Targeted Genome Editing for Gene Containment in Transgenic Black Ash

Black ash (Fraxinus nigra) is valued not only for commercial hardwood applications such as cabinets, paneling, flooring, and veneer, but also for food and habitat for wildlife. The wood is preferred by Native Americans for making splints for basketry. However, the emerald ash borer (EAB), an exotic wood-boring beetle from Asia, has killed millions of ash trees in Michigan since 2002, and EAB has spread to 22 states in the United States, and into Canada. Although several insecticides have been developed to control EAB, it has limitations. As a long-term alternative, development of transgenic black ash with EAB-resistance is urgently needed. A naturally occurring toxin gene from Bacillus thuringiensis (Bt) was introduced into the black ash genome through Agrobacterium-mediated transformation using hypocotyl explants. Adventitious shoots were regenerated from transformed cells showing kanamycinresistance, and the presence of the Bt-gene was confirmed. Once roots are formed on these shoots, the transgenic plantlets will be acclimatized to the greenhouse. However, transgenic trees are not allowed to be routinely planted because of the potential environmental impacts of transgene flow; movement of genes from a genetically modified organism to its wild or native relatives through pollen. With current molecular technologies, gene containment can be achieved by interfering with flowering. Transcription activator-like effector nuclease (TALEN) is one powerful tool for genome editing by inducing DNA double-strand breaks that stimulate non-homologous end joining or homologous recombination at specific genomic locations. TALENs are artificial restriction enzymes generated by fusing a TALE DNA binding domain of Xanthomonas to a DNA cleavage domain of FokI endonuclease. To disrupt black ash AGAMOUS, a C-class floral organ identity gene responsible for stamens and carpels, we can manipulate the DNA binding domain of TALEN based on the sequence of black ash AGAMOUS. Small insertion or deletion mutations at the target might be induced, so that the AGAMOUS gene would be disrupted. As a result, transgenic black ash would be sterile with no stamens and carpels. Our results will help improve our understanding of the usefulness of TALEN technology in the genetic modification of tree species.

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