Progress 07/01/06 to 06/30/11
Outputs
OUTPUTS: During the project, we developed and tested techniques for in vitro propagation and conservation of the "orphan" southeastern conifers, eastern hemlock (Tsuga canadensis) and Carolina hemlock (Tsuga caroliniana), using somatic embryogenesis and cryopreservation. In three culture initiation experiments over 3 years of the project, we demonstrated that embryogenic cultures of these threatened trees could be initiated using a protocol we had previously developed for southern pines. Briefly, we collected immature cones from eastern and Carolina hemlocks in Georgia and North Carolina at different times during May through August, dissected the cones to obtain seeds at a range of developmental stages and cultured whole megagametophytes with embryos or embryos dissected from them on three different induction media. Collection date and source tree had significant effects on induction of embryogenic callus from both species, with seeds collected in July giving the highest initiation rates. We also showed that we could produce mature somatic embryos from the cultures and that some of these could germinate and survive transfer from ex vitro conditions into potting mix and resume growth. Thus one output of the project was a defined protocol for embryogenic culture initiation and maintenance and production of somatic seedlings of the two hemlock species. Furthermore, we also demonstrated that embryogenic cultures of the two hemlock species could be cryostored and recovered, using a protocol we have used previously with embryogenic cultures of other forest tree species. Briefly, embryogenic cultures were pre-treated with sorbitol and cryostored using our standard procedure, testing the cryoprotectant dimethylsulfoxide (DMSO) at either 5 or 10 percent. All cryostored material of four of the five tested cultures recovered following cryostorage for over six months, regardless of DMSO treatment. Thus, a second output of the project was a defined protocol for cryostorage and recovery of hemlock embryogenic cultures. Taken together, the embryogenic culture and cryostorage work have defined a useful system for germplasm conservation and propagation of these threatened hemlock species. Results from this work have been disseminated in the form of oral and poster presentations to hemlock research community and the southern tree breeding research community. PARTICIPANTS: Individuals Scott Merkle was the PI on the project and designed culture initiation, somatic embryo production and cryostorage experiments Paul Montello (Research Professional III) performed culture initiation and cryostorage research Hannah Smith (Graduate Research Assistant) performed culture initiation research David Beleski (Graduate Research Assistant) performed culture initiation research and somatic embryo production research Partner organizations USDA Forest Service provided hemlock seeds for culturing CAMCORE (NC State University) provided hemlock seeds for culturing TARGET AUDIENCES: Target audiences for knowledge produced from the project include the hemlock and hemlock woolly adelgid research community, southern forest tree geneticists, conservation groups, undergraduate and graduate students in classes where guest lectures on threatened forest trees were delivered. PROJECT MODIFICATIONS: Given the urgency of the hemlock woolly adelgid problem and the likely loss of eastern and Carolina hemlocks to HWA, we decided to place the major emphasis of the project on the hemlock species and therefore did minimal work with longleaf pine and no work with Atlantic white cedar, as had been planned in the original proposal. While these two species have been ignored in tree improvement and propagation efforts, neither is threatened with extinction like the hemlocks are.
Impacts
The project aimed to contribute to conservation and restoration of threatened southern conifers that have largely been ignored with regard to mass propagation technology because they are not top commercial species. The development of tissue culture propagation and long term storage systems for the two threatened hemlocks added powerful new tools for conserving genetic diversity of these trees that might otherwise be lost and for mass propagating hemlock trees to use as planting stock in restoration efforts. The potential environmental impact of applying this technology lies with the ability to restore the hemlocks to the forest following devastating losses due to the hemlock woolly adelgid. By the end of the project, we showed that the same protocols used to initiate pine embryogenic cultures can be successfully applied to initiate embryogenic cultures from both hemlock species, and that production of hemlock somatic seedlings from them was possible. We also have now demonstrated that both hemlock germplasm can be successfully cryostored and recovered--another critical step if this approach is to be used for germplasm conservation.
Publications
- Montello, P.M., H.M. Smith and S.A. Merkle. 2008. Using Biotechnology to Conserve Eastern and Carolina Hemlock Germplasm. In: Proceedings of the 2007 Joint Meeting of the Southern Forest Tree Improvement Conference and the Western Forest Genetics Association, June 19-22, 2007, Galveston, TX. p.130.
- Montello, P.K., D.G. Beleski, H.M. Smith and S.A. Merkle. 2010. Somatic embryogenesis in eastern and Carolina hemlocks. In: Proceedings of the 30th Southern Forest Tree Improvement Conference, May 30-June 3, 2009, Blacksburg, VA. p. 114.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: During 2010, we had planned to initiate new embryogenic cultures from immature seeds of eastern and Carolina hemlocks. However, we were unable to initiate any new cultures because surviving mature trees with cones on them could not be identified after multiple attempts. The destruction caused by the hemlock wooly adelgid has made it increasingly difficult for us to obtain material from which to initiate cultures. During 2011, we will attempt to find new source trees with cones so that we can start cultures next season. Following our demonstration in 2009 that embryogenic hemlock cultures could be successfully cryostored and recovered using a standard protocol that we had used previously with pine embryogenic cultures, we applied the protocol to store the few new cultures we were able to initiate in 2009. Briefly, three embryogenic cultures of eastern hemlock and one of Carolina hemlock, which were maintained on semisolid EDM6 medium, were inoculated into liquid EDM6 and grown for 1 week on a gyratory shaker in the dark at 25 C. Then the suspension cultures were pre-treated overnight by shaking in liquid EDM6 with 0.4 M sorbitol. Cultures were cryoprotected with 5 percent DMSO. Freezing followed our standard published protocol and cryovials with embryogenic culture material were stored in liquid nitrogen. PARTICIPANTS: We have cooperated with the USDA Forest Service, the Georgia Department of Natural Resources and Camcore, which participated in the project by aiding with collection of hemlock material for us to culture. TARGET AUDIENCES: Target audiences have included tree breeders, conservation organizations and students in natural resources management. I have included information on our hemlock and longleaf pine research in lectures I have given to students in natural resources classes and presentations I have made to professional and community groups. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Our project aims to contribute to conservation and restoration of four southern conifers that have largely been ignored with regard to mass propagation technology because they are not top commercial species. The development of tissue culture propagation and long term storage systems for these species will add powerful new tools for conserving genetic diversity of these trees that might otherwise be lost and for generating seedling-like plants to use as planting stock in restoration efforts. The potential environmental impact of applying this technology lies with the ability to restore some of these species to the forest following devastating losses due to insect pests (hemlocks) or over-harvesting (Atlantic white cedar). With regard to economic impact, the ability to mass propagate longleaf pine should eventually help small landowners who wish to plant this species in the face of a limited supply of planting stock. We have already shown that the same protocols used to initiate pine embryogenic cultures can be successfully applied to initiate embryogenic cultures from both hemlock species, but we are still lacking evidence that this route will be useful for propagation, since we do not yet have a standard protocol that we know will produce mature somatic embryos and have only regenerated a few hemlock somatic seedlings. We also have now demonstrated that both hemlock and longleaf pine germplasm can be successfully cryostored and recovered--another critical step if this approach is to be used for germplasm conservation.
Publications
- Montello, P.K., D.G. Beleski, H.M. Smith and S.A. Merkle. 2010. Somatic embryogenesis in eastern and Carolina hemlocks. In: Proceedings of the 30th Southern Forest Tree Improvement Conference, May 30-June 3, 2009, Blacksburg, VA. p. 114. (abstract)
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: New hemlock cultures were initiated from seeds collected from one eastern hemlock and one Carolina hemlock tree in June 2009. Only 44 filled Carolina hemlock seeds were obtained and of these, only one was able to provide a proliferating embryogenic culture. Although cones were collected from 3 eastern hemlock trees, the cones of two were heavily infested with seed bugs and were not usable. While many cones of the third eastern hemlock tree were also infested, we obtained sufficient filled seed to use 27 seeds as explants. Six of these explants produced proliferating embryogenic cultures. A major goal of our research with "orphan" (non-commercial) southern coniferous species is to demonstrate that embryogenic cultures are a useful mechanism by which germplasm of these species can be conserved using cryopreservation. Thus, one experiment during 2009 was designed to demonstrate successful cryostorage and recovery of eastern and Carolina hemlock embryogenic cultures and to test two alternative cryoprotection treatments for the tissue with regard to their impact on regrowth following recovery from cryostorage. Three Carolina hemlock and two eastern hemlock embryogenic cultures, all initiated during 2007, were used for the experiment. Cultures, which were maintained on semisolid EDM6 medium, were inoculated into liquid EDM6 and grown for 1 week on a gyratory shaker in the dark at 25 C. Then the suspension cultures were pre-treated overnight by shaking in liquid EDM6 with 0.4 M sorbitol. Two cryoprotectant treatments were tested: 5 percent DMSO and 10% DMSO. Each treatment was replicated 3 times with each hemlock culture line. Freezing followed our standard published protocol and cryovials with embryogenic culture material were stored in liquid nitrogen. After 6 months, cryovials were removed from liquid nitrogen and embryogenic culture material was recovered and inoculated onto fresh semisolid EDM6 following our published protocol. All samples of four of the five tested embryogenic culture lines re-grew strongly following recovery from cryostorage. One eastern hemlock genotype showed slow regrowth and one replication (plate) of this line was lost to contamination. Differences between regrowth for the two DMSO treatments were not statistically significant. Six longleaf pine embryogenic culture lines, stored in liquid nitrogen between 2002 and 2004, were recovered from cryostorage and tested for regrowth following our published protocol in October 2009. All six longleaf pine embryogenic culture lines re-grew following recovery from cryostorage. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Our project aims to contribute to conservation and restoration of four southern conifers that have largely been ignored with regard to mass propagation technology because they are not top commercial species. The development of tissue culture propagation and long term storage systems for these species will add powerful new tools for conserving genetic diversity of these trees that might otherwise be lost and for generating seedling-like plants to use as planting stock in restoration efforts. The potential environmental impact of applying this technology lies with the ability to restore some of these species to the forest following devastating losses due to insect pests (hemlocks) or over-harvesting (Atlantic white cedar). With regard to economic impact, the ability to mass propagate longleaf pine should eventually help small landowners who wish to plant this species in the face of a limited supply of planting stock. During 2009, we confirmed that eastern and Carolina hemlock embryogenic cultures could be successfully cryostored and recovered and also demonstrated that longleaf pine cultures could be recovered from cryostorage after as long as 7 years in liquid nitrogen. These were critical steps needed to demonstrate that our approach can be used to conserve germplasm of these coniferous species.
Publications
- Merkle, S.A., P.M. Montello and H.M. Smith. 2009. Using biotechnology to conserve hemlock germplasm. Fourth Hemlock Wooly Adelgid Symposium, February 12-14, 2008, Hartford, CN. [poster]
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: During 2008, four experiments were conducted to test treatments for hemlock somatic embryo maturation. Each experiment used the same two eastern hemlock and two Carolina hemlock embryogenic culture lines. In the first experiment, three different abscisic acid (ABA) treatments were tested by supplementing the somatic embryo maturation medium (EMM2) with 56 uM (standard), 20 uM or 80 uM ABA. Both higher and lower levels of ABA than our standard have been used with spruce and other conifer embryogenic cultures, so we thought higher and lower concentrations should be tested with the hemlock cultures. While the 80 uM ABA treatment appeared to produce more early-stage embryos than the other treatments, none of the treatments produced mature somatic embryos that could be germinated. In the second experiment, a higher gellan gum concentration than our standard 4.5 g/l was tested in combination with replacing or not replacing the lids on plastic Petri plates following a period of desiccation using semi-permeable film. Two gellan gum concentrations, 4.5 g/l (standard) and 10 g/l were tested factorially with the re-lidding versus no re-lidding treatments. None of the tested treatments resulted in mature somatic embryos from any of the culture lines. The third experiment tested the use of polyethylene glycol (PEG) in EMM2. PEG has been used in many other conifer somatic embryo maturation protocols, although we have not used it previously in our lab. We tested 7.5 g/l PEG 6000 versus no PEG, in combination with the same re-lidding versus no re-lidding treatments tested in Experiment 3. Neither the standard nor PEG treatment resulted in the production of mature somatic embryos. In the fourth experiment, we tested a treatment that is standard in most spruce somatic embryo production protocols, which is the inclusion of a 1-week period between culture in 2,4-D-supplemented proliferation medium and culture in ABA-containing embryo maturation medium, during which time the embryogenic material is cultured in medium with no plant growth regulators to reduce the level of residual 2,4-D in the cultured material. In our experiment, we tested direct transfer of embryogenic hemlock material from proliferation medium (EDM6) to EMM2 versus transfer from EDM6 to EMM2 modified to contain no ABA, but supplemented with 1 g/l activated charcoal for 1 week, followed by transfer to EMM2 with the standard level of ABA (56 uM). Similar to the other experiments, neither treatment resulted in the production of mature somatic embryos. We think that the problem may be associated with the age of the cultures, which were almost a year old when the experiments were initiated. It is possible that like embryogenic pine cultures, embryogenic hemlock cultures rapidly lose their capability to produce mature, germinable embryos within 6-8 months, even though they still appear to be embryogenic. To address this possibility, we will initiate new embryogenic cultures during the summer of 2009 and get them into somatic embryo production experiments as soon as there is sufficient culture material to use for them, probably within 2 months. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Our research with conservation of eastern and Carolina hemlocks using biotechnology has been included in recent presentations on invasives and endangered species to University of Georgia students in FORS 1100 (Natural Resource Conservation), a course designed to introduce students potentially interested in Natural Resources to the field. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our project aims to contribute to conservation and restoration of four southern conifers that have largely been ignored with regard to mass propagation technology because they are not top commercial species. The development of tissue culture propagation and long term storage systems for these species will add powerful new tools for conserving genetic diversity of these trees that might otherwise be lost and for generating thousands of seedling-like plants to use as planting stock in restoration efforts. The potential environmental impact of applying this technology lies with the ability to restore some of these species to the forest following devastating losses due to insect pests (hemlocks) or over-harvesting (Atlantic white cedar). With regard to economic impact, the ability to mass propagate longleaf pine should eventually help small landowners who wish to plant this species in the face of a limited supply of planting stock. We have already shown that the same protocols used to initiate pine embryogenic cultures can be successfully applied to initiate embryogenic cultures from both hemlock species, but we are still lacking evidence that this route will be useful for propagation, since we do not yet have a standard protocol that we know will produce mature somatic embryos and have only regenerated a few hemlock somatic seedlings. We also have yet to demonstrate that hemlock or longleaf pine germplasm can be successfully cryostored and recovered, another critical step if this approach is to be used for germplasm conservation.
Publications
- No publications reported this period
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: During 2007, we conducted a large-scale culture initiation for two of the "orphan" southeastern conifers, eastern hemlock (Tsuga canadensis) and Carolina hemlock (Tsuga caroliniana), following up on the preliminary culture initiation study we conducted in 2006. We collected cones from four source trees of each native hemlock species (eastern and Carolina) on four dates during the summer, to get seeds at a range of developmental stages for explanting. Depending on the appearance of the seeds, we either cultured whole megagametophytes with embryos or dissected the embryos from the megagametophytes for culture without the megagametophyte. In all, over 2200 megagametophyte and zygotic embryo explants were cultured. Explants were cultured on semi-solid culture initiation media of three types: (1) Merkle et al.'s (2005) induction medium (IM), which was supplemented with 13.57 uM 2,4-D and 2.22 uM BA, (2) EDM6 medium (Walter et al. 1998), which is Smith's (1996) EDM supplemented
with 4.52 uM 2,4-D and 2.22 uM BA and (3) DCR medium (Gupta and Durzan 1985), which was supplemented with the same levels of plant growth regulators as Medium 1. While data analysis is not yet complete, it is apparent that cone collection date has a substantial effect on the induction of embryogenesis. For eastern hemlock, the peak date for embryogenesis appeared to be July 10 for North Carolina seeds (8%) and July 16 for Georgia seeds (52%). For Carolina hemlock, seeds collected in North Carolina on July 23 had the highest induction rates (17%). However, the only collection date resulting in embryogenesis induction for Georgia Carolina hemlock seeds was June 15 and even for this collection, only 1% of the explants produced callus that appeared to be embryogenic. Subsamples of each callus line that appeared to be embryogenic are now being screened to see which ones are capable of producing somatic embryos and plants. Also during 2007, we regenerated our first eastern and Carolina
hemlock somatic seedlings from embryogenic cultures initiated in 2006. A few of these cultures produced somatic embryos several months following transfer to Smith's (1996) EMM2 medium. Once somatic embryos reached the "bullet stage," they were transferred to germination medium and incubated under cool white fluorescent light at 22 C until they germinated. The first eastern hemlock somatic seedling was transferred from culture to potting mix and placed in a hardening off chamber in June 2007. Another three eastern hemlock and two Carolina hemlock somatic embryos germinated and were potted and transferred to the hardening off chamber in August 2007. So far, results have been disseminated to communities of interest via a poster on the project presented at the Joint Meeting of the Southern Forest Tree Improvement Conference and the Western Forest Genetics Association, June 19-22, 2007, in Galveston, TX.
PARTICIPANTS: M.S. level Graduate Student Hannah Smith worked on the project and gained training in conifer culture initiation and maintenance. We have cooperated with USDA Forest Service personnel and Georgia Department of Natural Resources personnel, who have participated in the project by aiding with collection of hemlock material for us to culture.
TARGET AUDIENCES: Target audiences have included professional tree geneticists and breeders, conservation organizations and students in natural resources management. I have included information about our efforts in hemlock conservation in lectures I have given to students in Natural Resources classes and in presentations I have given to professional groups and community groups.
Impacts
At this early stage of the project, the main impact of the research has been to demonstrate that techniques previously reported for initiating embryogenic cultures of pines and other conifers can be effectively extended to hemlock species, and thus have the potential to contribute to conservation and restoration of these threatened species. The success of this step provides us with the basis for continuing along this line of research to see how effective the approach will be for propagation of these species through somatic seedling production, and hemlock germplasm conservation through cryostorage.
Publications
- Montello, P.M., H.M. Smith and S.A. Merkle. Using biotechnology to conserve eastern and Carolina hemlock germplasm [Abstract]. In: Proceedings of the 2007 Joint Meeting of the Southern Forest Tree Improvement Conference and the Western Forest Genetics Association, June 19-22, 2007, Galveston, TX (in press).
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Progress 07/01/06 to 12/31/06
Outputs
During 2006, we initiated cultures of two of the "orphan" southeastern conifers, eastern hemlock (Tsuga canadensis) and Carolina hemlock (Tsuga caroliniana) in a preliminary study. With the help of USDA Forest Service and CAMCORE cooperators, we collected immature cones from eastern and Carolina hemlocks in North Carolina during July and August, 2006, dissected the cones to obtain immature seed explants and cultured the immature seeds or embryos dissected from them on two media containing 2,4-D that we have previously used for embryogenesis induction in southern pines. Of the 317 total explants cultured, several produced callus that appeared to have embryogenic potential, based on their similarity to embryogenic callus described for other conifers. Following transfer to maturation medium, one eastern hemlock and one Carolina hemlock culture, each derived from whole megagametophyte explants, produced somatic embryos. Embryos grew slowly, but upon transfer to
pre-germination medium in the light, they elongated and hypocotyls turned green. Following transfer to germination medium, cotyledons of a few Carolina hemlock somatic embryos expanded and radicles elongated into taproots.
Impacts
Our project aims to contribute to conservation and restoration of four southern conifers that have largely been ignored with regard to mass propagation technology because they are not top commercial species. The development of tissue culture propagation and long term storage systems for these species will add powerful new tools for conserving genetic diversity of these trees that might otherwise be lost and for generating thousands of seedling-like plants to use as planting stock in restoration efforts. The potential environmental impact of applying this technology lies with the ability to restore some of these species to the forest following devastating losses due to insect pests (hemlocks) or over-harvesting (Atlantic white cedar). With regard to economic impact, the ability to mass propagate longleaf pine should eventually help small landowners who wish to plant this species in the face of a limited supply of planting stock.
Publications
- No publications reported this period
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