Development of a New Genetic Transformation System for White and Green Ash Using Embryogenic Cultures

Abstract

All North American ash (Fraxinus spp.) species are threatened by the emerald ash borer (EAB; Agrilus planipennis), an exotic beetle which has already destroyed millions of ash trees in the U.S. and Canada. Although both chemical insecticides and biological control can be effective, and host resistance appears possible, the speed of the invasion has defied traditional management approaches. One potential, innovative approach to managing this destructive insect is to develop a host tree-induced gene silencing strategy using RNA interference (RNAi) constructs targeting EAB-specific genes. An important requirement for applying RNAi technology is a reliable transformation/regeneration system for the host tree species. We developed an Agrobacterium-mediated gene transfer system for white ash (F. americana) and green ash (F. pennsylvanica) using the embryogenic cultures of these species as target material. Embryogenic suspension cultures of multiple genotypes of both species were plated and inoculated with A. tumefaciens carrying the pFHI-GUSi expression vector, which carries the nptII selectable marker and intron-GUS reporter genes, followed by selection on a semi-solid medium containing geneticin. Putative transgenic events showed expression of the GUS gene at all tested developmental stages from callus to plantlets, and transgene presence in the leaves of regenerated plants was confirmed using PCR. The overall average transformation efficiency achieved was 14.5 transgenic events per gram of tissue. Transgenic somatic seedlings of two white ash and three green ash genotypes were produced and acclimated to greenhouse conditions.

Ingestion of Species-Specific dsRNA Alters Gene Expression and Can Cause Mortality in the Forest Pest, Ips calligraphus

“…Investigations into the behavior of dsRNA in plants have shown promising results for single tree protection in both deciduous [70,71] and coniferous seedlings [72], and success has been shown for soil drenches, foliar sprays, and trunk injections in citrus [73,74]. Scaling up application of dsRNA for forest protection poses additional challenges, but innovative techniques, such as those aiming to use microbial symbionts as a means for delivery [75,76] or developing transgenic trees that express pest-specific dsRNA [77], are examples of possible solutions to this barrier currently being investigated.…”

Will “Tall Oaks from Little Acorns Grow”? White Oak (Quercus alba) Biology in the Anthropocene

“…It is expected that Agrobacterium-mediated transformation systems based on binary plant expression vectors and inducible gene constructs should be adaptable to Q. alba transformation, as successfully tested in chestnut [311], poplar [312], Arabidopsis [313], and other systems. The AlcR/alcA system exhibits little or no basal expression in plants and permits the rapid, reversible induction of transgene expression [312,314].…”

Conservation of Green and White Ash Germplasm Using the Cryopreservation of Embryogenic Cultures

“…Infestations rapidly spread to 35 US states and several Canadian provinces, killing millions of ash trees in the process [4]. Minimal EAB resistance has been identified in native North American ash, although ongoing breeding and genetic transformation programs are seeking to address this [5,6]. Other mitigation strategies, such as long-term biological control using specialized parasitoid wasps, are being developed, but scalability and testing remain challenges [7].…”