GENE STAKING IN CROP PLANTS - A MOLECULAR APPROACH

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1 GENE STACKING IN CROP PLANTS : A MOLECULAR APPROACH

CONTENTS :

2 CONTENTS INTROUCTION STRATEGY FOR GENE STACKING POLYCYSTRONIC TRANSGENE AND ITS EXPRESSION STRATEGY CO- ORDINATING GENE EXPRESSION SELECTABLE MARKER REMOVAL SYSTEM GENE STACKING WITH MULTIPLE TRANSGENES INSECT RESISTANCE DISEASE RESISTANCE ABIOTIC STRESS TOLERANCE METABOLIC PATHWAY MANIPULATION CONCLUSION FUTURE TRUST

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3 Source: Helpin et al., 2005

Table:1 Global status of approved genetically modified (GM) crops with three or more stacked geneS.:

4 Table:1 Global status of approved genetically modified (GM) crops with three or more stacked gene S . Sr.no Trait Crop Genes Method Company event 1 Herbicide tolerance+ fertility restoration Canola Bar; barnase or barstar; neo Agrobacterium linked gene, 1 t-DNA Aventis crop science MS1, RF1, MS1, RF2, PHY14; PHY 35; PHY36 ; 2 Herbicide tolerance+ fertility restoration Chicory bar; barnase; neo Agrobactarium/linked gene, 1 T-DNA Bejo zaden BV RM3-3;RM3-4;RM3-6 3 Multiple virus resistance Squash Neo*; CP-CMV; CP-ZYMV;;CP-WMV2 Agrobactarium/linked gene, 1 T-DNA Asgrow (USA);seminis vegetable Inc.(canada) CZW-3 4 Multiple virus resistance Squash Neo*; CP-ZYMV;;CP-WMV2 Agrobactarium/linked gene, 1 T-DNA Upjohn (USA);seminis vegetable Inc.(canada) ZW20 5 Modifide colour + herbicide tolerant † Carnation surB †;dfr;hfl Agrobactarium/linked gene, 1 T-DNA Florigene Pty Ltd. 4,11,15,16 6 Modifide colour + herbicide tolerant † Carnation surB †;dfr;bp40 Agrobactarium/linked gene, 1 T-DNA Florigene Pty Ltd. 959A;988A;1226A;1351A;1363A;1400A; 7 Insect resistance + herbicide tolerance Cotton Bxn;cry1Ac;neo Agrobactarium/linked gene, 1 T-DNA Calgene Inc. 31807/31808 8 Insect + viruse resistant Potato cer3A;PLRVrep;neo or EPSPS Agrobactarium/linked gene, 1 T-DNA Monsento RBMT21-129;RBMT 21-50;RBMT22-082 9 Insect resistance + herbicide tolerance Maize Bar;cry1Ac;pin// ‡ Biolisics/3 plasmid co-transformation Dekalb genetics corporstion DBT481 10 Insect resistance + herbicide tolerance Maize cry1Ab;EPSPS;gox Biolisics/2 plasmid co-transformation Monsento MON802 MON809 MON810 MON832 ξ Source: Helpin et al., 2005

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5 Table:2 Bt cotton aproved Cry1 Ac & Cry2 Ab MON 15985 Event Bollguard II Source: http://www.envfor.nic.in/divisions/csurv/geac/bt_cotton_LST0506.pdf

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6 Source: http://www.envfor.nic.in/divisions/csurv/geac/bt_cotton_LST0506.pdf

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7 Source: http://www.envfor.nic.in/divisions/csurv/geac/bt_cotton_LST0506.pdf Cont…

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8 Title Reference Pyramiding of bacterial blight resistance genes in rice : MAS using RFLP and PCR N.Huang et al., 1997 A pyramid of loci for partial resistance to fusarium solani f. sp. Glycines maintains Myo -inositol-1-phosphate synthase expression in soybean roots Iqbal et al., 2002. Pyramiding genes affecting sprouting resistance in Rye by means of marker assisted selection Twardowska et al., 2005 Pyramiding quantitative trait locus (QTL) alleles determining resistance to barley stripe rust : effects on resistance at the seedling stage Castro et al., 2003 Strategies for pyramiding resistance genes against the barley yellow mosaic virus complex (BaMMV, BaYMV,BaYMV-2) Werner et al., 2005 Different crop plant pyramided through MAS

STRATEGY FOR GENE STACKING :

9 STRATEGY FOR GENE STACKING ITERATIVE PROCEDURE / SEXUAL HYBRIDIZATION RE- TRANSFORMATION CO- TRANSFORMATION

SEXUAL HYBRIDIZATION METHOD :

10 Limitations: Introduced transgene will be sited at different random loci in plant genome during segregation Each unlinked transgene introduced would double the size of breeding population Labour intensive and time consuming SEXUAL HYBRIDIZATION METHOD

Re-transformation :

11 Re-transformation Limitations: Re- transformation can induce transgene silencing Need for a range of selectable marker gene so that a different one can be used with each sequential transformation. Host cell

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12 Advantages: One step procedure for the introduction of the multiple “effect”gene Transgenes tend to co-intigrate at the same chromosomal position Limitations: High copy number integrating Multiple tandem repeat or inverted repeat – such complex integration pattern leads to transgene silencing Co- transformation Multiple transgenes either harborured within different T-DNA in single Agrobacterium strain or harboured separately within different strain.

Factors affecting Co - ordinated expression of the introduced genes:

13 Factors affecting Co - ordinated expression of the introduced genes Position effect Matrix Attachment Region (MARs) Number of transgenic loci Number of insertion at given locus and stability of each locus Promoter(s)

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14 Promotor homology can be avoided by Using diverse promoter Isolated from different plant and viral genomes Synthetic promoters Identified cis-elements of promoter can be placed In a synthetic stretch of DNA different from its own native DNA, context to create a functionally similar promoter with ‘novel’ DNA sequences ‘Domain swapping’-cis element of the promoter can be replaced with functionally equivalent regions form heterologous promoters

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15 A1 A TATA Bhular et al., 2003 India 35S

Polycistronic transgenes One way of overcoming the difficulties of co-ordinating the expression of different transgenes without duplicating the regulatory sequences is to express several ‘effect genes’ from a single promoter as a single transcription unit. :

16 Polycistronic transgenes One way of overcoming the difficulties of co-ordinating the expression of different transgenes without duplicating the regulatory sequences is to express several ‘effect genes’ from a single promoter as a single transcription unit. Gene 1 Gene 2 Gene 3 Promoter Polyprotein

Polyprotein expression system :

17 Polyprotein expression system IRES- Internal Ribosome Entry Site 2A polyprotein system NIa Protease sequence Protease-susceptible linker sequence

IRES- Internal Ribosome Entry Site:

18 IRES- Internal Ribosome Entry Site It is a common cap independent ribosome scanning system found in viruses like: Potyviridae, Comoviridae, Luteoviridae Crucifer-infecting tobamovirus (crTMV) CR CP,148 , IRES CR MP,75 IRES Dorokhov et al., 2002 Switzerland

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19 A B A A B A IRES IRES

2A polyprotein system :

20 2A polyprotein system It is novel polyprotein cleavage strategy from the FMDV (foot and mouth disease virus) Incorporate the 20 amino acid sequence of FMDV virus, which ensure the polyprotein cleavage. U.K Amrani et al., 2004

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21 1D 2A 2B 2C 1A Lpro 3A 3Cpro 3Dpol QLLNFDLLKLAGDVESNP G P FF 2A 2B 1B 1C

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22 A B A G G P P 2A 2A

NIa Protease sequence :

23 NIa Protease sequence Nuclear inclusion proteins (NIa) Plant potyviruses such as tobacco etch virus (TEV) and tobacco vein mottling virus (TVMV) having specific heptapeptide sequences which are responsible for processing of large viral polyproteins. A B 48kDa NIa protease sequences Source: Helpin et al., 2005

Protease-susceptible linker sequence:

24 Protease-susceptible linker sequence Francois et al., 2002 A B Host protease Host protease susceptible linker

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25 Scutt et al., 2002

Techniques for the removal of marker genes from transgenic plants:

26 Techniques for the removal of marker genes from transgenic plants Simple microbial recombinase-based system Transposable element system Co transformation system An Intrachromosomal Recombination (ICR) system The MAT vector system The CLX chemically inducible system Scutt et al., 2002

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27 MULTIPLE TRANSGENE

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28 Babilli & Beyer, 2005 Fig.A Pathway for β -carotene synthesis Phytoene desaturase Erwinia eredovora Phytoene synthase Lycopene β -cyclase Daffodil ( Narcissus pseudonarcissus )

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29 Fig:B Construct for transformation of β -carotene synthesis pathway in rice

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30 Wild type PSY from Daffodil PSY from Maize/Rice 37 μ g/g carotenoid (84% β -carotene)

Pyramiding transgenes Xa21,Rc7,and Bt for multiple resistance in rice against bactrial blight, sheath blight and yellow stem borer.:

31 Pyramiding transgenes Xa21,Rc7, and Bt for multiple resistance in rice against bactrial blight, sheath blight and yellow stem borer. 35S CaMV Actin 1 Promotor Xa21 Rc7 Bt Datta et al., 2002 Philippines.

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32 Datta et al., 2002 Philippines.

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33 Xa21 RC7 Bt Fig.A Southern blot analysis showing the presence of 3 genes in F 2 , same result was found in F 3 also.

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34 Pant no BB lesion (cm) PCR result (for Xa21 ) SB lesion (%) Southern result (for RC7 ) XYZ-7-10 a 3.2 + 15.6 + XYZ-7-14 3.3 + 20.0 + XYZ-7-16 3.6 + 25.0 + XYZ-7-12 a 4.4 + 6.6 + XYZ-7-18 4.6 + 14.4 + XYZ-7-20 7.4 + 5.9 + XYZ-7-25 8.1 + 15.6 + XYZ-7-21 9.5 + 5.7 + XYZ-7-27 12.9 + 25.0 + XYZ-8-5 7.9 + 22.5 + XYZ-8-2 8.0 + 25.0 + XYZ-8-17 a 8.2 + 15.0 + XYZ-8-26 10.9 + 23.1 + XYZ-8-28 12.2 + 35.0 + XYZ-8-24 12.9 + 13.8 + XYZ-19-1 7.9 + 24.4 + XYZ-19-32 8.7 + 29.4 + XYZ-19-3 10.5 + 26.3 + XYZ-19-15 15.4 + 8.8 + XYZ-21-5 5.2 + 41.3 + XYZ-21-19 a 6.9 + 11.9 + XYZ-21-10 8.1 + 14.8 + XYZ-21-1 10.0 + 33.8 + XYZ-21-11 10.1 + 23.5 + XYZ-21-15 10.4 + 22.5 + Table:3 Different lines carrying the Xa21 and RC7 genes showing a variable response to there respective bioassay All lines are Bt + & show 100% mortality Datta et al., 2002 Philippines.

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35 85% Resistance of transgenic tobacco containing β -hth + na-pi genes against Helicoverpa armigera HIGH MORTALITY 85% 50% 40% 28% Pest and disease protection conferred by expression of barley β - hordothionin( β -hth ) and Nicotiana alata proteinase inhibitor ( na-pi) genes in transgenic tobacco against Helicoverpa armigera , grey mold and bacterial wilt. Charity et al ., 2005 Australia Fig:A Fig: B

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36 β -hth na-pi β -hth + na-pi control Fig.A Fig.B Resistance of transgenic tobacco containing β -hth + na-pi genes against grey mold ( Botrytis cineria ) 72 hrs after inoculation 170mm 2 80% Charity et al ., 2005 Australia 80mm 2

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37 Resistance of transgenic tobacco containing β -hth + na-pi genes against bacterial wilt ( Pseudomonas solanacearum ) 7 day after inoculation 94% 75% 64% Fig.A Fig.B Charity et al ., 2005 Australia β -hth + na-pi β -hth Control

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38 INSECT RESISTANCE

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39 Table: 4 Toxicity effect of Cry1 B-Cry1 Ab fusion protein expressed in BT transgenic Cabbage plant on DBM larve Type Stage of larvae Released No. of larvae released Leaf damage Mortality (%) Time (hrs) taken to kill the larvae Leaf disc Bioassay Neonate 10 NIL 100 24 Second instar 10 NIL 100 48 detached Leaf Bioassay Second instar 15 NIL 100 48 third instar 15 NIL 100 48 fourth instar 15 NIL 100 48 Whale plant Bioassay Neonate 20 NIL 100 48 Second instar 40 NIL 100 48 third instar 40 NIL 100 48 Paul et al., 2005 India

Table: 5 Control of Cry 1AR or Cry 1CR neonate diamond back moth (DBM) larvae by detached leaves of Cry 1 Ac, Cry 1C, Cry1Ac+Cry1C brocolli plants 3 days after infestation. :

40 Table : 5 Control of Cry 1A R or Cry 1C R neonate diamond back moth (DBM) larvae by detached leaves of Cry 1 Ac, Cry 1C, Cry1Ac+Cry1C brocolli plants 3 days after infestation. Plant Bt gene Susceptible DBM Cry 1 A R DBM Cry 1 C R DBM Defoliation % Mortality % Defoliation % Mortality % Defoliation % Mortality % Green comet - 40 0 ± 0 30 10 ± 0 30 13 ± 12 Q23 cry1Ac 0 100 ± 0 25 23 ± 15 3-5 87 ± 6 Q7 cry1Ac 25 33 ± 6 30 27 ± 12 20 40 ± 10 H14 cry1C 0 100 ± 0 0 100 ± 0 30 37 ± 6 H12 cry1C 0 100 ± 0 0 100 ± 0 25 30 ± 10 H1 cry1C 0 100 ± 0 0 100 ± 0 15 30 ± 0 H3 cry1C 0 100 ± 0 0 100 ± 0 20 30 ± 10 Q23 X H1 cry1Ac + cry1C 0 100 ± 0 0 100 ± 0 0-5 90 ± 10 Q23 X H2 cry1Ac + cry1C 0 100 ± 0 0 100 ± 0 0-5 90 ± 10 Q7 X H12 cry1Ac + cry1C 0 100 ± 0 0 100 ± 0 10 70 ± 10 Q7 X H14 cry1Ac + cry1C 0 100 ± 0 0 100 ± 0 15 67 ± 12 Q23xH14-1* cry1Ac + cry1C 0 100 ± 0 0 100 ± 0 0 100 ± 0 Cao et al., 2002 USA

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41 DISEASE RESISTANCE

Table: 6 BACTERIAL BLIGHT R GENE PRESENT IN IN RICE LINES:

42 Table: 6 BACTERIAL BLIGHT R GENE PRESENT IN IN RICE LINES Lines R gene(s) present IRBB4 Xa4 IRBB5 Xa5 IRBB21 Xa21 IRBB56 Xa4,Xa5,Xa13 IRBB57 Xa4,Xa5,Xa21 IRBB58 Xa4,Xa13,Xa21 IRBB59 Xa5,Xa13,Xa21 IRBB60 Xa4,Xa5,Xa13, Xa21 MH2R Xa5 MH3R Xa5 IRBB60 XMH2600 F2 Xa4,Xa5,Xa13, Xa21 India Swamy et al., 2006

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43 India Swamy et al., 2006 Table: 7 Bacterial blight reaction of rice lines inoculated with Mxo isolates for disease reaction Line Isolate Mxo1 Mxo2 Mxo3 Mxo4 Mxo5 Mxo6 IRBB4 R MR R S S S IRBB12 R R R MR R MR IRBB56 R R R R R R IRBB57 R S R S S S IRBB58 R R R R R R IRBB59 R R R R R R IRBB60 R R R R R R IRBB60 x MH2R F2 R R R R R R TN 1 S S S S S S ( Xa4 ) ( Xa4,Xa5,Xa13 ) ( Xa4,Xa5,Xa21 ) ( Xa4,Xa5,Xa13 ) ( Xa5,Xa13,Xa21 ) ( Xa4,Xa5,Xa13,Xa21 ) ( Xa4,Xa5,Xa13,Xa21 ) R, 0-3 cm, MR, 3-6 cm, S, >6 cm, lesion length

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44 Isolate Line Mxo1 Mxo2 Mxo3 Mxo4 Mxo5 Mxo6 PB10-1 S S MR S S S PB10-3 S S MR S S S PB19-5 S S S S S S PB8-4 S S MR S S S PB8-5 S S MR S S S PB8-6 S S S S S S PB8-7 S S S S S S PB8-x S S S S S S PB8-9 S S S S S S PB8-10 S S MR S S S PB8-13 S S S S S S PB8-15 S S MR S S S PB8-17 S S MR S S S Pusa basmati 1 S S S S S S IR 72 R S MR S S S IR 72+ Xa21 R MR MR S R MR TNI S S S S S S India Swamy et al., 2006 Table: 8 Bacterial blight transgenic rice lines and corresponding controls inoculated with Mxo isolates. R, 0-3 cm, MR, 3-6 cm, S, >6 cm, lesion length

ABIOTIC STRESS TOLERANCE:

45 ABIOTIC STRESS TOLERANCE

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46 Fig:A Methylglyoxal treatment to leaf disk for 48 hrs Fig:B NaCl treatment to leaf disk Gly I -- Brassica Gly II -- rice Genetic engineering of the glyoxylase pathway in tobacco leads to enhanced salinity tolerance . India Singla-pareek et al., 2003 A

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47 200mM NaCl for 98 days Fig.A Fig.B

METABOLIC PATHWAY MANIPULATION :

48 METABOLIC PATHWAY MANIPULATION

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49 4-coumarate-CoA-ligase Coniferaldehyde 5-hydroxylase Combinatorial modification of multiple lignin trait in Aspen ( Populus tremuloides ) through multigenes cotransformation Li et al., 2003 Fig : Monolignol pathway in wood angiosperms

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50 70-90% reduced 4Cl activity 2.5-2.8 fold increase CAId5H 80-90% reduced 4CL 60-110% increaseCAId5H 30-40% reduced lignin No sig.effect on S/G ratio 2.5 fold increase in S/G ratio No influence on lignin content 38-52% reduced lignin 22-24% increased lignin ratio

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51 TRANSIT PEPTIDE MATURE PROTEIN LINKER THIOLASE REDUCTASE SYNTHASE Small subunit of Rubisco M A S.......... V K C M Q V.....................T R D S R V T D V ……… E R G T Q R……… H M G A T G …….. A K R 1 55 78 Targeting of the polyhydroxy butyrate biosynthetic pathway to the plastid of Arabidopsis thaliana Result in high level of polymer accumulation. Phb- A 3 ketothiolase Phb- B Acetoacetyle –Co-A reductase Phb- C PHB synthase Alcaligenes eutrophus USA Nawrath et al., 1994

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52 Fig. PHB accumulation in leaves of different hybrids USA Nawrath et al., 1994 Plastid transformed Nucleus transformed 20-30 day old 50-60 day old

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53 PHB granules Fig:A Transmission electron micrograph of PHB producing plastid

Conclusion:

54 Conclusion A number of conventional and more novel techniques already exist for the stacking of genes, no single method is ideal as yet. Co-transformation is an effective method for gene stacking as compared to re-transformation. Chimeric transgenes with fused sequences of several ‘effectgenes’ under the control of single promoter offer very significant advantages. Gene stacking technology is useful in achieving insect and disease resistance, multiple resistance, abiotic stress tolerance, quality enrichment and manipulation of metabolic pathways in crop plants

Future thrust:

55 Future thrust It is still require to expand our understanding about metabolic pathways and identification of gene involved. Refinement of the existing technique to be require for co-ordinated multigene manipulation in plant to provide more durable and cleaner transgene technologies that can simplify the route to regulatory approval and can reassure the consumers about safety and stability of GM product More suitable vector system should be design which can be transfer more than one gene with single transfer.

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56 Thank you

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