Marker free transgenic crops 602

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.

7. 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.

8. 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

9. 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.

10. 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)

11. 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.

12. 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).

13. CASE STUDY

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

15. Vector Construction

16. F0 progeny F1 progeny

17. 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

18. 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

19. Site- Specific Recombination

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

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

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

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

24. 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.

25. Transposition

26. (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.

27. (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;

28. 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 .

29. Homologous Recombination (HR)

30. 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 .

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