Establishment of an isolation and transfection protocol for Pisum sativum root protoplasts

Abstract

Pea (Pisum sativum) is a versatile and valuable crop with a wide array of applications, including its role as a food source, forage, and even a potential biofuel crop. Given the increasing interest in pea research, the development of an efficient method for transient transformation holds paramount importance. In this study, we introduce a comprehensive protocol for the isolation and transfection of pea primary root protoplasts. Through exploration of the protoplast isolation technique, enzyme compositions and concentrations, we have successfully achieved the isolation of pea root protoplasts, yielding 7.169E+03 protoplasts · root -1 after 6 hours of digestion. Moreover, we have identified conditions that sustain high protoplast viability for up to 16 hours. Furthermore, by optimizing the polyethylene glycol (PEG) calcium-mediated transformation process, we have achieved an average transfection efficiency of 15.48% by subjecting protoplasts to a 30% (w/v) PEG4000 solution for 5 minutes. In conclusion, we are confident that this protocol will find wide-ranging applications and prove highly beneficial for future research focused on the potential of pea crops for various purposes, enabling investigations such as gene expression and protein subcellular localization, and ultimately advancing our knowledge and utilization of this invaluable plant species.