A method for increasing resistant starch and dietary fibre in rice

Abstract

The present invention discloses mutations in the starch synthase genes associated with enhanced dietary fibre and resistant starch levels in the endosperm of a suitable variety of rice. The dietary fiber and resistant starch are enhanced to an extent to significantly reduce the hydrolysis index values of the rice grains to 35-40%. These rice varieties are in great demand for diabetic population and provide a number of other health benefits such as reduced body weight gain, cardiac health and colon health. As this strategy does not involve the use of genetic manipulation technologies, it can bedirectly employed in the rice breeding programmes without any restrictions.

Description

technical field

[0001] The present invention relates to rice plants with increased expression of dietary fiber and resistant starch. Specifically, the present invention relates to a method for chemically inducing mutations in genes encoding starch synthases in combination with mutations in genes encoding rice starch branching enzymes. By changing the structure of amylopectin, the amylose content, resistant starch As well as increased dietary fiber and decreased hydrolysis index. Background technique

[0002] Cereals, such as rice, are basic food components in the human diet and contain important nutrients such as dietary fiber and carbohydrates. Consumption of dietary fiber is especially important for digestion and has implications for the prevention or treatment of certain diseases such as diabetes, obesity and colon cancer. In general, dietary fiber is defined as the residue of plant material that is resistant to digestion by human digestive enzymes and includes non-star...

Examples

Embodiment 1

[0059] Example 1: RS Assessment Procedure

[0060] RS content was assessed using the Megazyme kit. The kit was obtained from M / s Megazyme International Ireland Ltd, Bray Business District, Wicklow, Ireland. Duplicate 100 ± 1 mg flour samples were taken in screw cap tubes and gently tapped to ensure that no sample stuck to the tube walls. Add 4 ml of amyloglucosidase (AMG) (3U ml -1) of pancreatic alpha-amylase (3 Ceralpha units / mg, 10 mg / ml). Cap the tube tightly so that it is completely dispersed on a vortex mixer and attached horizontally in a shaking water bath aligned with the direction of motion. Tubes were incubated for 16 hours at 37°C with continuous shaking (200 strokes / min). After incubation, tubes were treated with 4.0 mL of ethanol (99%) under vigorous mixing using a vortex mixer. Thereafter, the tubes were centrifuged at 1,500 xg (approximately 3,000 rpm) for 10 minutes (uncapped). The supernatant was decanted carefully and the pellet was resuspended in 8 ml...

Embodiment 2

[0061] Example 2: Degree of Polymerization of Amylopectin Chains

[0062] Pure starch was isolated from all mutants and wild type according to the method described by Lumdubwong and Seib (2000). The amylopectin chain length distribution of isolated starches was analyzed by fluorophore-assisted capillary electrophoresis (FACE) as described by Morell, Samuel and O'Shea (1998). Isolated starch was debranched (2 h at 37°C) using isoamylase (10 U) and labeled with 1-aminopyrene-3,6,8-trisulfonic acid (APTS). FACE was performed using a P / ACE system 5010 (Beckman Coulter, CA, USA) equipped with a 488 nm laser module. Debranched samples were separated using N-CHO (PVA) capillaries (Beckman Coulter, CA, USA) (50 m ID and 47 cm total length) with precombustion windows. Maltose was used as internal standard. Separation was performed at 10°C for 30 minutes. Based on the migration time of maltose, the degree of polymerization (DP) was assigned to the peak.

technical field

[0001] The present invention relates to rice plants with increased expression of dietary fiber and resistant starch. Specifically, the present invention relates to a method for chemically inducing mutations in genes encoding starch synthases in combination with mutations in genes encoding rice starch branching enzymes. By changing the structure of amylopectin, the amylose content, resistant starch As well as increased dietary fiber and decreased hydrolysis index. Background technique

[0002] Cereals, such as rice, are basic food components in the human diet and contain important nutrients such as dietary fiber and carbohydrates. Consumption of dietary fiber is especially important for digestion and has implications for the prevention or treatment of certain diseases such as diabetes, obesity and colon cancer. In general, dietary fiber is defined as the residue of plant material that is resistant to digestion by human digestive enzymes and includes non-star...

Examples

Embodiment 1

[0059] Example 1: RS Assessment Procedure

[0060] RS content was assessed using the Megazyme kit. The kit was obtained from M / s Megazyme International Ireland Ltd, Bray Business District, Wicklow, Ireland. Duplicate 100 ± 1 mg flour samples were taken in screw cap tubes and gently tapped to ensure that no sample stuck to the tube walls. Add 4 ml of amyloglucosidase (AMG) (3U ml -1) of pancreatic alpha-amylase (3 Ceralpha units / mg, 10 mg / ml). Cap the tube tightly so that it is completely dispersed on a vortex mixer and attached horizontally in a shaking water bath aligned with the direction of motion. Tubes were incubated for 16 hours at 37°C with continuous shaking (200 strokes / min). After incubation, tubes were treated with 4.0 mL of ethanol (99%) under vigorous mixing using a vortex mixer. Thereafter, the tubes were centrifuged at 1,500 xg (approximately 3,000 rpm) for 10 minutes (uncapped). The supernatant was decanted carefully and the pellet was resuspended in 8 ml...

Embodiment 2

[0061] Example 2: Degree of Polymerization of Amylopectin Chains

[0062] Pure starch was isolated from all mutants and wild type according to the method described by Lumdubwong and Seib (2000). The amylopectin chain length distribution of isolated starches was analyzed by fluorophore-assisted capillary electrophoresis (FACE) as described by Morell, Samuel and O'Shea (1998). Isolated starch was debranched (2 h at 37°C) using isoamylase (10 U) and labeled with 1-aminopyrene-3,6,8-trisulfonic acid (APTS). FACE was performed using a P / ACE system 5010 (Beckman Coulter, CA, USA) equipped with a 488 nm laser module. Debranched samples were separated using N-CHO (PVA) capillaries (Beckman Coulter, CA, USA) (50 m ID and 47 cm total length) with precombustion windows. Maltose was used as internal standard. Separation was performed at 10°C for 30 minutes. Based on the migration time of maltose, the degree of polymerization (DP) was assigned to the peak.

Images