Basin big sagebrush (Artemisia tridentata subsp. tridentata) is a keystone species of the sagebrush steppe, a widespread ecosystem of western North America threatened by climate change. The study’s goal was to develop an in vitro method of propagation for this taxon to support genome sequencing and genotype-by-environment research on drought tolerance. Such research may ultimately facilitate the reintroduction of big sagebrush in degraded habitats. Seedlings were generated from two diploid mother plants (2n = 2x = 18) collected in environments with contrasting precipitation regimes. The effects of IBA and NAA on rooting of shoot tips were tested on 45 individuals and 15 shoot tips per individual. Growth regulator and individual-seedling effects on percent rooting and roots per shoot tip were evaluated using statistical and clustering analyses. Furthermore, rooted shoot tips were transferred into new media to ascertain their continued growth in vitro. The results suggest that A. tridentata is an outbred species, as shown by individuals’ effect on rooting and growth. IBA addition was the most effective method for promoting adventitious rooting, especially in top-performing individuals. These individuals also have high survival and growth rates upon transferring to new media, making them suitable candidates for generating biomass for genome sequencing and producing clones for genotype-by-environment research.

Artemisia tridentata (big sagebrush) is an ecologically important shrub found in western North America. In vitro techniques can be applied to big sagebrush for the purpose of studying gene function, genotypic and phenotypic plasticity studies, cloning, genotypic preservation, and restoration. I performed experiments to develop an indirect organogenesis protocol to regenerate whole Wyoming big sagebrush plants from leaf explants. Callus formation frequency was 88% (±4.0%) in leaf explants cultured on medium containing 0.5 mg/l BAP and 1.0 mg/l NAA. Shoot formation frequency was variable between replicates and was the highest when callus tissue was cultured on medium containing 1.5 mg/l BAP and 0.1 mg/l NAA, 37% to 80%. I tested several auxin treatments to induce root formation and concluded the best to be 0.5mg/l IBA, which yielded 42% to 60% rooting. Taking into account all these variables, I estimate the total regeneration efficiency to range between 14% to 43% on this set of treatments. This protocol was also applied to basin big sagebrush. Callus formation was 100% in leaf explants. Shoot formation was 34% (±14.6%), but shoots exhibited a hyperhydric phenotype and were not transferred to root induction medium. The in vitro regeneration protocol developed is a crucial element that would be required to transform big sagebrush using molecular approaches. Experiments were also conducted to determine the feasibility of shoot tip and nodal cuttings to develop adventitious roots in vitro. This method can provide genetically identical material much faster than in vitro regeneration. Adventitious root formation in Wyoming big sagebrush cuttings cultured on two media types was inconsistent, ranging from 10% in some experiments to 80% in others. Limited success was achieved in nodal cuttings cultured on modified MS medium containing auxin and cytokinin 12.5% (±5.6%). No root formation was achieved in mature plant tissue collected in the field. Results indicated that genotypic influences were likely more responsible for variations in rooting than the medium or vessel conditions tested. Cloning experiments in basin big sagebrush further supported this notion. All material for these experiments came from half-sibling individuals that was maintained separately throughout the course of the experiments. Some half-siblings formed no adventitious roots on any treatments tested whereas others had high rates of formation on all treatments. Further studies, utilizing exogenous PGRs, such as auxins, may provide more successful adventitious root formation in shoot tips from both big sagebrush subspecies.

Premise: Determining the tolerance of plant populations to climate change requires the development of biotechnological protocols producing genetically identical individuals used for genotype-by-environment experiments. Such protocols are missing for slow-growth, woody plants; to address this gap, this study uses Artemisia tridentata, a western North American keystone shrub, as model. Methods and Results: The production of individual lines is a two-step process: in vitro propagation under aseptic conditions followed by ex vitro acclimation and hardening. Due to aseptic growth conditions, in vitro plantlets exhibit maladapted phenotypes, and this protocol focuses on presenting an approach promoting morphogenesis for slow-growth, woody species. Survival was used as the main criterion determining successful acclimation and hardening. Phenotypic changes were confirmed by inspecting leaf anatomy, and shoot water potential was used to ensure that plantlets were not water stressed. Conclusions: Although our protocol has lower survival rates (11-41%) compared to protocols developed for herbaceous, fast-growing species, it provides a benchmark for slow-growth, woody species occurring in dry ecosystems.