| Kentucky bluegrass (Poa Pratensis L.) is a typical cool season perennial grass commonly used in temperate and one of good materials for forage and turf. It is widely maintained lawn , sports ground, playing fields and public grounds, for the characters of strong resistance, beautiful quality of lawn, long period of green, better shear-bear, and trampling tolerance. 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,genetic 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 somatic cell hybridization, such as genetic transformation, provide a promising approach for breeding new tolerant cultivars of this species, good protoplast culture system is fundamental and restriction point. The experiment based on previous research used three species Midnight II , Nuglade and Rugby II of Poa Pratensis L. as explants, further studied the factors of protoplast culture of Kentucky bluegrass and taining procedures and related technical aspects. Explored the relationship between physiological characteristics of protoplast bulk materials of Kentucky bluegrass and regeneration of protoplast division.1. The callus induced and grow was studied using the mature seeds of Midnight II,Nuglade and Rugby II as explants. Focusing on waterlogging and browning status occurred during callus culture of Midnight II and Nuglade,and adjustment the culture conditions. As follows:(1) The optimal callus inducement medium of Midnight II at the initial stage was MS+1 mg·L-1 2,4-D + 0.3 mg·L-1 6-BA + 30 g·L-1 sucrose + 7 g·L-1agar, pH 5.8;Show much status of water-soaked after 5 generation, after adjustment the medium (MS+1mg·L-1 2,4-D+0.2 mg·L-1 6-BA+30 g·L-1 sucrose+7.5 g·L-1 agar,pH 6.0),the status of callus greatly improved.(2) The best callus inducement medium of Nuglade: 7 generation at the initial stage used basic MS medium(MS+30 g·L-1sucrose+7g·L-1 agar +3 mg·L-1 2,4-D+0.5 mg·L-1 6-BA,pH 5.8),MS medium of little change in some component started to use from the 8 th generation(add 0.4 mg·L-1 6-BA,7.5 g·L-1 agar, pH 6.0), others same as basic MS medium),can get callus of good quality.(3) The optimal callus inducement medium of Rugby II was MS+3 mg·L-1 2,4-D+0.1 mg·L-1 6-BA+30 g·L-1 sucrose+7 g·L-1 agar,pH 5.8. 2. The quality callus induced from Midnight II,Nuglade and Rugby II (Poa Pratensis L.) were used for protoplast preparation. The protoplasts were isolated through enzyme digestion. The effects of different penetrant and osmotic pressure on protoplast dissociate of Kentucky bluegrass, and effects of different culture methods on growth of protoplast division were focusing studied. The results showed that:(1) Embryogenic callus of Midnight II and Rugby II cultured 8 to 10d with 1.0% Cellulase OnozukaR-10, 1.0% Macerozyme R-10, 0.3% Driselase, 0.3% PectolaseY-23, and with Mannitol as penetrant. Highter yields and viabilities of protoplasts were obtained from embryogenic callus of Midnight II and Rugby II when the concentration is 13%, and from Nuglade when the concentration is11%.(2) Higher division frequency were obtained when protoplasts of three species cultured by Solid-liquid double mode culture method than by Shallow liquid culture. Division frequency of protoplasts of Midnight II and Rugby II will come to 42.8% and 29.4% respectively, which is the highest, when pH get to 5.8, that of protoplasts of Nuglade come to 50.1% which is the highest when pH get to 6.0. 3. Determine the physiological and biochemical of protoplasts materials of Midnight II,Nuglade and Rugby II (Poa Pratensis L.) which were subculture respectively. Discussed the relationship between these indicators and division,growth of protoplast regenerative cells. The results showed that:(1) The 8th to 10th day after replacement of fresh medium, soluble protein content of the callus of three species get to the maximum. There is tiny difference between the callus of Nuglade and Midnight II in soluble protein content, but that of Rugby II were the lowest.(2) Measured free amino acid amount, SOD and POX activity of various varieties callus which was fter 8 day changed new medium.The results that the callus of Rugby II are lower than the other species, those index of Nuglade is highest. That show there is a positive correlation between physiological state of starting materials and regeneration ability of protoplast. |
PDF Full Text Request