for cooking. After harvesting and drying, the seeds are run through a rice huller/husker for milling to remove the outer grain husks which gives the brown rice. Most people are accustomed to white rice rather than brown rice and the steps to turn brown rice into white rice leads to loss of almost 75 per cent of the dietary fibre content. Hence, decreasing the Gl of rice grains by decreasing its dietary fibre content in polished grain is an inevitable process.

Rice, being one of the primary dietary sources of carbohydrates worldwide, is of particular interest in starch digestibility. A better way to measure the digestibility of carbohydrates is the glycemic index (Gl). This index measures the changes in blood sugar levels after we eat a particular food. Foods that cause large increases in blood sugar levels have a high Gl, while those that don't cause large increases have a low Gl. Typically, foods that contain sugars rapidly boost blood sugar levels and so have a high Gl. Starchy foods are digested more slowly and tend to have a lower Gl. The low Gl foods are beneficial in management of diabetes.

The glycemic response of rice is known to be relatively high compared to other starchy foods. Previous reports on rice starch digestibility confirmed that rice should generally be classified as a high glycemic index food (Bjorck, 1996; Jenkins et al., 1984; Miller et al., 1992). There is a huge global demand for value added cereals especially rice to manage the profound risk of health due to higher consumption and low availability of dietary fibre, resistant starch and micronutrients. Unfortunately, the available rice is very low in nutritional quality, thus lead to serious health problems like diabetes, obesity and micronutrient deficiency. There are millions of people suffering with diabetes globally. Every country, including India, is spending billions of dollars for management of diabetes. Also, there is a significant chunk of people suffering with diabetes are living in low and middle income countries who has no option but to eat the rice as staple food.

Hence, there is an unmet need to develop value added cereals, including rice, with natural bio-fortification so as to cope with the needs of desired nutrient quality to manage the health risks associated with diabetes.

SUMMARY OF THE INVENTION

The present invention discloses a new non-GMO rice variety having low glycemic index and process of developing the same.

In one embodiment of the present invention, a rice variety - LRO-23 is disclosed. The present rice mutant (LRO-21 ) is a non-GMO giving better yields; having high dietary fibre (DF), High resistance starch, and Less rapidly digestible starch (LRDS); and having good cooking quality with appealing aroma and taste.

In another embodiment of the present invention, a process of developing the rice variety - LRO-23 is disclosed. The present rice variety - LRO-23 is developed, using forward genetics approach. Identification of the mutant line with desired qualities through Bio-chemical analysis was carried out. Developed value added rice variety prototype was validated under field conditions and its growth pattern, yield as well as desired qualities.

The present rice variety owing to its high dietary fibre (DF), Resistant starch (RS) and less rapidly digestible starch (LRDS) content could be really useful for the people suffering with diabetes in low and middle income countries in meeting their dietary needs.

The foregoing has outlined, in general, the various aspects of the invention and is to serve as an aid to better understanding the more complete detailed description which is to follow. In reference to such, there is to be a clear understanding that the present invention is not limited to the process or application of use described and illustrated herein. It is intended that any other advantages and objects of the present invention that become apparent or obvious from the detailed description or illustrations contained herein are within the scope of the present invention.

BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS The aforementioned aspects and other features of the present invention will be explained in the following description, taken in conjunction with the accompanying drawings, wherein:

Figure 1 shows position of mutation(s) identified with respect to genes SS I, SS I la., and SS Ilia, according one embodiment of the invention.

Figures 2A and 2B are graph plots depicting melting enthalpy of starch sample of mutant rice variety LRC and LR1 , according one embodiment of the invention.

The drawings described herein are for illustration purposes only and are not intended to limit the scope of the present disclosure in any way.

DETAILED DESCRIPTION OF THE INVENTION The embodiments of the present invention will now be described in detail with reference to the accompanying drawings. However, the present invention is not limited to the embodiments. The present invention can be modified in various forms. Thus, the embodiments of the present invention are only provided to explain more clearly the present invention to the ordinarily skilled in the art of the present invention.

The specification may refer to "an", "one" or "some" embodiment(s) in several locations. This does not necessarily imply that each such reference is to the same embodiment(s), or that the feature only applies to a single embodiment. Single features of different embodiments may also be combined to provide other embodiments.

As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless expressly stated otherwise. It will be further understood that the terms "includes", "comprises", "including" and/or "comprising" when used in this specification, specify the presence of stated features, integers, steps, operations, elements and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. As used herein, the term "and/or" includes any and all combinations and arrangements of one or more of the associated listed items.

Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

The present invention discloses a new non-GMO rice variety having high dietary fibre (DF), Resistant starch (RS) and less rapidly digestible starch (LRDS) content thus leading to sustained and slow release of glucose into blood stream while digestion which may confer low glycemic index (Gl). This solves the problem of low availability of dietary fibre, resistant starch and micronutrients in normally consumed rice and owing to these bio- fortifications the present non-GMO rice variety could be a suitable choice for the huge population suffering with serious health problems like diabetes, obesity and micronutrient deficiency.

The present invention also discloses a process of developing the said rice variety. The rice variety with such features could be really useful for the people suffering with diabetes in low and middle income countries in meeting their dietary needs.

In an embodiment of the present invention, a rice mutant/variety (LRO-21 ) having high dietary fibre (DF), Resistant starch (RS) and less rapidly digestible starch (LRDS) content is disclosed. The new rice variety (LRO- 21 ) is developed from the existing rice (BPT-5246) by adoption of mutation breeding and biochemical Screening approaches. The rice variety (LRO- 21 ) so developed owing to its high dietary fibre and RS leads to slow and sustained release of glucose (Low Gl) into blood stream during digestion.

Dietary fibre plays a crucial role starch metabolism and health management in humans. The high content of dietary fibre will contribute to the healthy life style and make people less prone to the chronic health risk like diabetes and obesity. It contributes to the colonization of probiotic micro flora in colon thus helps the immune system in fighting against colon cancers. Dietary fibre content in the rice mutant/variety (LRO-21 ) was determined in comparison with the wild type rice. It was found that mutant line was recorded with 5-fold increment in the dietary fibre content than the wild type un-mutated grains.

Normally the glycemic response of rice is known to be relatively high compared to other starchy foods. Previous reports on rice starch digestibility confirmed that rice should generally be classified as a high glycemic index food (Bjorck, 1996; Jenkins et al., 1984; Miller et al., 1992). However, the present invention of the rice mutant/variety (LRO-21 ) has less glycemic response thereby low glycemic index (Gl) as compared to normal rice.

Hydrolysis Index is direct measure of Glycemic Index. The grains having low HI are preferably fall under category of low-GI foods. In the present invention, the HI value for the grains of rice mutant/variety (LRO-21 ) was determined as 40, whereas HI value for wild type rice grains was found to be 76.

Bioinformatics analysis - Rice whole genome sequencing

Present invention, to elucidate the molecular mechanism behind the differential properties of the mutant rice comparison with wild type rice. Bioinformatics analysis of starch metabolic pathway genes including GBSS I, SS I, SS lla, SS Ilia, SBE la, SBE Mb, and GPT was carried out.

GBSS1 plays an important role in the starch biosynthesis, has no isoforms and is involved in synthesis of short chains (<10 DP). Abundance of short chains and shortage of long chains decrease the gelatinization temperature.

SSI and SSIIIa are the major enzymes in the developing rice endosperms and the activity of SSI is higher than that of the SSIIIa enzyme. The N- terminal extension of SSI is important for its proper binding with the starch granules (Imparl Radasevich et al., 2003). Amylopectin chains are synthesized by the coordinated actions of SSI, SSIIa and SSIIIa genes; and the activity of SSI is reported to be higher than that of the SSIIa and SSIIIa enzymes (Fuzita et al., 2006). The SSIIa gene plays a specific role in the synthesis of the medium size glucon chains (12-24 DP) by elongating short chains (<10 DP). Although, SSIIa gene is a minor contributor to the total SS enzyme activity in the endosperm as compared to SS-I and SS-I 11 genes, but loss/down regulation of SSIIa gene has the major impact on quantity and composition of starch in rice endosperm (Yu et al., 201 1 ). SSIIIa in rice, specifically expressed in the developing rice endosperm (Hirose and Terao, 2004; Ohdan et al., 2005; Fujita et al., 2007; Zhang et al., 201 1 ). The deficiency in the SS-llla, the second major SS enzyme in the developing rice endosperm, affected the structure of amylopectin, amylose content and the physicochemical properties of the starch granules in two ways: directly by the SSIIIa enzyme deficiency itself, resulting in the chain length increase (DP 10-15 and DP 20-25) and indirectly by the enhancement of both the SS-I and GBSS-I gene transcripts (Fuzita et al., 2007). The GBSS-I is encoded by waxy locus and is involved in the synthesis of long amylopectin (CL 85-180) chains (Takeda et al., 1987; Wang et al., 1995; Denyer et al., 1996; Cai et al., 1 998; Fu and Xue, 2010) in higher proportion, resulting in the absence of very long chains in the waxy rices (Hizukari et al., 1989). After elongation of the glucal chains by the SS enzyme, another enzyme namely SBE Mb generates ά-(1 -6) linkages by cleaving internal ά- (1 -4) bonds and transferring the released reducing ends to C6 hydroxyls to form the branched structure of the amylopectin molecule. SBEIIb proteins transfer shorter chains and show a higher affinity towards amylopectin as compared to the SBEIa, which show higher rates of branching with amylose (Guan and Preiss, 1993; Takeda et al., 1993; Tanaka et al., 2004; Tetlow et al., 2004; Tetlow, 2006; Nakamura et al., 2010). Down regulation or elimination of SBEIa gene activity showed minimal effects on the starch synthesis (Satoh etal., 2003b). GPT1 (Glucose-6-Phosphate Translocator) has a major association with resistant starch production in rice and it is highly associated with resistant-retrograded starch and amylose content (KM Ardashir et al. 2012).

Selected mutant rice lines were showed in filed and leaf samples were collected for DNA isolation. Isolated DNA was quantified and assessed for its purity through Agarose gel electrophoreses. Whole genome sequencing of rice was carried out by using NGS sequencing approach. Mutations in the target metabolic pathway genes including GBSS I, SS I, SS I la., SS Ilia, SBE la, SBE Mb, and GPT were identified in comparison with wild type samples using different bio-informatics software programmes. Discovered sequence variants were analyzed by the PARSESNP program (http://www.proweb.org/parsesnp/), which provides information on the location along with the details about amino acid changes and location of the amino acid changes and severity of mutations and provides information on the creation or loss of restriction sites caused by the induced polymorphisms. SIFT (Sorting Intolerant From Tolerant) is a sequence homology-based tool that sorts intolerant from tolerant amino acid substitutions (Sim et al., 2012) and predicts whether an amino acid substitution in a protein will have a phenotypic effect. SIFT is based on the premise that protein evolution is correlated with protein function. Positions important for function should be conserved in an alignment of the protein family, whereas unimportant positions should appear diverse in an alignment. SIFT score ranges from 0 to 1 . The amino acid substitution is predicted damaging is the score is <= 0.05, and tolerated if the score is > 0.05.

The rice mutant/variety (LRO-21 ) comprises the missense type mutations (substitution mutation resulting in an alternate codon, altering the amino acid at this position only) in the starch metabolic pathway genes SS I, SS lla and SS Ilia. The corresponding changes that has occurred in the nucleotide sequence(s) and resultant peptide sequence(s) as a result of the mutations are tabulated in Table 1. Figure 1 illustrates the position of mutation(s) identified on the corresponding genes encoding for enzymes starch synthase I, starch synthase lla and starch synthase Ilia, according to one embodiment of the present invention.

Table 1

It can be inferred from the Table 2, the gene starch synthase I (SS I) has a change that has occurred in the nucleotide sequence as a result of the mutation is c.220G>A (Substitution, position 220, G- A) while the change occurred in the peptide sequence as a result of the mutation is p.A74T (Substitution - Missense, position 74, A- T). Likewise, the other mutations listed in the Table 1 for the starch synthase lla (SS I la.) and starch synthase Ilia (SS Ilia) can be understood.

In another embodiment of the present invention, a process of developing the rice variety - LRO-23 is provided. The present rice variety - LRO-23 is developed, using forward genetics approach by subjecting rice variety of BPT-5246 to EMS mutagenesis. Identification of the mutant line with desired qualities through Bio-chemical analysis was carried out. DNA from the mutant line was extracted and whole genome sequencing of selected mutant line of the rice was carried out by using NGS sequencing approach. Mutations in the target metabolic pathway genes including GBSS I, SS I, SS lla, SS Ilia, SBE la, SBE Mb, and GPT were identified in comparison with wild type samples using different bio-informatics software programmes. Discovered sequence variants were analyzed to find out location along with the details about amino acid changes and location of the amino acid changes and severity of mutations. The selected non-GMO rice variety prototype based on the above analysis was validated under field conditions and its growth pattern, yield as well as desired qualities were confirmed.

One of the embodiments of method of developing the rice variety - LRO-23 uses classical mutational breeding approach.

Benefits of the consumption of the rice mutant/variety (LRO-21 ) are as following:

1 . Helps control blood sugar levels- In people with diabetes, the dietary fibre present in the rice can slow down the absorption of sugar and help improve blood sugar levels. A healthy diet that includes insoluble fibre may also reduce the risk of developing type 2 diabetes. This could result in a lower energy intake at the following meal and better body weight regulation also.

2. Normalizes bowel movements- Consumption of the rice mutant/variety increases the weight and size of stool and softens it. A bulky stool is easier to pass, decreasing your chance of constipation. The dietary fibre in people suffering with loose and watery stools, may help to solidify the stool because it absorbs water and adds bulk to stool. 3. Helps maintain bowel health- A high-fibre diet may lower the risk of developing haemorrhoids and small pouches in colon (diverticular disease).

4. Lowers cholesterol levels- Soluble fibre found in the rice mutant/variety may help lower total blood cholesterol levels by lowering low-density lipoprotein, or "bad," cholesterol levels. Studies also have shown that high-fibre foods may have other heart-health benefits, such as reducing blood pressure and inflammation. 5. Aids in achieving healthy weight- High-fibre foods, such as the present rice mutant/variety, tend to be more filling than low-fibre foods, so people are likely to eat less and stay satisfied longer. And high-fibre foods tend to take longer to eat and to be less "energy dense," which means they have fewer calories for the same volume of food.

The present invention is explained further in the following specific examples which are only by way of illustration and are not to be construed as limiting the scope of the invention. EXAMPLES

Example 1 : Generation of mutant population The rice variety of BPT-5246 was subjected to EMS mutagenesis. LD50 values were calculated using kill curve analysis. Upon successive LD50 value determination, a total of 1410 M2 families were raised to screen the mutant with desirable properties including, rapidly digestible starch (RDS) content, fibre content and resistance starch content after processing the rice.

Example 2: RDS analysis

RDS is rapidly digested and absorbed in the duodenum and proximal regions of the small intestine leading to a rapid elevation of blood glucose and usually a subsequent episode of hypoglycemia. The RDS is the digested portion within the initial 20 minutes of digestion. Rice having more of RDS leads to immediate raise in blood glucose generates high stress on the regulatory system of glucose homeostasis (Ludwig, 2002) that can further lead to cell, tissue, and organ damages (Brownlee, 2001 ). Less RDS leads to slow release of glucose while digestion and its controls the blood glucose level without any drastic change. By using AAOC method and Megazyme kit protocol, RDS percentage in the selected mutant lines was determined in comparison with wild type control grains (non-mutant). The RDS of the mutant was determined as 9% compared to its wild type which is recorded as 22%.

Example 3: Resistant Starch and Fibre analysis

The resistant starch (RS) content was estimated based on the method suggested by Goni et al. (1996) using the Megazyme kit. The kit was procured from M/s Megazyme International Ireland Ltd., Bray Business Park, Bray, Co. Wicklow, Ireland.

100 mg rice flour sample along with 100+1 mg RS control [52.5 per cent dry weight basis (dwb) RS] provided in the kit were taken in screw cap tubes in duplicates and gently tapped to ensure no sample adhered to the sides of the tube. 4 ml of pancreatic a-amylase (3 Ceralpha Units/mg, 10 mg/ml) containing amyloglucosidase (AMG) (3 U ml^-1) was added to each tube. The tubes were tightly capped, dispersed thoroughly on a vortex mixer, and attached horizontally in a shaking water bath aligned in the direction of motion. The tubes were incubated at 37°C with continuous shaking (200 strokes minute^-1). After shaking for exactly 16 hours, the tubes were taken out of the water bath, uncapped, and the contents were treated with 4.0 ml of ethanol (99 per cent) with vigorous mixing on a vortex mixer. After this, the tubes were centrifuged at 1 ,500 x g (approx. 3,000 rpm) for 10 minutes (non-capped). The supernatant was carefully decanted and the pellet re- suspended in 2 ml of 50 per cent ethanol and agitated using a vortex mixer. A further 6 ml of 50 per cent ethanol was added; the tubes were mixed and centrifuged again at 1 ,500 x g for 10 minutes. Again, the supernatant was decanted and the suspension and centrifugation steps were repeated once more. Finally, the supernatant was decanted and the tubes inverted on absorbent paper to drain excess liquid. A magnetic stirrer bar (5 x 15 mm) was added to each tube, followed by 2 ml of 2 M KOH solution. The pellet was re-suspended (and the RS dissolved) by stirring for about 20 minutes in an ice/water bath over a magnetic stirrer. Then, 8 ml of 1 .2 M sodium acetate buffer (pH 3.8) was added to each tube with stirring on a magnetic stirrer. Immediately, 0.1 ml of AMG (3300 U ml ¹) was added, the contents were mixed well under a magnetic stirrer, and the tubes were placed in a water bath at 50°C. The tubes were incubated for 30 minutes with intermittent mixing on a vortex mixer. Then they were directly centrifuged at 1 ,500 x g for 10 minutes. The final volume in each tube was approximately 10.3 (+0.05) ml. For the RS control, the contents of the tubes were transferred into a 100 ml volumetric flask and then diluted to 100 ml with distilled water. From this, 10.3 ( + 0.05) ml aliquot was taken and transferred into a screw cap tube. This was centrifuged together with the samples. From each tube, 0.1 ml aliquot (in duplicate) of the supernatant was transferred into glass test tubes, added with 3.0 ml of GOPOD reagent, and mixed well using a vortex mixer. A reagent blank was prepared by mixing 0.1 ml of 0.1 M sodium acetate buffer (pH 4.5) and 3.0 ml of GOPOD reagent. Glucose standards were prepared (in quadruplicate) by mixing 0.1 ml glucose (1 mg ml ¹) and 3.0 ml GOPOD reagent. The samples, blank and standards were incubated for 20 minutes at 50 °C, cooled, and the spectrophotometer was set to 0 using the reagent blank. The absorbance was measured at 510 nm against the reagent blank.

Table 2 shows the % of the Resistant Starch and Fibre content in the rice variety LRO-21 .

Table 2

Example 4: Gelatinization temperature

Gelatinization temperature reveals the quality of the rice in terms of cooking and texture. Present invention, Differential Scanning Colorimetric analysis was used to analysis the disassociation curve for the high fibre mutant as well as its wild. Results showed that, there is a significant difference in the Gelatinization temperature between mutant grains and wild type grains. The Gelatinization temperature for the mutant grains was determined as >100 °C whereas for wild type it was determined as 75 °C. Example 5: Analysis of Starch particle size

Starch particle size was assessed using bright field microscope. Starch particle size of the mutant rice was measured smaller than the wild type grains.

Example 6: Melting enthalpy determination

The melting enthalpy of each starch sample was assessed using a differential scanning calorimeter (Chung et al., 2006). A portion (about 10 mg) of the flour samples was transferred to DSC sample pans and 20 μΙ of water added. Then the sealed pans were heated from 30 °C to 130 °C, at a rate of 2 °C/min.

The result revealed during the DSC analysis, a new peak has been identified in the mutant high fibre line LR1 (refer Figures 2A and 2B). It is confirmed from the previous reports are available, the peak present in the LR1 due to the lipid and amylose complexion of the rice grains. In starch granules, amylose-lipid complex has been shown to retard granule swelling during heating in excess water, which reduces the enzyme accessibility to hydrolyze the starch granules (Cui & Oates, 1999; Tester & Morrison, 1990).

The present rice mutant (LRO-21 ) is a non-GMO giving better yields; having high dietary fibre (DF), High resistance starch, and Less rapidly digestible starch (LRDS); and having good cooking quality with appealing aroma and taste.

While the invention has been described with reference to preferred embodiments thereof, it will be apparent to one skilled in the art that various changes can be made, and equivalents employed, without departing from the scope of the invention.

Claims

We Claim:

1 . A non-GMO rice variety with high resistance starch and dietary fibre, comprises:

mutations in starch synthase I (SS I), starch synthase lla (SS I la) and starch synthase Ilia (SS Ilia) genes, wherein

SS I having mutation at gene position G220A in nucleotide sequence and thereby having amino acid substitution A74T in encoded peptide sequence,

SS lla having mutations C264G, C478A and A559C in nucleotide sequence and thereby having amino acid substitutions D88E, P1 17T and T144P, respectively in encoded peptide sequence, and

SS Ilia having mutation T8896A in nucleotide sequence and thereby having mutation H1460Q in encoded peptide sequence.

The non-GMO rice variety as claimed in claim 1 , wherein resistant starch content of the non-GMO rice variety ranges between 7.2 - 7.6 %.

The non-GMO rice variety as claimed in claim 1 , wherein fibre content of the non-GMO rice variety ranges between 8.3 - 8.9 %.

The non-GMO rice variety as claimed in claim 1 , wherein the non- GMO rice variety is developed from an existing rice (BPT-5246) by adoption of mutation breeding and biochemical screening approaches.