Masters Theses

Date of Award

5-2024

Degree Type

Thesis

Degree Name

Master of Science

Major

Plant Sciences

Major Professor

Charles N. Stewart

Committee Members

Charles N. Stewart, Reginald J. Miller, Feng Chen, Kimberly D. Gwinn

Abstract

Agrobacterium­-mediated transformation has long been utilized as a method of producing stable, transgenic plants. This project describes an attempt to develop a system of producing stably transformed hemp (C. sativa (L.)) plants. Because of its recalcitrance to in vitro tissue culture and regeneration, transformation studies of hemp have been severely limited. In this study, I combined media components that have been shown to improve tissue culture health and regeneration. Additionally, I used hemp cultivars and explant sources that have been reported to function well in in vitro systems. The results showed that the improved media components did not have an significant effect on plantlet regeneration. They did, however, show that the cultivars ‘Futura 75’ and ‘USO 31’ outperformed ‘Fedora 17’ in both the number of explants with shoots (responding explants) and the number of shoots per explant. Explant source was shown to have a significant effect on both the proportion of responding explants and the proportion of shoots per explant, with approximately 51% of hypocotyl explants producing shoots at a rate of 1.32 shoots per explant. I also witnessed novel organogenesis in cotyledon explants. Finally, I transformed the plant with a chimeric growth regulator shown to improve in vitro regeneration across several species. This growth regulator was transformed with mGFP as a reporter gene. While no regenerated plants were verified to have been transformed, several explants showed mGFP expression, with cotyledons exhibiting a low transformation rate of 6%. While these results were unfavorable (as no transformed plant was regenerated), I do believe that these experiments have laid important guidelines for future work in developing a stable transformation system for C. sativa (L.).

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