Somatic embryogenesis and cryostorage of eastern hemlock and Carolina hemlock for conservation and restoration
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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.
Eastern hemlock (Tsuga canadenesis) and Carolina hemlock (Tsuga caroliniana) are threatened with extirpation from their native ranges in eastern North America by the introduction of the hemlock woolly adelgid (HWA; Adelges tsugae), an exotic insect pest that has already killed millions of hemlock trees. Efforts to conserve and restore these members of the Pinaceae could be greatly enhanced by the availability of an in vitro propagation system. We conducted experiments to initiate embryogenic cultures from eastern and Carolina hemlock zygotic embryos at different stages of development using three media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-Benzylaminopurine (BA). Cone collection date, medium and source tree had significant effects on induction of embryogenic tissue from zygotic embryo explants of both species, which ranged as high as 52 % for eastern hemlock and 17 % for Carolina hemlock. Embryogenic hemlock cultures could be cryostored using a protocol employing sorbitol and DMSO, and recovered following several months of frozen storage. Transfer of embryogenic tissue from proliferation media containing 2, 4-D and BA to a Litvay medium with abscisic acid promoted the development of somatic embryos, which were stimulated to mature by slow drying under semi-permeable plastic film. Embryos moved to an imbibition-germination medium without plant growth regulators and incubated in the light elongated and subsequently germinated. A small number of germinated embryos survived transfer to ex vitro conditions and grew into somatic seedlings. The embryogenesis and cryostorage systems developed in the study are already being integrated with hemlock breeding efforts to develop clones with resistance or tolerance to HWA.
KeywordsTsuga canadensis Tsuga caroliniana Cryopreservation Forest restoration Hemlock woolly adelgid
Author contribution statement
SAM designed culture initiation experiments and helped design somatic embryo production experiments, conducted data analysis, took photos and wrote all drafts of the manuscript; PMM conducted culture initiation, somatic embryo production and cryopreservation experiments and took photos; HMR conducted culture initiation experiments; LK designed and conducted somatic embryo and somatic seedling production experiments and took photos. All authors approved the final draft of the manuscript.
The research reported here was supported by a grant from the USDA Forest Service—Forest Health Protection. The authors would like to thank the USDA Forest Service, the Georgia Department of Natural Resources, Blue Ridge Outdoor Education Center, Camcore, Rusty Rhea, Jim Compton, Chuck Gregory, Danny Tatum, Greg Yates, Bill Dvorak, Robert Jetton and Josh Rood for help with collecting hemlock material, Dale Smith for technical advice and Christine Holtz for help with statistical analysis.
Conflict of interest
The authors declare that they have no conflict of interest.
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