Science in Society Archive

Rice War Continues

Editor's note: The productivity of rice has been falling along with that of other food grains. Chief among the causes of the fall in productivity are severe water shortages due to over-irrigation and depletion of aquifers, eroded soils from over-application of chemical fertilizers and pesticides, and rising temperatures from global warming.

While innovative farmers have been addressing these problems with a range of effective measures to increase yields through regenerating degraded soils, conserving water and minimizing inputs (see many articles in SiS23), pro-GM scientists in the three major rice-growing countries, China, India and Japan, have all been researching and promoting GM rice with scant regard for safety or sustainability.

We are circulating Professor Joe Cummins' review on GM rice in China, and making available two others, on GM rice in India and Japan respectively on the I-SIS website:

GM Rice in Japan
GM Rice in India


GM Rice in Japan

Prof. Joe Cummins reviews genetically modified rice in Japan and points to overlooked dangers

Rice consumption in Japan

Japanese rice-consumption is eighth among nations, or about 7% that of China. Between 1970 and 2001, per capita consumption of rice decreased about 30% in Japan while consumption increased about 10% in China [1]. Japan is a leading nation in rice research, rivaling China, but with a somewhat different emphasis. Japan has had a very active research program developing genetically modified (GM) rice. Field trials of GM rice have been reported from 1993 to 2002, and those were engineered for rice stripe virus resistance, low allergen rice, low protein for saki-brewing, low gluterin (storage protein), human lactoferrin, herbicide tolerance and rice blast resistance. The largest numbers of tests were for Monsanto Japan’s GM rice tolerant to herbicide and for rice resistant to blast disease [2]. In 2003, Japan’s approvals for import and planting included GM rice for virus resistance, low allergenicity, low protein, low gluterin and herbicide tolerance [3].

Rice with human cytochrome p450 genes

There is a large volume of work on using human cytochrome p450 genes to produce tolerance to a range of herbicides. The cytochrome p450 enzymes are present in all organisms from bacteria to humans. There are a number of cytochrome p450 genes and alleles for a family of enzymes involved in detoxifying xenobiotic (artificial and hence unnatural) chemicals and in steroid metabolism. These enzymes are believed to have originated to prevent over-accumulation of fat-soluble chemicals in cell membranes. The cytochrome p450 enzymes in humans break down pharmaceutical drugs and also activate cancer-causing chemicals such as poly aromatic hydrocarbons (PAH) and aflatoxin. Interestingly, there does not seem to have been any attempt to adjust the codons of the human transgenes for those preferred by plants, so perhaps the relatively low level production of the enzymes proved satisfactory for the purpose.

GM rice plants expressing human cytochrome genes cyp2c9 and cyp2c19 were tolerant to a range of herbicides including the sulphonylurea herbicides; they were obtained by transformation with three separate plasmids simultaneously. The first plasmid contained the cyp2c9 gene driven by a CaMV promoter with seven enhancers, followed by an un-translated sequence from alfalfa mosaic virus and the Agrobacterium nos gene terminator tnos; accompanied by two genes for resistance to the antibiotics hygromycin and neomycin respectively. The second plasmid contained the cyp2c19 gene with the same regulatory sequences and markers as the first. The third plasmid contained the gus gene accompanied by the same regulatory genes and markers as the other two [4]. The CYP2C9 and CYP2C19 enzymes activate the PAH carcinogen Benzo(a)pyrene, a common air pollutant [5].

GM rice plants expressing the human cyp2b6 gene, obtained by transformation with a plasmid containing the gene with the same regulatory sequences and marker genes as described above, were tolerant to the herbicide ethofumesate, to which GM rice with other cyp genes were susceptible [6]. The CYP2B6 enzyme activates the water disinfection chemical bromodichloromethane to produce a carcinogen [7].

GM rice plants expressing human cyp1a1, with the same regulatory sequences and marker genes, were tolerant to a range of herbicides. Radioactively labelled herbicides - atrazine, chlortoluron and norflurazon - were used to study the breakdown products in the transgenic rice. These products, many of which are potential mutagens or carcinogens, were excreted into the soil, where they would persist in surface and groundwater [8]. The cyp1a1 gene product has been shown to activate many environmental carcinogens [5,9].

Rice with novel insect resistance genes

Along with the numerous commercial Bt rice strains field-tested, novel insect control genes have been used. For example, a trypsin-inhibitor was introduced into rice to interfere with the digestion of stem borer insects [10]. A synthetic trypsin-inhibitor gene derived from the winged bean with a reduced GC (guanine-cytosine) content to improve messenger RNA production in rice was placed under the enhanced CaMV promoter (see above) further boosted with a tobacco mosaic virus omega sequence and the first intron of a phaseolin gene, and terminated with tnos. In addition, a hygromycin resistance marker was also inserted.

GM rice bearing an insect pox virus gene was used to control army worm larvae. The pox virus gene product consumed by the army worm larvae made them susceptible to the common soil baculovirus, which are otherwise not virulent in the larvae. The synthetic insect pox gene had an altered DNA sequence driven by a CaMV promoter, further boosted by a non-coding region of the rice stripe virus RNA and transcription was terminated by tnos. A hygromycin resistance marker was also inserted. The army worm larvae were reported to be controlled by the baculovirus after feeding on the transgenic rice [11].

Rice to control bacterial blight

Cecropia moths have potent anti-bacterial peptides in the haemolymph (insect blood) of their larvae. Rice bacterial blight has been very difficult to control globally and novel antibacterial products are being sought. The larvae of the silk moth, Bombyx mori, provided a potent antibacterial peptide called cecropinB. The gene for that peptide was engineered into GM rice driven by another complicated version of the CaMV 35S promoter with enhancer 5p, the omega sequence from tobacco mosaic virus followed its promoter and the first intron of a phaseolin gene; a rice chitinase signal peptide was added to the cecropin sequence, terminated by tnos. A kanamycin-resistance marker was also introduced. The transgenic rice was reported to provide effective resistance to bacterial infection [12].

Rice with altered growth or metabolism

Rice has been modified to enhance metabolism. The most ambitious effort is to try to make photosynthesis more efficient. Plants are divided into two types - C3 and C4 plants - C3 photosynthesis being less efficient than C4. Most plants are C3, including sugar beet, rice and potatoes; while maize and sugarcane are C4 plants. Engineering rice to become a C4 plant may therefore increase the yield of rice crops.

The enzyme phosphoenolpyruvate carboxylase (PEPC) fixes carbon dioxide in C4 plants, while C3 plants fix carbon dioxide exclusively through an enzyme called Rubisco. PEPC acts as a pump to raise carbon dioxide concentration at the site of Rubisco in the chloroplast. In one effort to enhance expression of PEPC, the transgene for that enzyme was obtained from maize (a C4 plant) and accompanied by the maize PEPC promoter and all of the PEPC introns and exons. A hygromycin resistance marker was also inserted. The over-expression of PEPC failed to improve photosynthesis [13]. The gene for another enzyme phosphoenol pyruvate carboxylase (PCK) from a C4 weed, Urochloa panicoides (liver weed) was also used [14] in the attempt to create a C4 rice. But there is no guarantee that rice yields will be improved in the field even if a C4 rice is eventually created.

Dwarf rice is desirable because they resist lodging in wind and rain. The plant hormone gibberellin controls plant height, and reducing hormone levels will reduce plant height. Dwarf rice was created by incorporating the gene for an enzyme that degrades the hormone placed under the control of a strong rice promoter. Unfortunately the dwarf rice plants failed to set seed because the hormone also participates in seed set.

Dwarf rice that set seed and produced a good crop was produced using a tissue specific promoter for gibberellin synthesis. The rice was transformed with the hormone-degrading gene under control of the tissue specific promoter and terminated with tnos, together with a hygromycin resistance marker [15]. The semi dwarf transgenic rice has not yet been fully evaluated for field performance.

In Japan, 30% of the agricultural land is unsuitable for rice production because it is too alkaline. Rice suffers iron deficiency in alkaline soil. Iron uptake can be achieved in alkaline soil by the release of molecules called phytosiderophores from the roots of plants tolerant to alkaline soil. Barley secretes phytosiderophores through the action of an enzyme nicotianamine aminotransferase (NAAT). GM rice with the barley gene for NAAT showed enhanced tolerance to low iron availability and had a greater grain yield than conventional rice grown on alkaline soil. The barley NAAT transgene was driven by a CaMV promoter and terminated by tnos, and accompanied by hygromycin-resistance and neomycin-resistance marker genes [16].

Overlooked hazards

Japanese experiments in GM rice are technically sophisticated but the human and environmental safety of the GM crops has not yet been full evaluated. In particular, the human cytochrome p450 genes are already known to activate carcinogens. They should not be used in rice, which is an important food crop that is eaten widely as a staple in Japan and many parts of Asia. The extensive use of aggressive CaMV–based superpromoters untested for safety, and the incorporation of human genes will both increase the potential of transgenic DNA to invade human genomes through illegitimate and homologous recombination, with dangerous consequences including the creation/activation of new viruses or cancer [17].

Article first published 30/11/04



References

  1. Hossain M. FAO rice conference: Long term prospects for the global rice economy, 2004, http://www.fao.org/rice2004/en/pdf/hossain.pdf
  2. Biotechnology Safety Division Research Council Secretariat. The current status of transgenic crop plants in Japan, 2001, http://www.s.affrc.go.jp/docs/sentan/eguide/edevelp.htm
  3. USDA GAIN Report JA3002. Biotechnology Update on Japan’s Biotechnology safety and approval and labeling policies, 2003, http://www.fas.usda.gov/gainfiles/200302/145884801.pdf
  4. Inui H, Shiota N, Ido Y, Inoue T, Hirose S, Kawahigashi H, Ohkawa Y and Ohkawa H. Herbicide metabolism and tolerance in the transgenic rice plants expressing human CYP2C9 and CYP2C19. Pesticide Biochemistry and Physiology 2001, 71, 156–69.
  5. Yamazaki Y, Fujita K, Nakayama K, Suzuki A, Nakamura K, Yamazaki H and Kamataki T. Establishment of ten strains of genetically engineered Salmonella typhimurium TA1538 each co-expressing a form of human cytochrome P450 with NADPH-cytochrome P450 reductase sensitive to various promutagens. Mutat Res. 2004, 562(1-2), 151-62.
  6. Kawahigashi H, Hirose S, Hayashi E, Ohkawa H and Ohkawaa Y. Phytotoxicity and metabolism of ethofumesate in transgenic rice plants expressing the human CYP2B6 gene. Pesticide Biochemistry and Physiology 2002, 74,139–47.
  7. Allis J and Zhao G. Quantitative evaluation of bromodichloromethane metabolism by recombinant rat and human cytochrome P450s. Chemico-Biological Interactions 2002, 140, 137–53.
  8. Kawahigashi H, Hirose S, Hayashi E, Ohkawa H. and Ohkawa Y. Transgenic rice plants expressing human CYP1A1 exude herbicide metabolites from their roots. Plant Science 2003, 165, 373-81.
  9. Gábelová A, Binková B, Valovičová Z. and Šrám R. DNA adduct formation by 7H-Dibenzo[c,g]carbazole and its tissue- and organ-specific derivatives in Chinese hamster V79 cell lines stably expressing cytochrome P450 enzymes. Environmental and Molecular Mutagenesis 2004, 44, in press.
  10. Mochizuk A, Nishizawa Y, Onoder H, Tabe Y, Toki S, Habu Y, Ugak M and Ohashi Y. Transgenic rice plants expressing a trypsin inhibitor are resistant againstrice stem borers, Chilo suppressalis. Entomologia Experimentalis et Applicata 1999, 93, 173–78.
  11. Hukuhara T, Hayakawa T and Wijonarko A. Increased baculovirus susceptibility of armyworm larvae feeding on transgenic rice plants expressing an entomopoxvirus gene. Nature Biotech 1999, 17, 1122–4.
  12. Sharma A, Sharma R, Imamura M, Yamakawa M and Machii H. Transgenic expression of cecropin B, an antibacterial peptide from Bombyx mori, confers enhanced resistance to bacterial leaf blight in rice. FEBS Letters 2000, 484, 7-11.
  13. Agarie S, Miur A, Sumikur R, Tsukamoto S, Nose A, Arima S, Matsuoka M and Miyao-Tokutomi M. Overexpression of C4 PEPC caused O2-insensitive photosynthesis in transgenic rice plants. Plant Science 2002, 162, 257–65.
  14. Suzuki S, Murai N, Burnell J and Arai M. Changes in photosynthetic carbon flow in transgenic rice plants that express C4-Type phosphoenolpyruvate carboxykinase from Urochloa panicoides. Plant Physiology 2000,124, 163–72.
  15. Sakamoto T, Morinaka, Ishiyama K, Kobayashi M, Itoh H, Kayano T, Iwahori S, Matsuok M and Tanaka H. Genetic manipulation of gibberellin metabolism in transgenic rice. Nature Biotech 2003, 21, 909–13.
  16. Takahashi M, Nakanishi H, Kawasaki S, Nishizawa N and Mori S. Enhanced tolerance of rice to low iron availability in alkaline soils using barley nicotianamine aminotransferase genes. Nature Biotech 2001,19, 466–9.
  17. See Ho MW. Living with the Fluid Genome, ISIS & TWN, London and Penang, 2003, for a thorough review on horizontal gene transfer in general and the hazards of the CaMV 35S promoter in particular.

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