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Browsing by Subject "Plant transformation"

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    Investigating Novel de Novo Meristem Induction Through a Direct Delivery Gene Editing Technique in Greenhouse Grown Apple and Grape
    (2023-02) Spicer, Robert
    Apple and grape are woody fruit crops with a significant global market. The process of conventional breeding of woody fruit crops is a lengthy process that takes decades to produce commercially viable cultivars due to heterozygosity, self-incompatibility, and long juvenile periods. Genome editing technologies have become an alternative breeding method to traditional plant breeding techniques to bypass the time and resource limitation in current breeding programs. Currently, one of the most prominent gene editing technologies in plants is CRISPR/Cas9 that can be used to produce targeted changes for specific attributes comparable to conventional breeding but in a faster and more predictable manner. The efficiency of Cas9 for crop improvement has been studied in grape and apple as explants or somatic embryos but the efficiency of this technology in soil grown plants is understudied. This study investigates novel de novo meristem gene editing in soil grown apple and grape to develop and test methods for direct delivery of Cas9 and sgRNA for targeted gene knockouts in non-model species. Plasmids programmed with Cas9, sgRNA, developmental regulators, and bioluminescent reporter were injected into designated nodes in planta at different growth stages with varying Agrobacterium cell concentration treatments. We also investigated variations in preparing plants for injection by node ablation, tissue types, plant preparation, and experimental setups. After selecting for shoots with suspected gene edits, tissue was assayed for luciferase expression and knockout verification using PCR. No evidence of a gene knockout was observed through phenotypic observations or with luciferase and PCR assays. Early preliminary experiments had high rates of plants with inactive shoot growth but were useful in designing optimized experiments with substantially lower rates of inactivity. Abnormal shoot growth was observed in apple seedling experiments as well as gall-like formations. Both abnormal shoot growth patterns and gall-like formations were observed ubiquitously in all treatment groups. Young non-woody plant tissue was more susceptible to desiccation than lignified tissue, but some plants were capable of new shoot growth despite having desiccated stems. Method development remains important to improve the efficiency regeneration and increase the likelihood of gene edits.