9/16/2014

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Introduction

Need for marker free

transgenics

Approaches and applications of marker

free transgenics

content

Conclusions

Introduction4

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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

MAINSTRATEGIES

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

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Co-integration

T0

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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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2. Site-specific recombination mediated SMG removal

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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

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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

32Bala et al. (2013)

Cre gene

ASAL

hpt

Molecular analysis of marker gene excision.

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PCR analysis of F2 progeny plants.

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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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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

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R/RS recombination system from

Zygosaccharomyces rouxii

3. Transposon-based SMG removal

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3. Transposon-based SMG removal

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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(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

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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4. Positive selection

Kunze et al. (2001)

DOGR1 gene as alternative selectable marker

5. Methods of direct transformant screening

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