Wheat anther culture and genetic transformation of wheat microspore-derived embryos

The objectives of these studies were to simply determine ploidy levels of anther culture-derived plants, to improve haploid production system, and to develop stable transgenic plants in microspore-derived embryos using microprojectile bombardment. Using flow cytometry, the 2C nuclear DNA contents of total of 301 plants were estimated. Microspore-derived haploid (3x), doubled-haploid (6x), nineploid (9x), and twelveploid (12x) plants contained 15.44, 30.56, 45.57, 60.27 pg of DNA content. The frequency of haploids (43.6%) was similar to that for doubled haploids (43.0/o), and much larger than the frequency of endopolyploids [nineploid (1.3%), twelveploid (1.0%)] and various aneuploids (11.10/9). We improved a wheat haploid production system via anther culture in which plants are produced from microspore-derived embryos using one medium and one culture environment. In the improved protocol, 58% of the anthers were responsive with multiple embryos and 338 embryos were produced from 100 plated anthers. For plant regeneration, 60 plants were produced from 100 anthers. The frequencies of green plants were 43.2% of the regenerated plants and 49% plants were fertile and produced seeds. Wheat microspore-derived embryoids from anther culture were used as target tissues for genetic transformation. 0.6 M mannitol was optimal for transformation of wheat microspore-derived embryos. Two different plasmid DNA, pE7131-GUS and pUBI1NPTII-1, were used for stable genetic transformation. Plants regenerated from medium containing selection agents were analyzed using PCR. In the direct plant regeneration system, only 27 of 58 R0 plants, which were G418 tolerant, were the nptII gene positive, and over 50% of surviving plants were escapes. By southern hybridization, among 24 surviving plants from selection, two plants had the intact uidA gene and four plants had part of the uidA gene. In an indirect plant regeneration system, six plants were positive for the GUS determined by histochemical and fluorometric assay and eight plants were positive for the NPTII determined by ELISA. Six plants were positive for both the GUS and NPTII, and two plants were negative for the GUS gene but positive for the NPTII