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

In this study, the effects of different plant growth regulators on regeneration responses of various lentil explants through direct and indirect organogenesis and through somatic embryogenesis from calli and cell suspension cultures were investigated. Shoot regeneration was obtained in low frequencies from longitudinal embryonic axis explants and nodal buds of epicotyls, however whole plant regeneration was unsuccessful. Conditions provided for indirect organogenesis resulted only in swelling of hypocotyls and root directed ends of internodes and weak callus formation on leaves which were followed by tissue browning and necrosis. In somatic embryogenesis studies, the explants longitudinal embryonic axis and cotyledonary petioles produced soft and friable calli on MS media with Gamborg&rsquo / s vitamins supplemented with 0.75mg/L 2,4-D+0.5mg/L BA. The highest average number of embryos per explant, 12.36 was observed on media containing 0.75mg/L BA +0.5mg/L 2,4-D for cotyledonary petiole explants, whereas 3mg/L BA+1mg/L NAA was the only hormone combination that allowed embryo development to some extent, in both explants. Somatic callus failed to regenerate despite globular embryo formation and embryo development to some extent. Combination of sonication treatment with Agrobacterium transformation of three lentil explants / cotyledonary nodes, half cotyledons and cotyledonary nodes with intact shoots, had no effect on the improvement of transient gus gene expression on explants. Sonication treatment was also unable to form localized wounds on the petiole axils. The best gus gene expression on the axil region was obtained when cotyledonary nodes and KYRT1 strain were used in combination with vacuum infiltration and scalpel wounding of the axils. Gradual selection and repeated removal of regenerated shoots between selection cycles increased the number of gus expressing shoots significantly. The regenerated shoots were grafted on root stocks and whole plant regeneration was achieved in greenhouse conditions. By the use of the optimized Agrobacterium-mediated transformation protocol, 4 independent lines were obtained with 2.3% transformation efficiency. Southern blot analysis confirmed the integration of the gus gene into the genome of lentil plants. T0 plants were fertile and all plants showed Mendelian segregation of the gus gene in 3:1 ratio to their progenies except one line which carries three copies of the gene. Reverse transcription PCR has confirmed the expression of the genes in T0 and T1 generations. T0 plants and the following three generations strongly expressed gus gene uniformly in their tissues and the PCR amplifications of both gus and npt-II genes was successful through generations.

The objective of this study was to optimize an efficient selection system and Agrobacterium mediated transformation of chickpea (Cicer arietinum L.). Cotyledonary node explants of Turkish chickpea cultivar G&ouml / k&ccedil / e were used to determine the effects of selective agents, two antibiotics (Kanamycin, Hygromycin) and two herbicides (PPT, Glyphosate) as well as four antibiotics (Augmentin, Carbenicillin, Cefotaxime, Timentin) for eliminating Agrobacterium on multiple shoot and root induction. Selective agents and antibiotics were applied to explants at different concentrations for one month and numbers of regenerated shoots and roots were recorded. Kanamycin at 100 mg/L, Hygromycin at 20 mg/L, PPT at 3 mg/L and Glyphosate at 5 mg/L were found to be appropriate to select chickpea transformants. Lowest concentrations of all selective agents (50 mg/L Kanamycin, 10 mg/L Hygromycin, 3 mg/L PPT, 1 mg/L Glyphosate) totally inhibited rooting of the regenerated shoots. Among the Agrobacterium-eliminating antibiotics, Cefotaxime and Augmentin each up to 600 mg/L had no adverse effect on shoot induction, whereas Timentin (300 mg/L) significantly increased and Carbenicillin (300 mg/L) significantly decreased shoot induction after four weeks of culture. Augmentin was determined to have no effect on rooting capacities of chickpea shoots. However Cefotaxime at all concentrations significantly decreased root induction. On the other hand only high concentrations of Carbenicillin (300 mg/L) and Timentin (200 mg/L) significantly decreased rooting. Sulbactam in combination with Carbenicillin and Cefotaxime displayed effective inhibition of bacterial growth. Furthermore, Agrobacterium mediated transformation procedure for cotyledonary node explants of G&ouml / k&ccedil / e, was also optimized by monitoring transient uidA expression on 4th, 9th, and 16th days after transformation. Transformation procedure was improved via mechanical injury of axillary region of explants and application of vacuum infiltration at 200 mmHg for 40 minutes.