strategies to eleminate or avoid the use of selectable markers

1. Marker free transgenic crops
By: SHEETAL MEHLA
2018BS13D
COBS&H, CCS HAU, HISAR

2. MARKERS
• 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.

3. http://www.ag.ndsu.edu/pubs/plantsci/crops/a1219-2.gif

4. SELECTABLE MARKERS
• Selectable markers are those which allow the selection of
transformed cells, or tissue explants, by their ability to grow in
the presence of an antibiotic or a herbicide.
• Antibiotic and herbicide resistance genes are the most efficient
and widely-used selectable markers (Miki and McHugh, 2004).
• The selective agents are generally used in the initial stages of
transformation for an early selection of transgenic cells.
• Once transgenic plant is selected ,marker gene is no longer
necessary and remain as integral part of plant genome in
transgenic plants.

5. SCREENABLE MARKERS
• Genes that permit identification of transgenic
plants in the absence of a selective agent are
known as screenable markers.
• These marker employ a gene whose protein
product is detectable in the cell, either because it
produces a visible pigment or fluoresces under
appropriate conditions.
• Green fluorescent protein
• β glucuronidase

6. NEED FOR MARKER FREE TRANSGENIC
CROPS
• The protein products of such genes could be toxic to human/
animals.
• The food from transgenic crop will contain the antibiotic
resistance gene. When food from such crops is consumed,
the bacteria present in human intestine could acquire the
antibiotic resistance gene present in the food. This would
make the bacteria resistant to the antibiotic concern, and they
may become difficult to manage.
• These marker genes could be passed on from transgenic
crop to some other organism and could damage the
environment.
• For public acceptance of transgenics, keeping in mind
ecological and food safety.
• SMG removal lead to decrease in the insert size, which in
turn increases transformation frequency.

9. Marker Free Transgenics
• 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.

10. STRATEGIES OF MARKER FREE
TRANSGENICS
• Totally avoiding the use selectable marker genes
• Replacing selectable with screenable markers
• Co-transformation
• Excision of the selectable marker gene out of the
integrated transgene after successful selection
Marker-free transgenic plants Holger Puchta Botany II , Universitat Karlsruhe ,Received 10 July 2002; accepted in revised form 6
February 2003

11. Totally avoiding the use selectable
marker genes
• Totally avoiding the use selectable marker genes.
• Theoretically, it should be possible to identify
among a large number of cells the ones that are
carry a transgene, directly by molecular methods
particularly if transformation efficiencies can be
improved.
• However, even in the days of automated analysis
and polymerase chain reaction such a project is
still highly demanding.

12. Replacing Selectable With Screenable
Markers
• In this method non transformed cells are not killed
by rather the transformed cells experience a
metabolic or developmental advantage.
• β -glucuronidase (Joersbo and Okkels,1996),
• Xylose isomerase (Haldrup et al.,1998)
• Phosphomannose isomerase genes (Joersbo et al.,
1998; Negrotto et al.,2000) as well as the
• Isopentenyl transferase (ipt) gene (Ebinuma et al.,
1997)

13. Co-transformation
• 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. • In co-transformation experiments the desired
gene and the transformation marker can
supplied on two T-DNAs within the same binary
vector (Depicker et al., 1985; Komari et al.,
1996; Lu et al., 2001) or
• on two binary vectors within the same
Agrobacterium (Daley et al., 1998) or
• with two Agrobacterium strains (Depicker et al.,
1985; McKnight et al., 1987; De Block and
Debrouwer, 1991; Komari et al., 1996; De Neve
et al., 1997).

16. CASE STUDY

17. MATERIALS AND METHODS
• Vector construction
• Tobacco transformation
• Polymerase chain reaction (PCR)
• Southern blot analysis
• Progeny segregation analysis

18. Vector Construction

20. • F0 progeny F1 progeny

21. Excision of the selectable marker
gene out of the integrated transgene
after successful selection by
• Site- Specific Recombination
• Transposition
• Homologous Recombination (HR)
Marker-free transgenic plants Holger Puchta Botany II , Universitat Karlsruhe ,Received 10 July 2002; accepted in revised form 6
February 2003

22. Site-Specific Recombination 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

23. Site- Specific Recombination

24. Strategies for SSR
• The Cre-lox system from bacteriophage P1.
• The FLP/FRT system from S. cerevisiae.
• The R/Rs system from Zygosaccharomyces
rouxii.

25. • Basic strategy using Cre-mediated site-
specific recombination to marker gene
removal and nucleotide sequences of loxP
site

27. Material and method
• Plant material
• Vector construction
• Mustard transformation
• Screening through PCR
• Southern blotting analysis
• Western blotting
• ELISA
• In planta insect bioassay

28. T-DNA region of binary vectors used
for mustard transformations
A} pBKhgASAL B} pBK16.2

32. Tranposition
• 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.

33. Transposition

35. • (A) Schematic diagram of the
one-time transposon system
COKC and location of primers
(shown as solid triangle). LB,
left border; RB, right border;
5’ and 3’, Ac left and right
terminal-inverted repeat; PR-
1a, PR-1a inducible promoter;
HPT, hygromycin
phosphotransferase gene; pA,
poly(A) fragment; NOS,
nopaline synthase promoter;
A~E, transposase gene exon
1~exon 5; 1~8, epsps gene
exons;
• (B) RT-PCR analysis of
modified epsps expression in
transgenic rice lines (1-8) and
TNG67 (9);
• (C) RT-PCR analysis of
induced transposase gene
expression in transgenic rice
calli treated with water (1) or
5 mM SA (2 and 3). M, 100 bp
marker.

36. • (A) PCR analysis of COKC
transposition with the primers CF
and JR and the expected fragments;
(B) The control HPT specific
products which were amplified with
each sample;

37. • Glyphosate-tolerance analysis of the self-pollinated progeny
of COKC transposed (A) and untransposed line (B). Arrows
indicate the null COKC progeny, which are not resistant to
glyphosate.

38. Homologous Recombination (HR)

39. CONCLUSION
• 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. THANK YOU