A high throughput Brassica napus microspore culture system: influence of percoll gradient separation and bud selection on embryogenesis

Microspore culture for the purpose of developing doubled haploid plants is routine for numerous plant species; however, the embryo yield is still very low compared with the total available microspore population. The ability to select and isolate highly embryogenic microspores would be desirable for high embryo yield in microspore culture. To maximize the efficiency of canola microspore culture, a combination of bud size selection and microspore fractionation using a Percoll gradient was followed. This approach has consistently given high embryo yields and uniform embryo development. Microspores isolated from buds 1.5 to 4.4 mm in length of Brassica napus genotypes Topas 4079, DH12075, Westar and 0025 formed embryos at different frequencies. The most embryogenic bud size range varied with each cultivar: Topas 4079 3.5-3.9 mm, DH12075 2.0-2.4 mm, and Westar and 0025 2.5-2.9 mm. When the microspores from 2.0 to 2.4 mm buds of DH12075 were carefully layered on top of a discontinuous Percoll gradient of 10, 20 and 40%, and subsequently spun through the Percoll layers by centrifugation, bands were formed containing populations of microspores of uniform developmental stage. The middle layer of the gradient contained the late uninucleate and early binucleate microspores that were the most embryogenic. In addition, the relationship between the bud size, developmental stage of isolated microspores, Percoll gradient concentration and the embryogenic frequency of each cultivar were studied. Optimization of these factors is required for each genotype evaluated.