gene stacking in crop plants final

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GENE STACKING IN CROP PLANTS Himanshu Bhatt

Id No. 39684Advisor : Dr. Birendra PrasadGenetics & Plant Breeding

INTRODUCTION

GENE STACKING IN CROP PLANTS 03 oct 2015 2

WHY GENETIC VARIATION IS NECESSARY ?- GREATER CHANCES OF SURVIVAL AND FLOURISHING- REDUCES THE INCIDENCE OF UNFAVOURABLE INHERITED TRAITS.

• PLANT BREEDERS TAKE ADVANTAGE OF THESE GENETIC VARIANTS TO IMPROVE EXISTING PLANTS AND CREATE NEW VARIETIES. • THROUGH CROSS BREEDING THEY STRIVE TO BREED IN DISEASE

RESISTANCE, SUPERIOR FRUIT PRODUCTION, INCREASED COLD TOLERANCE, OR OTHER DESIRABLE TRAITS.

Local germplasms

Obsolete varieties

Wild spp./wild relatives

Interspp./intergenera

SOURCES OF VARIABILITY:

TRANSGENIC LEVEL

Isolation of genes

General Approach Fortransgenic development

GENE STACKING….??

• THE COMBINATION OF TWO OR MORE g.o.i. IN THE GENOME OF THE HOST PLANT i.e. THE CREATED GMO CARRIES TWO OR MORE DIFFERENT GENES AND TRAITS.•A GENETICALLY MODIFIED ORGANISM (GMO) AND ALL

SUBSEQUENT IDENTICAL CLONES RESULTING FROM A TRANSFORMATION PROCESS ARE CALLED COLLECTIVELY A TRANSFORMATION EVENT. IF MORE THAN ONE GENE FROM ANOTHER ORGANISM HAS BEEN TRANSFERRED, THE CREATED GMO HAS STACKED GENES (OR STACKED TRAITS), AND IS CALLED A GENE STACKED EVENT.

THE FIRST STACK THAT GAINED REGULATORY APPROVAL IN 1995 WAS A DUAL HYBRID COTTON STACK PRODUCED BY CROSSING BOLLGARD™ COTTON THAT EXPRESSES THE Bt TOXIN cry1ab AND ROUNDUP READY™ COTTON THAT PRODUCES THE epsps ENZYME CONFERRING RESISTANCE TO HERBICIDE GLYPHOSATE.

• ACCORDING TO THE ORGANIZATION FOR ECONOMIC CO-OPERATION AND DEVELOPMENT (OECD), STACKED TRANSFORMATION EVENTS ARE DEFINED AS “NEW PRODUCTS WITH MORE THAN ONE TRANSFORMATION EVENT” ( OECD, 2004 ).• TRANSFORMATION EVENT” - ACCORDING TO Halpin, 2005 , THE STACKED

TRAITS ARE CONFERRED BY THE EXPRESSION OF TWO OR MORE “EFFECT GENES”.

GM HYBRID V/S GENE STACKING EVENTS• IN A GM HYBRID, THE TRANSGENIC TRAIT ORIGINATES FROM THE

GM INBRED PARENTAL LINE THAT WAS CROSSED WITH ONE OR MORE NON-TRANSGENIC ELITE INBRED LINES. • IN A GM STAEV, TWO OR MORE TRANSGENIC TRAITS ARE

BROUGHT TOGETHER BY CROSSING GM INBRED LINES, EACH BEING DIFFERENT INITIAL EVENTS.

DE SCHRIJVER et al. (2007) DEFINE “ONEWAY GM STACKED EVENTS” AS STACKED EVENTS WHERE TWO TRANSGENIC TRAITS ARE COMBINED, WHILE “THREE-WAY GM STACKED EVENTS” CONTAIN THREE TRANSGENIC TRAITS.

•GENE PYRAMIDING : ASSEMBLING MULTIPLE DESIRABLE GENES FROM MULTIPLE PARENTS INTO A SINGLE GENOTYPE

•GENE STACKING : COMBINATION OF TWO OR MORE TRANS GENES OF INTEREST IN THE GENOME OF THE HOST PLANT.

GENE STACKING V/s GENE PYRAMIDING

Transgenic corn triple stacks, for instance containing a corn root worm (CRW) protection trait (e.g., Cry3B(b)1), a corn stalk-boring insect control trait (e.g., Cry1A(b)), and RR trait for herbicide tolerance.

STRATEGY FOR GENE STACKING

ITERATIVE PROCEDURE / SEXUAL HYBRIDIZATION

RE- TRANSFORMATION

CO- TRANSFORMATION

ITERATIVE PROCEDURE / SEXUAL HYBRIDIZATION

Plants containing several transgenes can be produced by crossing parents with different transgenes until all the required genes are present in the progeny.

e.g., Bt11xMIR604xGA21 maize that is corn borer and rootworm resistant and herbicide tolerant . • MON-87427-7 x MON-

89Ø34-3 x MON-ØØ6Ø3-6

• Two genes for a bacterial organic mercury detoxification pathway (mercuric reductase, merA , and organomercurial lyase, merB ) were combined by crossing in Arabidopsis , and plants expressing both genes were able to grow on 50-fold higher methyl mercury concentrations than wild-type plants ( Bizily et al ., 2000 ).

• An early example of the power of this strategy was the production of secretory IgA antibodies in plants by cross-breeding of tobacco to combine , in one plant, four genes encoding different immunoglobulin polypeptides (Ma et al. 1995 ).

LIMITATIONS:• TRANSGENES NOT LINKED & CAN SEGREGATE;• OBTAINING HOMOZYGOUS PLANTS FOR ALL TRANSGENES

DIFFICULT;• INCREASED BREEDING COSTS;• VARIETY OF SELECTABLE MARKERS NEEDED IN THE RE-

TRANSFORMATION STRATEGY;• MARKER REMOVAL SLOW, MULTISTEP PROCESS• LABOUR INTENSIVE AND TIME CONSUMING

RE- TRANSFORMATION

•Multi-trait or combined trait event with separate inserts.

•Gm plant produced by iterative event with separate inserts transformation with vectors containing different transgenes/traits. The transgenic inserts are integrated in multiple loci.•Multiple transgenes either harbored within different t-DNA in single

Agrobacterium strain or harbored separately within different strain. • Sequential transformation and retransformation of plants with

different individual transgenes in several rounds of transformation .•A plant harbouring a transgene is transformed with other

transgenes.

Host cell

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.

Cotton: Bollgard™ II (MON15985)

CO-TRANSFORMATION

Co-transformation events

single-plasmid co-transformation of linked transgenes

Multiple plasmid co transformation of unlinked transgenes.

• genes to be introduced are linked as a single piece of DNA, with each gene having its own promoter.

single-plasmid co-transformation of linked transgenes

Multiple plasmid co transformation of unlinked transgenes.

• consists of several plasmids or discrete fragments of DNA (if biolistics ), each carrying a different transgene (including a promoter), that are transformed together into a plant via. Agrobacterium mediated transformation or biolistic methods.

Transgenes tend to co-integrate at the same chromosomal position

One step procedure for the introduction of the multiple “effect” gene

Simultaneous introduction of multiple genes into the cell followed by integration of genes in cell genome.

Genes either present on same plasmid used in transformation(single plasmid co-transformation) or on separate plasmids (multiple plasmid co-transformation)

•Co-transformation , via particle bombardment, has also been used to simultaneously introduce three insecticidal genes (the Bt genes cry1ac and cry2a , and the snowdrop lectin gene gna ) into indica rice.• Transgenic plants containing all three genes showed significant

levels of protection against three of the most important insect pests of rice: Rice Leaf Folder (Cnaphalocrocis medinalis), Yellow Stem borer (Scirpophaga incertulas) and Brown Plant hopper (Nilaparvata lugens). ( Maqbool et al., 2001 ).

Maize: NaturGard™ Knockout™ (Bt176), Bt Xtra™ (DBT418), YieldGard™ (MON810, MON809, MON802)

• Integration of multiple transgenes, less transformation events, less time consuming;•Assembly of different expression cassettes is technically easier as

it is done on independently on different plasmids.( Komari et al., 1996)•Single-plasmid co-transformation offers an advantage over

multiple-plasmid co-transformation in that integration of both genes together into the same genomic location is ensured as they are linked as a single piece of DNA.

LIMITATIONS

•Difficulty to assemble complex plasmids with multiple gene cassettes (Francois et al, 2002.)•Problem of Gene silencing if same promoter is used with

each transgene.

•High copy number integrating •Undesirable incorporation of a complex T-DNA

molecules from multiple sources.•Transgenes derived from different sources typically

integrate at different locations in plant genome, which may lead to various expression patterns and possible segregation of the transgenes in the offspring.

HOW SELECTION IS DONE..?ITERATIVE PROCEDURE: selection at phenotypic level.

When for the different characters- on the basis of performance and response towards the desired character.

When for the same character- (e.g., disease)- molecular marker level.

RE-TRANSFORMATION/CO-TRANSFORMATION Selection –mainly with the help of markers assisted

selection Selection evaluation on the basis of phenotypic

characters.

Initial selection is better.

Existing methods for GS identification and detection– Single seed-based DNA analysis (real-time PCR):• Akiyama et al., 2005 (MON810 x GA21) multiplex rt PCR• Papazova et al., in preparation (MON810 x T25) individual seedpooling scheme

based on grinding of individual grains (MON810, GA21, MON810 x GA21) and multiplex qualitative real time PCR detection of SSIIb, P35S and GA21-construct in one tube.

Individual kernels contain either one of the transgenes (single events) or both transgenes(StaEv MON810xGA21), which can be distinguished based on amplification plots, end-point analysis (fluorophore emission intensities), or agarose gel separation of PCR products.

− Single seed based protein analysis:• Ma et al., 2005 (Bt x LL GS)

• use of protein flow strips• protein-based methods to detect LL, Bt and the stacked

Bt/LL events in seed and grain samples.

– Statistical approach:• Kobilinsky and Bertheau, 2005

– Sub-sampling approaches (sub sampling combined with rt PCR):• Allnut et al., 2006 (MS8 x RF3)

• Detection of GSs in seed pools by combining a sub-sampling strategy (control plan by multiple attributes, Laffont et al., 2005) with real-time PCR for detection of ms8 x rf3.

• The basic idea is to detect both event-specific sequences plus the bar gene which occurs in both events. The segregation pattern of the markers would then give an indication of the presence and abundance of the GS.

RECENTS

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 3Promoter

Polyprotein

POLYPROTEIN EXPRESSION SYSTEM

IRES- INTERNAL RIBOSOME ENTRY SITE

2A POLYPROTEIN SYSTEM

NIa PROTEASE SEQUENCE

PROTEASE-SUSCEPTIBLE LINKER SEQUENCE

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) -If bicistronic construct , IRES promotes the translation of a second cistron at 21%−31% of the levels of the first cistron. (Dorokhov et al.,2002). Thus, although both cistrons are co-ordinately regulated, they are expressed at different levels.

A B AA B AIRES IRES

2A POLYPROTEIN SYSTEM•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.

• mediate polyprotein ‘Cleavage’ by a unique non-proteolytic mechanism• a peptide bond is not formed between amino Acids 19 and 20 of

2A, yet translation continues (Donnelly et al., 2001). • Incorporation of the 2A peptide between two protein coding

sequences results in the translation of two polypeptides: (i) the first Protein incorporating a C-terminal extension of 19 amino Acids of 2A; (ii) the second protein including a single N-terminal proline from 2A.

1D 2A 2B 2C1ALpro 3A 3Cpro 3Dpol

QLLNFDLLKLAGDVESNPG PFF

A B AG GP P

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 B48kDa NIa protease sequences

Source: Helpin et al.,2005

Protease-susceptible linker sequence

Francois et al.,2002

Host protease

Host protease susceptible linker

•Kinal et al. 1995 produced transgenic tobacco plants expressing the KP6 preprotoxin from the fungal pathogen Ustilago maydis . • Processing of the preprotoxin results in the production and

activation of alpha and beta polypeptides.

• Two examples of linkers processed in this manner are the Kex2 and the AMP (antimicrobial peptide) linkers.

CASE STUDY

• A-lines : MH1A, MA2A, MH3A, MH4A• B-lines : MH1B, MH2B, MH3B, MH4B• R-lines: MH1R, MH2R, MH3R, MH4R, MH5R, MH6R• Controls for BB resistance testing : near isogenic IRBB lines containing combinations of Xa 21

genes • SUSCEPTIBLE CONTROL : TAICHUNG NATIVE 1 (TN1) • IR72 AND IR72 CARRYING THE XA21 GENE WERE TESTED FOR BB RESISTANCE.• GENERATION OF TRANSGENIC PUSA BASMATI LINES CARRYING xa21

Evaluation of bacterial blight resistance in rice lines carrying multiple resistance genes and Xa21 transgenic lines ( 2006)

Prashant Swamy, Ajay N. Panchbhai, Priti Dodiya, Vaishali Naik,S. D. Panchbhai, Usha B. Zehr, Kasi Azhakanandam and Bharat R. Char,

GENE STACKING IN CISGENIC WHEAT(2013)

Ainur Ismagul, E. Maltseva,

N. Aytkhozhina, G. Iskakova, N. Yang, G. Ismagulova, S.Lopato, S. Eliby and P. Langridge

• co-transformed three wheat genes – Acetohydroxy acid synthase (AHAS, als), Chitinase I and DREB3

• VARIETIES : Australian wheat cultivar Gladius, and four Kazakh spring wheat cultivars Saratovskaya 29, Kazakhstanskaya 19, Astana 2 and Tselinnaya 3C.

Cis-genic

Cis (same)

Trans-genic

Trans (across)

Etymology of cis and trans:From the Latin preposition cis -“on the same side as”, “on this side of”.

From Latin preposition trans - “across”, “on the far side”, “beyond”.

Cultivar

Number of regenerated

plants

Chitinase I C

AHAS+Chit.+DREB3 A+C+D

Co-transformation frequency (%),

St. 29 59 58 53 48 40 67.8Gladius 10 8 3 3 3 30.0

Kaz. 19 3 3 3 2 2 66.7

Astana 2 1 1 1 - - N/A

Ts. 3C 1 1 1 - - N/A

Pv. 93 2 0 0 0 0 N/AAktobe 39 0 0 0 0 0 N/A

Total: 74 71 61 53 45

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. (2005) Singla-pareek et al.,

200mM NaCl for 98 days

Fig.A Fig.B

Resistance of transgenic tobacco containing β-hth + na-pi genes against Helicoverpa armigera

HIGH MORTALITY

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

Fig:A Fig: B

• Linker peptide LP4/2A provided a more versatile and simple strategy for producing gene stacking in monocot plant and it allows for coordinated expression from a single promoter. LP4/2A is a superior linker for acquiring gene stacking in tobacco plants (Sun et al. 2012).

• The cleavage site of the LP4/2A sequence includes : enzyme-digested positions of the LP4 peptides and the self-cleaving position of 2A. The LP4/2A sequence possesses one more cleavage site than either the 2A sequence or the LP4 sequence alone, so the excess amino acid residues from the mature protein can be removed to avoid influencing the protein activity. In addition, removing the 2A sequence will reduce the risk of biosafety considerations in transgenic plant.

• development of lysine-rich maize is desirable:- it could decrease the additional cost of maize grain-based animal feed by reducing usage of supplemental lysine. Accordingly, transgenic maize line Y642 was developed as a GM crop whose grain contains higher concentrations of lysine.• Maize line Y642 produced by- insertion of the lysine-rich protein encoded by the

sb401 gene , originally isolated from the potato species S. berthaultii (Liu et al., 1997).

No dose-related adverse effects observed in rats consuming diets formulated with transgenic lysine-rich GM maize Y642 compared with the conventional QPM Nongda 108 diet and the AIN93G negative control diet.

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

‘EFFECT GENES’ 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:

- Biofortified mustard/golden mustard oil has the potential to alleviate VAD in India because of its high content of bioavailable provitamin A, and a comman man of society. (Concentration of vitamin A between 92.5 µg & 300 µg/g of oil.)

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 REQUIRED 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 TRANSFER MORE THAN ONE GENE WITH SINGLE TRANSFER.

SAME PROMOTER- REDUCES THE COMBINING ABILITY OF CODING REGION OF GENE & REDUCTION IN XPRESSION LEVEL

USE OF THE SAME PROMOTER CAN TRIGGER HOMOLOGY-BASED SILENCING AND THEREFORE IT IS POSSIBLE THAT THE INTRODUCED GENE MAY NOT BE STABLY EXPRESSED IN THE LONG-TERM (OVER MANY PLANT GENERATIONS).

Promotor homology can be avoided by

Using diverse promoter Isolated from different plant

and viral genomesSynthetic 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 to form heterologous promoters

Oncogenes(ipt, iaaM/H, rol )

of Agrobacterium

R/RS system

Transgenic plant A

Transgenic plant B

MAT vector system(multi-auto transformation)

Transgenic A+B

oncogenes control the endogenous levels of plant hormones and cell responses to plant growth regulators, to differentiate transgenic cells, and to select the marker-free transgenic plants.

• marker-free transgenic plants.

Hit and run insert

• A second promising marker excision system, termed clx (for Cre / lox DNA excision system) , uses chemical-regulated expression of the Cre recombinase to excise the marker gene.

gene stacking in crop plants final

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