Marker free transgenics for better and safer GMO for food and health security

1. 9/16/2014
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2. 2

3. 3
Introduction
Need for marker free
transgenics
Approaches and applications of marker
free transgenics
content
Conclusions

4. Introduction
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5. 5

6. Selectable markers (SMGs)
Selectable markers are those which allow the selection of
transformed cells, or tissue explants, by their ability to
grow in the presence of an antibiotic or a herbicide
The selective agents are generally used in the initial
stages of transformation for an early selection of
transgenic cells.
Once transgenic plant is selected ,marker gene is no
longer necessary and remain as integral part of plant
genome in transgenic plants.
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9. Controversy and disadvantages related
to SMG
1. Food safety , effect on natural ecosystem
2. Gene flow into non-GM crops, human
and animal bacteria, wild and weedy relatives
3. Inability for gene stacking in already
transformed plant with same SMG

10. ON TARGET
OUR AIM
• To eliminate selectable marker gene
• To avoid use of toxic selectable marker gene
Marker free transgenic

11. MAIN
STRATEGIES

12. 1. Co-transformation of marker
genes and gene of interest (GOI)

13. 13
Co-integration
T0

14. Schematic diagram of Co-transformation method for making marker free transgenic
plants. (a) Physical diagram of two T-DNA region showing gene of interest (GOI) and
marker gene. (b) Transformed calli having GOI and marker gene. (c) T0 plant having
GOI and marker gene. (d) Two T1 plants one with GOI and another with marker gene.
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Narendra Tuteja et al., 2012

15. Marker free sheath blight resistance rice by co transformation
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technique
Sripriya and Raghupathy. (2008)

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Southern blot analysis of T0 lines for chitinase gene
P- positive control, M -HindIII marker, E-empty lane, U undigested DNA from the
transgenic plants
Sripriya and Raghupathy. (2008)
Hind III

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CoT6 CoT23
Southern blot analysis of T1 populations from CoT6 & CoT23
Sripriya and Raghupathy. (2008)

18. Rice stripe virus (RSV)
The RSV genome consists of four single-stranded RNA segments,
designated as RNAs 1 to 4.
The complementary sense
• RNA 3 encodes the coat protein (CP)
• RNA 4 encodes the special-disease protein (SP).
18 Jaing et al. (2013)

19. PCR and leaf painting analysis of T0 transformation events derived
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from pDTRSVCP and pDTRSVSP
Jaing et al. (2013)

20. PCR analysis of T1 transformation events derived from pDTRSVCP and pDTRSVSP
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Jaing et al. (2013)

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22. Particle bombardment method
(leaf bacterial blight resistant rice)
Schematic maps of the source plasmid pCB1
and pCB4
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Act Rice actin-1 promoter, cB cecropinB gene encoding sequence, Pin potato
proteinase inhibitor II terminator,
Yan et al. (2007)

23. Varieties Transgenic
Lines
Test # of Basta
resistant
plants
# of cecropinB
PCR
(+) plants
Co-segregation
frequency (%)
Xiushui 04 XIF-41 30 30 100
XIF-42 30 27 90.0
Jia59 J4F-17 30 25 80.6
J4F-18 30 9 30.0
J4F-49 30 12 40.0
J4F-50 30 12 40.0
J4F-51 30 11 36.7
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The co-segregation frequency of bar and cecropinB gene cassettes in
transgenic rice lines of T1 generation
Yan et al. (2007)

24. The result of producing transgenic plants carrying cecropinB gene
cassette without selectable marker bar in T1 generation
Transgenic
plant lines
Germination
percentage of
T1 seeds (%)
T1 Basta-resistant
plant number
T1 Basta-sensitive
plant number
cecropinB PCR
(+) plant number
of T1
Basta-sensitive
plants
J4F-49 58 42 16 0
J4F-50 49 17 32 2
J4F-51 62 43 19 0
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Yan et al. (2007)

25. M DNA molecular weight markerIII, U untransformed rice plant control; 1 R0 plant of
J4F-50, 2 R0 plant of J4F-51, 3 and 4 marker-free transgenic plants of MFc-1and
MFc-2 carrying cecropinB gene cassette only.
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Yan et al. (2007)

26. Advantages
Simple and effective
Easier handling of the binary vectors because the two T-DNA
are separated
Disadvantages
It is time consuming and compatible only for sexually propagated
fertile plants.
The tight linkage between co-integrated DNAs may limit the
efficiency of co-transformation
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27. 2. Site-specific recombination
mediated SMG removal

28. (a) The T-DNA region showing Cre gene followed by the transcribed mRNA and Cre
protein expression. (b) T-DNA region showing GOI and marker gene merged between
loxP sites. (c) Resulting transgenic plants showing excision of marker gene.
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Narendra Tuteja et al., 2012

29. The Cre/lox system
Constitutive Expression of
Recombinase Gene
• Plant hybridization:
Transforme
d plant
GOI &
SMG
• Retranformation
Induced Expression of
Recombinase Gene
• Simultaneous transformations:
1st T-DNA (IP + Cre)
2nd T-DNA (SMG + GOI)
• Heat shock treatment
• Chemical treatment
• By activating the promoters with
inducers (heat or chemical), the
expression of recombinase gene
can be more tightly controlled.
• Autoexcision scheme
x
Transformed
F1 Plant containing both
transgene screened for SMG
deletion event
plant
RECOMBINAS
E CRE

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Aphid resistant marker free transgenic mustard.
Schematic representation of the T-DNA region of two binary
vectors used for mustard transformations.
A - pBKhgASAL showing ASAL gene & B - pBK16.2 showing the cre gene
Bala et al. (2013)

31. DNA blot analysis for confirming vector integration in T0
ASAL Cre
Lane 1 & 9 362 bp positive control for ASAL, Lane 2 & 10 negative control
31 Bala et al. (2013)

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Cre gene
ASAL
hpt
Molecular analysis of marker gene excision.
Bala et al. (2013)

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PCR analysis of F2 progeny plants.

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35. Auto excision mediated Cre/lox system with floral specificOsMADS45 promoter :
Bai et al. (2008)
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3.3kb gus

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FLP/FRT recombination system
Fig. FLB/frt site-specific recombination system. (a) The T-DNA region showing FLP gene controlled by heat
inducible promoter (hsp70) followed by the transcribed mRNA and FLP protein expression. (b) T-DNA region
showing GOI and marker gene merged between frt sites followed by resulting transgenic plants showing
excision of marker gene.

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Salt tolerant marker free transgenic maize
LB Promoter FLP Terminator RB
Li et al. (2010)
0.9 kb D1D2

39. Southern blotting analysis showing the presence of transgene
AtNHX1 and flp in the genome of the transgenic F1 plants.
BamH I Kpn I
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R/RS recombination system from
Zygosaccharomyces rouxii

42. 3. Transposon-based SMG removal

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3. Transposon-based SMG removal

44. marker-free rice plants expressing a Bt
Schematic representation of the Ds-cry1B T-DNA.
Olivier et al. (2002) 44
endotoxin gene

45. (A) Investigation of T-DNA organisation in T0
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Recovery of hph selectable marker free
Cry1B transgenic rice in T1 generation
Southern blot of EcoRI and BamHI of 30 T1 plants

49. Advantages
Suitable for removal of marker genes in vegetatively propagated
plants
Disadvantages
Variable rates of transposition
Labour and cost intensive
Mutations
Genomic instability
Decreased efficiency
Scutt et al., 2002
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50. 4. Positive selection

51. Kunze et al. (2001)
DOGR1 gene as alternative selectable marker

52. 5. Methods of direct transformant screening

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54. s