Effects of Biotin and Ascorbic Acid Applications on Haploid Embryo Induction in Semisolid and Double Layer Nutrient Media in Pepper (Capsicum annuum L.) Anther Culture

Abstract

Generation of homozygous double haploid (DH) lines by androgenesis is a promising alternative to selfpollination programs across generations. Despite the routine use of anther culture in peppers, there are still many bottlenecks and improvements in methodology are required. The aim of this study was to determine the effects of the structure of the nutrient medium (semi-solid and double layer) and the addition of biotin and ascorbic acid to the nutrient media on obtaining haploid embryos by anther culture method. MS (Murashige and Skoog 1962) medium containing 4 mg l -1 NAA, 0.1 mg l -1 BAP, 0.25% activated charcoal, 30 g l -1 sucrose, and 10 mg l -1 AgNO3 (silver nitrate) were used as the basal nutrient medium. A total of 8 nutrient media compounds were studied using 0.05 mg l-1 biotin and 0.5 mg l -1 ascorbic acid separately or together in semi-solid and bi-layer (double-phase) nutrient media. Solidification of nutrient media was achieved with 7 g 1 -1 agar. The cultured anthers were subjected to high-temperature pre-treatments at 35 °C in continuous dark conditions for 2 days. Then they were taken to a climate chamber at of 25 °C temperature adjusted to 16/8 hour photoperiod. It has been observed that the success of obtaining embryos of semi-solid medium was higher than double-layer medium. The addition of biotin and ascorbic acid to the nutrient medium provided 8.8 fold increases in embryo regeneration compared to the control medium. In the presence of only one of biotin or ascorbic acid in the nutrient medium, the number of embryos increased compared to the control.