Growth regulators and darkness increase efficiency in in vitro culture of immature embryos from peppers

Common pepper (Capsicum annuum L.) is one of the most important vegetables in the world, and extensive breeding efforts are being made to develop new improved strains of this species. In this regard, in vitro culture of immature embryos may help breeders accelerate breeding cycles and overcome interspecific barriers, among other applications. In this study, we have optimized a protocol for in vitro culture of immature embryos of C. annuum. Levels of indole-3-acetic acid (IAA) and zeatin have been tested to improve the efficiency (germination rates) of this technique in C. annuum embryos at the four main immature stages (i.e. globular, heart, torpedo, and early cotyledonary) from four varietal types of this species (California Wonder, Piquillo, Guindilla, and Bola). The effect of 5-day initial incubation in the dark was also tested on the most efficient hormone formulation. On average, relatively low levels of both IAA and zeatin (0.01 mg L−¹ each) (M1) provided the highest germination rates, particularly in the advanced stages (torpedo and cotyledonary). To a lesser extent, the lack of these growth regulators (M0) or high IAA (0.2 mg L−¹)/low zeatin (0.01 mg L−¹) (M2) combination also had a positive response. On the contrary, high zeatin levels (0.2 mg L−¹) produced very low germination rates or callus development (efficiency 0-7 %). Different responses were also found between genotypes. Thus, considering the best media (M0, M1, M2), Bola embryos had the highest rates. M1 plus 5-days of initial dark incubation (M1-D) improved the efficiency rates at all embryo stages, particularly in the earliest (globular) embryos which increased from 3 % to >; 20 %