Doubly stable and genetically homozygous

The breeders define the crosses from which the doubled haploid lines are to be developed.

The donor plants are grown in the greenhouse. The plants must be cultivated under special conditions; the starting material is very important.

As soon as the buds are the right size (approx. 3 mm), they are carefully harvested. There is only a narrow time window for removing the flower bud material suitable for the doubled haploid method. Around 120 to 160 buds are required.

The buds are treated with a surface disinfectant in order to eliminate microorganisms. They are then transferred to a sterile container containing nutrient solution.

The container with the buds and nutrient solution is placed in a homogenizer, which further separates the microspores containing the genetic material from the flower tissue.

The flower tissue is removed through a sieve. The microspores remain in the solution and are purified by means of centrifugation. That produces a highly concentrated solution of microspores, which settle at the bottom of the tube in the form of a pellet.

The solution is decanted. A nutrient solution containing growth regulators that stimulate the cells to divide is then applied to the pellet that remains.

The tube is placed on a shaker for two days and then centrifuged. A pellet of microspores forms again at the bottom.

The microspore pellet is mixed with nutrient solution, placed in a petri dish and stored in the dark in the culture room for 5 to 7 days. The dishes are then placed on a shaker, which further stimulates cell division with its continuous motion of the cells. Embryos are formed.

As soon as the embryos have reached a size of 2-3 mm, they are placed on a solid medium. The embryos grow into plantlets in a cell culture chamber (its light and temperature depend on the type of crop).

A ploidy measurement in the flow cytometer is used in the laboratory to check whether duplication has been successful so that breeders can obtain doubled haploid plants.

The traits are checked again using marker analysis and only the best doubled haploid plants are selected for further variety development. Thanks to molecular markers, these traits (genes) can be identified quickly and easily at early stages of plant development.

The breeders receive the selected plants for further breeding.