In vitro selection of microspore derived embryo genotypes based on molecular marker and oil quality analysis in oilseed rape (Brassica napus L.)

摘要

Microspore culture is frequently applied for the immediate regeneration of homozygous doubled haploid plants in oilseed rape. From the regenerated microspore derived embryos (MDE) usually only a smaller subset are used for plantlet regeneration and cultivation in the greenhouse until seed harvest, without any knowledge about their quality traits and agronomic performance.The random selection of MDE 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 MDE 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 did interfere with subsequent DNA isolation. The feasibility of the protocol was tested using MDE 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 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.