Mehran E. Shariatpanahi

Efficient embryogenesis and regeneration in freshly isolated and cultured wheat (Triticum aestivum L.) microspores without stress pretreatment

The major advantage of doubled haploids in plant breeding is the immediate achievement of complete homozygosity. Desired genotypes are thus fixed in one generation, reducing time and cost for cultivar or inbred development. Among the different technologies to produce doubled haploids, microspore embryogenesis is by far the most common. It usually requires reprogramming of microspores by stress such as cold, heat, and starvation, followed by embryo development under stress-free conditions. We report here the development of a simple and efficient isolated microspore culture system for producing doubled haploid wheat plants in a wide spectrum of genotypes, in which embryogenic microspores and embryos are formed without any apparent stress treatment. Microspores were isolated from fresh spikes in a nutrient-free medium by stirring and cultured in medium A2 in the dark at 25°C. Once embryogenic microspores were formed, ovaries and phytohormones were added directly to the cultures without changing the medium. The cultures were incubated in the dark at 25–27°C until the formation of embryos and then the embryos were transferred to regeneration medium. The regeneration frequency and percentage of green plants increased significantly using this protocol compared to the shed microspore culture method.

Isolated Microspore Culture and Its Applications in Plant Breeding and Genetics

Isolated microspore culture (IMC) represents a unique system of single cell reprogramming in plants wherein a haploid male gametophyte, the microspore, switches its default gametophytic developmental pathway toward embryogenesis by specific stress treatment. The application of a stress treatment(s) is necessary for efficient embryogenesis induction. Depending on species, microspores are often induced by cold and heat shock, osmotic stress, starvation, anti-microtubular agents, stress hormones, antibiotics, or polyamines. This technique (IMC) is likely to remain as a well-known method in plant breeding since it allows for the rapid production of completely homozygous lines while, in the context of developmental biology, it allows for in vitro embryogenesis to be explored in greater detail. Isolated microspores also represent ideal recipients for several gene transfer techniques including electroporation, microprojectile bombardment, and Agrobacterium-mediated transformation. IMC is also extensively used for genetic studies, i.e., studying inheritance of quantitative traits, quantitative trait loci (QTL) mapping, and genomics and gene identification, for mutation and selection and also used for producing reversible male-sterile lines. Male sterility avoids the labor costs of manual emasculation and serves as a molecular strategy for transgene containment by preventing pollen release to the environment. Combination of this technique with doubled haploid (DH) production leads to an innovative environmentally friendly breeding technology. In addition, the usefulness of DHs for reverse breeding program, an applied plant breeding technique introduced to directly produce parental lines for any hybrid plant, is also generally discussed.