Plant transformation II Neal Stewart 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?

Tissue culture + DNA delivery and integration = transgenic plants 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

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

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

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

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

Agroinfiltration-transient method of expressing transgenes Figure 10.5

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 article nature.com homepage Jump to navigation Jump to main content Login   Browse by subject Publications A-Z index 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

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