Preparation and Optimization of Pinus yunnanensis Protoplast

Purpose To establish the preparation and purification system of Pinus yunnanensis protoplasts, laying a foundation for the establishment of protoplasts culture, fusion and transient expression system of P. yunnanensis.

Methods Using P. yunnanensis seedlings as the experimental material, the effects of cellulose enzyme mass fraction, macerozyme mass fraction, mannitol concentration, vacuum treatment time, enzymatic hydrolysis conditions, and centrifugation conditions on the preparation of protoplast from P. yunnanensis needles were explored.

Results It was necessary to vacuum the young P. yunnanensis needles before enzymolysis, and after being vacuumized in the enzymolysis solution of 1.00% cellulase enzyme+0.80% segregation enzyme+0.3 mol/L mannitol for 10 minutes, the enzymolysis was carried out at 26 ℃, 130 r/min for four hours, and then centrifugation at 180 r/min for five minutes, the yield of protoplasts reached 5.98×10⁷ per/g, and the vitality was 72.22%. In addition, the protoplasts were prepared from the roots and stems of P. yunnanensis seedlings by using the optimized protoplast preparation program, and the yields were 4.35×10⁷ and 5.27×10⁷ per/g, respectively.

Conclusion In this study, the preparation and purification system of the protoplasts of P. yunnanensis is established, which will provide technical support for the studies of somatic hybridization and transient expression system of P. yunnanensis.