Impatiens walleriana, commonly grown as a herbaceous annual, is susceptible to Impatiens Necrotic Spot Virus (INSV). A lack of resistant cultivars leaves growers with the sole option of destroying infected plants before INSV spreads throughout their entire crop. Therefore, the introduction of INSV resistant cultivars would have the potential to save Impatiens growers a substantial amount of money. Virus resistance has been successfully conveyed in several crops by insertion of pathogen DNA into the host plant. One method of generating transgenic plants involves the use of Agrobacterium-mediated gene transfer. A commonly used technique involves transformation of explant tissue and subsequent regeneration in vitro under aseptic conditions. However, prior to our research there was no regeneration protocol suitable for Agrobacterium-mediated transformation of Impatiens walleriana available. Herein we report the development of a new method for regeneration of Impatiens walleriana using cotyledonary node culture. Using this technique, four regeneration media amended with 1, 3, 5, or 7µM of thidiazuron were evaluated for their ability to induce de novo shoot production in cotyledonary node explants, and evaluated for number of shoots produced per explant. Results showed a significantly greater frequency of regeneration and number of shoots per explant using media amended with 1µM of thidiazuron. This technique has shown to be repeatable and is not susceptible to ploidy instability. Unfortunately, damage to the cotyledonary node explants during Agrobacterium inoculation and transfection prevented regeneration of transformed shoots in several attempts. However, transient GFP expression after transfection of shoot pads derived from cotyledonary nodes with Agrobacterium strain LBA 4404 containing plasmid pHB2829 with nptII and S-GFP was obtained, indicating the possibility for this regeneration protocol to derive stably transformed Impatiens with INSV resistance. / Master of Science

vegetable crops. Micro-Tom is a model tomato cultivar used for research due to its small size and short time to fruiting. This project evaluated I. walleriana and Micro-Tom transformed with Agrobacterium. The construct contained GFP (green fluorescent protein) and hygromycin antibiotic-resistant selectable markers, and the antisense sequence of open reading frame of INSV nucleocapsid protein (N). The N gene is expected to confer INSV resistance by RNA interference or gene silencing. The presence of transgenes was confirmed by PCR. Transgenic Impatiens was selfed for two generations. Transgenic Micro-Tom was selfed for 4 generations. Spinach was used as an INSV reservoir. Impatiens, spinach and Micro-Tom were mechanically inoculated with INSV and evaluated visually, with assay tests, ELISA testing, and PCR. Spinach was successfully infected with INSV six times of seven attempts. Impatiens and Micro-Tom had no successful inoculations of three and five attempts, respectively. / Master of Science / Impatiens walleriana, also known as impatiens or ‘Bizzy Lizzy,’ is a popular ornamental plant. It has a wide variety of flower colors and grows well in shade. Impatiens necrotic spot virus (INSV) is an incurable virus that causes disfiguring dead spots on plants. Micro-Tom is miniature tomato used for research due to its small size and short time to fruiting. This project tested impatiens and Micro-Tom transgenic plants that had been genetically modified using bacteria. The bacteria had been modified to contain ‘markers’ which allow researchers to confirm the modifications were successful. It also contained a small piece of genetic material from the virus, which was expected to make the plants resistant to the virus by interfering with virus movement and reproduction. These transgenic plants were self-pollinated for multiple generations and tested to confirm the transgene was present. “Wild-type” (not genetically modified) spinach was infected with the virus by hand and infected spinach leaves were used to try to infect impatiens and Micro-Tom. The plants were inspected visually and leaves were tested for presence of the virus. Spinach was successfully infected with INSV six times of seven attempts. Impatiens and Micro-Tom had no successful inoculations of three and five attempts, respectively.