Standardization of Protocol for Maximizing Isolation Efficiency and Purity of Protoplasts and Lytic Vacuoles from Sugarcane Stalk

Understanding the biochemical and structural properties of sugarcane stalk lytic vacuoles is pivotal to unravel their role in plant metabolism and cellular homeostasis. The current study focuses on the optimization of the protocol for the isolation of protoplasts and vacuoles from matured sugarcane stalk parenchyma cells. One of the prerequisites for successful vacuole isolation is to ensure that the high-quality protoplasts get released through strategic enzymatic treatment-induced cellular breakdown. The optimal conditions, utilizing an enzyme concentration ‘D’ (of 2.5% Cellulase R-10 and 0.6% Macerozyme R-10), yielded 11.36 × 10⁴ protoplasts/g of fresh weight (FW), and its maximum viability was assessed with Evan’s blue staining. Thus, the study found 0.4 M mannitol concentration and 5 h of enzymolysis at 28 °C to be efficient conditions for maximum protoplasts’ yield. Further, the vacuole purification process executed via Ficoll density gradient-based centrifugation method produced intact vacuoles with high tonoplast integrity. The outcomes were validated by visualizing the vacuole fraction with neutral red staining specific for acidic compartments and using tonoplast-specific fluorescence dye MDY-64 under fluorescence microscope. Further, α-mannosidase, a vacuole-specific marker activity assay, revealed a remarkable 43-fold enrichment, thus confirming the purity of the vacuole. The enzyme activity assays with vacuole fraction confirmed the existence of minimal cross-contamination. Simultaneously, the biochemical analysis outcomes revealed differential levels of sucrose, amino acids, reducing sugars, and proteins between vacuoles and protoplasts, thus concluding their specialized metabolic roles. The results from the SDS-PAGE analysis demonstrated distinct protein profiles between the protoplasts and vacuole fractions. This optimized protoplast and vacuole isolation protocol resulted in enhanced yield, improved viability, and increased purity.