Agrobacterium-mediated direct transformation of wheat mature embryos through organogenesis.

Transgenic plant production in monocotyledonous species has primarily relied on embryogenic callus induction from both immature and mature embryos as the pathway for plant regeneration. We have efficiently regenerated fertile transgenic wheat plants through organogenesis after Agrobacterium-mediated direct transformation of mechanically isolated mature embryos from field-grown seed. Centrifugation of the mature embryos in the presence of Agrobacterium was found to be essential for efficient T-DNA delivery to the relevant regenerable cells. The inoculated mature embryos formed multiple buds/shoots on high-cytokinin medium, which directly regenerated into transgenic shoots on hormone-free medium containing glyphosate for selection. Rooted transgenic plantlets were obtained within 10-12 weeks after inoculation. Further optimization of this transformation protocol resulted in significant reduction of chimeric plants to below 5%, as indicated by leaf GUS staining and T1 transgene segregation analysis. Direct transformation of wheat mature embryos has substantial advantages over traditional immature embryo-based transformation systems, including long-term storability of the mature dry explants, scalability, and greatly improved flexibility and consistency in transformation experiments.

Osmotic induced stimulation of the reduction of the viability dye 2,3,5-triphenyltetrazolium chloride by maize roots and callus cultures.

Live cells can reduce colorless 2,3,5-triphenyltetrazolium chloride (TTC) to a red insoluble compound, formazan. Maize (Zea mays) callus, when osmotically stressed by 0.53 mol/L mannitol, produced 7-times or more formazan than untreated control callus. This result was seen with all osmotica tested and could not be attributed to differences in TTC uptake rate or accumulation, increased respiration rate as measured by O2 uptake, or to de novo protein synthesis. Increased formazan production could be detected after 2.5 h of exposure to osmotic stress and leveled off after 48 h of exposure. The increased formazan production was only detected when callus was moved from high osmotic medium to low osmotic, TTC-containing medium. Abscisic acid increased TTC reduction only when added in combination with 0.53 mol/L mannitol. Incubation of maize seedling roots with 0.53 mol/L mannitol also increased formazan production as seen visually. Further studies are needed to determine the cause of the increased formazan production. These results show that TTC viability measurements must be carefully evaluated with appropriate controls to confirm their validity.