Early, non-destructive selection of microspore-derived embryo genotypes in oilseed rape (Brassica napus L.) by molecular markers and oil quality analysis

In oilseed rape (Brassica napus L.) breeding, microspore culture is frequently applied for the immediate regeneration of homozygous doubled haploid (DH) plants. From the regenerated microspore-derived embryos (MDEs), usually only a smaller subset of around 200 are used for plantlet regeneration and cultivation in the greenhouse until seed harvest, without there being any knowledge about their quality traits and agronomic performance. The random selection of MDEs implies that valuable rare recombinant genotypes may be discarded at an early stage of in vitro culture. We report here on the development of a simple protocol for simultaneously extracting lipids (for oil quality analysis) and the isolation of DNA (for marker-assisted selection) from single cotyledons dissected from MDEs under aseptic conditions, thus keeping the rest of the embryo in vitro for plantlet regeneration. Neither the fatty acid extraction nor the transmethylation with sodium methylate at high pH interfered with subsequent DNA isolation. The feasibility of the protocol was tested using MDEs from a cross segregating for two linked transgenes, fae1 and plsC, affecting the fatty acid composition. Multiplex PCR was performed with specific PCR primers for the plsC gene and with locus-specific primers for a resident single copy fad2 gene. The amplification of the fad2 gene provided a control for the presence of DNA in sufficient quantity and quality, whereas the amplification of the plsC gene showed a 1:1 segregation expected for a single copy transgene in a segregating DH population. The early identification of the 50% MDE genotypes carrying the desired transgenes, along with a high expression of the trait, allows their early selection for plantlet regeneration.