Optimized protoplast isolation and PEG-mediated transient gene expression for high-altitude medicinal plant Picrorhiza kurrooa
摘要
Protoplast isolation and transient transformation has become indispensable tool for studying the subcellular localisation of proteins, identification of transcription factors, characterization of gene function, protein transport mechanisms and conducting single-cell omics research. Here, we optimized the protocol for isolation of protoplasts from Picrorhiza kurrooa, a Himalayan medicinal herb, followed by the transient transformation of PkWOX11, a member of the WOX (WUSCHEL-related homeobox) gene family. To achieve maximum yield and viability of protoplast, different enzymatic treatments and physical factors were explored. Enzymatic concentrations of cellulase, macerozyme and incubation time were optimized to isolate protoplast from micro propagated P. kurrooa. The effects of osmotic stabilizer, such as mannitol on protoplast integrity were also investigated. We observed, that 2% cellulase, 0.6% macerozyme followed by 0.6 M of mannitol and 4 h of dark incubation provides a maximum yield of 2.9 × 107 protoplasts per gram of fresh weight with 88.04% viability. The isolated protoplasts were visualised using microscopy and the viability was assessed to confirm their potential for use in plant genetic transformation. The optimisation of transient transformation resulting in a transformation efficiency of 27.33% using 40% PEG4000. Subcellular localization of the PkWOX11-GFP fusion protein in nucleus confirms the efficiency and reliability of this transient transformation system. Conclusively, our work provides an efficient platform for isolation of protoplast and transient transformation which can be harnessed for the generation of CRISPR ribonucleoprotein-mediated transgene-free genetically improved medicinal plants.