An efficient method for the isolation and transfection of Pea (Pisum sativum) mesophyll protoplasts

Abstract

Background Pea (Pisum sativum L.) is an important crop with a wide range of uses and benefits, including food source, forage, and potential use as a biofuel crop. Developing methods for the transformation in this species can provide opportunities to improve this important crop. The transient transformation of protoplasts serves as a method for plant engineering, providing a quick and cheap way to study gene and protein function in plants. Developing a protocol for protoplast transfection is especially important for species like pea, where stable transformation methods are laborious and inefficient. Results In this report, we established and optimized an efficient protoplast isolation method for pea mesophyll cells. Using the tape-sandwich method we were able to isolate an average of 6.7x10⁵ protoplasts per pea leaflet. Additionally, we determined the optimal conditions for the PEG4000 mediated transfection to achieve about 40% to 60% transfection efficiency in pea mesophyll protoplasts. Finally, we showed that with the protoplast transfection system, we were able to quickly assess protein subcellular localization in pea. Conclusion With the inclusion of this efficient and fast protoplast isolation and transfection system to the pea transformation toolbox, we hope to further aid many molecular and biochemical studies in this important legume crop.