An improved wheat microspore culture technique for the production of doubled haploid plants

Microspore culture is used to generate completely homozygous plants in a single generation, thereby reducing cost and time required for the production of doubled haploid (DH) plants for breeding and genetic studies. Many factors are known to influence green plant recovery including composition of regeneration media, donor plant genotype, microspore developmental stage, and pretreatment conditions. The objectives of this study were to: (i) develop an improved regeneration medium for wheat microspore culture and (ii) determine the optimal pretreatment conditions and regeneration media combination for increasing green plant recovery rates. Four wheat cultivars, two pretreatment methods (0.4 M mannitol at 4°C and solution B containing 0.3 M mannitol with inorganic components at room temperature), and five regeneration media were tested. Green plant recovery rates from each treatment combination were analyzed using the Proc Logistic model in SAS. Regeneration medium fortified with ascorbic acid produced the highest number of green plants across the different pretreatment conditions, regeneration media, and cultivars tested. Pretreating wheat (Triticum aestivum L.) spikes with 0.4 M mannitol at 4°C followed by embryoid induction and regeneration in a medium fortified with ascorbic acid resulted in the recovery of the highest number of green plants. This high-efficiency method may prove useful for producing DH populations for wheat improvement efforts.