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. 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. 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. 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. 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. 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. 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. 1-2 m of tungsten or gold particles (microprojectiles)coated
with DNA to be used for transformation are accelerated to
velocities using pressurized Helium gas
9. DNA solution is injected directly inside the cell using
capillary glass micropipetts .
10. 2 3 1T - DNA Ti - Plasmid
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. 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. 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. 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. 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. Gerbera (Elomaa 1993) Reduction of anthocyanin Petunia Rose
(Gutterson 1995), Chrysanthemum (Courtney- Gutterson et al.,1994) .
Carnation (Gutterson 1995) . (Krol et al.,1988). .
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. Attempted to overexpress chalone synthase (anthrocyanin
pigment gene) in petunia. (trying to darken flower color) Caused
the loss of pigment.
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. 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. 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. In petunia cyanidin and delphinidin derivatives but no
pelargonidin derivatives. Enzyme dihydro flavonol 4 reductase ( DFR
) A1 gene from maize encodes dihydro quercetin 4reductase- doesnt
show substrate specificity as doespetunia enzyme
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. 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. Later discovered - a chalcone 2-glucosyl transferase (C2GT)
enzyme - stabilizes the chemically un stable chalcone and is
necessary for producing chalcone-based yellow pigments. Carnation
C2GT gene has been cloned recently (Ishida et al. 2003, Okuhara et
al. 2004)
26. The most economically significant flowers Rose ,
Chrysanthemum, and Carnations - no blue color - no delphinidin -
lack of F35H in their flowers. Therefore, one can not produce a
blue rose or blue carnation by traditional breeding .
27. Petunia F35H 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. 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. '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. 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. 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. Genes isolated from Antirrhinum majus increased interest in
novel flower shapes through molecular manipulation.
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. 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. 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. Orchids Cut flowers Bulbs and corms Flowering pot plants
Foliage plants
42. Plant material Surface sterilization Morphogen esis
Induction Medium (MIM) Elongation Medium Rooting
Acclimatization
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 3040 days old plantlets Pelargonium peltatum cv. Aranjuez
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. 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. 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. 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. 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. 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. Promoter which induces transcription in male reproductive
specifically Gene which disrupts normal function of cell
Agrobacterium- mediated transformation regeneration male-sterile
plant
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. LBA4404 cells were electroporated to carry different
plasmids a pBIN19 binary vector .
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. Bacteria were grown at 28C 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 28C 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 28C
for 12 h. Inoculation of explants was conducted in bacterial
culture
55. Transformed explants were examined periodically for gfp
expression under a fluorescence stereomicroscope (Leica MZ FLIII)
.
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. 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. 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 samples expression level relative to Pelargonium x
hortorum PhACTIN7 is the mean of three biological repeats. C:
control WT leaves.
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. (a), 6 (b), 8 (c), 17 (d), 22 (e), 24 (f), 27 (g) and 34
(h) days of incubation in darkness.
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. (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. (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. 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. 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. 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.
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carotenoids: Pigments of photosynthetic biomenbranes. Met Enzymol
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57. Prez-Clemente RM, Prez A, Garca L, Beltrn JP, Caas LA:
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