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

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. annuumMLO 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.

Keywords: Agrobacterium tumefaciens; CRISPR/Cas9; CaMLO2; Capsicum annuum CM334; Capsicum annuum Dempsey; pBAtC binary vector.

Figure 1

CaMLO2 sgRNA1 cloned pBAtC binary vector. (a) Description of a pBAtC binary vector. RB, right border; Ter, tetracycline; CAS9hc:NLS: HA, human-codon-optimized Cas9 with the nuclear localization signal and an HA epitope; 35S, cauliflower mosaic virus (CaMV) 35S promoter; AtU6, Arabidopsis thaliana U6 promoter; Aar1, sgRNA cloning sites with two Aar1; sgRNA-s, the guide RNA-scaffold for Cas9; Nos, nos promoter; Bas-R, BASTA resistance gene; LB, left border; (b) Description of a pBAtC:CaMLO2–sgRNA1 binary vector. Green, the sgRNA1 sequence of CaMLO2 target locus; Blue, overhangs digested by Aar1 sites.

Figure 2

Comparison of callus induction ratios among the three Agrobacterium strains in Agrobacterium-mediated transformation of two pepper types. (a) Callus induction ratios of cv. Dempsey by three Agrobacterium strains (AGL1, n = 7 biological replicates (BR); EHA101, n = 8 BR; GV3101, n = 8 BR). Total number of explants was 265. Results are presented as mean ± SD; (b) Callus induction ratios of cv. CM334 by three Agrobacterium strains (AGL1, n = 9 BR; EHA101, n = 8 BR; GV3101, n = 8 BR). Total number of explants was 328. Results are presented as mean ± SD. Callus size ≥2.5 mm. ***, p < 0.001 based on analysis of variance (ANOVA).

Figure 3

Effects of PPT on selection of callus of explants transformed by three different strains of Agrobacterium. (a–c) Examination of a suitable concentration of PPT for cv. Dempsey leaf explants. Cultivar Dempsey leaf explants were placed on CIM without PPT (0 mg/mL) (a), and with PPT at 3 mg/L (b) and 5 mg/L (c); (d–f) Examination of a suitable concentration of PPT for cv. CM334 leaf explants. Cultivar CM334 leaf explants on CIM without PPT (0 mg/mL) (d), and with PPT at 0.5 mg/L (e) and 1 mg/L (f) are shown. All explants on the indicated PPT medium were examined for 10 days. Scale bars = 1 cm. PPT, phosphinothricin; CIM, callus induction medium.

Figure 4

PCR analyses of PPT-selected and transformed calli of cv. Dempsey and CM334. (a) Calli in cv. Dempsey induced by Agrobacterium strain AGL1, EHA101, and GV3101, respectively; (b) calli in cv. CM334 induced by Agrobacterium strain AGL1, EHA101, and GV3101, respectively. M, 100-bp DNA ladder; P, pBAtC:CaMLO2–sgRNA1 binary vector; N, non-transformed pepper callus. The indicated numbers (1 to 14) are the PPT-selected and transformed calli in cv. Dempsey and CM334.

Figure 5

Comparison of indel frequencies of selected pepper calli following Agrobacterium-mediated transformation. (a) Indel frequencies of selected cv. Dempsey calli (Control, n = 6; AGL1, n = 6; EHA101, n = 14; GV3101, n = 15); (b) Indel frequencies of selected cv. CM334 calli (Control, n = 7; AGL1, n = 41; EHA101, n = 22; GV3101, n = 32). Callus size ≥2.5 mm. The indel frequency (%) was calculated by dividing the number of sequencing reads containing indel at the target site by the number of total sequencing reads. *, p < 0.05; **, p < 0.01 based on analysis of variance (ANOVA); (c) Indel patterns of selected calli in both cv. Dempsey and CM334. Red, PAM sequence; Blue, Cas9 target sequence; Red dashed lines (-), a deleted nucleotide.