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F. Leaf section showing GUS expression (top), control leaf (bottom). G. Regenerated transformed plants in GM containment glasshouse. 8. 9. 10. II. 12. 13. 14. 15. 16. MnS04 may have various hydrated states which will alter the required weight. 95 gil. AgNO J is used to promote embryogenesis; Silver thiosulphate (a mix of silver nitrate and sodium thiosulphate) at 10 mg/I can be used as an alternative. Both are photosensitive so the stock solutions and any media plates containing them should be kept in the dark.
36 G. HENSEL AND 1. 3. Genetic Transformation of Barley Stable transfonnation of barley was first reported in 1994 (14), and since then there have been many reports of successful barley transfonnation (15). Most studies have used microparticle bombardment for trans gene insertion, but the use of Agrobacterium tumefaciens for transfonnation of barley (16) offers an alternative methodology. When conducting tissue culture of immature scutella as was developed for biolistic transfonnation, an Agrobacterium-based method can be considerably more efficient.
2. Add 5 III DNA (1 mg/ml in TE see Note 29) or water (see Note 30) and vortex briefly to ensure good contact of DNA with the particles (see Note 31). 3. 1 M spermidine into the lid of the Eppendorftube and mix together. Briefly vortex into the gold and DNA solution (see Note 32). Centrifuge 13,000 rpm for 3-5 sec in a microfuge to pellet the DNA-coated particles. Discard the supernatant. 4. Add 150 III 100% ethanol to wash the particles, re-suspending them as fully as possible (see Notes 33 and 34).