Wednesday, February 17, 2010

Field Testing Genetically Engineered Eucalyptus: Environment Assessment Still Inadequate

USDA/APHIS aims to support widely dispersed field trials with reckless disregard for safety and in flagrant violation of the precautionary principle
Prof. Joe Cummins
This report has been submitted to the USDA/APHIS on behalf of ISIS
Please circulate widely, and make your own submissions online at or by post: two copies to Docket No. APHIS-2008-0059, Regulatory Analysis and
Development, PPD, APHIS, Station 3A-03.8, 4700 River Road Unit 118,
Riverdale, MD 20737-1238, on or before 18 February 2010

The US forest biotechnology company ArborGen has had considerable success in getting permission from USDA/APHIS (United States Department of Agriculture/Animal and Plant Health Inspection Service) to undertake open field trials of the companies genetically modified (GM) eucalyptus (note: GM, transgenic, and genetically engineered are used interchangeably throughout). The first field test of modified eucalyptus was undertaken in Alabama and reached a Finding of No Significant Impact (FONSI). ISIS objected to the application for an earlier field test [1] ( GM Eucalyptus Environmental Assessment Irregular , SiS 35). Further field tests were carried out in Florida. Now, ArborGen proposes a gigantic follow-up by submitting two applications for field testing on 29 sites ranging in size from 0.5 to 20 acres located in Alabama, Florida, Texas and Louisiana [2].
USDA/APHIS has prepared a Revised Draft Environmental Assessment in response to the applications, to continue research on the transgenic Eucalyptus trees currently permitted, to be issued permits to plant additional trees, and for the environmental release of transgenic eucalyptus trees that will be allowed to flower on 28 of the 29 proposed sites. These plants are a clone, coded EH1, derived from a hybrid of Eucalyptus grandis X Eucalyptus urophylla , and have been genetically engineered with different constructs. The purpose of the environmental release is to assess the effectiveness of gene constructs intended to confer cold tolerance, to alter lignin biosynthesis; and to alter fertility. In addition, the trees have been engineered with a selectable marker gene.
The two combined permits requested by ArborGen would allow flowering on up to 330 acres across all 28 locations, in fields ranging from 0.5 to 20 acres [ 2]. .

Gene used as selectable marker

The kanamycin resistance selectable marker gene ( nptII ) engineered into the trees is accepted as being safe according to APHIS. In a number of instances, plants transformed with this gene have been deregulated by APHIS (e.g. corn, rapeseed, cotton, and papaya in past petitions). It should be pointed out that the food and feed crops deregulated by APHIS were not labelled and there has been no effort to study the impact of the antibiotic resistance gene on the human population or for that matter farm animals. Therefore, the gene is essentially untested, and APHIS has no ground in assuming that the use of that gene has no significant impact.

The cold hardiness modification

The most significant modification is the introduction of genes for cold hardiness. The genes introduced include a protein transcription factor called C-Repeat Binding Factor (CBF) driven by a cold hardy stress - inducible promoter. The transcription factor regulated the expression of a number of genes conferring frost hardiness. Using the stress-inducible promoter to drive CBF transgene expression significantly improves freeze tolerance without negatively impacting other agronomically important traits. The cold-inducible promoter causes the gene to be expressed under cold temperatures, thus mitigating the potential of reduced growth at low temperatures. A constitutive promoter such as that of CaMV used to drive CBF transgene would reduce plant growth. A patent application submitted by ArborGen reports the CBF gene sequences employed by ArborGen in GM Ecalyptus [3]. Given the propensity of the CBF transgenes to produce detrimental growth impacts when controlled by a constitutive promoter, it is advisable make a fuller study of the proteome (the entire set of proteins expressed by a genome, cell, tissue or organism) of the plants modified with CBF and the stress inducible promoter. The presumption that the effects of transgenic CBF and its cold inducible promoter has no harmful side effects is unwarranted without a fuller study.

Gene for altered fertility

According to APHIS, the barnase gene has been engineered into several other crops that have been previously reviewed and addressed in multiple environmental assessments by APHIS, and granted non-regulated status: Male sterile corn (USDA APHIS petitions for deregulation 95-288-01p, 97-342-01p and 98-349-01p), rapeseed (petitions 98-278-01p and 01-206-01p) and chicory (petition 97-148-01p). There is no reason to believe that the function and expression of this gene will be any different from the plants in which it has been previously assessed. In greenhouse tests using tobacco and an early flowering model Eucalyptus ( E. occidentalis ), the applicant has found that the barnase gene demonstrated 100 percent efficacy in preventing pollen formation. In developing flower buds from field grown transgenic Eucalyptus lines containing this cassette, 90 percent of lines showed complete pollen ablation. Recent observations from the replicated field study being conducted in Alabama under the approved BRS permit (BRS # 06-325-111r-a1) confirm that cold tolerant trees grown at the site and allowed to flower did not produce any viable pollen. APHIS concluded that barnase will have no significant impact on the environment.
APHIS ignores the fact that the product of the barnase gene is barnase ribonuclease, a powerful cell toxin poisoning humans, small mammals and bird [4]. The same toxin has been engineered to kill cancer cells [5]. A patent application submitted by ArborGen in 2009 [6] stated: “Accordingly, there exists a need for a reproductive ablation system having reduced barnase induced toxicity and minimal leaky expression in a plant's vegetative tissues.” The leakiness and toxicity of barnase was not mentioned in the APHIS assessment.

Gene for altered lignin

According to APHIS, in a small set of experiments the CBF and barnase genes are also being tested in combination with unspecified genes introduced to alter lignin biosynthesis (claimed as CBI, confidential business information). In addition, the trees have been engineered with a common selectable marker gene ( nptII ) that confers resistance to the antibiotic kanamycin. The lignin gene has been engineered into other crops that have been previously released into the environment under both notifications and permits. The gene engineered into the plants in these field tests has been previously tested in ArborGen field trials for more than two years. There might be a concern that altered lignin could lead to an increase in insect or disease susceptibility as lignin is often associated with resistance to insects and disease organisms , but the results of field tests with this particular gene have shown no differences in plant pest susceptibility. Growth measurements have indicated that trees containing this gene had normal to a moderately reduced growth phenotype. However, if during the tests there is evidence of increase disease or insect susceptibility, the applicant is required to report such unanticipated (including excessive mortality or morbidity) to APHIS under the terms of the permit [2].
The claim that the l ow lignin trees are not susceptible to diseases caused by insect predation, fungus virus , or bacteria needs independent verification. It would be advisable to subject some trees to pathogens and insect pests to evaluate their resistance to the pests or pathogens directly, and in controlled comparison to wild - type trees. Low lignin trees often have reduced mechanical strength, which causes problems during wind storms, ice, rain or heavy wet snow. That problem does not seem to have been considered in the environment assessment.
It is unfortunate that the genetic construction of the transgenic trees has been deemed CBI as is the locations of the field tests. The CBI designation seems inappropriate because a patent as well as four patent applications by ArborGen reveal the entire genetic modifications used to alter eucalyptus trees. Patents and patent applications are public information and the information within those applications should not be designated CBI(7) . Withholding public information under CBI designations is frivolous and prevents local authorities and neighbouring residents from recognizing and pinpointing untoward side effects of the genetically modified trees being tested.

Potential Impacts to Human Health

According to APHIS, the eucalyptus field tests could be a source of Cryptococcus neoformans gattii , a fungal pathogen hosted by a variety of Eucalyptus species and other tree species. It causes systemic fungal infections in humans, leading to fungal meningitis and death. C. neoformans gattii has been found on a number of Eucalyptus hosts, some of which are being grown in commercial plantations and imported and exported for ornamental use. People have contracted and died from cryptococcosis in India, Africa, Taiwan, South America and California. C. neoformans infections are found particularly in AIDS patients due to their weak immune systems. Infections with this fungus are rare in those with fully functioning immune systems. For this reason, C. neoformans is sometimes referred to as an opportunistic fungus. It is unlikely that the trees that are the subject of the proposed field release can be a source that might introduce the pathogen into the US, as the trees were derived from sterile tissue culture lines. APHIS concluded that an increase of additional acreage planted to Eucalyptus would not impact the likelihood that these field trials should lead to a higher incidence of C. gattii in the U.S. and therefore should not pose an unnecessary risk to human or animal health. APHIS did not consider the fact that the transgenic eucalyptus could subsequently become infected and serve as hosts to the fungus.
In realilty, Cryptococcus fungus (a yeast) is a serious pathogen that infects both plants and animals including humans. APHIS' discussion of the pathogen seemed to represent the approach of lawyers for the corporation developing the transgenic eucalyptus trees. APHIS did not take a precautionary approach but instead demanded absolute proof of hazard in the absence of which, they would gleefully release any and all transgenic organism of dubious safety to the environment.
For example, APHIS omitted an important study showing that pigeon droppings are a significant source of Cryptococcus pathogen causing human illness. Pigeons droppings can deposit pathogenic yeast on eucalyptus trees [8], and cryptococal disease is increasingly global [9]. The mating of the pathogenic yeast takes place on the eucalyptus leaf surfaces, and is enhanced by a plant hormone. The product of mating, the fungal hyphae is pathogenic to plants and animals [10]. APHIS has preferred to ignore and discount the role of eucalyptus in spreading the yeast infection and to ignore the possibility that extending the range of eucalyptus will extend the range of Cryptococcus yeast infection in the United States.

Horizontal Gene Transfer to Other Organisms

Horizontal gene transfer (HGT) is any process in which an organism incorporates genetic material from another organism without being the offspring of that organism. According to APHIS, HGT is a common phenomenon among bacteria but is not common between higher organisms and HGT and expression of DNA from these plant species to bacteria is unlikely. APHIS concludes that horizontal gene transfer poses no significant environmental risk. APHIS' position on HGT ignores a wealth of evidence in the scientific literature, which ISIS reviewed most recently in 2008 [11] ( Horizontal Gene Transfer from GMOs Does Happen , SiS 38).


APHIS' Revised Draft Environmental Assessment is highly inadequate. It is aimed at supporting widely dispersed field trials with reckless disregard for safety and in flagrant violation of the precautionary principle. APHIS is demanding high levels of proof of harm while ignoring or omitting findings that raise concerns over safety. ISIS remains opposed to the releases transgenic eucalyptus, as indeed of all transgenic trees, on account of the crucial role that forests plays in stabilizing climate [12 ] ( Moratorium on all GM Trees and Ban on GM Forest Trees , SiS 35), especially as new research has now shown that it is evapotranspiration pump of forest trees that draws rain from oceans to the land, in the tropics as well as temporate and boreal regions [13] (The Real Importance of the Amazon Rain Forest , SiS 46)
This is number 43 of ISIS' submission to the USDA.


  1. Cummins J and Ho MW. GM eucalyptus environmental assessment irregular. Science in Society 35 , 50, 2007.
  2. United States Department of Agriculture (USDA)/Animal and Plant Health Inspection Service (APHIS)Biotechnology Regulatory Services Permit applications 08-011-106rm and 08-014-101rm received from ArborGen LLC Field testing of genetically engineered Eucalyptus grandis X Eucalyptus urophylla Revised Draft Environmental Assessment December 17, 2009, 97 pages
  3. United States Patent Application 20090229008 Transcription Factors by Blocksberg LN et al., ArborGen, LLC, 10 September 2009.
  4. Prior TI, Kunwar S, Pastan I. Studies on the activity of barnase toxins in vitro and in vivo . Bioconjug Chem. 1996, 7(1), 23-9.
  5. Balandin TG, Edelweiss E, Andronova NV, Treshalina EM, Sapozhnikov AM, Deyev SM. Antitumor activity and toxicity of anti-HER2 immunoRNase scFv 4D5-dibarnase in mice bearing human breast cancer xenografts. Invest New Drugs 2009 Sep 30. [Epub ahead of print]DOI10.1007/s10637-009-9329-2
  6. United States Patent Application 20090075326 Reproductive ablation constructs , Rottman WH et al., ArborGen, LLC, 19 March 2009.
  7. United States Patent 7,456,338 Modification of plant lignin content Forster ,R. , et al. Arborgen, LLC (Summerville, SC) 5 November 2008.
  8. Garcia-Hermoso D, Mathoulin-PĂ©lissier S, Couprie B, Ronin O, Dupont B, Dromer F DNA typing suggests pigeon droppings as a source of pathogenic Cryptococcus neoformans serotype D . J Clin Microbiol . 1997, 35(10), 2683-5.
  9. Springer DJ, Chaturvedi V. Projecting global occurrence of Cryptococcus gattii . Emerg Infect Dis . 2010, 16(1), 14-20.
  10. Xue C, Tada Y, Dong X, Heitman J. The human fungal pathogen Cryptococcus can complete its sexual cycle during a pathogenic association with plants. Cell Host Microbe . 2007, 1(4), 263-73.
  11. Ho MW and Cummins J. Horizontal gene transfer from GMOs does happen Science in Society 38 , 22-24, 2008.
  12. Cummins J and Ho MW. Moratorium on all GM trees and ban on forest trees. Science in Society 35 , 32-34, 2007.
  13. Bunyard P. The real importance of the Amazon rain forest. Science in Society 46 (to appear).

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