The document discusses career opportunities in biotechnology, focusing on the development of marker-free transgenic plants and the various strategies employed to eliminate marker genes traditionally used in plant transformation. It examines methods such as positive and negative selection systems, co-transformation, and site-specific recombination to achieve this goal while addressing biosafety concerns and the ecological implications of genetic engineering. The overall aim is to enhance public acceptance of transgenic plants by ensuring they do not carry selectable marker genes that may pose risks to the environment and food safety.

1. SUBJECT : CAREER OPPORTUNITIES AND
ENTERPRENEURSHIP IN BIOTECHNOLOGY
SUBJECT CODE: MCE 713
SUBMITTED TO:
DR. GD RAM GURU
MCE DEPARTMENT
JIBB
SUBMITTED BY:
GAURAV AUGUSTINE
ID: 19MTBT001
M.TECH 2ND SEM.

2.  Introduction
 Why marker-free transgenic plants?
 Most commonly used marker
 Strategies to develop marker-free transgenics
 Strategies to eliminate marker genes from
transgenic
 Conclusion

3.  Molecular Marker :- In genetics, a molecular marker
(identified as genetic marker) is a fragment of DNA
that is associated with a certain location within the
genome. Molecular markers are used in molecular
biology and biotechnology to identify a particular
sequence of DNA in a pool of unknown DNA.
 Molecular markers are effective because they identify
an abundance of genetic linkage between identifiable
locations within a chromosome and are able to be
repeated for verification.
 The use of a marker gene in a transformation process
aims to give a selective advantage to the transformed
cells, allowing them to grow faster and better, and to
kill the non-transformed cells.

4.  In general, the selective gene is introduced into
the plant genome along with the genes of
interest.
 In some cases, the marker gene is the gene of
interest that will express an agronomic
characteristic, such as herbicide resistance.
 Although no adverse biosafety effects have
been reported for the marker genes that have
been adopted for widespread use, biosafety
concerns should help direct which markers will
be chosen for future crop development.

7.  Selectable marker genes (SMGs), such as antibiotic or
herbicide resistance genes, are used in nearly every plant
transformation protocol to efficiently distinguish
transformed from non-transformed cells.
 Marker genes generally have little agronomic value after
selection events .
 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.

8.  Positive selection system
 Negative selection system
 Abiotic stress related genes as selection markers
 Avoiding the use of selectable marker genes

9. Positive selection system :- Identification and
selection of transformed cell without injury or death of
non-transform cell. The selected marker genes code for
enzymes that give the transformed cells a capacity to
metabolize specific nutrient substance not usually
metabolized by normal plants.
Negative selection system :- in this method negative
selectable marker genes next to a positive marker gene
in the same construct and are a powerful means of
creating marker free transgenic plants. Transformed
plants are selected for the absence of negative selection
marker genes under the selection pressure of a negative
selection markers.
Avoiding the use of selectable marker genes :-
The simplest way to eliminate marker genes for
transgenic is to avoid their use in the transformation.

10. Abiotic stress related genes as selection
markers :-
 Various genes encode proteins that protect plants at
the time of several environmental stresses like
drought stress, salt stress and oxidative stress.
 So many genes that are well characterized in A.
thaliana or in several agronomically important
crops can be can be used for development of
marker free transgenic plants.
 The stress responsive gene for salt tolerance is
incorporated into the plant tissue or explants
without the selection marker gene.

11. Genes responsible for abiotic stress :-

12.  Co-transformation
 Site-specific recombination
 Multi-auto transformation vector
 Intra chromosomal recombination system
 Transposition system
 Use of screenable markers
 Auto excision strategy

13.  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.

14.  It cannot be used for vegetatively propagated
plants. So that, this method is only compatible
only for sexually propagated fertile plants.
 Very time consuming.
 The tight linkage between co-integrated DNAs
may limit the efficiency of co-transformation.
 This method is also not applicable to transgenic
trees with long generation times.
 The progeny selection with the desired genes may
also be very laborious.

15. Generation of marker-free transgenic plants by the co-
transformation method

16.  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.

17. The intrachromosomal recombination system for marker gene removal
from transgenic plants involving a phage- encoded λ integrase and a
bacterially encoded Integration Host Factor (IHF). TBS; Transformation
booster sequence, SMG, Selection marker gene, GOI; Gene of interest,
attP; attachment P region of bacteriophage.

18.  This process is quite similar to site specific
recombination where transposons or jumping
genes are used instead of a recombination and
recognition sites.
 This method involves Agrobacterium-mediated
transformation followed by intergenomic
replacement after the incorporation of two allied
genes and subsequent separation from the marker
gene in the progeny or straight deletion of marker
gene from the genome.

20.  There are two fundamental individuality possessed by
the autonomous Ac element for transposition that can
be physically separated: a transpossase coding gene
(Ac) and the reversed duplicate termini (Ds elements).
 The approach makes the use of the Ac/Ds transposition
system where Ds is a member of Ac/Ds family, can cut
out itself from its position and transpose to other sites
within a genome only in the existence of the
autonomous Ac element and Ac transposase activity is
vital for the expression of plant promoters
 The advantage of this system is that, the selection
marker gene will be lost in some somatic tissues due to
failure of the Ds element reintegration. This makes the
strategy suitable for removal of marker genes in
vegetatively propagated plants.

21.  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.

22. screenable markers

23.  Various approaches for removal of selectable
markers for transgeic have been described here but
appropriate choice depends on ease of use, crop
and methods of transformation and generation
time.
 the marker free methodologies are useful to
overcome the gene flow from the genetic
engineered plants to other living organism.
 The marker gene should be removed after next
generation in order to avoid their toxicity to other
living organisms.

24. THANK
YOU!