PROFESSOR JAYASHANKAR TELANGANA STATE AGRICULTURAL UNIVERSITY

College of Agriculture, Rajendranagar, Hyderabad- 500030

Golden Rice

Presented by,Ajay Kumar Chandra

RAM/14-97

M.Sc. (Ag) Mol. Biology & Biotechnology

What …… we going to discuss today?

• Introduction and Why....Rice? • Golden Rice… Project?• -Carotene Pathway: Problem in Plants.• Production of Golden rice: Genetic modification • The Golden Rice: Solution• How to make golden rice?

• Golden Rice-1: limits• Golden Rice 2• Controversy: Golden Rice…..Is it worth the risks?• Golden Rice: A boon or bane?• Clinical trials / food safety and nutrition research• Conclusion• Reference

Why....Rice? 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.

• For many countries, the infrastructure doesn’t exist to deliver vitamin pills.• Improved vitamin-A content in major staple consumed crops an attractive alternative to fight micronutrient deficiencies.

Solution

• In 2005, 190 million children and 19 million pregnant women, in 122 countries, were estimated to be affected by VAD.

Golden Rice… Project?• Idea first discussed at international conference at IRRI

in Philippines in 1984.

• 1999 Team of scientists, including Ingo Potrykus, Swiss Federal Institute of Technology, successfully genetically engineer rice to produce carotenoids, precursors to Vitamin-A.

• The hype begins: 2000 Time magazine cover story: “This rice could save a million kids a year.”

• May 2000: Adrian Dubock (Zeneca, now Syngenta): “One month delay = 50,000 blind children [a] month.”

• June 29, 2000 US special Congressional Forum, “Can Biotechnology Solve World Hunger?” Invitation stated, “ ‘golden rice’, which has been modified to include certain vitamins……….Is already saving the sight of thousands of children in the poorest parts of Asia.”

-Carotene Pathway: Problem in Plants IPP

Geranylgeranyl diphosphate

Phytoene

Lycopene

-carotene(vitamin A precursor)

Phytoene synthase

Phytoene desaturase

Lycopene-beta-cyclase

ξ-carotene desaturase

Problem:Rice lacks

these enzymes

NormalVitamin A

“Deficient”Rice

Production of Golden rice: Genetic modification

• Golden Rice technology is based on the simple principle that rice plants possess the whole machinery to synthesis of β-carotene, and while this machinery is fully active in leaves, parts of it are turned off in the grain endosperm.

• By adding only two genes, a plant phytoene synthase (psy) and a bacterial phytoene desaturase (crt I), the pathway is turned back on and β-carotene consequently accumulates in the grain.

• Golden rice was created by transforming rice with only two β-carotene biosynthesis genes:

1). psy (Phytoene synthase) from daffodil (Narcissus pseudonarcissus)

2). crtI (Carotene desaturase) from the soil bacterium Erwinia uredovora

Figure: Gene construct used to generate Golden Rice-1. RB, T-DNA right border sequence; Glu, rice endosperm-specific glutelin promoter;

tpSSU, pea ribulose bis-phosphate carboxylase small subunit transit peptide for chloroplast localisation; crtI Carotene desaturase from the soil bacterium Erwinia uredovora; nos, nopaline synthase terminator; Psy, phytoene synthase gene from Narcissus pseudonarcissus (GR1) or Zea mays (GR2); Ubi1, maize polyubiquitin promoter; Pmi, phosphomannose isomerase gene from E. coli for positive selection (GR2); LB, T-DNA left border sequence.

• Lycopene is then cyclized to beta-carotene by the endogenous cyclase in Golden Rice.

(The insertion of a lcy (lycopene cyclase) gene was thought to be needed, but further research showed it is already being produced in wild-type rice endosperm.)

• The psy and crtI genes were transformed into the rice nuclear genome and placed under the control of an endosperm-specific promoter, so they are only expressed in the endosperm.

• The exogenous lcy gene has a transit peptide sequence attached so it is targeted to the plastid, where geranylgeranyl diphosphate formation occurs.

• The end product of the engineered pathway is lycopene, but if the plant

accumulated lycopene, the rice would be red. Recent analysis has shown the plant's endogenous enzymes process the lycopene to β-carotene in the endosperm, giving the rice the distinctive yellow color for which it is named..

• The original golden rice was called SGR1, and under greenhouse conditions it produced 1.6 µg/g of carotenoids.

Genetic modification ......

The Golden Rice: SolutionIPP

Geranylgeranyl diphosphate

Phytoene

Lycopene

-carotene(vitamin A precursor)

Phytoene synthase

Phytoene desaturase

Lycopene-beta-cyclase

ξ-carotene desaturase

Daffodil gene

Single bacterial gene;performs both functions

Daffodil gene

-Carotene Pathway Genes Added

Vitamin APathway

is completeand functional

Golden Rice

Presence of pro-vitamin -A gives rice grains a yellowish-orange color, thus, the name ‘Golden Rice.’

Golden Rice-1: limits• Originial Golden Rice (GR1) does not produce enough ß-carotene (Provitamin A); it

produces “only 1.6 μg/gm of carotenoids; a child would have to eat more than 10kg/day to get sufficient dose”.

• Unexpected effect: GR1 was supposed to produce lycopene (as in tomatoes) and so be bright red; instead, it produced ß-carotene due to unexpected metabolic pathway.

Golden Rice 2• In 2005, Syngenta, produced a variety of golden rice called "Golden Rice 2".

• They combined the phytoene synthase(psy) gene from maize with carotene desaturase (crt1) from the original Golden rice-1.

• Both genes are under endosperm specific promoter control and the mannose act as selectable marker.

• Golden rice 2 produces 23 times more carotenoids than golden rice (up to 37 µg/g), and preferentially accumulates β-carotene (up to 31 µg/g of the 37 µg/g of carotenoids).

• To receive the Recommended Dietary Allowance (RDA), it is estimated that people who eat about 75g of golden rice per day.

• In June 2005, researcher Peter Beyer received funding from the Bill and Melinda Gates Foundation to further improve golden rice by increasing the levels of or the bioavailability of pro-vitamin A, vitamin E, iron, and zinc, and to improve protein quality.

Figure: Gene construct used to generate Golden Rice-2. RB, T-DNA right border sequence; Glu, rice endosperm-specific glutelin promoter;

tpSSU, pea ribulose bis-phosphate carboxylase small subunit transit peptide for chloroplast localisation; crtI Carotene desaturase from the soil bacterium Erwinia uredovora; nos, nopaline synthase terminator; Psy, phytoene synthase gene from Narcissus pseudonarcissus (GR1) or Zea mays (GR2); Ubi1, maize polyubiquitin promoter; Pmi, phosphomannose isomerase gene from E. coli for positive selection (GR2); LB, T-DNA left border sequence.

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

Golden Rice-2

Controversy: Golden Rice…..Is it worth the risks?

Health• May cause allergies or fail to perform desired effect.• Supply does not provide a substantial quantity as the recommended daily

intake. Environment

• Loss of Biodiversity. May become a super weed and endanger the existence of natural rice plants.

• Genetic contamination of natural, global staple foods.• Gene flow from GM to non- GM field crops.

Culture• Some people prefer to cultivate and eat only white rice based on traditional

values and spiritual beliefs on Veg. or Non-Veg.• Intervention in “Gods creation”.

• Critics of genetically engineered crops have raised various concerns.

Golden Rice: A boon or bane?• An early issue was that “golden rice originally did not have sufficient vitamin

A”.

• “The speed at which vitamin A degrades once the rice is harvested”, and “how much remains after cooking are contested.”

• Greenpeace opposes the use of any patented genetically modified organisms in agriculture and opposes the cultivation of golden rice, claiming “it will open the door to more widespread use of GMOs.”.

• Other groups argued that a varied diet containing foods rich in beta carotene such as sweet potato, leafy green vegetables and fruit would provide children with sufficient vitamin A.

• Keith West of Johns Hopkins, Bloomberg School of Public Health countered that “foodstuffs containing vitamin A are either unavailable, or only available at certain seasons, or that they are too expensive for poor families in underdeveloped countries”.

Vandana Shiva, an Indian anti-GMO activist, argued “the problem was not the plant per se, but potential problems with poverty and loss of biodiversity”. Shiva claimed “these problems could be amplified by the corporate control of agriculture.”

Golden Rice: A boon or bane?

Clinical trials / food safety and nutrition research

• IRRI also assures the public that “Golden Rice” will be available to farmers and consumers only after it has been determined to be safe for humans, animals, and the environment and authorized for propagation and consumption by the appropriate regulatory authorities. Therefore, “Golden Rice”, if and when released, will be deemed to be as safe as other rice.

• According to the information reported by the WHO, genetically modified products that are currently on the international market have all passed risk assessments conducted by national authorities." These assessments found no risk to human health”.

• The Food Allergy Resource and Research Program of the University of Nebraska (2006) that showed “the proteins from the new genes in Golden Rice did not show any allergenic properties”.

• In 2009, research results of a clinical trial of Golden Rice with adult volunteers from the USA were published in the American Journal of Clinical Nutrition. It concluded that "β-carotene derived from Golden Rice is effectively converted to vitamin A in humans is a safe".

• In August 2012, Tufts University and others published new research on Golden Rice in the American Journal of Clinical Nutrition showing that “the β-carotene produced by Golden Rice is as good as β- carotene in oil at providing vitamin A to children”.

“Public sentiment is everything. With public sentiment, nothing can fail. Without it, nothing can succeed.” – Abraham Lincoln.

Conclusion

Reference• BOOKS REFFERED:

GENE CLONING AND DNA ANALYSIS: AN INTRODUCTION - T. A. Brown; 5th edition; Blackwell publication.

GENE CLONING: PRINCIPLES AND APPLICATIONS - Julia lodge, Pete Lund & Steve Minchin; Taylor & Francis Publishing Group.

GENE VIII- Benjamin Lewin; Pearson Education, Inc.

• LITERATURE CITED:Al-Babili S, Hoa TTC, Schaub P (2006) Exploring the potential of the bacterial carotene desaturase

CrtI to increase the β-carotene content in Golden Rice. J Exp Bot 57:1007-1014.

Paine JA, Shipton CA, Chaggar S, Howells RM, Kennedy MJ, Vernon G, Wright SY, Hinchliffe E, Adams JL, Silverstone AL, Drake R (2005) A new version of Golden Rice with increased provitamin A content. Nature Biotechnology 23:482-487

Al-Babili S, Beyer P (2005) Golden Rice – five years on the road – five years to go? TRENDS in Plant Science 10:565-573.

Thank you