Study On Establishment Of Plant Regeneration System Of Creeping Bentgrass And Cryopreservation By Vitrification

Agrostis stolonifera L., a significant-grown cool season turf garss, with beautiful colour and the adventage of cut-resistance,Commonly used in the golf courses of the green and High-grade lawn. This study explores the best conditions of induction callus and establish high efficiency regeneration system. The procedure for cryopreservation by vitrification was preliminarily developed in Creeping bentgrass calli.It provide theoretical and practical basis for cell project and gene project of Creeping bentgrass. At the same time, it provided the basis for Long-term preservation of Agrostis stolonifera L. germplasm resources. The results showed that:1. The regeneration system of Creeping Bentgrass have been estabilished. With A- 4,A-1and pate for the three varieties of explant on callus induction, A-4 have the highest rate of callus induction; 2,4-D is the the most important hormone affecting callus induction, It has shown that 4 mg/L 2,4-D was optimal for callus induction. It can significantly improve the induction rate, which show that MS medium appended with 4mg/L2,4-D , 0.3mg/L 6-BA, 30g/L sucrose and 7g/L agar can give highest rate of callus induction; Callus subculture: MS medium supplemented with 2,4-D2.0 mg/L, 6-BA0.1 mg/L,ABA 0.3mg/Land CH 500mg/L is the most effective subculture medium. The best differentiation medium is MS+6-BA2.0 mg/L,the inductivity can reach 80%.;Roots induction: The suggested medium is 1/2MS+0.5 mg/LNAA2. The procedure for cryopreservation by vitrification:After subcultured twice, the callus was pre-cultured five days at 4℃,and then the excised calli were loaded with 2 mol/l glycerol and 0.4 mol/l sucrose.then the calli were exposed to PVS2 for 50 min at the zero temperature. after changing the solution with fresh PVS2, the calli were put into the liquid nitrogen at least one hour. the calli were thawed rapidly in a 30℃water bath. then they were washed with the MS medium with 1.0 mol/lsucrose for 3 times and each 10 minutes. The survival of cells can reached 85.6%. 3. Before and afte cryopreservation calli,The Peroxidase isozyme zymogram and chromosomes is not changed. The result provided a feasible proof of conservation Agrostis stolonifera L..through cryopreservation of in vitro shoot-tips by vitrification for long term...