Study On Protoplast Isolation Of Lilium Pumilum DC.

Lilium pumilum DC.is a perennial plant of Lilium in Liliaceae,which has the functions of ornamental,edible and medicinal,and it is valuable in Liliaceae breeding for its ability to resist cold and drought and salinity.However,the incompatibility between several different varieties greatly hinders the utilization efficiency of Lilium pumilum DC.gene resources.Protoplasts are living cells,which lack cell wall and have a complete set of plant genetic genes.Protoplast fusion is one of plant breeding methods,and protoplasts provide a good experimental platform for basic biological research on plant cell level and molecular level.The research of Lilium pumilum DC.protoplasts have important significance on genetic resources and basic research.In order to obtain high-yield and high quality protoplasts,isolation and purification conditions of protoplasts were optimized by using the callus from bulb of Lilium pumilum DC.The effects of concentration of enzyme and mannitol on the protoplasts separation of Lilium pumilum DC were studied by orthogonal test with L16?34?.The effects of enzymolysis time,purification method,pretreatment method and culture conditions on the separation and culture of Lilium pumilum DC protoplasts were studied by single factor test.Establishing a foundation for a high-efficiency and high-yield protoplast isolation and culture system,and supporting the efficient utilization of germplasm resources and theoretical research of Lilium pumilum DC.The specific research results are as follows:1.The optimal enzymolysis time was determined.Under the condition of this experiment,the enzymolysis time had a significant effect on the protoplast difference and activity,the optimal enzymolysis time was 6 hours,the protoplast yield was 3.46×10⁵ cells/mL,and the activity was 39.17%.The conditions of enzymatic concentration were 0.015 g/mL cellulase concentration+0.005g/mL pectinase concentration+0.65mol/L mannitol concentration,then adjusted pH to 5.7,the enzymatic hydrolysis was selected shaken in the dark,25?and 50 rpm shaker.2.In the three factors and four levels of L₁₆?3⁴?orthogonal experiments were designed to study different cellulase concentrations?0.00 g/mL,0.01 g/mL,0.02 g/mL and 0.03 g/mL?,pectinase concentrations?0.002 g/mL,0.004 g/mL,0.006 g/mL and0.008 g/mL?and mannitol concentrations?0.09 g/mL,0.11 g/mL,0.13 g/mL?0.15g/mL?.The effect of on the protoplast yield and activity of Lilium pumilum DC.Enzymolysis under the conditions of 3 hours,6 hours and 9 hours respectively.The range analysis of orthogonal test results showed the main and secondary factors of protoplast yield were cellulase concentration>pectinase concentration>mannitol concentration,and the main and secondary factors of protoplast activity were mannitol concentration>cellulase concentration>pectinase concentration.Further analysis of the orthogonal test results at different levels such as cellulase,pectinase and mannitol.Considering the requirements of protoplasts for yield and activity,we got the best combination of time-saving was cellulase concentration 0.03g/mL+pectinase concentration 0.006 g/mL+mannitol concentration 0.11 g/mL in 3hours,the best combination of the highest yield and activity was cellulase concentration0.02 g/mL,pectinase concentration 0.006 g/mL,mannitol concentration 0.11 g/mL in 6hours,and the best combination of the lowest cost was cellulase concentration 0.01g/mL,pectinase concentration 0.004 g/mL,mannitol concentration 0.13 g/mL in 9hours.3.Under the condition of this experiment,the best pretreatment was dark treatment for Lilium pumilum DC,which can significantly improve the yield and activity of protoplasts.The protoplasts yield of Lilium pumilum DC increased from 5.17×10⁵cells/mL to 9.67×10⁵ cells/mL,and the activity of protoplasts increased from 21.21%to 76.54%.The conditions of enzymatic hydrolysis were 0.02 g/mL cellulase concentration,0.008 g/mL pectinase concentration,0.11 g/mL mannitol concentration.Adjusted the pH of enzyme mixture to 5.7.The enzymatic hydrolysis was carried out for 6 hours in a dark,25?and 50 rpm shaking bed.4.Under the condition of this experiment,the optimal centrifugation condition was800¹000 r/min,centrifugation for 10min,protoplast yield was 9.67¹0×10⁵ pieces/mL,activity was 33.87⁴2.24%,enzymatic hydrolysis condition was 0.02 g/mL cellulase concentration,0.008 g/mL pectinase concentration,0.11 g/mL mannitol concentration.Adjusted the pH of enzyme mixture to 5.7.The enzymatic hydrolysis was carried out for6 hours in a dark,25?and 50rpm shaking bed.