Efficient isolation and transformation of protoplasts in coconut endosperm and leaves for gene function studies

To establish a rapid and efficient method for isolating and transforming protoplasts in coconut endosperm, coconut liquid endosperm and young leaves were utilized as starting materials. By comparing factors such as enzyme digestion time, quantity, activity, cell and nucleus morphology, ploidy, and subcellular localization, it was discovered that the optimal conditions for achieving the highest protoplast yield and activity were 45 minutes of enzyme digestion for liquid endosperm and 2.5 hours for leaves. Furthermore, the endosperm cells were found to be 2−3 times larger than leaf cells and exhibited triploid characteristics. Moreover, DAPI staining and subcellular localization revealed the presence of significantly enlarged nuclei in protoplasts from coconut liquid endosperm, occupying nearly the entire cell. Using PEG-mediated transformation, the exogenous gene NAC96 was successfully overexpressed in protoplasts derived from both coconut leaves and endosperm. This study has established an efficient method for isolating and transforming protoplasts in coconut while identifying the distinct characteristics of protoplasts in liquid endosperm. These findings provide a crucial experimental platform for verifying coconut gene function and exploring gene expression regulation.