Transcript ppt - Department of Plant Sciences

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
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 Tstrand, 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.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
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