Golden Rice Golden Crops Transgenic Plants for Food

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Golden Rice & Golden Crops Transgenic Plants for Food Security in the Context of

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

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

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: Ø Supplementation Ø Industrial fortification Ø Education All necessary and very valuable

Intervention strategies: Ø Supplementation Ø Industrial fortification Ø Education All necessary and very valuable but there are drawbacks: Ø Distrubution, educated medical staff Ø Centrally processed food items Ø Only partially applicable Economically sustainable?

Biofortification is an alternative to classical interventions to fight micronutrient deficiencies Improve the nutritional

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

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

Golden Rice cannot be bred …. the application of recombinant DNA technology is necessary

Why engineering ß-carotene (provitamin A) biosynthesis into rice endosperm? Ø Milled rice is provitamin-a-free

Why engineering ß-carotene (provitamin A) biosynthesis into rice endosperm? Ø Milled rice is provitamin-a-free Ø Symptoms of a provitamin-a-free diet • Night-blindness • Xerophthalmia • Fatal susceptibility to childhood diseases (e. g. measles) and general infections (diarrhoea, respiratory diseases) Ø 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

Xerophthalmia

Why engineering ß-carotene (provitamin A) biosynthesis into rice endosperm? Ø Milled rice is provitamin-a-free

Why engineering ß-carotene (provitamin A) biosynthesis into rice endosperm? Ø Milled rice is provitamin-a-free Ø Symptoms of a provitamin-a-free diet • Night-blindness • Xerophthalmia • Fatal susceptibility to childhood diseases (e. g. measles) and general infections (diarrhoea, respiratory diseases) Ø 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? E 8 Assembly-line technologies E 7 E 6 E 5 E 4 E

HOW? E 8 Assembly-line technologies E 7 E 6 E 5 E 4 E 3 E 2 E 1 Precursor Intermediates Expressed genes DNA, m. RNA Product

(HOW? ) Starting Point: Wild-type E 1 PP IPP PP DMAPP E 2 GGPP-Synthase

(HOW? ) Starting Point: Wild-type E 1 PP IPP PP DMAPP E 2 GGPP-Synthase E 3 Phytoene-Synthase Phytoene Synthase E 4 Phytoene Desaturase E 5 (Cis/trans Isomerase? ) E 6 -Carotene Desaturase E 7 Lycopene cis/trans Isomerase E 8 , -Lycopene Cyclase PP GGPP Phytoene Phytofluene -Carotene Neurosporene Lycopene -Carotene All missing (not expressed) in rice endosperm? ? ?

(HOW? ) Towards a concept: PSY (E 3) transformation E 1 PP IPP Wild-type

(HOW? ) Towards a concept: PSY (E 3) transformation E 1 PP IPP Wild-type rice endosperm PP E 2 GGPP-Synthase E 3 Phytoene-Synthase Phytoene Desaturase Phytofluene E 4 -Carotene E 5 (Cis/trans Isomerase? ) E 6 -Carotene Desaturase E 7 Lycopene cis/trans Isomerase E 8 , -Lycopene Cyclase DMAPP PP GGPP Phytoene Neurosporene Lycopene -Carotene Wild-type endosperm can produce a precursor molecule, GGPP!

Assembly-line technologies E 8 E 7 E 6 E 5 E 4 E 3

Assembly-line technologies E 8 E 7 E 6 E 5 E 4 E 3 E 2 E 1 Precursor Intermediate Only two appeared at work! Product

(HOW? ) E 1 PP IPP Wild-type rice endosperm PP DMAPP E 2 GGPP-Synthase

(HOW? ) E 1 PP IPP Wild-type rice endosperm PP DMAPP E 2 GGPP-Synthase E 3 Phytoene-Synthase Phytoene Synthase E 4 Phytoene Desaturase E 5 (Cis/trans Isomerase? ) E 6 -Carotene Desaturase E 7 Lycopene cis/trans Isomerase E 8 , -Lycopene Cyclase PP GGPP Phytoene Phytofluene -Carotene Neurosporene Lycopene -Carotene It is a nightmare to transform six transgenes. Luckily there is Crt. I !

Luckily there is Crt. I transformed E. coli Pantoea ananatis carotenoid gene cluster crt.

Luckily there is Crt. I transformed E. coli Pantoea ananatis carotenoid gene cluster crt. E ORF 2 ORF 3 ORF 4 crt. X crt. Y crt. I crt. B ORF 6 Crt. I substitutes for 4 plant genes crt. Z ORF 12

Carotene Desaturases Complex vs. „simple“ Cyanobacteria and Plants Bacteria 15 -cis-Phytoene E 4 E

Carotene Desaturases Complex vs. „simple“ Cyanobacteria and Plants Bacteria 15 -cis-Phytoene E 4 E 5 15 -cis-Phytoen PDS Z-ISO ? ? 9, 15, 9‘-tri-cis-z-Carotene CRTI E 6 E 7 ZDS 9, 9‘-di-cis-z-Carotene CRTISO 7, 9, 9‘, 7‘-tetra-cis-Lycopene all-trans-Lycopin

The Crt. I gene product provides a shortcut B Plant Desaturation pathway Crt. I

The Crt. I gene product provides a shortcut B Plant Desaturation pathway Crt. I shortcut A

(HOW? ) Towards a prototype: The concept E 1 PP IPP Wild-type rice endosperm

(HOW? ) Towards a prototype: The concept E 1 PP IPP Wild-type rice endosperm PP DMAPP GGPP-Synthase E 3 Phytoene-Synthase E 4 Phytoene Desaturase E 5 (Cis/trans Isomerase? ) E 6 -Carotene Desaturase E 7 Lycopene cis/trans Isomerase E 8 , -Lycopene Cyclase PP GGPP E 3 E 2 Phytoene Phytofluene Crt. I -Carotene Neurosporene Lycopene E 7 -Carotene ´Three instead of six!!

Pathway Complementation in Rice, Co-Transformation Gt 1 p 35 Sp (1) p. ZPs. C

Pathway Complementation in Rice, Co-Transformation Gt 1 p 35 Sp (1) p. ZPs. C E 3 (PSY) aph IV E, 4, 5, 6, 7 (tp-Crt. I) 35 Sp Gt 1 p (2) p. ZLcy. H E 8(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

Control

hpc 11

hpc 11

But there was a second construct without lycopene cyclase ! aph IV Gt 1

But there was a second construct without lycopene cyclase ! aph IV Gt 1 p 35 Sp p. B 19 hpc E 3 (PSY) E 3, 4, 5, 6, 7 (tp-Crt. I) 0. 018 0. 016 Single transformant hpc 2 b 0. 008 0. 006 -Carotene 0. 010 Zeaxanthin Lutein 0. 012 0. 004 …why is Golden Rice golden (yellow) instead of red? ? ? 0. 002 0. 000 -0. 002 0 20 40 …Lesson learned: no need for lycopene ß-cyclase ß-Carotene 0. 014 60 80 100 Ye et al. , 2000; Science 287: 303

We just need to bridge a gap!! E 1 PP PP IPP E 2

We just need to bridge a gap!! E 1 PP PP IPP E 2 GGPP-Synthase E 3 Phytoene-Synthase Phytoene Synthase E 4 Phytoene Desaturase E 5 (Cis/trans Isomerase? ) Neurosporene E 6 -Carotene Desaturase Lycopene E 7 Lycopene cis/trans Isomerase DMAPP Wild-type rice endosperm PP E 3 GGPP Phytoene Phytofluene Crt. I -Carotene E 8 -Carotene Xanthophylls -Carotene , -Lycopene Cyclase Only two transgenes are necessary!! Schaub et al. (2005), Plant Physiol. 138: 441

Assembly-line technologies E 8 E 7 E 6 E 5 E 4 E 3

Assembly-line technologies E 8 E 7 E 6 E 5 E 4 E 3 E 2 E 1 Precursor Intermediate Product Only two transgenes are necessary to fill the gap!!

Prototypes: Not apt for product development Construct ill-defined Integration ill-defined Antibiotic selectable marker Low

Prototypes: Not apt for product development Construct ill-defined Integration ill-defined Antibiotic selectable marker Low amount of b. C (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

Improved Golden Rice variants came in two versions In the public and in the private sector (Syngenta - Orynova) Gt 1 p PSY (Np) Gt 1 p tp-Crt. I (from Narcissus) No selectable marker gene (co-transformed and removed) Almost 1000 events Deregulation-amenable integration Crt. I 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 T 2 Rice grains C D E F G H Hoa et

A B T 2 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……

The preselected events (PS&S) underwent 2 field trials at Louisiana State University……

…where the GR 1 events showed 4, 8 – 7, 1 µg/g

…where the GR 1 events showed 4, 8 – 7, 1 µg/g

Improvements: The past years were dominated by efforts to increase the amount of ß-carotene

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 C 3 -Carbon Metabolism IPP/DMAPP Phytoene synthase (E

Potential bottlenecks to higher carotenoid levels C 3 -Carbon Metabolism IPP/DMAPP Phytoene synthase (E 3) activity? GGDP Phytoene Desaturation (Crt. I) activity? Carotenoid storage? -Carotene Lycopene ß-Carotene Zeaxanthin Lutein Precursor shortage?

Achieved!!! But no significantly improved ß-carotene accumulation. Carotene desaturation is not rate-limiting in Golden

Achieved!!! But no significantly improved ß-carotene accumulation. Carotene desaturation is not rate-limiting in Golden Rice Crt. I PSY Cacar 48 -67 -8 -7 (T 3) Western Cacar 48 -67 -4 -9 (T 3) p. Fun 3 promoter change & codon optimized p. Car. New promoter change Car. New E 4 -4 (T 1) Glu. Bp Synth tp crt. I WT PSY (Np) + control PMI 35 Sp Car. New E 1 -19 (T 1) Improving Crt. I (E 4, 5, 6) expression

Phytoene synthase was investigated by Rachel Drake (Syngenta) Because PSY expression is good in

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 Crt. I Seed promoter Daffodil Psy Ubi promoter hyg. R Maize Psy Rice Psy Transformation into a japonica short-grain rice, (Asanohikare) 20+ plants each Tomato Psy Pepper Psy Rice and Maize PSY (E 3) Best. Proportion of ß-carotene increased.

Assembly-line technologies OK E 7 OK Crt. I OK Too slow!!! E 1 E

Assembly-line technologies OK E 7 OK Crt. I OK Too slow!!! E 1 E 2 E 3 Crt. I Precursor Zwischenprodukt Produkt

Golden Rice 2 was made for implementation GT 1 p. I tp-Crt. I GT

Golden Rice 2 was made for implementation GT 1 p. I tp-Crt. I GT 1 p. I Zm. PSY ubi 1 p PMI p. SYN 12424 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

Improved provitamin A Accumulation in Golden Rice I and II

GR 2… Contains the bacterial Crt. I and PSY (but from maize) just like

GR 2… Contains the bacterial Crt. I 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 GR 1 and about 25 -fold over the prototype Golden Rice is mainly a breeding project today: • Philippines (IRRI, Phil. Rice) • Vietnam (CLDRI) • India (IARI, TNAU, DRRI) • Bangladesh (BRRI) Introgressing 8 events into 11 varieties Event selection completed, moving towards deregulation

GR 2 GR 1 Wild-Type

GR 2 GR 1 Wild-Type

1. Some crop plants do not show adequate trait variability Maize, for instance Maize

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-Crt. I combination, as used in GR boosts ß-carotene production in an African white cultivar to 60 µg/g ! Breeding approaches (ongoing - lyc. E 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,

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 (Provit. A 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-Apsy

Transient testing of the transgenes using direct Agrotransformation of banana fruits NT NT Ubi-Apsy 2 a Ubi-Crt. I Ubi-Apsy 2 a+Crt. I Preliminary HPLC data indicated increased -carotene, -carotene and lutein

2. Some crop plants cannot be (easily) bred Cassava, for instance • 250 million

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 5 th 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; Vit. E, 1 µg/g; Iron 5 ppm, Zinc 1 ppm (fresh weight) • Varietal recovery 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 CP 2 - crt. B Line #12 …more lines epressing multiple genes

Cassava promoter CP 2 - crt. B Line #12 …more lines epressing multiple genes coming this year

2. Some crop plants cannot be (easily) bred Potato, for instance potato ranks fourth,

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 p. K-I p. K-BI p. K-YBI p. P-BI p. P-YBI 35 S TP Crt. I Nos Pat 1 TP Crt. B Ocs 35 S Crt. Y TP 35 S Pat 1 Nos Pat 2 TP Pat 1 TP Pat 2 TP Crt. I Nos Crt. B Ocs Crt. Y Nos Pat 2 Pat 1 TP TP Crt. I Nos Crt. B Ocs 35 S Pat 2 TP TP Crt. I Nos only this one: A mini-pathway

„Golden Potato“ Diretto et al. , Plos. One, 2007

„Golden Potato“ Diretto et al. , Plos. One, 2007

All of the here-mentioned examples bear significant potential: They represent the major staples The

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 : Golden. Rice and other “Golden Crops” are a

The problem of VAD remains : Golden. Rice 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: ETH / Swiss Federal Funds European Commission

To all our sponsors since before 1990: ETH / Swiss Federal Funds European Commission Harvest. Plus 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 Rice Teams & budgets @ • IRRI • Phil Rice- Philippines • CLRRI- Vietnam • DBT, IARI, DRR, TNAU- India