Post on 31-Dec-2015
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?
Methods of delivering DNA into plant cells
• Biological– Agrobacterium– Other bacteria– Viruses
• Physical– Particle bombardment– Electroporation – Silicon carbide whiskers– Carbon nanofibers
Causal agent of crown gall disease
microbewiki.kenyon.edu/index.php/Agrobacterium http://pubs.caes.uga.edu/caespubs/pubcd/images/B1286-17.jpg
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
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.
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
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
Most plants still need tissue culture for transformation and regeneration
http://wwww.cirad.fr/presentation/programmes/biotrop/resultats/images/agrobac.gif