4. 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 .
5.
6. 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
7. 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.
8. 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)
9. 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.
11. 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
12.
13. 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
14. DNA solution is injected directly inside the cell
using capillary glass micropipetts .
16. 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
17. 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.
18. 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.
19. 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 .
20. 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).
22. 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)
23. Attempted to overexpress chalone synthase (anthrocyanin
pigment gene) in petunia.
(trying to darken flower color)
Caused the loss of pigment.
24. 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
25. 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
26. 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
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42. 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
43.
44. 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)
45. 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).
46. 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)
47.
48. 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 .
49. 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
50.
51. 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.
52. '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
53. 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
54. 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'.
55.
56. Genes isolated from Antirrhinum
majus increased interest in novel
flower shapes through molecular
manipulation.
57.
58. 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)
59.
60.
61. 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)
62. 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
76. 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
78. 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.
79. 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 )
80. Promoter which
induces transcription
in male reproductive
specifically
Gene which disrupts
normal function of cell
Agrobacterium-
mediated
transformation
regeneration
male-sterile
plant
81. (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
82.
83.
84. • LBA4404 cells were electroporated
to carry different plasmids a pBIN19 binary vector .
86. 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
87.
88.
89.
90.
91. Transformed explants were examined periodically for
gfp expression under a fluorescence stereomicroscope
(Leica MZ FLIII) .
92.
93.
94.
95. 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).
96. 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).
97. 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.
98. 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.
99.
100.
101.
102.
103.
104. (a), 6 (b), 8 (c), 17 (d), 22 (e), 24 (f), 27 (g) and 34 (h) days of incubation in darkness.
105. 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].
106. (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 .
107. (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.
108.
109. 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.
110. 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. .
111. 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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Manual 1994, D1:1–8.
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53. Lichtenthaler HK: Chlorophylls and carotenoids: Pigments of photosynthetic biomenbranes. Met Enzymol
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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.
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green fluorescent protein (GFP) improves Agrobacterium-mediated transformation of ‘Spadona’ pear (Pyrus
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