Common pepper (Capsicum annuum L.) is among the most important vegetables in the world and huge breeding efforts are made every year to develop new improved materials of this species. In this regard, in vitro culture of immature embryos may help breeders to accelerate breeding cycles, to overcome interspecific barriers, among other applications. In this work, we have optimized a protocol for in vitro culture of immature embryos of C. annuum. Different levels (0.01 and 0.2 mg L-1) 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 different varietal types of this species (California Wonder, Piquillo, Guindilla, and Bola). The effect of 5-days initial incubation in 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) (M 1) provided the highest germination rates (33%), particularly in the advanced stages (torpedo and cotyledonary). To a lower extent, the lack of these growth regulators or high IAA (0.2 mg L-1)/low zeatin (0.01 mg L-1) combination also showed a positive response of embryos (24%). On the contrary, high zeatin levels (0.2 mg L-1) provoked very low germination rates or callus development (efficiency 0-7% at any stage and genotype). Finally, M 1 plus 5-days of initial dark incubation (M 1-D) improved the efficiency rates at any embryo stage, particularly in the earliest (globular) embryos which increased from 3% to >20%.
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