Developing an Optimized Protocol for Regeneration and Transformation in Pepper

Abstract

Capsicum annuum L. is extensively cultivated in subtropical and temperate regions globally, respectively, when grown in a medium with 8 holding significant economic importance. Despite the availability of genome sequences and editing tools, gene editing in peppers is limited by the lack of a stable regeneration and transformation method. This study assessed regeneration and transformation protocols in seven chili pepper varieties, including CM334, Zunla-1, Zhongjiao6 (ZJ6), 0818, 0819, 297, and 348, in order to enhance genetic improvement efforts. Several explants, media compositions, and hormonal combinations were systematically evaluated to optimize the in vitro regeneration process across different chili pepper varieties. The optimal concentrations for shoot formation, shoot elongation, and rooting in regeneration experiments were determined as 5 mg/L of 6-Benzylaminopurine (BAP) with 5 mg/L of silver nitrate (AgNO₃), 0.5 mg/L of Gibberellic acid (GA₃), and 1 mg/L of Indole-3-butyric acid (IBA), respectively. The highest regeneration rate of 41% was observed from CM334 cotyledon explants. Transformation optimization established 300 mg/L of cefotaxime for bacterial control, with a 72-h co-cultivation period at OD₆₀₀ = 0.1. This study optimizes the protocols for chili pepper regeneration and transformation, thereby contributing to genetic improvement efforts.

Keywords: Agrobacterium tumefaciens; Capsicum annuum; explants; in vitro culture.

Figure 1

Schematic diagram of the vectors of pGH, pGH-GRF-GIF, and pGH-rGRF-GIF, respectively (Modified from Feng et al. [28]). GFP, Green Fluorescent Protein; GRF4-GIF1, Growth Regulating Factor 4-Growth Interacting Factor 1; HygR, Hygromycin Resistance gene; LB, Left Border of T-DNA; NosT, Nopaline Synthase Terminator; polyA, Polyadenylic acid; RB, Right Border of T-DNA; 4×Ala, A sequence with four alanine residues.

Figure 2

Effect of plant growth regulators (PGRs) and explants on callus induction in different varieties. (a) effects of 5 mg/L of BAP + 1 mg/L of IAA + 2 mg/L of ZT (Callus Induction Medium I) on ZJ6, CM334, Zunla-1, 0819, 0818, 297, and 348; (b) effect of 5 mg/L of BAP + 1 mg/L of IAA + 2 mg/L of ZR (Callus Induction Medium II) on ZJ6, CM334, Zunla-1, 0819, 0818, 297, and 348; (c) effect of 6 mg/L of BAP + 2 mg/L of ZR + 1 mg/L of NAA (Callus Induction Medium III) on ZJ6, CM334, and Zunla-1; (d) effect of 2 mg/L of ZR + 1 mg/L of IAA on ZJ6, CM334 and Zunla-1 (Callus Induction Medium IV); (e) effect of 4 mg/L of ZR + 5 mg/L of BAP + 1 mg/L of IAA (Callus Induction Medium V) on ZJ6, CM334 and Zunla-1; (f) effect of 5 mg/L of BAP + 1 mg/L of IAA (Callus Induction Medium VI) on ZJ6, CM334 and Zunla-1; and explants (cotyledons, hypocotyls, and roots) on callus induction. The ANOVA statistical analysis indicates that there are significant differences in the different letters (A, B, C, D, and E) among the varieties of explants (p < 0.05), whereas no significant differences are observed in the same letters among the varieties of explants in treatments.

Figure 2

Effect of plant growth regulators (PGRs) and explants on callus induction in different varieties. (a) effects of 5 mg/L of BAP + 1 mg/L of IAA + 2 mg/L of ZT (Callus Induction Medium I) on ZJ6, CM334, Zunla-1, 0819, 0818, 297, and 348; (b) effect of 5 mg/L of BAP + 1 mg/L of IAA + 2 mg/L of ZR (Callus Induction Medium II) on ZJ6, CM334, Zunla-1, 0819, 0818, 297, and 348; (c) effect of 6 mg/L of BAP + 2 mg/L of ZR + 1 mg/L of NAA (Callus Induction Medium III) on ZJ6, CM334, and Zunla-1; (d) effect of 2 mg/L of ZR + 1 mg/L of IAA on ZJ6, CM334 and Zunla-1 (Callus Induction Medium IV); (e) effect of 4 mg/L of ZR + 5 mg/L of BAP + 1 mg/L of IAA (Callus Induction Medium V) on ZJ6, CM334 and Zunla-1; (f) effect of 5 mg/L of BAP + 1 mg/L of IAA (Callus Induction Medium VI) on ZJ6, CM334 and Zunla-1; and explants (cotyledons, hypocotyls, and roots) on callus induction. The ANOVA statistical analysis indicates that there are significant differences in the different letters (A, B, C, D, and E) among the varieties of explants (p < 0.05), whereas no significant differences are observed in the same letters among the varieties of explants in treatments.

Figure 3

Callus induction in various explants from different varieties. This Figure shows callus formation in CM334, Zunla-1, ZJ6, 297, and 0818 varieties, using cotyledons (a–e) and hypocotyls (f–j) as explants, respectively, in callus induction media after 36–48 days. The observed callus formations are displayed on these two specific mediums due to their optimal conditions for callus induction. Scale bars represent 2.1 cm in (a–j).

Figure 4

Effect of PGRs and explants on shoot formation in different varieties. (a) Effect of 5 mg/L of BAP + 1 mg/L of IAA (Shoot Formation Medium I) on ZJ6, CM334, Zunla-1, 0818, 0819, and 297; (b) Effect of 5 mg/L of BAP + 1 mg/L of IAA + 5 mg/L of AgNO₃ (Shoot Formation Medium II) on ZJ6, CM334, Zunla-1, 0818, 0819, and 297; (c) Effect of 6 mg/L of BAP + 1 mg/L of IAA + 5 mg/L of AgNO₃ (Shoot Formation Medium III) on ZJ6, CM334, Zunla-1, 0818, 0819, and 297; (d) Effect of 8 mg/L of BAP + 1 mg/L of IAA + 5 mg/L of AgNO₃ (Shoot Formation Medium IV) on ZJ6, CM334, Zunla-1, 0818, 0819, and 297; and explants (cotyledons and hypocotyls) on shoot formation. The ANOVA statistical analysis indicates that there are significant differences in the different letters (A, B, and C) among the varieties of explants (p < 0.05), whereas no significant differences are observed in the same letters among the varieties of explants in treatments.

Figure 5

Shows shoot formation in different varieties. Shoot formation initiation of the CM334 (a,b) occurs after 48–60 days; Zunla-1 (c,d) after 48–80 days; and ZJ6 (e) after 30–60 days, for varieties derived from cotyledons and hypocotyls, respectively, in the shoot formation medium (5 mg/L BAP + 1 mg/L IAA + 5 mg/L AgNO₃). Scale bars, 2.1 cm in (a,b), 4.3 cm (c), and 2.6 cm (d,e).

Figure 6

Effect of PGRs and explants on shoot elongation in different varieties. (a) The effect of 0.5 mg/L of GA₃; (b) effects of 1 mg/L of GA₃ on CM334 and Zunla-1 varieties using cotyledon and hypocotyl explants, respectively. This Figure represents the average shoot length for each variety. The ANOVA statistical analysis indicates that there are significant differences in the different letters (A and B) among the varieties of explants (p < 0.05), whereas no significant differences are observed in the same letters among the varieties of explants in treatments.

Figure 7

Effect of different PGRs on shoot elongation. This Figure illustrates the shoot lengths of the CM334 (a,b), and Zunla-1 (c,d) from cotyledon explants after of 60–90 days of culture in shoot elongation medium supplemented with 0.5 mg/L of GA₃. Scale bars, 2.6 cm in (a–d).

Figure 8

Root induction observed with the application of 1 mg/L of IBA. An amount of 1 mg/L of IBA and explants (cotyledons and hypocotyls) were used on root induction in CM334 and Zunla-1 varieties; The ANOVA statistical analysis indicates that there are significant differences in the different letters (A and B) among the varieties of explants (p < 0.05).

Figure 9

Different stages of the rooting and acclimatization and the regeneration rate. The described roots produced in rooting medium (a,b) after 20–30 days; Seedlings in tap water for one week (c); Seedlings in a Zip pot filled with peat moss soil (d,e) after 60–120 days. The regeneration rate of cotyledons and hypocotyls of the CM334 and Zunla-1 varieties, respectively (f). This Figure represents the regeneration rate on CM334 and Zunla-1 varieties using cotyledon and hypocotyl explants, respectively. ANOVA statistical analysis indicates that there are significant differences in the different letters (A and B) among the varieties of explants (p < 0.05). Scale bars, 2.6 cm in (a,b); 1.5 cm (c); 3.3 cm (d); and 6.3 cm (e).

Figure 10

Effect of PGRs and explants on shoot regeneration in the transformation of the different chili pepper varieties. (a) Effect of 5 mg/L of BAP on CM334 and Zunla-1 and explants (cotyledons, hypocotyls, and leaves); (b) Effect of 4 mg/L of TDZ on CM334 and Zunla-1 and explants (cotyledons, hypocotyls, and leaves); (c,d) Indicate the shoot formation of the CM334 from leaf and hypocotyl explants, respectively; The ANOVA statistical analysis reveals that different letters (A and B) represent significant differences among the varieties of explants (p < 0.05). In contrast, the same letters denote no significant differences among the varieties of explants in treatments.

Figure 11

The transformation efficiency. (a,b) These Figures represent the transformation efficiency of CM334 and Zunla-1 varieties using pGH (Blue), pGH-GRF-GIF (Red), and pGH-rGRF-GIF (Green) vectors, and leaf, cotyledon, and hypocotyl explants, respectively.

Figure 12

Detection of GFP and GGF in transformed plant tissues. (a) GFP signals on the leaves of non-transgenic plants (Negative control) using a Leica Microscope (Leica Microsystems Co., Ltd., Wetzlar, Germany). (b) GFP signals on the leaves of CM334 observed using a Leica Microscope. The red arrow refers to the GFP positive signal. (c) PCR analysis of GFP and GGF in CM334 leaves. M, Marker; GGF refers to GRF4 and GIF1; Samples 1–8 represent leaves of CM334; CK is the control, that is, ddH₂O is used as the PCR template; V3, pGH+rGRF-GIF. Scale bars, 1 mm in (a,b). The PCR fragments observed correspond to the expected sizes of 543 bp for GFP and 249 bp for GGF.