ZHONGYI LI, CSIRO AGRICULTURE AND FOOD, CANBERRA

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1 ZHONGYI LI, CSIRO AGRICULTURE AND FOOD, CANBERRA

2 CSIRO Agriculture & Food Our advantage Integrated Agricultural and Food Research 2

3 CSIRO capability relevant to today s talk CSIRO Agriculture and Food Cereal Quality Traits Innovative Food Centre CSIRO Health and Biosecurity Nutritional and Health Substantiation

4 Some BARLEYmax products in Australia BARLEYmax oat soughdough Spin-out Japan first products launched August 2016 USA first products launched May 2017

5 Marcus Newberry Ahmed Regina control HAW Resistant starch % of wholemeal flour HAW control Amylose % >>10 x resistant starch 3x TDF in refined flour 2x TDF in wholemeal Excludes RS 5

6 Hordein reduced >10,000x 56,000ppm to 3-5ppm WHO Gluten-free target 20 ppm First the beer. Commercial release April 2016 Crispin Howitt Greg Tanner Malcolm Blundell

7 Today s presentation: Cereal Grain Traits What we are doing: Resistant starch barley Resistant starch rice Micronutrient dense rice Long shelf life rice

8 Resistant starch barley BARLEYmax Zhongyi Li

small intestine Reduces glycemic index and glycemic load Improves satiety Fermented to short chain fatty acids in large bowel Acetate,

9 Why resistant starch? a fermentable fibre High amylose (>60%) Modifies starch granule structure to produce resistant starch Resistant starch Resists digestion in the small intestine Reduces glycemic index and glycemic load Improves satiety Fermented to short chain fatty acids in large bowel Acetate, proprionate, butyrate preferred energy for colonocytes Lower bowel ph - increases good bugs and reduces the pathogenic Enhanced Mg and Ca absorption Healthier colonocytes Reduced rates of bowel cancer Reduced absorption of pathogens Glycaemic Load

10 Chronic disorders in Australia Cardiovascular disease 18% of Australians $3.9 billion a year Colorectal cancer 4,400 death pa, cost $2 billion a year overweight or obese 64% men, 47% women Type II diabetes 8% of Australians, cost $3 billion a year

11 Adequate RS intake in Australia RS (g/day) Male Female Required Current

More fibre than any other cereal grain 30 High Resistant starch, fructan and fibre 25 % of total weight 20 15 10

12 More fibre than any other cereal grain 30 High Resistant starch, fructan and fibre 25 % of total weight Rye Oats Wheat Barley BarleyMax Rye Oats Wheat Barley BARLEYmax Resistant Starch Solube Fibre Insoluble Fibre

13 SSIIa mutants in barley increase resistant starch, β-glucan, fructan and total fibre Grain composition (%) % 162% 430% 171% 4200% wildtype barley SSIIa mutant

ph in the large bowel (pig trial) Increased SCFA

14 Clinical substantiation Reduced blood glucose concentration lower GI (humans) Reduced plasma lipids (pig trial) Reduced ph in the large bowel (pig trial) Increased SCFA concentration in the large bowel (pig trial) Tony Bird David Topping

15 BARLEYmax path to market and impact The Healthy Grain CSIRO F&N CSIRO Agriculture CSIRO F&N INDUSTRY Health concept Genotype Composition Processing Performance End Product Quality Nutritional benefit PRODUCT Biochemical analysis Genes Mutants Markers Breeding Rheology Small scale Sensory Analysis Substantiation trials BARLEYmax

BARLEYmax 1, 2 and 3 Commercial rights with The Healthy Grain http://www.

16 BARLEYmax 1, 2 and 3 Commercial rights with The Healthy Grain CEO: Robert Burbury Technical Business Manager: Dr Paul Sims

17 Resistant starch rice

18 Increasing amylose content in rice grain through down-regulating the expression of SBEIIb by RNAi and amirna approaches Vito Butardo

19 Increase amylose in rice Amylopectin ADPG PPase Sucrose G1P ATP PPi ATP G1P ADPG PPase PPi ADPG SSI ADP SSIVa SSIIIa SSIIa GBSSI X DBEI BEIIb BEIIa BEI Pul Pho D-enzyme ADPG Amylose Cytoplasm Amyloplast

20 RNAi and amirna were used

21 Increasing amylose content in rice grain through combining different starch synthetic gene alleles Jixun Luo

22 RILs following cross between IR64 (indica) and Nipponbare (japonica) Genotypes of RILs of six starch biosynthetic enzyme groups Genotype Wxi Wxj SSIi SSIj SSIIai SSIIaj SBEIi SBEIj SBEIIai SBEIIaj SBEIIbi SBEIIbj Wx i j i i j i j SSI i i j j i j i SSIIa i i i j i i i SBEI i i i i j i i SBEIIa j i j i i j i SBEIIb i j j i j i j i' and j are abbreviations for indica (IR64) and japonica (Nipponbare) genotypes of each enzyme

23 Producing higher amylose rice grain through combining different starch synthetic gene alleles Amylose content % a d b c c b c d c c d d b d Black columns indicate lines containing Wxi allele; Grey columns indicate lines containing Wxj allele.

24 RILs following cross between A203 and 3-12 Genotypes of RILs of four starch biosynthetic enzyme groups A203 SSIIa jap SSIIa jap / SBEIIb DR SBEIIb DR (opaque) 3-12 SSIIa ind SSIIa jap / SBEIIb WT SBEIIb WT (translucent) Parents SSIIa SBEIIb Wx Seed colour G1: ind - ve jap opaque G2: jap - ve jap opaque G3: ind + ve jap translucent G4: jap + ve jap translucent F3

25 The impact of the indica SSIIa allele on the starch properties of high amylose starch from rice grains Amylose content (%) a a b b 0 G1 G2 G3 G Swelling power (folds) a a a b G1 G2 G3 G4 SSIIa SBEIIb G1: ind - ve G2: jap - ve G3: ind + ve G4: jap + ve

26 Two different mechanisms for increasing amylose content in rice grains A 400 Peak2 40 Peak1 4 G-1 G-2 G-3 G B Peak2 40 Peak1 4 A203 Nip IR36ae IR36 SSIIa SBEIIb Wx G1: ind - ve jap G2: jap - ve jap G3: ind + ve jap G4: jap + ve jap SSIIa jap SBEIIb Wx jap A203: jap -ve jap Nip: jap +ve jap : jap +ve jap 3-5-2: ind +ve jap IR36ae: ind -ve ind IR36: ind +ve ind

27 Summary for manipulating starch in rice grain Both suppression of SBE IIb expression and gene recombination can increase amylose content. High amylose trait can be achieved through two different mechanisms. The indica SSIIa can improve starch properties for high amylose starch from rice grain through down-regulation of SBEIIb.

28 Micronutrient dense rice

New type of rice - thick aleurone mutants Rice

th most important contributors to disease in

B group vitamins Minerals and phytic acid

29 New type of rice - thick aleurone mutants Rice dominant diets: Zn and Fe deficient 5 th and 6 th most important contributors to disease in developing countries (WHO, 2002) Starchy endosperm Aleurone layer Dietary fibre Proteins, enzymes Phenolics, lignans Vitamin E B group vitamins Minerals and phytic acid Lipids, plant sterols Aleurone important sink for micronutrients

30 ta-1 and ta-2 showed increased aleurone layers ZH11 ta1 ta2 27 days after pollination

31 Nutritional analyses of ta2 mutant wholemeal flour compared to normal wholemeal flour 50% increase in lipid 20% increase in Zn, Fe, Mg, K, P, S 60% increase in Vitamin B6 & B9 80% increase antioxidant capacity 30% increase in fibre 20% increase in Vitamin B3

32 Summary for micronutrient dense rice Micronutrient dense rice has been generated through increasing aleurone sink for micronutrients in rice grain. Thick aleurone rice grain also contains more dietary fibre and vitamins. Patent application is in place.

33 Long shelf life rice

Oil oxidation Oxidation stages Oleic acid Linoleic acid Linolenic

process Bad Oil Hydroperoxides Aldehydes Carboxylic Alcohol

For Linoleic acid 9-Hydroperoxide 13-Hydroperoxide eg.

34 Oil oxidation Oxidation stages Oleic acid Linoleic acid Linolenic acid Autoxidation Stage 1 Autoxidation Stage 2 Good Oil Oxidation process Bad Oil Hydroperoxides Aldehydes Carboxylic Alcohol hydrocarbons Oxidized flavor Products eg. For Linoleic acid 9-Hydroperoxide 13-Hydroperoxide eg. For Linoleic acid Pentanal Hexanal 2,4-decadiental eg. soybean and corn oil contain 23.5% Hexanal in volatile compounds

35 PCR screening of OsFAD2-1 RNAi T 0 plants (Nipponbare) A ZLBx173 OsFAD2-1 Rint9 OsFAD2-1 Bx17 promoter pfad2-r NOS terminator hpt gene B

36 Linear relationship between linoleic acid and oleic acid content for OsFAD2-1 RNAi and negative segregant T 1 grains Linoleic acid (%) Negative segregant FAD2 RNA y = -0.86x + 68 R 2 = Oleic acid (%) OsFAD2-1 RNAi

Increased oleic acid and reduced linoleic acid contents of T 4 grains for OsFAD2-1 RNAi lines 60 50 Content

37 Increased oleic acid and reduced linoleic acid contents of T 4 grains for OsFAD2-1 RNAi lines Content (%) OsFAD2-1 RNAi Negative segregant 0 Palmitic acid oleic acid Linoleic acid Hong Wang Ming Huang

38 Summary Producing BARLEYmax, HA wheat and Kebari grains containing novel traits that are commercially available. Developed rice grain with increased RS and low GI. Developed micronutrient dense rice. Producing long shelf life rice grain. Looking for the opportunities to work with other partners for improving rice grain quality.

39 Acknowledgement Russell Heywood Mark Cmiel Oscar Larroque Behjat Kosar-Hashemi Emmett Leyne Chris Konik-Ross Sadiq Rahman Qing Liu Peter Chandler Steve Jobling Matthew Morell ACVL Ltd (BARLEYmax JV, BARLEYmax Enterprises, The Healthy Grain) Austgrains Popina Food Services (Goodness Superfoods, Freedom Foods Group) Limagrain (ARISTA) Rob Defeyter Lindsay Adler Paul Sims CSIRO Plant Industry CAISO Food Future Flagship CSIRO Agriculture Flagship

40 Thank you CSIRO Agriculture Flagship Presenter Name: Zhongyi Li Presenter Title: Manipulating starch in cereal grains Cereal quality traits Group t e zhongyi.li@csiro.au w CSIRO AGRICULTURE FLAGSHIP

Next Generation Healthy Grains: Developing grains to combat diet-related chronic diseases

Next Generation Healthy Grains: Developing grains to combat diet-related chronic diseases Steve Jobling, CSIRO Agriculture and Food, Canberra, Australia Modifying grain for health outcomes High amylose