Introduction ◦ The process of developing transgenic plants without the presence of selectable markers (or) by use of more acceptable marker genesis regarded as Clean Gene Technology. ◦ Also called “Marker Free Approach” for transgenic plant. Need of CGT ◦ The products of some marker genes may be toxic or allergic. ◦ The antibiotic resistance might be transferred to pathogenic microorganisms in the soil. ◦ There is a possibility of creation of superweeds that are resistant to normally used herbicides. Methods of CGT ◦1. Co-Transformation ◦2. Site Specific Recombination ◦3. Transposon‐Based Marker Method ◦4. Intra Chromosomal Homologous Recombination 1.Co-transformation ◦ Co-transformation is a method for production of marker free transformants based on Agrobacterium- or biolistic mediated transformation in which a SMG and gene of interest are on separate constructs. ◦ SMGs can subsequently be removed from the plant genome during segregation and recombination that occurs during sexual reproduction by selecting on the transgene of interest and not the SMG in progeny. 2. Site Specific Recombination ◦ Also called “mediated marker deletion”. ◦ The deletion of short, recognized DNA sequence with the activity of recombinase enzymes has been a major step to acquire the marker free transgenic plant. ◦ Systems for removal of marker gene 1. Cre/loxP: 2. FLP/FRT: 3. R/RS: E.coli Bacteriophage Saccharomyces cerevisiae Zygosaccharomyces 3. Transposon‐based marker method ◦ This process is quite similar to site specific recombination where transposons or jumping genes are used instead of a recombination and recognition sites. ◦ Steps: •(i) Insertion of the marker gene onto a transposon, a segment of DNA that “hops” around in the plant’s genome; •(ii) co-transformation with gene of interest •(iii) Segregation of the marker gene. 4. Intrachromosomal recombination system ◦Non‐recombinase ◦ Spontaneous excision attp: attachment P region of bacteriophage λ ICRS ◦ Recombination sites are engineered into the plant, but no recombinase is expressed. ◦Intrachromosomal recombination in plants is obtained by insertion of SMG between two direct repeats of attP that facilitates spontaneous excision. ◦ Base composition of the attP site sequence is A + T rich, which is conjectured to play a recombination-stimulating role. .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 program.

1. Clean Gene
Technology
Guided By:- Dr. Bishun Deo Prasad
Prepared By:- Asjad Raza
Biotech. 412 Principles and Applications of Plant Genetic Transformation

2. Introduction
◦ The process of developing transgenic plants without the
presence of selectable markers (or) by use of more acceptable
marker genesis regarded as Clean Gene Technology.
◦ Also called “Marker Free Approach” for transgenic plant.

3. Need of CGT
◦ The products of some marker genes may be toxic or allergic.
◦ The antibiotic resistance might be transferred to pathogenic
microorganisms in the soil.
◦ There is a possibility of creation of superweeds that are resistant
to normally used herbicides.

4. Methods of CGT
◦1. Co-Transformation
◦2. Site Specific Recombination
◦3. Transposon‐Based Marker Method
◦4. Intra Chromosomal Homologous Recombination

5. 1. Co-transformation
◦ Co-transformation is a method for production of marker free
transformants based on Agrobacterium- or biolistic mediated
transformation in which a SMG and gene of interest are on
separate constructs.
◦ SMGs can subsequently be removed from the plant genome
during segregation and recombination that occurs during sexual
reproduction by selecting on the transgene of interest and not
the SMG in progeny.

6. Co-transformation
Narendra Tuteja et al., 2012

7. 2. Site Specific Recombination
◦ Also called “mediated marker deletion”.
◦ The deletion of short, recognized DNA sequence with the activity of
recombinase enzymes has been a major step to acquire the marker free
transgenic plant.
◦ Systems for removal of marker gene
1. Cre/loxP: 2. FLP/FRT: 3. R/RS:
E.coli Bacteriophage Saccharomyces cerevisiae Zygosaccharomyces

8. Narendra Tuteja et al., 2012

9. 3. Transposon‐based marker method
◦ This process is quite similar to site specific recombination where
transposons or jumping genes are used instead of a
recombination and recognition sites.
◦ Steps:
• (i) Insertion of the marker gene onto a transposon, a segment
of DNA that “hops” around in the plant’s genome;
• (ii) co-transformation with gene of interest
• (iii) Segregation of the marker gene.

10. 4. Intrachromosomal recombination system
◦ Non‐recombinase
◦ Spontaneous excision
attp: attachment P region of bacteriophage λ
Meyer at el., 2002

11. ICRS
◦ Recombination sites are engineered into the plant, but no
recombinase is expressed.
◦ Intrachromosomal recombination in plants is obtained by
insertion of SMG between two direct repeats of attP that
facilitates spontaneous excision.
◦ Base composition of the attP site sequence is A + T rich, which is
conjectured to play a recombination-stimulating role.

12. 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 program.