Peppers (Capsicum spp.) are recalcitrant to in vitro culture regeneration, making the application of in vitro-based breeding strategies difficult. We evaluated the impact of different combinations of auxins, cytokinins and micronutrients on the induction of direct organogenesis in cotyledon and hypocotyl explants of C. annuum, C. baccatum and C. chinense. We found variation in the regeneration response among species and type of explant. In this way, the average numbers of shoots per cotyledon and hypocotyl explant were, respectively, 1.44 and 0.28 for C. annuum, 4.17 and 3.20 for C. baccatum and 0.08 and 0.00 for C. chinense. Out of the six media, the best overall results were obtained with the medium Pep1, which contained 5 mg/L BAP (6-benzylaminopurine), 0.5 mg/L IAA (indole-3-acetic acid) and 0.47 mg/L CuSO₄, followed by a subculture in the same medium supplemented with 10 mg/L AgNO₃ (medium Pep1.2). The best result for the Pep1 + Pep1.2 medium was obtained for C. baccatum using cotyledon explants, with 8.87 shoots per explant. The explants grown in medium Pep1 + Pep1.2 were the ones with greener tissue, while overall the hypocotyl explants were greener than the cotyledon explants. Our results indicate that there is wide variation among Capsicum species in terms of regeneration. Our results suggest that the synergistic effect of copper and silver resulted in a higher regeneration rate of Capsicum explants. Explants with shoots were transferred to different media for elongation, rooting and acclimatization. Although acclimatized plantlets were obtained for C. baccatum and C. chinense, an improvement in these latter stages would be desirable for a high throughput regeneration pipeline. This work contributes to the improvement of Capsicum regeneration protocols using specific combinations of medium, explant and genotype, reaching the levels of efficiency required for genetic transformation and of gene editing technologies for other crops.

Eggplant (Solanum melongena L.) displays extensive fruit color diversity, in which chlorophyll-related pigmentation contributes to both external appearance and market value. Previous genetic studies identified SmAPRR2 and SmGLK2 as major candidate genes controlling uniform green pigmentation and green netting in fruit, respectively, but their individual and combined functional contributions had not been validated through targeted mutagenesis in a common genetic background. Here, we established a multiplex CRISPR/Cas12a system in eggplant accession MEL3, representing, to our knowledge, the first application of this nuclease for genome editing in eggplant. Transformation efficiency was 2.0%, but all 15 genotyped regenerants were edited, yielding four SmAPRR2 and six SmGLK2 alleles. Segregation and crossing enabled the recovery of six transgene-free lines carrying single or combined edited alleles. Disruption of SmGLK2 abolished the reticulated green netting pattern while preserving a uniformly green peel and the internal green ring. Conversely, disruption of SmAPRR2 reduced the background uniform peel pigmentation and eliminated the green ring while retaining green netting. Double mutants carrying disruptive alleles at both loci produced white fruits lacking internal green pigmentation, whereas putatively hypomorphic SmAPRR2 and SmGLK2 alleles generated intermediate phenotypes. Whole-genome resequencing identified only two predicted off-target sites under a canonical TTTV PAM search allowing up to four mismatches. Both were fully covered, and no edited-line-specific candidate variants were detected. These findings establish complementary and partially separable roles for SmAPRR2 and SmGLK2 in fruit peel and flesh chlorophyll pigmentation and demonstrate the potential of Cas12a for functional genomics, allele engineering, and precision breeding in eggplant.

Solanum retroflexum is cultivated for both its fruit and leaves in Africa, China, India, and Indonesia. However, it is still considered an underutilized crop due to limited genetic and genomic resources to support improvement. To enable gene function studies for future improvement by new breeding technologies, this study established efficient plant regeneration and Agrobacterium tumefaciens –mediated transformation approaches. Cotyledon and hypocotyl explants were used to test plant regeneration on a Murashige and Skoog salts-based medium supplemented with 0.5 mg L ⁻¹ zeatin. Both explant types showed 100% regeneration; however, cotyledons produced a greater number of whole plants than hypocotyls. For transformation experiments, cotyledon explants were infected with A. tumefaciens AGL1 carrying the pJL33 binary vector containing the Green Fluorescent Protein ( GFP ) reporter and the Neomycin Phosphotransferase-II ( nptII ) selectable marker genes. Following cocultivation, firstly, kanamycin at various concentrations (75.0 to 400.0 mg L ⁻¹ ) was evaluated to determine the optimal concentration for maximizing transgenic line recovery. However, a significant number of escapes even at 400.0 mg L ⁻¹ kanamycin were observed. Because the nptII gene also confers resistance to G418, in this study’s subsequent experiments the current authors tested the effectiveness of a range of G418 concentrations (25.0 to 150.0 mg L ⁻¹ ). Fluorescence microscopy and PCR analysis showed that 25.0 mg L ⁻¹ G418 resulted in the highest transformation efficiency at 88% and the fewest escapes. Ploidy levels of regenerated, transgenic, and wild-type plants were assessed using flow cytometry, confirming stable ploidy. The optimized selection medium facilitated an efficient and stable transformation system that will be utilized to support crop improvement efforts of S. retroflexum .

Eggplant ( Solanum melongena ) research has been hindered by the lack of suitable model plants. The development of Micro-Mel and Mini-Mel, two short-cycle dwarf model lines with introgressions from the wild relative S. anguivi , represents a breakthrough in eggplant research. Both lines were characterized with 30 phenotypic descriptors and compared to their parental lines and the Micro-Tom tomato. Micro-Mel has determinate growth, early flowering (41.0 days post-transplantation), and physiological fruit maturity at 103.7 days. Mini-Mel exhibits indeterminate growth, later flowering (57.2 days) and fruit maturity (114.7 days), with a more conventional architecture. Micro-Mel and Mini-Mel complete 3.5 and 3.0 full seed-to-seed cycles per year, respectively. Their compact size makes them ideal for space-limited environments, controlled growth chambers, speed breeding, ornamental horticulture, and urban horticulture. To validate their potential for gene editing, in vitro regeneration experiments were performed on a medium containing zeatin riboside. Regeneration was similar to parental S. melongena (1.2 shoots/explant), with Micro-Mel developing 1.0 and Mini-Mel 0.8. Whole-genome sequencing (23X coverage) identified 179,653 SNPs between the parental lines, revealing heterozygosity rates of 0.00098% for Micro-Mel and 0.00123% in Mini-Mel. Approximately 8.50% and 10.79% of their genomes were introgressed from S. anguivi , respectively. Analysis identified 18 shared introgressions from S. anguivi among Micro-Mel, Mini-Mel, and four backcross dwarf materials derived from the same original plant. Notably, three orthologues of genes associated with dwarfism in tomato ( SlDREB1, SlER , and SlSERK1 ) were identified within these introgressions. Micro-Mel and Mini-Mel represent transformative tools for eggplant research, accelerating genetic, physiological, and breeding studies.

Eggplant (Solanum melongena L.) is a major horticultural crop for which genetic transformation and genome editing could accelerate functional genomics and precision breeding, but their routine use remains constrained by regeneration recalcitrance, strong genotype dependence, and fragmented methodological reporting. This review critically assesses Agrobacterium-mediated transformation protocols, genome-editing studies, and emerging in planta virus-based approaches across the complete workflow, from explant preparation and nucleic-acid delivery to regeneration, selection, plant recovery, and outcome assessment. The major variables shaping transformation success are examined, including explant type and age, pre-culture, bacterial strain, infection and co-culture conditions, acetosyringone, regeneration medium, selection pressure, shoot recovery, rooting, and efficiency assessment. The evidence indicates that reported performance differences are difficult to interpret because studies often rely on genotype-specific optimization, incomplete methodological description, and non-equivalent efficiency metrics. Complementary strategies are also discussed, including virus-induced gene silencing, virus-induced gene editing, biolistics, protoplast delivery, floral dip-inspired approaches, highly regenerable model genotypes, and morphogenic regulators. Finally, the review outlines priorities to move eggplant biotechnology from proof-of-concept toward reproducible breeding pipelines: standardized reporting, direct protocol comparisons, genotype-aware optimization, mechanistic studies of recalcitrance, and transformation-competent reference lines. Although centered on eggplant, the methodological framework and reporting priorities identified here are relevant to other horticultural crops in which genotype-dependent regeneration limits genome engineering. Together, these priorities provide a roadmap from isolated proof-of-concept experiments to reproducible and breeding-relevant genome-editing pipelines.