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Merkle Lab

Research in the Merkle Lab

Research in our lab has focused on adapting the in vitro phenomenon known as somatic embryogenesis (SE) for mass clonal propagation and genetic manipulation of southern forest species. With SE, structures resembling seed embryos can be produced by the thousands in culture. These “somatic embryos” are clonal copies of each other and can be germinated to produce seedling-like plants called “somatic seedlings.” Up until the past 10 years, the main motivation for this research theme in the lab had been the improvement of southern hardwoods and conifers for industrial and ornamental purposes, including biomass energy.  Thus, since our first report of somatic embryogenesis in yellow-poplar (Liriodendron tulipifera) in 1986, our laboratory has reported somatic embryogenesis for commercial species, such as loblolly pine (Pinus taeda) and sweetgum (Liquidambar styraciflua), popular ornamental trees such as southern magnolia (Magnolia grandiflora), rare species such as pyramid magnolia (Magnolia pyramidata) and trees with high potential for woody biomass production, such as black locust (Robinia pseudoacacia) and hybrid sweetgum (L. styraciflua x L. formosana; see below).

More recently, the focus of the lab has shifted to the application of 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. In this connection, 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 20th 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.  Most 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.  Details of our current research with each of these forest species can be found below.

Using biotechnology to develop blight-resistant American chestnut

American chestnut growing in the Great Smoky Mountains of North Carolina around 1900
American chestnut growing in the Great Smoky Mountains of North Carolina around 1900

American chestnut tree killed by chestnut blight
American chestnut tree killed by chestnut blight

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). For the past seven years, we have been collaborating with scientists at SUNY-ESF, Penn State, Clemson University and the U.S. 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 have been 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 have been regenerated (Holtz et al., in press).

American chestnut embryogenic culture
American chestnut embryogenic culture 
 

Transgenic American chestnut somatic embryos expressing the GUS reporter gene
Transgenic American chestnut somatic embryos expressing the GUS reporter gene

American chestnut embryogenic cultures growing in air-lift bioreactors
American chestnut embryogenic cultures growing in air-lift bioreactors

American chestnut somatic seedling
American chestnut somatic seedling


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

Carolina hemlock
Carolina hemlock

Eastern hemlock
Eastern hemlock

HWA-infested hemlock foliage
HWA-infested hemlock foliage

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.

T. caroliniana x T. chinensis hybrid somatic embryos
T. caroliniana x T. chinensis hybrid somatic embryos

T. caroliniana x T. chinensis hybrid somatic seedlings
T. caroliniana x T. chinensis hybrid somatic seedlings


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

White ash
White ash 

Emerald ash borer adult
Emerald ash borer adult 

Green ash
Green ash 

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% (Koch et al. 2015).  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) and have since extended this work to produce somatic seedlings from cultures initiated from lingering white ash parents, working with collaborators at Ohio State University. These trees can eventually be employed in clonal screens for resistance to EAB, and production of promising clones could be scaled-up for mass clonal propagation of EAB-resistant planting stock to aid forest restoration in areas affected by EAB.

White ash embryogenic culture
White ash embryogenic culture
Green ash and white ash somatic seedlings
Green ash and white ash somatic seedlings
Green ash embryogenic culture
Green ash embryogenic culture

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 as single embryogenic cultures 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.

Hybrid sweetgum somatic embryos following fractionation and plating of suspension culture
Hybrid sweetgum somatic embryos following fractionation and plating of suspension culture
Some hybrid sweetgum clones grow very fast
Some hybrid sweetgum clones grow very fast
ArborGen production of hybrid sweetgum clones via rooted cuttings
ArborGen production of hybrid sweetgum clones via rooted cuttings

People in Merkle Lab

Paul Montello Research Professional
Paul Montello Research Professional
Ryan Tull Research Technician
Ryan Tull Research Technician
Heather Gladfelter Research Professional and PhD student
Heather Gladfelter Research Professional and PhD student
Ryan McNeill PhD student
Ryan McNeill PhD student
  Maisie MacKnight Student Intern
Maisie MacKnight Student Intern