MARKER FREE TRANSGENICS DEVELOPMENT

Guided by- Dr. Kanchan S. Bhan

Assistant Proffessor

Presented By

Arpita MahobiaPh.D. 1st Year

Dept. of Plant Molecular Biology &

Biotechnology

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

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.

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.

04/15/2023Dept. of PMBB

04/15/2023Dept. of PMBB

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

STARATEGIES TO PRODUCE RESISTANT MARKER‐FREETRANSGENIC PLANTS

•Use of screenable markers•Co-transformation•Site-specific recombination •Multi-autotransformation vector•Intrachromosomal recombination system•Transposition system

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.

•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

Cannot be used for vegetative propagated plants

(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.

• 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

Site‐specific recombination‐mediatedmarker deletion

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

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• 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

MAT SYSTEM

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•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

MAT SYSTEM

04/15/2023Dept. of PMBB

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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.

INTRACHROMOSOMAL RECOMBINATION SYSTEM•Recombination resulting from crossing over

between two linked gene pairs.

• 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

Transposon‐based marker methods

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.

An inducible transposon system to terminate thefunction of a selectable marker in transgenic rice

(Tu et al., 2008)

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.

THANK YOU