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9 12 .


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9 12 .

  1. 1. Ornamental floriculture is becoming an important industry . Ornamentals include a large variety of crop plants Cut flowers, Bulbs and corms, Foliage and Flowering pot plants. All the present day ornamental varieties and novelties are as a result of extensive hybridization, induced mutation and selection .
  2. 2. Genetic engineering:The technology of preparing recombinant DNA in vitro by cutting up DNA molecules and splicing together fragments from more than one organism. Genetic engineering is a laboratory technique for gene manipulation. Genetic engineering brings about novel combination of genes by using recombinent DNA technology which is not
  3. 3. Genetic engineering of plants is much easier than animals. there is natural transformation system for plants(Agrobacterium). plant tissue can redifferentiate. plant transformation and regeneration are relatively easy for a variety of plants. Agrobacterium tumefaciens can infect wounded plant tissue, transferring a large plasmid, the Ti plasmid, to the plant cell.
  4. 4. Important methods in recombinant DNA technology are Isolation of desired gene Insertion of isolated gene into a suitable vector Introduction of recombinant vector in to host Selection of transformed host cells (A.C.Dutta 2005)
  5. 5. Digestion of the cell wall by enzymatic action, dissolution of the biological membranes by detergent losses, centrifugation to isolate pure DNA. DNA cut into no. of fragments by restriction endonulcleases “molecular scissors” with sticky ends.
  6. 6. Isolating Genomic DNA Fragmenti ng DNA Screening DNA fragments Insertion of DNA in vector Introducin g DNA in host Culturing the cells Transformatio n of host cell
  7. 7.  Most widely used  More economical  More efficient Agrobacterium mediated gene transfer  Particle bombardment or micro projectile .  Direct DNA delivery by PEG . Electroporation .  Microinjection .Chandler and Brugliera, 2011
  8. 8. 1-2 µm of tungsten or gold particles (microprojectiles)coated with DNA to be used for transformation are accelerated to velocities using pressurized Helium gas
  9. 9. DNA solution is injected directly inside the cell using capillary glass micropipetts .
  10. 10. 2 3 1T - DNA Ti - Plasmid
  11. 11. 2 The same restriction enzymes cut the same base sequences in plasmid DNA. 5 Recombinant DNA inserted into host cells is copied each time the host cells divide. 1 Restriction enzymes cut specific base sequences. 4 The result is recombinant DNA molecules with both Target and plasmid DNA. 3 The plasmid DNA and the target DNA fragments are mixed in a solution with enzymes that link them together. Recombinant DNA Technology
  12. 12. For a modern and industrialized horticulture there is always demand and necessity for new varieties. To develop new varieties through genetic manipulation , there are several plant breeding techniques.
  13. 13. However combining large parts of parental genomes in rather uncontrolled fashion is a miss process to a larger extent. Genetic engineering on the other hand allows transfer of very specific genes in to plants.
  14. 14. This transgenic technology can be used to generate Flower crops resistant to biotic and a biotic stresses Flowers with new colors, Flowers with improved size, shape and floral scent , Flowers having long vase life .
  15. 15. Flavonoids are one of the main determinants of flower colors. Flavonoid compounds are produced by the phenyl propanoid pathway. Primary function of flavonoid pigments in flowers is to attract insects and other animals which help in cross pollination (Brouillard and Dangles 1993).
  16. 16. Gerbera (Elomaa 1993) Reduction of anthocyanin Petunia Rose (Gutterson 1995), Chrysanthemum (Courtney- Gutterson et al.,1994) . Carnation (Gutterson 1995) . (Krol et al.,1988). .
  17. 17. Wild-type petunia producing purple anthocyanin pigments Chalcone synthase (CHS) is the enzyme at the start of the biosynthetic pathway for anthocyanins Photo credit Richard Jorgensen; Aksamit-Stachurska et al. (2008) BMC Biotechnology 8: 25. Anthocyanins Chalcone synthase (CHS)
  18. 18. Attempted to overexpress chalone synthase (anthrocyanin pigment gene) in petunia. (trying to darken flower color) Caused the loss of pigment.
  19. 19. Small RNAs are a pool of 21 to 24 nt RNAs that generally function in gene silencing . Small RNAs contribute to post-transcriptional gene silencing by affecting mRNA stability or translation AAAAA RNA Pol Histone modification, DNA methylation
  20. 20. Sense RNA Antisense RNA Sense construct: PRO CHS Endogenous gene mRNA Transgene PRO CHS mRNA Protein translated mRNA mRNA Extra protein translated Antisense construct: PRO CHS Transgene Sense-antisense duplex forms and prohibits translation
  21. 21. Surprisingly, both antisense and sense gene constructs can inhibit pigment production Photo credit Richard Jorgensen Plants carrying CHS transgene CaMV 35S pro : CHS CaMV 35S pro : CHS Sense Antisense OR
  22. 22. In petunia cyanidin and delphinidin derivatives but no pelargonidin derivatives. Enzyme dihydro flavonol 4 reductase ( DFR ) A1 gene from maize encodes dihydro quercetin 4reductase- doesn’t show substrate specificity as doespetunia enzyme
  23. 23. RL01 mutant petunia line - accumulates dihydrokaempferol - no pigmentation Insertion of Maize A1 gene as a chimeric constuct withca MV35s promoter (Schwarz –somner et al., 1987) encodes dihydroquercetin 4 reductase. Over expression of A1 gene + abundant substrate due to petunia mutation – synthesis of novel brick red colored petunia (Meyer et al., 1987)
  24. 24. Chalcones contribute to the yellow colors in Dianthus caryophyllus (Forkman and Dangel meyer 1980). In petunia and Lisianthus aimed at accumulating chalcones, and produce yellow pigments in flowers as expected (Van bockland et al., 1993).
  25. 25. Later discovered - a chalcone 2′-glucosyl transferase (C2′GT) enzyme - stabilizes the chemically un stable chalcone and is necessary for producing chalcone-based yellow pigments.  Carnation C2′GT gene has been cloned recently (Ishida et al. 2003, Okuhara et al. 2004)
  26. 26. The most economically significant flowers – Rose , Chrysanthemum, and Carnations - no blue color - no delphinidin - lack of F3′5′H in their flowers. Therefore, one can not produce a blue rose or blue carnation by traditional breeding .
  27. 27. Petunia F3′5′H gene was expressed in the same carnation line – dramatic improvement in the level of delphinidin - shift in the flower color from a pink and red to mauve and purple. Florigene's new lilac - and mauve - hued carnations-'Moondust' and 'Moonglow', now dominate the North and South American carnation cut-flower markets
  28. 28. No blue rose - naturally – incapable of synthesizing delphinidin • Molecular geneticists with Florigene and Suntory achievedby combining something old, something new, Something borrowed, and something blue.
  29. 29. 'something blue' the delphinidin gene cloned from a pansy. 'something borrowed an iris gene for an enzyme, DFR, required to complete the delphinidin- synthesis reaction 'something new' man-made gene designed by geneticists exploited a powerful new developed technology - to switch off a rose gene . 'something old ' Roses are very old garden subjects
  30. 30. Use of RNAi technology to switch off DFR gene in a red rose to block cyanidin pathway, and then install the delphinidin gene – plus a new DFR gene to complete delphinidin synthesis
  31. 31. The three-gene package (pansy delphinidin, iris DFR, anti - rose DFR )package worked: Suntory's transgenic rose produced very high levels of delphinidin in its petals, and a small residue of cyanidin. The new rose is an attractive shade of mauve - lilac roses like 'Blue Moon' and 'Vol de Nuit'.
  32. 32. Genes isolated from Antirrhinum majus increased interest in novel flower shapes through molecular manipulation.
  33. 33. Constitutive expression of Antirrhinum majus B genes DEF and GLO in transgenic torenia resulted in the conversion of sepals to petals . (Dr. Takashi Handa, personal communication) expression of the C gene from Rosa rugosa In torenia resulted in a carpeloid structure in place of sepals (Kitahara et al. 2004, plant science:166)
  34. 34. Post harvest longevity determines value of a cut flower. Senescence of a flower is highly controlled process requiring active gene expression and protein synthesis – amenable to manipulation (Woodson1987)
  35. 35. Rapid clonal in vitro propagation of plants from cells,tissues or organs cultured aseptically on defined media contained in culture vessels maintained under controlled conditions of light and temperature
  36. 36. Orchids Cut flowers Bulbs and corms Flowering pot plants Foliage plants
  37. 37. • Arachnis • Aranda • Aranthera • Cattleya • Cymbidium • Dendrobium • Lycaste • Paphiodelphium • Miltonia • Odontoglossum
  38. 38. • Chrysanthemum • Gerbera • Anthurium • Rose • Carnation
  39. 39. Gladiolus Tulips Lilies Tuberose Amaryllis Iris
  40. 40. García-Sogo et al. BMC Plant Biology 2012, 12:156 http://www.biomedcentral.c om/1471-2229/12/156
  41. 41. Kingdom: Plantae Subkingdom: Tracheobionta Superdivision: Spermatophyta Division: Magnoliophyta Class: Magnoliopsida Subclass: Rosids Order: Geraniales Family: Geraniaceae Genus: Pelargonium
  42. 42. Plant material Surface sterilization Morphogen esis Induction Medium (MIM) Elongation Medium Rooting Acclimatization
  43. 43. Surface sterilization Morphogen esis Induction Medium (MIM) Calculate activity cost drivers’ rates Rooting Acclimatization Plant material Pelargonium zonale cv. 370 Explants: Young leaf explants from 30–40 days old plantlets Pelargonium peltatum cv. Aranjuez
  44. 44. Plant material Morphogen esis Induction Medium (MIM) Elongation Medium Rooting Acclimatization Rinsed three times with sterile distilled water. Iimmersion in a 2.5% solution of sodium hypochlorite for 20 min. Surface sterilization
  45. 45. Plant material Surface sterilization Calculate activity cost drivers’ rates Rooting Acclimatization leaves was cut into 1 cm2 pieces and cultured on MIM MS basal medium and Shahin [46] vitamins Morphogen esis Induction Medium (MIM) supplemented with 50 mg l-1 kanamycin Regeneration in Pelargonium zonale was carried out via direct organogenesis and in Pelargonium peltatum via somatic embryogenesis.
  46. 46. Plant material Surface sterilization Morphogen esis Induction Medium (MIM) Rooting Acclimatization •After 2.5 - 3 months in culture, calli showing well developed morphogenetic structures (shoots in the case of P. zonale and somatic embryos in P. peltatum) were transferred to a selective Elongation Medium . • Elongation Medium (EM: MS basal medium and Shahin vitamins, supplemented with 50 mg l-1 kanamycin) • All explants were subculture every 2 weeks onto the same fresh medium until shoots were long enough to be separated .. Elongation Medium
  47. 47. Plant material Surface sterilization Assign costs to activity cost pools Elongation Medium Acclimatization • After 1 – 1.5 months in EM, the shoots were cut and cultivated in Rooting Medium (RM). Rooting
  48. 48. Plant material Surface sterilization Morphogen esis Induction Medium (MIM) Elongation Medium Rooting •and acclimatized in growth chambers under (16-h light/8-h dark photoperiod) and then transferred to a greenhouse until they flowered.. • Regenerated plantlets with welldeveloped roots were transferred to plastic pots containing peat moss and perlite (3:1). Acclimatization Transformation efficiency was estimated as the number of independent transformation events (one transgenic plant per explant) in relation to the total number of inoculated explants.
  49. 49. Cytokinins have been implicated in several aspects of plant development, including plant senescence [15- 20], and are thought to be synthesized mainly in the roots and transported to the shoots via the xylem. Overexpression of the ipt gene in transgenic plants led to elevated foliar cytokinin concentrations and delayed leaf senescence, but high cytokinin levels have been reported to be detrimental to growth and fertility [26 30]. To circumvent these effects : Specificgene promoter (pSAG12 )
  50. 50. Promoter which induces transcription in male reproductive specifically Gene which disrupts normal function of cell Agrobacterium- mediated transformation regeneration male-sterile plant
  51. 51. (A portmanteau of "BActerial" "RiboNucleASE") is a bacterial protein that consists of 110 amino acids and has ribonuclease activity. It is synthesized and secreted by the bacterium Bacillus amyloliquefaciens, but is lethal to the cell when expressed without its inhibitor barstar . The inhibitor binds to and occludes the ribonuclease active site, preventing barnase from damaging the cell's RNA
  52. 52. • LBA4404 cells were electroporated to carry different plasmids a pBIN19 binary vector .
  53. 53. nptII nos •GFP •report er gene CaMV •Barnase •barstar TA29ipt pSAG 12GUS 35SCa MV •npt •marker gene nos T- DNA region Bacterial DNA Virulence region Oregion of replication
  54. 54. Bacteria were grown at 28°C on solid LB plates supplemented with 40 mg l-1 rifampicin and 100 mg l-1 kanamycin Single colony was used to inoculate 25 ml of LB liquid medium with the same antibiotics , maintained at 28°C and 200 rpm for 24 h Inoculate a liquid MS medium supplemented 0.2 mM acetosyringone dissolved in 70% ethanol (sterilized by filtration), which was cultured at 28°C for 12 h. Inoculation of explants was conducted in bacterial culture
  55. 55. Transformed explants were examined periodically for gfp expression under a fluorescence stereomicroscope (Leica MZ FLIII) .
  56. 56. Identification of the ipt transgene (460 bp fragment) by PCR in different P. zonale pSAG12::ipt transgenic plants. C + (positive control: pVDH393-pSAG12::ipt) and TI (negative control).
  57. 57. Identification of the barnase-barstar transgene (544 bp fragment) by PCR in different P.zonale male sterile plants. C + (positive control: pBI101-PsEND1::barnase-barstar) and TI (negative control).
  58. 58. Realtime RT-PCR analysis of pSAG12::ipt transcript levels in detached leaves from the transgenic lines 3.4, 3.9, 4.3 and 4.12. Each sample’s expression level relative to Pelargonium x hortorum PhACTIN7 is the mean of three biological repeats. C: control WT leaves.
  59. 59. Measurements were taken in the greenhouse on transgenic plants and WT control plants :  Plant height (distance from soil line to top of the tallest growing point),  leaf length and width (average measurements from five fully expanded leaves),  leaf petiole length,  internodal length  Number of inflorescences per plant were evaluated.  Morphological measurements were taken over the course of several days on each plant as its first five flowers reached anthesis .  Means differing significantly were compared at a 5% probability level.  Data variability was expressed as the mean ± SE.
  60. 60. (a), 6 (b), 8 (c), 17 (d), 22 (e), 24 (f), 27 (g) and 34 (h) days of incubation in darkness.
  61. 61. Analysis of leaf senescence was conducted by extraction of chlorophyll in detached leaves incubated in darkness from WT control and pSAG12::ipt plants respectively. Using a porcelain mortar cooled with liquid nitrogen, samples were crushed to a fine powder. In 10 ml centrifuge tubes the samples were mixed with 100 mg of MgCO3 and 5 ml of 100% (v/v) acetone. Bleached leaf material was removed by centrifugation (5 min; 2,000 g) and 1 ml aliquots of supernatants transferred to new tubes. Chlorophyll (a + b) content of extracts was determined spectrophotometrically [53].
  62. 62. (i) Mean concentration (±SE) of chlorophyll a + b (mg/g fresh weight) from detached leaves of control (WT) and pSAG12::ipt (TRG) plants at 0, 6 and 8 days of incubation in darkness .
  63. 63. (j) Senescence delay of detached leaves from pSAG12::ipt plants. Fresh weight changes in detached leaves of WT P. zonale and a transgenic line carrying the pSAG12::ipt chimaeric gene over the time course analyzed. Data are the means of sixteen leaves ± SE. Bars: 1 cm.
  64. 64. The chimaeric pSAG12::ipt construct useful in Pelargonium spp. to delay the senescence process and to produce long-lived plants, which could have commercial interest. Transgenic pSAG12::ipt plants showed delayed leaf senescence, increased branching and reduced internodal length as compared to non-transformed plants. Transgenic pSAG12::ipt plants showed a more compact architecture than the WT.
  65. 65. Expression of the barnase gene under control of PsEND1 promoter caused specific ablation of the tissues, necrotic at early stages of anther development. No pollen grains were observed in the ablated anthers from the male-sterile plants, indicating that barnase effectively destroys specific cell lines that form the structural tissues of the anther , preventing pollen development. .
  66. 66. The use of engineered male sterility would be especially useful to eliminate pollen allergens and to produce environmentally friendly transgenic plants carrying new traits by preventing gene flow between the genetically modified ornamentals and related plant species.
  67. 67. 50. Rogers SO, Bendich AJ: Extraction of total cellular DNA from plants, algae and fungi. Plant Mol Biol Manual 1994, D1:1–8. 51. He J, Gray J, Leisner S: A Pelargonium ARGONAUTE4gene shows organspecific express 53. Lichtenthaler HK: Chlorophylls and carotenoids: Pigments of photosynthetic biomenbranes. Met Enzymol 54. Elliot AR, Campbell JA, Dugdale B, Brettell RIS, Grof CPL: Green fluorescent protein facilitates rapid in vivo detection of genetically transformed plant cells. Plant Cell Rep 1999, 18:707–714. 55. Escobar MA, Park JI, Polito VS, Leslie CA, Uratsu SL, Mc Granahan GH, Dandekar AM: Using GFP as a scorable marker in walnut somatic embryo transformation. Ann Bot 2000, 85(6):831–835. 56. Ghorbel R, Juárez J, Navarro L, Peña L: Green fluorescent protein as a screenable marker to increase the efficiency of generating transgenic woody fruit plants. Theor Appl Genet 1999, 99:350–358. 57. Pérez-Clemente RM, Pérez A, García L, Beltrán JP, Cañas LA: Transformation and regeneration of peach plants (Prunus persica L.) from embryo sections using the green fluorescent protein (GFP) as a vital marker. Mol 58. Rakosy-Tican E, Aurori CM, Dijkstra C, Thieme R, Aurori A, Davey MR: The usefulness of the gfp reporter gene for monitoring Agrobacteriummediated transformation of potato dihaploid and tetraploid genotypes. 59. Yancheva SD, Shlizerman LA, Golubowicz S, Yabloviz Z, Perl A, Hanania U, Flaishman MA: The use of green fluorescent protein (GFP) improves Agrobacterium-mediated transformation of ‘Spadona’ pear (Pyrus 60. Baranski B, Klocke E, Schumann G: Green fluorescent protein as an efficient selection marker for Agrobacterium rhizogenes mediated carrot transformation. Plant Cell Rep 2006, 25:190