This document discusses marker-free transgenics, which aim to generate transgenic plants without selectable marker genes. It describes various strategies to produce marker-free plants, including using screenable markers, co-transformation, site-specific recombination, multi-autotransformation vectors, intrachromosomal recombination, and transposon-based methods. The document concludes that developing marker-free transgenic crops could help advance crop improvement efforts and increase public acceptance of transgenic technologies.

1. MARKER FREE TRANSGENICS
DEVELOPMENT
Presented
By
Arpita Mahobia
Ph.D. 1st Year
Dept. of Plant Molecular Biology &
Biotechnology
IGKV, Raipur

2. TRANSGENIC PLANT
• 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

3. MARKER GENES
• 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.

4. Need for Marker Free Transgenics
• Marker genes generally have little agronomic value after
selection events
• Retention of the HT 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 transgenics,
keeping in mind ecological and food safety
•Marker free transgenics should be
developed.

5. 6/8/2015Dept. of PMBB

6. 6/8/2015Dept. of PMBB

7. 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.
MARKER FREE TRANSGENIC

8. STARATEGIES TO PRODUCE
RESISTANT MARKER‐FREE
TRANSGENIC PLANTS
• Use of screenable markers
• Co-transformation
• Site-specific recombination
• Multi-autotransformation vector
• Intrachromosomal recombination system
• Transposition system

9. SCREENABLE MARKERS
• Screenable markers encode gene products whose
enzyme activity can be easily assayed
• Can detect transformants
• Also estimation of the levels of foreign gene expression
in transgenic tissue done.
• Ex. β-glucuronidase (GUS), luciferase or β-
galactosidase genes, phytohormone metabolism
isopentenyl transferase (ipt) gene from the T-DNA of
Agrobacterium.
• Can be used to study cell-specific as well as
developmentally regulated gene expression.

10. • Involves transformation with two plasmids that target
insertion at two different plant genome loci. One
plasmid carries a SMG and the other carries the GOI
• In this system, SMG and target genes are not loaded
between the same pair of T-DNA borders.
• Instead, they are loaded into separate T-DNAs, which
are expected to segregate independently in a
Mendelian fashion.
Co-transformation Method

11. Cannot be used for vegetative
propagated plants

12. (a) Physical diagram of two T-DNA region showing gene of interest (GOI) and marker gene. (b)
Transformed calli having GOI and marker gene. (c) T0 plant having GOI and marker gene. (d) Two T1
plants one with GOI and another with marker gene.

13. • 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
SITE SPECIFIC RECOMBINATION
(SSR)SYSTEM

14. Site‐specific recombination‐mediated
marker deletion

15. Cre/loxp‐mediated marker gene excision in
transgenic maize (Zea mays L.) plants
-(Gilbertson et al., 2003)

16. • 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
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MAT SYSTEM

17. • Together with the R recombinase gene , entire
assembly is situated within a T-DNA element for the
Agrobacterium-mediated transformation.
• Neither antibiotic- nor herbicide-resistance genes are
necessary as a selection marker. In addition, it allows
for repeated transformation of genes of interest in a
plant (Sugita et al. 2000).
• Principle of MAT uses oncogene (ipt) for selection of
transgenic plants and a SSR system
17
MAT SYSTEM

18. Recombinase (R) catalyses
recombination between two
directly oriented recognition
sites (Rs) and removes a ‘hit
and run’ cassette from a plant
genome.
Recombinase (R) gene
expression is under the
chemically inducible promoter
(IP) in order to avoid early
removal of ipt gene.
P; promoter, T; terminator,
GOI; gene of interest, LB; left
border, RB; right border.
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19. INTRACHROMOSOMAL
RECOMBINATION SYSTEM
• Recombination resulting from crossing over between
two linked gene pairs.

20. • 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 the T-DNA and SMG.
Transposon‐based marker methods

21. Transposon‐based marker methods

22. Transposon based marker free
transgenics
Fig: Schematic diagram of the Ac-Ds transposon system. (a) T-DNA region showing GOI
merged between Ac sites and marker gene, reporter gene and AcTpase region is outside the
Ac sites. (b) Diagram showing the T-DNA region having GOI merged in Ac region excised out
from marker and reporter gene.

23. An inducible transposon system to terminate the
function of a selectable marker in transgenic rice
(Tu et al., 2008)

24. Conclusion and future prospects
• 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 marker-
free transgenic crop.
• But development of marker free transgenics would
further increase the crop improvement programme.