Using Biotechnology to Develop Blight-Resistant American Chestnut

Until the beginning of the Twentieth Century, American chestnut [Castanea dentata )(Marshall) Borkh.] 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. around 1900 resulted in the death of most mature trees in the natural range of the species, such 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 were able to initiate embryogenic American chestnut cultures and to use microprojectile bombardment to introduce foreign DNA into the cells (Merkle et al. 1990, Carraway et al. 1994, Carraway and Merkle 1997), but for several years, we were unable to regenerate trees from our cultures.

Maturing American chestnut somatic embryos

Transgenic American chestnut somatic embryos expressing the GUS gene

Recently, using funding from ArborGen LLC and the Institute for Forest Biotechnology, we have begun collaborating with the SUNY-ESF scientists in a renewed effort to regenerate transgenic American chestnut trees engineered with candidate anti-fungal genes.  Supplementing the embryo development medium with specific amino acids allowed us to regenerate our first American chestnut somatic seedlings in 2002, although only a handful of plantlets survived hardening off (Robichaud et al. 2004).  More recently, by applying a number of new treatments, including cold storage, activated charcoal and suspension cultures, we have improved American chestnut somatic seedling production efficiency by over 100-fold (Andrade and Merkle 2005).  In addition, we have developed a high-frequency system for producing transgenic American chestnut trees using Agrobacterium-mediated transformation of the embryogenic cultures (manuscript in preparation).  We expect to collaborate with SUNY-ESF scientists and others to begin inserting anti-fungal candidate genes into the tree during 2006.

Development of transgenic trees for phytoremediation of contaminated soil and water

Millions of hectares of soil and water in the U.S. and throughout the world are contaminated with pollutants, which include both organic compounds and heavy metals, several of which are highly toxic to humans and other animals. Phytoremediation, the use of plants to stabilize, reduce or detoxify pollutants, offers an alternative approach to traditional engineering-based remediation approaches, which are both expensive and completely destructive of the sites they are used on. While some plants are naturally endowed with the ability to take up and handle pollutants, others can be adapted via engineering with genes that will greatly enhance their ability to be used for remediation. For example, no plants are currently known to naturally hyperaccumulate mercury.

In collaboration with Dr. Richard Meagher’s lab in the UGA Genetics Department, we examined the ability of yellow poplar (Liriodendron tulipifera)) tissue cultures and plantlets to express modified mercuric reductase (merA)) gene constructs. Mercury resistant bacteria express merA to convert highly toxic, ionic mercury, Hg(II), to much less toxic, elemental mercury, Hg(0). Expression of merA in transgenic plants might provide an ecologically compatible approach for the remediation of mercury pollution. Yellow poplar proembryogenic masses (PEMs) were transformed with three modified merA constructs via microprojectile bombardment. Each construct was synthesized to have altered flanking regions with stepwise increases (0%, 9%, and 18% blocks) of modified coding sequence to generate the constructs merA0 , merA9 and merA18). All merA) constructs were shown to confer resistance to toxic, ionic mercury for independently transformed PEM colonies. Stability of merA transgene expression increased in parallel with the extent of gene coding sequence modification. Regenerated plantlets containing the most modified merA gene (merA18 )) germinated and grew vigorously in media containing normally toxic levels of ionic mercury. The merA18) plantlets released elemental mercury at approximately 10X the rate of untransformed control plantlets (Rugh et al. 1998).

To further investigate if a genetic engineering approach for mercury phytoremediation can be effective in trees with a greater potential in riparian ecosystems, again in collaboration with Dr. Meagher’s lab, we generated transgenic eastern cottonwood (Populus deltoides) trees expressing modified merA9 and merA18 genes. Leaf sections from transgenic cottonwood plantlets produced adventitious shoots in the presence of 50µ M mercuric chloride (HgCl2), which inhibited shoot induction from leaf explants of wild-type plantlets. Transgenic shoots cultured in medium containing 25µ M HgCl2 showed normal growth and rooted, while wild-type shoots were killed. When the transgenic cottonwood plantlets were exposed to Hg(II), they evolved 2-4 times the amount of Hg(0) relative to wildtype plantlets. Transgenic merA9 and merA18 plants accumulated significantly higher biomass than control plants on a Georgia Piedmont soil contaminated with 40 ppm mercuric ion (Che et al., 2003).

These results indicate that forest trees expressing modified merA constructs may provide a means for the phytoremediation of mercury pollution.

Propagation of fast-growing hybrid southerm hardwoods for biomass energy

Both sweetgum (Liquidambar styraciflua) and yellow-poplar (Liriodendron tulipifera) have counterparts native to eastern Asia, which have been separated by continental drift from the North American species for at least 10 million years. The Formosan sweetgum (Liquidambar formosana Hance) is found in temperate forests of eastern Asia, and is interfertile with L. styraciflua.

While production of a few Liquidambar hybrids has been accomplished, in order for the products of a hybrid breeding program to be useful, scores of hybrid genotypes need to be generated and tested and the most desirable genotypes selected for mass clonal propagation. In vitro culture may provide a useful approach for clonal propagation of hybrid Liquidambar. 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). Long-term plans call for field-testing the trees regenerated from these cultures to provide performance data as a basis for selecting clones to be scaled-up for production, which will require the ability to store the cultures while the clones are field-tested. Therefore, cryopreservation was tested as a means for long-term storage of the hybrid cultures (Vendrame et al. 2000). Random amplified polymorphic DNA (RAPD) analysis provided confirmation of the hybrid genotypes of cultures and regenerated trees.

Propagation of mature sweetgum trees via somatic embryogenesis

Floral and inflorescence tissues have proven to be useful explants for embryogenic culture initiation for a number of woody perennials via indirect embryogenesis. The ability to use such tissues, rather than those of genetically unproven seeds or seedlings, offers an important advantage for rapid improvement of forest trees, since it makes possible mass cloning of trees that are sufficiently mature to be evaluated for superior growth and other qualities.

Sweetgum is one of the most important commercial hardwoods in the United States. We have shown that embryogenic sweetgum cultures can be initiated from staminate and pistillate floral and inflorescence tissues isolated from dormant buds and cultured on medium with thidiazuron, NAA or no PGRs at all, and that plantlets can be regenerated from the cultures (Merkle et al. 1997, 1998, Merkle and Battle 2000).

These studies indicated that genotype (source tree), inflorescence developmental stage and plant growth regulator treatments all influenced embryogenesis induction. Dormant buds stored for two months at -15 C were still capable of producing embryogenic cultures, although frozen storage decreased this ability by over one-half, and was especially detrimental to induction from staminate inflorescences (Merkle and Battle 2000).

Improvement of somatic seedling production for southern pines

Early stage slash pine somatic embryos

Our lab, in collaboration with visiting scientist Dr. Dale Smith of MetaGenetics (New Zealand) has developed protocols for somatic embryogenesis of slash pine (Pinus elliottii ), loblolly pine (Pinus taeda ) and longleaf pine (Pinus palustris ). Embryogenic tissue initiation, cryopreservation (storage of tissue in liquid nitrogen) development and maturation of somatic embryos, and production of somatic seedlings of these three species has been accomplished.

However, while mature somatic embryos and plants in soil were produced in small numbers, the protocols need to be improved before they will be of use to forest industry. Industrial clients will wish to see somatic seedlings delivered to the forests in large numbers, at an acceptable cost and, perhaps most importantly, on a predetermined timetable. One of the most serious production bottlenecks standing in the way of wide forestry industry adoption of this technology is the low frequencies of somatic embryo maturation and conversion to somatic seedlings. Recently published research with southern pines focused on testing variables just prior to and during germination of pine somatic embryos. Our experiments with light quality have indicated that using red wavelengths not only results in higher germination frequencies for loblolly, slash and longleaf pine somatic embryos compared to the standard cool white fluorescent lights, but that it allows embryos of some clones to germinate that failed to germinate at all under cool white light.  Furthermore, somatic seeding quality, as measured by numbers of first order lateral roots, was enhanced under red wavelengths (Merkle et al. 2005).

Other Research Areas

Research in our lab has focused on adapting the in vitro phenomenon known as somatic embryogenesis for mass clonal propagation and genetic manipulation of southern forest species. With somatic embryogenesis, 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.

The motivation for this research theme has been the improvement of southern hardwoods and conifers for industrial and ornamental purposes, as well as for non-traditional uses such as remediation of polluted soil and water. Since our first report of somatic embryogenesis in yellow-poplar in 1986, our laboratory has reported somatic embryogenesis for 10 forest tree species and 2 hybrids.

Trees for which our lab developed embryogenic regeneration systems include important commercial species, such as yellow-poplar (Liriodendron tulipifera) and sweetgum (Liquidambar styraciflua), popular ornamental trees such as southern magnolia (Magnolia grandiflora), rare species such as pyramid magnolia (Magnolia pyramidata), a tree with high potential for woody biomass production (black locust; Robinia pseudoacacia), one species under attack by a devastating disease (American chestnut, Castanea dentata) and two hybrids with potential ornamental and industrial uses (yellow-poplar x Chinese tuliptree and sweetgum x Formosan sweetgum). More recently, we have initiated research on improving somatic seedling production from embryogenic cultures of southern pines.

Lab Personnel

Paul Montello, Research Professional

Gisele Andrade, Research Professional

Taryn Kormanik, Ph.D. Student

Siteria Gregory, M.S. Student

Hannah Smith, Graduate Research Assistant

Lake Maner, Undergraduate Research Assistant