1. Plant Gene Transfer John J. Finer http://www.oardc.ohio-state. edu/plantranslab/

2. For successful production of transgenic plants, 1)DNA delivery 2)Marker genes (GFP) are useful 3)Regeneration from single cells

3. DNA Delivery Agrobacterium Particle gun

4. Agrobacterium Agrobacterium is a soil borne gram- negative bacterium, that has a unique ability to introduce part of its DNA into plant cells.

5. Agrobacterium Most of the native transferred bacterial DNA is replaced with genes of interest.

6. Agrobacterium In the laboratory, bacteria are co- cultured or inoculated with plant tissue and the bacteria transfer part of their DNA into plant cells.

7. Particle gun For particle bombardment, tungsten or gold particles are coated with DNA and accelerated towards target plant tissues. In the early days, the force used to accelerate the particles was a.22 caliber blank. Today, most devices use compressed helium.

8. Particle gun The particles punch holes in the plant cell wall and usually penetrate only 1-2 cell layers. Particle bombardment is a physical method for DNA introduction and the biological incompatibilities associated with Agrobacterium are avoided.

9. Particle gun The DNA-coated particles can end up either near or in the nucleus, where the DNA comes off the particles and integrates into plant chromosomal DNA.

10. GFP - Jellyfish Green Fluorescent Protein

11. GFP is a marker gene used in DNA transfer studies. The jellyfish green fluorescent protein gene has been modified for optimum expression in plants. The protein from the gene will fluoresce green when illuminated with high intensity blue light.

12. GFP - Jellyfish Green Fluorescent Protein Chlorophyll can fluoresce red under the same conditions which cause GFP to appear green. GFP fluorescence occurs as spots if individual cells are targeted or the whole tissue can be green if all of the cells within the tissue contain the gfp gene.

13. GFP - Jellyfish Green Fluorescent Protein GFP is only useful as a marker or indicator of successful gene transfer. It has little application beyond this.

14. Introduction of the gfp gene into different target tissues Petunia petal - cells Wheat callus - cells Soybean seed – whole seedling (on right)

15. GFP expression in wheat seeds (left seed, on left) and roots (below)

16. No gfp – red chlorophyll fluorescence GFP – green fluorescence GFP expression in soybean tissues

17. gif animation of GFP expression in soybean tissue Shows variability in expression pattern standard illumination on left – gfp illumination on right gif animation of GFP expression in soybean tissue Shows variability in expression pattern standard illumination on left – gfp illumination on right

18. Plant Regeneration Plants can be generated from single cells using “tissue culture”, where parts of the plants are placed in Petri dishes. The specific response of the plant tissue depends on the starting plant materials and the medium in the Petri dish.

19. Plant Regeneration Under the right conditions, plant tissues regenerate into whole plants via two distinct processes; somatic embryogenesis and shoot morphogenesis.

20. Plant Regeneration For somatic embryogenesis, embryos or artificial seeds form, which can germinate into a whole plant.

21. Soybean Embryogenesis Induction Proliferation Development Germination Plant Recovery Starting Material Immature seeds

22. Plant Regeneration For shoot morphogenesis, shoots form, which must generate roots before they can be transferred to the soil.

23. Production of Transgenic Plants When DNA delivery, plant regeneration and selection for transgenic cells are merged, transgenic plants can be produced. The idea is to introduce DNA into cells, which can be selected and generated into whole transgenic organisms.

24. For further information or suggestions, contact: John J. Finer Dept Horticulture and Crop Science OARDC/The Ohio State University 1680 Madison Ave. Wooster, OH 44691 USA Finer.1@osu.edu Finer.1@osu.edu http://www.oardc.ohio-state. edu/plantranslab/