Date of Award

7-2023

Rights

© 2023 Tyler Riendeau

Document Type

Thesis

Degree Name

Master of Science in Environmental Sciences

Department

Environmental Science

First Advisor

Thomas Klak

Second Advisor

Noah Perlut

Third Advisor

Bethany Woodworth

Abstract

The American chestnut (Castanea dentata) was once a prominent hardwood species of the eastern United States forests. From Maine to Alabama, the chestnut provided many ecosystem and economic services to wildlife and humans alike. After an accidental importation of chestnut blight (Cryphonectria parasitica) from Asia, billions of American chestnuts succumbed to the disease. Since the 1980s, researchers have been working to develop a fungal blight-tolerant chestnut in hopes of restoring the species. By the early 1990s, Dr. William Powell and his fellow scientists at the State University of New York College of Environmental Science and Forestry (SUNY-ESF) successfully transformed an American chestnut that codes for the enzyme oxalate oxidase (OxO), a gene expression that provides resistance to oxalic acid by an oxalate-producing fungi (Powel et al. 2019). Oxalate oxidase reduces the acidity, and therefore the deadliness of the fungus’s oxalic acid used to kill the American chestnut, thereby protecting the species against severe damage from blight infection. This genetically engineered tree has been rigorously studied in the laboratory and is now increasingly in the field and is poised to be approved by the federal government for widespread restoration.

This thesis addresses three questions fundamental to using transgenic American Chestnut trees in restoration: First, how does the viability of transgenic pollen change over time? Secondly, how do transgenic American chestnut trees perform in a field setting compared to other types of chestnuts? Third, how can Geographic Information Systems (GIS) and aerial drone technology aid in conducting spatially explicit field experiments such as this one?

Pollen from transgenic trees is critical to restoration. Transgenic pollen carries over the OxO enzyme gene from the father to their offspring during outcrossing. Blight tolerant offspring that inherit the OxO gene through Mendelian genetics are more likely to survive in the wild, as fungal blight is widespread in the environment. The OxO gene provides offspring with a natural chemical defense mechanism against threats posed by blight. Transgenic pollen produced in the laboratory was shipped throughout the country to produce blight resistant offspring during controlled outcrossing. I studied pollen viability in the laboratory and the field to help guide these efforts.

Transgenic pollen was collected from greenhouse-grown transgenic trees, desiccated in granular desiccant at 4°C, and freezer stored at -80°C from 19 June, 2020 to 20 July, 2021. Pollen was used in controlled pollinations in Maine and shipped across the tree’s native range. Pilkey (2021) found that pollen stored at -80 °C remained viable for up to 8 months after collection. I tested pollen stored from 1 to 13 months to ascertain viability and source variability between 2020 and 2021. I tested pollen viability using a sucrose-based germination medium (as she did) and in controlled field pollinations. For pollen tube development (a viable pollen grain produces a tube), Pilkey (2021) set a ballpark estimate for successful results at 30%. Drawing from Pilkey (2021), I too consider pollen tube development near or exceeding the 30% level to be an adequate viability level. In my research, pollen viability varied substantially by age and source, but all ages and sources were shown to be effective at producing both pollen tubes and fertile nuts, even after 13 months in storage.

As of 2021, a one-acre field in Cape Elizabeth, Maine was granted permission by the United States Department of Agriculture (USDA) to plant genetically engineered American chestnuts for the first time in New England. At this field site, I studied the height growth of transgenic chestnuts over their first growing season compared with their non-transgenic full siblings and other controls. In this orchard, the University of New England (UNE) transgenic chestnut team compares transgenic, non-transgenic full siblings, Chinese, f1 hybrid or first-generation Chinese American hybrid, and backcrossed advanced-generation hybrid trees (see Table 1.1). Seedlings were raised from seed in two greenhouses at UNE and planted in the field in a randomized design between 14 May to 18 May 2021, when trees were 5.5 to 6 months old. We examined the roles of the type of seedling, greenhouse conditions, and seedling conditions at planting on the growth rates of seedlings.

Despite varying seedlings’ initial health conditions, they grew similar heights before being outplanted in the field. After the first field season, healthier seedlings depicted as pathogen – seedlings (refer to Chapter 3 Methods) grew statistically larger in height than sick seedlings depicted as pathogen + seedlings. Additionally, seedlings bred in more southern sites grew successfully in Maine during year one, suggesting growth in historically northern chestnut native range possible. Transgenic seedlings grew similarly to their non-transgenic full siblings, suggesting that, thus far, the inserted gene from wheat does not impede growth in the field. Lastly, offspring with some European sativa genes inherited from Maine mothers ULL, UNU, and USU supported strong height growth trends in Maine, like Ashdale seedlings in New York. The two greatest factors on height growth for seedlings after year one were the influence on sativa gene inheritance by offspring and seedlings’ initial growth in a pathogen – environment.

Geographic Information Systems (GIS) is a tool that holds great promise for applications to spatially explicit problems. At the same time, drone-captured aerial imagery provides high resolution spatial data that can be used to advance research efforts. However, little work is being done combining drone-captured aerial images and ArcGIS. I incorporated drone-captured aerial images with ArcGIS Pro to map the seedlings’ performances for the Cape Elizabeth field site. I created two maps to help visualize the growth trends of seedlings after the first year’s field season and as a baseline for multi-year future analysis and model for the parallel SUNY-ESF orchard.

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Master's thesis

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