marker free transgenics: concept and approaches
TRANSCRIPT
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Introduction
Need for marker free
transgenics
Approaches and applications of marker
free transgenics
content
Conclusions
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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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
Controversy and disadvantages related to SMG
ON
TA
RG
ET
• To eliminate selectable marker gene
• To avoid use of toxic selectable marker gene
OUR AIM
Marker free transgenic
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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.
Narendra Tuteja et al., 2012
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Marker free sheath blight resistance rice by co transformation
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 plantsSripriya and Raghupathy. (2008)
Hind III
17Sripriya and Raghupathy. (2008)
CoT6 CoT23
Southern blot analysis of T1 populations from CoT6 & CoT23
18Jaing et al. (2013)
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).
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PCR and leaf painting analysis of T0 transformation events derived
from pDTRSVCP and pDTRSVSP
Jaing et al. (2013)
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PCR analysis of T1 transformation events derived from pDTRSVCP and pDTRSVSP
Jaing et al. (2013)
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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)
Varieties TransgenicLines
Test # of Bastaresistantplants
# of cecropinBPCR(+) plants
Co-segregationfrequency (%)
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)
Transgenicplant lines
Germination percentage ofT1 seeds (%)
T1 Basta-resistantplant number
T1 Basta-sensitiveplant 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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The result of producing transgenic plants carrying cecropinB gene
cassette without selectable marker bar in T1 generation
Yan et al. (2007)
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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.
Yan et al. (2007)
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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(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.
Narendra Tuteja et al., 2012
Constitutive Expression of Recombinase Gene
• Plant hybridization:
• Retranformation
• 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
Induced Expression of
Recombinase Gene
The Cre/lox system
x
F1 Plant containing both transgene screened for SMG
deletion event
Transforme
d plant
GOI &
SMG
Transformed
plant
RECOMBINAS
E CRE
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Schematic representation of the T-DNA region of two binary
vectors used for mustard transformations.
Bala et al. (2013)
A - pBKhgASAL showing ASAL gene & B - pBK16.2 showing the cre gene
Aphid resistant marker free transgenic mustard.
31Bala et al. (2013)
DNA blot analysis for confirming vector integration in T0
Lane 1 & 9 362 bp positive control for ASAL, Lane 2 & 10 negative control
ASAL Cre
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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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RBTerminator FLPPromoterLB
Li et al. (2010)
Salt tolerant marker free transgenic maize
0.9 kb D1D2
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Southern blotting analysis showing the presence of transgene
AtNHX1 and flp in the genome of the transgenic F1 plants.
BamH I Kpn I
Schematic representation of the Ds-cry1B T-DNA.
44Olivier et al. (2002)
marker-free rice plants expressing a Bt
endotoxin gene
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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
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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