Date of Award

5-2025

Rights

© 2025 The Author

Document Type

Thesis

Degree Name

Master of Science in Environmental Sciences

Department

Environmental Science

First Advisor

Thomas Klak

Second Advisor

Ek Han Tan

Third Advisor

Steven Travis

Fourth Advisor

Pamela Morgan

Abstract

The American chestnut (Castanea dentata) was a dominant, foundational forest canopy species in eastern North America until an imported chestnut blight (caused by Cryphonectria parasitica) rendered it functionally extinct across its native range. Biotechnological approaches, like the Darling 54 transgenic line, have potential for future restoration, but need both lab and field evaluations of their breeding and blight tolerance. This thesis adds to the body of knowledge on the Darling 54 American chestnut. This research reports three novel advancements made through indoor breeding and the first systematic field test for blight tolerance of transgenic American chestnuts.

Historically, field-based breeding has been hampered by long generation times, ≤50% transgene inheritance, and regulatory restrictions on outdoor breeding of transgenic trees. However, this work demonstrates that long generational time and field constraints can be circumvented through refined indoor speed breeding. The research produced quantities of both male pollen and receptive female flowers in controlled, indoor facilities. More specifically, this paper presents three novel contributions to research on the Darling 54 (D54) American chestnut; a transgenic line with an inserted oxalate oxidase (OxO) gene which detoxifies the blight. (1) The refinement of indoor speed breeding methodology for transgenic American chestnuts yielding quantities of male and female receptive flowers. (2) The documentation of generational advances (pollen and fertile nuts) from transgenic and wild-type individuals under speed breeding conditions. (3) The production of full-term burs crossed within and between two transgenic lines (D54 and DarWin). These findings demonstrate that an obligate outcrossing and slow-maturing forest tree can reach sexual maturity rapidly and abundantly in a controlled, indoor environment.

Advances in evaluating transgenic trees for blight tolerance are, like breeding, slowed by the years it takes for seedlings to grow to saplings and then to trees, which can live hundreds of years. Current regulatory restrictions further constrain the planting and growth of transgenic chestnuts in the wild. Given these constraints, this study focused on field testing blight tolerance among approximately 200 diverse T3 and T4 transgenic and control chestnut saplings in a permitted orchard in Maine, near the northern end of the native range. These saplings were branch-inoculated with a highly virulent strain of the fungal blight, EP-155. Statistical analysis of inoculated transgenic Darling 54 saplings compared to controls suggested that the transgenic chestnuts produced cankers that are comparable to the naturally-tolerant Chinese (C. mollissima) and F1 (C. dentata x C. mollissima) controls and significantly smaller than cankers on their wild-type, susceptible American chestnut counterparts. Results demonstrate that early field testing techniques to gauge the relative fungal blight tolerance of transgenic chestnut saplings can reveal significant patterns blight tolerant of transgenic D54 lines. This field test of transgenic chestnuts contributes to the goal of restoring this iconic species to its eastern North American native range.

Comments

Master's thesis

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