Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.

Cisgenics- Clean marker assisted Technology


Published on

Cisgenics as a next generation GMO crops. This concept is new and alternative to transgenic crops...can avoid fear of transgenics w.r.t health and environment problems.

Published in: Education
  • Be the first to comment

Cisgenics- Clean marker assisted Technology

  1. 1. Welcome
  2. 2. Cisgenesis strongly improves Introgression and Induced Translocation breeding of plants.2
  3. 3. Introduction Why cisgenic approach? Prerequisites for cisgenic approach Method to develop cisgenic plant Conclusion3
  4. 4. Concept of cisgenesis introduced by Dutch researchers Schouten, Krens and Jacobsen (2006)4
  5. 5. How can we go for genetic improvement? g PLANT VARIETIES d in ree Modern lb insights GMO ca &techniques REGULATIONS including ssi biotechnologyCla 5
  6. 6. Overview of existing Technologies6
  7. 7. Key terms • Cisgenesis is the genetic modification of a recipient plant with a natural gene (includes its introns and is flanked by its native promoter and terminator in the normal sense orientation) from a crossable sexually compatible plant • A cisgene is a natural gene, coding for an (agricultural) trait, from the crop plant itself or from a sexually compatible donor plant that can be used in conventional breeding.7
  8. 8. Contd...  The gene pool for cisgenesis is identical to the gene pool available for classical breeding.  Cisgenic plants can harbor one or more cisgenes, do not contain any transgenes.  No foreign DNA, such as selection marker genes and vector- backbone sequences, should remain in the final cisgenic plant. The author of ‘Invasion of Genes - Genetic Heritage of India’ Dr B. S. Ahloowalia said: “Cisgenics removes all fears associated with transgenic crops.”8
  9. 9. Contd…• Transgenesis is the genetic modification of a recipient plant with one or more genes from any non-plant organism, or from a donor plant that is sexually incompatible with the recipient plant.• This includes gene sequences of any origin in the anti-sense orientation, any artificial combination of a coding sequence and a regulatory sequence, such as a promoter from another gene, or a synthetic gene. 9
  10. 10. Contd… • Traditional or conventional breeding: uses all classical and modern insights and techniques, including those related to biotechnology, however, without genetic modification. • It includes a number of techniques that are not regarded as genetic modification or that are exempted from the GMO- legislation. • Particularly the European Directive 2001/18/EC on the deliberate release of GMOs into the environment (European Parliament, 2001)10
  11. 11. Contd… • Translocations or interchanges: structural changes in chromosomes, where segments of non homologous chromosomes have exchanged positions.  Why interchanges important for plant breeders, geneticists & evolutionists?  Interchanges bring about changes in linkage relationship, changes in chromosome structure & behavior by which variability is created.11
  12. 12. Traditional breeding 12
  13. 13. Why cisgenic approach?  Some plant spp. difficult to breed by classical method e.g. woody plants- don’t flower for many years, intolerant to inbreeding, highly heterozygous.  Some plant spp. are naturally sterile / are part of a highly desired and commercially widespread clone whose genotype needs to remain intact. e.g. potato, apple, grape, and banana.13
  14. 14. To appreciate cisgenesis…… 1st we need to understand the problems related to… 1. Transgenic approach 2. Traditional breeding and 3. Translocation breeding.14
  15. 15. What is the problem with transgenesis? B.t Transferred gene usually derives from an alien species.• Extends the gene pool of the recipient species.• Such a novel gene might provide the target plant with a new trait that neither occurs in the recipient species in nature nor can be introduced through traditional breeding.15
  16. 16. Contd…  In recipient species fitness may change in various ways:  Through gene flow between a GM crop and its wild relatives potentially creating shifts in natural vegetation.  The generation of these new ‘unnatural’ gene combinations is regarded as both unethical and having potential long-term risks for health and environment.(non-targeted organisms/soil ecosystems) den Nijs, 200416
  17. 17. T a r g e t p l a n t GENE FLOW17 FITNESS OF THE POPULATION
  18. 18. How cisgenic plants can overcome problems of transgenic plants? Transgenesis18
  19. 19. What is the problem with introgression breeding? High-quality genotype (cultivar) X wild plant (gene of interest). The wild plant, passes genes of interest to the progeny, but also deleterious genes. This ‘linkage drag’ tremendously slow down the breeding process, esp. if the gene of interest is genetically tightly linked to one or more deleterious genes. Quality of crop is ruined. To reduce linkage drag, need successive generations of recurrent backcrossing with the cultivated plant and simultaneous selection for the trait.19
  20. 20. Contd..  As apple cultivars are self-incompatible and highly heterozygous, the phenotype of a cultivar is unique and breeding produces genotypes with new and distinct characteristics.  Limited Popularity of the new cultivars carrying disease resistance genes since originality of cultivar lost. (Gardiner et al., 2007)20
  21. 21. How cisgenesis can overcome problems of introgression breeding? Introgression breeding21
  22. 22. Induced Translocation breeding  Chromosome pairing and recombination in common wheat is largely governed by the gene Phi, located on the long arm of chromosome 5B, which ensures that only homologous chromosomes can pair and recombine.  Sears (1956) used ionizing radiation treatment to induce chromosome breaks and transferred a gene conditioning resistance to:  leaf rust caused by Puccinia recondita f. sp. tritici from Ae. umbellulata Zhuk. to wheat. Riley & Chapman,1958; Sears & Okamoto, 1958; Sears, 1976;22
  23. 23. Major problem in induced translocation breeding.  Radiation treatment causes random chromosome breaks.  The majority of translocations resulting from radiation treatments were formed between non-homoeologous chromosome arms.  These non-compensating translocations are genetically unbalanced, and lead to reduced agronomic performance. The radiation-induced Sr26 transfer, derived from A. elongatum, the translocation causes a reduction of about 10% in yield23 The et al., 1988
  24. 24. 24
  25. 25. Is Cisgenesis efficient method in crops? Yes…  Particularly efficient method for cross-fertilizing heterozygous plants that propagate vegetatively, such as potato, apple and banana.  Cisgenesis might also supplement classical breeding for improving traits with. limited natural allelic variation in cultivars and wild species  E.g. expression of an endogenous phytase gene in barley through the insertion of extra gene copies of the endogenous phytase gene isolated from barley itself25
  26. 26. The prerequisites for cisgenesis…. Sequence information of the plant. The isolation and characterization of genes of interest from crossable relatives.26
  27. 27. Web site address for data base info..27
  28. 28. 28
  29. 29. Solutions via cisgenesis29
  30. 30. Development of a cisgenic apple plant. Thalia Vanblaere,201130
  31. 31. Present day Apple (Malus x domestica) cultivars are susceptible to Apple scab which is caused by the fungus Venturia inaequalis early symptoms31
  32. 32. Apple growers spray on an average 15 times in one season to control the disease. Spraying fungicides in Apple orchard Patocchi et al., 200432
  33. 33. Any natural resistance? Yes……..  Source natural resistance to diseases is known .  Classical breeding has developed scab resistant cultivars, mostly by introgression of Vf resistance from Malus floribunda 821 (Lespinasse,1989; MacHardy, 1996)33
  34. 34. Several apple scab resistance genes Identified and were mapped on different linkage groups of the Apple genome.34
  35. 35. Impact of Vf genes in conventionally bred Apple35
  36. 36.  Only Vf locus has been positionally cloned ( Vinatzer et al., 2001)  Proved to consists of a gene cluster with four paralogs : 1. HcrVf1 2. HcrVf2 3. HcrVf3 and 4. HcrVf4 ( Xu and Korban 2002)36
  37. 37.  Initially, only the HcrVf2 gene was tested by overexpression using the CaMV35S promoter & also own promoter and nos terminator,in scab susceptible Apple plants.  Only one resistance encoding gene, HcrVf2, has been isolated and proven functional to date in cvs. Gala and Elstar. Belfanti et al., 2004; Szankowski et al., 2009; Joshi,37 2010.
  38. 38.  In Transgenic Apple, for successful transformation marker gene nptII & other regulators added. Joshi, (2010)38
  39. 39. But people don’t need Transgenic Apple……..  A large proportion of the consumers in Europe view genetically modified foods as a risk to both health and the environment (Gaskell et al., 2000) CISGENIC39
  40. 40. Cisgenic plants are produced by the same transformation techniques as transgenic plants.40
  41. 41. Construction of vector 17434 bp41
  42. 42.  In order to develop marker-free plants the chemically inducible recombinase system reported in strawberry was applied to apple. Schaart et al., (2004)42
  43. 43. Method to develop cisgenic plants  Technique used: 2 independent regeneration step with 1 binary vector Transformation with stable integration using positive selection e.g. on kanamycin (nptII) Removal of marker by chemical induction of Recombinase R activity ( Decamethosone treatment) Selection for marker free plants using negative selection (codA) on 5-Fluro cytosine (toxic 5-Fluro uracil)43 Schaart,2004
  44. 44. Clean vector systemT-DNA insert in transgenic line RB RS R-LBD Recombinase CodA-NptII fusion RS LB prom HcrVf2 term Recombination RB RS R-LBD Recombinase CodA-NptII fusion RS LB prom HcrVf2 term T-DNA insert after cisgenic line RS LB RB prom term HcrVf244
  45. 45. 10 transgenic lines were regenerated through selection on Kanamycin medium 8 out of 10 lines have backbone integration (nptII)45
  46. 46. ‘Gala’ scab susceptible cultivarHcrVf2 Florina,classical bred cv. Vf resistance 3 cisgenic lines are derivedCodA marker gene pMF vectornptII (backbone) 46
  47. 47. Gene expression analysis by PCR of cDNA Transgenic Transgenic Cisgenic lines Cisgenic lines47
  48. 48. Copy number by southern Blot1 copy of nptII in all transgenic lines 1 copy of HcrVf2 in C7.1.49 & C 11.1.53 2 copies of HcrVf2 in T12.1 (C12.1.49)48
  49. 49. 49
  50. 50. A cisgenic GM strategy for durable resistance based on R and Avr genes The new concept : HEALTHY POTATO Haverkort, A. J. et al.,200750
  51. 51. Late blight of potato deadly disease in potato. Phytophthora resistance in potato is easily broken. A better strategy is needded for Sustainable resistance Many R-genes are available in crossable wild species, enabling the development of a resistance strategy With useful molecular knowledge Avr-genes from the pathogen.51
  52. 52. 52
  53. 53. List of R genes and Avr genes53
  54. 54. Approach: Cloning of R genes Marker free transformation Cisgene field tests54
  55. 55. 55
  56. 56. Endogenous Fungal Resistance Genes Plants have been genetically engineered with either:  Endogenous gene 1: V. vinifera thaumatin-like protein gene (vvtl-1) for fungal disease resistance or  Endogenous gene 2: 2S albumin gene (alb) from grapevine56
  57. 57. Genes are expressed via bidirectional promoter system57
  58. 58. 58
  59. 59. Cisgenic Approach for Improving the Bioavailability of Phosphate in the Barley Grain. Holme I B et al.,201259
  60. 60. leading to water pollution…….60
  61. 61. PAPhy (purple acid phosphatase)  Two types: HvPAPhy_a and HvPAPhy_b.  HvPAPhy_a is preferentially synthesized during seed development and stored as preformed phytase in the mature grain,  HvPAPhy_b is preferentially synthesized during germination. Therefore ,the HvPAPhy_a gene as a candidate for our cisgenic approach.61
  62. 62. 62
  63. 63. Phytase Activities in Cisgenic Barley Seeds Revealed a positive correlation between gene dosage and gene expression of the phytase gene.63
  64. 64. Modifying Plant Growth using GA-associated cisgenes. POPLAR TRE ES Han K,201064
  65. 65.  The transfer of entire native genes that play roles in biosynthesis or signaling of gibberellic acids (GAs), including their 5’ and 3’ proximal regulatory regions, impart changes in growth rate and stature in poplar trees.65
  66. 66.  Five different cisgenes : GA20ox7,GA2ox2, GAI1, RGL 1_1, and RGL 1_2 .  GA20oxidase catalyzes the penultimate step in the biosynthetic GA pathway, in turn promote cell division and elongation.  The other genes tend to repress or attenuate active GA actions. a) GA degradation by GA2ox2. b) DELLA domain proteins that attenuate GA signals.66
  67. 67.  Plants transformed with GA20ox7 cisgene had a higher rate and frequency of regeneration of transgenic shoots during antibiotic selection.  The average stem volume of the GA20ox7 transformed plants increased by 40% compared with the transgenic (empty vector) controls.  GA20ox7 gene expression was also statistically associated with the growth enhancement.67
  68. 68. 68
  69. 69. 69
  70. 70. Thank you