| Kentucky bluegrass (Poa Pratensis L.) is a typical cool season perennial grass with facultative apomictic mode of reproduction, commonly used of for forage and turf in temperate. It is widely maintained on parks, golf courses, playing fields and public grounds, for the characters of beautiful color, high shoot density, cold tolerance and better shear-bear. Nevertheless it is sensitive to drouhgt and heat stress, and is easily aggrieved from diseases and insecst. Due to its facultative apomictic nature of reproduction,genetie improvement by conventional breeding methods has been difficult and genetic manipulation for desirable traits may be used as an alternative method in the improvement of this specis.Technical improvements in plant cellular and genetic engineering , such as genetic transformation, provide a promising approach for breeding new tolerant cultivars of this species.The experiment used three species Midnight II , Nuglade and Rugby II of Poa Pratensis L.as explants, and set up callus regeneration system,base on this, we set up the system of protoplast culture and somatic hybridization.1. In order to establish the basis for protoplast culture and cell fusion of Poa pratensis L., the plant regeneration was studied using the mature seeds of MidnightII,Nuglade and Rugby II as explants. Results indicate that:(1) The optimal callus inducement medium of Midnight II was MS+1mg/L 2,4-D + 0.1mg/L 6-BA + 3% sucrose + 0.7% agar and the callus inducement rate was 52.3%; the best medium of subculture was MS + 1mg/L 2,4-D + 0.3mg/L 6-BA + 3% sucrose + 0.7% agar; the best medium of differentiation was MS + 0.5mg/L NAA + 1mg/L 6-BA + 3% sucrose + 0.6% agar and the callus differentiation rate was 57.5%.(2) The best callus inducement medium of Nuglade was MB5 + 3mg/L 2,4-D + 0.5mg/L 6-BA + 3% sucrose + 0.7% agar; the callus inducement rate was 75%; the optimal medium of subculture was MS+3mg/L 2,4-D+0.5mg/L 6-BA+3% sucrose + 0.7% agar; the optimal medium of differentiation was MS+3mg/L 2,4-D+0.5mg/L 6-BA+3% sucrose + 0.6% agar and the callus differentiation rate was 55%.(3) The optimal callus inducement medium and medium of subculture of Rugby II was MS + 3mg/L 2,4-D + 0.1mg/L 6-BA + 3% sucrose + 0.7% agar; the callus inducement rate was 80.5%; the best medium of differentiation was MS + 0.5mg/L NAA +2mg/L 6-BA + 3% sucrose + 0.6% agar and the callus differentiation rate was 52.5%.2. The friable calli induced from mature seeds of Midnight II,Nuglade and Rugby II (Poa Pratensis L.) were used for protoplast preparation. The protoplasts were isolated through enzyme digestion. The plant regeneration had been established by protoplasts culture. The effects of different composition of enzyme, time of enzyme digestion, calli age and osmoticum on protoplast isolation were studied. The results showed that:(1) Highter yields and viabilities of protoplasts were obtained from embryogenic calli 8 to 10d with 1.0% Cellulase OnozukaR-10, 1.0% Macerozyme R-10, 0.3% Driselase, 0.5% PectolaseY-23 and 14～16h of enzyme digestion.(2) The first divisions occurred in 3 days and small cell clusters could be seen after 2 to 3 weeks in the culture. At this moment , the addition of the protoplast culture medium with 0.1mol/ L～0.2mol/L mannitol 2 or 3 time was needed for the continuous protoplasts division to form calli.(3) The plant regeneration system from protoplasts of Midnight II and Nuglade were developed successfully. Microcalli visiable to the nakes eyes were obtained of Rugby II but they failed to develop any further.3. Fusion of Midnight II prtoplasts and Nuglade prtoplasts of Poa Pratensis L.by means of ployethylene glycol (PEG) method has been resulted to callus. Study the effects the viability of protoplasts of parents with pretreatment and the optimal fusion condition. The results showed that:(1) The protoplast of Nuglade was tread by IOA with different concentrations, the best of this experiment was shown as follows: 5mmol/ mL IOA was used to kill the protoplast of Nuglade by treating10 minutes.(2) The protoplast of Midnight II was treated by R-6G with different concentrations,the best of this experiment was shown as follows: 40μg/ mL R-6G was used to kill the protoplast of Midnight II by treating 5 minutes.(3) Hybrid cells of Poa Pratensis L.were obtained using protoplast fusion by PEG method,The best fusion condition 35%PEG(6000)can obtain the hybrid cell,the fusion rate is 9.8%. |
