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Marker free transgenics: concept and approaches

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Marker free transgenics for better and safer GMO for food and health security

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Marker free transgenics: concept and approaches

  1. 1. 9/16/2014 1
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  3. 3. 3 Introduction Need for marker free transgenics Approaches and applications of marker free transgenics content Conclusions
  4. 4. Introduction 4
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  6. 6. Selectable markers (SMGs) 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 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. 6
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  9. 9. Controversy and disadvantages related to SMG 1. Food safety , effect on natural ecosystem 2. Gene flow into non-GM crops, human and animal bacteria, wild and weedy relatives 3. Inability for gene stacking in already transformed plant with same SMG
  10. 10. ON TARGET OUR AIM • To eliminate selectable marker gene • To avoid use of toxic selectable marker gene Marker free transgenic
  11. 11. MAIN STRATEGIES
  12. 12. 1. Co-transformation of marker genes and gene of interest (GOI)
  13. 13. 13 Co-integration T0
  14. 14. Schematic diagram of Co-transformation method for making marker free transgenic 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. 14 Narendra Tuteja et al., 2012
  15. 15. Marker free sheath blight resistance rice by co transformation 15 technique Sripriya and Raghupathy. (2008)
  16. 16. 16 Southern blot analysis of T0 lines for chitinase gene P- positive control, M -HindIII marker, E-empty lane, U undigested DNA from the transgenic plants Sripriya and Raghupathy. (2008) Hind III
  17. 17. 17 CoT6 CoT23 Southern blot analysis of T1 populations from CoT6 & CoT23 Sripriya and Raghupathy. (2008)
  18. 18. Rice stripe virus (RSV) The RSV genome consists of four single-stranded RNA segments, designated as RNAs 1 to 4. The complementary sense • RNA 3 encodes the coat protein (CP) • RNA 4 encodes the special-disease protein (SP). 18 Jaing et al. (2013)
  19. 19. PCR and leaf painting analysis of T0 transformation events derived 19 from pDTRSVCP and pDTRSVSP Jaing et al. (2013)
  20. 20. PCR analysis of T1 transformation events derived from pDTRSVCP and pDTRSVSP 20 13 18 Jaing et al. (2013)
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  22. 22. Particle bombardment method (leaf bacterial blight resistant rice) Schematic maps of the source plasmid pCB1 and pCB4 22 Act Rice actin-1 promoter, cB cecropinB gene encoding sequence, Pin potato proteinase inhibitor II terminator, Yan et al. (2007)
  23. 23. Varieties Transgenic Lines Test # of Basta resistant plants # of cecropinB PCR (+) plants Co-segregation frequency (%) Xiushui 04 XIF-41 30 30 100 XIF-42 30 27 90.0 Jia59 J4F-17 30 25 80.6 J4F-18 30 9 30.0 J4F-49 30 12 40.0 J4F-50 30 12 40.0 J4F-51 30 11 36.7 23 The co-segregation frequency of bar and cecropinB gene cassettes in transgenic rice lines of T1 generation Yan et al. (2007)
  24. 24. The result of producing transgenic plants carrying cecropinB gene cassette without selectable marker bar in T1 generation Transgenic plant lines Germination percentage of T1 seeds (%) T1 Basta-resistant plant number T1 Basta-sensitive plant number cecropinB PCR (+) plant number of T1 Basta-sensitive plants J4F-49 58 42 16 0 J4F-50 49 17 32 2 J4F-51 62 43 19 0 24 Yan et al. (2007)
  25. 25. M DNA molecular weight markerIII, U untransformed rice plant control; 1 R0 plant of J4F-50, 2 R0 plant of J4F-51, 3 and 4 marker-free transgenic plants of MFc-1and MFc-2 carrying cecropinB gene cassette only. 25 Yan et al. (2007)
  26. 26. Advantages Simple and effective Easier handling of the binary vectors because the two T-DNA are separated Disadvantages It is time consuming and compatible only for sexually propagated fertile plants. The tight linkage between co-integrated DNAs may limit the efficiency of co-transformation 26
  27. 27. 2. Site-specific recombination mediated SMG removal
  28. 28. (a) The T-DNA region showing Cre gene followed by the transcribed mRNA and Cre protein expression. (b) T-DNA region showing GOI and marker gene merged between loxP sites. (c) Resulting transgenic plants showing excision of marker gene. 28 Narendra Tuteja et al., 2012
  29. 29. The Cre/lox system Constitutive Expression of Recombinase Gene • Plant hybridization: Transforme d plant GOI & SMG • Retranformation Induced Expression of Recombinase Gene • Simultaneous transformations: 1st T-DNA (IP + Cre) 2nd T-DNA (SMG + GOI) • Heat shock treatment • Chemical treatment • By activating the promoters with inducers (heat or chemical), the expression of recombinase gene can be more tightly controlled. • Autoexcision scheme x Transformed F1 Plant containing both transgene screened for SMG deletion event plant RECOMBINAS E CRE
  30. 30. 30 Aphid resistant marker free transgenic mustard. Schematic representation of the T-DNA region of two binary vectors used for mustard transformations. A - pBKhgASAL showing ASAL gene & B - pBK16.2 showing the cre gene Bala et al. (2013)
  31. 31. DNA blot analysis for confirming vector integration in T0 ASAL Cre Lane 1 & 9 362 bp positive control for ASAL, Lane 2 & 10 negative control 31 Bala et al. (2013)
  32. 32. 32 Cre gene ASAL hpt Molecular analysis of marker gene excision. Bala et al. (2013)
  33. 33. 33 PCR analysis of F2 progeny plants.
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  35. 35. Auto excision mediated Cre/lox system with floral specificOsMADS45 promoter : Bai et al. (2008) 35
  36. 36. 36 3.3kb gus
  37. 37. 37 FLP/FRT recombination system Fig. FLB/frt site-specific recombination system. (a) The T-DNA region showing FLP gene controlled by heat inducible promoter (hsp70) followed by the transcribed mRNA and FLP protein expression. (b) T-DNA region showing GOI and marker gene merged between frt sites followed by resulting transgenic plants showing excision of marker gene.
  38. 38. 38 Salt tolerant marker free transgenic maize LB Promoter FLP Terminator RB Li et al. (2010) 0.9 kb D1D2
  39. 39. Southern blotting analysis showing the presence of transgene AtNHX1 and flp in the genome of the transgenic F1 plants. BamH I Kpn I 39
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  41. 41. 41 R/RS recombination system from Zygosaccharomyces rouxii
  42. 42. 3. Transposon-based SMG removal
  43. 43. 43 3. Transposon-based SMG removal
  44. 44. marker-free rice plants expressing a Bt Schematic representation of the Ds-cry1B T-DNA. Olivier et al. (2002) 44 endotoxin gene
  45. 45. (A) Investigation of T-DNA organisation in T0 45
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  48. 48. 48 Recovery of hph selectable marker free Cry1B transgenic rice in T1 generation Southern blot of EcoRI and BamHI of 30 T1 plants
  49. 49. Advantages Suitable for removal of marker genes in vegetatively propagated plants Disadvantages Variable rates of transposition Labour and cost intensive Mutations Genomic instability Decreased efficiency Scutt et al., 2002 49
  50. 50. 4. Positive selection
  51. 51. Kunze et al. (2001) DOGR1 gene as alternative selectable marker
  52. 52. 5. Methods of direct transformant screening
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  54. 54. s

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