Establishment Of Tissue Culture Regeneration System And Preliminary Study On Genetics Transformation Of Creeping Bentgrass

Agrostis stolonifera L., a widely-grown cool season turf garss, with beautiful colour and the adventage of cut-resistance, is used in planting-lawn in north china and the whole globe.Mature seeds of creeping bentgrass were applied as explant material in all experiments. The effect of different medium, different concentrations of phytohormones on callus inducing , shoots differentiation and roots inducing were studied. The optimum conditions were found, providing a basis for gene transformation. A full regeneration system of Agrostis stolonifera L. was achieved. The main results of this research were listed as following:1. Disinfection of explants: The seeds were washed in water for 30 minutes, before putting in 70% ethanol for 30s. Then the seeds were surface-disinfected for 12～15 minutes in HgCl₂(0.1%). This kind of treatment can achieve good disinfection effect.2. Callus induction: The effects of different medium and phytohormones on callus induction were studied, which show that MS medium appended with 2mg/L2,4-D , 0.1mg/L 6-BA, 30g/L sucrose and 7g/L agar can give highest rate of callus induction, L-93 was 78.7%, pennA-1 was 60.8%, pennA-4 was 59.8%。3. Callus subculture: MS medium supplemented with 2,4-D2.0 mg/L, 6-BA0.1 mg/L and sucrose 30g/L is the most effective subculture medium.4. The best medium for shoot differentiation is MSO medium supplemented with sucrose 15g/L without phytohormones, and the shoot differentiation frequency is as high as 85%.5. Roots induction: The suggested medium is MSO+IBA0.2mg/L+sucrose 15g/L, which give 70% upwards, and L-93's roots induction rate is up to 88.8%.6. Tube-plants transfer: The strong tube-plants with more roots were selected and acclimated in green house for 15～20 days, before transferred into the field. The surviving rate is up to 90%.7. Different bacteriophages were studied in controlling agrobacterium LBA4404.