Doubled haploidy (DH) methodology is used in many plant species to accelerate crop improvement and cultivar development; however not all species are amenable to the tissue culture technique. Experiments were undertaken to develop DH protocols for three perennial grasses [crested wheatgrass (Agropyron cristatum (L.) Gaertn.), hybrid bromegrass (Bromus riparius x B. inermis), and meadow bromegrass (Bromus riparius Rehm.)]. The initial experiment screened these forage grass species to established wheat (Triticum aestivum L.) microspore culture protocols. Following the initial screen, several factors influencing microspore embryogenesis were evaluated. These included genotype, donor plant conditions, developmental stage of the microspore, pretreatments, media composition, and culture conditions. For regeneration of the embryos to plants, media composition and culture conditions were assessed. Microspore-derived embryos/calli as well as green haploid/doubled haploid plants were regenerated from all three forage grasses. Differences were observed between species and genotypes within species in terms of embryogenic response. Modifications to the initial wheat DH protocol included the donor plant conditions, developmental stage of the microspore to late uninucleate to early binucleate and media composition. Regenerated plants were grown in the greenhouse.
