Marker Free Transformation in Plants

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DNA, Selectable marker, antibiotic resistance, herbicide resistance, P-DNA, T-DNA, Marker-free transformation

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Marker-free Transformation:

Marker-free Transformation GEETIKA 2012A41D GENETICS AND PLANT BREEDING 21.11.2015 (3.00pm)

Introduction:

Introduction What is marker-free transformation? Process of developing transgenic plants containing only target trait gene (TG) devoid of selectable marker gene(SMG) (for example – antibiotic resistance gene , herbicide resistance gene etc .) . SELECTABLE MARKER GENE TARGET GENE TRANSFORMATION + REMOVAL OF SMG + MFT

Introduction:

Introduction A major focus of modern plant breeding over the last few years is the development of: Specific plant sequences that express desirable traits. This is achieved mainly by: Inserting new sequences in plant genome (i.e. gene targeting) or Removal of undesirable gene sequences from plant genome (removal of selectable markers)

Introduction:

Introduction Selectable marker genes (SMGs) have been very useful in identifying transgenic plants. The most used SMGs encode resistance to antibiotics or herbicides and are used for selection of desirable transgenic combinations by killing non transgenic tissue. However, there are several risks involved by using SMG-transgenic plants, and therefore, efforts are being made to develop marker-free systems. Transgenic Non transgenic Add antibiotic

SMG Concerns (Disadvantages) :

SMG Concerns (Disadvantages) Since SMGs are integrated into the plant genome, there are concerns about widespread occurrence of transgenes in novel ecosystems (e.g., antibiotic resistance in crops and their agro ecosystems). It makes the consumer of the product resistant to these antibiotics. TRANSGENE ANTIBIOTIC RESISANCE CONSUMED BY HUMAN BEING RESISTANT TO ANTIBIOTIC UNPREDICTABLE hazard to the ecosystem or to human health

SMG Concerns (Disadvantages) :

SMG Concerns (Disadvantages) A B C Species Gene transfer A to A B to B C to C VERTICAL GENE TRANSFER CONVENTIONAL A B C Species HORIZONTAL GENE TRANSFER A to B B to A A to C C to A B to C C to B NON-CONVENTIONAL SMG will get transmitted to 1. the same species through pollen in the field (VGT)or another crossable species. 2. another cross incompatible species in the lab (HGT)

Methods of marker-free transformation:

Methods of marker-free transformation Elimination of marker gene by Co-transformation P-DNA Mediated Transformation Transformation by Replacing selectable with screenable markers Site-specific recombination Transposon -based systems

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Co-transformation 1

Co-transformation:

Co-transformation To separate selectable marker genes from the transgene of interest is to separate them at the stage of transformation. Usually Agrobacterium -mediated transformation is used for this purpose, because separate integration events occur more regularly using this method than with direct gene delivery methods. In principle, in co-transformation experiments the desired gene and the transformation marker can be supplied on two T-DNAs within the same binary vector on two binary vectors within the same Agrobacterium with two different Agrobacterium strains

……..Co-transformation:

……..Co-transformation The  binary vector  is a shuttle  vector , so-called because it is able to replicate in multiple hosts ( E. coli and Agrobacterium tumefaciens ). Systems in which T-DNA and vir genes are located on separate replicons are called T-DNA  binary  systems TG SMG TG SMG on two T-DNAs within the same binary vector on two binary vectors within the same Agrobacterium with two different Agrobacterium strains vir

………Co-transformation:

………Co-transformation In all of these variants, 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. How these SMGs are separated ?

Limitations of co-transformations:

Limitations of co-transformations Therefore, co-transformation methods cannot be used for vegetatively propagated plants . These procedures not only require fertile plants , but are also very time consuming . Since Segregation and Recombination are required for removal of SMGs Reports of all kinds are available w.r.t. its success rate

Requirements for Co-transformation:

Requirements for Co-transformation Tight linkage between co-integrated DNAs limits the efficiency of co-transformation. Integration of an SMG and the transgene of interest on separate loci are required . Moreover, it is not applicable to transgenic trees with long generation times.

EXAMPLE :

EXAMPLE Two T-DNAs were efficiently introduced into separate loci using selectable marker gene cassettes consisting In the present study, the CaMV35S:GUS(marker gene) and CaMV35S:GFP(target gene) gene cassettes were co-introduced into the 'Koshihikari' genome Sulfonylurea herbicide resistance to transgenic rice callus was incorporated and the other gene ( CaMV35S:GUS )was removed . Agrobacterium -mediated co-transformation of rice using two selectable marker genes derived from rice genome components. Wakasa et al ., (2012) ' Koshihikari Susceptible to Sulfonylurea herbicide Cotransformation ' Koshihikari RESISTANT to Sulfonylurea herbicide

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2 P-DNA Mediated Transformation

Using P-DNA instead of T-DNA:

Using P-DNA instead of T-DNA P-DNA originated from the T-DNA that was inserted long back into the plant genome through infection of Agrobacterium Plant genome has sequences from Ti Plasmid without open reading frames and rich in A/T infection P-DNA T-DNA ORIGIN PLANT

P-DNA:

P-DNA We can use P-DNA instead of using T-DNA for transformation . P-DNA is found in plants so question of biosafety does not arise. Examples are there which demonstrate that P-DNA can be used to replace the Agrobacterium T-DNA mediated transformation Co-transformation of the inserted desired transgene into P-DNA and SMG-containing T-DNA can produce marker-free transgenic plants. T-DNA P-DNA SMG Desired transgene Co-transformation Marker-free transgenic plants No biosafety questions

Use of mutated virD2 gene :

Use of mutated virD2 gene If T-DNA has virD2 mutation , it is lost in subsequent generations . This property of mutated T-DNA is used for marker free transformation . Agrobacterium with virD2 gene into T-DNA Agrobacterium without virD2 gene into T-DNA + P-DNA with transgene + P-DNA with transgene Mostly P-DNA with transgene P-DNA with transgene + T-DNA MFT

EXAMPLE :

EXAMPLE Double T-DNA" binary vector system pDLBRBbarm was used. It carried two independent T-DNAs, one containing a selectable marker neomycin phosphotransferase ( nptll ) gene and the other a bar gene (inserted), was constructed. Transgenic tobacco ( Nicotiana tabacurn L.) plants were then produced by Agrobacterium -mediated transformation with this vector. Nptll was deleted in segregating generations Zhoug Hong et al. (2003) Generating Marker-Free Transgenic 'tobacco Plants by Agrobactertum -mediated Transformation with Double T-DNA Binary Vector

EXAMPLE :

EXAMPLE The  ipt -type MAT vector uses the  ipt  gene for regeneration of marker-free transgenic plants. When scutellum tissues of rice seeds were transformed, marker-free transgenic rice plants directly regenerated from 25.5% infected scutellum tissues without forming  ipt -intermediates within 4 weeks after an infection. Excision of the ipt  gene occurred in embryogenic tissues. Therefore, this system needs no sexual crossing for identification of transgenic plants not containing a selectable marker gene. Endo  et al (2002) Single-step transformation for generating marker-free transgenic rice using the  ipt -type MAT vector system

EXAMPLE :

EXAMPLE To develop transgenic rice resistant to lepidopteran insects, a marker-free binary vector harboring the  Bacillus thuringiensis   cyr1Ac  gene was constructed and used for Agrobacterium tumefaciens -mediated transformation of rice calli. The transgene expression of cry1Ac  was stable and effective against rice leaf folder. Generation and molecular characterization of marker-free Bt transgenic rice plants by selectable marker-less transformation Hee-Jong  Woo et al. (2015)

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3 Replacing selectable with screenable markers

Replacing selectable with screenable markers :

Replacing selectable with screenable markers In general, untransformed cells are killed by the presence of antibiotics or spray of herbicides ( selective agent ) because they are susceptible to antibiotics and herbicides. A new set of markers is has been developed by which identification of transgenic plants in the absence of a selective agent is done using screenable markers The transformed cells also get the metabolic or developmental advantage . Here biosafety ethics are followed.

EXAMPLE:

EXAMPLE Recently, an Escherichia coli -derived phosphomannose isomerase (PMI) was used to convert mannose-6-phosphate to fructose-6-phosphate for positive selectable marker in plant transformation. Only transformed cells are capable of utilizing mannose as a carbon source. PMI has been used as a screenable marker for transformation of many plant species, such as sugar beet, maize, wheat, rice, pearl millet, canola, apple and plum . SOURCES : Daniell et al ., 2001, Joersbo et al ., 1998, Negrotto et al., 2000; Wright et al., 2001, Reed et al ., 2001 and Gadaleta et al ., 2006

EXAMPLE:

EXAMPLE Other screenable markers have been used successfully in transgenic plant production were: bacterial b – glucuronidase yeast 2-decoxyglucose-6-phosphate phosphatase Kunze et al ., 2001

EXAMPLE :

EXAMPLE It was found that ipt  gene with a  35 S promoter can be a visually selectable/screenable marker for transformation of tobacco plants and hybrids. ipt  gene raised the cytokinin level of plants (potato, cucumber, tobacco,  Arabidopsis ) that promoted shoot formation. Shoot formation was used to identify transgenic plants. Selection of marker-free transgenic plants using the isopentenyl  transferase gene ( ipt  gene ) in Tobacco Hiroyasu Ebinuma et al .(1997) ipt  gene 35 S promoter

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4 Site-specific recombination

Site-specific recombination:

Site-specific recombination Homologous DNA molecules participates in recombination. This recombination is a universal phenomenon that can occur at any place along these two homologous DNA molecules. x x x x However,site-specific recombination occurs between defined excision sites in the phage and in the bacterial chromosome.

Site-specific recombination Use of pMF-vectors for marker-free technology:

Site-specific recombination Use of pMF -vectors for marker-free technology pMF is a new plant transformation vector for marker-free transformation. Use of the pMF vectors for plant transformation leads to removal of undesired DNA sequences, like antibiotic resistance genes.

Site-specific recombination Use of pMF-vectors for marker-free technology:

Site-specific recombination Use of pMF -vectors for marker-free technology Principle The pMF vector provides an inducible site-specific recombination system for removal of undesired DNA sequences. A negative selection step using the cod A (cytosine deaminase) gene, ensures the ultimate production of completely marker-free plants. Cod A is a conditionally lethal dominant gene encoding an enzyme that converts non-toxic 5-fluorocytosine (5-FC) to cytotoxic 5-fluorouracil (5-FU). MARKER GENE IS DELETED

EXAMPLE :

EXAMPLE Marker free approach to develop Salt Tolerant Brassica Rajwanshi et al., (2007) LB RB gly I Inducible promoter T-DNA cre recombinase hsp promoter The over expression of the gly I gene imparts salt and heavy metal stress tolerance loxP npt II loxP Inducible promoter cre recombinase loxP npt II loxP The heat shock induction resulted in the excision of the intervening region between the loxP sites, which contained the npt II marker gene and the hsp-cre gene Heat shock induction LB RB gly I + MFT Salt Tolerant

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5 Transposon-based expelling system

What are transposons?:

What are transposons? Besides site-specific recombination, transposable elements can also be used to obtain marker-free transgenic plants. The most widely studied transposon is the P element from the fruitfly ( Drosophila melanogaster ). Use of transposable elements for marker gene removal involves several steps: insertion of the marker gene onto a transposon, co-transformation with gene of interest; and segregation of the marker gene

……Transposons:

……Transposons The advantage of this system is not only to unlink the marker-gene, but also to create a series of plants with transgene loci different from one original transformant . This re-positioning allows expression of the transgene at different genomic positions and consequently at different levels of expression. However, different species have variable rates of transposition efficiency.

EXAMPLE :

EXAMPLE Efficient generation of marker-free transgenic rice plants using an improved transposon-mediated transgene reintegration strategy .  It has been shown that Ac-Ds transposable elements can be used to generate marker-free transgenic plants in maize. To test the worth of this strategy, the Bacillus thuringiensis (Bt) δ- endotoxin gene was cloned into the Ds elements and transformed transposon vectors into rice ( Oryza sativa ) cultivars via Agrobacterium tumefaciens. Ds element helped in producing marker-free transgenic rice by eliminating marker gene but retain ing striped stem borer resistance. Striped stem borer Chilo suppressalis.  Gao et al., (2015)

CONCLUSION:

CONCLUSION Different approaches for elimination of selectable marker genes have been developed over the last several years, and further improvements are now underway. Each approach has its plus as well as minus points. Refinement is still required for any method to be precise and perfect. However Site specific recombination and co-transformations have proved their worth in many crops. Screenable markers and P-DNA have also advantages over SMGs w.r.t . biosafety concerns.

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