Abstract
Production of doubled haploids (DHs) is a convenient
tool to obtain pure lines for breeding purposes. Until
now, the easiest and most useful approach to obtain pepper
DHs is via anther culture. However, this method has an
associated possibility of producing calli from anther wall
tissues that would be coexisting in the anther locule with
embryos derived from microspores. Using two established
protocols for anther culture, Dumas de Vaulx et al. (Agronomie
2:983988, 1981) and Supena et al. (Sci Hort
107:226232, 2006a; Plant Cell Rep 25:110, 2006b) callus
and embryo development was assessed in four sweet pepper
cultivars. For all genotypes tested, the protocol of Dumas de
Vaulx et al. (Agronomie 2:983988, 1981) promoted both
embryo development and callus growth, whereas the protocol
of Supena et al. (Sci Hort 107:226232, 2006a; Plant Cell
Rep 25:110, 2006b) produced no callus but only embryos.
However, differences in embryo production were observed
among these genotypes. In parallel, anthers were exposed to
a 35 C inductive heat shock for 4, 8, 12 and 16 days, prior to
culture at 25 C. The duration of the heat shock had significant
effects in embryo production, but also in callus generation.
Callus generation increased with prolonged
exposures to 35 C. Embryo and callus origin was analyzed
by flow cytometry, light microscopy and molecular markers.
Tests conducted demonstrated a gametophytic origin for all
of the embryos tested, and a sporophytic origin for all of the
calli. Together, our results reveal that culture conditions have
a significant influence on the presence of calli derived from
anther walls, which could be minimized by reducing heat
shock exposure and/or using a shed-microspore approach.