Pea is the second most important grain legume in the world (1). It is an annual plant belonging to the Leguminosae and its seeds play a crucial role as a food source due their high content of proteins, starch, fibers, and minerals (2). Despite the great commercial interests in pea, the molecular research on this important crop is limited due to lack of robust and optimized transformation methods (3). The generation of transgenic lines is relatively expensive and time-consuming and in the case of pea, particularly challenging due an inefficient plant regeneration system (3). Transient transformation systems, like the transfection of protoplasts, serve as quick and efficient alternative methods to study a wide range of cellular and molecular processes in plants (4).
Protoplasts are plant cells that have their cell walls removed by cell wall digesting enzymes. They often maintain the general features of intact cells, providing a unique opportunity to study cellular and molecular events in plants (5). Additionally, protoplasts can be easily transformed with the introduction of a foreign DNA or protein, allowing researchers to quickly observe in vivo gene expression, protein sub-cellular localization, DNA-protein interaction, or protein-protein interaction (4, 5). More recently, protoplasts have been used as the source material for single-cell genomics in plants (6, 7), highlighting the versatility and usefulness of plant protoplasts in modern plant research.
The utility of plant protoplasts as a method for crop research is exemplified by the extensive development of protocols for the isolation and transformation of protoplasts in many crop species like rice (8), maize (9), cassava (10), sunflower (11), including legumes like soybean (12), common bean (13), and chickpea (14). The inclusion of a protocol for protoplast isolation and transfection to the transformation toolbox in these species is further aiding the studies of biochemical and molecular processes in these important crops (5, 15–17). In this sense, the development of a reliable and efficient method for the isolation and transfection in pea can create opportunities to further aid studies that aim to improve this important crop.
To address this need, we aimed to develop and optimize and efficient method for the isolation and transient transformation of pea mesophyll protoplasts. By optimizing the distinct steps, we developed a protocol that yield large amounts of viable leaf protoplasts with a high transformation efficiency. Additionally, we showed that this method is suitable to evaluate the sub-cellular localization of proteins in pea cells. We hope that this protocol will further enhance pea research community by providing a cheap and quick transient transformation system in this important crop.