Golden Rice: A Perfect Example of GM Unintended Effects


Dear Friends and Colleagues

Golden Rice: A Perfect Example of GM Unintended Effects

Genetically modified Golden Rice has long been promoted as a potent tool to alleviate Vitamin A deficiency. However, recent research in India shows that the genes needed to produce it have unintended effects. When the researchers introduced the engineered DNA, their high-yielding and agronomically superior Indian rice variety became pale and stunted, flowering was delayed and the roots grew abnormally. Yields were so reduced that it was unsuitable for cultivation.

The researchers identified several reasons for these detrimental effects (see TWN Biosafety Info: Detrimental Genomic Effects Found in Golden Rice Offspring, 9 March 2017): the new gene constructs interfered with the plant’s own gene for producing growth hormones, and the additional gene constructs were not, as intended, active solely in the kernels, but also in the leaves. This led to a substantial reduction in the content of chlorophyll, which is essential for vital functions in the plants. In other words, the GR genes caused a “metabolic meltdown”.

This illustrates the fundamental challenge of GMO metabolic engineering: to not make unintended changes by disrupting the many intersecting biochemical pathways and thereby disrupting the complex plant processes that depend on them. Golden Rice stands out as a perfect example of failure in this respect, throwing a substantial shadow over the general feasibility of nutritional enhancement by GM technology.

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Allison Wilson, PhD
Independent Science News
25 October 2017

GMO Golden Rice is promoted as a potent tool to alleviate vitamin A deficiency. However, Indian researchers now report that the genes needed to produce it have unintended effects. When they introduced the engineered DNA, their high-yielding and agronomically superior Indian rice variety became pale and stunted, flowering was delayed and the roots grew abnormally. Yields were so reduced that it was unsuitable for cultivation (Bollinedi et al. 2017).

Rice is a good source of certain nutrients but it lacks Vitamin A. In low-income households in certain countries, a rice-based diet can therefore result in vitamin A and other nutrient deficiencies.

According to Syngenta and certain public sector biotechnologists, vitamin A nutrition can be improved by introducing transgenes that specify enzymes in the ?-carotene biosynthesis pathway (Ye et al. 2000; Bollinedi et al. 2014). ?-carotene is one of several carotenoid precursors to vitamin A.

A short history of Golden Rice

The first GMO Golden Rices had either two or three introduced genes (in addition to a transgene specifying antibiotic resistance as a selectable marker). Plants with two had a daffodil phytoene synthase (psy) transgene and a phytoene desaturase (ctrI) transgene from the bacterium Erwinia uredovora. These were transferred together as a gene cassette. Those with three had in addition a daffodil lycopene ?–cyclase (lcy) transgene, co-transferred in a separate cassette (Yeet al. 2000). Each independently generated event consisted of one or more gene cassettes integrated at a particular location in the plant genome.

The public sector group who developed these subsequently created a new set of events using just the psy and ctrI transgenes. They also changed the vector, the selectable marker and used different rice varieties. These changes were intended to make Golden Rice “amenable to deregulation” (Hoaet al. 2003).

As critics pointed out, the rice from both sets of events had very low carotenoid levels, less than 1.6ug/g.

Simultaneously, Syngenta Corporation produced a third set of Golden Rice events (called GR1). For GR1, Syngenta replaced the viral promoter of the ctrI transgene used by the public sector group with a promoter designed to give enzyme activity just in the rice grain. They also removed the selectable marker. Total carotenoid levels in GR1 rice grains were higher: 6ug/g (Al-Babili and Beyer 2005).

The hulled grains of these different iterations of Golden Rice were yellow due to the ?-carotene. However, even the best GR1 events still had ?-carotene levels too low to alleviate deficiencies (Bollinedi et al. 2014).

Engineering Golden Rice GR2

Syngenta then made the fourth and current version of Golden Rice, called GR2. They introduced three genes as a single cassette. As before, two specified enzymes in the ?-carotene biosynthesis pathway — a bacterial ctrI gene and, this time, a maize version of the psy gene (Paine et al. 2005). The third gene specified a (non-antibiotic) selectable marker gene.

In GR2, the ctrI and psy genes were again placed under the control of an endosperm-specific promoter. The GR2 cassette was introduced into the genome of the American rice variety Kaybonnet.

Syngenta researchers selected 23 independent GR2 events with between 9 and 37ug/g total carotenoid in the endosperm (Paineet al. 2005).

Golden Rice for India?

Through its Humanitarian Board on Golden Rice (HumBo), Syngenta made six of these GR2 events available to public sector breeding programs (Chikkappaet al. 2011). The Indian Researchers chose GR2-R1, presumably the most promising event, to breed a Golden Rice variety suitable for India (Bollinediet al. 2017).

However, when they introduced GR2-R1 into the high-yielding variety Swarna, the plants with the GR2-R1 event were “drastically altered” (Bollinedi et al. 2017). GR2-R1 Swarna rice had pale green leaves, various root defects, and extra side shoots (tillers). The plants also flowered later, were half the height, and half as fertile. Yield was one-third of non-GMO Swarna.
Most of these abnormalities, it transpired, had been present in the original GR2-R1 plants donated by Syngenta.

Molecular-Genetic origins of GR2-R1 defects

DNA analysis revealed a partial explanation for these defects. The GR2-R1 DNA had inserted into and disrupted a native rice gene called OsAux1. OsAux1 specifies a transporter for the important plant hormone auxin. The researchers suggested this disruption explains some of the root and shoot defects.

A second contributor to the defects seems to be that, although the psy and ctrIgenes had been specifically engineered so that their enzyme products would be present only in grains, the researchers found evidence the enzymes were unexpectedly functioning in GR2-R1 leaves.
Chemical analysis of leaves, stems, and flowering parts showed GR2-R1 plants had altered levels of three other key plant hormones: abscisic acid (ABA), gibberellin (GA3), and cytokinin.

To explain this the researchers proposed that the presence of the genetically engineered PSY and CTRI enzymes in leaves depletes a compound (GGDP) needed to make other plant biochemicals, in particular hormones and chlorophylls. Lack of chlorophyll would explain the pale leaves, while altered hormone levels would explain the other growth defects and the yield loss seen in GR2-R1 plants.

The researchers did not report whether metabolic and hormonal disruptions also occurred in the rice endosperm, where PSY and CTRI were intended to function.

The End for Golden Rice?

Golden rice has for over 20 years stood as the exemplar of a “good GMO” and proponents have blamed its failure to reach the market on “over-regulation” of GMOs and on “anti-GMO” opposition (Lee and Krimsky 2016).

This latest research suggests a different narrative. It shows that problems intrinsic to GMO breeding are what have prevented researchers from developing Golden Rice suitable for commercialization (Schubert 2002; Wilson et al. 2006).

The second great significance of this research, is that it implies engineering sufficient levels of ?-carotene is disruptive to the basic metabolism of the plants.

“What the Indian researchers show is that the Golden Rice transgenes given to them by Syngenta caused a metabolic meltdown,” says Jonathan Latham, Executive Director of the Bioscience Resource Project. “The classic criticisms of genetic engineering as a plant breeding tool have always been, first, that introduced DNA will disrupt native gene sequences and, second, that unpredictable disruption of normal metabolism may result from introducing new functions. Golden Rice exemplifies these flaws to perfection.”

This then is the fundamental challenge of GMO metabolic engineering. It seems that making the intended metabolic changes (in this case increasing ?-carotene levels) is the easy part (Giuliano 2017). The real challenge is to not make unintended changes by disrupting the many intersecting biochemical pathways—and thereby disrupting the complex plant processes that depend on them (Schubert 2008).

With their BioBricks approach to biology, Syngenta and their public sector allies have shown negligible understanding of these complexities, leaving it once again to non-GMO breeders to successfully enhance nutrient levels in plants (Andersson et al. 2017).

For years the quintessential example used to support GMO plant breeding, Golden Rice may now become “Exhibit A” for those wishing to critique it.

Highly Recommended Further Reading: The place of Golden Rice in the broader politics of rice, including its prospects in the Philippines (or elsewhere) are well explained in an important new paper by Stone, G. D., & Glover, D. (2016).”Disembedding grain: Golden Rice, the Green Revolution, and heirloom seeds in the Philippines” This paper is full of insights and up-to-date information.
Stone, G. D., & Glover, D. (2017). Disembedding grain: Golden Rice, the Green Revolution, and heirloom seeds in the Philippines. Agriculture and Human Values, 34(1), 87-102.

Andersson, M. S., Saltzman, A., Virk, P. S., & Pfeiffer, W. H. (2017). Progress update: crop development of biofortified staple food crops under HarvestPlus. African Journal of Food, Agriculture, Nutrition and Development, 17(2), 11905-11935.
Al-Babili, S., & Beyer, P. (2005). Golden Rice–five years on the road–five years to go?. Trends in plant science, 10(12), 565-573.
Bollinedi, H., Gopala, K. S., Sundaram, R. M., Sudhakar, D., Prabhu, K. V., & Singh, N. K. (2014). Marker assisted biofortification of rice with pro-vitamin A using transgenic Golden Rice lines: progress and prospects. Indian Journal of Genetics, 74(4), 624-630.
Bollinedi, H., Prabhu, K. V., Singh, N. K., Mishra, S., Khurana, J. P., & Singh, A. K. (2017). Molecular and Functional Characterization of GR2-R1 Event Based Backcross Derived Lines of Golden Rice in the Genetic Background of a Mega Rice Variety Swarna. PloS one, 12(1), e0169600.
Chikkappa, G. K., Tyagi, N. K., Venkatesh, K., Ashish, M., Prabhu, K. V., Mohapatra, T., & Singh, A. K. (2011). Analysis of transgene (s)(psy+ crtI) inheritance and its stability over generations in the genetic background of indica rice cultivar Swarna. Journal of plant biochemistry and biotechnology, 20(1), 29-38.
Giuliano, G. (2017). Provitamin A biofortification of crop plants: a gold rush with many miners. Current Opinion in Biotechnology, 44, 169-180.
Hoa, T. T. C., Al-Babili, S., Schaub, P., Potrykus, I., & Beyer, P. (2003). Golden Indica and Japonica rice lines amenable to deregulation. Plant physiology, 133(1), 161-169.
Lee, H., & Krimsky, S. (2016). The Arrested Development of Golden Rice: The Scientific and Social Challenges of a Transgenic Biofortified Crop. International Journal of Social Science Studies, 4(11), 51-64.
Paine, J. A., Shipton, C. A., Chaggar, S., Howells, R. M., Kennedy, M. J., Vernon, G., … & Drake, R. (2005). Improving the nutritional value of Golden Rice through increased pro-vitamin A content. Nature biotechnology, 23(4), 482-487.
Schubert, D. (2002). A different perspective on GM food. Nature biotechnology, 20(10), 969-969.
Schubert, D. R. (2008). The problem with nutritionally enhanced plants. Journal of medicinal food, 11(4), 601-605.
Wilson, Allison K., Jonathan R. Latham, and Ricarda A. Steinbrecher. “Transformation-induced mutations in transgenic plants: analysis and biosafety implications.” Biotechnology and Genetic Engineering Reviews 23.1 (2006): 209-238.
Ye, X., Al-Babili, S., Klöti, A., Zhang, J., Lucca, P., Beyer, P., & Potrykus, I. (2000). Engineering the provitamin A (?-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science, 287(5451), 303-305.

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