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.

Fruit weight is an important agronomic trait in pepper production and is closely related to yield. At present, many quantitative trait loci (QTL) related to fruit weight have been found in pepper; however, the genes affecting fruit weight remain unknown. We analyzed the fruit weight-related quantitative traits in an intraspecific Capsicum annuum cross between the cultivated species blocky-type pepper, cv. Qiemen, and the bird pepper accession, “129−1” (Capsicum annuum var. glatriusculum), which was the wild progenitor of C. annuum. Using the QTL-seq combined with the linkage-based QTL mapping approach, QTL detection was performed; and two major effects of QTL related to fruit weight, qFW2.1 and qFW3.1, were identified on chromosomes 2 and 3. The qFW2.1 maximum explained 12.28% of the phenotypic variance observed in two F₂ generations, with the maximum LOD value of 11.02, respectively; meanwhile, the qFW3.1 maximum explained 15.50% of the observed phenotypic variance in the two F₂ generations, with the maximum LOD value of 11.36, respectively. qFW2.1 was narrowed down to the 1.22 Mb region using homozygous recombinant screening from BC₂S₂ and BC₂S₃ populations, while qFW3.1 was narrowed down to the 4.61Mb region. According to the transcriptome results, a total of 47 and 86 differentially expressed genes (DEGs) in the candidate regions of qFW2.1 and qFW3.1 were identified. Further, 19 genes were selected for a qRT-PCR analysis based on sequence difference combined with the gene annotation. Finally, Capana02g002938 and Capana02g003021 are the most likely candidate genes for qFW2.1, and Capana03g000903 may be a candidate gene for qFW3.1. Taken together, our results identified and fine-mapped two major QTL for fruit weight in pepper that will facilitate marker-assistant breeding for the manipulation of yield in pepper.

Efficient regeneration of pepper (Capsicum annuum L.) is essential for genetic transformation and improving agronomic traits. However, the recalcitrant nature of pepper to in vitro manipulation has led to relatively low regeneration efficiencies. This study presents an optimized regeneration protocol for the Zunla-1 variety, focusing on genotype, explant type, and growth regulator combinations. Cotyledon and hypocotyl explants showed superior callus induction with 1 mg/L IAA or 1 nM CaREF1 peptide compared to 2,4-D treatments, while root explants exhibited limited callus formation. Shoot regeneration was most effective with 5 mg/L AgNO₃ and 1 nM CaREF1 for cotyledon explants, and shoot elongation was enhanced with GA₃ (0.5 mg/L) and CaREF1 (1 nM) in Zunla-1. Rooting efficiency was improved with IBA combined with CaREF1, yielding higher rooting percentages than IBA or IAA alone. Supplementation with CaREF1 peptide increased the overall ratio of regenerated plants from 27.2% to 55.0%. Histological analysis showed improved cellular organization in explants treated with CaREF1, indicating active cell division and differentiation. This study provides an optimized protocol for efficient regeneration of Zunla-1, highlighting the role of CaREF1 in overcoming regeneration recalcitrance.

Background/Objectives: Pepper (Capsicum annuum L.) is a widely grown vegetable and spice crop worldwide. This study aims to reveal the differences of metabolites among pepper accessions from different regions and explore candidate genes related to metabolites of pepper. Methods: The metabolome and transcriptome of 36 pepper accessions were determined by widely targeted metabolomics analysis and RNA sequencing technology, and the differential metabolites and differential genes among C. annuum from four important pepper production and consumption provinces of China, Hunan, Guizhou, Yunnan and Sichuan, were analyzed. Results: Flavonoids are the main characteristic metabolites that distinguish pepper accessions from Yun_Gui_Chuan Group and Hunan Group. The aglycones of characteristic flavonoids in each group are different; in Yun_Gui_Chuan Group mainly are luteolin, quercetin, chrysoeriol and isorhamnetin; in Hunan Group mainly are apigenin. Transcriptome data showed that two genes related to flavonoid 3′-monooxygenase differed significantly between the two groups of chili peppers, and we speculated that they may be the core enzymes regulating their flavonoid profile. And an SNP mutation located in gene Cgla06g001871 showed a strong correlation with pepper accessions from Yun_Gui_Chuan Group, which can be used as a DNA marker to identify pepper accessions from Yun_Gui_Chuan Group, and provide strong support for regional specialty variety conservation. In addition, we also analyzed the metabolites related to the taste and nutrition of pepper accessions in the four provinces, and the results showed that the sugar content of pepper accessions from Guizhou was low and the capsaicinoids content of pepper accessions from Sichuan was low, while no significant difference was found in acid and vitamin contents among pepper accessions from the four provinces. Conclusions: The metabolome and transcriptome of 36 pepper accessions from four important pepper production and consumption provinces of China were determined, and the characteristic metabolites and expressed genes of pepper accessions from each province were analyzed.