PLNT Unit 9d Applications of Plant Biotechnology in Agriculture. Golden Rice

Transcription

1 PLNT Unit 9d Applications of Plant Biotechnology in Agriculture Golden Rice Plant Biotechnology Adrian Slater, Nigel Scott and Mark Fowler Chapters 5-10 Unless otherwise cited or referenced, all content of this presenataion is licensed under the Creative Commons License Attribution Share-Alike 2.5 Canada 1

2 Metabolic Engineering of Secondary Plant Metabolytes Photosynthesis carbohydrates Reductive pentose phosphate cycle Glycolysis Reductive pentose phosphate cycle phosphoenolpyruvate glyceraldehyde3-phosphate + erythrose-4-phosphate pyruvate shikimic acid acetyl-coa mevalonic acid malonyl-coa Terpenoids (mono-, di-, tetra-) (plastid) (β-carotene) (~40,000 cmpds) amino acids aromatic amino acids Alkaloids (~12,000) Terpenoids (sesqui-, tri-) (cytoplasm) Phenylpropanoids 2

3 Food Enhancement Vitamin A (retinol) Required for sight, healing, immune response World Health Organization (WHO) estimates that up to 500,000 children suffer vision damage each year in developing countries from lack of Vitamin A Vitamin A is made in the body from ingesting various carotenoids but most efficiently from β-carotene Rice is a staple in most of the countries where vitamin A deficiency is a problem 3

4 Vitamin A and Vitamin A equivalents (from carotenoids) Food Vitamin A (IU)* Carrot juice, canned, ½ cup 22,567 Carrots, boiled, ½ cup slices 13,418 Spinach, frozen, boiled, ½ cup 11,458 Carrots, 1 raw (7½ inches) 8,666 Cantaloupe, 1 cup cubes 5,411 Spinach, raw, 1 cup 2,813 Apricots with skin, juice pack, ½ cup 2,063 Papaya, 1 cup cubes 1,532 Tomato juice, canned, 6 ounces 819 Polar bear liver (100 g) 1,000,000 %DV** toxic level * IU = International Units. ** DV = Daily Value. DVs are reference numbers based on the Recommended Dietary Allowances (RDAs). 4

5 Vitamin A and β-carotene β-carotene COOH Retenoic acid CHO Retinal Retinol (Vitamin A) CH2OH 5

6 CH3 Isopentenyl pyrophosphate CH2 CH3 β-carotene synthesis in plants OPP geranylgeranyl diphosphate PPO OPP Phytoene synthase Phytoene (colorless) desaturases lycopene (red) Lycopene β-cyclase β-carotene (orange) 6

7 Golden Rice - An Initiative of the Rockefeller Foundation Profs Ingo Potrykus (ETH Zurich) and Peter Beyer (Univ. of Freiberg) Initiated in conjunction with the Rockefeller Foundation Syngenta donated Intellectual Property Rights for transgenic technologies; Syngenta also negotiated additional IPR with Novartis, Bayer, Monsanto and Japan Tobacco Breeders from developing nations can obtain use licenses for breeding with local rice varieties Resultant rice varieties can be grown royalty free by farmers earning less than $US 10,000 annually. 7

8 Golden Rice 1 Released: genes for production of β-carotene in endosperm Phytoene synthase: Daffodil psy gene Desaturase/β carotene synthetase: Level of total carotenoids (mg/g): 1.6 mg/g % of daily required level from 72g of rice intake: ~2.2 Erwinia uredovora crtl gene (60 g is the estimated rice intake of a child/meal) 8

9 Why was there so little β-carotene in golden rice 1? Adequate geranylgeranyl diphosphate? Efficacy of phytoene synthase? Efficacy of desaturase? Ability to store carotenoids in non-carotenogenic tissue (endosperm)? Work on Golden Rice 2 conducted by Rachael Drake with Syngenta in Britain 9

10 Psy genes from various plants were tested Genes stably transformed into carotenogenic maize callus to examine potential of each gene source and then into rice - all under same promoter Maize Ubi-1 psy coding region NOS 3 Ranking gene sources for carotenoid production levels in transgenic callus and rice: Maize high Rice high Carrot intermediate Tomato intermediate Bell pepper intermediate Arabidopsis thaliana intermediate Daffodil low Agrobacteria transformation used for both maize and rice Some difference in copy insertion number but no correlation with levels 10

11 Golden Rice 2 Rice glutelin promoter Intron near start of coding region Nuclear encoded plastid enzyme Rice Glut Maize psy coding region Rice Glut SSU NOS 3 Erwinia uredovora crtl NOS 3 carotene synthetase 1 Encoding plastid transit peptide from RUBISCO small subunit Many highly expressed genes in monocots (rice maize etc) have an early intron Transformed for nuclear genome integration but both proteins are targeted to the plastid. 11

12 Golden rice 1 Released: vs 2000 Golden rice 2 March 2005 Both added 2 gene for production of β-carotene in endosperm Phytoene synthase: Daffodil psy gene Phytoene desaturase: Erwinia uredovora crtl gene Level of total carotenoids (mg/g): 1.6 mg/g Maize psy gene Erwinia uredovora crtl gene 37 mg/g (23 x ) (31 mg β-carotene/g) % of daily required level from 72g of rice intake: ~2.2 ~50 (60 g is the estimated rice intake of a child/meal) Nature Biotechnology 23, (2005) doi: /nbt

13 White rice Golden rice 2 13

14 Outstanding issues Approval in countries where it might be used Efficiency of conversion of β-carotene from rice to vitamin A Acceptance of yellow rice when normal is white or brown Intellectual property - no claims by Syngenta What is the next most limiting constitutent? 14