Key message
Temporary immersion bioreactors can improve hybrid sweetgum somatic embryo conversion and somatic seedling growth rates compared to semisolid medium, significantly improving somatic ...seedling production efficiency for hybrid sweetgum, an emerging woody biomass crop.
Fast-growing hybrid southern hardwood trees should make excellent material for woody biomass production in the Southeastern US, if elite clones can be identified and efficiently propagated. We have enhanced the potential of sweetgum (
Liquidambar styraciflua
) as a biomass species by generating hybrids between the tree and its Chinese relative,
Liquidambar formosana
, and propagating the most promising clones via somatic embryogenesis. Some of the hybrid clones have already demonstrated superior biomass productivity compared to elite
L. styraciflua
trees. However, production of somatic seedlings from these clones remains labor-intensive. Bioreactors, specifically temporary immersion designs, such as the RITA
®
, have been applied to improve the efficiency of in vitro propagation of a number of woody species. We tested RITA
®
bioreactors for their potential to improve the production efficiency of high-quality hybrid sweetgum somatic seedlings. In one tested genotype, a RITA
®
with 50 somatic embryos had about 66% higher conversion frequency (
p
< 0.05) and produced about 40% more “high-quality” somatic seedlings (
p
< 0.05) than when somatic embryos were germinated on semisolid medium in GA-7 vessels. In all the genotypes we tested, somatic seedlings produced in RITA
®
bioreactors had higher survival percentages by the end of acclimatization than somatic seedlings produced on semisolid medium in GA-7 vessels.
Stewartia
is a genus of flowering shrubs and trees in the family Theaceae.
Stewartia
species are native to eastern Asia, with the exception of
S
.
ovata
and
S
.
malacodendron
, which are indigenous ...to southeastern North America. Despite having outstanding ornamental value and unique horticultural features,
Stewartias
are not readily available for landscaping in the horticultural trade due to difficulty with mass propagation. In addition to commercial propagation, there is a need to develop propagation techniques for the conservation of the North American species, which are rare (
S
.
ovata
) or endangered (
S
.
malacodendron
). This research examined somatic embryogenesis as a means to mass propagate Asian and North American
Stewartia
species and to provide material for cryopreservation of valuable
Stewartia
germplasm. By testing a variety of plant growth regulators (PGRs), somatic embryogenesis was induced from immature zygotic embryo explants of seven
Stewartia
species—five Asian and two North American. Additionally, embryogenic cultures were obtained from three North American cultivars,
S
.
malacodendron
‘Delmarva’,
S
.
ovata
‘Red Rose’, and ‘Royal Purple’. For two
Stewartia
species,
S
.
ovata
and
S
.
sinensis
, somatic embryogenesis was induced from immature endosperm. Picloram (0.05 or 0.1 mg/L) and 2,4-dichlorophenoxyacetic (2 or 4 mg/L) were the most effective PGRs for inducing somatic embryogenesis of North American and Asian
Stewartia
species, respectively. All seven
Stewartia
species examined produced somatic embryos that converted into plants that could be transferred to the greenhouse. Embryogenic tissue from five
Stewartia
species were successfully put into cryo-storage and recovered, providing a means of long-term germplasm storage. This is the first report of somatic embryogenesis in the genus
Stewartia
.
Key message
This is the first report of somatic embryogenesis from immature zygotic embryos in the genus
Stewartia
and it was accomplished with seven different species.
Green ash (
) and white ash (
) populations are currently experiencing major declines across their native ranges in North America due to infestation by the exotic insect pest emerald ash borer (
). ...The development of a reliable method for the long-term storage of green and white ash germplasm in the form of embryogenic cultures using cryopreservation would be a considerable aid to ash conservation efforts. We compared recovery percentages of cryopreserved green and white ash embryogenic cultures using vitrification versus slow cooling methods. Three Plant Vitrification Solution 2 (PVS2) exposure durations (40, 60, and 80 min) for vitrification and three DMSO concentrations (5%, 10%, and 15%) for slow cooling were tested for their effects on the percentage of cultures that regrew following cryostorage. Vitrification resulted in a higher overall culture recovery percentage (91%) compared to cultures that were cryostored using the slow cooling approach (39%), and a more rapid initiation of regrowth (5 days versus 2-3 weeks) resulted. Recovery from cryostorage by cultures using the slow cooling approach varied significantly (
< 0.05) between experiments and with genotype (
< 0.05). The recovery of vitrified tissue from cryostorage did not vary with genotype, species, or PVS2 exposure duration (
> 0.05). The vitrification cryopreservation protocol provides a reliable and versatile alternative to the traditional slow cooling method, strengthening our ability to preserve valuable ash germplasm for conservation and 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.
Introduced pests (insects and pathogens) have rapidly increased the numbers of at-risk native forest tree species worldwide. Some keystone species have been functionally extirpated, resulting in ...severe commercial and ecological losses. When efforts to exclude or mitigate pests have failed, researchers have sometimes applied biotechnology tools to incorporate pest resistance in at-risk species to enable their reintroduction. Often erroneously equated solely with genetic engineering, biotechnology also includes traditional and genome informed breeding—and may provide a holistic approach toward applying genomic-based information and interventions to increase tree species’ pest resistance. Traditional tree breeding is responsible for successes to date, but new technologies offer hope to increase the efficiency of such efforts. Remarkable recent progress has been made, and for some at-risk species, novel biotechnological advances put reintroduction within reach. The high costs of reintroduction of at-risk species at necessary scale, however, will initially limit the pursuit to a few species. Successful deployment of pest resistant material may require improved species-specific knowledge and should integrate into and leverage existing reforestation systems, but these operations are sometimes rare where pest threats are greatest. While use of some biotechnologies, such as traditional tree breeding, are commonplace, others such as genetic engineering are controversial and highly regulated, yet may be the only viable means of achieving reintroduction of some at-risk species. Efforts to modify policy toward allowing the use of appropriate biotechnology, especially genetic engineering, have lagged. Provided that risk-benefits are favorable, policy is likely to follow with public opinion; in some countries, society is now increasingly open to using available biotechnologies. Continued engagement using the most recent advances in social science to build public trust, combined with a science-based collaboration among land managers and regulators, will generate the collective momentum needed to motivate policymakers to act rapidly given the speed at which forest health threats unfold and the large areas they affect.
All North American ash (Fraxinus spp.) species are threatened by the emerald ash borer (EAB; Agrilus planipennis), an exotic beetle which has already destroyed millions of ash trees in the U.S. and ...Canada. Although both chemical insecticides and biological control can be effective, and host resistance appears possible, the speed of the invasion has defied traditional management approaches. One potential, innovative approach to managing this destructive insect is to develop a host tree-induced gene silencing strategy using RNA interference (RNAi) constructs targeting EAB-specific genes. An important requirement for applying RNAi technology is a reliable transformation/regeneration system for the host tree species. We developed an Agrobacterium-mediated gene transfer system for white ash (F. americana) and green ash (F. pennsylvanica) using the embryogenic cultures of these species as target material. Embryogenic suspension cultures of multiple genotypes of both species were plated and inoculated with A. tumefaciens carrying the pFHI-GUSi expression vector, which carries the nptII selectable marker and intron-GUS reporter genes, followed by selection on a semi-solid medium containing geneticin. Putative transgenic events showed expression of the GUS gene at all tested developmental stages from callus to plantlets, and transgene presence in the leaves of regenerated plants was confirmed using PCR. The overall average transformation efficiency achieved was 14.5 transgenic events per gram of tissue. Transgenic somatic seedlings of two white ash and three green ash genotypes were produced and acclimated to greenhouse conditions.
Franklinia alatamaha
is a small tree in the tea family (Theaceae) that has been valued for its ornamental qualities since 1765, when it was first recorded by John and William Bartram on the Georgia ...Coastal Plain. The only known population of
F. alatamaha
became extinct in the wild by the early 1800s. However,
F. alatamaha
has been cultivated as an ornamental for nearly 250 years. The plants existing today are derived from seeds collected from the Georgia population by the Bartrams. To examine the genetic variation of the cultivated
F. alatamaha
population, leaves from live and herbarium accessions were obtained from 42 sites worldwide. Genotyping-by-sequencing (GBS) was used to determine the genetic diversity and structure of 76
F. alatamaha
accessions, including a 178-year-old herbarium specimen. STRUCTURE analysis with 9604 high-quality single-nucleotide polymorphisms (SNPs) identified two subpopulations within the cultivated accessions. This result was supported by UPGMA (unweighted pair group method with arithmetic mean) and principal component analyses. F statistics indicated that there was a moderate level of genetic diversity among the cultivated accessions (
F
ST
= 0.09), with more genetic diversity among accessions within a subpopulation than between the two subpopulations. The inbreeding coefficient of the cultivated accessions was low (
F
IS
= − 0.4902), indicating that the sampled trees represent what was once a highly outcrossing population. Polymorphism was also detected by GBS analysis of 19
F. alatamaha
plants grown from gamma-irradiated seeds. The genetic differentiation identified in this study may be useful for further development of new horticultural traits of
F. alatamaha
.
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.
KEY MESSAGE : The developmental sequences of zygotic embryos of green ash collected from the same tree were widely asynchronous and an intermediate developmental stage was the best explant for ...inducing somatic embryogenesis. All North American ash (Fraxinus) 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 U.S. states and Canada. We tested treatments aimed at initiating embryogenic cultures from seeds of green ash (F. pennsylvanica), with the long-term goal of using these cultures to aid in research to generate EAB-resistant ash trees for restoration. In preparation for somatic embryogenesis induction experiments, we first defined specific stage(s) of green ash zygotic embryo development using time-tracing sampling by collecting samaras of two green ash trees from May to August in 2012. Seed development was divided into seven stages according to both seed and embryo size, and the numbers of seeds and embryos in each stage were recorded for each collection date. Surprisingly, a broad range of seed and embryo developmental stages could be found in samaras collected from the same tree on the same date, in particular for the later collection dates. Using this information, single-date collections of seeds with embryos at various stages of development were made from three local Athens, GA green ash trees and one horticultural cultivar and cultured on two different basal media with different combinations of plant growth regulators (PGRs). A low percentage of zygotic embryo explants at an intermediate stage of development from all three local source trees produced proembryogenic masses (PEMs) when cultured on a modified Woody Plant Medium with 2,4-dichlorophenoxyacetic acid and benzyladenine. Although embryogenesis was also induced from explants of the horticultural cultivar, these cultures failed to produce germinable somatic embryos. Transfer of PEMs to PGR-free medium resulted in highly dense production of somatic embryos, some of which were germinated to produce somatic seedlings.
