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Plant Genetic Transformation

Plant Genetic Transformation. All stable transformation methods consist of three steps:. Delivery of DNA into a single plant cell. Integration of the DNA into the plant cell genome. Conversion of the transformed cell into a whole plant. Methods of delivering DNA into plant cells. Biological

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Plant Genetic Transformation

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  1. Plant Genetic Transformation

  2. All stable transformation methods consist of three steps: • Delivery of DNA into a single plant cell. • Integration of the DNA into the plant cell genome. • Conversion of the transformed cell into a whole plant.

  3. Methods of delivering DNA intoplant cells • Biological • Agrobacterium • Other bacteria • Viruses • Physical • Particle bombardment • Electroporation • Silicon carbide whiskers • Carbon nanofibers

  4. Agrobacterium-mediated Transformation

  5. Biology of the Agrobacterium-plant interaction The only known natural example of inter-kingdom DNA transfer

  6. Infects at root crown or just below the soil line. • Can survive independent of plant host in the soil. • Infects plants through breaks or wounds. • Common disease of woody shrubs, herbaceous plants, dicots. • Galls are spherical wart-like structures similar to tumors.

  7. The genus Agrobacterium has a wide host range: • Overall, Agrobacterium can transfer T-DNA to a broad group of plants. • Yet, individual Agrobacterium strains hve a limited host range. • The molecular basis for the strain-specific host range is unknown. • Many monocot plants can be transformed (now), although they do not form crown gall tumors. • Under lab conditions, T-DNA can be transferred to yeast, other fungi, and even animal and human cells.

  8. Why is Agrobacterium used for producing transgenic plants? • The T-DNA element is defined by its borders but not the sequences within. So researchers can substitute the T-DNA coding region with any DNA sequence without any effect on its transfer from Agrobacterium into the plant.

  9. Ti Plasmid

  10. Key steps from natural Agrobacterium to “useful”Agrobacterium • Some vir genes deleted—disarmed • Opines not going to be produced • Deleting tumorogenesis function • Choosing strains that transfer DNA in lab • Clone in genes of interest, antibiotic resistance genes, etc. • Binary system-- two plasmids are better than one Ti plasmid

  11. Arabidopsis Floral Dip

  12. Biolistic Transformation Gene Gun

  13. AgroinfiltrationTransient method of expressing transgenes

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