1. Lecture 25 The future of transgenic plants Chapter 16 Neal Stewart

2. 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?

3. 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.

5. Problems in plant biotechnology: might 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.

6. The case of “Terminator” technology AKA Technology Protection System AKA Gene Use Restriction Technology

7. 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
Recombinase gene
Promoter
Toxin 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.
Recombinase gene
Promoter
Toxin gene
Toxin protein
Stewart 2004, Genetically Modified Planet Fig 5.2

8. Figure 16.1
Figure 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. A.
(BP’)
(PB’) 1 2 3 4 3 2
(PP’) 1 4
(BB’) B.
(BB’)
(PP’) 1 2 3 4 1
(BP’) 3 2
(PB’) 4 C.
(BB’)
(BP’) 1 2 1 4 3
(PP’) 4 3
(PB’) 2
This figure is slightly different from the one in the book—correct.

9. Figure 16.2
Figure Renessen’s 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
cordapA
LY038

10. Site-specific recombinase-mediated transgene excision
loxP
Transgene
Cre
Transgene
loxP
loxP lo

11. Figure 16.3
trait
nptII
rec inducible
Recombinase gene induced
by developmental cues
Figure 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.

12. Site-specific recombinase-mediated transgene excision in pollenRS LB RB
Pollen genome
Pollen-specific promoter LAT52 activates recombinase in polle
excision
LAT52 pro
Recombinase
NOS ter
GUS- NPTII
35S pro
35S ter
GUS-NPTII
Luo et al Plant Biotechnol J 5:263

13. GM-gene-deletor system (Luo et al. 2007 Plant Biotechnol J 5:263)
No recombinase vector
Cre-loxP/FRT vector

14. Fused recombination sites increase efficiency of excision
Luo et al Plant Biotechnol J 5:263

15. Hudson et al 2001 Mol Ecol Notes 1:321

16. GFP marker for field trialsRB
LAT52 pro
Recombinase
NOS ter RS
Bar LB
NOS pro
GFP
LAT59 pro
35S ter
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

17. Zinc finger nucleases

18. ZFNs in gene therapy
Nature 435:577

19. Double-strand break by zinc finger nuclease
Promoter activates ZFN
5’-TTCTTCCCCGAATTCGGGGAAGAA-3’
ZFN recognition sites
Promoter
Zinc finger Nuclease
Ter
3’-AAGAAGGGGCTTAAGCCCCTTCTT-5’
ZFN cutting sites
Plant genome
5’-TTCTTCCCCG
3’-AAGAAGGGGCTTAA
Double-strand Break
GCCCCT TCT T-5’
AATTCGGGGAAGAA-3’
Double-strand break occurs between ZFN recognition sites

20. Zinc finger nuclease-mediated transgene excision in pollen
Pollen genome
Pollen-specific promoter LAT52 activates ZFN in pollen
excision LB RB R
LAT52 pro
ZFN
NOS ter
NPTII
35S pro
35S ter
Excision sites

21. ZFN constructs
5’-TTCTTCCCCGAATTCGGGGAAGAA-3’
3’-AAGAAGGGGCTTAAGCCCCTTCTT-5’
QQR ZFN recognition sites
LAT52 pro
QQR ZFN
Ter
35S pro
GUS::NPTII RB LB
5’-TTCTTCCCCGAATTCGGGGAAGAA-3’
3’-AAGAAGGGGCTTAAGCCCCTTCTT-5’
QQR ZFN recognition sites
LAT52 pro
Ter
35S pro
GUS::NPTII RB LB
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 PNAS 102:2232

22. Figure 16.4
CTCCCTGTC
GCCACTCTC 1 2 3 4 2’ 3’ 1 2 3 4 1 2’ 3 4
Figure 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.

23. Last questions
Is food too emotionally hot to be addressed by biotechnology? Where on earth?
What is the scientist’s role here?
What about non-food plant biotechnology such as bioenergy?

24. “Ordinary tomatoes do not contain genes, while genetically modified ones do”
People in different countries have varied knowledge about the facts of genetics and biotechnology.
Slide courtesy of Tom Hoban

25. American consumers’ trust in biotechnology information sources
Slide courtesy of Tom Hoban

26. Source of information trusted most to tell the truth about biotechnology (includes all European countries)
Slide courtesy of Tom Hoban

27. Path to cellulosic ethanol

28. Bioenergy and plant genomics: Expanding the nation’s renewable energy resources
Tomorrow
Carbon
allocation
Today
Short rotation
hardwoods
High yield
wood crops
Accelerated Domestication
Conventional
Forestry
Yesterday
Whole Genome Microarrays
Yesterday
In the eighties and early nineties ORNL identified the most promising species for biomass production. Leading a partnership with Universities and the USDA, ORNL undertook a major effort to take two essentially wild species - poplar and switchgrass - and improve their productivity. Cutting edge plant physiology that sought to understand the mechanisms that influenced productivity and survival of these species guided the traditional breeding efforts. Allocation of photosynthate to above and below ground parts, the control of plant hormones on branch angle and subsequently plantation productivity, and osmotic regulation under drought conditions were elucidated in combined field and lab experiments.
Today
The success of these past studies is evident in the 60,000+ acres of commercial poplar crops now being grown in the US for fiber and bioenergy demonstration facilities fueled with switchgrass. While field trials and traditional physiological studies are of course still important, ORNL is applying the new tools of genomics and metabolic profiling to the task of increasing productivity and modifying plant chemical characteristics for easier conversion into new bioproducts and biofuels such as ethanol. With JGI, ORNL is sequencing the the first tree genome – poplar and is organizing an international consortium of scientists to guide this effort. Concurrent with the sequencing efforts are functional genomic studies exploring the genetic basis of carbon allocation and partitioning in poplar trees.
Tomorrow
With the new tools of genomics, it may be possible to domesticate poplar within decades rather than the thousands of years it took to domesticate corn. If the US dedicated 39 million acres of farmland ( less acreage than that currently devoted to corn) to the production of a domesticated poplar with yields double those of today, the US could provide enough raw biomass for ethanol production to replace ALL projected oil imports from the Persian Gulf. By combining fundamental plant physiology with traditional tools of breeding and new insights of genomics, ORNL scientists are leading the US to a new, renewable energy future.
Metabolic
Profiling
Brian Davison ORNL 28

29. Cell wall structure
Nature Reviews Molecular Cell Biology 2, (2001) 29

30. Dixon and Chen 2007 Nature Biotechnology 25: 759-761

31. Dixon and Chen 2007 Nature Biotechnology 25: 759-761

32. 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