Ingo Potrykus Golden Rice Kameron Starr

Problem Many developing countries suffer from Vitamin A deficiency due to the predominant consumption of rice. -Rice lacks Vitamin A and pro-vitamin A in the edible endosperm. This deficiency leads to blindness, especially in children

Problem Many developing countries suffer from Vitamin A deficiency due to the predominant consumption of rice. -Rice lacks Vitamin A and pro-vitamin A in the edible endosperm. This deficiency leads to blindness, especially in children Solution Genetically engineer rice to produce pro-vitamin A, β-carotene.

Chlorophyll dominating Carotenoid dominating Carotenoid organic pigment (red/orange/yellow color)

The Biosynthesis Pathway Enzymes involved 1. IPP/DMAPP-Isomerase 2. GGPP-Synthase 3. Phytoene synthase (psy) 4. phytoene desaturase 5. ζ-carotene desaturase 6. lycopene –cyclase (lcy) Catalyze introduction of two double bonds each (shown on next slide) bacterial phytoene desaturase (crtI) Catalyze introduction of all 4 double bonds

Where did the foreign genes come from? Daffodil (psy), (lcy) Erwinia uredovora (crtI)

The Biosynthesis Pathway

Ti plasmid-mediated gene transfer using Agrobacterium tumafaciens Recall: Agrobacterium: Plant pathogen that delivers DNA to plants. Can be used in genetic engineering - Delete the phytohormone genes - Add cloning site for YFG

Single Transformation Initially this plasmid was used to insure that lycopene (red) was being synthesized. Contains genes for two enzymes - Phytoene-Synthase (psy) - Bacterial Phytoene Desaturase (crtI)

Co-Transformation LB Gt1p psy nos! 35sp tp crtI nos! RB These plasmid were used to complete the synthesis of β-Carotene (Yellow) pZPsC is very similar to pB19hpc - provides machinery for the synthesis of Lycopene pZLcyH was added to introduce Lycopene β-Cyclase which should complete the pathway

SURPRISE! To the researcher’s surprise, the single transformation produced β-carotene without the accumulation of lycopene. - α,β-cyclase may be constitutively expressed in endosperm - formation of lycopene turns these genes on This makes transformation much more simple. Only two genes must be added. Pb19hpc was used and modified in the following two ways - PMI selection replaced hygromycin - nptII selectable marker removed Results The transgene was inherited by offspring with a 3:1 Segregation, (Golden:WT) 1.6 μg carotenoid/g dry rice endosperm - A child would need 3,330g(7.3lbs)/day - Usually eat a max of 120g/day Proof of concept Modified pB19hpc

Results A.Control seeds B.Single transformant C.Co-tranformant D.Co-transformant Seed content analyzed by high performance liquid chromatography (HPLC) Co-transformants much more variable Lutein and zeaxanthin also synthesized\ -other carotenoids contributing to eye health

Golden Rice 2 Psy was found to be the limiting factor - hypothesis based on the lack of lycopene accumulation Psy genes from various other plants were used in an attempt to increase carotenoid synthesis. These include Maize, Pepper, and Tomato Maize yielded the best results

Results The transgene was inherited by offspring with a 3:1 Segregation, (Golden:WT) 37 μg carotenoid/g dry rice endosperm - A child would need 144g/day - compares to the max of 120g/day Still no lycopene accumulation, further optimization possible

Controversy People are reluctant to consume this due to its genetic alteration Farmers fear this could contaminate WT rice Riots and “rice picking’s” have occurred