Development of transgenic North American ash trees expressing a Bacillus thuringiensis protein for management of the emerald ash borer
Ash timber is valued for applications requiring strong, hard wood. Green ash is used for both solid wood applications (crating, boxes, and tool handles) and for fiber in the manufacture of high grade paper. White ash is the primary commercial hardwood used in the production of baseball bats, tool handles, furniture, flooring, doors, cabinets, and other specialty products such as canoe paddles and boats. Black ash is typically used for interior furniture, cabinets, and Native Americans require this species for the art of basketry. Ash trees also play an important role in the urban landscape because of their historical resistance to pests and tolerance of adverse growing conditions, such as soil compaction and drought. Ash trees are considered vital to the urban forest environment because they sequester gaseous air pollutants, help conserve energy by providing shade, provide shelter belts for urban fauna, and contribute aesthetic pleasure to people. An aggressive exotic pest from Asia, the EAB, [Agrilus planipennis Fairmaire; Coleoptera: Buprestidae], is attacking and killing all ash (Fraxinus spp.) trees in North America. First identified in Michigan in 2002, the EAB also established in Windsor, Ontario, has since been detected in Ohio in 2003, northern Indiana in 2004, northern Illinois and Maryland in 2006, western Pennsylvania and West Virginia in 2007, Wisconsin, Missouri, and Virginia in 2008, and Minnesota and New York in 2009. The pest is fatal to an infested tree, there are no known innate resistance genes in native species of ash or means of complete eradication, and evidence exists that the EAB is continually spreading throughout North America causing considerable economic and environmental damage. The EAB has cost municipalities, property owners, nursery operators, and forest products industries tens of millions of dollars. An estimated six-million trees in Michigan alone have died or are dying from this exotic wood-boring beetle infestation. In addition to the multi-million dollar U.S. market consumption of ash wood, the U.S. has exported ash lumber to 78 countries and ash logs to 45 countries. The development of transgenic Fraxinus spp. exhibiting resistance to attack by the EAB is urgently needed.
Bacillus thuringiensis (Bt) is a naturally occurring bacterium that produces spores with proteinaceous parasporal inclusion bodies composed of d–endotoxins. Its spores and inclusion bodies express insecticidal activity, and are used in commercial biological preparations to control larval forms of agriculturally important insect pests. Bt has widespread use to control forest pests in the U.S. and Canada as a result of its environmentally benign nature, when compared to chemical pesticides. The Bt toxin is specific to groups of insects while lacking toxicity to mammals. The main target pests of Bt insecticides include various lepidopterous, dipterous, and individual coleopterous species (such as EAB). The insecticidal action of Bt is enhanced by a concurrent induction of bacteraemia following the binding of toxin proteins to the intestinal wall of the insect leading to the formation of perforations. The crystal proteins (Cry and Cyt) of Bt have been extensively studied because of their pesticidal properties and their high natural levels of production. Preliminary laboratory bioassays of adult EAB with both the Cry8Da protoxin and Cry8Da activated toxin demonstrated relatively high toxicities (Leah Bauer, unpublished data). Expression of an effective Bt gene (Cry8Da) in transgenic ash trees would impart resistance to the aggressive EAB.
The objectives of our research are to: 1) determine the toxicity of proteins expressed from the cry8Da constructs in EAB larvae and adults; 2) develop protocolc for adventitious shoot regeneration and rooting of green, white, and black ash for use in Agrobacterium-mediated genetic transformation studies using the cry8Da gene; 3) transform these ash species, regenerate, acclimatize, and propagate transgenic ash plants containing the Bt toxin specific to the EAB; 4) analyze normal and transgenic ash lines for Bt expression; 5) conduct laboratory bioassays of transgenic lines against the EAB to confirm efficacy; and 6) establish transgenic ash trees in a field trial.
The development of transgenic Fraxinus spp. exhibiting resistance to attack by the EAB will have great economic benefits to landowners, the nursery and forest products industries, and to the U.S. foreign trade of ash lumber and logs. This research will result in the development of an Agrobacterium-mediated transformation system for Fraxinus spp. with recovery of transgenic plants resistant to the EAB. In addition, this research will initiate studies of effective deployment strategies for transgenic ash trees that will provide data for future risk assessment that is necessary for commercial release. Another major focus of future studies will be to examine the use of sterility genes to further modify transgenic ash in a way that would prevent their hybridization with native ash populations once introduced into the landscape. Robust genetic sterility of transgenic ash would prevent the escape of transgenes into the native ash population, and greatly reduce the possibility of unforeseen negative impacts on natural ecosystems. Knowledge gained through this research could be applicable to other high-value tree species. Ultimately, the improved ash germ plasm will become a valuable resource to the entire nursery and hardwood industries.
Du, N. and Pijut, P.M. Isolation and characterization of an AGAMOUS homolog from Fraxinus pennsylvanica. (Manuscript in Preparation for Publication)
Du, N. and Pijut, P.M. 2009. Agrobacterium-mediated transformation of Fraxinus pennsylvanica hypocotyls and plant regeneration. Plant Cell Reports 28: 915-923.
Du, N. and Pijut, P.M. 2008. Regeneration of plants from Fraxinus pennsylvanica hypocotyls and cotyledons. Scientia Horticulturae 118: 74-79.
- Paula M. Pijut, USDA Forest Service, Northern Research Station (NRS), Hardwood Tree Improvement and Regeneration Center, Research Plant Physiologist
- Leah S. Bauer, USDA Forest Service, NRS, Research Entomologist
- John Libs, Phyllom LLC, Director Operations
Last Modified: 06/12/2013