1) Ornamental floriculture is an important industry that includes cut flowers, bulbs, potted flowering and foliage plants produced through extensive hybridization, induced mutation, and selection. 2) Genetic engineering techniques like Agrobacterium-mediated transformation and particle bombardment can be used to transfer genes into ornamental plants and generate novel traits. 3) Researchers genetically engineered blue roses by combining genes from different plants - a delphinidin gene from pansy, an enzyme gene from iris, and a gene to silence a rose's own gene. This produced blue roses for the first time.

5. 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 .

7. 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

8. 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.

9. 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)

10. 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. 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

12.  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

14. 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

15. 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.

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).

21. Gerbera
(Elomaa 1993)
Reduction of
anthocyanin
Petunia
Rose
(Gutterson 1995),
Chrysanthemum
(Courtney-
Gutterson et al.,1994)
.
Carnation
(Gutterson 1995)
.
(Krol et al.,1988).
.

22. 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

23. 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

24. 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

25. No blue rose - naturally – incapable of
synthesizing delphinidin
• Molecular geneticists with
Florigene and Suntory achieved by
combining something old,
something new,
Something borrowed,
and something blue.

26. '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

27. 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

28. 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'.

31. 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)

32. 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

33. Orchids
Cut flowers
Bulbs and corms
Flowering pot plants
Foliage plants

34. • Arachnis
• Aranda
• Aranthera
• Cattleya
• Cymbidium
• Dendrobium
• Lycaste
• Paphiodelphium
• Miltonia
• Odontoglossum

35. • Chrysanthemum
• Gerbera
• Anthurium
• Rose
• Carnation

36. Gladiolus
Tulips
Lilies
Tuberose
Amaryllis
Iris

37. García-Sogo et al. BMC
Plant Biology 2012, 12:156
http://www.biomedcentral.c
om/1471-2229/12/156

38. Kingdom: Plantae
Subkingdom: Tracheobionta
Superdivision: Spermatophyta
Division: Magnoliophyta
Class: Magnoliopsida
Subclass: Rosids
Order: Geraniales
Family: Geraniaceae
Genus: Pelargonium

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 30–40 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

54. • LBA4404 cells were electroporated
to carry different plasmids a pBIN19 binary vector .

55. 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

56. 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

61. Transformed explants were examined periodically for
gfp expression under a fluorescence stereomicroscope
(Leica MZ FLIII) .

65. 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).

66. 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).

67. 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.

68. 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.

74. (a), 6 (b), 8 (c), 17 (d), 22 (e), 24 (f), 27 (g) and 34 (h) days of incubation in darkness.

75. 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].

76. (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 .

77. (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.

79. 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.

80. 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. .

81. 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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