Gene stacking refers to combining two or more transgenes into a single plant genome. It provides broader crop traits than single genes alone. Various techniques for gene stacking include sexual hybridization, retransformation, and cotransformation. Cotransformation allows integrating multiple transgenes together, making it more efficient than other methods. Coordinated expression of stacked genes requires addressing issues like promoter homology and position effects. Future work includes refining techniques for stable multigene expression and identifying new gene combinations to improve crop traits.

1. Gene Stacking
Submitted by:
B.Rachana
RAD/2018-18
Ph.D 1st year
(GPBR)
MBB-602
Advances in Genetic Engineering
Submitted to:
Dr. K.N. Yamini
Associate Professor
Institute of
Biotechnology (IBT)

2. Introduction
• Variability is the foremost important requirement
for any crop improvement programme.
• The source of variability may be a local variety of
that crop or wild variety, weedy species or
obsolete variety.
• If there is no desired variability present in these
variety then breeder has to create the desired
variability by two way, i.e. either he goes for inter
specific or inter-generic cross by conventional
hybridization method or producing the transgenic
plant with the help of genetic engineering.
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3. What is Gene Stacking?
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• Gene stacking is a type of gene cloning that refers to the
process of combining two or more genes of interest into a
single plant.
• The emerging combined traits from this process are called
stacked traits.
• A genetically engineered crop variety that bears stacked
traits is called a biotech stack or simply stack.
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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.

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Global status of approved genetically modified (GM) crops with three or more stacked genes
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;140
0A;
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;ne
o 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
MON832ξ
Source: Helpin et al., 2005
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5. Need for gene stacking
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• Stacks offer broader agronomic enhancements that allow farmers to meet their
needs under complex farming conditions.
• Biotech stacks are engineered to have better chances of overcoming the
numerous of problems in the field such as insect pests, diseases, weeds, and
environmental stresses so that farmers can increase their productivity.
• Gene stacking boost up and simplifies pest management for biotech crops as
demonstrated by multiple insect resistances based on Bt gene technology.

6. GENE STACKING V/s GENE PYRAMIDING
•Gene Pyramiding : assembling multiple desirable
genes from multiple parents into a single genotype.
•Gene Stacking : combination of two or
more transgenes of interest in the genome
of the host plant.
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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.

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Gene
Stacking
Hybrid Stacking
Molecular
stacking
Sexual Hybridisation
Re-transformation and
Co- transformation

8. STRATEGY FOR GENE STACKING
SEXUAL HYBRIDIZATION
RE- TRANSFORMATION
CO- TRANSFORMATION

9. Sexual Hybridisation
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Plants containing several transgenes can be produced by crossing
parents with different transgenes until all the required genes are
present in the progeny.
Ex-
 The production of secretory IgA antibodies in plants by cross-
breeding of tobacco to combine four genes encoding different
immunoglobulin polypeptides in to one plant (Ma et al. 1995 ).
 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 ).
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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.
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11. Re-Transformation
•In this process a plant harbouring a transgene is
transferred again with other gene.
•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.
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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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Co- transformation

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Advantages:
One step procedure for the
introduction of the multiple
“effect”gene
Transgenes tend to co-
integrate at the same
chromosomal position.
Integration of multiple
transgenes,less
transformation events, less
time consuming.
Limitations:
High copy number
Multiple tandem repeat or
inverted repeat – such
complex integration pattern
leads to transgene silencing.
Undesirable incorporation of
a complex T-DNA molecules
from multiple sources.
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Factors affecting Co - ordinated expression of the
introduced genes
Position effect
Matrix Attachment Region (MARs)
Number of insertion at given locus
Stability of each locus
Promoter(s)
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Overcoming the difficulties of
Co - ordinated expression of
the introduced genes

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Polycistronic transgenes
Gene 1 Gene 2 Gene 3Promoter
Polyprotein
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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.

21. 21
Polyprotein expression system
• IRES- Internal Ribosome Entry Site
• 2A polyprotein system
• NIa Protease sequence
• Protease-susceptible linker sequence
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IRES- Internal Ribosome Entry Site
• A more novel method utilised for expressing multiple transgenes in
planta is the use of internal ribosome entry sites (IRES).
• An IRES is a sequence internal to a mRNA which recruits the ribosome
to an initiation codon downstream of the capped 5¢-end of the mRNA.
• Translation of the first open reading frame (ORF) occurs from the first
AUG start codon in a cap-dependent manner. Translation of the second
open reading frame is initiated from the IRES in a cap-independent
manner
• The location for IRES elements is often in the 5'UTR, but can also
occur elsewhere in mRNAs.
• It is a common cap independent ribosome scanning system found in
viruses like: Potyviridae, Comoviridae, Luteoviridae, Crucifer-infecting
tobamovirus (crTMV)
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A B AA B A
IRES IRES
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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,
ediates 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:
1. the first Protein incorporating a C-terminal extension of 19
amino Acids of 2A
2. the second protein including a single N-terminal proline
from 2A.
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1D 2A 2B 2C1ALpro 3A 3Cpro 3Dpol
QLLNFDLLKLAGDVESNPG PFF
2A 2B
1B 1C
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A B A
G GP P
2A 2A
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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
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Protease-susceptible linker sequence
Francois et al.,2002
A B
Host protease
Host protease susceptible linker
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Same promoter- reduces the combining ability of coding
region of gene & reduction in expression 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).
Promoters

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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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• The emergence of new strains of M. oryzae is associated with
recurrent failure of resistance response mediated by single
resistance (R) gene in rice.
• Stacking or combining of multiple R genes could improve the
durability of resistance against multiple strains of M. oryzae.
• In the present study, intragenic stacking of rice blast resistance
orthologue genes Pi54 and Pi54rh was performed through co-
transformation approach.
• The two genes were expressed under the control of independent
promoters and blast susceptible indica rice line IET17021 was
used for transformation.
• The stacked transgenic IET17021 lines (Pi54 + Pi54rh) have shown
complete resistance to Mo-ei-ger1 strain in comparison to non-
transgenic lines.

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These two R gene stacked indica transgenic lines could serves as a novel
germplasm for rice blast resistance breeding programmes.

37. Conclusion
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• 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.
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38. Future thrust
• It is still required to expand our understanding about
metabolic pathways and identification of genes
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 designed which
can transfer more than one gene with single transfer.
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