Bread Wheat Anther Culture.
Abstract
Doubled haploid (DH) technology can rapidly deliver fixed lines for breeding and research purposes, especially in crops like wheat (Triticum aestivum L.). In Western Australia, we have developed an anther culture protocol that generates more than 12,000 bread wheat DH lines annually. Key aspects of the protocol include the growth conditions of donor plants, microspore developmental stage, stress pretreatment, induction and regeneration medium and culture conditions. Using controlled environment rooms for donor plant growth is important in Australia, enabling year-round production of high-quality plants. We select spikes for anther culture when microspores are at the mid-late to late uninucleate stage. Several stress pretreatments are applied to excised anthers to reprogram microspores from a pathway of gametogenesis to embryogenesis and plant development. We use a combination of mannitol and n-butanol stress pretreatments to induce embryogenesis and these are critical steps in our protocol. Following stress pretreatments, anthers are coincubated with ovaries in a liquid induction medium (LIM) for 4-6 weeks. The inclusion of ovaries during the induction phase is another important step. Resulting embryos are then transferred to a solid regeneration medium for shoot and root development. Plants are subsequently transferred to a soil medium and grown to maturity. We rely on spontaneous chromosome doubling to restore fertility in our wheat DH lines.