Abstract
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.
Highlights
Efficient direct organogenesis induction would have a large impact on the breeding of and research on peppers (Capsicum spp.)
For C. annuum, no significant differences were observed among cotyledon and hypocotyl explants for the number of cut edges with calli, with the mode and the median set as 2 for both explants (Table 3)
We have demonstrated the significant effect of the culture medium, the type of explant and genotype on the organogenesis of pepper, a crop that is very recalcitrant to in vitro regeneration
Summary
Efficient direct organogenesis induction would have a large impact on the breeding of and research on peppers (Capsicum spp.). Despite the great advances achieved in the breeding and genomics of peppers [2,3], most Capsicum materials are considered as recalcitrant to in vitro culture techniques, making the application of conventional and modern breeding techniques that require in vitro regeneration more difficult [3,4]. Horticulturae 2021, 7, 261 demonstrated the difficulties of pepper regeneration in in vitro culture, including natural morphogenetic recalcitrance, rosette bud formation, sensitivity to ethylene, and differences among genotypes [3,6]. There are several studies of regeneration from different explants showing some success [6,7,8,9,10,11], they do not reach the required efficiency for genetic engineering applications
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