The document presents on gene stacking, pathway engineering, and marker-free transgenic development strategies. It discusses various methods for combining multiple genes/traits into plants, including gene stacking, gene pyramiding, sexual hybridization, re-transformation, co-transformation, and marker-free techniques. The goal is to develop crops with improved agronomic traits through plant genome engineering approaches.

1. Assignment Presentation
On
Gene Stacking :Pathway Engineering : Marker- free transgenic development
stratagies.
Course Title: Plant Genome Engineering
(MBB-602) -3(3+0)
Presented By
Mr. Rahul Kumar Maurya
Ph. D. Agril. Biotechnology
ID. No. A-11164/19/22
Presented To
Associate Professor, Department of PMB&GE,
ANDUA&T, Kumarganj, Ayodhya-224229
Dr. Shambhoo Prasad
Dr. Adesh Kumar
Assistant Professor, Department of PMB&GE,
ANDUA&T, Kumarganj, Ayodhya-224229

2. 1. Introduction
2. What is Gene
3. Need For Gene
4. GENE STACKING V/s GENE PYRAMIDING
5. STRATEGY FOR GENE
6. Sexual Hybridisation Plants
7. Re- transformation
8. Co- transformation
9. Marker free transgenic development
10. Conclusion and future prospects

3.  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.

4.  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.
 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.

5. • 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 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.
 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.

7.  Gene Stacking Hybrid
 Sexual Hybridisation
 Re-transformation and
 Co- transformation

9.  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 into one plant (Ma et al. 1995 ).
 Two genes for a bacterial organic mercury
detoxification pathway (mercuric reductase, merA
and organomer curial 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 ).

11.  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.

12.  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

15. • 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.

17.  Co-transformation with a single plasmid:
 This method can be performed by introducing a single
genetic construction with all genes, each with its own
promoter and terminator, in the same plasmid.
 This can be used for genetic transformation by
bombardment or Agrobacterium tumefaciens, and all
genes tend to be integrated together in the same plant
genome site(s).

21.  Plants that have been genetically engineered,
an approach that uses recombinant DNA
techniques to create plants with new
characteristics. Also known as Genetically
Modified Organism (GMO).
 Plant developed after successful gene
transfer have stably integrated foreign gene.

22.  Monitoring and detection of plant transformation
systems in order to know DNA successfully transferred
in recipient cells or not.
 A set of genes introduced along with the target gene
into the plasmid known as Marker genes.
 Antibiotic and herbicide resistance genes successfully
used as marker genes.
 Allow the transformed cell to tolerate the antibiotic or
herbicide and regenerate into plants while the
untransformed ones get killed.

23.  Free transgenics Marker genes generally have little
agronomic value after selection events.
 Retention of the marker gene in the genome may be
problematic. In situations requiring more transformations
into cultivars the presence of a particular marker gene in
a transgenic plant - use of the same marker in subsequent
transformation.
 Use of a different marker system is required for each
transformation round or event.
 For public acceptance of trans-genics, keeping in mind
ecological and food safety. Marker free trans-genics
should be developed.

27.  The generation of transgenic plants by the
elimination of the “problematic” selectable
marker genes from the genome of the
transgenic plants or avoiding the use of
selectable marker genes in the beginning of
transformation by a marker-free vector.

28.  To eliminate selectable marker gene.
 To avoid use of toxic selectable marker gene.
 (a) Physical diagram of two T-DNA region showing
gene of interest (GOI) and marker gene.
 (b)Transformed call having GOI and marker gene.
 (c)To plant having GOI and marker gene.
 (d)TwoT1 plants one with GOI and another with marker
gene.

30.  A positive selection system.
 Unique as it uses morphological changes caused by
oncogene [ipt gene] or rhizogene (the rol gene) of A.
Tumefaciens which control the endogenous levels of
plant hormones and the cell responses to PGR as the
selection marker.
 A chosen GOI is placed adjacent to a multigenic
element flanked by RS recombination sites.
 A copy of the selectable ipt gene from
A.tumefaciens is inserted between these sites.

31.  SITE SPECIFIC RECOMBINATION(SSR) SYSTEM .
In this approach, SMG is flanked with direct repeats of
recognition sites for a site specific recombinase, which
allows the enzyme to excise the marker gene from the
plant genome by enzyme mediated site specific
recombination.
 A common feature of the system is that after a first
round of transformation, transgenic plants are produced
that contain the respective recombinase and the sequence
to be eliminated between two directly oriented
recognition sites.
 After expression of the single chain recombinase, the
recombination reaction is initiated resulting in transgenic
plants devoid of the selectable marker.

32.  (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.

35.  The maize Ac/Ds transposable element system has been
used to create novel T-DNA vectors for separating genes
that are linked together on the same T-DNA after insertion
into plants.
 Once integrated into the plant genome, the expression of
the Ac transposase within the T-DNA can induce the
transposition of the GOI from the T-DNA to another
chromosomal location.
 This results in the separation of the gene of interest from
theT-DNA and SMG.

38.  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.

39. • The removal of marker gene from the transgenic
plants supports multiple transformation cycles for
transgene pyramiding.
• It is clear that several viable methods for the
removal of unwanted marker genes already exist.
• It seems highly likely that continued work in this
area will soon remove the question of publicly
unacceptable marker genes.
• At present there is no commercialization of
markerfree transgenic crop.
• But development of marker free transgenics would
further increase the crop improvement programme.

40.  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.