Efficient preparation of maritime pine (Pinus pinaster) protoplasts suitable for transgene expression analysis

Establishment of protoplast techniques for conifers is of crucial importance to forest biotechnology. Protoplasts can be used to regenerate whole trees and to study transcriptional regulation in woody plants by promoter analysis. Here we describe a technique for isolating protoplasts fromPinus pinaster shoots. Protoplast viability was determined by fluorescein diacetate staining. Transient gene expression was studied after electroporation of protoplasts with plasmid DNA containing a β-glucuronidase (GUS) reporter gene. The 35S promoter and a pine (cytosolic glutamine synthetase) promoter both were able to driveGUS expression, indicating applicability of conifer protoplasts as a model to study gene promoters using transient expression.