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Graduate Defense: Rachael Barron

July 6 @ 10:00 am - 11:00 am MDT

Thesis Information

Title: In Vitro Regeneration, Rooting, and Cloning of Artemisia tridentata

Program: Master of Science in Biology

Advisor: Dr. Marcelo Serpe, Biological Sciences

Committee: Dr. Julie Tinker, Biological Sciences, and Dr. Troy Weeks, Biological Sciences

Abstract

Artemisia tridentata (big sagebrush) is an ecologically important shrub found in western North America. In vitro techniques could be applied to big sagebrush to study gene function, genotypic and phenotypic plasticity, and for cloning and germplasm preservation. 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%. Subsequently, I tested several auxin treatments to induce root formation in the regenerated shoots. The highest root formation was observed in medium containing 0.5 mg/l IBA, which yielded 42% to 60% rooting frequency. The overall efficiency of regeneration from leaf explant to rooted shoots ranged between 14% to 43%. The protocol to regenerate Wyoming big sagebrush was also applied to basin big sagebrush. Callus formation was 100% in leaf explants, and shoot formation from callus was 34% (±14.6%). However, these shoots exhibited a hyperhydric phenotype and were not transferred to root induction medium. I also conducted experiments to determine the feasibility of rooting nodal cuttings and shoot tips in vitro. This method can provide genetically identical material much faster than in vitro regeneration and reduces the chances of mutations during propagation. With nodal cuttings of Wyoming big sagebrush, limited rooting success (12.5 ±5.6%) was achieved by first culturing the cuttings in a callus induction medium and then transferring them into a root induction medium. With shoot tip cuttings, adventitious root formation was variable on two types of media, ranging from 10% in some experiments to 80% in others. Results suggest that this variation in rooting frequency reflects differences in intrinsic characteristics of the seedlings rather than the media or environmental conditions tested. Attempts to clone basin big sagebrush from shoot tips further supported this notion. All material for these experiments came from half-sibling individuals that were maintained separately throughout the experiments. Some half-siblings formed no adventitious roots on any of the treatments tested, whereas others had high rates of formation on all treatments. Overall, through this research, a protocol was established to regenerate Wyoming big sagebrush in vitro. This protocol is an important step towards the development of an approach to transform this subspecies using molecular approaches. The study also demonstrates that in vitro cloning of big sagebrush seedlings via shoot tip culture is feasible. Further studies aimed at identifying the basis for the differences in adventitious rooting competence among half-siblings and seedlings, in general, may help optimize the protocol for big sagebrush cloning thus far developed.