Lecture 25 The future of transgenic

plantsChapter 16

Neal Stewart

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?

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.

http://www.youtube.com/watch?v=QVeVIM1yRMU

http://www.rt-pcr.com/

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.

The case of “Terminator” technologyAKA Technology Protection System

AKA Gene Use Restriction Technology

http://cls.casa.colostate.edu/TransgenicCrops/terminator.html

Promoter Toxin geneRecombinase gene

Recombinase protein

Promoter Toxin gene

Blocking DNA

Recombinase gene

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.

2.

1.

Toxin protein

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.

Stewart 2004, Genetically Modified Planet Fig 5.2

Ethanol-inducible promoter

Figure 16.1

41

3 2

1 2 3 4

1 2 3 4

1

23

4

1 2

3 4

1 4

3 2

A.

B.

C.

(BB’)

(PP’)

(BP’) (PB’)

(BB’)(PP’)

(PB’)(BP’)

(BB’)

(PP’)

(BP’)

(PB’)

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.

This figure is slightly different from the one in the book—correct.

Figure 16.2

LY038cordapA

cordapA nptII

cross in cre gene

segregate away cre gene

Figure 16. 2 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.

Site-specific recombinase-mediated transgene excision

Transgene Cre loxPloxP

Cre Transgene loxPloxP

loxPlo

Figure 16.3

trait nptII

Recombinase gene induced by developmental cues

rec inducible

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.

Site-specific recombinase-mediated transgene excision in pollen

RS

LBRBPollen genome

Pollen-specific promoter LAT52 activates recombinase in polle

excision

LAT52 pro Recombinase NOS ter RS GUS- NPTII35S pro 35S terRS

LAT52 pro Recombinase NOS terRS

LB

RS GUS-NPTII35S pro 35S ter

RBPollen genome

Luo et al. 2007 Plant Biotechnol J 5:263

GM-gene-deletor system(Luo et al. 2007 Plant Biotechnol J 5:263)

Cre-loxP/FRT vector

No recombinase vector

Fused recombination sites increase efficiency of excision

Luo et al. 2007 Plant Biotechnol J 5:263

Hudson et al 2001 Mol Ecol Notes 1:321

GFP marker for field trials

RB

LAT52 pro Recombinase NOS terRS Bar NOS ter

LB

RSNOS pro GFPLAT59 pro 35S ter

RB

LAT52 pro NOS terRS Bar NOS ter

LB

RSNOS pro GFPLAT59 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

Zinc finger nucleases

www.bmb.psu.edu, www.wpclipart.com, www.faculty.ucr.edu

ZFNs in gene therapy

Nature 435:577

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

Promoter Zinc finger Nuclease Ter 5’-TTCTTCCCCG

3’-AAGAAGGGGCTTAA

ZFN recognition sites

Double-strand Break

GCCCCT TCT T-5’

AATTCGGGGAAGAA-3’Plant genome

Double-strand break occurs between ZFN recognition sites

Zinc finger nuclease-mediated transgene excision in pollen

Pollen genome

Pollen-specific promoter LAT52 activates ZFN in pollen

excision

LBRBPollen genomeR R

LAT52 pro ZFN NOS ter NPTII35S pro 35S terR R

LAT52 pro ZFN NOS ter NPTII35S pro 35S ter

RB LB

R R R R

Excision sites

ZFN constructs

5’-TTCTTCCCCGAATTCGGGGAAGAA-3’

3’-AAGAAGGGGCTTAAGCCCCTTCTT-5’

QQR ZFN recognition sites

LAT52 pro QQR ZFN Ter 35S pro GUS::NPTII Ter 5’-TTCTTCCCCGAATTCGGGGAAGAA-3’

3’-AAGAAGGGGCTTAAGCCCCTTCTT-5’

QQR ZFN recognition sites

RB LB

5’-TTCTTCCCCGAATTCGGGGAAGAA-3’

3’-AAGAAGGGGCTTAAGCCCCTTCTT-5’

QQR ZFN recognition sites

LAT52 pro Ter 35S pro GUS::NPTII Ter 5’-TTCTTCCCCGAATTCGGGGAAGAA-3’

3’-AAGAAGGGGCTTAAGCCCCTTCTT-5’

QQR ZFN recognition sites

RB LB

• ZFN domain under the control of pollen specific promoter LAT52• ZFN recognition sites • GUS and NPTII fusion under the control of 35SLloyd et al. 2005 PNAS 102:2232

Figure 16.4

1 2 3 4

2’ 3’

1 2’ 3 4

1 2 3 4

CTCCCTGTC GCCACTCTC

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.

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?

1996 - 1998

40

48

46

51

35

36

32

34

45

52

38

21

24

27

44

20

39

22

45

33

22

31

30

22

21

44

29

44

10

15

0 20 40 60 80 100

United Kingdom

Switzerland

Sweden

Netherlands

Italy

Germany

France

Austria

United States

Canada

Percent Response

False (Correct) Don't Know True

“Ordinary tomatoes do not contain genes, while genetically modified ones do”

Slide courtesy of Tom Hoban

People in different countries have varied knowledge about the facts of genetics and biotechnology.

American consumers’ trust in biotechnology information sources

4

6

7

11

16

20

20

28

32

41

43

48

52

52

54

61

66

59

51

46

52

46

41

37

30

19

14

12

17

13

0% 20% 40% 60% 80% 100%

Activist Groups

Chefs

Food Manufacturers

Biotechnology Companies

TV News Reporters

Farmers

Registered Dietitians

University Scientists

Food and Drug Administration

American Medical Association

A Lot Some None Slide courtesy of Tom Hoban

7

9

4

8

9

16

17

20

6

6

4

7

7

21

14

26

0 5 10 15 20 25 30

None (Spontaneous)

Don't Know

Mass Media

Government

Universities

Medical Profession

Environmental Groups

Consumer Organizations

Percent Response

1999

1996

Source of information trusted most to tell the truth about biotechnology(includes all European countries)

Slide courtesy of Tom Hoban

Path to cellulosic ethanol

Whole Genome Microarrays

Yesterday

TodayTomorrow

Accele

rated

Domestica

tion

MetabolicProfiling

Carbon allocation

ConventionalForestry

Short rotation hardwoods

High yield wood crops

Bioenergy and plant genomics:Expanding the nation’s renewable

energy resources

Brian Davison ORNL

Cell wall structure

Nature Reviews Molecular Cell Biology 2, 33-39 (2001)

Dixon and Chen 2007 Nature Biotechnology 25: 759-761

Dixon and Chen 2007 Nature Biotechnology 25: 759-761

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