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?

3. What are some ways that the biological method for DNA introduction (Agrobacterium) has been improved over the years?

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?

Formula

Tissue culture + DNA delivery and integration = transgenic plants

Methods of delivering DNA into plant cells

• Biological– Agrobacterium– Other bacteria– Viruses

• Physical– Particle bombardment– Electroporation – Silicon carbide whiskers– Carbon nanofibers

Transformation overview

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

Agrobacterium tumefaciens

Nature’s little plant transformer

Causal agent of crown gall disease

microbewiki.kenyon.edu/index.php/Agrobacterium http://pubs.caes.uga.edu/caespubs/pubcd/images/B1286-17.jpg

Figure 10.1

Figure 10.2

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

Figure 10.4

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

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

http://www.nature.com/nature/journal/v433/n7026/images/433583a-f2.2.jpg

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

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

Biotechnologist of the dayMaud 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

Figure 10.5

Agroinfiltration-transient method of expressing transgenes

Agroinfiltration—forcing Agrobacterium with transgenes into

leaves

Agroinfiltration--tobacco

Power T via agroinfiltration

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

From the following articleAgrobacterium-mediated

transformation of Arabidopsis thaliana using

the floral dip methodXiuren Zhang, Rossana

Henriques, Shih-Shun Lin, Qi-Wen Niu and Nam-Hai

ChuaNature Protocols 1, 641 -

646 (2006)doi:10.1038/nprot.2006.97

Floral dipping Arabidopsis

Arabidopsis floral dip

: www.plantmethods.com/content/2/1/16/figure/F1

Most plants still need tissue culture for transformation and regeneration

http://wwww.cirad.fr/presentation/programmes/biotrop/resultats/images/agrobac.gif

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