Refinement of shed-microspore culture protocol to increase normal embryos production in hot pepper (Capsicum annuum L.)

A low percentage of normal-looking embryos (20%) is still a problem in an efficient shed-microspore culture of hot pepper (Capsicum annuum L.) and it is more a serious problem in other androgenesic culture systems of pepper. Therefore, several factors were investigated to refine the protocol in order to increase the percentage of normal-looking embryos. The most important factors which improved the protocol and resulted in a significantly higher percentage of normal-looking embryos produces (>50%), were delayed enrichment of the liquid upper layer medium with 2.5 μM Zeatin and 5 μM IAA, and reduced incubation temperature from 28 °C to 21 °C, both after 3 weeks of culture. Addition of 1% activated charcoal in the solid lower layer of the medium enhanced the total embryo yield only, while an application of abscisic acid and increased osmolality medium had a detrimental effect on embryo production. The use of doubled haploid lines as anther donor plants has clearly decreased the variability and improved the statistical analysis of treatment effects. A higher percentage of normal embryos are produced with this refined protocol, and is therefore more suitable for implementation in the breeding programs of hot pepper.

► Enrichment of liquid medium with 2.5 μM Zea + 5 μM IAA after 3 weeks improves embryo quality.
► Reduction of incubation temperature from 28 to 21 °C after 3 weeks improves embryo quality.
► Addition of 1% activated charcoal in the solid medium improves embryo yield.
► Use of doubled haploid has decreased variability and improved analysis of treatment.
► The refined protocol is more efficient and increase normal embryos produces up twofold.