Petition for Determination of Non-regulated Status for Freeze Tolerant Hybrid Eucalyptus Lines The undersigned submit this petition under 7 CFR Part 340.6 to request that the Administrator make a determination that the article should not be regulated under 7 CFR Part 340. Prepared and Submitted by: Narender S. Nehra, PhD Senior Manager, Regulatory Affairs And Leslie Pearson, PhD Director, Regulatory Affairs ArborGen Inc. P.O. Box 840001 Summerville, SC 29484 Date Submitted: January 17, 2011 ArborGen Reference # ARB-FTE1-11 No CBI Contributors: Maud. A. Hinchee, PhD; William H.Rottmann, PhD; Chunsheng Zhang, PhD; Shujun Chang, PhD; Jeff. A. Wright, PhD; William J. Hammond, PhD; Lauren M. Chupp; Samantha A. Miller; Anita M. Thomas; Ron T. Kothera; Nathan E. Ramsey; Peter J. Raymond; Donald J. Kaczmarek, PhD; Victor C. Steel; Melissa S. Wolff Executive Summary ArborGen Inc. is submitting this Petition to USDA-APHIS-BRS to request a determination of non- regulated status for Freeze Tolerant Eucalyptus (FTE) lines 427 and 435 and plants propagated from these lines under 7 CFR Part 340. The pulp and paper industry is a major economic sector in the southeastern United States, with annual global shipments of paper products valued at almost $60 billion. Hardwood trees in the Southeast are a critical feedstock component for this industry. A reliable, high quality and cost-effective hardwood supply is necessary to sustain the pulp and paper industry in the United States, both to meet domestic demands and retain a competitive position in global markets. Hardwood supplies in the United States are projected to experience increasing demands, both from the pulp and paper sector as well as emerging new bioenergy applications. Despite this, hardwoods are not extensively planted and managed in dedicated stands due in part to the cost of plantation establishment, and their relatively slow growth and corresponding long rotation time to harvest. The development of purpose-grown hardwood trees with fast growth rates and short harvest cycles is one of the effective solutions to address hardwood supply challenges anticipated in the southeastern United States. Eucalyptus species are among the fastest growing woody plants in the world and represent about 8% of all planted forests (~18 million hectares) grown in 90 countries (FAO, 2007). While there are over 700 Eucalyptus species identified, only a limited number are grown commercially. Eucalyptus is a preferred fiber source for the global pulp and paper industry, both for its fiber qualities and productivity. It has been the focus of extensive breeding and tree improvement programs aimed at enhancing desirable wood properties such as basic density, cellulose content, fiber length and improved growth (Raymond, 2002). There is a range in freeze sensitivity among the Eucalyptus species, however the most productive Eucalyptus species favor tropical to sub-tropical conditions, and the preferred fast-growing pulp species show very limited tolerance to freezing temperatures. Attempts have been made to grow a wide variety of Eucalyptus species in several parts of the southeastern US but in many cases these species have been unable to withstand the dramatic and sudden drops in temperature that are typical of the region. Efforts to improve the freezing tolerance of fast growing species through controlled crossing with inherently freeze tolerant (but slower growing) temperate Eucalyptus species have in the past not been very successful. Currently, large scale plantings of Eucalyptus in the southeastern US are limited to regions of central and southern Florida. However, non-genetically engineered Eucalyptus is actively being developed by a number of research programs as an alternative fiber and biomass source for the U.S. south and can reasonably be expected to be established in forest plantations across the region in the near future. Scientific advancements in understanding the cold acclimation process allowed the discovery of transcription factor genes common to the plant cold-response pathway (Jaglo-Ottsen et al., 1998; Stockinger et al.; 1997, Gilmour et al., 1998; Liu et al., 1998; Kasuga et al., 1999). The discovery of cold tolerant genes combined with the development of efficient Argobacterium-mediated gene transfer methods for Eucalyptus species has allowed the development of genetically engineered FTE lines as described in this Petition. FTE lines included in this Petition were developed by the introduction of the C- Repeat Binding Factor (CBF2) gene from Arabidopsis into a fast growing but freeze susceptible commercial hybrid genotype of E. grandis x E. urophylla. The potential for reduced growth by over- expression of CBF genes in the FTE lines has been significantly mitigated by the use of a cold-inducible promoter that limits the expression of the CBF gene under conditions where this would not be desirable. In addition to the CBF2 gene, these FTE lines contain a selectable marker used extensively in plant transformation and a gene expression cassette that prevents pollen development. This pollen control cassette provides an additional level of confinement by restricting gene flow from the FTE lines. However, the inclusion of pollen control mechanism has only limited bearing on the consideration for ArborGen Inc. ARB-FTE1-11 Page 3 deregulation of FTE lines because the existing biological limitations of Eucalyptus species, when grown in the southeastern US, would in themselves serve as an effective barrier to gene flow. The FTE lines included in this Petition were subject to detailed molecular characterization of the inserted DNA. These analyses confirm the insertion of a single T-DNA insert with intact gene cassettes integrated at a single locus within the Eucalyptus genome. The results also indicate the absence of any notable backbone sequence from the plasmid used for transformation. Analyses performed on the translines using Western blots indicated that CBF2 protein expression is too low to detect which is consistent with the scientific literature. However, RNA analysis confirmed detectable levels of transcription in response to cold. The field data for the FTE lines under a freeze-stress environment provides convincing evidence that the freeze tolerant phenotype is correlated with the induced expression of the inserted CBF2 gene. Field performance of FTE lines 427 and 435 was assessed under authorized APHIS-BRS Notifications and Permits at multiple sites representing both freeze stress and freeze stress-free environments across the southeastern US. Performance of selected freeze tolerant lines 427 and 435 was assessed in 21 field trials established at 8 different locations representing USDA Hardiness Zones 8a (potential kill zone), 8b (target freeze stress zone) and 9a ( freeze stress-free zone) across the southeastern US. The data collected from these trials over five winter/growing seasons clearly show that translines 427 and 435 are substantially equivalent to EH1 control trees for growth characteristics under freeze stress-free conditions and prior to a significant freeze event in freeze stress environments. The cumulative multi-season data obtained from these trials demonstrate conclusively that the freeze tolerant trait in line 427 and 435 provided protection against temperature fluctuations typical of those expected at this location in USDA Hardiness Zone 8b. In addition, the data collected from these trials also demonstrate that in mild winters, minimal damage occurred to both the translines and the EH1 control trees while in more severe winters there was clear differentiation between the control and transgenic trees. It is evident from these studies that translines 427 and 435 are able to withstand the winters that are likely to occur in the target freeze stress environment represented by the USDA Hardiness Zone 8b in the southeastern US. We can therefore conclude that the selected translines 427 and 435 would be preferably planted for commercial production in USDA Hardiness Zone 8b and in the regions south of this Zone where there is an occasional risk for occurrence of a significant freeze event. From data collected from trials established in USDA Hardiness Zone 8a, where temperatures routinely fell below 15°F, both translines showed severe or total dieback each winter together with an associated reduction in survival. It is therefore not expected that these translines will be planted for commercial production in the Hardiness Zone 8a. The non-transformed control variety has been grown for over a decade in Brazil over many thousands of acres and has not demonstrated any plant pest characteristics. Since translines have been imported under strict quarantine measures these are not expected to be a source for introducing any new pests and diseases of Eucalyptus or other plants into the USA. Through extensive monitoring of field trials there is no evidence that FTE lines have increased susceptibility to pest or diseases compared to the non- genetically engineered controls. Introduction of FTE lines therefore would not result in significant biological impacts from pests or diseases associated with these trees. Compositional analyses of wood samples using standard industry analytical methods for several commonly assessed wood quality parameters indicated that the transgenic trees are comparable to the untransformed controls. Therefore, there is no evidence to suggest that these FTE lines express any phenotypes other than those expected based on the introduced genes. The detailed comparisons