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 theU.S. and Canada. Although both chemical insecticides and biological control can be effective, andhost resistance appears possible, the speed of the invasion has defied traditional management ap‐proaches. One potential, innovative approach to managing this destructive insect is to develop ahost tree‐induced gene silencing strategy using RNA interference (RNAi) constructs targeting EAB‐specific genes. An important requirement for applying RNAi technology is a reliable transfor‐mation/regeneration system for the host tree species. We developed an Agrobacterium‐mediatedgene transfer system for white ash (F. americana) and green ash (F. pennsylvanica) using the embryo‐genic cultures of these species as target material. Embryogenic suspension cultures of multiple gen‐otypes of both species were plated and inoculated with A. tumefaciens carrying the pFHI‐GUSi ex‐pression vector, which carries the nptII selectable marker and intron‐GUS reporter genes, followedby selection on a semi‐solid medium containing geneticin. Putative transgenic events showed ex‐pression of the GUS gene at all tested developmental stages from callus to plantlets, and transgenepresence in the leaves of regenerated plants was confirmed using PCR. The overall average trans‐formation efficiency achieved was 14.5 transgenic events per gram of tissue. Transgenic somaticseedlings of two white ash and three green ash genotypes were produced and acclimated to green‐house conditions.

Citation

Tull, Andrew Ryan; Gladfelter, Heather; Pampolini, Flavia; Rieske, Lynne; Nelson, C. Dana; Merkle, Scott. 2022. Development of a New Genetic Transformation System for White and Green Ash Using Embryogenic Cultures. Forests. 13(5): 671-. https://doi.org/10.3390/f13050671.