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Lecture 15 Chapter 10

Lecture 15 Chapter 10. Plant transformation II Neal Stewart. Discussion questions. 1. What is a transgene/transgenic plant? 2. What are some differences between physical and biological methods for DNA introduction into plant cells?

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Lecture 15 Chapter 10

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  1. Lecture 15 Chapter 10 Plant transformation II Neal Stewart

  2. Discussion questions 1. What is a transgene/transgenic plant? 2. What are some differences between physical and biological methods for DNA introduction into plant cells? 3. What are some ways that the biological method for DNA introduction (Agrobacterium) has been improved over the years?

  3. Discussion questions 4. How is gene introduction performed with the model plant, Arabidopsis? Is this technique widely applied to other plants? 5. How do the DNA integration patterns differ in plant cells, transformed via Agrobacterium and particle bombardment? 6. Can you think of additional methods for DNA introduction into plant cells?

  4. Formula Tissue culture + DNA delivery and integration = transgenic plants

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

  6. Transformation overview http://www.ag.ndsu.edu/pubs/plantsci/crops/a1219-2.gif

  7. Agrobacterium tumefaciens Nature’s little plant transformer

  8. Causal agent of crown gall disease microbewiki.kenyon.edu/index.php/Agrobacterium http://pubs.caes.uga.edu/caespubs/pubcd/images/B1286-17.jpg

  9. Figure 10.1

  10. Figure 10.2

  11. 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

  12. Figure 10.4 http://www.youtube.com/watch?v=L7qnY_GqytM

  13. http://openlearn.open.ac.uk/file.php/2808/S250_1_005i.jpg

  14. Agrobacterium contains a tumour-inducing (Ti) plasmid, which includes virulence (vir) genes and a transferred-DNA (T-DNA) region. Genes of interest can be inserted into the T-DNA. Wounded plant cells produce phenolic defence compounds, which can trigger the expression of the Agrobacterium vir genes. The encoded virulence (Vir) proteins process the T-DNA region from the Ti-plasmid, producing a 'T-strand'. After the bacterium attaches to a plant cell, the T-strand and several types of Vir proteins are transferred to the plant through a transport channel. Inside the plant cell, the Vir proteins interact with the T-strand, forming a T-complex. This complex targets the nucleus, allowing the T-DNA to integrate into the plant genome and express the encoded genes. http://www.nature.com/nature/journal/v433/n7026/images/433583a-f2.2.jpg

  15. http://research.cip.cgiar.org/confluence/download/attachments/3023/FIG3-BAC.JPGhttp://research.cip.cgiar.org/confluence/download/attachments/3023/FIG3-BAC.JPG

  16. www.mindfully.org/GE/Vitaly-Citovsky-Projects.htm

  17. UC-Davis (BS & PhD) Univ Wash (MS) At Monsanto for nearly 20 years—developed “…methods to specifically target our genetic engineering tool, Agrobacterium, to the right cells at the right time.” Produced Roundup Ready Soybean Now Chief Technology Officer--ArborGen Biotechnologist of the dayMaud Hinchee

  18. Agroinfiltration-transient method of expressing transgenes Figure 10.5

  19. Agroinfiltration—forcing Agrobacterium with transgenes into leaves

  20. Agroinfiltration--tobacco

  21. Power T via agroinfiltration

  22. Stable transformation using Agrobacterium • Floral dip transformation of Arabidopsis • Seems to transform ovule • Not easily conducive for other species • Most species: using organogenesis or embryogenesis-based tissue culture methods to regenerate transgenic plants

  23. nature.com homepage • Jump to main content • Jump to navigation • Login • Publications A-Z index • Browse by subject Floral dipping Arabidopsis From the following article Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method Xiuren Zhang, Rossana Henriques, Shih-Shun Lin, Qi-Wen Niu and Nam-Hai Chua Nature Protocols 1, 641 - 646 (2006) doi:10.1038/nprot.2006.97

  24. Arabidopsis floral dip : www.plantmethods.com/content/2/1/16/figure/F1

  25. Most plants still need tissue culture for transformation and regeneration http://wwww.cirad.fr/presentation/programmes/biotrop/resultats/images/agrobac.gif

  26. Key steps for traditional Agrobacterium-mediated transformation • Infection (cocultivation) and DNA transfer—Agrobacterium strain and acetosyringone • Kill off unwanted Agrobacterium after gene transfer • Selection methods to prevent escapes • Plant regeneration

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