Isolation Of Protoplasts From Mesophyll Cells Of Sophora Japonica

Sophora japonica,originated in the north of China,is a kind of tree species which integrates edible,medicinal,timber,urban landscape greening and ornamental.Plant protoplasts are bare living cells encapsulated only by the protoplasmic membranes.They are widely used in basic life sciences and crop breeding improvement because they have no cytoderm obstruction and have the characteristics of cell totipotency,independence and high uniformity among individuals.At present,there is no research report on the isolation of protoplasts from Sophora japonica.This study established a protoplast isolation system for the mesophyll cells of the Sophora japonica by isolating the protoplast isolation process,the optimal isolation period of the leaves,the optimization of the concentration of the enzyme solution and the isolation conditions.To provide a basis for further research on genetics and other molecular biology in Sophora japonica.The main results are as follows:1.Determination of protoplast isolation process and optimization isolation period of leavesBy referring to the isolation method of different plant mesophyll protoplasts,the protoplast isolation was carried out by using the leaves of Sophora japonica,and the isolation process was improved according to the isolation effect.Finally,the suitable methods for protoplast isolation of Sophora japonica mesophyll cells were determined,including enzyme preparation,protoplast isolation and purification,protoplast counting and activity determination.The seedlings of Sophora japonica were sown and cultured in the greenhouse,and the leaves of different leaf ages were selected for isolation test.The results showed that the protoplasts yield and viability of the leaves isolated from 10 to 15 days were higher,and there was no significant difference in the isolation effect among these leaf ages.The protoplast yield of Sophora japonica decreased significantly when the leaf age was more than 15 days,and single cells with cytoderm could not be degraded.Therefore,10-15days leaf age was the best isolation period for Sophora japonica.2.Optimum concentration of enzyme solution combinationIsolation experiments were carried out in greenhouses in September 2018,March 2019and April 2019,respectively.Based on cellulase R-10（1.0%,1.5%,2.0%and 2.5%）,pectolyase Y-23（0.2%,0.3%,0.4%and 0.5%）and macerozyme R-10（0.8%,1.0%,1.2%and 1.4%）,orthogonal experiments were carried out on L₁₆（4⁵）.The final concentration of the optimized enzyme solution was determined as follows:September 2018,2.0%cellulase R-10+0.5%pectolyase Y-23+1.0%macerozyme R-10;March 2019,1.5%cellulase R-10+0.3%pectolyase Y-23+0.8%macerozyme R-10;April 2019,1.5%cellulase R-10+0.4%pectolyase Y-23+1.4%macerozyme R-10.3.Selection of conditions for protoplast isolationThe single factor experiment was carried out on the osmotic pressure of enzyme solution,enzymatic hydrolysis time and temperature,pH of enzyme solution and material pretreatment,the isolation conditions of mesophyll protoplasts of Sophora japonica in different experimental periods were determined as follows:In September 2018,after plasmolysis was carried out for 1 hour in CPW containing 0.5M mannitol,placed in 2.0%cellulase R-10+0.5%pectolyase Y-23+1.0%macerozyme R-10,0.4mol/L mannitol,pH5.7 enzyme solution,enzymatic hydrolysis at 26℃for 2.5h.The protoplast yield and viability were 8.02×10⁶gFW^-1 and 84.1%,respectively.In March 2019,after plasmolysis in the CPW wash solution containing 0.5M mannitol for 1 hour,1.5%cellulase R-10+0.3%pectolyase Y-23+0.8%macerozyme R-10 was placed,0.4mol/L mannitol,pH 5.7 enzyme solution,enzymatic hydrolysis at 26℃for 3h,protoplast yield and viability were8.48×10⁶gFW^-1 and 84.4%,respectively.In April 2019,after 0.5M mannitol osmotic plasmolysis treatment for 1h,1.5%cellulase R-10+0.4%pectolyase Y-23+1.4%macerozyme R-10,0.4 mol/L was placed,an enzyme solution with a pH of 5.7,was hydrolyzed at 26°C for 3h,and its protoplast yield and viability were 8.37×10⁶ gFW^-1 and83.1%,respectively.In April 2019,the protoplast yield and were 8.37×10⁶gFW^-1 and 83.1%,respectively.4.The difference of the enzymes concentration and isolation conditions of protoplast isolation of Sophora japonica in different growing seasons were due to differences in temperature and light conditions during the test period,which resulted in different plant cell physiological conditions and plant cell wall components.