Establishment Of Embryogenic Cell Suspension System And Isolation Of Protoplast In Tall Fescue(Festuca Arundinacea Schreb.)

Four varieties of Tall Fescue(Festuca arundinacea Shreb.) were used as material,and optimization of plant regeneration system of Tall Fescue,establishment of embryogenic cell suspension system,the different methods of monocell culture,and optimization of protoplast isolation condition were discussed,in order to obtain a lor of high quality materials in a short time and provide theoretical and practical basis for cell project and gene project of Tall Fescue.Conclusions are showed as following:1.In order to establish the high efficiency plant regeneration system of tall fescue(Festuca arundinacea Schreb.),four varieties of Tall Fescue:Millennium,Houndog V,Aridâ…¢and Crossfireâ…¡were studied in this experiment.The results indicated that:Millennium performed better than other varieties of tall fescue in both callus induction and subculture.The callus induction rate and embryogenic callus induction rate of Millennium were 63%and 36.8%.2.During different stage of plant regeneration of Tall Fescue,the suited medium is different. A1(MS+2,4-D 9mg/L+KT 0.1mg/L+Cu₂SO₄·5H₂O 2.5mg/L) and B1(MS+2,4-D 9mg/L+CH 300mg/L) medium are suited to obtain the original callus,the callus induction rate of A1 and B1 were 64.2%and 65%;B2(MS+2,4-D 5mg/L+CH 400mg/L+sucrose 60g/L+agar 12g/L) medium is suited to obtain embryogenic callus,the embryogenic callus induction rate was 32%;C3(MS+6-BA 2.0mg/L+NAA 0.5mg/L+Pro 500mg/L) medium is suited to obtain regenerated plantlet,the frequency of green plant regeneration and albino plant were 85.71%and 14.29%;1/2MS is suited to obtain plantlet with root.3.Three suspension cell lines from two varieties of Tall Fescue were established.In order to maintain the suspension cell system,1.0-3.0ml/40ml density(PCV),pH 5.0-5.7,and 5-7 days' subculture period were appropriate for cell suspension culture of Tall Fescue. 4.A single cell size of Tall Fescue is 50-100μm.Using suspension cell in exponential phases as material,monocell suspensions could be obtained by screening with nickel net(75μm).Comparing three culture methods,the results indicated that suspension culture is better than others.The product of the cell suspension culture line,round cell induction rate and the frequency of cell division were 150%,69.57%and 57.01%.The callus which can be seen by eyes was formed at the third day.5.Using suspension cells as material,the various influencing factors on protoplast isolation were studied,such as the concentration and combination of enzyme,isolation-time and pretreatment condition.The results showed that the embryogenic cell suspensions after cultured by 3 days were the best materials.The suited pretreatment time and enzyme-isolation time were 2h and 8h.The best combination of enzyme and mannital is 2.5%Cellulase Onozuka R-10+0.6%Pectolyase Y-23+11% mannitol.6.Using filtrate-centrifugal with low speed method to purified protoplast.Before centrifugal, filtrated enzyme solution must be diluted by CPW-11M solution,centrifugal speed is 1000rpm.