Development of a Micropropagation Protocol for the Ex Situ Conservation of Nuttall's Scrub Oak (Quercus dumosa).

Worldwide, oak species are threatened with extinction due to habitat loss, pathogens, and changing fire regimes. Ex situ conservation through tissue culture may protect the remaining genetic diversity of Quercus dumosa, or the coastal sage scrub oak, from further loss. We designed three basal salt formulations based on the mineral composition of shoot tips and first leaves from mature Q. dumosa and explored carbohydrate source, stress-mitigating compounds, and plant growth regulator concentrations to develop a method of cultivating many Q. dumosa culture lines in vitro. All three novel basal salt formulations led to decreased necrosis compared with commercial basal salt formulas WPM, MS, and DKW. Substitution of 30 g L-1 sucrose with glucose and adding 250 mg L-1 ascorbic acid, 5.2 mg L-1 SNP sodium nitroprusside, and 103 mg L-1 y-aminobutyric acid improved culture health overall. In an experiment involving 115 culture lines, 0.66 mg L-1 6-benzylaminopurine produced the highest average shoots per explant, but 0.33 mg L-1 produced the greatest proportion of shoots 2 cm or greater. Incubation for 24 h in 20 mg L-1 indole-3-butyric acid led to the most rooting. These methods show promise for the ex situ conservation of many genotypes of endangered Q. dumosa.

Plant Cryopreservation: Principles, Applications, and Challenges of Banking Plant Diversity at Ultralow Temperatures.

Progressive loss of plant diversity requires the protection of wild and agri-/horticultural species. For species whose seeds are extremely short-lived, or rarely or never produce seeds, or whose genetic makeup must be preserved, cryopreservation offers the only possibility for long-term conservation. At temperatures below freezing, most vegetative plant tissues suffer severe damage from ice crystal formation and require protection. In this review, we describe how increasing the concentration of cellular solutes by air drying or adding cryoprotectants, together with rapid cooling, results in a vitrified, highly viscous state in which cells can remain viable and be stored. On this basis, a range of dormant bud-freezing, slow-cooling, and (droplet-)vitrification protocols have been developed, but few are used to cryobank important agricultural/horticultural/timber and threatened species. To improve cryopreservation efficiency, the effects of cryoprotectants and molecular processes need to be understood and the costs for cryobanking reduced. However, overall, the long-term costs of cryopreservation are low, while the benefits are huge.

Improving culture initiation of mature oak shoots through use of silver thiosulfate.

Premise: Of the approximately 430 species of oaks (Quercus spp.) that have been assessed, 31% are threatened with extinction and in need of safeguarding. However, oak seeds cannot be seed banked, and thus rely on alternative strategies such as in vitro culture for ex situ conservation. One challenge to this approach is low culture initiation rates. Our objective was to identify factors that may improve the establishment of shoot cultures in vitro using new growth collected from mature trees. Methods: Shoot cuttings were harvested from individuals of five different oak species (Q. alba, Q. bicolor, Q. macrocarpa, Q. muehlenbergii, and Q. palustris). Shoots were cultured onto medium with or without 50 µM silver thiosulfate (STS), a known inhibitor of the stress hormone ethylene. Cultures were grown for one month, at which point shoots were assessed for survival. Results: Shoot survival was significantly greater in shoots cultured on medium containing STS compared to the control group, with the overall survival rate increasing from 65% to 73%. Discussion: Increasing the survival rate of newly established cultures is important in ensuring that material collected from endangered species has the best chance for survival, which is critical for successful ex situ conservation.

Harnessing the power of botanical gardens: Evaluating the costs and resources needed for exceptional plant conservation.

Premise: The effective ex situ conservation of exceptional plants, whether in living collections or cryo-collections, requires more resources than the conservation of other species. Because of their expertise with rare plants, botanical gardens are well positioned to lead this effort, but a well-developed strategy requires a clear understanding of the resources needed. Methods: Grant funding was obtained from the Institute of Museum and Library Services to support a three-year project on cryobanking, and to provide smaller grants to 10 other botanical gardens for one-year projects on either (1) seed behavior studies or (2) the development of protocols for in vitro propagation or cryopreservation. Results: Nine of the partner gardens worked on 19 species (one was unable to continue due to the COVID-19 pandemic), while the larger project focused on 14 species. A point system was developed for tasks accomplished, and the average costs per point of the larger and smaller projects were similar. Labor accounted for half the costs. Projects focused on species in the Asteraceae and Orchidaceae had lower costs per point than other species. Discussion: Both large and small projects can contribute to a strategy for exceptional plant conservation for similar costs. Prioritizing species with lower costs could help advance the field while allowing time for work on more difficult species to develop.

The Tip of the Iceberg: Cryopreservation Needs for Meeting the Challenge of Exceptional Plant Conservation.

Cryopreservation is increasingly important as a conservation tool, particularly for threatened exceptional species. The goal of this study was to investigate the current knowledge of plant cryopreservation through a search of the literature in Web of Science and align that with the 775 species currently identified on the Working List of Exceptional Plants. While there is a good foundation in plant cryopreservation research, particularly with economically important species, there are significant gaps in research on families that contain the largest numbers of currently known exceptional species, including the Dipterocarpaceae, Rhizophoraceae, and Pittosporaceae. Even families well represented in both in the literature and on the List of Exceptional Plants had much less overlap at the level of genus. Tropical trees, a significant portion of exceptional species, were not as well represented in the literature as herbaceous species. Over 70% of all articles dealt with in vitro cryopreservation, with much less emphasis on other methods (seed, embryo, dormant bud, and pollen) that will be more cost-effective for species where they can be applied. While the research on plant cryopreservation to date provides a strong foundation and is being utilized effectively for conserving the diversity of a number of economically important species, this study revealed significant gaps that can help prioritize future research to more effectively conserve the diversity of threatened exceptional species.

Survival and growth of embryo axes of temperate trees after two decades of cryo-storage.

Liquid nitrogen (LN) storage is recommended for conserving plants with seeds for which standard seed banking is not feasible, but there are few empirical reports confirming its long-term effectiveness. In this study we evaluated in vitro viability and ex vitro growth of embryo axes of a species that is short-lived in seed bank conditions (Juglans nigra) and two recalcitrant seeded species (Aesculus hippocastanum and A. glabra) that were stored 11-23 years in LN. Viability of J. nigra axes did not decrease significantly after 23 years and produced normal-appearing plantlets. Similarly, viability of A. hippocastanum axes did not decrease after 23 years. However, A. glabra axes showed a significant decline in viability after 23 years, from 80% when freshly harvested to 33%. These results demonstrate that LN storage can preserve embryo axes of J. nigra and Aesculus sp. for over two decades, providing a workable conservation tool for these and similar species.

Tools for the ex situ conservation of the threatened species, Cycladenia humilis var. jonesii.

Ex situ conservation is critical for hedging against the loss of plant diversity. For those species (exceptional species) that cannot be conserved long-term in standard seed banks, alternative methods are required, often involving in vitro culture and cryopreservation, or storage in liquid nitrogen. Cycladenia humilis var. jonesii is a federally threatened perennial native to Utah and Arizona. It is classified as an exceptional species, because it produces few seeds, and, thus, in vitro propagation and cryopreservation were investigated as tools for its propagation and preservation. Shoot-propagating cultures were established from both seedling and wild-collected shoots, but cultures from both sources displayed an extreme form of the physiological disorder, hyperhydricity. This phenotype could be at least partially normalized by the use of vented closures, as well as by using agar, rather than gellan gum, in the medium. The hyperhydric (HH) phenotype had a lower dry weight, more branching, minimal leaf development and more poorly developed vascular tissue than the more normal (MN) phenotype. Only more normalized shoots could be rooted and the resulting plants acclimatized. Both HH and MN shoots also provided shoot tips capable of surviving cryopreservation using the droplet vitrification method. These in vitro and cryopreservation methods provide tools that can be used for propagating plants of C. humilis var. jonesii for research and restoration, as well as for supplying shoot tips for the ex situ conservation of this species. The two distinct phenotypes also provide a useful system for studying factors involved in the HH response of this dryland species in vitro.

Differential effects of abscisic acid on desiccation tolerance and carbohydrates in three species of liverworts.

Tissues of three species of in vitro grown liverworts, Riccia fluitans, Pallavicinia lyellii, and Marchantia polymorpha, were subjected to rapid drying with and without preculture for 1 week on medium containing 10 microM ABA. ABA preculture initiated total desiccation tolerance in R. fluitans, whereas control tissues were killed after 30 min of drying. Survival was also improved in P. lyellii, whereas ABA did not affect survival of M. polymorpha after rapid drying. ABA treatment did, however, reduce the rate of water loss in M. polymorpha. Total soluble carbohydrates were increased in ABA-treated R. fluitans and P. lyellii, but not in M. polymorpha, although there was no correlation between survival and changes in the percentage of these carbohydrates as reducing sugars. These differences in response to ABA and desiccation likely reflect different adaptations of these three species to conditions in situ.