Development of A Unique Protocol for the Production of Doubled Haploids in Hot Pepper

Abstract

Hot pepper ranks seventh among the most produced vegetables in the world. This crop is famous for its color and pungency. Hot pepper contains many important phytonutrients that enable it to fight against cancer. Conventional breeding in Hot Pepper is labor-consuming and a long-term process. This can be overcome by using novel approaches, such as, in vitro doubled haploid plant production. This method not only accelerate breeding programs but also facilitates recovery of recessive mutations.  There are many articles published on doubled haploid development in Capsicum annuum; it reports both anther culture and shed-microspore culture methods, but both these methods require either subculture of anthers or the addition of new media, which makes both protocols laborious. The present study aims to develop a single-step direct embryogenesis protocol to produce doubled haploids in Capsicum annuum. We analyzed effectiveness of microspore embryogenesis on ten Hot pepper genotypes with six media combinations including shed-microspore culture protocol (Supena et.al 2006) and two-step anther culture protocol (Dumas de vaulx et.al 1981 and Para-Vega et.al 2016). Among six, one media combination responded very well to single-step direct embryogenesis compared to other protocols with more than 20 % plant regeneration frequency. We observed an average embryogenesis of 7.53% and plant regeneration frequency of 3.81%. We achieved a survival index of 97.61% after acclimatization of plants in the polyhouse.  The results of these experiments show that, one-step direct embryogenesis can be achieved with unique combination of plant growth regulators.