The United States Department of Agriculture Animal and Plant Health Inspection Service (USDA/APHIS) Prepared a Draft Environmental Assessment  in response to University of Florida Petition 04-337-01P seeking a Determination of Nonregulated Status for X17-2 Papaya Resistant to Papaya Ringspot Virus . USDA/APHIS solicited public comments by 3 November 2008 at: http://www.regulations.gov/fdmspublic/component/main?main=DocumentDetail&o=09000064806cf5de
Our comment is submitted on behalf of The Institute of Science in Society, London , UK ( www.i-sis.org.uk ).
The USDA/AHIS assessment  concluded that: “National Environmental Policy Act (NEPA) implementing regulations require consideration of the degree to which the possible effects on the human environment are highly uncertain or involve unique or unknown risk None of the effects on the human environment identified are highly controversial, highly uncertain, or involve unique or unknown risks. The effects are similar in kind to (and no worse than) those already observed for currently commercially available and widely planted genetically engineered papaya varieties in agriculture production systems. APHIS is not aware of any means by which the proposed action (a determination of nonregulated status for X17-2 papaya) would threaten or violate Federal, State, or local law requirements.” That conclusion, though consistent with the information provided in the petition for nonregulated status, is not consistent with evidence from well known scientific studies that calls into question USDA/APHIS’ environment assessment.
There are at least two areas where the environmental assessment is faulted. The first is the lack of a conclusive safety analysis of the coat protein gene that confers resistance to the papaya ring spot virus; the second is a failure to consider conflicting evidence about the impact of the transgenic papaya on soil biota.
The coat protein gene
The line X17-2 was constructed with a kanamycin resistance coding sequence from E. coli with Nopaline synthase (NOS) promoter and terminator from Agrobacterium , and the coding sequence of a mutant coat protein (CP) from the papaya ringspot virus driven by the cauliflower mosaic virus 35S promoter and the a beta-glucuronidase (uid) enhancer, and terminated by the NOS terminator. The parent of the X17-2 line was transformed with T-DNA containing the CP gene with a frameshift mutation that would have prevented it from being translated into a protein. However the current X17-2 line somehow ‘repaired’ the frameshift, and the CP gene is now translatable and expressed. Nevertheless, a satisfactory level of resistance to virus infection appears to be maintained, presumably due to activation of an interfering RNA molecule that has not yet been identified.
As X17-2 produces measurable quantities of the viral coat protein, it should be noted that the coat protein has been found to contain amino acid sequences that are active as allergen epitopes, and should require full clinical studies on allergenic activity of the protein , as we have already highlighted previously [4, 5] ( Allergenic GM Papaya Scandal ,. SiS 18; Are Transgenic Proteins Allergenic? SiS 25).
Papaya X17-2 is presumed to resist the papaya ring spot virus by means of small virus inhibiting RNA molecules originating from the host papaya, or from the inserted transgene and elicited by the coat protein transgene, or originating from a portions of the inserted transgene [1, 2]. Small putative silencing RNA molecules were observed in cantaloupe transformed with inverted repeats of the papaya ring spot virus coat protein gene  A transgenic papaya mainly active against the ring spot virus in Hawaii was given nonregulated status in 1996 . That coat protein transgenic papaya was theorized to resist virus infection by means of an elicited small inhibitory RNA . USDA/APHIS maintains that inhibitory RNA such as the small interfering RNA elicited in the coat protein plum-pox resistant transgenic plum trees is harmless to human and animals, as are all RNA and DNA molecules .That assertion, as we have informed USDA, is based on an old US Food and Drug Administration opinion, since proven wrong , that the molecules are generally recognized to be safe  ( USDA Proposes to Deregulate Its Own Transgenic Plum , SiS 31). There is clear evidence that interfering RNA may be fatal to mice [10, 11] ( Gene Therapy Nightmare for Mice, SiS 31). Feeding animals with plant material expressing double stranded silencing RNA has been found to trigger RNA interference in insect pests . These considerations should make it compulsory to identify the interfering RNA species in the transgenic papaya and to test them for toxicity in animals or humans that consume the plant material.
Impact of transgenic papaya on soil biodiversity
The draft environmental assessment  suggest that the soil microbes are not significantly affected by plantings of transgenic papaya, and no conflicting studies were mentioned.. However, there are a number of publications reporting significant impacts of transgenic papaya on the soil microbes. Significantly increased populations of bacteria, actinomycetes, and fungi were found in soils on which transgenic papaya was grown . There were also significantly increased populations of kanamycin resistant bacteria and actinomycetes. A second study corroborated the finding that bacteria, actinomycetes, and fungi were increased in soil in which transgenic papaya was growing compared with non-transgenic papaya . Transgenic DNA added to the soil was found to decay rapidly in the first week then more slowly over the next 7 weeks , confirming that Horizontal Gene Transfer from GMOs Does Happen , and is one of the greatest hidden hazards of releasing GMOs into the environment, and which USDA has persistently ignored.
There is no way in which granting transgenic papaya X17-2 nonregulated status can be justified. We have opposed its release into the environment in the first place. We hold USDA and the individual risk assessors responsible for any harm resulting from granting the transgenic papaya X17-2 nonregulated status. This is number 42 of ISIS ‘ submissions to the USDA.
1. U.S. Department of Agriculture Animal and Plant Health Inspection Service Biotechnology Regulatory Services USDA/APHIS Draft Environmental Assessment In response to University of Florida Petition 04-337-01P seeking a Determination of Nonregulated Status for X17-2 Papaya Resistant to Papaya Ringspot Virus 2008
2. Davis M. Petition for Determination of nonregulated status for the X17-2 line of payapa: A papaya ringspot virus-resistant papaya 2008
3. Kleter GA, Peijnenburg AA. Screening of transgenic proteins expressed in transgenic food crops for the presence of short amino acid sequences identical to potential, IgE – binding linear epitopes of allergens. BMC Struct Biol. 2002; 2, 8-12
4. Cummins J. Allergenic GM papaya scandal Science in Society 18 , 32, 2003.
5. Ho MW, Pusztai A, Bardocz S, Cummins J. Are transgenic proteins allergenic? Science in Society 25 , 4, 2005.
6. Krubphachaya P, Jurícek M, Kertbundit S. Induction of RNA-mediated resistance to papaya ringspot virus type W. J Biochem Mol Biol. 2007;40(3), 404-41
7. Gonsalves D, Manshardt R. Petition for Determination of Regulatory Status Transgenic Papaya Lines 55-1 and 63-1 and Their Derivatives 96-051-01p 1996, http://www.aphis.usda.gov/brs/aphisdocs/96_05101p.pdf
8. Fuchs M, Gonsalves D. Safety of virus-resistant transgenic plants two decades after their introduction: lessons from realistic field risk assessment studies. Annu Rev Phytopathol. 2007, 45,173-202.
9. Cummins J and Ho MW. USDA proposes to deregulate Its Own Transgenic Plum Science in Society 31 , 5-7, 2006.
10. Grimm D, Streetz KL, Jopling CL, Storm TA, Pandey K, Davis CR, Marion P, Salazar F, Kay MA. Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways. Nature 2006; 441(7092), 537-41.
11. Ho MW. Gene Therapy Nightmare for Mice: Could Humans be Next? S Science in Society 31 , 25, 2006.
12. Mao YB, Cai WJ, Wang JW, Hong GJ, Tao XY, Wang LJ, Huang YP, Chen XY. Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol.. Nat Biotechnol . 2007, 25(11), 1307-13
13. Wei XD, Zou HL, Chu LM, Liao B, Ye CM, Lan CY. Field released transgenic papaya effect on soil microbial communities and enzyme activities. Plant Soil 2006, 285, 347-58.
14. Hsieh YT , Pan TM. Influence of planting papaya ringspot virus resistant transgenic papaya on soil microbial biodiversity.J Agric Food Chem. 2006 Jan 11;54(1):130-7
15. Lo CC, Chen SC, Yang JZ. Use of real-time polymerase chain reaction (PCR) and transformation assay to monitor the persistence and bioavailability of transgenic genes released from genetically modified papaya expressing nptII and PRSV genes in the soil. J Agric Food Chem . 2007; 55(18), 7534-40.
16. Ho MW and Cummins J. Horizontal gene transfer from GMOs does happen. Science in Society 38 , 22-24, 2008.