Establishment of the cotyledon protoplast transient expression system of Ammopiptanthus mongolicus and subcellular localization of the AmDREB1 protein

Using young cotyledons of Ammopiptanthus mongolicus (Maxim. ex Kom.) Cheng f. seedlings as donor material, the technical system for the isolation, purification, and transient expression of the cotyledon protoplasts was studied. Results showed that the optimal enzyme solution for the protoplast isolation was CPW solution + 3.0% cellulase R-10 + 0.5% macerozyme R-10 + 0.3% hemicellulose + 9.0% mannitol (pH5.8), and the optimal enzymolysis conditions were gentle shaking of the enzyme solution containing cotyledon tissues at 40 r/min in the dark for 14 h at room temperature. Using the W5 solution as a washing solution, the enzymatic hydrolysate was diluted and filtered, with the filtrate then centrifuged at 4℃ and 700 r/min for 5 min. The purified protoplast yield was approximately 2.50×10⁶ cells/g and the protoplast viability reached 90%. Using the PEG-mediated method, the protoplasts were successfully transformed with plant transient expression vector pBI-GFP and the transformation efficiency was 50.8%. Moreover, using the established protoplast transient expression system, a dehydration-responsive transcription factor of A. mongolicus, namely AmDREB1, was found in the nucleus.