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Golden Rice & Golden Crops Transgenic Plants for Food Security in the Context of Development Peter Beyer University of Freiburg Germany Pontifical Academy of Science, Vatican, May 2009 Nutritional Diversity Iron, Zinc Folate Provit A Vit. E Rice (grain) - - - - Tomato - - (+) + Beans + + - + Spinach + + + + Nutritional Diversity Iron, Zinc Folate Facts: Provit A Vit. E Three billion - live on-less than -2 $ per day, 1.5 billion on less than 1 $ per day and cannot afford a Tomato - industrially (+) supplements + diversified diet or produced Beans + + + Rice Spinach Meat + + + + Vit A + + + • Millions are chronically micronutrient malnourished Intervention strategies: Supplementation Industrial fortification Education All necessary and very valuable but there are drawbacks: Distrubution, educated medical staff Centrally processed food items Only partially applicable Economically sustainable? Biofortification is an alternative to classical interventions to fight micronutrient deficiencies Improve the nutritional value of agronomically important crop tissues through 1. Breeding 2. Recombinant DNA technologies is all achievable through breeding ? Simple answer: NO!! 1. Some crop plants do not show adequate trait variability Rice, (polished grains) for instance Provitamin A: Germplasm screening did not reveal any „yellow grains“ with β-carotene Folate: Practically absent Iron: low variability, ranging from 1 – 8 ppm (Final breeding target 14 ppm) Zinc: much more important variability, ranging from 16 – 28 ppm (Target: 24 ppm) Golden Rice cannot be bred ….the application of recombinant DNA technology is necessary Why engineering ß-carotene (provitamin A) biosynthesis into rice endosperm? Milled rice is provitamin-a-free Symptoms of a provitamin-a-free diet • Night-blindness • Xerophthalmia • Fatal susceptibility to childhood diseases (e.g. measles) and general infections (diarrhoea, respiratory diseases) Epidemiology • 124 million children are deficient in vitamin A • 1-2 million deaths annually (1-4 years) • 0.25-0.5 million deaths (5-10 years) UNICEF; Humphrey et al., 1992) • A severe public health problem in (118) countries (WHO) Xerophthalmia Why engineering ß-carotene (provitamin A) biosynthesis into rice endosperm? Milled rice is provitamin-a-free Symptoms of a provitamin-a-free diet • Night-blindness • Xerophthalmia • Fatal susceptibility to childhood diseases (e.g. measles) and general infections (diarrhoea, respiratory diseases) Epidemiology • 124 million children are deficient in vitamin A • 1-2 million deaths annually (1-4 years) • 0.25-0.5 million deaths (5-10 years) UNICEF; Humphrey et al., 1992) • A severe public health problem in (118) countries (WHO) HOW? E8 Assembly-line technologies E7 E6 E5E4E3 E2 E1 Precursor Intermediates Expressed genes DNA, mRNA Product (HOW?) Starting Point: Wild-type E1 PP IPP PP E2 GGPP-Synthase E3 Phytoene-Synthase Phytoene Synthase E4 Phytoene Desaturase E5 (Cis/trans Isomerase?) Neurosporene E6 -Carotene Desaturase Lycopene E7 Lycopene cis/trans Isomerase DMAPP PP GGPP Phytoene Phytofluene -Carotene E8 -Carotene -Carotene All missing (not expressed) in rice endosperm??? , -Lycopene Cyclase (HOW?) Towards a concept: PSY (E3) transformation E1 PP IPP Wild-type rice endosperm PP E2 GGPP-Synthase E3 Phytoene-Synthase Phytoene Desaturase Phytofluene E4 -Carotene E5 (Cis/trans Isomerase?) Neurosporene E6 -Carotene Desaturase Lycopene E7 Lycopene cis/trans Isomerase DMAPP PP GGPP Phytoene E8 -Carotene , -Lycopene Cyclase -Carotene Wild-type endosperm can produce a precursor molecule, GGPP! Assembly-line technologies E8 E7 E6 E5E4E3 E2 E1 Precursor Intermediate Only two appeared at work! Product (HOW?) E1 PP IPP Wild-type rice endosperm PP E2 GGPP-Synthase E3 Phytoene-Synthase Phytoene Synthase E4 Phytoene Desaturase E5 (Cis/trans Isomerase?) Neurosporene E6 -Carotene Desaturase Lycopene E7 Lycopene cis/trans Isomerase DMAPP PP GGPP Phytoene Phytofluene -Carotene E8 -Carotene , -Lycopene Cyclase -Carotene It is a nightmare to transform six transgenes. Luckily there is CrtI ! Luckily there is CrtI transformed E. coli Pantoea ananatis carotenoid gene cluster crtE ORF2 ORF3 ORF4 crtX crtY crtI crtB ORF6 CrtI substitutes for 4 plant genes crtZ ORF12 Carotene Desaturases Complex vs. „simple“ Cyanobacteria and Plants Bacteria 15-cis-Phytoene E4 E5 15-cis-Phytoen PDS Z-ISO ?? 9, 15, 9‘-tri-cis-z-Carotene CRTI E6 E7 ZDS CRTISO 9, 9‘-di-cis-z-Carotene 7, 9, 9‘, 7‘-tetra-cis-Lycopene all-trans-Lycopene all-trans-Lycopin The CrtI gene product provides a shortcut B Plant Desaturation pathway CrtI shortcut A (HOW?) Towards a prototype: The concept E1 PP IPP Wild-type rice endosperm PP E2 GGPP-Synthase E3 Phytoene-Synthase E4 Phytoene Desaturase E5 (Cis/trans Isomerase?) Neurosporene E6 -Carotene Desaturase Lycopene E7 Lycopene cis/trans Isomerase DMAPP PP GGPP E3 Phytoene Phytofluene CrtI -Carotene E7 E8 -Carotene , -Lycopene Cyclase -Carotene ´Three instead of six!! Pathway Complementation in Rice, Co-Transformation Gt1p 35Sp (1) pZPsC E3 (PSY) aph IV E,4,5,6,7 (tp-CrtI) 35Sp Gt1p (2) pZLcyH E8(ß-LCY) With the selectable marker gene only in (2), all yellow transgenic seeds expressed lycopene-ß-cyclase. They all contained ß-carotene; this was the expected outcome. Control hpc11 But there was a second construct without lycopene cyclase ! aph IV Gt1p 35Sp pB19hpc E3 (PSY) E3,4,5,6,7 (tp-CrtI) 0.018 0.016 …Lesson learned: no need for lycopene ß-cyclase 0.008 -Carotene 0.010 Zeaxanthin Lutein 0.012 0.006 0.004 ß-Carotene Single transformant hpc 2b 0.014 …why is Golden Rice golden (yellow) instead of red??? 0.002 0.000 -0.002 0 20 40 60 80 100 Ye et al., 2000; Science 287:303 We just need to bridge a gap!! E1 PP PP IPP E2 GGPP-Synthase E3 Phytoene-Synthase Phytoene Synthase E4 Phytoene Desaturase E5 (Cis/trans Isomerase?) Neurosporene E6 -Carotene Desaturase Lycopene E7 Lycopene cis/trans Isomerase DMAPP Wild-type rice endosperm PP GGPP E3 Phytoene Phytofluene CrtI -Carotene E8 -Carotene Xanthophylls -Carotene , -Lycopene Cyclase Only two transgenes are necessary!! Schaub et al. (2005), Plant Physiol. 138: 441 Assembly-line technologies E8 E7 E6 E5E4E3 E2 E1 Precursor Intermediate Only two transgenes are necessary to fill the gap!! Product Prototypes: Not apt for product development Construct ill-defined Integration ill-defined Antibiotic selectable marker Low amount of bC (1.6 µg/g) Start from scratch include Indica rice varieties. Happy Easter Improved Golden Rice variants came in two versions In the public and in the private sector (Syngenta - Orynova) Gt1p PSY (Np) Gt1p tp-CrtI (from Narcissus) No selectable marker gene (co-transformed and removed) Almost 1000 events Deregulation-amenable integration CrtI controlled by an endosperm-specific promoter In Cocodrie (Javanica) Amount up to 6 µg/g Three events preselected Known as Golden Rice 1 Technology works in Indica varieties A B T2 Rice grains C D E F G H Hoa et al., Plant Physiol. 133, 2003 The preselected events (PS&S) underwent 2 field trials at Louisiana State University…… …where the GR1 events showed 4,8 – 7,1 µg/g Improvements: The past years were dominated by efforts to increase the amount of ß-carotene in GR both, in the public sector as well as at Syngenta Potential bottlenecks to higher carotenoid levels C3-Carbon Metabolism Precursor shortage? IPP/DMAPP Phytoene synthase (E3) activity? GGDP Phytoene Desaturation (CrtI) activity? Carotenoid storage? -Carotene Lycopene ß-Carotene -Carotene Zeaxanthin Lutein Inefficient Inefficient transgene transgene expression? expression? CrtI Achieved!!! But no significantly improved ß-carotene accumulation. Carotene desaturation is not rate-limiting in Golden Rice PSY Cacar 48-67-8-7 (T3) Western Cacar 48-67-4-9 (T3) pFun3 promoter change & codon optimized pCarNew promoter change CarNew E4-4 (T1) GluBp Synth tp crtI WT PSY (Np) + control PMI 35Sp CarNew E1-19 (T1) Improving CrtI (E4,5,6) expression Phytoene synthase was investigated by Rachel Drake (Syngenta) Because PSY expression is good in GR, different versions of the PSY gene were assayed. Seed promoter CrtI Seed promoter Daffodil Psy Ubi promoter hygR Maize Psy Rice Psy Tomato Psy Carotenoid content (mg g-1 dwt) Transformation into a japonica short-grain rice, (Asanohikare) 20+ plants each 20 18 16 14 12 10 8 6 4 2 0 Pepper Psy Rice and Maize PSY (E3) Best. Proportion of ß-carotene increased. rice Psy/crtI maize Psy/crtI pepper Psy/crtI tomato Psy/crtI Individual transgenic plant (event) daffodil Psy/crtI Assembly-line technologies OK E7 OK OK Too slow!!! CrtI CrtI E1 E2 E3 CrtI Precursor Zwischenprodukt Produkt Golden Rice 2 was made for implementation GT1pI tp-CrtI GT1pI ZmPSY ubi1p PMI pSYN12424 Transform long grain rice variety (Kaybonnet) Sugar selectable marker 619 individual GM rice plants Screen for seed colour, gene copy number, fertility Select 6 “Golden Rice 2” events for further screening and development Improved provitamin A Accumulation in Golden Rice I and II GR 2… Contains the bacterial CrtI and and PSY (but from maize) just like the previous versions. Both genes are under endosperm specific promoter control; the selectable marker agent is mannose. Increase in provitamin A content is about 10-fold over GR1 and about 25-fold over the prototype Golden Rice is mainly a breeding project today: • Philippines (IRRI, PhilRice) • Vietnam (CLDRI) • India (IARI,TNAU, DRRI) • Bangladesh (BRRI) Introgressing 8 events into 11 varieties Event selection completed, moving towards deregulation GR2 GR1 Wild-Type 1. Some crop plants do not show adequate trait variability Maize, for instance Maize is the world´s third most important staple crop. In maize, the pathway proceeds beyond beta carotene. The genetic variability for high carotenoid levels is very substantial, but low for provitamin A carotenoids. A Psy-CrtI combination, as used in GR boosts ß-carotene production in an African white cultivar to 60 µg/g ! Breeding approaches (ongoing - lycE polymorphisms identified) have yielded so far ca.14 µg/g ß-carotene. Naqvi et al., PNAS, 2009 Harjes et al., Science 2. Some crop plants show adequate trait Variability but cannot be (easily) bred Bananas, for instance • Bananas are a staple in 50 (+) countries (Uganda; 222 kg/person year) • East Africa Highland Bananas are very low in micronutrients (ProvitA 2.7 µg/g; Vit E 1 µg/g; Iron 2.6 ppm, fresh weight) • Conventional breeding: extremely difficult as bananas are essentially sterile Most current cultivars are sterile triploids selected from the wild • Have not been genetically improved for thousands of years • Huge challenges from global movement of devastating diseases James Dale, QUT Australia, Grand Challenges in Global Health Transient testing of the transgenes using direct Agrotransformation of banana fruits NT NT Ubi-Apsy2a Ubi-CrtI Ubi-Apsy2a+CrtI Preliminary HPLC data indicated increased -carotene, -carotene and lutein 2. Some crop plants cannot be (easily) bred Cassava, for instance • 250 million sub-Saharan Africans and 600 millions globally rely on cassava as their major source of calories •Ranks 5th among crops directly consumed by humans (No. 1 in Sub-Saharan Africa). Provides food security. • Very low in micronutrients Provit A (mostly) 1-5 µg/g; VitE, 1 µg/g; Iron 5 ppm, Zinc 1 ppm (fresh weight) • Varietal recovery very difficult upon breeding (vegatatively propageted) • Very long breeding cycle Richard Sayre, Danforth Center, St. Louis, USA, Grand Challenges in Global Health, BMGF University of Freiburg – CIAT, Harvest Plus Cassava promoter CP2 - crtB Line #12 …more lines epressing multiple genes coming this year 2. Some crop plants cannot be (easily) bred Potato, for instance potato ranks fourth, among the staple foods of mankind, after wheat, rice and maize pK-I pK-BI pK-YBI pP-I pP-BI pP-YBI 35S TP CrtI Nos Pat1 TP CrtB Ocs 35S CrtY TP 35S Pat1 Nos Pat2 TP Pat1 TP Pat2 TP CrtI Nos CrtB Ocs CrtY CrtY Nos Pat2 Pat1 TP TP TP TP CrtI Nos CrtB Ocs CrtI Nos CrtB Ocs 35S Pat2 TP TP CrtI CrtI Nos Nos only this one: A mini-pathway „Golden Potato“ Diretto et al., PlosOne, 2007 All of the here-mentioned examples bear significant potential: They represent the major staples The provitamin A bioavailability is very good! Bioavailability: Golden Rice: 3.8:1, Human (maybe even better) Tang et al., Am J Clin Nutr 2009 Maize: 3:1 Gerbil, human study is underway Howe and Tanumihardjo, J. Nutr. 2006 Cassava: 3.7:1, Gerbil Howe et al., British Journal of Nutrition (2009) Spinach: 20:1, Human Tang et al., Am J Clin Nutr 2005; Very good bioavailability of ß-carotene from simple starchy food matrices (Like banana and potato?) The problem of VAD remains : GoldenRice and other “Golden Crops” are a potentially significant contribution to alleviation. Genetic modification is an indispensible tool Breeding where possible Genetic modification where necessary To all our sponsors since before 1990: Rice Teams & budgets @ •IRRI •Phil Rice- Philippines ETH / Swiss Federal Funds European Commission HarvestPlus USAID Syngenta Company Syngenta Foundation National Institutes of Health (USA) Bill and Melinda Gates Foundation The Golden Rice Humanitarian Board Bayer, Mogen, Novartis, Monsanto, Orynova, Zeneca •CLRRI- Vietnam •DBT, IARI, DRR,TNAU- India