Tissue culture and genetic transformation of tall fescue

Tall fescue is an open-pollinated, perennial, cool-season forage and turfgrass of great economical importance. The objectives of this research were to improve tall fescue tissue culture responses to facilitate of tall fescue genetic transformation, to develop efficient transformation procedure of tall fescue, and to enhance tall fescue brown patch resistance by transforming tall fescue with disease resistance genes. The major achievements of this research are summarized below.; Twenty-five turf-type tall fescue cultivars were evaluated for their callus induction and plant regeneration responses. Significant differences were found among the 25 cultivars for both callus induction and plant regeneration. The results indicated that significant variability exists in tissue culture response among tall fescue cultivars. The best cultivars identified in this study are useful candidates for research in tall fescue genetic transformation.; The effects of five culture medium supplements on tissue culture responses of an elite cultivar, ‘Coronado’, were investigated with immature embryos and mature seeds, as explant tissues. For both explants, calli induced on BAP-containing medium had significantly improved regeneration ability. In addition, by slicing mature seeds into 2halves longitudinally, the callus induction frequencies, as well as the corresponding overall plant regeneration frequencies, were increased approximately three to six fold in all three combinations of 2,4-D and BAP in callus induction media.; To understand more of the regeneration event of tall fescue calli, scanning electron microscopy was performed and somatic embryogenesis was revealed in this species. Somatic embryogenesis initiated from compact, granular sectors of a callus. The scutellum became obvious during development, and the coleoptile formed later at the base of the scutellum. The scutellum of somatic embryos of tall fescue exhibited a leafy nature as indicated by trichome formation on the surface of the tissue.; Approximately 100 independent transgenic turf-type tall fescue plants were obtained by microprojectile bombardment of embryogenic suspension cells of two elite turf-type cultivars, ‘Coronado’ and ‘Virtue’. Expression of the transgenes in transgenic plants was revealed by resistance to the herbicide “Finale” or antibiotic hygromycin B and by GUS activity assays. Stable integration of the transgenes into plant genomes was demonstrated by Southern hybridization analysis. Transgenic tall fescue plants with the bar transgene are fully resistant to the commercially applied concentration of the herbicide “Finale”.; For long-term storage of elite materials, tall fescue suspension culture cells were cryopreserved in liquid nitrogen. Two different cryoprotectants, along with the presence or absence of osmotic treatment, were compared. Cryopreserved cells were able to multiply when plated on culture medium after thawing. Suspension cultures were re-established from cryopreserved material and transformation experiments were conducted using these materials. Green shoots regenerated from antibiotic resistant calli upon selection. The best cryopreservation effect was achieved by using a solution of 0.5 M DMSO, 0.5 M glycerol, and 1 M sucrose as the cryoprotectant.; Preliminary results of two other experiments, silicon carbide whisker transformation and evaluation of transgenic plants for brown patch resistance, are also reported in this thesis.