Rhus michauxii is a perennial rhizomatous shrub native to the southeastern United States that is found mainly in sunny, dry, open rocky or sandy woodlands. Moreover, it is found on ridges or river bluffs in the inner coastal plane and lower piedmont of Virginia, Georgia, and the Carolinas. Habitat conversion to agriculture, suppression of fires, and low reproduction have caused R. michauxii to become rare and it is now federally listed as threatened. Methods are needed to multiply and conserve R. michauxii. Protocols were developed for seed cryopreservation, in vitro germination, and micropropagation for R. glabra and R. michauxii. Seed scarification in concentrated sulfuric acid for 6 h and germination on ½ MS medium resulted in germination up to 96% for control and cryopreserved seeds of R. glabra and 70 and 40% for control and cryopreserved seeds of R. michauxii. Shortly after germination in vitro, young seedlings were established in a greenhouse potting mix providing new plants from the endemic Georgia R. michauxii populations. Several of the findings meet goals within the R. michauxii recovery plan by providing methods for sexual and asexual multiplication and long-term seed storage under cryogenic conditions. The protocols developed will assist in the safeguarding and conservation of dwindling natural R. michauxii populations.

Xerophyllum asphodeloides (Xerophyllaceae), known as eastern turkeybeard, is an herbaceous perennial found in eastern North America. Due to decline and destruction of its habitat, several states rank X. asphodeloides as “Imperiled” to “Critically Imperiled”. Protocols for seed cryopreservation, in vitro germination, sustainable shoot micropropagation, shoot establishment in soil, and seed germination are presented. Seeds from two tested sources were viable after 20 months of cryopreservation. Germination of isolated embryos in vitro was necessary to overcome strong seed dormancy. Shoot multiplication and elongation occurred on ½ MS medium without PGRs. Shoots rooted in vitro without PGRs or with 0.5 mg/L NAA or after NAA rooting powder treatment and placement in potting mix. When planted in wet, peaty soil mixes, shoots grew for two months and then declined. When planted in a drier planting mix containing aged bark, most plants continued growth. In the field, plant survival was 73% after three growing seasons. Safeguarding this species both ex situ and in situ is possible and offers a successful approach to conservation. Whole seeds germinated after double dormancy was overcome by incubation under warm moist conditions for 12 weeks followed by 12 weeks cold at 4 °C and then warm.

Apios priceana, commonly known as Price’s potato-bean, is a perennial species native to the Southeastern US. Habitat destruction has caused A. priceana to become rare, and it is federally listed as threatened. Protocols developed for in vitro germination, shoot micropropagation, in vitro rooting, shoot establishment in soil, and seed cryopreservation will assist in the safeguarding and conservation of dwindling natural populations. Seeds were germinated in vitro on plant growth regulator (PGR)-free Murashige and Skoog (MS) medium after seed sterilization in H2O2 and seed nicking. Greatest shoot multiplication occurred on MS medium with BAP/IBA/GA3 at 2.22/0.49/1.44 μM with 2.0 g/l Phytagel and pH adjusted to 5.7. Shoots rooted in vitro in MS medium with 3.2 μM IBA. Shoots rooted in vitro rapidly established in greenhouse potting mix, usually showing new growth within 2 wk, with tuber formation by the end of the growing season. Plants transferred to a forest setting in late winter survived, grew throughout the summer, and became dormant in the fall. In small post cryopreservation tests with A. americana and A. priceana seeds, air or desiccant-dried to water contents below 10% but above 2.5%, germination reached 87–90%.

Aims Several Sarracenia species are endangered due to habitat destruction. Knowledge of their microbiomes may better inform reintroduction efforts. We characterized bacterial communities within rhizomes of three species with an emphasis on diazotrophs. Methods Microbiomes of wild and greenhouse-grown Sarracenia were characterized using cultivation-independent and cultivation-dependent methods, including Illumina MiSeq, 16S rRNA and nifH qPCR, colony identification via 16S rRNA sequencing, and nifH colony PCR. Rhizomes were tested for biological nitrogen fixation using acetylene reduction assays. Results Diazotrophs make up a notable proportion of the S. oreophila microbiome according to qPCR. Microbiomes of greenhouse S. oreophila varied from wild ones by having greater proportions of diazotrophs as indicated by qPCR. Wild and greenhouse plant microbiomes also differed with respect to several phylogenetic groups. Acetylene reduction assays indicated that nitrogen fixation occurred in some rhizomes. Multiple colonies from S. leucophylla and S. purpurea were nifH+, and 16S rRNA sequencing likewise revealed dissimilarities between wild and greenhouse-grown plant microbiomes. Conclusion Our study is the first to characterize bacterial communities within Sarracenia rhizomes and demonstrates that substantial differences exist between wild and greenhouse plants. We provide evidence that Sarracenia can contain large populations of diazotrophs that may help explain how these plants survive in oligotrophic environments.

Somatic embryogenesisSomatic embryogenesis (SE) technology has the potential to be the lowest-cost method to rapidly produce large numbers of high-value seedlings with desired characteristics for plantationPlantation forestryForestry. SE is expected to play an important role in the future to increase forest productivity, sustainability and uniformity. SE technology has the advantages of: (1) shortening time to produce desired Planting stock, (2) allowing control of genetic variation, (3) permitting commercial hybrids, and (4) facilitating genetic engineering efforts for desirable traits. Conifer SE proceeds through four steps: initiation, multiplication, maturation and germination and cryopreservation when storage of cultures is desired. This report will focus on the initiation step. When research began, initiation rates for loblolly pine were often below 1%. Early improvements occurred through combinations of optimal embryo stages, half-strength P6 salts, ovule osmotic profile research, modeling activated carbon (AC) uptate of 2,4-D and research to understand the effect of pH and AC on mineral availability. Many improvements in loblolly pine initiation over the past 30 years have resulted from careful study of the developing seed and embryo. Medium supplements and environmental conditions are available to improve imitiation and somatic embryo development that have resulted from analytical studies of seed tissues, the seed environment and gene experssion in the megagametophyte, zygotic embryos and somatic embryos.

Fraser firFraser fir ( Abies fraseri [Pursh] Poir.) is a coniferous species native to the Southern Appalachian Mountains of the Eastern United States. Fraser fir has high economic and recreational value but is vulnerable to extinction due to introduced pests and global warming. Somatic embryogenesis may assist in the clonal production of Christmas trees and conservation of rare and valuable germplasm via cryopreservation. Improved protocols for embryogenic tissue initiation, culture capture and growth, somatic embryo development and maturation and cryogenic storage are presented based on the findings of Pullman (47:453–480, 2016).

Fraser fir (Abies fraseri [Pursh] Poir.) is a coniferous species native to the Southern Appalachian Mountains of the eastern United States. The species has high economic and recreational value but is vulnerable to extinction due to introduced pests and global warming. Somatic embryogenesis technology may assist in the clonal production of desired lines of Christmas trees and safekeeping of rare and valuable germplasm via cryopreservation. We have developed a highly effective medium for initiation of embryogenic tissue from immature or mature seeds of Fraser fir that contains AL salts (Kvaalen et al., in Can J For Res 35:1053–1060, 2005), brassinolide, paclobutrazol and abscisic acid. Using dominant embryos attached to the female gametophyte placed on medium, the highest initiation percentages occurred with precotyledonary stage 3 embryos. When tested with 11 high-value open-pollinated families over 5 years, initiation tests for medium containing brassinolide and paclobutrazol averaged 6–62 % initiation. A maintenance medium was developed that contained AL salts and 1.1 mg L−1 BAP and was able to capture approximately 50 % of the initiations. A maturation medium was developed containing AL salts, maltose, polyethylene glycol 8000 and abscisic acid that produced cotyledonary embryos capable of germination to produce a root and shoot. Culture cryopreservation and retrieval was also demonstrated.

A major barrier to the commercialization of somatic embryogenesis technology in loblolly pine (Pinus taeda L.) is recalcitrance of some high-value crosses to initiate embryogenic tissue (ET) and continue early-stage somatic embryo growth. Developing initiation and multiplication media that resemble the seed environment has been shown to decrease this recalcitrance. Glutathione (GSH), glutathione disulfide (GSSG), ascorbic acid and dehydroascorbate analyses were performed weekly throughout the sequence of seed development for female gametophyte and zygotic embryo tissues to determine physiological concentrations. Major differences in stage-specific oxidation-reduction (redox) agents were observed. A simple bioassay was used to evaluate potential growth-promotion of natural and inorganic redox agents added to early-stage somatic embryo growth medium. Compounds showing statistically significant increases in early-stage embryo growth were then tested for the ability to increase initiation of loblolly pine. Low-cost reducing agents sodium dithionite and sodium thiosulfate increased ET initiation for loblolly pine and Douglas fir (Mirb) Franco. Germination medium supplementation with GSSG increased somatic embryo germination. Early-stage somatic embryos grown on medium with or without sodium thiosulfate did not differ in GSH or GSSG content, suggesting that sodium thiosulfate-mediated growth stimulation does not involve GSH or GSSG. We have developed information demonstrating that alteration of the redox environment in vitro can improve ET initiation, early-stage embryo development and somatic embryo germination in loblolly pine.

Helonias bullata, commonly known as swamp pink, is a perennial rhizomatous herb native to the eastern coast of the United States that is found mainly in forested wetlands, swamps, and mountain bogs. Habitat destruction has caused H. bullata to become rare, and it is federally listed as threatened. Protocols were developed for in vitro germination, shoot micropropagation, shoot establishment in soil, and seed cryopreservation that will assist in the safeguarding and conservation of dwindling natural populations. Seeds were germinated in vitro on plant growth regulator (PGR)-free 1/3-strength Murashige and Skoog (MS) medium after seed sterilization in H2O2 and used for establishment of shoot cultures. Shoot multiplication and elongation occurred on 1/3-strength MS medium containing 0.25–1 mg/l kinetin or 1–2 mg/l trans-zeatin followed by growth on PGR-free medium. All shoots (100%) rooted when planted directly into potting media. Rapid-immersion seed cryopreservation resulted in statistically significant increases in germination in vitro or in potting mix compared to control treatments. H. bullata seeds share characteristics of both orthodox and recalcitrant seeds including rapid loss of viability over time, survival at 5°C, and survival after cryopreservation. Therefore, they are most accurately classified as intermediate-type seed.

The shift from vegetative to embryogenic growth requires tissue to enter a radically different program of development and can be studied in vitro through the development of somatic embryos. From an applied perspective somatic embryogenesis (SE) is expected to play an important role in increasing productivity, sustainability, and uniformity of future forests. For commercial use, SE technology must work with a variety of genetically diverse trees. Since the first reports of SE in Picea abies and Larix decidua in 1985, many different coniferous species have shown the ability to produce embryogenic tissue. However, initiation frequency is often low, many desired seed sources are recalcitrant, and culture survival is often poor, raising costs of somatic seedlings produced from successful genotypes. A number of tools are now available to improve embryogenic tissue initiation and somatic embryo development in vitro that have resulted from analytical studies of seed tissues, the seed environment and gene expression in megagametophyte, zygotic embryos and somatic embryos. Benefits have occurred from medium supplementation with hormones, plant growth regulators, hormone inhibitors and polyamines. Somatic embryo growth has been enhanced with medium supplementation of nutritional components including specific sugar types, vitamins, organic acids, and redox potential modifiers. Control of environmental factors including, water potential, pH, adsorption of medium components by activated carbon and liquid versus gelled medium have also led to SE protocol improvements. The use of analytical studies to duplicate the seed environment in vitro is improving protocol development resulting in increased initiation, improved yields and higher-quality somatic embryos.

BACKGROUNDHabitat loss and over collection have caused North American pitcher plants to become rare, including U.S. federally endangered Sarracenia alabamensis and S. oreophila, and S. leucophylla, S. psittacina and S. purpurea spp. venosa, endangered in several states.OBJECTIVETo develop reliable seed cryopreservation protocols for endangered Sarracenia species enabling similar germination percentages before and after storage in liquid nitrogen (LN) either in vivo or using in vitro tools.METHODSSeed germination pre- and post-cryopreservation were compared following seed drying with germination in soil, aseptic environment with wet filter paper or enriched medium, and using scarification or stratification for dormancy removal.RESULTSAfter cryostorage, germination in vitro (1/6- or 1/3-strength MS medium) increased compared to germination on peat moss. Germination pre- and post-cryopreservation was similar for S. alabamensis and S. oreophila when seeds were stratified and grown in vitro. S. leucophylla and S. psittacina also showed high germination after cryopreservation when germinated on medium following stratification.CONCLUSIONRapid liquid nitrogen exposure and rewarming induced seed coat cracking that damaged seeds, likely allowing internal damage during acid scarification and microbial entry during germination in non-sterile environments.

Symphyotrichum georgianum (Asteraceae), commonly known as Georgia aster, is a candidate for listing under the Federal Endangered Species Act in the four southeastern U.S. states where it lives. Rarity of this species is thought to be attributable in part to small population sizes and limited seed production. Protocols for in vitro germination, sustainable shoot micropropagation, shoot establishment in soil, and seed cryopreservation are presented that will assist in the safeguarding and augmentation of dwindling natural populations. Germination in vitro on growth regulator-free half-strength Murashige and Skoog (MS) medium after sterilization in H 2 O 2 initiated the development of shoot cultures. Shoot multiplication and elongation occurred on half-strength MS salts containing 0.1 mg·L –l benzylaminopurine and 0.2 mg·L –l gibberellic acid, producing an average of 18 new shoots over a 6- to 8-week subculture cycle. Shoots rooted easily when planted into cutting mix after treatment with rooting powder containing indole-3-butyric acid (IBA) or in vitro rooting in medium with or without N-acetyl-L-aspartic acid (NAA). Plant survival after 1 month was 90% or higher for all treatments. Cryopreservation tests with seeds from three populations averaged 46.7% germination compared with control seed (no cryostorage) germination of 43%; differences were not statistically significant. Fresh seeds and seeds equilibrated for 1 to 4 weeks at room temperature and 12% relative humidity did not differ significantly in germination post-cryopreservation. Initial observations suggest that Georgia aster rapidly loses seed viability over 1 to 2 years when stored at room temperature. The ability to increase seed longevity through cryopreservation storage may be a critical step in the conservation of this species.

Torreya taxifolia Arn., an ancient evergreen tree, is on the brink of extinction from attack by a fungal disease, recently reported to be caused by a novel isolate of Fusarium. We report the development of a somatic embryogenesis tissue culture system that can be used for cryogenic storage of T. taxifolia cultures and subsequent plant regeneration. Initiation of embryogenic tissue from immature zygotic embryos occurred on a conifer tissue culture medium containing 0.25 % activated charcoal, 43.8 mM maltose, 0.5 mM 2,4-dichlorophenoxacetic acid, 0.2 mM 6-benzylaminopurine, 0.2 mM kinetin, 0.1 μM brassinolide, 3.8 μM abscisic acid, 20.5 μM biotin, 1.13 μM folic acid, 1.28 mM 2(n-morpholino)ethanesulfonic acid and 0.69 mM pyruvic acid. Embryo induction ranged from 60 to 100 % across six seed sources. Somatic embryo development occurred on a medium containing 43.8 mM maltose, 1 % activated charcoal, 37.8 μM abscisic acid, 20.5 μM biotin, 0.1 μM brassinolide, 0.205 mM folic acid, 1.28 mM 2(n-morpholino)ethanesulfonic acid and 0.69 mM pyruvic acid. Germination of somatic embryos ranged from 64 to 82 %. Embryogenic tissue cultures from 30 genotypes representing seed from six mother trees were cryopreserved, and culture recovery was demonstrated after freezing. In contrast to many other coniferous tree seeds, the measured water potential (−MPa) of T. taxifolia megagametophyte tissue rose greatly during seed after-ripening. Duplication of this rise in vitro allowed development of somatic embryos to the cotyledonary stage.

The genus Sarracenia forms a group of carnivorous pitcher plants native to North America. Habitat destruction and overcollection have caused pitcher plants to become rare, including U.S. federally endangered S. oreophila as well as S. leucophylla and S. purpurea spp. venosa (Raf.) Wherry, both listed as endangered in several states. Protocols for in vitro germination, sustainable shoot micropropagation, shoot establishment in soil, and seed cryopreservation are presented. Six-min sulfuric acid scarification treatments coupled with appropriate tissue culture media resulted in germination in vitro within 3 weeks, often reaching greater than 50%. Best germination for S. leucophylla and S. purpurea occurred on one-third strength Murashige and Skoog (MS) salts, whereas S. oreophila germinated best on one-sixth strength MS salts. Adjustment of pH to 4.5 to simulate a bog environment further increased germination for S. leucophylla. Shoot multiplication occurred at optimal levels when explants were placed on media in the presence of a cytokinin without auxin with greatest multiplication on 6-benzylaminopurine (BAP) or trans-zeatin and best shoot quality on trans-zeatin. Plant establishment in soil required both an in vitro rooting treatment and use of shoot clusters resulting in greater than 80% survival in soil. Seed cryopreservation tests with all three species suggest storage in liquid N-2 followed by in vitro micropropagation and plant establishment can be used to preserve material long term.

Xyris tennesseensis is a critically endangered species native to the southeastern USA. A micropropagation protocol was developed which may assist in the safeguarding and augmentation of dwindling natural populations of this ecologically and medically valuable plant. Four different batches of seeds were sterilized using hydrogen peroxide and germinated in vitro on modified one third-strength Murashige and Skoog medium. Shoot multiplication from seedling tissue was obtained using modified one third-strength Murashige and Skoog medium containing 1 mg/l kinetin and 0.1–0.5 mg/l α-naphthaleneacetic acid. Optimal shoot size and sustainable multiplication rates of three to five per 2-mo subculture occurred on medium containing 0.3–0.4 mg/l α-naphthaleneacetic acid. Shoots rooted successfully when placed on growth regulator-free medium for 10 d followed by transfer to greenhouse soil under high humidity. Use of seed cryopreservation resulted in significant increases in germination compared to control treatments with average germination rates of 97%. Shoot tip cultures from soil-grown plants of X. tennesseensis and Xyris spathifolia were also developed using the above protocols. Plant tissue culture tools will assist in the multiplication, long-term storage, and conservation of these rare and valuable plants as well as provide a template for the micropropagation of other Xyris species.

Summary Myo‐inositol hexakisphosphate (InsP6), abundant in animals and plants, is well known for its anticancer activity. However, many aspects of InsP6 function in plants remain undefined. We now report the first evidence that InsP6 can inhibit cellular proliferation in plants under growth conditions where phosphorus is not limited. A highly anionic molecule inhibitory to early‐stage somatic embryo growth of loblolly pine (LP) was purified chromatographically from late‐stage LP female gametophytes (FGs), and then characterized structurally using mass spectrometry (MS) and nuclear magnetic resonance (NMR) analyses. Exact mass and mass spectrometry‐mass spectrometry (MS‐MS) fragmentation identified the bioactive molecule as an inositol hexakisphosphate. It was then identified as the myo‐isomer (i.e. InsP6) on the basis of 1H‐, 31P‐ and 13C‐NMR, 1H‐1H correlation spectroscopy (COSY), 1H‐31P heteronuclear single quantum correlation (HSQC) and 1H‐13C HSQC. Topical application of InsP6 to early‐stage somatic embryos indeed inhibits embryonic growth. Recently evidence has begun to emerge that InsP6 may also play a regulatory role in plant cells. We anticipate that our findings will help to stimulate additional investigations aimed at elucidating the roles of inositol phosphates in cellular growth and development in plants.

Glutathione/ glutathione disulfide and ascorbic acid /dehydroascorbate are two major redox pairs that control the redox-state in a developing seed. Recently these redox compounds have been shown to exert strong positive effects on embryo development in several plants including white spruce. A picture is emerging that early-stage embryo development occurs best in the presence of a reducing environment while late-stage development requires a shift to an oxidizing environment. Knowledge of physiological concentrations of these compounds during pine embryo development would allow us to better mimic the seed environment and improve somatic embryo development. Triplicate analysis for the seed redox chemicals ascorbic acid (AsA), dehydroascorbate (DHA), glutathione (GSH) and glutathione disulfide (GSSG) were completed for developmentally staged zygotic embryos and female gametophytes over the sequence of loblolly pine (Pinus taeda L) seed development. The two trees tested generally showed similar concentrations and patterns of change through time. The redox potential changed significantly through the progression of seed development. These profiles suggest that earlystage embryo development occurs best in the presence of a reducing environment while late-stage development requires a shift to an oxidizing environment. The redox chemical profiles obtained provide guidelines and targets to mimic in the tissue culture environment. Recent and ongoing tests have shown that alteration of the in vitro redox environment through medium supplementation with glutathione or glutathione disulfide during specific developmental stages can indeed enhance loblolly pine somatic embryo development and germination.

Somatic embryogenesis (SE) has the potential to be the lowest-cost method to rapidly produce large numbers of high-value somatic seedlings with desired characteristics for plantation forestry. At least 24 of the 115-120 known Pinus species can undergo SE. Initiation for most species works best with immature megagametophytes as starting material, although a few pines can initiate SE cultures from isolated mature seed embryos. Successful initiation depends heavily on explant type, embryo developmental stage, and medium salt base. Most first reports of initiation used 2,4-D and BAP or a combination of cytokinins. More recent reports have optimized initiation for many Pinus spp., but still use mostly the combinations of auxin and cytokinins. Initiation can be stimulated with medium supplements including abscisic acid (ABA), brassinosteroids, ethylene inhibitors, gibberellin inhibitors, organic acids, putrescine, specific sugar types (maltose, galactose, D-chiro-inositol, and D-xylose), triacontanol, vitamins (B12, biotin, vitamin E, and folic acid), or manipulation of environmental factors including pH, water potential, cone cold storage, gelling agent concentration, and liquid medium. Embryo development and maturation usually occur best on medium containing ABA along with water potential reduction (with sugars and polyethylene glycol) or water availability reduction (with raised gelling agent increasing gel-strength). Activated carbon and maltose may also improve embryo maturation. The main issues holding SE technology back are related to the high cost of producing a somatic seedling, incurred from low initiation percentages for recalcitrant species, culture loss, and decline after initiation and poor embryo maturation resulting in no or poor germination. Although vast progress has been made in pine SE technology over the past 24 years, fundamental studies on seed and embryo physiology, biochemistry, and gene expression are still needed to help improve the technology to a point where large-scale commercialization is economically viable for a broad range of pine species.

Stage-specific measurements of female gametophyte (FG) and embryo pH (hydrogen ion concentration) were made through the sequence of loblolly pine (Pinus taeda L.) seed development. The FG tissue from two open-pollinated trees showed similar pH profiles starting at 5.5 shortly after fertilization, increasing to about 6.1 at stage 7, levelling off at 6.3-6.5 towards the end of development and dropping to 6.0 just before cone opening. Measurements of the chalazal end were 0.05-0.2 pH units less than the micropylar end through early-to-mid-development. In contrast, embryo pH maintained a nearly constant value near 7.0 through development. Profiles of pH through seed development were similar whether portrayed by date or stage of embryo present in the seed. The pH profiles assisted in the development of improved embryogenic tissue initiation techniques. When post-autoclaving maturation medium pH was raised from about 5.3 in control medium to 5.7 or 5.5-5.7 with 2(n-morpholino)ethanesulphonic acid, cotyledonary embryo yields increased.

A major barrier to the commercialization of somatic embryogenesis technology in loblolly pine (LP, Pinus taeda L.) is recalcitrance of some high-value crosses to initiate embryogenic tissue and to continue early-stage somatic embryo growth. Developing initiation and multiplication media that resemble the seed environment may decrease this recalcitrance. Sugar and sugar alcohol analyses were performed weekly throughout the sequence of seed development for female gametophyte and zygotic embryo tissues to determine physiologic concentrations (Pullman, G.S. and M. Buchanan. 2008. Identification and quantitative analysis of stage-specific carbohydrates in LP (Pinus taeda) zygotic embryo and female gametophyte tissues. Tree Physiol. 28:985-996). Major differences in stage-specific sugars were observed. A simple bioassay was used to evaluate the potential growth promotion of individual carbohydrates added to initiation or multiplication media at physiologic concentrations. Seventeen sugars were screened. Compounds showing statistically significant increases in early-stage embryo growth were then tested for the ability to increase the initiation of LP. d-xylose and d-chiro-inositol produced statistically significant increases in early-stage embryo growth. When tested for improved initiation in P. taeda, Pseudotsuga menziesii (mirb) Franco and Picea abies L., Karst., d-xylose increased the averages of initiation by 6.5%, 7.3% and 16.7%, respectively. d-chiro-inositol increased the initiation in P. taeda by 7.3% in one test but not in the other, whereas in P. menziesii the initiation increases averaged 8.4% in two tests. Analyses of sugars and sugar alcohols in the seed environment coupled with a bioassay to screen potential media supplements for protocol improvement resulted in statistically significant increases in embryogenic tissue initiation for several coniferous species.