Agrobacterium-Mediated Capsicum annuum Gene Editing in Two Cultivars, Hot Pepper CM334 and Bell Pepper Dempsey.

Sung-Il Park,Hyeran Kim,Hyun-Bin Kim,Hyun-Ji Jeon

doi:10.3390/ijms22083921

Sung-Il Park, Hyeran Kim + Show 2 more

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https://doi.org/10.3390/ijms22083921

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Abstract

Peppers (Capsicum annuum L.) are the most widespread and cultivated species of Solanaceae in subtropical and temperate countries. These vegetables are economically attractive worldwide. Although whole-genome sequences of peppers and genome-editing tools are currently available, the precision editing of peppers is still in its infancy because of the lack of a stable pepper transformation method. Here, we employed three Agrobacterium tumefaciens strains—AGL1, EHA101, and GV3101—to investigate which Agrobacterium strain could be used for pepper transformation. Hot pepper CM334 and bell pepper Dempsey were chosen in this study. Agrobacterium tumefaciens GV3101 induced the highest number of calli in cv. Dempsey. All three strains generated similar numbers of calli for cv. CM334. We optimized a suitable concentration of phosphinothricin (PPT) to select a CRISPR/Cas9 binary vector (pBAtC) for both pepper types. Finally, we screened transformed calli for PPT resistance (1 and 5 mg/L PPT for cv. CM334 and Dempsey, respectively). These selected calli showed different indel frequencies from the non-transformed calli. However, the primary indel pattern was consistent with a 1-bp deletion at the target locus of the C. annuum MLO gene (CaMLO2). These results demonstrate the different sensitivity between cv. CM334 and Dempsey to A. tumefaciens-mediated callus induction, and a differential selection pressure of PPT via pBAtC binary vector.

Highlights

Owing to global climate change and the increase in participation of the older adult population in agriculture, facility agriculture is gaining importance
We examined the proper selection pressure by PPT on calli induced by pBAtC in transgenic hot pepper and bell pepper
CM334 leaves were completely brown without induced callus (Figure 3f). These results showed that hot pepper CM334 was more sensitive to PPT than bell pepper Dempsey (Figure 3)

Summary

Introduction

Owing to global climate change and the increase in participation of the older adult population in agriculture, facility agriculture is gaining importance. Horticulture facilities are shifting to conventional plant–microbe interactions, resulting in several newly emerging plant diseases, such as powdery mildew infection in tomato and pepper [1]. Symptoms of the infection can be macroscopically observed as white-covered epithelial mycelia of powdery mildew pathogens on leaves and fruits [2]. Some biotrophic plant pathogens, including powdery mildew fungi, display properties that pose challenges to conventional infection prevention methods, such as protectant fungicides, so resistant cultivars are needed. Powdery mildew resistance conferred by mildew resistance locus O (MLO) genes has been reported in various plant species, such as barley, Arabidopsis, and wheat [3,4,5]

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