Lecture 25 The future of transgenic plants Chapter 16 Neal Stewart

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Lecture 25 The future of transgenic
plantsChapter 16Neal Stewart
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Discussion questions

• What is the main dichotomy between innovation and
  caution (or risk, or the perception of risk)?
• What is real-time PCR and why is it better than
  regular PCR?
• Describe site-specific recombination and how it
  could lead to greater precision in plant
  transformation.
• How might site-specific recombination enhance
  biosafety?
• What are zinc-finger nucleases, and how might
  they alter the future of plant biotechnology?
• How do feelings and trust influence plant
  biotechnology?
• What are key issues in future applications in
  bioenergy?

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Real-time PCR or Quantitative PCR

• Real-time PCR uses fluorescence as an output for
  DNA amplification in real-time.
• The amount of starting template DNA (or cDNA for
  RNA measurement (real-time RT-PCR) is correlated
  with the Ct number.
• More DNA lower Ct Ct is the cycle number when
  a threshold amount of DNA is produced.

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http//www.rt-pcr.com/
http//www.youtube.com/watch?vQVeVIM1yRMU
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Problems in plant biotechnologymight be
addressed with new technologies

• Agrobacterium- and especially biolistics-mediated
  transformation are imprecise
• Transgenic plants are regulated because they are
  transgenic
• Gene flow (hybridization and introgression)
  remains to be a major issue in regulation.

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The case of Terminator technologyAKA
Technology Protection SystemAKA Gene Use
Restriction Technology
http//cls.casa.colostate.edu/TransgenicCrops/term
inator.html
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1. A recombinase gene is under the control of an
ethanol inducible promoter. In this case no
ethanol is applied. Result toxin gene is not
expressed since blocker DNA remains in place and
seeds can germinate.
1.
Ethanol-inducible promoter
Blocking DNA
Toxin gene
Promoter
Recombinase gene

2. Ethanol is applied and turns on expression of
recombinase gene. The recombinase acts to remove
the blocking DNA from the toxin gene. Result
toxin gene is expressed and kills embryo in seeds
so they cannot germinate.
Recombinase protein

2.
Promoter


Toxin gene
Recombinase gene
Toxin protein
Stewart 2004, Genetically Modified Planet Fig 5.2
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Figure 16.1
Figure 16.1 Recombination between recombination
sites (arrowheads) leading to (A) deletion
(excision of circular molecule 2,3 from molecule
1,2,3,4 or integration (insertion of molecule
2,3 into molecule 1,4 (B) inversion (of DNA
segment 2,3 flanked by recombination sites of
opposite orientation) or (C) translocation (of
DNA of different molecules). Some recombination
systems use recombination sites that differ in
sequence generally known as attB, attP, attL and
attR, here shown as BB, PP, BP and PB,
respectively. In these systems, recombination
between attL and attR requires an excisionase
protein in addition to an integrase protein.
(BP)
(PB)
A.
1
2
3
4
3
2
(PP)
4
1
(BB)
(PP)
B.
(BB)
1
2
3
4
1
2
3
4
(BP)
(PB)
C.
(BB)
(BP)
1
2
1
4
3
4
3
2
(PP)
(PB)
This figure is slightly different from the one in
the bookcorrect.
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Figure 16.2
Figure 16. 2 Renessens high lysine corn line
LY038 used site-specific recombination to remove
the transformation selectable marker, the
kanamycin resistance gene nptII, after stable
incorporation of cordapA that directs high lysine
production in seed. Cre recombinase, introduced
from hybridization with a cre transgenic plant,
excised the nptII marker flanked by directly
oriented lox recombination sites. The cre gene
was subsequently segregated away in the following
generation.
cordapA
nptII
cross in cre gene
segregate away cre gene
LY038
cordapA
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Site-specific recombinase-mediated transgene
excision
Transgene
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Figure 16.3
trait
nptII
rec inducible
Recombinase gene induced by developmental cues
Figure 16.3 Recombination sites that flank the
entire transgenic locus permits removal of
transgenic DNA upon induced expression of a
recombinase gene. For instance, if the
recombinase gene is placed under the control of
sperm-specific or fruit-specific promoters, the
excision of transgenic DNA may help reduce the
outcross of transgenes, or minimize the
production of transgene-encoded proteins needed
elsewhere in the plant but not in the edible
portions of food.
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Site-specific recombinase-mediated transgene
excision in pollen
Luo et al. 2007 Plant Biotechnol J 5263
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GM-gene-deletor system(Luo et al. 2007 Plant
Biotechnol J 5263)
No recombinase vector
Cre-loxP/FRT vector
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Fused recombination sites increase efficiency of
excision
Luo et al. 2007 Plant Biotechnol J 5263
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Hudson et al 2001 Mol Ecol Notes 1321
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GFP marker for field trials

• Cre recombinase with loxP recognition sites
• ParA recombinase with MRS recognition sites
• CinH recombinase with RS2 recognition sites
• Cre recombinase with fused loxP-FRT recognition
  sites
• No recombinase with loxP recognition sites

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Zinc finger nucleases
www.bmb.psu.edu, www.wpclipart.com,
www.faculty.ucr.edu
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ZFNs in gene therapy
Nature 435577
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Double-strand break by zinc finger nuclease
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Zinc finger nuclease-mediated transgene excision
in pollen
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ZFN constructs

• ZFN domain under the control of pollen specific
  promoter LAT52
• ZFN recognition sites
• GUS and NPTII fusion under the control of 35S

Lloyd et al. 2005 PNAS 1022232
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Figure 16.4
CTCCCTGTC
GCCACTCTC
1
2
3
4
2
3
1
2
3
4
1
2
3
4
Figure 16.4 A possible approach for homologous
gene replacement in plants. Example shows
replacement of gene 2 by gene 2, mediated by two
heterologous zinc finger nucleases, each binding
a unique 9 bp sequence separated by a spacer of
6 bp. Each zinc finger (triangle) recognizes a
3-nucleotide sequence. Cleavage at the spacer
DNA promotes DNA repair and a higher rate of
homologous recombination.
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Last questions

• Is food too emotionally hot to be addressed by
  biotechnology? Where on earth?
• What is the scientists role here?
• What about non-food plant biotechnology such as
  bioenergy?

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Ordinary tomatoes do not contain genes, while
genetically modified ones do
1996 - 1998
People in different countries have varied
knowledge about the facts of genetics and
biotechnology.
Slide courtesy of Tom Hoban
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American consumers trust in biotechnology
information sources
Slide courtesy of Tom Hoban
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Source of information trusted most to tell the
truth about biotechnology(includes all European
countries)
Slide courtesy of Tom Hoban
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Path to cellulosic ethanol
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Bioenergy and plant genomicsExpanding the
nations renewable energy resources
Tomorrow
Carbon allocation
Today
Short rotation hardwoods
High yield wood crops
Accelerated Domestication
Conventional Forestry
Yesterday
Metabolic Profiling
Brian Davison ORNL
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Cell wall structure
Nature Reviews Molecular Cell Biology 2, 33-39
(2001)
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Dixon and Chen 2007 Nature Biotechnology 25
759-761
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Dixon and Chen 2007 Nature Biotechnology 25
759-761
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Biomass/bioenergy crops

• Should not be food crops
• Should not interfere with food production
• Must be sustainable
• Will probably require biotechnology for better
  yield and cell wall digestion
• Major biosafety issue with transgenic switchgrass
  will be gene flow
• An opportunity to do it right from the beginning