gene stacking in crop plants final

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
For
transgenic
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 COLLECTIVELYA
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.
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.

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 seed
pooling 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.

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 A
IRES 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
2A 2B
1B 1C

A B A
G GP P
2A 2A

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
A B
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.

• 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 ofXa 21 genes
• SUSCEPTIBLE CONTROL : TAICHUNG NATIVE 1 (TN1)
• IR72 AND IR72 CARRYING THE XA21 GENE WERE TESTED FOR BB RESISTANCE.
• GENERATION OF TRANSGENICPUSA 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
regenerate
d plants
AHAS
A
Chitina
se I
C
DREB3
D
AHAS+Chit.
+DREB3
A+C+D
Co-transformation
frequency (%),
A+C+D
St. 29 59 58 53 48 40 67.8
Gladius 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/A
Aktobe 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.,
A

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

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
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 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
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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