1. Plant transformation II Neal Stewart
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. Tissue culture + DNA delivery and integration = transgenic plants
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

7. Agrobacterium tumefaciens
Nature’s little plant transformer

8. Causal agent of crown gall disease
microbewiki.kenyon.edu/index.php/Agrobacterium

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

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.

15. http://research. cip. cgiar

17. Biotechnologist of the day Maud Hinchee
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

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. From the following article
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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, (2006)
doi: /nprot

24. Arabidopsis floral dip:

25. Most plants still need tissue culture for transformation and regeneration

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