The Merkle Lab

About our work

Using somatic embryogenesis (SE), the focus of the lab has shifted from propagating southern hardwoods and conifers to using SE and other in-vitro propagation approaches for conservation and restoration of threatened North American trees, in particular those under attack by exotic pests and pathogens.

We were the first lab to report somatic embryogenesis in American chestnut (Castanea dentata), which was devastated by chestnut blight in the first half of the 20^th century, and in eastern and Carolina hemlocks (Tsuga canadensis and Tsuga caroliniana), important coniferous species native to the Appalachian Mountains that have been attacked by hemlock woolly adelgid.

More recently, we have developed embryogenic culture systems for green ash and white ash (Fraxinus pennsylvanica and Fraxinus americana), which are being wiped out by emerald ash borer.

Current Research

Using biotechnology to develop blight-resistant American chestnut

Until the beginning of the Twentieth Century, American chestnut (Castanea dentata) was one of the most prevalent and valuable trees in our eastern forests. The accidental introduction of the chestnut blight fungus (Cryphonectria parasitica) into the U.S. resulted in the death of most mature trees in the natural range of the species, so that today it mainly exists as an understory shrub. A number of approaches have been taken to combat the blight over the last century, but to date, none has succeeded in restoring the tree to its place in the forest. In 1990, we began testing protocols to establish embryogenic cultures of American chestnut, with the goals of developing a system for mass clonal propagation of the tree and providing a means by which the tree might be engineered with genes that might provide resistance to the blight, as such genes became available for testing. We initiated the first embryogenic American chestnut cultures over 25 years ago (Merkle et al. 1990), but plantlet production remained a bottleneck for several years.  More recently, by applying a number of new treatments, including cold storage, activated charcoal and suspension cultures, we improved American chestnut somatic seedling production efficiency by over 100-fold (Andrade and Merkle 2005) and developed an Agrobacterium-mediated gene transfer system for the species (Andrade et al. 2009). During 2010 - 2018, we collaborated with scientists at SUNY-ESF, Penn State, Clemson

University and the USDA Forest Service in an effort called the Forest Health Initiative to regenerate transgenic American chestnut trees engineered with candidate anti-fungal genes. Part of this effort involved scaling up production of embryogenic culture material using air-lift bioreactors (Kong et al. 2014). Hundreds of these transgenic trees were planted in field tests to be screened for resistance to chestnut blight. Over the past few years, we have also collaborated with scientists at The American Chestnut Foundation (TACF) to test our embryogenic culture system for its potential to propagate TACF's hybrid backcross material, including selected BC3F3 genotypes.  Although pure Chinese chestnut and F1 hybrids could not be propagated using our system, embryogenic cultures could be started from BC3F3 material and dozens of BC3F3 somatic seedlings were regenerated (Holtz et al. 2017).  Finally, we have also applied our chestnut SE protocol to successfully propagate Ozark chinquapin (Castanea pumilavar. ozarkensis).

Conservation of hemlock germplasm and propagation of putatively HWA-resistant hemlocks by somatic embryogenesis

The eastern North American hemlock species, eastern hemlock (Tsuga canadensis) and Carolina hemlock (Tsuga caroliniana), were important components of eastern forest ecosystems, but over the past few decades, they have been devastated by the hemlock woolly adelgid (Adelges tsugae; HWA). We are testing biotech approaches to conserving these trees and restoring them to the forest. We have developed embryogenic systems for both native hemlocks.  These cultures can be cryostored to facilitate hemlock germplasm conservation. In addition, working with breeders from the Forest Research Alliance, we have generated embryogenic cultures from hybrids between Carolina hemlock and the HWA-resistant Asian species, Chinese hemlock (Tsuga chinensis) and southern Japanese hemlock (Tsuga sieboldii). Recently, the first putative hybrid somatic seedlings were produced for HWA tolerance screening and potential use in restoration plantings.

Propagation of emerald ash borer-resistant green ash and white ash trees by somatic embryogenesis

Ash trees, in particular white ash (Fraxinus americana) and green ash (F. pennsylvanica) are among the most abundant hardwood species in the eastern U.S. and are integral to the ecology of many ecosystems in the

region.  Not only are ash trees valued as urban tree and landscape species, but ash wood, which is strong, straight-grained and dense, is used for a variety of products, including tool handles, baseball bats, furniture, flooring and cabinets. All North American ash species are under threat of extirpation from their native ranges by the emerald ash borer (EAB; Agrilus planipennis), an exotic wood-boring beetle that has already destroyed millions of ash trees in 15 US states and Canada. EAB has been spreading rapidly since it was first discovered in Michigan in 2002. The development of EAB-resistant or EAB-tolerant ash trees will be critical for ash reforestation in both urban and natural forests. Genetically-based resistance or tolerance to EAB existing in the native ash population may offer a one route to restoration of these valuable trees. Individual native white ash and green ash trees have been identified as potentially EAB-resistant by their persistence in populations where EAB-induced mortality exceeds 99%.  These so called “lingering ash” trees constitute a potential source of resistance genes that could be used in selection and breeding programs. We reported propagation of green ash via somatic embryogenesis (Li et al. 2014). More recently, working with Dr. Kamal Gandhi in the Warnell School, as well as collaborators from Ohio State University and the USDA Forest Service, we have produced embryogenic cultures derived from seeds collected from lingering white ash parents (Merkle et al. 2017). The first lingering white ash somatic seedlings were planted in a small field test in Athens, GA, along with EAB-susceptible and EAB-resistant control trees, in April 2018. These trees will provide clonal screens for resistance to EAB, and clones that perform well could potentially be scaled-up for mass propagation of EAB-resistant planting stock to aid forest restoration in areas affected by EAB.

Propagation of fast-growing hybrid sweetgum for pulp and paper

Sweetgum (Liquidambar styraciflua) is a common southern hardwood that has become an important feedstock for the southern pulp and paper industry, particular in the production of fine papers. Despite the tree’s abundance, availability to mills is sometimes problematic, prompting some to consider establishing plantations of the tree.

The Formosan sweetgum (Liquidambar formosana), found in temperate forests of eastern Asia, is interfertile with L. styraciflua, even following 10 million years of separation between the two species. We developed an approach for clonally propagating hybrid sweetgum genotypes via somatic embryogenesis from hybrid seed explants (Vendrame et al. 2000; Dai et al., 2003). Thousands of hybrid sweetgum somatic embryos can be produced from a single embryogenic culture flask, following size fractionation and plating of the suspension culture. Field tests of trees regenerated from some of the hybrid sweetgum clones by collaborators at ArborGen Inc. resulted in the identification of some clones with biomass productivity superior that of either parent species, due to faster growth rates and higher wood density. These clones have been commercialized by ArborGen and hundreds of thousands of trees have been sold to landowners over the past few years. These trees are expected to contribute to the supply of hardwood fiber for the pulp and paper industry.

Restoring the extinct (in nature) Franklinia to Southern landscapes

Franklinia, Franklinia alatamaha, was discovered growing in a single location on the Altamaha River in Georgia by the naturalists John and William Bartram in 1765 while traveling through the southeastern U.S. on an ecological expedition. On another excursion in 1773, William collected seeds and brought them back to his hometown of Philadelphia, where he cultivated Franklinia on his estate. No one has seen the Franklinia tree in the wild since 1803. Today, the tree only exists in cultivation. The exquisite ornamental characteristics of Franklinia, along with its intriguing botanical history and discovery, has have made it a coveted tree in landscapes, botanical gardens and arboretums. Unfortunately, the tree is very difficult to grow in the southeastern U.S. because of it extreme susceptibility to Phytophthora root rot, which is caused by the oomycete Phytophthora cinnamomi. We have developed methods for propagating Franklinia via adventitious shoot production and axillary shoot multiplication. The shoot cultures are currently being used in an experiment to try to produce Phytophthora root rot-resistant clones via mutation breeding.  

We have also developed in vitro propagation systems for multiple species of trees in the genus Stewartia, which is closely related to Franklinia. Like Franklinia, Stewartia species are small trees with beautiful flowers. The two species native to Georgia, S. malecodendron and S. ovata, are rare. We have clonally propagated both species via somatic embryogenesis.