An efficient doubled‐haploid production technology that induces homozygosity can greatly reduce the time and cost of cultivar development. Low efficiency of doubled‐haploid production previously has limited exploitation of this method for crop improvement. This study aimed to develop a more efficient and effective isolated microspore culture system for generating doubled‐haploid wheat (Triticum aestivum L.) plants. We report here the development and testing of a new chemical formulation for its efficiency to induce microspore embryogenesis, and the development of a system for double haploid production, in which the induction of embryogenesis in microspores was followed by isolating embryogenic microspores, and culturing them under optimized growth conditions to produce high embryoid yields. Up to 50% of the total treated microspores in the whole spike were converted from their preprogrammed gametophytic to a sporophytic pathway by a chemical inducer formulation consisting of 0.1 g L−1 of 2‐hydroxynicotinic acid, 10−6 mol L−1 2,4‐dichlorophenoxyacetic acid, and 10−6 mol L−1 6‐benzylaminopurine. The isolated embryogenic microspores were cocultivated with live wheat ovaries in a liquid NPB 99 media with an osmolality of about 300 mOsmol kg−1 H2O, resulting in the regeneration of 50 to 5500 green plants per single spike of eight wheat genotypes. The high efficiency and simplicity make the system practical for biological research and for accelerating cultivar development in wheat breeding programs.
Microspores were isolated from wheat (Triticum aestivum L.) spikes of various genotypes following an effective pretreatment that induced microspore embryogenesis. The isolated microspores were cultured with or without live ovaries, and with or without medium pre-conditioned by ovaries for varying periods of time. Live ovaries alone increased androgenic embryoid yields up to 4.5-fold over the control for microspores isolated from responsive genotypes. While live ovary co-culture alone was not effective for microspores isolated from recalcitrant genotypes, the addition of medium preconditioned by ovaries to microspore cultures increased embryoid yield by more than 100-fold. Without ovary-conditioned medium, no embryoids could be obtained from some recalcitrant genotypes. Ovary-conditioned medium apparently functions to increase embryoid yields by providing essential substance(s) for elaboration of the embryogenic program already triggered during pretreatment.
Although a high efficiency of microspore embryogenesis can be achieved for a wide range of genotypes, some genotypes regenerate a high proportion of albino plants. Significant improvements in embryo regeneration and green plant formation were achieved by adding 10% NPB98 medium to a microspore pretreatment solution containing a chemical inducer formulation. For the wheat genotype WED 202-16-2, which is known to produce a moderate frequency of albinos, the plant regeneration rate was 15% higher, and the green plant percentage increased 27% following the addition of this nutrient supplement during embryo initiation. There were no negative effects on the responsive genotype Svilena. Embryo production in both genotypes was not affected. Our results indicate that providing readily available nutrients during microspore embryo initiation is effective in promoting plant regeneration and enhancing green plant development in a wheat genotype with a propensity for albinism.
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