| Tall Fescue(Festuca arundinacea Schreb)is a part of the Gramineae perennial cool-season grass,because of planting fast,drought-resistant,high temperature resistance,trampling tolerance,to adapt to a wide rage of soil and climatic conditions,etc,and have been widely used in playgrounds,landscaping,airports and slope protection.However in the course of using,there are some shortage,such as leaves rough,no stolon,lawn poor scalability,slow-growing in summer,etc.This experiment used mature seeds of Festuca arundinacea Schreb(New Horizons and the Legendary Luminous Pearl 2)as experimental material,adopt the method of removal of part of endosperm,and use of tissue culture on study of callus and adventitious bud formation,finally established stable and high frequence regeneration system of Festuca arundinacea Schreb.At the same time,genetic transformation was studied by using dip method of Agrobacterium,firstly established transformation system,and lay the foundation for mutation breeding,cell engineering and genetic engineering.The results are as following: 1. Establish regeneration system of mature seedsThis experiment used mature seeds of New Horizons and the Legendary Luminous Pearl 2 as experimental material,and systemic researched the methods of tissue culture,and debated the effect of combinations of different plant growth regulators and concentration on callus induction and differentiation.The result indicated that the culture medium contenting 2,4-D,NAA,6-BA had big significant effect on callus induction,the range of induction rate are 9-92%,12-94%,the two variety had strong sensitivity of 2,4-D,the best induction medium to the Legendary Luminous Pearl 2 was 5.0mg/L2, 4-D+0.1mg/L6-BA,the induction rate reached 92%;the best induction medium to New Horizons were 5.0mg/L2, 4-D+0.1mg/L6-BA and 5.0mg/L2,4-D+0.1mg/LNAA,but the callus induction rate of the former higher than the latter,the rate up to 94%;6-BA was an important factor in callus differentiation,the best differentiation medium to the Legendary Luminous Pearl 2 was 2.0mg/L6-BA,and to New Horizons was 1.0mg/L6-BA,the differentiation rate were 78% and 76%;two variety of the best rooting medium was 1/2MS+0.5mg/LNAA,rate was 100%. 2. Atumefaciences-mediated transformation system of callus of the Legendary Luminous Pearl 2It was studied that several factor affected genetic transformation of Tall Fescue(Festuca arundinacea Schreb),and finally established genetic transformation system.Following the experiment results,it was confirmed the optimal transformation condition:callus were precultured on MS+0.5mg/L6-BA+0.5mg/LNAA for 2-3d;the A.Tumefaceins cells which OD600 comes up to about 1.0 or so were collected by centrifugation and resuspended in same volume of liquid MS containing 100umol/L AS,then it was made the OD600 came up to 0.6-0.7 or so。The callus were transferred into A.Tumefaceins suspension as described above for 10-15min.After the A.Tumefaceins cells on the surface of callus were removed by filter paper,the explants were co-cultured on MS+5.0mg/L2 , 4-D+0.1mg/L6-BA+100umol/LAS with a filter paper for 3-4d for whole night illumination.When the co-culture was over,the explants were washed three times in liquid MS,then washed in liquid MS containing 500mg/L Cef for 2 hours.After removing the liquid on the surface of the explants,the explants were transferred to MS+5.0mg/L2, 4-D+0.1mg/L6-BA+30mg/LCef to de-culture for 2-3d.After that,the explants were transferred to MS+5.0mg/L2, 4-D+0.1mg/L6-BA+20mg/LG418+100mg/LCef to induce resistant calli.The explants were transferred to MS+1.0mg/L6-BA+30mg/LG418+100mg/LCef when the shoots formed.Regenerated shoots were inoculated into l/2MS+0.5mg/LNAA+30mg/LG418 for rooting salection.Using the optimized transformation system,gene were introduced into Festuca arundinacea Schreb,and 2 Kan-resistant plantlets were obtained. |
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