Lecture 10
Plant transformation
Neal Stewart
Discussion questions
1. What is a transgene/transgenic plant?
2. What part or parts of the plant cell provide
the most resistance to DNA introduction?
3. In the case of a successful DNA
introduction, where in the target cell does
the foreign DNA end up?
4. What are some differences between
physical and biological methods for DNA
introduction into plant cells?
Discussion questions
5. What are the size and composition of the
particles that are used for the particle
bombardment method?
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
Keys
•
•
•
•
DNA must be moved into nucleus
DNA must be integrated into the genome
DNA must be stable
Tissue culture techniques are needed for
– Preparation of cells amenable to receive DNA
– Selecting transgenic cells and tissues
– Regenerating transgenic plants
– Transgene(s) inherited in Mendelian fashion
Transformation overview
http://www.ag.ndsu.edu/pubs/plantsci/crops/a1219-2.gif
Physical methods
Naked DNA into cells—most
important and common method is
particle bombardment
Switchgrass transformation
BANG!
Figure 11.7
Figure 11.7 Two different particle bombardment devices: The
commercially available PDS1000 He (BioRad) (left) and the noncommercial Particle Inflow Gun (right).
Figure 11.6
Figure 11.6 Tungsten (left) and gold particles (right) used for particle
bombardment, prior to DNA precipitation. Gold particles are more uniform
and spherical than tungsten particles.
Schematic of the PDS 1000-He
http://www.youtube.com/watch?v=crmj7A1zQ2k
But ignore the last 10 seconds of the video, which consists of nonsense.
Figure 11.8
Figure 11.8 Particle bombardment-mediated transient GFP expression in lima
bean cotyledonary tissues. This target tissue is flat, non-pigmented and ideally
suited for tracking GFP expression in individual transiently transformed cells.
John Sanford
Biotechnologist of the day
• New Yorker (Bronx
native)
• Always wanted to
engineer soybean
• As a graduate student
with John Sanford at
Cornell he was the
first person to
engineer plants using
the gene gun
• A soybean genetic
engineer at
DuPont/Pioneer
Electroporation of protoplasts
Protoplasts are cells stripped of their cell walls and maintained in culture
Switchgrass protoplasts isolated from:
Leaves
Roots
Figure 11.9
Figure 11.9 Maize protoplasts, electroporated with a gfp gene,
showing bright field (left) and with GFP filters (right). Images
provided by JC Jang.
Silicon carbide whiskers
Figure 11.10
Figure 11.10 Nanofiber array with single fiber at higher magnification
(inset). Images provided by Tim McKnight.
Figure 11.11
Figure 11.11 Nanofiber array
introduction of DNA into onion
cells. The white arrows in the
top panel point to dislodged
nanofibers while the arrow in
the bottom panel shows one
fiber embedded in the nucleus
of a cell, expressing an
introduced green fluorescent
protein gene.
Agrobacterium tumefaciens
Nature’s little plant transformer
Figure
11.1
Figure 11.1 Agrobacteriuminduced tumor formation on
dahlia. White arrows show
tubers, black arrows show
formation of large crown galls.
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 11.2
Figure 11.2 Agrobacterium growing
on soybean tissue.
Figure 11.4
Helper plasmid
Figure 11.4 Simple schematic of Agrobacterium-mediated transformation of
a plant cell, showing production of acetosyringone by the plant cell, induction
of the vir genes on the Ti plasmid, generation of the T-strand from the binary
vector, transport through the bacterial pilus, and integration into plant
chromosomal DNA.
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 Science Officer,
Agricen Sciences
Agroinfiltration-transient method of
expressing transgenes
Figure 11.5
Agroinfiltration—forcing
Agrobacterium with transgenes into
leaves
Agroinfiltration--tobacco
Power T via agroinfiltration
Agroinfiltration is useful to
overproduce therapeutic
proteins, such as vaccines
https://www.youtube.com/watch?v=oCGFW1WOFTY
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