Efficient Stable Genetic Transformation of Pea (Pisum sativum)

Pea (Pisum sativum) has emerged as a major protein source for meat substitutes due to its high nutritional value, low production costs, and short life cycle. The generation of elite pea cultivars can be achieved via genetic engineering and CRISPR-based gene editing. However, this approach has lagged due to the low efficiency, lack of reproducibility, and cultivar dependency of the reported pea transformation protocols. Due to the challenges in the genetic engineering of pea, we employed a transient expression approach to identify optimal conditions for gene expression with the expectation that these conditions would enhance the efficiency of stable transformation. The highest transient expression was achieved when the Agrobacterium suspension was used at 1.6 optical density, combined with a co-cultivation time of one hour. With the optimized conditions and a staggered antibiotic selection protocol, genetic perturbations, including ectopic and antisense expression and CRISPR/Cas9 editing of the flavanone 3-hydroxylase (F3H) gene, were performed in a purple-seeded pea line. We report an efficient, stable transformation protocol for pea with a mean efficiency of 2.9%. Greenhouse-adapted seed-bearing transgenic plants were obtained in eight months. The T2 transgenic lines were verified using PACE-PCR and RT-qPCR analysis, which confirmed the transgenic status of the plant and altered expression of the F3H gene demonstrating successful genetic engineering in Pisum sativum .