Supplementary information Present Status and Market Potential … · 2015-01-14 · Supplementary Table 2 Overview of successful biofortification reports through metabolic engineering

Supplementary information Present Status and Market Potential of Transgenic Biofortified Crops.

De Steur, H., Blancquaert, D., Strobbe, S., Lambert, W., Gellynck, X., and D. Van Der Straeten.

Nature Biotechnology, 33(1), 25-29. Supplementary Glossary

Biofortification

Biofortification = the enhancement of the micronutrient content of staple crops, through the application of conventional breeding (with or without biotechnological approaches, such as QTL-mapping and marker-assisted breeding) or metabolic engineering;

Multi-biofortification = the enhancement of the level of several micronutrients simultaneously, through the application of conventional breeding and/or metabolic engineering approaches.

R&D

Conventional breeding = the crossing of closely-related (sexually compatible) plants in order to create new individuals with desired characteristics;

Intragenic GM crops = crops that are genetically modified by transferring genes from closely related species capable of sexual hybridization (e.g. from a wild rice variety into another rice variety), allowing in vitro recombination of elements isolated from different genes within the sexually compatible gene pool (as opposed to cisgenic GM crops, in which insertion of a reorganized gene is not allowed, the gene has to contain its introns and is flanked by its native promoter and terminator in the normal orientation);

Metabolic engineering = engineering metabolic pathways by genetic modification to enhance or decrease the accumulation of (a) target compound(s);

Transgenic crops = crops in which genetic material from sexually compatible or incompatible organisms has been artificially introduced. In first generation GM crops, transgenes originated from sexually incompatible organisms. In second generation GM crops, transgenic crops were created by introduction of genetic material from sexually compatible organisms (coined cisgenic or intragenic, see above).

Market potential

Consumer acceptance = a construct that reflects a positive predisposition towards an (hypothetical) object (e.g. GM food);

Cost-benefit analysis = a common decision-making tool for evaluating health interventions (e.g. biofortified crops), in which health benefits are converted into monetary values and compared with the costs;

Cost-effectiveness analysis = a common decision-making tool for evaluating health interventions (e.g. biofortified crops), in which health benefits are compared to the costs, often expressed as the cost per additional health unit (e.g. cost per DALY saved);

Disability-Adjusted Life Year (DALY) = a health measure that describes both mortality and morbidity associated with a health condition (e.g. vitamin deficiency) as a single index: years of life lost + years lived with disability;

Willingness-to-pay = a construct that reflects one’s economic value for a (non-market) good, usually stated as a price premium (e.g. the highest amount one is prepared to pay more for a biofortified crop as compared to a conventional crop). Related constructs refer to consumers’ purchase intention or preference (choice).

Supplementary note on GMO regulation

Although an increasing number of studies report successful biofortification attempts, second-generation

staple GM crops are still unavailable for consumers. Metabolic engineers often work on laboratory

varieties to provide a proof-of-concept, which then needs to be transferred to locally adapted and

consumed varieties. The latter can be achieved by crossing biofortified germplasm with those

agronomically important crop varieties or by transformation with the target genes necessary for nutrient

enhancement. The path from proof-of-concept to a commercially available engineered crop is inhospitable

and full of hurdles.

Nevertheless, the regulatory pipeline is nowadays well defined1,2

. First of all, a regulatory clean transgenic

event needs to be found. Therefore, a large screening of many independent transformation events needs

to be conducted to identify a transformed line with a single copy of the T-DNA insert without any

rearrangements, ballast DNA, nor read-through across the borders of the construct. This process is time-

consuming and expensive and therefore often requires a public-private collaboration. Moreover, the

creation of a metabolically engineered crop generally involves patented technologies, which could possibly

hamper a release onto the market. Luckily, as was the case for GR, these patents are usually restricted in

time and companies are, when aiming for humanitarian purposes, willing to provide them free of charge.

Next, a biosafety assessment needs to be performed. This can be divided in event-independent and -

dependent studies. The former include bioavailability tests and characterization of the protein encoded by

the introduced genes, to make sure that the gene products do not elicit toxin or allergen accumulation.

The event-dependent studies include a molecular analysis to ensure that the introduced T-DNA is well-

defined, restricted to the minimum, inheritably stable and its insertion place into the host genome

characterized. In addition, expression analyses of the transgenes (proving that the genes are expressed to

a desirable level and restricted to the target tissue) need to be conducted over a number of generations

(or after vegetative propagation for crops such as potatoes and bananas). Last but not least, field trials

need to be conducted to ensure that the introduced traits do not compromise crop yield nor disease

resistance as compared to non-transformed controls, as well as a compositional analysis over two growth

seasons and an environmental risk assessment.

Although these regulatory steps are now well-defined, the release of GM biofortified crops is perpetually postponed by a distrusting public opinion, often fed by non-scientifically based arguments. Nevertheless, Golden Rice acts as a pioneer which, once released onto the market, will open the door for other second-generation GM crops. References 1. König, A. et al. Food Chem. Toxicol. 42, 1047-1088 (2004). 2. Bertebos Foundation. Kungl. Skogs- och Lantbruksakademiens TIDSKRIFT 7, 1-116 (2008).

Supplementary Table 1 Comparison of conventional and transgenic breeding techniques for biofortification of staple crops with β-carotene. Compound level, per crop, in µg/g dry weight (DW)

Conventional breeding

Metabolic engineering

Compound Crop Compound level Reference Compound level Reference

β-carotene corn 13,6 µg/g DW Harjes et al., 20081 59,3 µg/g DW Naqvi et al., 2009

2

rice 0

/ 31 µg/g DW Paine et al., 2005

3

cassava 16,3 µg/g DW Welsch et al., 2010

4 6,67 µg/g DW Welsch et al., 2010

4

potato 2,19 µg/g DW Andre et al., 2007

5 47 µg/g DW Diretto et al., 2007

6

wheat* 0,46* µg/g DW total carotenoids Cong et al., 20097 4,96 µg/g DW total carotenoids Cong et al., 2009

7

DW, Dry weight; *no reports on natural variation found, value reported is a wild type (WT) value

References 1. Harjes, C.E. et al. Natural genetic variation in lycopene epsilon cyclase tapped for maize biofortification. Science (New York, NY) 319, 330 (2008). 2. Naqvi, S. et al. Transgenic multivitamin corn through biofortification of endosperm with three vitamins representing three distinct metabolic pathways.

Proceedings of the National Academy of Sciences 106, 7762-7767 (2009). 3. Paine, J.A. et al. Improving the nutritional value of Golden Rice through increased pro-vitamin A content. Nature biotechnology 23, 482-487 (2005). 4. Welsch, R. et al. Provitamin A accumulation in cassava (Manihot esculenta) roots driven by a single nucleotide polymorphism in a phytoene synthase

gene. The Plant Cell Online 22, 3348-3356 (2010). 5. Andre, C.M. et al. Antioxidant profiling of native Andean potato tubers (Solanum tuberosum L.) reveals cultivars with high levels of β-carotene, α-

tocopherol, chlorogenic acid, and petanin. Journal of agricultural and food chemistry 55, 10839-10849 (2007). 6. Diretto, G. et al. Metabolic engineering of potato carotenoid content through tuber-specific overexpression of a bacterial mini-pathway. PLoS One 2,

e350 (2007). 7. Cong, L. et al. Expression of phytoene synthase1 and carotene desaturase crtI genes result in an increase in the total carotenoids content in

transgenic elite wheat (Triticum aestivum L.). Journal of agricultural and food chemistry 57, 8652-8660 (2009).

Supplementary Table 2 Overview of successful biofortification reports through metabolic engineering in staple crops.

Target compound Target crop Target tissue Target gene(s) Compound level Fold increase Reference

Ascorbate corn seed Ta dhar 160 nmol/g FW 1.9 Chen et al., 20031

Os dhar 110 µg/g DW 6 Naqvi et al., 20092

potato tuber Fa GalUR 3.2 µmol/g FW 2 Hemavathi et al., 2009

3

rat GLOase 3 µmol/g FW 1.4 Hemavathi et al., 20104

St VTC2B 1.83 mg/g DW 2.7 Bulley et al., 20125

β-carotene rice seed Np psy, Pa crt1, Np lcy 1.6 µg/g DW total carotenoids / Ye et al., 20006

Zm psy1, Pa crt1 31 µg/g DW / Paine et al., 20057

potato tuber Pa crtB 11 µg/g DW / Ducreux et al., 20058

silencing LCY-e 44 ng/g DW 14 Diretto et al., 20069

Ba Or 24 µg/g DW total carotenoids 6 Lu et al., 200610

silencing CHY1 and CHY2 85.30 ng/g DW 38 Diretto et al., 2007a11

Pa crtB, Pa crt1, Pa crtY 47 µg/g DW 3600 Diretto et al., 2007b12

Ba Or 5.01 µg/g DW / Lopez et al., 200813

corn seed Eh crtB, Eh crtl 4.5 µg/g DW / Aluru et al., 200814

Zm psy1, Pa crt1, P crtW, Gl lycb 57.35 µg/g DW 410 Zhu et al., 200815

Zm psy1, Pa crt1 59.32 µg/g DW 169 Naqvi et al., 20092

wheat seed Zm y1, Pa crt1 4.96 µg/g DW total carotenoids 10.8 Cong et al., 200916

cassava root bacterial crtB 6.67 µg/g DW 16 Welsch et al., 201017

Pa crtB, At DXS 30 µg/g dW 30 Sayre et al., 201118

sorghum seed Zm psy1, Pa crt1, At DXS 34.5 µg/g DW 8 Lipkie et al., 201319

Folate rice seed At GTPCHI, At ADCS 1723 µg/100g FW 100 Storozhenko et al., 200720

corn seed Ec folE (GTPCHI) 1.94 µg/g DW 2 Naqvi et al., 2009

2

γ-tocopherol corn seed At HPPD, At MPBQ MT 8.8 µg/g DW 3 Naqvi et al., 201121

α-tocotrienol rice seed At γ-TMT 33 µg/g DW 3 Zhang et al., 201322

tocopherol+tocotrienol corn seed barley HGGT approx. 900 nmol/g seed weight 6 Cahoon et al., 200323

iron rice seed Pv ferritin / 3 Goto et al., 199924

synthetic HLF 1.93 mg/100g FW 2 Nandi et al., 200225

Pv ferritin / 2 Lucca et al., 200226

Pv ferritin 34.7 µg/g DW 2 Vasconcelos et al., 200327

soybean ferritin SoyferH-1 20 µg/g DW 1.5 Qu et al., 200528

OsNAS3 34 µg/g DW 2.9 Lee et al., 200929

At NAS, Pv ferritin, Af phytase / 6.3 Wirth et al., 200930

OsNAS2 64 µg/g DW 3 Johnson et al., 201131

OsNAS2 26 µg/g DW 3 Lee et al., 201232

Pv ferritin, Hv NAS1, Os YSL2 7 µg/g DW 6 Masuda et al., 201233

corn seed Pv ferritin 35 µg/g 1.7 Drakakaki et al., 200534

barley seed AtZIP1 58 µg/gDW 2 Ramesh et al., 200435

zinc rice seed Pv ferritin 55.5 µg/g DW 1.6 Vasconcelos et al., 200327

OsNAS3 43 µg/g DW 2.2 Lee et al., 200929

OsNAS2 91 µg/g DW 2 Johnson et al., 201131

Pv ferritin, Hv NAS1, Os YSL2 20 µg/g DW 1.6 Masuda et al., 201233

barley seed AtZIP1 84,5 µg/g DW 2/2.5 Ramesh et al., 200435

copper rice seed OsNAS3 2.2 µg/g DW 1.7 Lee et al., 200929

ADCS, aminodeoxychorismate synthase; Af, Aspergillus fumigatus; At; Arabidopsis thaliana; Ba, Brassica oleraceae; CHY, non-heme β-carotene hydroxylase; crt1, carotene desaturase 1; crtB, phytoene synthase; crtY, lycopene β-cyclase; dhar; dehydroascorbate reductase; DW, dry weight; DXS, 1-deoxyxylulose-5-phosphate synthase; Ec, Escherichia coli; Eh, Erwinia herbicola; Fa, Fragaria x ananassa; FW, fresh weight; GalUR, D-galacturonic acid reductase; Gl, Gentiana lutea; GLOase, L-gulono-γ-lactone oxidase; GTP, guanosine triphosphate; GTPCHI, GTP cyclohydrolase 1; HLF, human lactoferrin; HPPD, ρ-hydroxyphenylpyruvate dioxygenase (HPPD); Hv, Hordeum vulgare; lcy, lycopene β-cyclase; LCY-e, lycopene epsilon cyclase; MPBQ MT, 2-methyl-6-phytylplastoquinol methyltransferase; NAS, nicotianamine synthase; Np, Narcissus pseudonarcissus; Or, orange; Os, Oryza sativa; P, Paracoccus ssp.; Pa, Pantoea ananatis (= Erwinia uredovora); psy1, phytoene synthase 1; Pv, Phaseolus vulgaris; St VTC2B, Solanum tuberosumGDP-L-galactose phosphorylase gene; Ta, Triticum aestivum; γ-TMT, γ-tocopherol methyltransferase; y1, phytoene synthase 1; Ysl, iron(II)-nicotianamine transporter; Zm, Zea mays

References

1. Chen, Z., Young, T.E., Ling, J., Chang, S.-C. & Gallie, D.R. Increasing vitamin C content of plants through enhanced ascorbate recycling. Proceedings of the National Academy of Sciences 100, 3525-3530 (2003).

2. Naqvi, S. et al. Transgenic multivitamin corn through biofortification of endosperm with three vitamins representing three distinct metabolic pathways. Proceedings of the National Academy of Sciences 106, 7762-7767 (2009).

3. Hemavathi et al. Over-expression of strawberry D-galacturonic acid reductase in potato leads to accumulation of vitamin C with enhanced abiotic stress tolerance. Plant science 177, 659-667 (2009).

4. Hemavathi et al. Enhanced ascorbic acid accumulation in transgenic potato confers tolerance to various abiotic stresses. Biotechnology letters 32, 321-330 (2010).

5. Bulley, S. et al. Enhancing ascorbate in fruits and tubers through over‐expression of the l‐galactose pathway gene GDP‐l‐galactose phosphorylase. Plant biotechnology journal 10, 390-397 (2012).

6. Ye, X. et al. Engineering the provitamin A (β-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science 287, 303-305 (2000). 7. Paine, J.A. et al. Improving the nutritional value of Golden Rice through increased pro-vitamin A content. Nature biotechnology 23, 482-487 (2005). 8. Ducreux, L.J.M. et al. Metabolic engineering of high carotenoid potato tubers containing enhanced levels of β-carotene and lutein. Journal of experimental Botany 56,

81-89 (2005). 9. Diretto, G. et al. Metabolic engineering of potato tuber carotenoids through tuber-specific silencing of lycopene epsilon cyclase. BMC Plant Biology 6, 13 (2006).

10. Lu, S. et al. The cauliflower Or gene encodes a DnaJ cysteine-rich domain-containing protein that mediates high levels of β-carotene accumulation. The Plant Cell Online 18, 3594-3605 (2006).

11. Diretto, G. et al. Silencing of beta-carotene hydroxylase increases total carotenoid and beta-carotene levels in potato tubers. BMC Plant Biology 7, 11 (2007). 12. Diretto, G. et al. Metabolic engineering of potato carotenoid content through tuber-specific overexpression of a bacterial mini-pathway. PLoS One 2, e350 (2007).

13. Lopez, A.B. et al. Effect of the cauliflower Or transgene on carotenoid accumulation and chromoplast formation in transgenic potato tubers. Journal of experimental Botany 59, 213-223 (2008).

14. Aluru, M. et al. Generation of transgenic maize with enhanced provitamin A content. Journal of experimental Botany 59, 3551-3562 (2008). 15. Zhu, C. et al. Combinatorial genetic transformation generates a library of metabolic phenotypes for the carotenoid pathway in maize. Proceedings of the National

Academy of Sciences 105, 18232-18237 (2008).

16. Cong, L. et al. Expression of phytoene synthase1 and carotene desaturase crtI genes result in an increase in the total carotenoids content in transgenic elite wheat (Triticum aestivum L.). J. Agric. Food Chem. 57, 8652-8660 (2009).

17. Welsch, R. et al. Provitamin A accumulation in cassava (Manihot esculenta) roots driven by a single nucleotide polymorphism in a phytoene synthase gene. The Plant Cell Online 22, 3348-3356 (2010).

18. Sayre, R. et al. The BioCassava plus program: biofortification of cassava for sub-Saharan Africa. Annu. Rev. Plant Biol. 62, 251-272 (2011). 19. Lipkie, T.E. et al. Bioaccessibility of carotenoids from transgenic provitamin A biofortified sorghum. J. Agric. Food Chem. 61, 5764-5771 (2013). 20. Storozhenko, S. et al. Folate fortification of rice by metabolic engineering. Nat. Biotechnol. 25, 1277-1279 (2007).

21. Naqvi, S. et al. Simultaneous expression of Arabidopsis ρ-hydroxyphenylpyruvate dioxygenase and MPBQ methyltransferase in transgenic corn kernels triples the tocopherol content. Transgenic research 20, 177-181 (2011).

22. Zhang, G.-Y. et al. Increased α-tocotrienol content in seeds of transgenic rice overexpressing Arabidopsis γ-tocopherol methyltransferase. Transgenic research 22, 89-

99 (2013). 23. Cahoon, E.B. et al. Metabolic redesign of vitamin E biosynthesis in plants for tocotrienol production and increased antioxidant content. Nat. Biotechnol. 21, 1082-1087

(2003). 24. Goto, F., Yoshihara, T., Shigemoto, N., Toki, S. & Takaiwa, F. Iron fortification of rice seed by the soybean ferritin gene. Nature biotechnology 17, 282-286 (1999). 25. Nandi, S. et al. Expression of human lactoferrin in transgenic rice grains for the application in infant formula. Plant science 163, 713-722 (2002). 26. Lucca, P., Hurrell, R. & Potrykus, I. Fighting iron deficiency anemia with iron-rich rice. Journal of the American College of Nutrition 21, 184S-190S (2002). 27. Vasconcelos, M. et al. Enhanced iron and zinc accumulation in transgenic rice with the< i> ferritin</i> gene. Plant science 164, 371-378 (2003).

28. Qu, L.Q., Yoshihara, T., Ooyama, A., Goto, F. & Takaiwa, F. Iron accumulation does not parallel the high expression level of ferritin in transgenic rice seeds. Planta 222, 225-233 (2005).

29. Lee, S. et al. Iron fortification of rice seeds through activation of the nicotianamine synthase gene. Proc. Natl. Acad. Sci. U. S. A. 106, 22014-22019 (2009). 30. Wirth, J. et al. Rice endosperm iron biofortification by targeted and synergistic action of nicotianamine synthase and ferritin. Plant biotechnology journal 7, 631-644

(2009). 31. Johnson, A.A. et al. Constitutive overexpression of the OsNAS gene family reveals single-gene strategies for effective iron-and zinc-biofortification of rice endosperm.

PLoS ONE 6, e24476 (2011). 32. Lee, S. et al. Activation of rice nicotianamine synthase 2 (OsNAS2) enhances iron availability for biofortification. Molecules and cells 33, 269-275 (2012). 33. Masuda, H. et al. Iron biofortification in rice by the introduction of multiple genes involved in iron nutrition. Scientific reports 2 (2012).

34. Drakakaki, G. et al. Endosperm-specific co-expression of recombinant soybean ferritin and Aspergillus phytase in maize results in significant increases in the levels of bioavailable iron. Plant molecular biology 59, 869-880 (2005).

35. Ramesh, S.A., Choimes, S. & Schachtman, D.P. Over-expression of an Arabidopsis zinc transporter in Hordeum vulgare increases short-term zinc uptake after zinc deprivation and seed zinc content. Plant Mol. Biol. 54, 373-385 (2004).

Supplementary Table 3 Key characteristics of studies on acceptance of, and willingness-to-pay for GM biofortified crops

Crop, Micronutrient Country, region

Method of data collection

Year of data collection Sample size

e

Key outcome indicator References

Rice

Pro-Vitamin A United States Telephone survey 2001 1203 Acceptance Hallman et al., 20021

United States, Mississippi Mail survey 2001 574 WTP Lusk, 20032

China, Beijing In-person interview 2002 599 WTP/WTA Curtis & Moeltner, 2006 ;Li, Q et al., 20033; 4

United States Mail survey 2004 501 WTP Lusk & Rozan, 20055

France Mail survey 2004 200 Acceptance, WTP Rozan et al., 20076

Philippines, Laguna Experiment 2006 60f;100

f,g WTP Corrigan et al., 2009 ; Depositario et al., 2009

7; 8

India; India, Ahmedabad, Gujarat

Mixed methodsd 2006 2937; (602;110) WTP, preference Deodhar et al., 2008

9

Folate (Vitamin B9)

China, Shanxic In-person interview 2008 944 Acceptance, WTP De Steur et al., 2010

10

China, Shanxic Experiment 2011 252

h (120

f; 132) WTP De Steur et al., 2012 & 2013

11; 12;

China, Shanxic Experiment 2011 126 WTP, preference De Steur et al., 2014

13

Vitamin C Malaysia, Klang Valley In-person survey 2009 434 Acceptance Amin et al., 201114

Cassava

Pro-vitamin A Brazil, North-East regiond In-person survey 2006 414 WTP, preference Gonzalez et al., 2009

15

Apple

Pro-vitamin A New Zealand, Auckland Experiment 2005 123 WTP Kassardjian et al., 201216

Vitamin C New Zealand, Auckland Experiment 2005 146 WTP Kassardjian et al., 201216

Cookie

Vitamin E Italy Telephone survey 2004 366 WTP Canavari & Nayga, 200917

Broccoli

Vitamin Ea United States,

Iowa & Pennsylvania Experiment 2007 190 WTP Colson et al., 2011

18

Tomato

Vitamin Ea United States,

Iowa, & Pennsylvania Experiment 2007 190 WTP Colson et al., 2011

18

Potato

Vitamin Ea United States,

Iowa & Pennsylvania Experiment 2007 190 WTP Colson et al., 2011

18

Vegetablesb

Vitamin Ea United States,

Iowa & Pennsylvania Experiment 2007 190 WTP, preference Colson & Huffman, 2011

19

WTA, willingness-to-accept; WTP, willingness-to-pay Note: Unless otherwise stated, all studies use a randomized sample of adults. For an overview of methods, see supplementary discussion. a These studies examined differences in consumer preference between intragenic and transgenic applications.

b The analysis of this study focuses on the average of broccoli, tomato and potato.

c High-risk region of the targeted vitamin deficiency.

d The applied method depends on the targeted sample: 602 in-person interviews (population based sample) versus 110 online surveys (university based sample).

e Sample sizes in parentheses refer to differences in research location

9 or target group

11, 12.

f Based on a student sample.

g Depositario et al.

8 only report findings on the largest sample.

h The sample consists of women of childbearing age (120 students versus 132 non-students), which are key beneficiaries of folate interventions.

References

1. Hallman, W.K., Adelaja, A.O., Schilling, B.J. & Lang, J.T. (Rutgers, The State University of New Jersey, 2002). 2. Lusk, J.L. Effects of cheap talk on consumer willingness-to-pay for golden rice. American Journal of Agricultural Economics 85, 840-856 (2003). 3. Curtis, K.R. & Moeltner, K. Genetically Modified Food Market Participation and Consumer Risk Perceptions: A Cross-Country Comparison. Canadian Journal of

Agricultural Economics 54, 289-310 (2006). 4. Li, Q., Curtis, K.R., McCluskey, J.J. & Wahl, T.I. Consumer Attitudes toward Genetically Modified Foods in Beijing, China. AgBioForum 5, 145-152 (2003). 5. Lusk, J.L. & Rozan, A. Consumer acceptance of biotechnology and the role of second generation technologies in the USA and Europe. Trends in Biotechnology 23,

386-387 (2005). 6. Rozan, A., Lusk, J.L. & Campardon, M. Consumer acceptance of a genetically modified organism of the second generation: The Golden Rice. Revue D Economie

Politique 117, 843-852 (2007).

7. Corrigan, J.R., Depositario, D.P.T., Nayga, R.M., Jr., Wu, X. & Laude, T.P. Comparing Open-Ended Choice Experiments and Experimental Auctions: An Application to Golden Rice. American Journal of Agricultural Economics 91, 837-853 (2009).

8. Depositario, D.P.T., Nayga, R.M., Jr., Wu, X. & Laude, T.P. Effects of Information on Consumers' Willingness to Pay for Golden Rice. Asian Economic Journal 23, 457-

476 (2009). 9. Deodhar, S.Y., Ganesh, S. & Chern, W.S. Emerging markets for GM foods: an Indian perspective on consumer understanding and the willingness to pay. Int. J.

Biotechnol. 10, 570-587 (2008). 10. De Steur, H. et al. Willingness-to-accept and purchase genetically modified rice with high folate content in Shanxi Province, China. Appetite 54, 118-125 (2010).

11. De Steur, H., Gellynck, X., Feng, S.Y., Rutsaert, P. & Verbeke, W. Determinants of willingness-to-pay for GM rice with health benefits in a high-risk region: Evidence from experimental auctions for folate biofortified rice in China. Food Quality and Preference 25, 87-94 (2012).

12. De Steur, H., Buysse, J., Feng, S. & Gellynck, X. The role of information on consumers’ willingness-to-pay for GM rice with health benefits. An application to China. Asian Economic Journal 27, 391-408 (2013).

13. De Steur, H., Feng, S., Xiaoping, S. & Gellynck, X. Consumer preferences for micronutrient strategies in China. A comparison between folic acid supplementation and folate biofortification. Public Health Nutr. 17, 1410–1420 (2014).

14. Amin, L., Azlan, N.A.A. & Hashim, H. Ethical perception of cross-species gene transfer in plant. African Journal of Biotechnology 10, 12457-12468 (2011). 15. Gonzalez, C., Johnson, N. & Qaim, M. Consumer Acceptance of Second-Generation GM Foods: The Case of Biofortified Cassava in the North-East of Brazil. Journal

of Agricultural Economics 60, 604-624 (2009).

16. Kassardjian, E., Robin, S. & Ruffieux, B. L'hostilite aux OGM survit-elle a des produits attractifs? (Does Consumer's Aversion to GM Food Survive a Yummy Apple? With English summary.). Revue Francaise d'Economie 26, 121-150 (2012).

17. Canavari, M. & Nayga, R.M., Jr. On Consumers' Willingness to Purchase Nutritionally Enhanced Genetically Modified Food. Applied Economics 41, 125-137 (2009).

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19. Colson, G. & Huffman, W.E. Consumers' Willingness to Pay for Genetically Modified Foods with Product-Enhancing Nutritional Attributes. American Journal of Agricultural Economics 93, 358-363 (2011).

Supplementary Table 4 Results from micro-level analyses on the market potential of Transgenic biofortified crops: willingness-to-pay and acceptance Crop Rice Rice Cassava Cookie Broccoli (1) Tomato (2) Potato (3) (1)+(2)+(3) Apple Apple

Micronutrient Pro-vitamin A Folate Pro-vitamin A Vitamin E Vitamin E Vitamin E Vitamin E Vitamin E Vitamin C Pro-vitamin A

Country

China1, 2

, France3, India

4,

Philippines5, 6

, US21, 23, 24

China7-10c

Brazil11

Italy12

US13

US13

US13

US14

New Zealand

15

New Zealand

15

WT

P

Value (% premium)

TG 33.3, 40.0, 66.7a (P);

38.0 (C); 19.5 (I); 19.5, 38.0 (US)

25.8a; 33.7, 34.0 64.0, 70.0 36.1 32.6 24.5 62.3 26.0 48.0 13.9 (vs infiltration)

25.2 (vs irradiation)

IG 34.8 40.3 60.8 31.0

Purchase intention (% sample)

TG 43.9 (C) ; 16.9 (F) 82.5, 84.9; 77.5a 57.4

IG

Preference (% sample)

TG 65.9, 71.1 (indifferent: +1.9, 5.2) (I)

b

66.7 (indifferent: +18.2)

40.0, 75.0 (indifferent: 5.0, 6.0)

37.0 (indifferent: +28.0)

IG 48.0 (indifferent: +32.0)

Crop Rice Rice Rice Micronutrient Vitamin A Folate Vitamin C Country France

3, US

16 China

10 Malaysia

17

Accept. Market share

(% approval) TG 50.0 (F); 80.7 (US) 62.2

(indifferent: +26.6)

Score (1-7)

3.67 (E) - 2.89 (R)

C, China; E, ethical; F, France; I, India; IG, Intragenic; P, Philippines; R, religious; TG, Transgenic; US, United States a Based on a student sample.

b Figures refer to the two large, population based samples. The preference rate of the university based sample, i.e. 37.3% (+22.7% indifferent), is not presented.

c Based on sample of women of childbearing age.

References 1. Curtis, K.R. & Moeltner, K. Genetically Modified Food Market Participation and Consumer Risk Perceptions: A Cross-Country Comparison. Canadian Journal of

Agricultural Economics 54, 289-310 (2006). 2. Li, Q., Curtis, K.R., McCluskey, J.J. & Wahl, T.I. Consumer Attitudes toward Genetically Modified Foods in Beijing, China. AgBioForum 5, 145-152 (2003). 3. Rozan, A., Lusk, J.L. & Campardon, M. Consumer acceptance of a genetically modified organism of the second generation: The Golden Rice. Revue D Economie

Politique 117, 843-852 (2007).

4. Deodhar, S.Y., Ganesh, S. & Chern, W.S. Emerging markets for GM foods: an Indian perspective on consumer understanding and the willingness to pay. Int. J. Biotechnol. 10, 570-587 (2008).

5. Corrigan, J.R., Depositario, D.P.T., Nayga, R.M., Jr., Wu, X. & Laude, T.P. Comparing Open-Ended Choice Experiments and Experimental Auctions: An Application to Golden Rice. American Journal of Agricultural Economics 91, 837-853 (2009).

6. Depositario, D.P.T., Nayga, R.M., Jr., Wu, X. & Laude, T.P. Effects of Information on Consumers' Willingness to Pay for Golden Rice. Asian Economic Journal 23, 457-

476 (2009).

7. De Steur, H., Buysse, J., Feng, S. & Gellynck, X. The role of information on consumers’ willingness-to-pay for GM rice with health benefits. An application to China. Asian Economic Journal 27, 391-408 (2013).

8. De Steur, H., Feng, S., Xiaoping, S. & Gellynck, X. Consumer preferences for micronutrient strategies in China. A comparison between folic acid supplementation and folate biofortification. Public Health Nutr. 17, 1410–1420 (2014).

9. De Steur, H., Gellynck, X., Feng, S.Y., Rutsaert, P. & Verbeke, W. Determinants of willingness-to-pay for GM rice with health benefits in a high-risk region: Evidence from experimental auctions for folate biofortified rice in China. Food Quality and Preference 25, 87-94 (2012).

10. De Steur, H. et al. Willingness-to-accept and purchase genetically modified rice with high folate content in Shanxi Province, China. Appetite 54, 118-125 (2010). 11. Gonzalez, C., Johnson, N. & Qaim, M. Consumer Acceptance of Second-Generation GM Foods: The Case of Biofortified Cassava in the North-East of Brazil. Journal

of Agricultural Economics 60, 604-624 (2009). 12. Canavari, M. & Nayga, R.M., Jr. On Consumers' Willingness to Purchase Nutritionally Enhanced Genetically Modified Food. Applied Economics 41, 125-137 (2009).

13. Colson, G.J., Huffmann, W.E. & Rousu, M.C. Improving the Nutrient Content of Food through Genetic Modification: Evidence from Experimental Auctions on Consumer Acceptance. Journal of Agricultural and Resource Economics 36, 343-364 (2011).

14. Colson, G. & Huffman, W.E. Consumers' Willingness to Pay for Genetically Modified Foods with Product-Enhancing Nutritional Attributes. American Journal of Agricultural Economics 93, 358-363 (2011).

15. Kassardjian, E., Robin, S. & Ruffieux, B. L'hostilite aux OGM survit-elle a des produits attractifs? (Does Consumer's Aversion to GM Food Survive a Yummy Apple? With English summary.). Revue Francaise d'Economie 26, 121-150 (2012).

16. Hallman, W.K., Adelaja, A.O., Schilling, B.J. & Lang, J.T. (Rutgers, The State University of New Jersey, 2002). 17. Amin, L., Azlan, N.A.A. & Hashim, H. Ethical perception of cross-species gene transfer in plant. African Journal of Biotechnology 10, 12457-12468 (2011).

Supplementary Table 5 Key characteristics of studies on the potential health impact and cost-effectiveness of GM biofortified crops

Product, Micronutrient Country, region Time horizon Key outcome indicator References

Rice

Multia

China 30 years Burden, health impact, cost, CEA De Steur et al, 20121

Pro-vitamin A China 30 years Burden, health impact, cost, CEA Steur et al, 20121

India 30 years Burden, health impact, cost, CEA/CBA Stein et al, 20062

Philippines 30 years Burden, health impact, cost, CBA Zimmermann & Qaim, 20043

Folate China 30 years Burden, health impact, cost, CEA De Steur et al, 20121

China 30 years Burden, health impact De Steur et al, 20104

China, Shanxib 30 years Burden, health impact, cost, CEA De Steur et al, 2012

5

Mustard oil

Vitamin A India 16 years Health impact, costs, CEA Chow et al, 20106

CBA, cost-benefit analysis; CEA, cost-effectiveness analysis a Pro-vitamin A + Folate (Vitamin B9) + zinc + iron.

b Shanxi Province is considered one of the world most important risk regions of folate deficiency

7.

References 1. De Steur, H. et al. Potential impact and cost-effectiveness of multi-biofortified rice in China. New Biotech. 29, 432-442 (2012). 2. Stein, A.J., Sachdev, H.P.S. & Qaim, M. Potential impact and cost-effectiveness of Golden Rice. Nat. Biotechnol. 24, 1200-1201 (2006). 3. Zimmermann, R. & Qaim, M. Potential health benefits of Golden Rice: a Philippine case study. Food Policy 29, 147-168 (2004). 4. De Steur, H. et al. Health impact in China of folate-biofortified rice. Nat. Biotechnol. 28, 554-556 (2010).

5. De Steur, H., Blancquaert, D., Gellynck, X., Lambert, W. & Van Der Straeten, D. Ex-ante Evaluation of Biotechnology Innovations: the Case of Folate Biofortified Rice in China. Curr. Pharm. Biotechnol. 13, 2751-2760 (2012).

6. Chow, J., Klein, E.Y. & Laxminarayan, R. Cost-Effectiveness of "Golden Mustard" for Treating Vitamin A Deficiency in India. PLoS One 5, e12046 (2010).

7. Ren, A. et al. Awareness and use of folic acid, and blood folate concentrations among pregnant women in northern China - An area with a high prevalence of neural tube defects. Reprod. Toxicol. 22, 431-436 (2006).

Supplementary Table 6 Results from macro-level analyses on the market potential of transgenic biofortified crops: disease burden, health impacts and cost-effectiveness/cost-

benefits

Product Rice Rice Rice Mustard oil Micronutrient

Multi (pro-vitamin A + folate + zinc + iron)

a Pro-vitamin A

Folate

Pro-vitamin A

Country China1 China

1, India

2, Philippines

3 China

1, 4 (& Shanxi

5, d) India

6

Burden of micronutrient deficiency

Current burden (mi. DALYs lost/year)

10.64 1.99 (C), 2.33 (I), 0.27 (P) 0.31 (C); 0.11 (S)

0.31

Health impacts Low - High (mi. DALYs saved/year)

1.19 - 4.91 0.33 - 1.20 (C); 0.20 - 1.38 (I); 0.02 - 0.08 (P)

0.12 - 0.26; 0.06 - 0.19 (C); 0.02 - 0.07

(S)

0.91 - 1.69

Low - High (% burden reduction/year)

11.0 - 46.0 17.0 - 60.0 (C); 8.8 - 59.4 (I); 5.7 - 31.5 (P)

36.9 - 81.9 (C); 20.0 - 60.0 (C, S)

Low - High (lives saved/year)

5 500 - 39 700 (I) 113 000 - 654 000

Cost-effectiveness

Low - High ($/DALY saved/year)

9.6 - 2.3 7.9

b - 1.9

b

18.1 - 5.0 (C); 19.4 - 3.1 (I)

64.2 - 21.4 (C); 120.3 - 40.1 (S)

Low - High ($/live saved/year)

358 - 54 (I)

Low - High (ICER, $/DALY saved/year)

450 - 403e

Low - High (ICER, $/live saved/year)

303 300 - 53 000e

Cost-benefit Low - High (IRR, %)

29 - 66, 35 - 77, 44 - 93 (I)c;

66 - 133 (P)

Low - High (BCR)

26 - 163, 52 - 327, 129 - 816 (I)

c

BCR, benefit-cost ratio; C, China; DALY, Disability-Adjusted Life Year, I, India; ICER, incremental cost-effectiveness ratio; IRR, internal rate of return; P, Philippines; S, Shanxi Note: Except for the burden figures, a distinction is made between a low and high impact scenario. Additional data, including cost figures, are included in the Supplementary Data. a Iron and zinc are considered ‘conventional’ traits as their levels – unlike pro-vitamin A en folate, can be increased in rice with conventional breeding methods.

b Figures refer to a scenario of gene stacking, i.e. where the different micronutrient traits are combined in one gene construct and transferred into a new stacked event.

c Figures based on a DALY valued at standard values for a DALY (500$, 1000$) or at the national per capita income (2500$), respectively.

d Based on regional data for Shanxi Province, one of the world most important risk regions of folate deficiency.

e ICER figures as compared to vitamin A supplementation.

References 1. De Steur, H. et al. Potential impact and cost-effectiveness of multi-biofortified rice in China. New Biotech. 29, 432-442 (2012). 2. Stein, A.J., Sachdev, H.P.S. & Qaim, M. Potential impact and cost-effectiveness of Golden Rice. Nat. Biotechnol. 24, 1200-1201 (2006). 3. Zimmermann, R. & Qaim, M. Potential health benefits of Golden Rice: a Philippine case study. Food Policy 29, 147-168 (2004). 4. De Steur, H. et al. Health impact in China of folate-biofortified rice. Nat. Biotechnol. 28, 554-556 (2010).

5. De Steur, H., Blancquaert, D., Gellynck, X., Lambert, W. & Van Der Straeten, D. Ex-ante Evaluation of Biotechnology Innovations: the Case of Folate Biofortified Rice in China. Curr. Pharm. Biotechnol. 13, 2751-2760 (2012).

6. Chow, J., Klein, E.Y. & Laxminarayan, R. Cost-Effectiveness of "Golden Mustard" for Treating Vitamin A Deficiency in India. PLoS One 5, e12046 (2010).

Supplementary Figure 1 Comparison of conventional and transgenic breeding techniques for biofortification of staple crops with β-carotene. The blue bars indicate the natural variation of β-carotene in staple crops, which can be obtained by conventional breeding. The red bars indicate β-carotene levels reached thus far by metabolic engineering. All values are reported in µg/g dry weight (DW). * Total carotenoid content. For calculations, see Supplementary Table 1.

0

10

20

30

40

50

60

70

corn rice cassava potato wheat*

β-c

aro

ten

e (

µg

/g D

W)

Conventional breeding

Metabolic engineering

Present Status and Market Potential of Transgenic Biofortified Crops. Nature Biotechnology, 33(1), 25-29.

Hans De Steur, Dieter Blancquaert, Simon Strobbe, Willy Lambert, Xavier Gellynck, Dominique Van Der Straeten

Search strategy and procedures

Study selection

Data extraction

Focus

Key words

Focus

Key words

Focus

Key words "GM", "genetically modified", "transgenic", "biofortified" (crop/food) or similar, "golden" AND ("health impact", "cost-effectiveness",

cost-benefit" or similar

Methods

We conduct three independent reviews, as shown below, through an extensive search for published articles in ISI Web of Knowledge and, in the case of

socio-economic research, also EconLit and AgEcon Search (working papers). The search syntax (free text words) was developed in Web of Knowledge

and, if needed, adapted to the other electronic databases. As not all socio-economic studies on GM crops with elevated micronutrient levels specifically

refer to terminology like "biofortification", a broader syntax was used to include all relevant research. All records were subsequently merged into three

separate databases (Endnote, version X5, Thomson Reuters, NY, USA).

- The selection of studies broadly consists of three steps. First, duplicates were removed (regarding socio-economic research, this also includes

conference papers that are available in AGECON but also published as a journal article e.g. in Web of Knowledge). Second, title and abstracts were

screened. Finally, full text papers were read to decide upon their inclusion.

Depending on the reviewed topic, data was extracted on the product attributes (targeted product, nutrient, applied technology), study characteristics (data

collection, study location, sample) and outcome indicators (e.g. economic valuation, preference or acceptance, burden analysis, health impact

assessment, cost analysis, economic evaluation).

"Biofortification" or similar; "enhancement AND target compound" or similar.

- Common criteria for excluding studies for the reviews are: (a) editorials, comments, book reviews and manuals; (b) languages other than English, French

or Spanish; and (c) lack of specification of the targeted transgenic biofortified crop or micronutrient. A specific inclusion criteria for the biotech studies was

the success (>2-fold) of engineering attempts of staple crops, except for ascorbate, where a treshold value of 1.4 fold increase was used. A specific

inclusion criteria for socio-economic studies refers to the presence of key outcome indicators (e.g. economic valuation, preference or acceptance, burden

analysis, health impact assessment, cost analysis, economic evaluation).

- Due to the specific scope and objective of these review studies, our data extraction aims to provide an exhaustive overview of the current status of socio-

economic research in the field of transgenic biofortification rather than to look for findings that are consistent across studies, like meta-analysis. Therefore,

one needs to take into account the potential risk of study-specific biases, e.g. measurement bias, selection bias and secondary data biases. Caution is

needed when interpreting the findings.

Biotechnology research

Socio-economic research

Biotechnology studies on transgenic biofortification

Willingness-to-pay studies (premium, purchase intention, preference) and consumer acceptance studies on transgenic biofortified

crops

Health impact, cost-effectiveness and cost-benefit analyses on transgenic biofortified crops

Macro-level analysis

Micro-level analysis

"GM", "genetically modified", "transgenic", "biofortified" (crop/food) or similar, AND "acceptance", "willingness-to-pay" , "purchase

intention", "preference", "valuation", or similar

Supplementary Dataset

ID Reference Outcome Outcome

Compound Crop Tissue Gene Compound level Fold increase

1 Chen et al ., 2003 Ascorbate corn seed Ta dhar 160 nmol/g FW 1,9

2 Naqvi et al ., 2009 Ascorbate corn seed Os dhar 110 µg/g DW 6

3 Hemavathi et al ., 2009 Ascorbate potato tuber Fa GalUR 3.2 µmol/g FW 2

4 Hemavathi et al ., 2010 Ascorbate potato tuber rat GLOase 3 µmol/g FW 1,4

5 Bulley et al ., 2012 Ascorbate potato tuber St VTC2B 1.83 mg/g DW 2,7

6 Ye et al. , 2000 β-carotene rice seed Np psy, Pa crt1, Np lcy 1.6 µg/g DW total carotenoids /

7 Paine et al ., 2005 β-carotene rice seed Zm psy1, Pa crt1 31 µg/g DW /

8 Ducreux et al ., 2005 β-carotene potato tuber Pa crtB 11 µg/g DW /

9 Diretto et al ., 2006 β-carotene potato tuber silencing LCY-e 44 ng/g DW 14

10 Lu et al ., 2006 β-carotene potato tuber Ba Or 24 µg/g DW total carotenoids 6

11 Diretto et al ., 2007a β-carotene potato tuber silencing CHY1 and CHY2 85.30 ng/g DW 38

12 Diretto et al ., 2007b β-carotene potato tuber Pa crtB, Pa crt1, Pa crtY 47 µg/g DW 3600

13 Lopez et al ., 2008 β-carotene potato tuber Ba Or 5.01 µg/g DW /

14 Aluru et al ., 2008 β-carotene corn seed Eh crtB, Eh crtl 4.5 µg/g DW /

15 Zhu et al ., 2008 β-carotene corn seed Zm psy1, Pa crt1, P crtW, Gl lycb 57.35 µg/g DW 410

2 Naqvi et al ., 2009 β-carotene corn seed Zm psy1, Pa crt1 59.32 µg/g DW 169

16 Cong et al ., 2009 β-carotene wheat seed Zm y1, Pa crt1 4.96 µg/g DW total carotenoids 10,8

17 Welsch et al., 2010 β-carotene cassava root bacterial crtB 6.67 µg/g DW 16

18 Sayre et al., 2011 β-carotene cassava root Pa crtB, At DXS 30 µg/g dW 30

19 Lipkie et al., 2013 β-carotene sorghum seed Zm psy1, Pa crt1, At DXS 34.5 µg/g DW 8

20 Storozhenko et al ., 2007 Folate rice seed At GTPCHI, At ADCS 1723 µg/100g FW 100

2 Naqvi et al ., 2009 Folate corn seed Ec folE (GTPCHI) 1.94 µg/g DW 2

21 Naqvi et al ., 2011 γ-tocopherol corn seed At HPPD, At MPBQ MT 8.8 µg/g DW 3

22 Zhang et al ., 2013 α-tocotrienol rice seed At γ-TMT 33 µg/g DW 3

23 Cahoon et al., 2003 tocopherol+tocotrienol corn seed barley HGGT approx. 900 nmol/g seed weight 6

24 Goto et al ., 1999 iron rice seed Pv ferritin / 3

25 Nandi et al ., 2002 iron rice seed synthetic HLF 1.93 mg/100g FW 2

26 Lucca et al ., 2002 iron rice seed Pv ferritin / 2

27 Vasconcelos et al ., 2003 iron rice seed Pv ferritin 34.7 µg/g DW 2

28 Qu et al., 2005 iron rice seed soybean ferritin SoyferH-1 20 µg/g DW 1.5

29 Lee et al ., 2009 iron rice seed OsNAS3 34 µg/g DW 2,9

30 Wirth et al ., 2009 iron rice seed At NAS, Pv ferritin, Af phytase / 6,3

31 Johnson et al., 2011 iron rice seed OsNAS2 64 µg/g DW 3

29 Lee et al ., 2012 iron rice seed OsNAS2 26 µg/g DW 3

32 Masuda et al ., 2012 iron rice seed Pv ferritin, Hv NAS1, Os YSL2 7 µg/g DW 6

33 Drakakaki et al ., 2005 iron corn seed Pv ferritin 35 µg/g 1,7

34 Ramesh et al., 2004 iron barley seed AtZIP1 58 µg/gDW 2

27 Vasconcelos et al ., 2003 zinc rice seed Pv ferritin 55.5 µg/g DW 1,6

29 Lee et al ., 2009 zinc rice seed OsNAS3 43 µg/g DW 2,2

31 Johnson et al. , 2011 zinc rice seed OsNAS2 91 µg/g DW 2

32 Masuda et al ., 2012 zinc rice seed Pv ferritin, Hv NAS1, Os YSL2 20 µg/g DW 1,6

34 Ramesh et al., 2004 zinc barley seed AtZIP1 84,5 µg/g DW 2/2.5

35 Lee et al ., 2009 copper rice seed OsNAS3 2.2 µg/g DW 1,7

Product attributes

Biotechnology research

Remark

Multibiofortification studies: 2, 27, 29, 31, 32, 34

Abbreviations

ADCS, aminodeoxychorismate synthase

Af, Aspergillus fumigatus

At; Arabidopsis thaliana

Ba, Brassica oleraceae

CHY, non-heme β-carotene hydroxylase

crt1, carotene desaturase 1

crtB, phytoene synthase

crtY, lycopene β-cyclase

dhar; dehydroascorbate reductase

DW, dry weight

DXS, 1-deoxyxylulose-5-phosphate synthase

Ec, Escherichia coli

Eh, Erwinia herbicola

Fa, Fragaria x ananassa

FW, fresh weight

GalUR, D-galacturonic acid reductase

Gl, Gentiana lutea

GLOase, L-gulono-γ-lactone oxidase

GTP, guanosine triphosphate

GTPCHI, GTP cyclohydrolase 1

HLF, human lactoferrin

HPPD, ρ-hydroxyphenylpyruvate dioxygenase (HPPD)

Hv, Hordeum vulgare; lcy, lycopene β-cyclase

LCY-e, lycopene epsilon cyclase; MPBQ MT, 2-methyl-6-phytylplastoquinol methyltransferase

NAS, nicotianamine synthase

Np, Narcissus pseudonarcissus

Or, orange; Os, Oryza sativa

P, Paracoccus ssp.

Pa, Pantoea ananatis (= Erwinia uredovora)

psy1, phytoene synthase 1

Pv, Phaseolus vulgaris

St VTC2B, Solanum tuberosumGDP-L-galactose phosphorylase gene

Ta, Triticum aestivum

γ-TMT, γ-tocopherol methyltransferase

y1, phytoene synthase 1

Ysl, iron(II)-nicotianamine transporter

Zm, Zea mays

ID Reference OutcomePreference (GM

biofortified over Remark

Crop Micro

nutrient

Technology* Method Nature of

study*

Year UN subregion Country Region/

State/ City

Description Size Key indicators Measure Method Value (% premium for

GM biofortified crops)*

Purchase intention

(% sample)

Market share (%

sample)

Measurement

method

Market share

(% sample)

Awareness

(% sample)*

Perception (%

sample)*

Reluctance

1 Amin et al., 2011*Rice Vitamin C Transgenic In-person survey Hypothetical 2009 Southeastern AsiaMalaysia Klang Valley Random, adults 434 Acceptance Likert scale (1-7) 3.67 (E), 2.89 (R)*

2 Canavari & Nayga, 2009Cookie* Vitamin E Transgenic Telephone survey Hypothetical 2004 Southern Europe Italy Random, adults 366* Valuation WTP Discrete choice modelling 40.5% (purchase intention, incl. discount) 59.5%

WTP • 36.1% (premium)*

• 2.7% (equal price)*

• 1.7% (discount)*

3A Colson & Huffman, 2011Average of Broccoli, Tomato and PotatoVitamin E* Intragenic Experiment Non-hypothetical2007 Northern AmericaUnited StatesDes Moines, Iowa; Harrisburg, PennsylvaniaRandom, adults 190 Valuation, Preference WTP Exp. auction (uniform-price)31.0% 48 % (+32% indifferent)

Transgenic 26.0% 37% (+28% indifferent)

3B Colson et al., 2011Broccoli Vitamin E* Intragenic Experiment Non-hypothetical2007 Northern AmericaUnited StatesDes Moines, Iowa; Harrisburg, PennsylvaniaRandom, adults 190 Valuation WTP Exp. auction (random nth price)34.8%

Tomato Intragenic 40.3%

Potato Intragenic 60.8%

Broccoli Transgenic 32.6%

Tomato Transgenic 24.5%

Potato Transgenic 62.3%

5A Corrigan et al. , 2009Rice Vitamin A Transgenic* Experiment Non-hypothetical2006 Southeastern AsiaPhilippines University of Las Baños, LagunaRandom, students 60 Valuation WTP Choice experiment (OECE)33.3% 78.0% 63.0% (S)

100 Exp. auction (uniform-price)66.7% 70.0% 55.0% (S)

5B Depositario et al., 2009Rice Vitamin A Transgenic* Experiment Non-hypothetical2006 Southeastern AsiaPhilippines University of Las Baños, LagunaRandom, students 100 Valuation WTP Exp. auction (uniform-price)40.0% 70.0% 55.0% (S)

7A Curtis & Moeltner, 2006Rice Vitamin A Transgenic* In-person interview Hypothetical 2002 Eastern Asia China Beijing Random, adults 599 Valuation WTP/WTA Contingent valuation (double-bounded dichotomous choice) 81.3% (purchase intention)

7B Li, Q et al., 2003 Rice Vitamin A Transgenic* In-person interview Hypothetical 2002 Eastern Asia China Beijing Random, adults 599 Valuation WTP/WTA Contingent valuation (double-bounded dichotomous choice)38.0% 86.0% (purchase intention, incl. discount) 14.0%

• 43.9% (premium)

• 37.4% (equal price)

• 4.7% (discount)

9 De Steur et al., 2010Rice Folate Transgenic** In-person interview Hypothetical 2008 Eastern Asia China Shanxi ProvinceRandom, adults 944 Acceptance, Valuation WTP Contingent valuation (open-ended)34.0% Categorical (yes, indifferent, no)62.2% (of which 26.6% indifferent) 11.2%

10A De Steur et al., 2012Rice Folate Transgenic** Experiment Non-hypothetical2011 Eastern Asia China Taiyuan, Shanxi ProvinceRandom, women of cba 252 Valuation WTP Exp. auction (2nd price) 33.7% 82.5% (premium) 17.5%

• student sample 120 25.8% 77.5% (premium) 22.5%

• non-student sample 132 40.0% 87.1% (premium) 12.9%

10B De Steur et al., 2013Rice Folate Transgenic** Experiment Non-hypothetical2011 Eastern Asia China Taiyuan, Shanxi ProvinceRandom, women of cba 252 Valuation WTP Exp. auction (2nd price) 33.7% 82.5% (premium) 17.5%

10C De Steur et al., 2014Rice Folate Transgenic** Experiment Non-hypothetical2011 Eastern Asia China Taiyuan, Shanxi ProvinceRandom, women of cba 126* Valuation, Preference WTP Exp. auction (2nd price) 33.9%** 84.9% (premium) 66.7% (18.2 % indifferent)* 15.1%*

13 Deodhar et al., 2008Rice Vitamin A Transgenic* Mixed methods Hypothetical 2006 Southern Asia India Ahmedabad, Gujarat & IndiaRandom, adults 2937** Valuation, Preference WTP Random utility method 19.5% 65.9% (+5.2% indifferent) 28.9%

In-person interview Ahmedabad, Gujarat 602 71.1 % (+1.9% indifferent) 26.9%

Online survey Academic institutes, Ahmedabad, Gujarat 110 37.3% (+22.7% indifferent) 40.0%

14 Gonzalez et al., 2009Cassava Vitamin A Transgenic In-person survey Hypothetical 2006 South America Brazil Araripina, Lagoa Grande, Correntes and Itambe, Pernambuco State, North-East BrazilRandom, adults 414 Valuation, Preference WTP Contingent valuation (double-bounded dichotomous choice)64.0% 75.0% (+5% indifferent)** 20.0%

Choice modeling (contingent ranking)70.0% 40.0% (+6% indifferent)***

15 Hallman et al., 2002**Rice Vitamin A Transgenic* Telephone survey Hypothetical 2001 Northern AmericaUnited States Random, adults 1203 Acceptance Categorical* 80.7% (of which 46.8% strong approval) 17.5%

16 Kassardjian et al., 2012Apple Vitamin C Transgenic Experiment Non-hypothetical2005 Australia & New ZealandNew ZealandAuckland Random, adults 146 Valuation WTP Exp. auction (2nd price) 48.0% 57.4% (premium) 11.0%

Vitamin A 123 25.2% (vs irradiation)

13.9% (vs infiltration)

17 Lusk, 2003 Rice Vitamin A Transgenic* Mail survey Hypothetical 2001 Northern AmericaUnited StatesMississippi Random, adults 574 Valuation WTP Contingent valuation (double-bounded dichotomous choice) 19.5%*** 26.7%

18 Lusk & Rozan, 2005***Rice Vitamin A Transgenic* Mail survey Hypothetical 2004 Northern AmericaUnited States Random, adults 501 Valuation WTP Contingent valuation 38.0%

19 Rozan et al., 2007Rice Vitamin A Transgenic* Mail survey Hypothetical 2004 Western Europe France Random, adults 200 Acceptance, Valuation WTP Contingent valuation 25,5% (intention to purchase) Dichotomous (yes, no)50.0% 5.0% 74.5%

16.9% (premium)**

36.1% (equal price)**

28.2% (discount)**

*See also Amin, et al. (2011) for a similar analysis on "synthetic biology"*based on GM wheat*Including antioxidants * Because trans- and intragenic biofortified products are not (yet) allowed for human consumption, the experimental auction studies (3, 5, 9, 17) have informed the participants about the non-GM nature after the experiment, by which the winner received a non-GM alternative. Source: http://millenniumindicators.un.org/unsd/methods/m49/m49regin.htm *Part of the total sample *When based on a study with information treatments, values refer to mean differences after the 'no GM information' treatment. Unless otherwise stated, the non-GM, non-biofortified product is used as a benchmark to calculate the values. Due to the application of different valuation methods, we derived the outcomes of several studies (2,3B,5A,5B) based on (own) additional calculations.*Based on a price sensitivity analysis*Comparison of FBR with FAR, i.e. non-GM rice supplemented with Folic Acid pills*5 categories: Strongly Approve, Mildly Approve, Mildly Disapprove, Strongly Disapprove and Don’t Know*Degree of acceptance, rather than acceptance in terms of the percentage of people in favor of the GM crop. *Dummy variable (yes,no) refers to "informed, heard of, or aware of"*Dummy variable (yes, no) *When offered a discount, 84.2% of the initially reluctant participants are willing to buy it.

**See also Schilling, et al. (2003) **Golden Rice **Pooled sample (initial & follow-up study) **Due to the lower sample size, slightly different from De Steur et al. (2012).**Refers to a subsample, i.e. those who received an offer above, equal to, or lower than the conventional rice price.**Technology support, based on 4 point likert scale. Preference = moderate or strong support; Rejection = moderate or strong opposition.

***Results on the French data is not included, as it is more elaborated in Rozan et al. (2007) (21) ***Folate Biofortified Rice ***As calculated in Lusk et al. (2005) ***Preference of Vitamin A biofortification methods (GM, conventional, indifferent) in cassava

Remarks

- Studies based on the same dataset have the same reference number.

- While most studies inform their participants orally about the characteristics of the transgenic biofortified crops, few studies (3A,B) use labeling.

Abbreviations

cba, childbearing age

E, ethical

FAR, rice supplemented with free folic acid pills

FBR, Folate Biofortified Rice

GM, genetically modified

OECE, open-ended choice experiment

R, religious

S, safety

UN, United Nations

WTA, willingness-to-accept (a compensation)

WTP, willingness-to-pay (a premium)

Acceptance Other

Socio-economic research : micro-level analysis

Product attributes Data collection Study location Sample Valuation

ID Reference Outcome Burden analysis*

Crop Micro

nutrient

Technology Nature of

study

Time

horizon

UN subregion Country Region /

State

Key indicators mi. DALYs lost/yr Indicator Health benefits (per

scenario/yr)*

Reduction in

burden (%/yr)

Basic R&D Country-

specific

Social

marketing

Maintenance

breeding

Total Method Indicator Outcome

(per scenario)

1 Chow et al., 2010 Mustard oil Vitamin A Transgenic Hypothetical 20 years Southern Asia India 16 States Health impact, costs, CEA mi. DALYs saved 0.91 (low) CEA ICER, $/DALY saved 450 (low)*

1.69 (high) 403 (high)*

lives saved 113,000 (low) CEA ICER, $/live saved 303,300 (low)*

654,000 (high) 53,000 (high)*

2 De Steur et al. , 2010Rice Folate Transgenic** Hypothetical 30 years Eastern Asia China 31 Provinces Burden, health impact 0.31 mi. DALYs saved 0.12 (low) 36.9 (low)

0.26 (high) 81.9 (high)

3 De Steur et al., 2012Rice Folate Transgenic** Hypothetical 30 years Eastern Asia China Burden, health impact, cost, CEA0.31 mi. DALYs saved 0.06 (low) 20 (low) CEA $/DALY saved 64.2 (low)

0.19 (high) 60 (high) 21.4 (high)

Vitamin A 1.99 0.33 (low) 17 (low) 18.1 (low)

1.20 (high) 60 (high) 5.0 (high)

Multi* 10.64 1.19 (low) 11 (low) 9.6 (low), 7.9 (low, stacking)**

4.91 (high) 46 (high) 2.3 (high), 1.9 (high, stacking)**

4 De Steur et al., 2013Rice Folate Transgenic** Hypothetical 30 years Eastern Asia China Shanxi ProvinceBurden, health impact, cost, CEA0.11 mi. DALYs saved 0.02 (low) 20 (low) CEA $/DALY saved 120.3 (low)

0.07 (high) 60 (high) 40.1 (high)

5 Stein et al., 2006* Rice Vitamin A Transgenic* Hypothetical 30 years Southern Asia India Burden, health impact, cost, CEA/CBA2.33 mi. DALYs saved 0.20 (low) 8.8 % (low) 7.5 1.2 + 2.5 15.6 2.1 21.4 CEA $/DALY saved 19.4 (low)

1.38 (high) 59.4 % (high) 3.3 0.8 + 2.2 30.7 1.9 27.9 3.1 (high)

CBA IRR 29%, 35%, 44% (low)***

66%, 77%, 93% (high)***

BCR 26, 52, 129 (low)***

163, 327, 816 (high)***

lives saved 5,500 (low) CEA $/live saved 358 (low)

39,700 (high) 54 (high)

6 Zimmerman & Qaim, 2004Rice Vitamin A Transgenic* Hypothetical 16 years Southeastern AsiaPhilippines Burden, health impact, cost, CBA0.27 mi. DALYs saved 0.02 (low) 5.7% (low) 7.0 5.0 15.7 CBA IRR 66% (low)

0.08 (high) 31.5% (high) 133% (high)

* See also Stein et al. (2008) *Folate, vitamin A, zinc and iron*Golden Rice Source: http://millenniumindicators.un.org/unsd/methods/m49/m49regin.htm *Burden of respective micronutrient deficiency, see column 'D'*A distinction is made between a low and high impact scenario. It is important to note, however, that the assumptions for each scenario differ between the studies. *A lower cost in the case of gene stacking, i.e. when different micronutrient traits are combined in one gene construct and be transferred such that a new stacked event is generated.*A one-time regulatory approval cost *Based on administrative and regulatory costs, costs for training, promotional and educational materials, program monitoring and evaluation costs($0.002 per kg of oil); bottling costs ($0.09 per l of oil) and a one-time regulatory approval cost. *As compared to industrial fortification and supplementation

**Folate Biofortified Rice **A lower cost in the case of gene stacking, i.e. when different micronutrient traits are combined in one gene construct and be transferred such that a new stacked event is generated. *A lower cost in the case of gene stacking, i.e. when different micronutrient traits are combined in one gene construct and be transferred such that a new stacked event is generated. **A higher cost-effectiveness in the case of gene stacking, i.e. when different micronutrient traits are combined in one gene construct and be transferred such that a new stacked event is generated.

***BCR's based on a DALY valued at standard values for a DALY (500$, 1000$) or at the national per capita income measured at purchasing power parity (2500$), respectively.

Remarks

- Asterisks are explained below the respective column.

- Health impact analyses on Golden Rice in Bangladesh are underway, see Demont & Stein (2013)

Abbreviations

BCR, benefit-cost ratio

CEA, cost-effectiveness analysis

DALY, Disability-Adjusted Life Year

ICER, incremental cost-effectiveness ratios

IRR, internal rate of return

UN, United Nations

Socio-economic research: macro-level analysis

Product attributes Data collection Study location Health impact assessment* Economic evaluation

5.6* 3,103*

5.7 8.0 + 1.5 15.0 2.1 31.6

Cost estimation (mi. US$)

19.8

3.7

7.6 8.0 + 1.5 30.7 2.1

5.7 8.0 + 1.5 4.3 0.3

48.9

28.1 (22.5*) 24.0 (16.0**) 30.7 2.6 85.4 (71.8**)

Reference Full reference Abstract Year Authors Title Journal

Aluru et al., 2008 Aluru, M. et al. Generation of transgenic maize with enhanced provitamin A content. J. Ex. Bot. 59, 3551-3562 (2008).Vitamin A deficiency (VAD) affects over 250 million people worldwide and is one of the most prevalent nutritional deficiencies in developing countries, resulting in significant socio-economic losses. Provitamin A carotenoids such as β-carotene, are derived from plant foods and are a major source of vitamin A for the majority of the world's population. Several years of intense research has resulted in the production of ‘Golden Rice 2’ which contains sufficiently high levels of provitamin A carotenoids to combat VAD. In this report, the focus is on the generation of transgenic maize with enhanced provitamin A content in their kernels. Overexpression of the bacterial genes crtB (for phytoene synthase) and crtI (for the four desaturation steps of the carotenoid pathway catalysed by phytoene desaturase and ζ-carotene desaturase in plants), under the control of a ‘super γ-zein promoter’ for endosperm-specific expression, resulted in an increase of total carotenoids of up to 34-fold with a preferential accumulation 2008 Aluru, M., Y. Xu, R. Guo, Z. Wang, S. Li, W. White, K. Wang, and S. RodermelGeneration of transgenic maize with enhanced provitamin A content Journal of Experimental Botany

Bulley, S. et al., 2012 Bulley, S. et al. Enhancing ascorbate in fruits and tubers through over-expression of the L-galactose pathway gene GDP-L-galactose phosphorylase. Plant Biotechnol. J. 10, 390-397 (2012).Ascorbate, or vitamin C, is obtained by humans mostly from plant sources. Various approaches have been made to increase ascorbate in plants by transgenic means. Most of these attempts have involved leaf material from model plants, with little success reported using genes from the generally accepted l-galactose pathway of ascorbate biosynthesis. We focused on increasing ascorbate in commercially significant edible plant organs using a gene, GDP-l-galactose phosphorylase (GGP or VTC2), that we had previously shown to increase ascorbate concentration in tobacco and Arabidopsis thaliana. The coding sequence of Actinidia chinensis GGP, under the control of the 35S promoter, was expressed in tomato and strawberry. Potato was transformed with potato or Arabidopsis GGP genes under the control of the 35S promoter or a polyubiquitin promoter (potato only). Five lines of tomato, up to nine lines of potato, and eight lines of strawberry were regenerated for each construct. Three lines of tomato had a threefold to sixfold2012 Bulley, S., M. Wright, C. Rommens, H. Yan, M. Rassam, K. Lin‐Wang, C. Andre, D. Brewster, S. Karunairetnam, and A.C. AllanEnhancing ascorbate in fruits and tubers through over-expression of the L-galactose pathway gene GDP-L-galactose phosphorylasePlant Biotechnology JournalCahoon et al., 2003 Cahoon, E.B. et al. Metabolic redesign of vitamin E biosynthesis in plants for tocotrienol production and increased antioxidant content. Nat. Biotechnol. 21, 1082-1087 (2003).Tocotrienols are the primary form of vitamin E in seeds of most monocot plants, including cereals such as rice and wheat. As potent antioxidants, tocotrienols contribute to the nutritive value of cereal grains in human and livestock diets. cDNAs encoding homogentisic acid geranylgeranyl transferase (HGGT), which catalyzes the committed step of tocotrienol biosynthesis, were isolated from barley, wheat and rice seeds. Transgenic expression of the barley HGGT in Arabidopsis thaliana leaves resulted in accumulation of tocotrienols, which were absent from leaves of nontransformed plants, and a 10- to 15-fold increase in total vitamin E antioxidants (tocotrienols plus tocopherols). Overexpression of the barley HGGT in corn seeds resulted in an increase in tocotrienol and tocopherol content of as much as six-fold. These results provide insight into the genetic basis for tocotrienol biosynthesis in plants and demonstrate the ability to enhance the antioxidant content of crops by introduction of an enzyme that redire2003 Ramesh, S. A., Choimes, S., and D.P. SchachtmanMetabolic redesign of vitamin E biosynthesis in plants for tocotrienol production and increased antioxidant content Nature Biotechnology

Chen, Z. et al., 2003 Chen, Z. et al. Increasing vitamin C content of plants through enhanced ascorbate recycling. Proc. Natl. Acad. Sci. USA 100, 3525-3530 (2003).Vitamin C (L-ascorbic acid, AsA) has important antioxidant and metabolic functions in both plants and animals. Once used, ascorbic acid can be regenerated from its oxidized form in a reaction catalyzed by dehydroascorbate reductase (DHAR, EC 1.8.5.1). To analyze the physiological role of DHAR catalyzing the reduction of DHA to ascorbate in environmental stress adaptation, we examined whether increasing the level of AsA through enhanced AsA recycling would limit the deleterious effects of oxidative stress. A chimeric construct consisting of the double CaMV35S promoter fused to the Myc-dhar gene was introduced into Arabidopsis thaliana. Transgenic plants were biochemically characterized and tested for responses to oxidative stress. Western blot indicated that the dhar-transgene was successfully expressed. In homozygous T(4) transgenic seedlings, DHAR overexpression was increased up to 1.5 to 5.4 fold, which enhanced foliar ascorbic acid levels 2- to 4.25-fold and ratio of AsA/DHA about 3- to 16-fold relative to2003 Chen, Z., T.E. Young, J. Ling, S.-C. Chang, and D.R. GallieIncreasing vitamin C content of plants through enhanced ascorbate recycling Proceedings of the National Academy of Sciences

Cong, L. et al., 2009 Cong, L. et al. Expression of phytoene synthase1 and carotene desaturase crtl genes result in an increase in the total carotenoids content in trangenic elite wheat (Triticum aestivum L.). J. Agric. Food Chem. 57, 8652-8660 (2009).Dietary micronutrient deficiencies, such as the lack of vitamin A, are a major source of morbidity and mortality worldwide. Carotenoids in food can function as provitamin A in humans, while grains of Chinese elite wheat cultivars generally have low carotenoid contents. To increase the carotenoid contents in common wheat endosperm, transgenic wheat has been generated by expressing the maize y1 gene encoding phytoene synthase driven by a endosperm-specific 1Dx5 promoter in the elite wheat (Triticum aestivum L.) variety EM12, together with the bacterial phytoene desaturase crtI gene from Erwinia uredovora under the constitutive CaMV 35S promoter control. A clear increase of the carotenoid content was detected in the endosperms of transgenic wheat that visually showed a light yellow color. The total carotenoids content was increased up to 10.8-fold as compared with the nontransgenic EM12 cultivar. To test whether the variability of total carotenoid content in different transgenic lines was due to differences in t2009 Cong, L., C. Wang, L. Chen, H. Liu, G. Yang, and G. HeExpression of phytoene synthase1 and carotene desaturase crtl genes result in an increase in the total carotenoids content in trangenic elite wheat (Triticum aestivum L.)Journal of Agricultural and Food Chemistry

Diretto, G. et al., 2006 Diretto, G. et al. Metabolic engineering of potato tuber carotenoids through tuber-specific silencing of lycopene epsilon cyclase. BMC Plant Biol. 6,13 (2006).Background: Potato is a major staple food, and modification of its provitamin content is a possible means for alleviating nutritional deficiencies. beta-carotene is the main dietary precursor of vitamin A. Potato tubers contain low levels of carotenoids, composed mainly of the xanthophylls lutein, antheraxanthin, violaxanthin, and of xanthophyll esters. None of these carotenoids have provitamin A activity.Results: We silenced the first dedicated step in the beta-epsilon- branch of carotenoid biosynthesis, lycopene epsilon cyclase (LCY-e), by introducing, via Agrobacterium-mediated transformation, an antisense fragment of this gene under the control of the patatin promoter. Real Time measurements confirmed the tuber-specific silencing of Lcy-e. Antisense tubers showed significant increases in beta-beta-carotenoid levels, with beta-carotene showing the maximum increase (up to 14-fold). Total carotenoids increased up to 2.5-fold. These changes were not accompanied by a decrease in lutein, suggesting that LCY-e 2006 Diretto, G., S. Al-Babili, R. Tavazza, V. Papacchioli, P. Beyer, and G. GiulianoMetabolic engineering of potato tuber carotenoids through tuber-specific silencing of lycopene epsilon cyclase PLoS One

Diretto, G. et al., 2007a Diretto, G. et al. Silencing of beta-carotene hydroxylase increases total carotenoid and beta-carotene levels in potato tubers. BMC Plant Biol. 7,11 (2007a).Background: Beta-carotene is the main dietary precursor of vitamin A. Potato tubers contain low levels of carotenoids, composed mainly of the xanthophylls lutein (in the beta-epsilon branch) and violaxanthin (in the beta-beta branch). None of these carotenoids have provitamin A activity. We have previously shown that tuber-specific silencing of the first step in the epsilon-beta branch, LCY-e, redirects metabolic flux towards beta-beta carotenoids, increases total carotenoids up to 2.5-fold and beta-carotene up to 14-fold.Results: In this work, we silenced the non-heme beta-carotene hydroxylases CHY1 and CHY2 in the tuber. Real Time RT-PCR measurements confirmed the tuber-specific silencing of both genes . CHY silenced tubers showed more dramatic changes in carotenoid content than LCY-e silenced tubers, with beta-carotene increasing up to 38-fold and total carotenoids up to 4.5-fold. These changes were accompanied by a decrease in the immediate product of beta-carotene hydroxylation, zeaxanthin, but not of t2007 Diretto, G., R. Tavazza, R. Welsch, D. Pizzichini, F. Mourgues, V. Papacchioli, P. Beyer, and G. GiulianoSilencing of beta-carotene hydroxylase increases total carotenoid and beta-carotene levels in potato tubers BMC Plant Biology

Diretto, G. et al., 2007b Diretto, G. et al. Metabolic engineering of potato carotenoid content through tuber-specific overexpression of a bacterial mini-pathway. PLoS ONE 2,e350 (2007b).Background: Since the creation of “Golden Rice”, biofortification of plant-derived foods is a promising strategy for the alleviation of nutritional deficiencies. Potato is the most important staple food for mankind after the cereals rice, wheat and maize, and is extremely poor in provitamin A carotenoids.Methodology: We transformed potato with a mini-pathway of bacterial origin, driving the synthesis of beta-carotene (Provitamin A) from geranylgeranyl diphosphate. Three genes, encoding phytoene synthase (CrtB), phytoene desaturase (CrtI) and lycopene beta-cyclase (CrtY) from Erwinia, under tuber-specific or constitutive promoter control, were used. 86 independent transgenic lines, containing six different promoter/gene combinations, were produced and analyzed. Extensive regulatory effects on the expression of endogenous genes for carotenoid biosynthesis are observed in transgenic lines. Constitutive expression of the CrtY and/or CrtI genes interferes with the establishment of transgenosis and with the accumu2007 Diretto, G., R. Welsch, R. Tavazza, F. Mourgues, D. Pizzichini, P. Beyer, and G. GiulianoMetabolic engineering of potato carotenoid content through tuber-specific overexpression of a bacterial mini-pathway BMC Plant Biology

Drakakaki, G. et al., 2005 Drakakaki, G. et al. Endosperm-specific co-expression of recombinant soybean ferritin and Aspergillus phytase in maize results in significant increases in the levels of bioavailable iron. Plant Mol. Biol. 59, 869-880 (2005).We have generated transgenic maize plants expressing Aspergillus phytase either alone or in combination with the iron-binding protein ferritin. Our aim was to produce grains with increased amounts of bioavailable iron in the endosperm. Maize seeds expressing recombinant phytase showed enzymatic activities of up to 3 IU per gram of seed. In flour paste prepared from these seeds, up to 95% of the endogenous phytic acid was degraded, with a concomitant increase in the amount of available phosphate. In seeds expressing ferritin in addition to phytase, the total iron content was significantly increased. To evaluate the impact of the recombinant proteins on iron absorption in the human gut, we used an in vitro digestion/Caco-2 cell model. We found that phytase in the maize seeds was associated with increased cellular iron uptake, and that the rate of iron uptake correlated with the level of phytase expression regardless of the total iron content of the seeds. We also investigated iron bioavailability under more com2005 Drakakaki, G., S. Marcel, R.P. Glahn, E.K. Lund, S. Pariagh, R. Fischer, P. Christou, and E. StogerEndosperm-specific co-expression of recombinant soybean ferritin and Aspergillus phytase in maize results in significant increases in the levels of bioavailable ironPlant Molecular Biology

Ducreux, L.J.M. et al., 2005 Ducreux, L.J.M. et al. Metabolic engineering of high carotenoid potato tubers containing enhanced levels of β-carotene and lutein. J. Ex. Bot. 56, 81-89 (2005).In order to enhance the carotenoid content of potato tubers, transgenic potato plants have been produced expressing an Erwinia uredovora crtB gene encoding phytoene synthase, specifically in the tuber of Solanum tuberosum L. cultivar Desiree which normally produces tubers containing c. 5.6 microg carotenoid g(-1) DW and also in Solanum phureja L. cv. Mayan Gold which has a tuber carotenoid content of typically 20 microg carotenoid g(-1) DW. In developing tubers of transgenic crtB Desiree lines, carotenoid levels reached 35 microg carotenoid g(-1) DW and the balance of carotenoids changed radically compared with controls: beta-carotene levels in the transgenic tubers reached c. 11 microg g(-1) DW, whereas control tubers contained negligible amounts and lutein accumulated to a level 19-fold higher than empty-vector transformed controls. The crtB gene was also transformed into S. phureja (cv. Mayan Gold), again resulting in an increase in total carotenoid content to 78 microg carotenoid g(-1) DW in the most affe2005 Ducreux, L.J.M., W.L. Morris, P.E. Hedley, T. Shepherd, H.V. Davies, S. Millam, and M.A. TaylorMetabolic engineering of high carotenoid potato tubers containing enhanced levels of β-carotene and lutein Journal of Experimental Botany

Goto, F. et al., 1999 Goto, F. et al. Iron fortification of rice seed by the soybean ferritin gene. Nat. Biotechnol. 17, 282-286 (1999).To improve the iron content of rice, we have transferred the entire coding sequence of the soybean ferritin gene into Oryza sativa (L. cv. Kita-ake) by Agrobacterium-mediated transformation. The rice seed-storage protein glutelin promoter, GluB-1, was used to drive expression of the soybean gene specifically in developing, self-pollinated seeds (T1 seeds) of transgenic plants, as confirmed by reverse transcription PCR analysis. Stable accumulation of the ferritin subunit in the rice seed was demonstrated by western blot analysis, and its specific accumulation in the endosperm by immunologic tissue printing. The iron content of T1 seeds was as much as threefold greater than that of their untransformed counterparts.1999 Goto, F., T. Yoshihara, N. Shigemoto, S. Toki, and F. TakaiwaIron fortification of rice seed by the soybean ferritin gene Nature Biotechnology

Hemavati, et al., 2009 Hemavati, et al. Over-expression of strawberry D-galacturonic acid reductase in potato leads to accumulation of vitamin C with enhanced abiotic stress tolerance. Plant Sci. 177, 659-667 (2009).Vitamin C (ascorbic acid) is an essential component for collagen biosynthesis and also for the proper functioning of the cardiovascular system in humans. Unlike most of the animals, humans lack the ability to synthesize ascorbic acid on their own due to a mutation in the gene encoding the last enzyme of ascorbate biosynthesis. As a result, vitamin C must be obtained from dietary sources like plants. In this study, we have developed transgenic potato plants (Solanum tuberosum L. cv. Taedong Valley) over-expressing strawberry GalUR gene under the control of CaMV 35S promoter with increased ascorbic acid levels. Integration of the GalUR gene in the plant genome was confirmed by PCR and Southern blotting. Ascorbic acid (AsA) levels in transgenic tubers were determined by high-performance liquid chromatography (HPLC). The over-expression of GalUR resulted in 1.6–2-fold increase in AsA in transgenic potato and the levels of AsA were positively correlated with increased GalUR activity. The transgenic lines with enha2009 Hemavati, Upadhyaya, C.P., K.E. Young, N. Akula, J.J. Heung, O.M. Oh, C.R. Aswath, S.C. Chun, D.H. Kim, and S.W. ParkOver-expression of strawberry D-galacturonic acid reductase in potato leads to accumulation of vitamin C with enhanced abiotic stress tolerancePlant Science

Hemavati, et al., 2010 Hemavati, et al. Enhanced ascorbic acid accumulation in transgenic potato confers tolerance to various abiotic stresses. Biotechnol. Lett. 32, 321-330 (2010). L-ascorbic acid (Vitamin C, AsA) is an important component of human nutrition. Plants and several animals can synthesize their own ascorbic acid, whereas humans lack the gene essential for ascorbic acid biosynthesis and must acquire from their diet. In the present study, we developed transgenic potato (Solanum tuberosum L. cv. Taedong Valley) over-expressing L-gulono-gamma-lactone oxidase (GLOase gene; NCBI Acc. No. NM022220), isolated from rat cells driven by CaMV35S constitutive promoter that showed enhanced AsA accumulation. Molecular analyses of four independent transgenic lines performed by PCR, Southern and RT-PCR revealed the stable integration of the transgene in the progeny. The transformation frequency was ca. 7.5% and the time required for the generation of transgenic plants was 6-7 weeks. Transgenic tubers showed significantly enhanced AsA content (141%) and GLOase activity as compared to untransformed tubers. These transgenics were also found to withstand various abiotic stresses caused by Methyl2010 Hemavati, Upadhyaya, C.P., N. Akula, K.E. Young, S.C. Chun, D.H. Kim, and S.W. ParkEnhanced ascorbic acid accumulation in transgenic potato confers tolerance to various abiotic stresses Biotechnology Letters

Johnson et al., 2011 Johnson, A.A.T., et al. Constitutive overexpression of the OsNAS gene family reveals single-gene strategies for effective iron- and zinc-biofortification of rice endosperm. PLoS ONE 6, e24476 (2011).Rice is the

primary 2011 Johnson, A. A., B. Kyriacou, D. L. Callahan, L. Carruthers, J. Stangoulis, E. Lombi and M. TesterConstitutive overexpression of the OsNAS gene family reveals single-gene strategies for effective iron- and zinc-biofortification of rice endospermPLoS One

Lee, S. et al., 2009 Lee, S. et al. Iron fortification of rice seeds through activation of the nicotianamine synthase gene. Proc. Natl. Acad. Sci. USA 106, 22014-22019 (2009).The most widespread dietary problem in the world is mineral deficiency. We used the nicotianamine synthase (NAS) gene to increase mineral contents in rice grains. Nicotianamine (NA) is a chelator of metals and a key component of metal homeostasis. We isolated activation-tagged mutant lines in which expression of a rice NAS gene, OsNAS3, was increased by introducing 35S enhancer elements. Shoots and roots of the OsNAS3 activation-tagged plants (OsNAS3-D1) accumulated more Fe and Zn. Seeds from our OsNAS3-D1 plants grown on a paddy field contained elevated amounts of Fe (2.9-fold), Zn (2.2-fold), and Cu (1.7-fold). The NA level was increased 9.6-fold in OsNAS3-D1 seeds. Analysis by size exclusion chromatography coupled with inductively coupled plasma mass spectroscopy showed that WT and OsNAS3-D1 seeds contained equal amounts of Fe bound to IP6, whereas OsNAS3-D1 had 7-fold more Fe bound to a low molecular mass, which was likely NA. Furthermore, this activation led to increased tolerance to Fe and Zn deficienci2009 Lee, S., U.S. Jeon, S.J. Lee, Y.-K. Kim, D.P. Persson, S. Husted, J.K. Schjørring, Y. Kakei, H. Masuda, and N.K. NishizawaIron fortification of rice seeds through activation of the nicotianamine synthase gene Proceedings of the National Academy of Sciences

Lee, S. et al., 2012 Lee, S. et al. Activation of rice nicotianamine synthase 2 (OsNAS2) enhances iron availability for biofortification. Mol. Cells 33, 269-275 (2012).Because micronutrients in human diets ultimately come from plant sources, malnutrition of essential minerals is a significant public health concern. By increasing the expression of nicotianamine synthase (NAS), we fortified the level of bioavailable iron in rice seeds. Activation of iron deficiency-inducible OsNAS2 resulted in a rise in Fe content (3.0-fold) in mature seeds. Its ectopic expression also increased that content. Enhanced expression led to higher tolerance of Fe deficiency and better growth under elevated pH. Mice fed with OsNAS2-D1 seeds recovered more rapidly from anemia, indicating that bioavailable Fe contents were improved by this increase in OsNAS2 expression.2012 Lee, S., Y.-S. Kim, U.S. Jeon, Y.-K. Kim, J.K. Schjoerring, and G. AnActivation of rice nicotianamine synthase 2 (OsNAS2) enhances iron availability for biofortification Molecules and CellsLipkie et al., 2013 Lipkie, T.E. et al. Bioaccessibility of carotenoids from transgenic provitamin A biofortified sorghum. J. Agric. Food Chem. 61, 5764-5771 (2013).Biofortified sorghum (Sorghum bicolor (L.) Moench) lines are being developed to target vitamin A deficiency in Sub-Saharan Africa, but the delivery of provitamin A carotenoids from such diverse germplasms has not been evaluated. The purpose of this study was to screen vectors and independent transgenic events for the bioaccessibility of provitamin A carotenoids using an in vitro digestion model. The germplasm background and transgenic sorghum contained 1.0–1.5 and 3.3–14.0 μg/g β-carotene equivalents on a dry weight basis (DW), respectively. Test porridges made from milled transgenic sorghum contained up to 250 μg of β-carotene equivalents per 100 g of porridge on a fresh weight basis (FW). Micellarization efficiency of all-trans-β-carotene was lower (p < 0.05) from transgenic sorghum (1–5%) than from null/nontransgenic sorghum (6–11%) but not different between vector constructs. Carotenoid bioaccessibility was significantly improved (p < 0.05) by increasing the amount of coformulated lipid in test porridges 2013 Lipkie, T. E., F. F. De Moura, Z.-Y. Zhao, M. C. Albertsen, P. Che, K. Glassman and M. G. FerruzziBioaccessibility of carotenoids from transgenic provitamin A biofortified sorghum Journal of Agricultural and Food Chemistry

Lopez, A.B. et al., 2008 Lopez, A.B. et al. Effect of the cauliflower Or transgene on carotenoid accumulation and chromoplast formation in transgenic potato tubers. J. Ex. Bot. 59, 213-223 (2008).Transgenic plants have facilitated our understanding of the functional roles of genes and the metabolic processes affected in plants. Recently, the Or gene was isolated from an orange cauliflower mutant and it was shown that the Or gene could serve as a novel genetic tool to enrich carotenoid content in transgenic potato tubers. An in-depth characterization of these Or transgenic lines is presented here. It was found that the Or transgene may facilitate the identification of potential rate-limiting step(s) of the carotenoid biosynthetic pathway. The Or transgenic tubers accumulated not only increased levels of carotenoids that normally are present in the controls, but also three additional metabolite intermediates of phytoene, phytofluene, and zeta-carotene, indicating that the desaturation steps became limiting following the expression of the Or transgene. Moreover, we observed that long-term cold storage greatly enhanced carotenoid content in the Or transgenic tubers to a level of 10-fold over controls. Exp2008 Lopez, A.B., J. Van Eck, B.J. Conlin, D.J. Paolillo, J. O'Neill, and L. LiEffect of the cauliflower Or transgene on carotenoid accumulation and chromoplast formation in transgenic potato tubers Journal of Experimental Botany

Lu et al., 2006 Lu, S. et al. The cauliflower Or gene encodes a DnaJ cysteine-rich domain-containing protein that mediates high levels of β-carotene accumulation. Plant Cell 18, 3594-3605 (2006).Despite recent progress in our understanding of carotenogenesis in plants, the mechanisms that govern overall carotenoid accumulation remain largely unknown. The Orange (Or) gene mutation in cauliflower (Brassica oleracea var botrytis) confers the accumulation of high levels of beta-carotene in various tissues normally devoid of carotenoids. Using positional cloning, we isolated the gene representing Or and verified it by functional complementation in wild-type cauliflower. Or encodes a plastid-associated protein containing a DnaJ Cys-rich domain. The Or gene mutation is due to the insertion of a long terminal repeat retrotransposon in the Or allele. Or appears to be plant specific and is highly conserved among divergent plant species. Analyses of the gene, the gene product, and the cytological effects of the Or transgene suggest that the functional role of Or is associated with a cellular process that triggers the differentiation of proplastids or other noncolored plastids into chromoplasts for carotenoid ac2006 Lu, S., J. Van Eck, X. Zhou, A.B. Lopez, D.M. O'Halloran, K.M. Cosman, B.J. Conlin, D.J. Paolillo, D.F. Garvin, and J. VrebalovThe cauliflower Or gene encodes a DnaJ cysteine-rich domain-containing protein that mediates high levels of β-carotene accumulationThe Plant Cell Online

Lucca, P. et al., 2002 Lucca, P. et al. Fighting iron deficiency anemia with iron-rich rice. J. Am. Coll. Nutr. 21, 184S-190S (2002).Objective: Iron deficiency is estimated to affect about 30% of the world population. Iron supplementation in the form of tablets and food fortification has not been successful in developing countries, and iron deficiency is still the most important deficiency related to malnutrition. Here we present experiments that aim to increase the iron content in rice endosperm and to improve its absorption in the human intestine by means of genetic engineering.Methods: We first introduced a ferritin gene from Phaseolus vulgaris into rice grains, increasing their iron content up to twofold. To increase iron bioavailability, we introduced a thermo-tolerant phytase from Aspergillus fumigatus into the rice endosperm. In addition, as cysteine peptides are considered major enhancers of iron absorption, we over-expressed the endogenous cysteine-rich metallothionein-like protein.Results: The content of cysteine residues increased about sevenfold and the phytase level in the grains about one hundred and thirtyfold, giving a ph2002 Lucca, P., R. Hurrell, and I. PotrykusFighting iron deficiency anemia with iron-rich rice Journal of American College of Nutrition

Masuda et al., 2012 Masuda, H. et al. Iron biofortification in rice by the introduction of multiple genes involved in iron nutrition. Sci. Rep. 2, 543 (2012).To address the problem of iron-deficiency anemia, one of the most prevalent human micronutrient deficiencies globally, iron-biofortified rice was produced using three transgenic approaches: by enhancing iron storage in grains via expression of the iron storage protein ferritin using endosperm-specific promoters, enhancing iron translocation through overproduction of the natural metal chelator nicotianamine, and enhancing iron flux into the endosperm by means of iron(II)-nicotianamine transporter OsYSL2 expression under the control of an endosperm-specific promoter and sucrose transporter promoter. Our results indicate that the iron concentration in greenhouse-grown T2 polished seeds was sixfold higher and that in paddy field-grown T3 polished seeds was 4.4-fold higher than that in non-transgenic seeds, with no defect in yield. Moreover, the transgenic seeds accumulated zinc up to 1.6-times in the field. Our results demonstrate that introduction of multiple iron homeostasis genes is more effective for iron bio2012 Masuda, H., Y. Ishimaru, M.S. Aung, T. Kobayashi, Y. Kakei, M. Takahashi, K. Higuchi, H. Nakanishi, and N.K. NishizawaIron biofortification in rice by the introduction of multiple genes involved in iron nutrition Scientific Reports

Nandi et al., 2002 Nandi, S. et al. Expression of human lactoferrin in transgenic rice grains for the application in infant formula. Plant Sci. 163, 713-722 (2002).Breast-fed infants are healthier than formula-fed infants, which may in part be due to the lack of components like human lactoferrin (HLF) in infant formula. Thus, fortification of infant formula with bioactive proteins such as recombinant HLF (rHLF) may provide health benefits. A synthetic HLF gene linked to a rice glutelin 1 promoter and signal sequence was transformed to rice cells to produce rHLF. The expression level was up to 0.5% of dehusked rice grain weight. The rHLF was specifically expressed in the rice grain but not in other tissues. Rice grains expressing rHLF were advanced for four generations and expression levels were stable. Biochemical and physical characterization showed that rHLF is identical to HLF in N-terminal sequence, pI, iron-binding capacity, antimicrobial activity against a human pathogen and resistance to protease digestion. Receptor binding activity assessed by Caco-2 cells, a human small intestinal cell line, was also retained for rHLF. Since rice may be used in infant formula, 2002 Nandi, S., Y.A. Suzuki, J. Huang, D. Yalda, P. Pham, L. Wu, G. Bartley, N. Huang, and B. LönnerdalExpression of human lactoferrin in transgenic rice grains for the application in infant formula Plant Science

Naqvi et al., 2009 Naqvi, S. et al. Transgenic multivitamin corn through biofortification of endosperm with three vitamins representing three distinct metabolic pathways. Proc. Natl. Acad. Sci. U. S. A. 106, 7762-7767 (2009).Vitamin deficiency affects up to 50% of the world's population, disproportionately impacting on developing countries where populations endure monotonous, cereal-rich diets. Transgenic plants offer an effective way to increase the vitamin content of staple crops, but thus far it has only been possible to enhance individual vitamins. We created elite inbred South African transgenic corn plants in which the levels of 3 vitamins were increased specifically in the endosperm through the simultaneous modification of 3 separate metabolic pathways. The transgenic kernels contained 169-fold the normal amount of β-carotene, 6-fold the normal amount of ascorbate, and double the normal amount of folate. Levels of engineered vitamins remained stable at least through to the T3 homozygous generation. This achievement, which vastly exceeds any realized thus far by conventional breeding alone, opens the way for the development of nutritionally complete cereals to benefit the world's poorest people.2009 Naqvi, S., C. Zhu, G. Farre, K. Ramessar, L. Bassie, J. Breitenbach, D.P. Conesa, G. Ros, G. Sandmann, and T. CapellTransgenic multivitamin corn through biofortification of endosperm with three vitamins representing three distinct metabolic pathwaysProceedings of the National Academy of Sciences

Naqvi et al., 2011 Naqvi, S. et al. Simultaneous expression of Arabidopsis rho-hydroxyphenylpyruvate dioxygenase and MPBQ methyltransferase in transgenic corn kernels triples the tocopherol content.Transgenic Res. 20, 177-181 (2011).The quantity and composition of tocopherols (compounds with vitamin E activity) vary widely among different plant species reflecting the expression, activity and substrate specificity of enzymes in the corresponding metabolic pathway. Two Arabidopsis cDNA clones corresponding to ρ-hydroxyphenylpyruvate dioxygenase (HPPD) and 2-methyl-6-phytylplastoquinol methyltransferase (MPBQ MT) were constitutively expressed in corn to further characterize the pathway and increase the kernel tocopherol content. Transgenic kernels contained up to 3 times as much γ-tocopherol as their wild type counterparts whereas other tocopherol isomers remained undetectable. Biofortification by metabolic engineering offers a sustainable alternative to vitamin E supplementation for the improvement of human health.2011 Naqvi, S., G. Farré, C. Zhu, G. Sandmann, T. Capell, and P. Christou Simultaneous expression of Arabidopsis rho-hydroxyphenylpyruvate dioxygenase and MPBQ methyltransferase in transgenic corn kernels triples the tocopherol contentTransgenic Research

Paine et al., 2005 Paine, J.A. et al. Improving the nutritional value of Golden Rice through increased pro-vitamin A content. Nat. Biotechnol. 23, 482-487 (2005)."Golden Rice" is a variety of rice engineered to produce beta-carotene (pro-vitamin A) to help combat vitamin A deficiency, and it has been predicted that its contribution to alleviating vitamin A deficiency would be substantially improved through even higher beta-carotene content. We hypothesized that the daffodil gene encoding phytoene synthase (psy), one of the two genes used to develop Golden Rice, was the limiting step in beta-carotene accumulation. Through systematic testing of other plant psys, we identified a psy from maize that substantially increased carotenoid accumulation in a model plant system. We went on to develop "Golden Rice 2" introducing this psy in combination with the Erwinia uredovora carotene desaturase (crtI) used to generate the original Golden Rice. We observed an increase in total carotenoids of up to 23-fold (maximum 37 microg/g) compared to the original Golden Rice and a preferential accumulation of beta-carotene.2005 Paine, J.A., C.A. Shipton, S. Chaggar, R.M. Howells, M.J. Kennedy, G. Vernon, S.Y. Wright, E. Hinchliffe, J.L. Adams, and A.L. SilverstoneImproving the nutritional value of Golden Rice through increased pro-vitamin A content Nature BiotechnologyQu et al., 2005 Qu, L.Q. et al. Iron accumulation does not parallel the high expression level of ferritin in transgenic rice seeds. Planta 222, 225-233 (2005).To answer the question whether iron accumulation in transgenic rice seeds depends on the expression level of exogenous soybean ferritin, we generated two kinds of ferritin hyper-expressing rice lines by introducing soybean ferritin SoyferH-1 gene under the control of the rice seed storage glutelin gene promoter, GluB-1 and the rice seed storage globulin gene promoter, Glb-1, (GluB-1/SoyferH-1 and Glb-1/SoyferH-1, DF lines), and by introducing the SoyferH-1 gene under the control of Glb-1 promoter alone (Glb-1/SoyferH-1, OF lines). Ferritin expression was restricted to the endosperm in both lines and protein levels determined by western blot analysis were up to 13-fold higher than in a construct previously reported FK22 (GluB-1/SoyferH-1, in genetically Kitaake background); [corrected] however, the maximum iron concentrations in seeds of both of the new lines were only about 30% higher than FK22. The maximum iron concentration in the OF and DF lines was about threefold higher than in the non-transformant. The 2005 Qu, L. Q., T. Yoshihara, A. Ooyama, F. Goto and F. TakaiwaIron accumulation does not parallel the high expression level of ferritin in transgenic rice seeds Planta

Ramesh et al., 2004 Ramesh, S.A. et al. Over-expression of an Arabidopsis zinc transporter in Hordeum vulgare increases short-term zinc uptake after zinc deprivation and seed zinc content. Plant Mol. Biol. 54, 373-385 (2004).Increasing the zinc content of cereal grains will be important for improving human nutrition. Improved plant zinc efficiency will lead to increased yields when available zinc is limiting plant growth. The aim of our work was to test how the over-expression of zinc transporters in cereals affects plant growth, seed mineral content, and zinc transport rates. Known zinc transporters from Arabidopsis were over-expressed in Hordeum vulgare cv. Golden Promise by means of a ubiquitin promoter. Multiple transgenic lines were obtained, and the locus number and expression levels were verified. Transgenic lines were tested in long-term growth and short-term uptake experiments. Seeds from transgenic lines grown in soil had higher zinc and iron contents than controls. Short-term uptake rates were higher in the transgenic lines after zinc deprivation. Resupply of zinc after a period of deprivation resulted in the rapid decrease in zinc uptake even in transgenic lines in which a zinc transporter gene was constitutively expr2004 Ramesh, S. A., S. Choimes and D. P. SchachtmanOver-expression of an Arabidopsis zinc transporter in Hordeum vulgare increases short-term zinc uptake after zinc deprivation and seed zinc contentPlant Molecular Biology

Sayre et al., 2011 Sayre, R. et al. The Bio-Cassava-Plus Program: Biofortification of cassava for Sub-Saharan Africa. Annu. Rev. Plant Biol. 62, 251-272 (2011).More than 250 million Africans rely on the starchy root crop cassava (Manihot esculenta) as their staple source of calories. A typical cassava-based diet, however, provides less than 30% of the minimum daily requirement for protein and only 10%-20% of that for iron, zinc, and vitamin A. The BioCassava Plus (BC+) program has employed modern biotechnologies intended to improve the health of Africans through the development and delivery of genetically engineered cassava with increased nutrient (zinc, iron, protein, and vitamin A) levels. Additional traits addressed by BioCassava Plus include increased shelf life, reductions in toxic cyanogenic glycosides to safe levels, and resistance to viral disease. The program also provides incentives for the adoption of biofortified cassava. Proof of concept was achieved for each of the target traits. Results from field trials in Puerto Rico, the first confined field trials in Nigeria to use genetically engineered organisms, and ex ante impact analyses support the efficacy 2011 Sayre, R., J. R. Beeching, E. B. Cahoon, C. Egesi, C. Fauquet, J. Fellman, M. Fregene, W. Gruissem, S. Mallowa and M. ManaryThe Bio-Cassava-Plus Program: Biofortification of cassava for Sub-Saharan Africa Annual Review of Plant Biology

Storozhenko et al., 2007 Storozhenko, S. et al. Folate fortification of rice by metabolic engineering. Nat. Biotechnol. 25, 1277-1279 (2007).Rice, the world's major staple crop, is a poor source of essential micronutrients, including folates (vitamin B9). We report folate biofortification of rice seeds achieved by overexpressing two Arabidopsis thaliana genes of the pterin and para-aminobenzoate branches of the folate biosynthetic pathway from a single locus. We obtained a maximal enhancement as high as 100 times above wild type, with 100 g of polished raw grains containing up to four times the adult daily folate requirement.2007 Storozhenko, S., V. De Brouwer, M. Volckaert, O. Navarrete, D. Blancquaert, G.-F. Zhang, W. Lambert, and D. Van Der StraetenFolate fortification of rice by metabolic engineering Nature Biotechnology

Vasconcelos et al., 2003 Vasconcelos, M. et al. Enhanced iron and zinc accumulation in transgenic rice with the ferritin gene. Plant Sci. 164, 371-378 (2003).In this report, we show that the expression of the soybean ferritin gene, driven by the endosperm-specific glutelin promoter, leads to higher iron and zinc levels in transgenic indica rice grains. Brown rice is rarely consumed, and polishing of the rice grain brings considerable loss of micronutrients by removing its outer layers. No data until now have shown that after commercial milling the micronutrient concentration remains higher than that of the control. In our experiment, expression of the soybean ferritin gene under the control of the glutelin promoter in rice has proven to be effective in enhancing grain nutritional levels, not only in brown grains but also in polished grains. Besides determining the iron levels in transgenic rice grains, we also checked for zinc concentration, and it was found to be higher in transgenic seeds than in the control. Moreover, we introduced this gene in an elite indica rice line that has highly desirable agronomic and field-performance traits. Prussian blue staining rea2003 Vasconcelos, M., K. Datta, N. Oliva, M. Khalekuzzaman, L. Torrizo, S. Krishnan, M. Oliveira, F. Goto, and S.K. DattaEnhanced iron and zinc accumulation in transgenic rice with the ferritin gene Plant Science

Welsch et al., 2010 Welsch, R. et al. Provitamin A accumulation in Cassava (Manihot esculenta) roots driven by a single nucleotide polymorphism in a phytoene synthase gene. The Plant Cell 22, 3348-3356 (2010).Cassava (Manihot esculenta) is an important staple crop, especially in the arid tropics. Because roots of commercial cassava cultivars contain a limited amount of provitamin A carotenoids, both conventional breeding and genetic modification are being applied to increase their production and accumulation to fight vitamin A deficiency disorders. We show here that an allelic polymorphism in one of the two expressed phytoene synthase (PSY) genes is capable of enhancing the flux of carbon through carotenogenesis, thus leading to the accumulation of colored provitamin A carotenoids in storage roots. A single nucleotide polymorphism present only in yellow-rooted cultivars cosegregates with colored roots in a breeding pedigree. The resulting amino acid exchange in a highly conserved region of PSY provides increased catalytic activity in vitro and is able to increase carotenoid production in recombinant yeast and Escherichia coli cells. Consequently, cassava plants overexpressing a PSY transgene produce yellow-fleshed2010 Welsch, R., J. Arango, C. Bär, B. Salazar, S. Al-Babili, J. Beltrán, P. Chavarriaga, H. Ceballos, J. Tohme, and P. BeyerProvitamin A accumulation in Cassava (Manihot esculenta) roots driven by a single nucleotide polymorphism in a phytoene synthase geneThe Plant Cell Online

Wirth et al., 2009 Wirth, J. et al. Rice endosperm iron biofortification by targeted and synergistic action of nicotionamine synthase and ferritin. Plant Biotechnol. J. 7, 631-644 (2009).Nearly one-third of the world's population, mostly women and children, suffer from iron malnutrition and its consequences, such as anaemia or impaired mental development. Iron fortification of food is difficult because soluble iron is either unstable or unpalatable, and non-soluble iron is not bioavailable. Genetic engineering of crop plants to increase iron content has therefore emerged as an alternative for iron biofortification. To date, strategies to increase iron content have relied on single genes, with limited success. Our work focuses on rice as a model plant, because it feeds one-half of the world's population, including the majority of the iron-malnourished population. Using the targeted expression of two transgenes, nicotianamine synthase and ferritin, we increased the iron content of rice endosperm by more than six-fold. Analysis of transgenic rice lines confirmed that, in combination, they provide a synergistic effect on iron uptake and storage. Laser ablation-inductively coupled plasma-mass spec2009 Wirth, J., S. Poletti, B. Aeschlimann, N. Yakandawala, B. Drosse, S. Osorio, T. Tohge, A.R. Fernie, D. Günther, and W. GruissemRice endosperm iron biofortification by targeted and synergistic action of nicotionamine synthase and ferritin Plant Biotechnology Journal

Ye et al., 2000 Ye, X. et al. Engineering the provitamin A (β-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science 287, 303-305 (2000).Rice (Oryza sativa), a major staple food, is usually milled to remove the oil-rich aleurone layer that turns rancid upon storage, especially in tropical areas. The remaining edible part of rice grains, the endosperm, lacks several essential nutrients, such as provitamin A. Thus, predominant rice consumption promotes vitamin A deficiency, a serious public health problem in at least 26 countries, including highly populated areas of Asia, Africa, and Latin America. Recombinant DNA technology was used to improve its nutritional value in this respect. A combination of transgenes enabled biosynthesis of provitamin A in the endosperm.2000 Ye, X., S. Al-Babili, A. Klöti, J. Zhang, P. Lucca, P. Beyer, and I. PotrykusEngineering the provitamin A (β-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm Science

Zhang et al., 2013 Zhang, G.-Y. et al. Increased α-tocotrienol content in seeds of transgenic rice overexpressing Arabidopsis γ-tocopherol methyltransferase. Transgenic Res. 22, 89-99 (2013).Vitamin E comprises a group of eight lipid soluble antioxidant compounds that are an essential part of the human diet. The α-isomers of both tocopherol and tocotrienol are generally considered to have the highest antioxidant activities. γ-tocopherol methyltransferase (γ-TMT) catalyzes the final step in vitamin E biosynthesis, the methylation of γ- and δ-isomers to α- and β-isomers. In present study, the Arabidopsis γ-TMT (AtTMT) cDNA was overexpressed constitutively or in the endosperm of the elite japonica rice cultivar Wuyujing 3 (WY3) by Agrobacterium-mediated transformation. HPLC analysis showed that, in brown rice of the wild type or transgenic controls with empty vector, the α-/γ-tocotrienol ratio was only 0.7, much lower than that for tocopherol (~19.0). In transgenic rice overexpressing AtTMT driven by the constitutive Ubi promoter, most of the γ-isomers were converted to α-isomers, especially the γ- and δ-tocotrienol levels were dramatically decreased. As a result, the α-tocotrienol content was great2013 Zhang, G.-Y., R.-R. Liu, G. Xu, P. Zhang, Y. Li, K.-X. Tang, G.-H. Liang, and Q.-Q. LiuIncreased α-tocotrienol content in seeds of transgenic rice overexpressing Arabidopsis γ-tocopherol methyltransferase Transgenic Research

Zhu et al., 2008 Zhu, C. et al. Combinatorial genetic transformation generates a library of metabolic phenotypes for the carotenoid pathway in maize. Proc. Natl. Acad. Sci. USA 105, 18232-18237 (2008).Combinatorial nuclear transformation is a novel method for the rapid production of multiplex-transgenic plants, which we have used to dissect and modify a complex metabolic pathway. To demonstrate the principle, we transferred 5 carotenogenic genes controlled by different endosperm-specific promoters into a white maize variety deficient for endosperm carotenoid synthesis. We recovered a diverse population of transgenic plants expressing different enzyme combinations and showing distinct metabolic phenotypes that allowed us to identify and complement rate-limiting steps in the pathway and to demonstrate competition between beta-carotene hydroxylase and bacterial beta-carotene ketolase for substrates in 4 sequential steps of the extended pathway. Importantly, this process allowed us to generate plants with extraordinary levels of beta-carotene and other carotenoids, including complex mixtures of hydroxycarotenoids and ketocarotenoids. Combinatorial transformation is a versatile approach that could be used to mo2008 Zhu, C., S. Naqvi, J. Breitenbach, G. Sandmann, P. Christou, and T. CapellCombinatorial genetic transformation generates a library of metabolic phenotypes for the carotenoid pathway in maize Proceedings of the National Academy of Sciences

Biotechnology research

Reference Full reference Abstract Year Authors Title Journal

Amin et al., 2011 Amin, L., Azlan, N.A.A., Hashim, H., 2011. Ethical perception of cross-species gene transfer in plant. African Journal of Biotechnology, 10(58), 12457-12468.Plants can be genetically modified through a variety of methods in the hope that it will be improved in some way to increase the yield and quality of a crop, or to add nutritional value or shelf life. The development of genetically modified (GM) rice to enrich its nutritional value, such as Vitamin C might involve gene transfer across different species. The purpose of this paper is to examine how the public in the Klang Valley region of Malaysia, perceive the development of GM rice which contain mice gene to increase its vitamin C content. A survey was carried out using self constructed multi-dimensional instrument measuring ethical perception of GM rice. The respondents (n = 434) were stratified according to stakeholders groups. Results from the survey on 434 respondents have shown the Malaysian stakeholders were not very familiar with GM rice and perceived it as having moderate risk, its benefits to the society would not be much denied if it is not developed and the ethical aspects were considered as not ac2011 Amin, L., Azlan, N.A.A., Hashim, HEthical Perception of Cross-species Gene Transfer in Plant African Journal of Biotechnology

Canavari & Nayga, 2009 Canavari, M., Nayga, R.M., Jr., 2009. On Consumers' Willingness to Purchase Nutritionally Enhanced Genetically Modified Food. Applied Economics, 41(1-3), 125-137.This study assesses consumers' willingness to purchase genetically modified (GM) food products with two different types of benefits: an input (i.e., reduced pesticides) and an output trait benefit (i.e., nutritionally enhanced). Data were collected using a telephone survey of an Italian households sample. Discrete choice approach is used to elicit the purchase intentions of the respondents. Four separate probit models are estimated to examine the effect of various factors on choices. Results suggest that majority of Italians are not willing to buy GM food products even if they are nutritionally enhanced. However, more consumers are willing to buy a nutritionally enhanced plant based GM product than a traditional plant based GM (with input trait benefit). Willingness to buy for a nutritionally enhanced animal based GM product and for a traditional animal based GM product with input trait benefit are similar. Consumers unwilling to buy GM food would not buy it even if it is nutritionally enhanced and has lower 2009 Canavari, M., Nayga, R.M., JrOn Consumers' Willingness to Purchase Nutritionally Enhanced Genetically Modified Food Applied Economics

Colson & Huffman, 2011 Colson, G., Huffman, W.E., 2011. Consumers' Willingness to Pay for Genetically Modified Foods with Product-Enhancing Nutritional Attributes. American Journal of Agricultural Economics, 93(2), 358-363., 93(2), 358-363. / 2011 Colson, G., Huffman, W.E.Consumers' Willingness to Pay for Genetically Modified Foods with Product-Enhancing Nutritional Attributes American Journal of Agricultural Economics

Colson et al., 2011 Colson, G.J., Huffmann, W.E., Rousu, M.C., 2011. Improving the Nutrient Content of Food through Genetic Modification: Evidence from Experimental Auctions on Consumer Acceptance. Journal of Agricultural and Resource Economics, 36(2), 343-364.This paper assesses consumers' acceptance of nutritionally enhanced vegetables using a series of auction experiments administered to a random sample of adult consumers. Evidence suggests that consumers are willing to pay significantly more for fresh produce with labels signaling enhanced levels of antioxidants and vitamin C achieved by moving genes from within the species, as opposed to across species. However, this premium is significantly affected by diverse information treatments injected into the experiments.2011 Colson, G.J., Huffmann, W.E., Rousu, M.C.Improving the Nutrient Content of Food through Genetic Modification: Evidence from Experimental Auctions on Consumer AcceptanceJournal of Agricultural and Resource Economics

Corrigan et al. , 2009 Corrigan, J.R., Depositario, D.P.T., Nayga, R.M., Jr., Wu, X., Laude, T.P., 2009. Comparing Open-Ended Choice Experiments and Experimental Auctions: An Application to Golden Rice. American Journal of Agricultural Economics, 91(3), 837-853.We use two experimental valuation methods to estimate consumer demand for genetically modified golden rice. The first is an open-ended choice experiment (OECE) where participants name the quantities of golden rice and conventional rice demanded at each of several price combinations, one of which will be randomly chosen as binding. This allows us to estimate market demand by aggregating demand across participants. This estimate of market demand also allows us to estimate own-price elasticity and consumer surplus for golden rice. Comparing willingness-to-pay (WTP) estimates from the OECE with those from a uniform-price auction, we find that OECE WTP estimates exhibit less affiliation across rounds, and the effects of positive and negative information under the OECE are more consistent with prior expectations and existing studies. We also find that, while auction WTP estimates more than double across five rounds, OECE WTP estimates are stable across rounds and are always roughly equal to those from the final auc2009 Corrigan, J.R., Depositario, D.P.T., Nayga, R.M., Jr., Wu, X., Laude, T.P.Comparing Open-Ended Choice Experiments and Experimental Auctions: An Application to Golden Rice American Journal of Agricultural Economics

Depositario et al., 2009 Depositario, D.P.T., Nayga, R.M., Jr., Wu, X., Laude, T.P., 2009. Effects of Information on Consumers' Willingness to Pay for Golden Rice. Asian Economic Journal, 23(4), 457-476.We examine the effects of information on consumer bidding behavior using a uniform-price auction with four units supply for golden rice. Our findings show that mean willingness to pay (WTP) bids are highest under positive information, followed by no information, negative information, and unexpectedly lowest with two-sided information. Participants might have put more weight on the negative when faced with both positive and negative information. There is also a minor difference in WTP with respect to the reference price between positive information and no information. Furthermore, the marginal effect on WTP of positive information vis-a-vis no information is minimal. This suggests that the positive information faced by consumers might not be compelling enough to drastically increase WTP bids for a genetically modified product such as golden rice.2009 Depositario, D.P.T., Nayga, R.M., Jr., Wu, X., Laude, T.P.Effects of Information on Consumers' Willingness to Pay for Golden Rice Asian Economic Journal

Curtis & Moeltner, 2006 Curtis, K.R., Moeltner, K., 2006. Genetically Modified Food Market Participation and Consumer Risk Perceptions: A Cross-Country Comparison. Canadian Journal of Agricultural Economics, 54(2), 289-310.As developing nations look to become more competitive in world agricultural markets, genetically modified (GM) crops are one avenue of pursuit. However, fears of primary export market loss, negative media attention, and adverse government regulations often hinder GM crop implementation and increase GM food risk perceptions among domestic consumers. In this study we analyze consumer surveys of GM food purchase propensity conducted in the developing countries of Romania and China. Through the examination of marginal effects and the drivers of purchase propensity, we find that in spite of demographic and psychographic similarities, consumer willingness to purchase GM foods is quite different between the two samples. Consumer preferences are largely dependent on risk perceptions, which are high in the Romanian sample, but low in the Chinese sample. Additionally, the effect of regressors on GM purchase propensity is invariant across foods in Romania, but distinctly different across foods in China, possibly due to 2006 Curtis, K.R., Moeltner, K.Genetically Modified Food Market Participation and Consumer Risk Perceptions: A Cross-Country Comparison Canadian Journal of Agricultural Economics

Li, Q et al., 2003 Li, Q., Curtis, K.R., McCluskey, J.J., Wahl, T.I., 2003. Consumer Attitudes toward Genetically Modified Foods in Beijing, China. AgBioForum, 5(4), 145-152.A consumer survey in Beijing, China, was conducted in August 2002. Although the majority of surveyed consumers reported that they had little or no knowledge of biotechnology, their attitudes toward genetically modified (GM) foods was generally positive, especially for GM foods with product-enhancing attributes. Using dichotomous choice contingent valuation methodology, Chinese consumers' willingness to pay (WTP) for GM rice and GM soybean oil in our sample was positively affected by respondents' positive opinion toward GM foods for both products and by higher levels of self-reported knowledge for soybean oil. However, for GM rice, WTP was negatively affected by the respondent's age--the older respondents were less likely to choose GM rice. These results imply that, unlike Europe and Japan, there is a potential market for GM foods in China. GM food producers and exporters can use this information to design effective marketing strategies.2003 Li, Q., Curtis, K.R., McCluskey, J.J., Wahl, T.I.Consumer Attitudes toward Genetically Modified Foods in Beijing, China AgBioForum

De Steur et al., 2010 De Steur, H., Gellynck, X., Storozhenko, S., Liqun, G., Lambert, W., Van Der Straeten, D., Viaene, J., 2010. Willingness-to-accept and purchase genetically modified rice with high folate content in Shanxi Province, China. Appetite, 54(1), 118-125.Neural-tube defects (NTDs) are considered to be the most common congenital malformations. As Shanxi Province, a poor region in the North of China, has one of the highest reported prevalence rates of NTDs in the world, folate fortification of rice is an excellent alternative to low intake of folate acid pills in this region. This paper investigates the relations between socio-demographic indicators, consumer characteristics (knowledge, consumer perceptions oil benefits, risks, safety and price), willingness-to-accept and willingness-to-pay genetically modified (GM) rice. The consumer survey compromises 944 face-to-face interviews with rice consumers in Shanxi Province, China. Multivariate analyses consist of multinomial logistic regression and multiple regression. The results indicate that consumers generally are willing-to-accept GM rice, with an acceptance rate of 62.2%. Acceptance is influenced by objective knowledge and consumers' perceptions on benefits and risks. Willingness-to-pay GM rice is influenced 2010 De Steur, H., Gellynck, X., Storozhenko, S., Liqun, G., Lambert, W., Van Der Straeten, D., Viaene, J.Willingness-to-accept and purchase genetically modified rice with high folate content in Shanxi Province, China Appetite

De Steur et al., 2012 De Steur, H., Gellynck, X., Feng, S.Y., Rutsaert, P., Verbeke, W., 2012. Determinants of willingness-to-pay for GM rice with health benefits in a high-risk region: Evidence from experimental auctions for folate biofortified rice in China. Food Quality and Preference, 25(2), 87-94.Provision of folate biofortified rice (FBR), a GM rice variant with higher folate content, has been recently proposed as an alternative health strategy to address folate deficiency and its main adverse outcomes, such as neural-tube defects. Based on experimental auctions with 252 women of childbearing age, split up between student (n = 120) and non-student auctions (n = 132), this study investigates the determinants of willingness-to-pay for this GM rice with health benefits in Shanxi Province, a high-risk region of China. The study shows that female Shanxi rice consumers are prepared to pay a premium of (sis) 1.73 or 33.7%, which corresponds with a switch to a rice variety that is one price/quality level higher than regular rice. Offering a GM-free folate substitute did not reveal significant differences in bidding behavior. Besides a significant target group effect, by which students are less likely to buy and pay more for FBR, willingness-to-pay is mainly determined by consumers' GM food acceptance and obj2012 De Steur, H., Gellynck, X., Feng, S.Y., Rutsaert, P., Verbeke, W.Determinants of willingness-to-pay for GM rice with health benefits in a high-risk region: Evidence from experimental auctions for folate biofortified rice in ChinaFood Quality and Preference

De Steur et al., 2013 De Steur, H., Buysse, J., Feng, S., Gellynck, X., 2013. The role of information on consumers’ willingness-to-pay for GM rice with health benefits. An application to China. Asian Economic Journal, 27(4), 391-408.Despite their potential health benefits, the controversial nature surrounding the use of GM technology in biofortified crops remains a subject of discussion. This paper investigates the role of information on willingness-to-pay for folate-biofortified rice in a Chinese folate-deficient region. We conducted second price auctions with 252 women of childbearing age, split up into two target groups based on street(n = 132, non-student sample) or school auctions (n = 120, student sample). Notwithstanding a general positive effect of folate-related information (i.e. content, benefits and regional situation), evidence reveals that the non-student sample is less concerned about GM technology. The results also provide insight into serial position effects in conflicting GM information. Whereas participants in the non-student sample are more prone to a primacy bias when receiving both positive and negative information, an alarmist reaction is observed in the student sample. This study highlights the need for segmented,2013 De Steur, H., Buysse, J., Feng, S., Gellynck, X.The Role of Information on Consumers’ Willingness-to-pay For GM Rice with Health Benefits. An Application to China Asian Economic Journal

De Steur et al., 2014 De Steur, H., Feng, S., Xiaoping, S., Gellynck, X., 2014. Consumer preferences for micronutrient strategies in China. A comparison between folic acid supplementation and folate biofortification. Public Health Nutrition, in press. Objective: Despite public health efforts, folate deficiency is still largely prevalent in poor, rural populations and continues to cause a large burden of disease. The present paper determines and compares consumer preferences for two folate strategies: folic acid supplementation v. folate biofortification, i.e. the enhancement of the folate content in staple crops.Design: Experimental auctions with non-repeated information rounds are applied to rice in order to obtain willingness-to-pay for folate products. Thereby, GM or non-GM folate-biofortified rice (FBR) is auctioned together with rice that is supplemented with free folic acid pills (FAR).Setting: Shanxi Province (China) as a high-risk region of folate deficiency.Subjects: One hundred and twenty-six women of childbearing age, divided into a school (n 60) and market sample (n 66).Results: Despite differences according to the targeted sample, a general preference for folate biofortification is observed, regardless of the applied breeding technology. P2014 De Steur, H., Feng, S., Xiaoping, S., Gellynck, X.Consumer preferences for micronutrient strategies in China. A comparison between folic acid supplementation and folate biofortificationPublic Health Nutrition

Deodhar et al., 2008 Deodhar, S.Y., Ganesh, S., Chern, W.S., 2008. Emerging markets for GM foods: an Indian perspective on consumer understanding and the willingness to pay. International Journal of Biotechnology, 10(6), 570-587.A random utility approach is used to estimate logit equations which indicate what factors affect the likelihood of consumption of non-GM and GM foods, and, whether or not consumers are willing to pay a premium for non-GM/GM foods. Ceteris paribus as the price difference between non-GM and GM food rose, people were more likely to consume GM foods. Likelihood of GM food consumption was higher in the middle income brackets. Consumers were willing to pay an expected premium of 19.5% and 16.12% for golden rice and GM edible oil respectively. In case of chicken, consumers seemed to pay a very negligible premium for non-GM fed chicken. Overall, it appears that GM foods may be acceptable in the Indian market. However, basic awareness about the GM foods may have to be created among the consumers through government ministries, consumer interest groups, and biotech food-crop companies.2008 Deodhar, S.Y., Ganesh, S., Chern, W.S.Emerging markets for GM foods: an Indian perspective on consumer understanding and the willingness to pay International Journal of Biotechnology

Gonzalez et al., 2009 Gonzalez, C., Johnson, N., Qaim, M., 2009. Consumer Acceptance of Second-Generation GM Foods: The Case of Biofortified Cassava in the North-East of Brazil. Journal of Agricultural Economics, 60(3), 604-624.Biofortified staple foods are currently being developed to reduce problems of micronutrient malnutrition among the poor. This partly involves use of genetic modification. Yet, relatively little is known about consumer acceptance of such second-generation genetically modified (GM) foods in developing countries. Here, we analyse consumer attitudes towards provitamin A GM cassava in the north-east of Brazil. Based on stated preference data, mean willingness to pay is estimated at 60-70% above market prices for traditional cassava. This is higher than the results from similar studies in developed countries, which is plausible given that micronutrient malnutrition is more severe in developing countries. GM foods with enhanced nutritive attributes seem to be well received by poor consumers. However, the results also suggest that acceptance would be still higher if provitamin A were introduced to cassava through conventional breeding. Some policy implications are discussed.2009 Gonzalez, C., Johnson, N., Qaim, M.Consumer Acceptance of Second-Generation GM Foods: The Case of Biofortified Cassava in the North-East of Brazil Journal of Agricultural Economics

Hallman et al., 2002 Hallman, W.K., Adelaja, A.O., Schilling, B.J., Lang, J.T., 2002. Public Perceptions of Genetically Modified Foods Americans Know Not What They Eat. Rutgers, The State University of New Jersey.Biotechnology stands to be a defining technology in the future of food and agriculture. Proponents argue that science and industry are poised to bring consumers a wide variety of products that have potential for meeting basic food needs, as well as delivering a wide-range of health, environmental and economic benefits. Opponents counter that the potential exists for unintended consequences, ranging from ecological disruption to adverse human health implications, and that these risks are not fully understood. Fundamental questions exist, however, regarding the general public's position on food products derived with the use of biotechnology. To address these questions, the Food Policy Institute addressed consumers using computer-assisted telephone interviews (CATI) system, a public phone survey of a sample selection of 1203 U.S. residents was administered between March and April 2001. The questionnaire was developed to address perceived gaps in the current literature on American consumer awareness, acceptance, 2002 Hallman, W.K., Adelaja, A.O., Schilling, B.J., Lang, J.T.Public Perceptions of Genetically Modified Foods Americans Know Not What They Eat Food Policy Institute Report, Rutgers University

Kassardjian et al., 2012 Kassardjian, E., Robin, S., Ruffieux, B., 2012. L'hostilite aux OGM survit-elle a des produits attractifs? (Does Consumer's Aversion to GM Food Survive a Yummy Apple? With English summary.). Revue Francaise d'Economie, 26(3), 121-150.We know (e.g., Noussair et alii [2004]) that consumers devalue first-generation GM food. Here, using a framed field experiment method, we reveal consumer's willingness to pay for various second-generation GM apples, defined as differentiated products containing an innovative attribute, desirable for the consumer. Values for such second-generation GM apples are compared with (a) a standard conventional apple value, and (b) values for identically differentiated products, processed with alternative technologies. Some of our GM differentiated apples appear to be more valued than the standard apple; therefore a desirable innovative attribute is sufficient to balance the loss of value due to the GM technology. Nonetheless, when an alternative technology to GM is available, consumers prefer it.2012 Kassardjian, E., Robin, S., Ruffieux, BL'hostilite aux OGM survit-elle a des produits attractifs? (Does Consumer's Aversion to GM Food Survive a Yummy Apple?)(French)Revue Francaise d'Economie

Lusk, 2003 Lusk, J.L., 2003. Effects of cheap talk on consumer willingness-to-pay for golden rice. American Journal of Agricultural Economics, 85(4), 840-856.A large body of literature suggests willingness-to-pay is overstated in hypothetical valuation questions as compared to when actual payment is required. Recently, "cheap talk" has been proposed to eliminate the potential bias in hypothetical valuation questions. Cheap talk refers to process of explaining hypothetical bias to individuals prior to asking a valuation question. This study explores the effect of cheap talk in a mass mail survey using a conventional value elicitation technique. Results indicate that cheap talk was effective at reducing willingness-to-pay for most survey participants; however, consistent with previous research, cheap talk did not reduce willingness-to-pay for knowledgeable consumers.2003 Lusk, J.L. Effects of cheap talk on consumer willingness-to-pay for golden rice American Journal of Agricultural Economics

Lusk & Rozan, 2005 Lusk, J.L., Rozan, A., 2005. Consumer acceptance of biotechnology and the role of second generation technologies in the USA and Europe. Trends in Biotechnology, 23(8), 386-387. / 2005 Lusk, J.L., Rozan, A.Consumer acceptance of biotechnology and the role of second generation technologies in the USA and Europe Trends in Biotechnology

Rozan et al., 2007 Rozan, A., Lusk, J.L., Campardon, M., 2007. Consumer acceptance of a genetically modified organism of the second generation: The Golden Rice. Revue D Economie Politique, 117(5), 843-852.In this paper we present the results of a study which focuses on a second generation genetically modified organism - golden rice genetically modified to contain b-carotene. We report the results of a contingent valuation survey conducted with a demographically representative sample of French households. If the majority of the sample are not aware of Golden Rice and judge this food product unacceptable, however we observe a quarter of the sample would be willing to buy this product if the price is less than or equal to that of non-genetically modified rice.2007 Rozan, A., Lusk, J.L., Campardon, M.Consumer acceptance of a genetically modified organism of the second generation: The Golden Rice Revue D Economie Politique

Macro-level studiesReference Full reference Abstract Year Authors Title Journal

Chow et al., 2010 Chow, J., Klein, E.Y., Laxminarayan, R., 2010. Cost-Effectiveness of "Golden Mustard" for Treating Vitamin A Deficiency in India. Plos One, 5(8).Background: Vitamin A deficiency (VAD) is an important nutritional problem in India, resulting in an increased risk of severemorbidity and mortality. Periodic, high-dose vitamin A supplementation is the WHO-recommended method to preventVAD, since a single dose can compensate for reduced dietary intake or increased need over a period of several months.However, in India only 34 percent of targeted children currently receive the two doses per year, and new strategies areurgently needed.Methodology: Recent advancements in biotechnology permit alternative strategies for increasing the vitamin A content ofcommon foods. Mustard (Brassica juncea), which is consumed widely in the form of oil by VAD populations, can begenetically modified to express high levels of beta-carotene, a precursor to vitamin A. Using estimates for consumption, wecompare predicted costs and benefits of genetically modified (GM) fortification of mustard seed with high-dose vitamin Asupplementation and industrial fortification of mustar2010 Chow, J., Klein, E.Y., Laxminarayan, R.Cost-Effectiveness of "Golden Mustard" for Treating Vitamin A Deficiency in India. Plos One

De Steur et al. , 2010 De Steur, H., Gellynck, X., Storozhenko, S., Ge, L.Q., Lambert, W., Van Der Straeten, D., Viaene, J., 2010. Health impact in China of folate-biofortified rice. Nature Biotechnology, 28(6), 554-556./ 2010 De Steur, H., Gellynck, X., Storozhenko, S., Ge, L.Q., Lambert, W., Van Der Straeten, D., Viaene, J.Health impact in China of folate-biofortified rice. Nature Biotechnology

De Steur et al., 2012 De Steur, H., Gellynck, X., Blancquaert, D., Lambert, W., Van Der Straeten, D., Qaim, M., 2012. Potential impact and cost-effectiveness of multi-biofortified rice in China. New Biotechnology, 29(3), 432-442. Unlike the other major crops, no genetically modified (GM) varieties of rice have been commercialized at a large scale. Within the next 2-3 years new transgenic rice varieties could be ready for regulatory approval and subsequent commercialization, though. Given the importance of rice as staple crop for many of the world's poorest people, this will have implications for the alleviation of poverty, hunger and malnutrition. Thus, policy-makers need to be aware of the potential benefits of GM rice. We provide an overview of the literature and discuss the evidence on expected agronomic and consumer benefits of genetically engineered rice. We find that while GM rice with improved agronomic traits could deliver benefits similar to already commercialized biotechnology crops, expected benefits of consumer traits could be higher by an order of magnitude. By aggregating the expected annual benefits, we estimate the global value of GM rice to be US$64 billion per year. This is only an indicative value, as more GM varie2012 De Steur, H., Gellynck, X., Blancquaert, D., Lambert, W., Van Der Straeten, D., Qaim, M.Potential impact and cost-effectiveness of multi-biofortified rice in China. New Biotechnology

De Steur et al., 2013 De Steur, H., Blancquaert, D., Gellynck, X., Lambert, W., Van Der Straeten, D., 2012. Ex-ante Evaluation of Biotechnology Innovations: the Case of Folate Biofortified Rice in China. Current Pharmaceutical Biotechnology, 13(15), 2751-2760.In order to valorize novel biotechnology innovations, there is a need to evaluate ex-ante their market potential. A case in point is biofortification, i.e. the enhancement of the micronutrient content of staple crops through conventional or genetic breeding techniques. In a recent article in Nature Biotechnology, for example, De Steur et al. (2010) demonstrated the large potential consumer health benefits of folate biofortified rice as a means to reduce folate deficiency and Neural-Tube Defects. By focusing on a Chinese high-risk region of Neural-Tube Defects, the current study defines the potential cost-effectiveness of this genetically modified crop where the need to improve folate intake levels is highest. Building on the Disability-Adjusted Life Years (DALY) approach, both the potential health impacts and costs of its implementation are measured and benchmarked against similar innovations. The results show that this transgenic crop could be a highly cost-effective product innovation (US$ 120.34 -US$ 40.1 2013 De Steur, H., Blancquaert, D., Gellynck, X., Lambert, W., Van Der Straeten, D.Ex-ante Evaluation of Biotechnology Innovations: the Case of Folate Biofortified Rice in China Current Pharmaceutical Biotechnology

Stein et al., 2006 Stein, A.J., Sachdev, H.P.S., Qaim, M., 2006. Potential impact and cost-effectiveness of Golden Rice. Nature Biotechnology, 24(10), 1200-1201./ 2006 Stein, A.J., Sachdev, H.P.S., Qaim, M.Potential impact and cost-effectiveness of Golden Rice. Nature Biotechnology

Zimmerman & Qaim, 2004 Zimmermann, R., Qaim, M., 2004. Potential health benefits of Golden Rice: a Philippine case study. Food Policy, 29(2), 147-168.Golden Rice has been genetically modified to produce beta-carotene in the endosperm of grain. It could improve the vitamin A status of deficient food consumers, especially women and children in developing countries. This paper analyses potential impacts in a Philippine context. Since the technology is still at the stage of R&D, benefits are simulated with a scenario approach. Health effects are quantified using the methodology of disability-adjusted life years (DALYs). Golden Rice will not completely eliminate the problems of vitamin A deficiency, such as blindness or increased mortality. Therefore, it should be seen as a complement rather than a substitute for alternative micronutrient interventions. Yet the technology could bring about significant benefits. Depending on the underlying assumptions, annual health improvements are worth between US$ 16 and 88 million, and rates of return on R&D investments range between 66% and 133%. Due to the uncertainty related to key parameters, these results should be trea2004 Zimmermann, R., Qaim, M.Potential health benefits of Golden Rice: a Philippine case study. Food Policy

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Micro-level studies

Socio-economic research