Justin Whitehill

Coniferous giants are some of the most majestic, yet vulnerable tree species on Earth. Long life cycles and slow generation times limit our ability to improve and deploy genetically elite trees across the landscape. The cascading impacts of accelerated climate change threaten existing elite genetic resources as forest fires become more frequent and intense. To ensure the long-term preservation of elite germplasm and facilitate the rapid deployment of genetically improved individual genotypes requires the development of novel tools that offer solutions to these new challenges. Here we propose to initiate somatic embryogenic (SE) cultures of elite sugar pine (Pinus lambertiana) germplasm. SE is an alternative and synergistic approach used to preserve important coniferous genetic material. Ultimately, we plan to facilitate rapid deployment of improved conifers through clonal propagation of elite germplasm.

Greenhouse, field, and AI optimization of germplasm for poplar and hemp are needed to address economic and environmental challenges to sustainable bioeconomies in the South's highland or mountain regions.

I am proposing to request funds to support a post-doc and sequencing costs associated with genome sequencing of Fraser fir.

Our proposal aims to develop bioluminescent Fraser fir Christmas trees using our existing somatic embryogenic elite Fraser fir lines. We plan to transform these existing Fraser fir lines with 4 genes recently identified and patented from a mushroom.

This project will be a collaboration between the Christmas Tree Genetics Program and the Molecular Tree Breeding Lab in the Department of Forestry and Environmental Resources at North Carolina State University. Our goal is to accelerate the genetic improvement of Fraser fir against the important regulatory pest Elongate Hemlock Scale (EHS). Fraser fir is one of North Carolina������������������s most important specialty crops generating annual revenues exceeding $100 million. The development of novel genomic tools and technologies will have a positive, transformative impact on the North Carolina Christmas tree industry. Our project builds on resources developed by the NCSU Christmas Tree Genetics Program in collaboration with the NCDA and NC Christmas tree growers over the past 4+ decades. We propose four major objectives in this proposal: (1) evaluation of genetic variability in Fraser fir and select Abies spp. response to Elongate Hemlock Scale infestation; (2) histological evaluation of EHS feeding on Fraser fir and Abies spp. foliage; (3) biochemical and molecular response of Abies spp. to Elongate Hemlock Scale infestation; (4) development of molecular resources to identify defense characteristics of EHS resilient Abies genotypes; and (5) synthesis and dissemination of results to NC Christmas tree stakeholders. Subsequent to the funding period, these efforts will benefit the North Carolina Christmas tree community and contribute to the genetic conservation of native Fraser fir populations in the Appalachian Mountains. We expect project deliverables will help address key knowledge gaps of pest resilience in Fraser fir and push conventional conifer breeding strategies and integration with genomic information into a new era.

This project will be a collaboration between the Christmas Tree Genetics Program, the Forest Health and Conservation Program, and the Molecular Tree Breeding Lab in the Department of Forestry and Environmental Resources at North Carolina State University. Our goal is to accelerate the genetic improvement of Fraser fir against the tree-killing pathogen Phytophthora root rot and insect pest balsam woolly adelgid. Fraser fir is one of North Carolina������������������s most important specialty crops generating annual revenues exceeding $100 million. The development of novel genomic tools and technologies will have a positive, transformative impact on the North Carolina Christmas tree industry. Our project builds on resources developed by the NCSU Christmas Tree Genetics Program in collaboration with the NCDA and NC Christmas tree growers over the past 4+ decades. We propose five major objectives in this proposal: (1) genomic resource development of Fraser fir responses to Phytophthora and BWA; (2) identification of Phytophthora and BWA elicitors; (3) evaluation of Fraser fir responses to isolated elicitors; (4) population level analysis of key pest responsive genes in existing NCSU Fraser fir breeding program resources; and (5) synthesis and dissemination of results to NC Christmas tree stakeholders. Subsequent to the funding period, these efforts will benefit the North Carolina Christmas tree community and contribute to the genetic conservation of native Fraser fir populations in the Appalachian Mountains. We expect project deliverables will help address key knowledge gaps of pest resilience in Fraser fir and push conventional conifer breeding strategies and integration with genomic information into a new era.

This project will be a collaboration between the Forest Biotechnology Group in the Department of Forestry and Environmental Resources and the Forest Restoration Alliance in the Department of Entomology and Plant Pathology at North Carolina State University. We propose a integrative approach to understanding the genetic response to hemlock woolly adelgid (HWA) infestation in susceptible and resistant hemlock species, and how these genetic regulations are transduced to alterations in phenotypic traits associated with HWA susceptibility. The proposed project builds upon ongoing research in developing a CRISPR genome editing system for hemlocks funded by the SCBGP in 2020-21. Comparative transcriptomics and phenomics of hemlock variants with varying extent of HWA susceptibility will produce genetic insights that facilitate identification of candidate gene targets for editing using CRISPR-Cas to enhance HWA resistance. This project will focus on four key objectives: (1) controlled HWA infestation in putatively susceptible and resistant genotypes of hemlock species, (2) assessment of phenotypic response to infestation in hemlocks, (3) full transcriptomic analysis of hemlock response to HWA infestation, and (4) integration of transcriptomic and phenotypic responses to identify putative gene targets associated with HWA resistance. The putative genes identified in this project will be targeted for hemlock genome editing in a subsequent research that is beyond the scope of this project period.

The Center for Plant Conservation received a grant from the Institute for Museum and Library Services (IMLS) for a project entitled, ����������������RNA integrity as a powerful metric of aging in preserved seed collections of wild rare plant species��������������� (MG-245983-OMS-20). To satisfy grant commitments, CPC has the need for a subcontractor to collect a ����������������fresh accession��������������� of rare plant seed from the same wild population previously collected 15 years ago or more and subsequently preserved in orthodox seed storage. The older seed accession currently in orthodox storage will hereafter be known as the ����������������original accession���������������. Contractor represents that they are able and willing to undertake this work. As the contractor, NC State University will make one ����������������fresh��������������� seed collection from populations of Fraser fir (Abies fraseri) in western North Carolina. Fresh collections will either be made from the same population as the ����������������original accessions���������������; from ex-situ plants of shared wild provenance; or from plants grown or bulked from seeds of a seed collection held in long term storage for 15 years or more ������������������ known as the ����������������original accession.��������������� The specific quantity of seed required for testing may vary depending on the size of seed and inclusion in the different experimental groups. Seed quantities for target species are the estimated quantity of seed needed to achieve 75mg of material plus 100 seeds for a germination trial. The bolded species will need multiple 75mg replicates from the freshly collected accession for inclusion in an advanced aging study.

The Equipment Grants Program (EGP) serves to increase access to shared special purpose equipment for scientific research for use in the food and agricultural sciences programs in our Nation������������������s institutions of higher education, including State Cooperative Extension System

This project will be a collaboration between the Forest Biotechnology Group and the Christmas Tree Genetics Program in the Department of Forestry and Environmental Resources at North Carolina State University. Our goal is to develop novel CRISPR-based genome editing technology that would accelerate the genetic improvement of Fraser fir. The proposed technology would enable the rapid production of new variants of Christmas trees edited for traits of ecological and economic values such as disease tolerance and post-harvest quality. Fraser fir is one of North Carolina������������������s most important specialty crops. Developing novel genomic tools and genome editing technology for Fraser fir will have a transformative impact on the North Carolina Christmas tree industry. This project builds on our recently established somatic embryogenic system and cell transfection method for Fraser fir (funded by SCBGP in 2018), which lay the foundation for optimizing efficient and robust CRISPR-Cas9 delivery and regeneration of enhanced Fraser fir from genome-edited somatic embryos. We propose three major objectives in this proposal: (1) Optimize the delivery of CRISPR-SpCas9 in Fraser fir somatic embryogenic protoplasts: we will test several experimental parameters to maximize transfection efficiency; (2) Regenerate and maturate CRISPR-SpCas9 edited protoplasts into Fraser fir plantlets: we will optimize an integrated protocol for regeneration and maturation of CRISPR-edited protoplasts originated from Fraser fir SE; (3) Validation of target gene editing in regenerated Fraser fir plantlets: we will genotype CRISPR-driven editing events in the regenerated fir plantlets. Subsequent to the funding period, the transgene-free CRISPR-based SE system will be used to edit superior clonal seedlings for Christmas tree field trials in the North Carolina Mountains.