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 degrees 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 degrees 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.

Background: The major birch pollen allergen Bet v 1 (or Bet v 1a) is one of the main causes of seasonal type I allergies. Various environmental factors such as light, temperature and air pollution may influence the activity of the Bet v 1a gene. The creation of a model system to evaluate the role of environmental factors affecting the Bet v 1a gene expression would be highly desirable. We suggest the use of transgenic tobacco plants carrying a Bet v 1a promoter-reporter gene fusion as such a system. Methods: The promoter of the Bet v 1a gene was isolated with the use of the Universal Genome Walker kit (BD Biosciences Clontech, USA). Web Software was used to search for putative cis-regulatory elements within the promoter. Transgenic tobacco plants harboring the promoter-β-glucuronidase (GUS) reporter gene fusion were obtained via Agrobacterium tumefaciens-mediated transformation. Promoter activity was examined with histochemical and quantitative assays. Results: Structural analysis predicted elements responsible for pollen-specific, light-, stress- and hormone-mediated induction within the Bet v 1a promoter. The evaluation of GUS activity in transgenic tobacco plants showed that the Bet v 1a promoter is pollen-specific. Moreover, the Bet v 1a promoter is considered to be the strongest isolated pollen-specific promoter reported to date. It was shown that temperature and abscisic acid positively regulate the activity of the Bet v 1a promoter during pollen development, providing evidence for environment-dependent regulation of the Bet v 1a gene. Conclusions: A model system to study the effect of environmental factors on the expression of the Bet v 1a gene encoding the major birch allergen in pollen was generated. Additionally, we suggest that this system could be used to search for factors that inhibit the activity of the gene in pollen in order to reduce the potential allergenicity of birch trees.

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