The Establishment Of Regeneration Systems And PBI121-Lyz-GFP Gene Transformation Of Creeping Bentgrass

Agrostis stolonifera,the English name is creeping bentgrass.it has strong cold tolerance, drought tolerance, shade tolerance and low lever cutting tolerance. Transgenic technology is the best method to obtained new variety of creeping bentgrass for disease resistance in breeding of turf grass. The purpose of this research was to improve the disease-resistance of creeping bentgrass by means of transfering the resistance gene into it.After comparison of different regeneration systems in 3 creeping bentgrass varieties including PennA-1, Seaside and Putter, an efficient creeping bentgrass regeneration system was established based on the organogenesis,according to which, resistance gene was transferred into creeping bentgrass mediated by Agrobacterium, and transgenic plants were obtained. The main results are as follows:1.The establishment of regeneration systems. The main factors which influence the callus induction, differentiation and rooting of PennA-1,Seaside and Putter (Agrostis.stolonifera cv.) from mature seeds were studied. The results showed that mature seeds were cultured on MS medium supplemented with different 2, 4-D concentrations, the rate of callus induction of Putter is higher than PennA-1 and Seaside. MS medium supplemented with 2mg/L 2, 4-D and 0.1 mg/L 6-BA was the best callus induction medium for PennA-1 and Seaside, MS medium supplemented with 3mg/L 2, 4-D and 0.1 mg/L 6-BA was the best callus induction medium for Putter. Callus inducement rate were 45.2%, 76.5%, 87.0%; MSO medium was suitable for differentiation. Callus differentiation rate were 50.0%, 59.0%, 57.6%. The optimum condition for rooting was 1/2MS+1.0mg/L IBA. It indicated that the regeneration ability of Seaside and Putter are stronger than PennA-1 after comparison .2.Agrobacterium mediated gene transformation of resistance gene. Cultivated 40 days Callus of Seaside and Putter as explants and agrobacterium tumefaciens strain as mediator , the optimized genetic transformation system is as follows: Kanamycin selectors has no effect to creeping bentgrass because of natural resistance to kanamycin in creeping bentgrass. 300 mg·L-1 Carb could restrained growing of agrobacterium effectively. 10 minutes infection time and OD 0.3～0.5 agrobacterial density,3 days coculture was suitable. In a series of replicated experiment, 46 transgenic plants were obtained,moreover , 19 Putter and 23 Seaside. The result of PCR and fluorescence microscope analysis stated that the detected transgenic plants were all negative.