Creating saponin‐free yellow pea seeds by <i>CRISPR/Cas9</i>‐enabled mutagenesis on β‐amyrin synthase

Abstract

Dry pea (<i>Pisum sativum</i>) seeds are valuable sources of plant protein, dietary fiber, and starch, but their uses in food products are restricted to some extent due to several off-flavor compounds. Saponins are glycosylated triterpenoids and are a major source of bitter, astringent, and metallic off-flavors in pea products. <i>β-amyrin synthase</i> (<i>BAS</i>) is the entry point enzyme for saponin biosynthesis in pea and therefore is an ideal target for knock-out using CRISPR/Cas9 genome editing to produce saponin deficient pea varieties. Here, in an elite yellow pea cultivar (CDC Inca), LC/MS analysis identified embryo tissue, not seed coat, as the main location of saponin storage in pea seeds. Differential expression analysis determined that <i>PsBAS1</i> was preferentially expressed in embryo tissue relative to seed coat and was selected for CRISPR/Cas9 genome editing. The efficiency of CRISPR/Cas9 genome editing of <i>PsBAS1</i> was systematically optimized in pea hairy roots. From these optimization procedures, the AtU6-26 promoter was found to be superior to the CaMV35S promoter for gRNA expression, and the use of 37°C was determined to increase the efficiency of CRISPR/Cas9 genome editing. These promoter and culture conditions were then applied to stable transformations. As a result, a bi-allelic mutation (deletion and inversion mutations) was generated in the <i>PsBAS1</i> coding sequence in a T₁ plant, and the segregated <i>psbas1</i> plants from the T₂ population showed a 99.8% reduction of saponins in their seeds. Interestingly, a small but statistically significant increase (~12%) in protein content with a slight decrease (~5%) in starch content was observed in the <i>psbas1</i> mutants under phytotron growth conditions. This work demonstrated that flavor-improved traits can be readily introduced in any pea cultivar of interest using CRISPR/Cas9. Further field trials and sensory tests for improved flavor are necessary to assess the practical implications of the saponin-free pea seeds in food applications.