in this ppt, I cover up the the maker genes free methods to develop transgenic plant and animal species.

1. GURU GHASIDAS VISHWAVIDYALAYA
BILASPUR CHHATTISGARH
Subject: Plant & Animal Biotechnology
Marker Free Methodologies
Presented By
Parth Sharma
MSc 1st semester
DEPARTMENT OF BIOTECHNOLOGY

2. WHAT ARE THE MARKER GENE?
• A marker gene is a gene used to determine if a nucleic acid sequence (GOI) has
been successfully inserted into an organism’s DNA.
• There are two sub-types of these marker genes: a selectable marker and a marker
for screening.
• A selectable marker protects the organism from a selective agent that would
normally kill it or prevent its growth. Antibiotic resistant genes are mostly used
as selectable marker.
• A screenable marker will make cells containing the gene look different.

3. HOW THEY ARE USE?

4. PROBLEMS RELATED TO SELECTABLE MARKER:
• The main perceived risk is horizontal gene transfer of antibiotic resistance genes to pathogenic organisms
or the transfer of herbicide resistance genes to weeds.
• In situations requiring more transformation, the presence of a particular marker gene in a transgenic plant
use of the same marker in subsequent transformation.
• Marker gene can flow into non GM crops, animals and humans.
• Their presence may have some technological drawbacks. It has been reported that some genes (selectable
markers included) may induce pleiotropic effects under certain conditions.
• The absence of resistance genes in transgenic plants could also lower the costs for developing and speed
up the commercial release of new products.

5. ANTIBIOTICS & HERBICIDE RESISTANCE GENE USED
AS SELECTABLE MARKER
• Kanamycin: Protein translation inhibitors.
• Neomycin
• Spectromycin
• Phosphinothricin : It is an analogue of glutamine, which inhibits glutamine
synthetase.
• Glyphosate
• Gabaculine

6. MARKER FREE METHODOLOGIES
• Co transformation
• Transposone based marker
• Site specific marker
• Antibiotic free marker

7. CO-TRANSFORMATION
• Co-transformation is the simultaneous introduction of
multiple genes in a cell and the gene can be present
on same plasmid or on separate plasmid.
• The SMG and the gene of interest can be delivered by:
(i) two different Agrobacterium strains each containing
a binary plasmid carrying a single T-DNA region (ii) a
single Agrobacterium strain, either containing one
plasmid with two separate T-DNAs (iii) containing two
separate plasmids each containing a T-DNA
• Based on mandel’s law of segregation
• GOT and markers are separated during the sagrigation
formation in the next generation.
Image Refrences : Strategies for Developing Marker-
free Transgenic Plants Hee-Jong Woo, Seok-Cheol
Suh, and Yong-Gu Chor

8. TRANSPOSON BASED MARKER
• Transposones are class of genetic element that can
jumps to different location within a genome.
• The gene of interest (GOI) is placed on the
transposable element. Thus, the GOI will be excised
and can be reinserted in a locus that is not linked to
the locus in which the selectable marker gene is
located they can be segregated in the next
generation.
• The Ac/Ds transposable element system has been
used for relocation and elimination of a selectable
marker in tomato and rice. Image References : DNA transposons in vertebrate
functional genomics Csaba Miskey1, Zsuzsanna

9. SITE SPECIFIC RECOMBINATION
• It is based on microbial site specific recombinase to cleave DNA at specific site and ligate to the cleaved DNA is
used to manipulating DNA.
• The most used recombination systems are Cre/lox from bacteriophage P1, FLP/FRT from Saccharomyces
cerevisiae and R/RS from Zygosaccharomyces rouxii.
• A recombination site (lox, FRT or RS) is remaining in the genome and it could potentially serve as a site for
integrative recombination.
• In a first category of strategies, the recombinase gene and the selectable marker are on a different vector and
the recombinase gene is delivered to the plant containing the SMG by re-transformation or by sexual crosses.
• In a second category of methods using site-specific recombination, the selectable marker and the recombinase
genes are on the same vector between the recombination sites. This system is often referred to as “auto-
excision” or self-excision.

10. Image refernces : https://en.wikipedia.org/wiki/Site-specific_recombination

11. ANTIBIOTIC FREE MARKER
• An alternative method to produce transformants without any antibiotic herbicide
marker gene.
• For this method specific kind of enzymes are used which shows any specific activity
inside a specific transformed cells.
• For ex.
1. Phosphomanose isomerase for E.coil. It converts manose-6-phosphate to fructose-
6-phosphate.
2. Xylpse isomerase for Streptomyces rubiginosus. It catalysed isomerization reaction
of D-xylose to D-xylulose.

12. REFERENCES :
• Strategies for Generating Marker-Free Transgenic Plants; Pérez BC and Angenon
G.
• Methods to produce marker-free transgenic plants; Darbani1B, Eimanifar A, C.
Neal Stewart, Jr. and Camargo W N.
• Marker-free transgenic plants Holger Puchta.
• Transformation Methods for Obtaining Marker-Free Genetically Modified Plants;
Schaart J G, Frans A. Krens, Anne-Marie A. Wolters, and Richard G.F. Visser

13. THANK YOU