Environmental Interactions with Transgenic American Chestnuts Andrew E. Newhouse and William A. Powell, with gracious acknowledgement to many research collaborators SUNY College of Environmental Science & Forestry, Syracuse, NY

Background Bumble Bees and Pollen Darling 58 transgenic American chestnuts have shown significantly enhanced tolerance to the Chestnuts are often considered to be wind-pollinated, but recent research has chestnut blight fungus, and will soon be evaluated by federal regulatory agencies before potential shown that insects (including native bees) contribute to successful public distribution. The oxalate oxidase enzyme expressed in Darling transgenic American chestnuts pollination13-15. Bumble bees were reared in is naturally found in many food crops and wild plants, and it protects the tree by degrading a toxin microcolonies (photo at right) and supplied rather than by killing the fungus, so it is unlikely to present novel risks when expressed in American with chestnut pollen containing the oxalate oxidase enzyme. chestnut. However, along with data from molecular, growth, and nutritional tests, it is valuable for There were no differences in survival, body size, pollen use, regulators and prudent for environmental scientists to carefully evaluate potential environmental risks or reproduction when bees were exposed to a field-realistic before deploying new restoration material. Here we summarize several comparative risk assessments concentration of oxalate oxidase in pollen16. of environmental interactions with transgenic American chestnuts and non-transgenic controls.

Leaf Herbivory by Insects Transgene Inheritance Aquatic mayfly larvae performed better on American chestnut leaves than oaks If or when transgenic American chestnuts are and other trees in a preliminary test1, so chestnut re-introduction may benefit deployed for restoration purposes, their ecological stream ecosystems. Subsequent studies have shown that caddisfly larvae survival significance ultimately will rely on inheritance of was not significantly different on transgenic vs. wild-type American chestnut the transgene by offspring. Meaningful genetic leaves2. Separate studies of terrestrial (gypsy moth) diversity in a potential restoration population will caterpillars showed leaf consumption differences between be accomplished primarily by outcrossing 17 Chinese and American leaves, but similar consumption transgenic trees with surviving wild chestnuts . between wild-type American and transgenic or B3F3 leaves. Since the transgene is only present on one half of the chromosome, about half the offspring from a cross with one transgenic parent are expected to be transgenic. This has been confirmed in analyses of two generations of Darling 58 offspring: 56 of 115 nuts tested to date show transgene activity. Growth rates and survival of these Germination of Nearby Native Seeds transgenic offspring are equivalent to their non-transgenic siblings (that didn’t Seeds of several native plant types from American chestnut habitats inherit the transgene), and Cryphonectria parasitica inoculations indicate that (Elymus/grass, Cichorium/forb, Gaultheria/shrub, Acer/deciduous tree, transgenic offspring have blight tolerance similar to their transgenic parent. Pinus/conifer) were germinated in potting mix with different types of chestnut leaf litter3. Pinus seeds showed reduced germination in the presence of one wild-type control leaf type compared to no-leaf trays, and Cichorium showed reduced biomass in B3F3 leaves compared to Darling 58 leaves, but there were no significant differences in seedling germination rates or total biomass Tadpoles in Vernal Pools when grown in transgenic vs wild-type American chestnut leaf litter. Wood frog tadpoles were raised in individual jars with different types of deciduous leaf litter to simulate an interaction that could take place in vernal pools18. Chestnut leaves (transgenic American, wild-type American, hybrid, or Chinese) were not detrimental to tadpole survival compared to sugar maple controls; only American beech leaves increased Leaf Litter Decomposition tadpole mortality. Development and growth rates were Leaf decomposition rates were compared by placing different chestnut leaf types (wild- similar on most leaf types, but American chestnut leaves type and transgenic American, and BC1 hybrid) in mesh bags on the forest floor. All (both transgenic and wild type) allowed slightly increased leaf types lost ~90% of their mass after 1 year; rates were not significantly different4. growth rates compared to other leaf types when supplemental food was not present, suggesting American Transgenic chestnut leaves were also tested for persistence of oxalate oxidase enzyme chestnut restoration could conceivably benefit amphibians. activity after leaf drop in the fall. Enzyme activity essentially ceased as soon as leaves turned brown (approximately one week after leaf drop in outdoor conditions), though activity could be preserved for more than a month in artificial freezer conditions5. Conclusions Some environmental interaction experiments show differences between Chinese and American chestnuts, which is not surprising given the differences between these species. However, neither B3F3 Mycorrhizal Fungi nor transgenic OxO-expressing American chestnuts have shown significant ecological differences References: Danielsen, L., Lohaus, G., et al. (2013). Ectomycorrhizal ESF. Newhouse, A.E., Oakes, A.D., et al. (2018). Transgenic Colonization and Diversity in Relation to Tree Biomass and Kaldorf, M., Fladung, M., et al. (2002). Mycorrhizal American Chestnuts Do Not Inhibit Germination of Native Brown, A.J. (2017). Comparative EfficacySeveral of Entomopathogens experiments have examined mycorrhizal associations with transgenic American chestnut Nutrition in a Plantation of Transgenic Poplars with Modified colonization of transgenic aspen in a field trial. Planta 214, Seeds or Colonization of Mycorrhizal Fungi. Front. Plant Sci. compared to wild-type American chestnuts, apart from blight tolerance. Thus Darling 58 transgenic and Parasitoids of Lepidopteran Larvae Among Transgenic Lignin Biosynthesis. PLOS ONE 8, e59207. 653–660. 9, 1–9. Blight-Resistant American Chestnut and Conventionally Bred roots. Most recently,Goldspiel, H., Newhouse, Darling A.E., et al. (2018). 58Effects ofroots were observed to be colonizedSteiner, K.C., Westbrook, at the J.W., etsame al. (2017). Rescue rate of (>95% of Cultivars. M.S. Thesis. State University of New York College Lelmen, K.E., Yu, X., et al. (2010). Mycorrhizal colonization of Transgenic American Chestnut Leaf Litter on Growth and American chestnut with extraspecific genes following its American chestnuts do not appear to present greater ecological risks than traditional breeding. of Environmental Science and Forestry (SUNY-ESF). transgenic Eucalyptus camaldulensis carrying the mangrin gene3 Survival of Wood Frog Larvae. Restor. Ecol. for salt tolerance. Plant Biotechnol. 27, 339–344. destruction by a naturalized pathogen. New For. 48, 317–336. D’Amico, K.M., Horton, T.R., et al.root (2015). Comparisons tips colonized)of as wild-type American chestnut controls . These results corroborate older doi:10.1111/rec.12879. Tourtellot, S.G. (2013). The impact of transgenic American Ectomycorrhizal Colonization of Transgenic American Newhouse, A.E., Schrodt, F., et al. (2007). Transgenic Gray, A. (2015). Investigating the Role of Transgenic American 6 chestnuts (Castanea7 dentata) on ectomycorrhizal fungi in open- Chestnut with Those of the Wild Type, a Conventionally Bred American elm shows reduced Dutch elm disease symptoms and studies on transgenicChestnut (Castanea dentata) chestnut Leaf Litter in Decomposition, (including both greenhouse andfield field and mature forestexperiments) sites. M.S. Thesis. SUNY-ESF. and studies on Hybrid, and Related Fagaceae Species. Appl. Environ. normal mycorrhizal colonization. Plant Cell Rep. 26, 977–987. 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