Emerald ash borer (Agrilus planipennis; EAB) has devastated populations of ash (Fraxinus spp.) trees in dozens of U.S. states and Canada over the past few decades. The continued survival of scattered ash trees known as "lingering ash" in heavily infested natural stands, however, offers evidence of genetic resistance or tolerance to EAB. These surviving or "lingering" ash individuals may form the basis for reforestation programs in EAB-impacted areas, and clonal mass-propagation of these genotypes can help accelerate these efforts. Between 2013 and 2018, we initiated embryogenic cultures by culturing immature zygotic embryos from open-pollinated (OP) seeds collected from several surviving white ash and green ash trees in Michigan and Pennsylvania. In addition, in 2018, we initiated cultures from crosses made between lingering green ash parents from the USDA Forest Service ash breeding program in Ohio. Somatic embryos were produced by growing cultures in liquid suspension, followed by fractionation and plating on semisolid medium to produce developmentally synchronous populations of somatic embryos. Somatic embryo germination and conversion were enhanced by a combination of pre-germination cold treatment and inclusion of activated charcoal and gibberellic acid in the germination medium. Ash somatic seedlings derived from OP explants grew rapidly following transfer to potting mix and somatic seedlings representing nine ash clones were acclimatized, grown in the greenhouse and planted in a preliminary field test, along with EAB-resistant Manchurian ash (F. mandshurica) and EAB-susceptible control seedlings. Somatic seedlings have now been produced from cultures that originated from seeds derived from the progeny of lingering green ash parents and an ex vitro germination protocol has shown some promise for accelerating early somatic seedling growth. Results of this research could provide the basis for scaled-up production of EAB-resistant ash varieties for seed orchard production for forest restoration and cultivar development for urban tree restoration.

Atlantic white cedar (AWC; Chamaecyparis thyoides), an aromatic evergreen conifer native to swamps and bogs along the Atlantic and Gulf coasts of the eastern United States was once an important species for timber production due to its durable wood. However, native populations have declined over the past two centuries. We established an in vitro propagation system for AWC via somatic embryogenesis (SE) without the use of plant growth regulators (PGRs). Whole megagametophytes with zygotic embryos from immature AWC cones were cultured on a modified half-strength embryo maturation (EM) medium with three different PGR treatments, including one devoid of PGRs. Both PGR treatment and cone collection date had significant effects on embryogenesis induction, with EM with no PGRs giving the highest embryogenesis induction, which ranged as high as 27%. We also conducted experiments to determine the effects of activated carbon (AC) and abscisic acid (ABA) in the maturation medium on production of mature somatic embryos. AC significantly affected this variable, with 2 g l−1 producing more embryos than 0 g l−1. Application of exogenous ABA not only failed to improve production of mature somatic embryos, the highest level tested (200 µM), apparently lowered production of mature embryos compared to the 0 ABA control. The highest numbers of mature somatic embryos per ml of plated embryogenic suspension (32–37) were produced on medium with 2 g l−1 AC and levels of ABA at 100 µM or lower. The SE system described here has the potential to contribute the restoration of Atlantic white cedar to its native habitat.

Embryogenic cultures of eastern and Carolina hemlocks could be initiated, and somatic embryos and plantlets produced using standard conifer protocols and media. Embryogenic hemlock cultures were cryostored and recovered.

Arsenic is a metalloid that occurs naturally at parts per million (ppm) levels in the earth's crust. Natural and human activities have contributed to arsenic mobilization and increased concentration in the environment, such that World Health Organization guidelines for arsenic levels in drinking water are exceeded at many locations, worldwide. This translates into an increased risk of arsenic-related illnesses for millions of people. Recent studies demonstrate that increasing thiol-sinks in transgenic plants by overexpressing the bacterial gamma-glutamylcysteine synthetase (ECS) gene results in a higher tolerance and accumulation of metals and metalloids such as cadmium, mercury, and arsenic. We used Agrobacterium-mediated transformation to genetically engineer eastern cottonwood with a bacterial ECS gene. Eastern cottonwood plants expressing ECS had elevated thiol group levels, consistent with increased ECS activity. In addition, these ECS-expressing plants had enhanced growth on levels of arsenate toxic to control plants in vitro. Furthermore, roots of ECS-expressing plants accumulated significantly more arsenic than control roots (approximately twice as much), while shoots accumulated significantly less arsenic than control shoots (approximately two-thirds as much). We discuss potential mechanisms for shifting the balance of plant arsenic distribution from root accumulation to shoot accumulation, as it pertains to arsenic phytoremediation.

Somatic embryogenesis may have greater potential for propagation of forest trees for ornamental purposes than for production forestry. Somatic embryogenesis-derived hybrid sweetgum (Liquidambar styraciflua x Liquidambar formosana) clones, originally developed for pulp and paper and biomass energy, have displayed a range of growth rates and other phenotypic variation. While some of the fast-growing clones show promise for fiber production, others offer potential as ornamental trees. We investigated variables to try to improve somatic seedling quality of the most interesting hybrid sweetgum clones. A pre-germination cold treatment of at least eight weeks improved both germination frequency and conversion frequency of the somatic embryos to close to 100%, and produced more vigorous plantlets than embryos given a four-week cold treatment or no cold treatment. Germinating embryos vertically in test tubes rather than on plates of gelled medium helped eliminate a problem with crooked root collars in the resulting somatic seedlings, which had previously been found to lead to a higher chance of stem breakage. The potential for a group of eight-year-old hybrid clones that had displayed outstanding growth rates in a test planting to be propagated via somatic embryogenesis using mature tree tissues was studied using staminate inflorescence explants collected from dormant buds. Embryogenesis induction for the three clones ranged in the study from 1.6% to 12.8%, depending on clone and plant growth regulator treatment, with NAA providing a higher induction frequency than TDZ. A demonstration planting of somatic seedlings representing seven hybrid sweetgum clones revealed a number of potentially useful ornamental phenotypes after one season of growth, with some clones growing over 1.1 m in 4 months and others with dwarf or shrub phenotypes. Some clones also displayed striking fall leaf color. The manipulation of embryogenic suspension cultures of the hybrid clones will enable the synchronous production of thousands of propagules of the most desirable clones for horticultural and other applications.

Effects of photoautotrophic and photomixotrophic growth conditions on adventitious shoot regeneration from leaf explants of eastern cottonwood (Populus deltoides Bartr. ex Marsh.) were investigated. Rooting and proliferating shoot cultures (Stage I) were grown in either an elevated (1500 ppm) CO(2) concentration ([CO(2)]) at high photosynthetic photon flux (PPF; similar to 150 mu mol m(-2) s(-1)) (photoautotrophic condition) with 0, 10 or 30 g l(-1) sucrose or under standard conditions (ambient (360 ppm) [CO(2)] at low PPF (similar to 60 mu mol m(-2) s(-1)) with 30 g l(-1) sucrose). Leaves harvested from these Cultures were analyzed for Soluble sugars and were used as explants for adventitious shoot regeneration (Stage II), which was also carried Out under photoautotrophic and standard conditions. Photoautotrophic conditions during Stage I promoted growth of rooting shoots but inhibited axillary shoot proliferation. Photoautotrophic conditions during Stage II suppressed callus and adventitious bud production from leaf explants compared with standard conditions. The regeneration environment appeared to be more important in controlling bud formation than the conditions under which the donor shoots were grown. Regardless of Stage I treatment, bud production Was Lip to 100-fold higher for leaves cultured under standard conditions than under photoautotrophic conditions. Once adventitious buds were differentiated from the leaf tissues, however, their elongation was faster under photoautotrophic conditions than that under standard conditions, with some shoots reaching 10 mm in length on leaf explants cultured under photoautotrophic conditions. Because total leaf soluble sugar concentration was always lowest in shoots under standard conditions, which also yielded the highest bud production, the results Suggest that endogenous starvation enhanced shoot production.

Eastern cottonwood (Populus deltoides Bartr. ex Marsh.) trees were engineered to express merA (mercuric ion reductase) and merB (organomercury lyase) transgenes in order to be used for the phytoremediation of mercury-contaminated soils. Earlier studies with Arabidopsis thaliana and Nicotiana tabacum showed that this gene combination resulted in more efficient detoxification of organomercurial compounds than did merB alone, but neither species is optimal for long-term field applications. Leaf discs from in vitro-grown merA, nptII (neomycin phosphotransferase) transgenic cottonwood plantlets were inoculated with Agrobacterium tumefaciens strain C58 carrying the merB and hygromycin resistance (hptII) genes. Polymerase chain reaction of shoots regenerated from the leaf discs under selection indicated an overall transformation frequency of 20%. Western blotting of leaves showed that MerA and MerB proteins were produced. In vitro-grown merA/merB plants were highly resistant to phenylmercuric acetate, and detoxified organic mercury compounds two to three times more rapidly than did controls, as shown by mercury volatilization assay. This indicates that these cottonwood trees are reasonable candidates for the remediation of organomercury-contaminated sites.

Cryopreservation has become anessential tool for operational application offorest tree embryogenic cultures, due to thelong evaluation periods needed for treesregenerated from these cultures. Fiveyellow-poplar (Liriodendron tulipifera)and seven sweetgum (Liquidambar spp.)embryogenic culture lines werestored in liquid nitrogen for 48 hours, afterwhich they were thawed and tested for regrowthand ability to produce somatic seedlings.Combinations of two sorbitol pretreatments andthree dimethylsulfoxide (DMSO) cryoprotectantlevels were evaluated for their impact onrecovery following cryogenic storage. The bestresults were obtained with 0.4 M sorbitol and5% DMSO, which provided 100% recovery.Somatic seedlings were regenerated from allculture lines and treatments, except for atransgenic sweetgum line.