GENETIC ENGINEERING IN FLORICULTURE

1. GENETIC ENGINEERING IN FLORICULTURE
Speaker : Zaman Mariya S.
Course No. : MBB 591
Major Sub. : Plant Biotechnology
Reg. No. : 04-1273-2010
ANAND AGRICULTURAL UNIVERSITY
Major Guide : Dr. G. C. Jadeja
Minor Guide : Dr. J. G. Talati
Date :15 /09/2012
Time : 14:00 Hrs
wel come

2. The Floriculture Industry……….
Biotechnology In Floriculture
Current Status Of Genetic Modification In
Floriculture
Transformation
Color Modification
Vase life
Flower Scent
Modified Plant Structure and Architecture
Disease Resistance
Case studies
Conclusion
Future Prospects
2

3. THE FLORICULTURE INDUSTRY
• Floriculture is considered to include the cut flowers, potted
plants, and ornamental bedding plants and garden plant
industries (Chandler, 2003).
• The worldwide production value of flowers and floricultural
plants is approx. 50 billion EURO.
• From the per capita consumption data provided by the Dutch
flower council (Flower Council of Holland, 2007), it can be
extrapolated that the global consumption value at consumer
level is somewhere between 100 to 150 billion EURO.
• The main areas of production and consumption of floricultural
products are in the United States and Europe, with a
significant industry in Japan. The area of production in China
is also increasing rapidly.
• The highest consumption per head is in the Netherlands,
Luxembourg, Germany, Austria, and France.
3

4. Orchids
Arachnis
Aranda
Aranthera
Cattleya
Cymbidium
Dendrobium
Lycaste
Paphiodelphium
Miltonia
Odontoglossum
Chrysanthemum
Gerbera
Antirrhinum
Rose
Carnation
Cut flowers
Bulbs and corms
Gladiolus
Tulips
Lilies
Tuberose
Amaryllis
Iris
4

5. World Flower Production and Consumption
Production Consumption
China
Kenya
Columbia
Japan
Europe
USA
$40 billion
(Getu, 2009)
Market grow
20% annually
Japan
EU
USA
5

6. The global flower industry thrives on novelty.
Genetic engineering is providing a valuable means of
expanding the floriculture gene pool so promoting the
generation of new commercial varieties.
Engineered traits are valuable to either the consumer or
the producer.
At present, only consumer traits appear to provide a
return capable of supporting what is still a relatively
expensive molecular breeding tool.
The goal of genetic engineering is to improve the
characteristics of flowers such as, flower colour, vase life,
floral scent, flower morphology, disease as well as pest
resistance, flower productivity, timing and synchrony of
flowering.
Biotechnology In Floriculture
6

7. Transgenic
Technology
Resistant To
Biotic Stresses
7

8. Genetic modification can be used to transfer
new
specific traits into the plant
Conventional Breeding
many gene and limited by genetic
incompability
Plant biotechnology
single gene with no specific to plant
species
Genetic engineering: Manipulation of plant genome
through recombinant DNA technology to alter plant
characteristics.
8

9. Gene transfer methods
Indirect Direct
Most widely used
More economical
More efficient
Transformation success is 80-85%
Agrobacterium mediated
gene transfer
 Particle bombardment or
micro projectile
 Direct DNA delivery by
Microinjection or
PEG mediated uptake
 Ultrasonication
 Electroporation
Chandler and Brugliera, 2011
9

10. Gene transformation
Bacterium mixed
with plant cells
GENE
identified and
isolated
Gene inserted
into Ti plasmid
Gene replication
Gold particles
coated with
DNA
Cells shot with gene gun and
DNA incorporated into plant cell
chromosome
Ti plasmid moves
into plant cell and
inserts DNA into
plant chromosome
Cells screened
for transgene
Transformed cells
selected with
selectable marker
Transgenic plant
regenerated from single
transformed cell 10

11. Color and color patterns
Flower color: most important trait, dictating consumer
attraction
Role of color:
• Attraction of pollinators
• Function in photosynthesis
• In human health as antioxidants and precursors of
vitamin A
• Protecting tissue against photooxidative damage
• Resistant to biotic and abiotic stress
• Symbiotic plant-microbe interaction
• Act as intermediary for other compounds
Color pattern: Differential accumulation of pigment(s)
11

12. Pigments Compound Types Compound Examples Typical Colors
Porphyrins Chlorophyll Chlorophyll a & b Green
Flavonoids Anthocyanins Pelargonidin, Cyanidin, Delphinidin,
Peonidin
Red, Blue, Violet
Anthoxanthins Flavonols Kaempferol, Quercetin, Fisetin,
Kaempferide, Morin, Myricetin,
Myricitrin, Rutin
Yellow
Flavones Apigenin, Biacalein, Chrysin, Diosmetin,
Flavone, Luteolin
Yellow
Isoflavones Diadzin, Genistein, Enterodiol,
Coumestrol, Biochanin
Flavonones Eriodictyol, Hesperidin, Naringin,
Naringenin
Colorless Co-
pigments
Flavans Biflavan, Catechin, Epicatechin Colorless Co-
pigments
Carotenoids Carotenes Lycopene, α-carotene, β-carotene,
γ-carotene
Yellow, Orange, Red
Xanthophylls Lutein, Cryptoxanthin, Zeaxanthin,
Neoxanthin, Rhodoxanthin, Violaxanthin,
Canthaxanthin, Astaxanthin
Betalains Betacyanins Reddish To Violet
Betaxanthins Miraxanthin & Portulaxanthin Yellow To Orange
12

13. Chlorophylls and
carotenoids are in
chloroplasts
Anthocyanins
are in the
vacuole
13

14. Factors on flower color perception
pH of the vacuole:
pH of the vacuole is acidic
Small changes of pH have visible effects on flower color
Metal ions:
Metal complexing has a blueing effect on flower color
Co-pigmentation:
Co-pigmentation of anthocyanins with the colourless flavonols and
flavones is an important factor influencing pigmentation.
Co-pigmented flowers give a mauve colour, whereas in the absence
of flavonols maroon flowers are formed.
Co-occurrence of anthocyanins and yellow pigments
Mixtures of Ans and yellow flavonoids were found in the orange-
yellow or orange-red flowers of antirrhinum and bronze flower colour
of helichrysum.
Tanaka et al., 2009
low pH to high pH

15. Protein (Enzyme)
Red Pigment
Springob et al., 2003
Genes contain regulatory region
and coding region
Regulatory region Coding region
The genes consist of DNA which is
made up of four chemical letters
The chromosome is
made up of genes
Chromosomes-23 pairs
A cell
15

16. Genes involved in pigment synthesis
Structural (enzyme) gene: Gene that codes for any
RNA or protein product other than a regulatory protein.
Enzyme Gen
e
Species
CHS (Chalcone synthase) Chs Antirrhinum, Chrysanthemum, Orchid, Rosa,
Dianthus
CHI (Chalcone-flavanone
isomerase)
Chi Antirrhinum, Petunia, Eustoma, Dianthus
F3H (Flavone 3-hydroxylase) F3h Antirrhinum, Calistephus, Chrysanthemum,
Dianthus, Orchid
F3’H (Flavonoid 3’ hydroxlase) F3’h Antirrhinum, Dianthus, Petunia
F3’5’H (Flavonoid 3’,5’-
hydroxlase)
F3’5’h Calistephus, Eustoma, Petunia
FLS (Flavonol synthase) Fls Petunia, Rosa
FNS (Flavone synthase) FnsII Antirrhinum, Gerbera
DFR (Dihydroflavonol-4-
reductase)
Dfr Antirrhinum, Calistephus, Gerbera, Orchid,
Dianthus, Petunia Vainstein, 2004
Regulatory Genes
Structural (Enzyme) Genes

17. Regulatory genes: Influence the type, intensity and pattern of
flavonoid
accumulation but do not encode flavonoid enzyme.
Two classes of regulatory genes are
identified:
 TF with MYB domain
 TF with MYC/bHLH motif
(Vainstein, 2004)
Plant Gene
Myb Myc
Petunia Rosea, mixta Delia
Gerbera Gmyc I
Perilla MybpI
Petunia Phmyb3, An2,
An4
An1
17

18. Genes involved in carotene pigment synthesis
(Vainstein, 2004)
Gene Enzyme
Dxs Deoxyxylulose 5- phosphate synthase
Dxr Deoxyxylulose 5- phosphate
reducoisomerase
Lpi LytB protein
Gps Geranyl diphosphate synthase
Fps Fernsyl diphosphate synthase
Ggps Geranyl geranyl diphosphate synthase
Psy Phytotene synthase
Zds β-Carotene dessaturase
Lcy-b Lycopene β-cyclase
Lcy-c Lycopene β-cyclase
Nsy Neoxanthin synthase
Ccs Capsanthin capsorubin synthase
Ptox Plastid terminal oxysidase 18

19. 19
B
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ti
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p
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w
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fl
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20. Colour modification done by:
Over expression of structural genes
Use of sense or antisense enzyme construct
Inhibition of key biosynthetic enzyme
Addition of an enzyme in a particular biosynthetic step
20

21.  FLORIGENE scientists Isolated the blue gene in 1991 and
patented in 1992.
 Petunia gene didn’t work in roses, so FLORIGENE
scientists used their techniques on carnations— a much
easier species to manipulate than roses.
 In 1996, Florigene developed mauve-coloured
carnation, FLORIGENE Moondust and it was the
world's first genetically modified flower on sale.
 In 1997, developed second genetically-modified
carnation, FLORIGENE Moonshadow with a richer and
true purple colour.
 Successfully developed a range of transgenic violet
carnations by introduction of a F3′5′H gene together
with a petunia DFR gene into a DFR-deficient white
carnation.
Fukui et al., 2003
mooniquemoonpearlmoonvelvetmoonberry
21

22. • Why a natural rose could not have the true blue colour?
 "Flavonoid 3', 5'- hydroxylase" is one of the key enzymes
involved in the flavonoid biosynthesis for blue colour
development deficient in rose.
 Natural rose did not have delphinidin.
 pH of cell sap is 4.0-4.5 (acidic).
 Cell sap is govern by 7 genes and each gene contributes 0.5
pH Anthocyanidin
The price for a single blue rose is
about $22 to $33. Source: The
Japan Today
The transgenic rose variety
‘‘Applause’’ was commercially
released in Japan in 2009 (Tanaka
et al., 2009)
22

23. Blue Gene Technology
www.suntory.com and www.florigene.com.au
 In April of 2005, Suntory Ltd. and Florigene Ltd. announced the
production of a blue rose by introducing three transformation constructs
simultaneously into roses:
To make a blue
roses:
(1) Turn off’ the
rose DFR
gene
(2) Insert pansy
gene to open the
‘blue’ door
(3) Insert iris DFR gene
to make blue
pigment
23

24. Silencing anthocyanin biosynthetic pathway by
a) Transcriptional down-regulation
b) By inactivating the key enzymes
CHS
gene
F3’5’H
gene
DFR
gene
More sensitive to ranges of stresses
Alternative targets, better
and increased fragrance
Zuker et al., 200224

25. Gerbera
(Courtney-Gutterson et al. 1994),
Genetic engineering for white flower
(Elomaa 1993),
ChrysanthemumPetunia
(Krol et al. 1988),
Rose
(Gutterson 1995),
Carnation
(Gutterson 1995).
25

26. Genetic engineering for red/
orange flowers
•Cyanidin and delphinidin derivatives occur
but no pelargonidin derivatives in petunia.
•Petunia DFR cannot able to reduce
dihydrokaempferol because it shows
substrate specificity which lead to the
synthesis of leucoanthocyanidins.
•Over expression of A1 gene + abundant
substrate –opens a new pathway leading
to the synthesis of novel brick red colour
petunia.
Maize A1 gene
encodes
dihydroquercetin
4 reductase
Meyer et al., 198726

27. Genetic engineering for yellow
flowers
• Aurones are bright yellow
flavonoids found in species
such as snapdragon, dahlia
etc..
• Aurone synthase, more specifically
aureusidin synthase (AS), was
purified from yellow snapdragon
petals and the cDNA encoding the
enzyme was cloned.
• A pale yellow petunia expressing a
Lotus japonica PKR gene.
27
•Chalcone and aurones contribute to yellow
colours
• The most common chalcone, THC, is
yellow but is spontaneously
isomerized to naringenin in vitro and
rapidly isomerized in vivo by CHI.
• Discovery of chalcone 2′-
glucosyltransferase (C2′GT)
enzyme - stabilizes the
chemically unstable chalcone.
• Recent activity: (Okuhara et al.
2004)
Lack of CHI activity + presence
of UDP-glucose: THC 2’
glucosyltransferase (C2’GT)
activity = yellow carnation

28. Plant species Original
colours
Gene sources Methods Produced
flower colors
References
Cyclamen persicum Purple Endogenous F3’5’H Antisense Red to pink Boase et al. (2010)
Gentiana sp. Blue Endogenous CHS Antisense White Nishihara et al.
(2006)
Blue Endogenous F3’5’H RNAi Lilac to purple blue Nakatsuka et al.
(2008b)
Nierembergia sp. Violet Endogenous F3’5’H Antisense Pale blue Uyema et al. (20006)
Ostespermum
hybrida
Magenta Endogenous F3’5’H RNAi reddish Seitz et al. (2007)
Gerbera hybrida DFR Over expression
Petunia hybrida Blue Mazus psonilum CHS Dominant negative Pale blue Hanumappa et al.
(2007)
Red Lotus japonicus PKR Over expression Variegated red Shimada et al. (2006)
Rosa hybrida Red to pale cyanic Viola sp. F3’5’H Over expression Bluish Katsumoto et al.
(2007)
Torenia hybrida Blue Endogenous ANS RNAi Whitish to pale blue Nakamura et al.
(2006)
Blue Endogenous ANS Antisense Pale blue Nakamura et al.
(2006)
Blue Antirrhinum majus
AS
Over expression yellow Ono et al. (2006)
Flower colour modifications by regulating flavonoid biosynthesis
28

29. value of a cut flower =Post harvest longevity
Senescence  highly controlled process  requires active gene
expression & protein synthesis for programmed cell death.
Increased respiration and ethylene production induction of
catabolic enzymes resulting in decreased proteins
Genetic engineering for
longer vase life
Aging of
petals
Inhibiting or
by blocking
perception
of ethylene
29

30. Florigene has developed carnation flowers with
enhanced vase life using antisense RNA technology.
Down regulated
petal ACC
synthase
Control STS Transgenic
30

31. A high level of resistance in osmotin, pr-1 and/or
chitinase genes to a major carnation pathogen
(fusarium oxysporum f. Sp. Dianthi ) was
demonstrated in greenhouse tests. (Zuker et al.,
2005)
Genetic Engineering for biotic stress
Transformation of chrysanthemum cultivar 'shuho-
no-chikara' was modified by delta-endotoxin gene
cry1ab (mcbt) from bacillus thuringiensis 
biological activity against lepidopteran insects into
chrysanthemum.
Protection of flower crops against coat protein
viruses (William R. Woodson 1991)
Transgenic chrysanthemum showing resistance
against chrysanthemum stunt viroid (csvd) and
TSWV.
31

32. Genetic engineering for improved shape,
size
ABC MODEL
32
The combination of three genes that
give rise to the flower parts.
A  sepals
A + B  petals
B + C  stamens
C  carpels
• The ABC model (Coen and
Meyerowitz 1991) and its modified
version (Theißen 2001) are known to
be applicable to a broad range of
plants (Kim et al. 2005).

33. 33
Genetic engineering for improved shape,
size
Constitutive expression of Antirrhinum majus
B genes DEF And GLO in transgenic torenia
resulted in the conversion of sepals to petals.
Constitutive expression of the C gene from
Rosa rugosa in torenia resulted in a carpeloid
structure in place of sepals (Kitahara et al.
2004)
Studies on homeotic mutants have clarified
many important aspects of genetic control on
flower development.
Deficiencies genes and AGAMOUS genes
isolated from Antirrhinum majus increased
interest in novel flower shapes through
molecular manipulation.
Transformant Wild type

34. Genetic engineering
for floral scent
may enhance the value of cut flowers to consumers…
Fragrance numerous
volatile aromatic organic
substances present in the
flower.
Such as,
hydrocarbons,
alcohols,
aldehydes,
ketones, esters,
ethers
Manipulation of fragrance in
flowers chemicals contributing to
the fragrance of roses, their
pathways of synthesis and
enzymes controlling these
pathways to be identified.34

35. Genetic engineering to modify
plant architecture
Control of plant height is of great importance in floriculture
35
• A petunia plant transformed with
Arabidopsis gai-1 (right).
• Genetic modification may replace
chemical growth retardants in
future.

36. Limitation of Plant gene transformation
• Improvement of traits controlled by many gene.
• Improvement of traits controlled by gene that has not
been identified.
• Requires high technology knowledge and equipment.
• Expensive cost.
• Government regulation.
36

37. o Flower colour changed from purple to almost white by the
down-regulation of the CHS gene
Surfinia Purple Mini
Tsuda et al., 2004
Surfinia Pure White
37Japan
1
Case studies

38. Flowers of transgenic Surfinia Purple Mini plant harboring
antisense DFR gene
Expression of DFR gene change the expression of the flavonol
synthase and flavone synthase gene
Contd…..
C
38Japan Tsuda et al., 2004

39. Vector construction
Figure 2. Schematic representation of the expression cassettes in T-DNA
of binary vectors constructed for colour modification.
39Japan

40.  Generation of orange petunia expression of rose DFR gene
+ Suppression of F3H gene by antisense or RNAi method
 Generation of red petunia expression of rose DFR gene
 T-DNA copy number analysis of transgenic plants show that
three most stable lines, two plants had a single copy insert,
and the other one had two.
Contd…..
40Japan Tsuda et al., 2004

41.  Modification of co-pigment by Suppressing flavonol biosynthesis resulted in
darker and slightly redder colour.
 A flower of a transgenic plant expressing rose FLS gene has a paler flower
Surfinia Violet Mini
transgenic petunia
expressing torenia
FNSII gene
Creeping character did not change
41Japan Tsuda et al., 2004

42. Engineering of the Rose Flavonoid Biosynthetic
Pathway Successfully Generated Blue-Hued Flowers
Accumulating Delphinidin
Katsumoto et. al (2007)Japan
o Rosa hybrida lacks violet to blue colour due to the absence of
flavonoid 3’,5’-hydoxylase (F3’5’H) enzyme which produces
delphinidin-based anthocyanins.
o Other factors such as co-pigments and vacuolar pH also affect
flower colour.
o Expression of the viola F3’5’H gene  accumulates (~95% high)
delphinidin  a novel bluish flower colour.
42
2

43. • For more exclusive and dominant accumulation of
delphinidin irrespective of the hosts, the endogenous
dihydroflavonol 4-reductase (DFR) gene was down-
regulated and overexpressed the Iris3hollandica DFR
gene in addition to the viola F3’5’H gene in a rose
cultivar.
• The resultant roses exclusively accumulated delphinidin
in the petals, and the flowers had blue hues not achieved
by hybridization breeding.
• Moreover, the ability for exclusive accumulation of
delphinidin was inherited by the next generations.
43 Katsumoto et. al (2007)Japan

44. Bi
o
sy
n
t
h
et
ic
p
at
h
w
a
y
O
f
fl
a
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o
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oi
d
44

45. WKS77 WKS82 WKS100
WKS116 WKS124 WKS140
Rose Varieties transformed with pSPB130 and their flower colour changed are shown
Schematic representation of T-DNA region of binary vectors constructed for colour
modification  for the constitutive over expression of the viola F3’5’H BP40 gene
and the torenia 5AT gene in rose.
45

46. o Correlation of delphinidin content and petal colours in transgenic Lavande.
o Pure red and blue have hue values of 360° and 290° in the hue angle.
46
Japan Katsumoto et. al (2007)

47. Host flower
violet-coloured
transgenic flower
98% delphinidin
98% delphinidin Madam Violet Seiryu
 The vector pSPB919 is to down-regulate the endogenous rose DFR gene using RNA
interference (RNAi) and to over express the iris DFR and the viola F3’5’H genes.

48. • Northern blot analysis of LA/919-4-10.
• The expected sizes of the transcripts of
viola F3’5’H BP40 (1.8 kb) and iris DFR (1.7
kb) genes & smaller size was detected for
rose DFR mRNA (A).
• A rose DFR probe detected about 23 bp
small sized RNA, which was supposed to be
a degraded endogenous rose DFR transcript
with RNAi (B).
• Delphinidin contents was confirmed
in all transgenic (KmR) progeny of
LA/919-4-10.
• The flowers of F1 and F2 progeny
contained exclusively delphinidin.
48
(si RNA)

49. Nakatsuka et.al (2009)Japan 49
(A)A binary vector, pSMABR-rolCpro-intF3’5’H:5/3’ATir
(B)The typical flower of wild-type gentian cv. ‘Albireo’
(C) 5/3’AT – suppressed transgenic gentian clone no.1.
(D) 5/3’ATandF3’5’H double suppressed transgenic gentian clone no.15.
3

50. •Expressions of anthocyanin
biosynthetic genes in
transgenic gentian plants.
•The membranes were
hybridised with DIG-labelled
probes for GtCHS, GtF3’5’H
and 5/3’AT, respectively.
•WT indicates untransformed
wild-type gentian cv. ‘Albireo’.
Ethidium bromide-stained
ribosomal RNA bands (rRNA)
are shown as a control.
•Accumulation of anthocyanidin and flavone derivatives
in transgenic gentian plants.
(B) Flavone conc. in the petal
of an untransformed
control plant (WT) and
transgenic plants were
determined by measuring
the absorbance at 330 nm
using HPLC analysis with
apigenin and luteolin as
the standards.
• Data are the average ±SD
of five replicate flowers.
(A) Anthocyanidin conc. in the
petal of an untransformed
control (WT) and transgenic
plants were determined.
• Data are the average ±SD of
five replicate flowers.
50
RNA gel blot analysis

51. Mercuri et. al (2001)Italy
 GFP detection in Eustoma
(Lisianthus) flower petals. (A)
 GFP detection in
Osteospermum ligules
flower petals. (A)
GFP = Green Fluorescent Protein
51
4

52. Block schemes of the gfp-expression constructs used in this work.

53. GFP detection in leaves and roots of transgenic
plants. (A, B) Leaf trichomes from Osteospermum
transfected with vector alone. (C, D) Leaf
trichomes from Osteospermum transfected with
mGFP5. (E, F) Roots from Osteospermum
transfected with vector alone (left) or mGFP5
(right). (A, C, E) were illuminated with white
light; (B, D, F) were illuminated with UV light.53
GFP detection in flower and leaf extracts
PAGE analysis of plant extracts
Western blot analysis
A B
C D
E F

54. (A) Phenotypes of transgenic Torenia flowers.
• (Upper) Flower color under white light.
• (Lower) Cellular fluorescence from adaxial side of petal in each line.
(B) Expression analysis of each transgenic line by RT-PCR/Southern blotting
Coexpression of the Am4CGT and AmAS1 genes was sufficient for the accumulation
of aureusidin 6-O-glucoside in transgenic flowers (Torenia hybrida).
54Japan Ono et. al (2006)
A
B
5

55. 55
Pathway Of
Aurone flavonoid
Synthesis

56. Production of picotee-type flowers in Japanese
gentian by CRES-T
A B
Wild type flower‐Solid color suppression of pigment
production generates picotee
type flower
Nakatsuka et al., 2011Japan
CRES-TChimeric repressor gene-silencing technology
56
6

57. • (CRES-T) is an efficient gene suppression system which
worked successfully in Japanese gentian.
• A chimeric repressor of the anthocyanin biosynthetic
regulator gene GtMYB3, under the control of the
Arabidopsis actin2 promoter, was introduced into blue-
flowered gentian.
• Of 12 transgenic lines, 2 exhibited a picotee flower
phenotype with a lack of pigmentation in the lower part of
the petal.
• HPLC analysis showed that the petals of these lines
contained less anthocyanin and more flavone than the
wild-type.
• The expressions of ‘late’ flavonoid biosynthetic genes,
including F3H, F35H, DFR and ANS, were strongly
suppressed in petals of these transgenic plants.
57

58. • Expression of flavonoid biosynthetic genes in transgenic
gentian plants.
• The expression levels of GtMYB3-SRDX and endogenous
flavonoid biosynthetic genes were determined by semi-
quantitative RT-PCR analysis in wild-type and GtMYB3-
SRDX expressed transgenic gentian clone nos. 7 & 11
• Schematic representation of pSMABR-AtACT2pro-
GtMYB3-SRDX.
• Bar  herbicide bialaphos resistance gene as a
selectable marker.
• NOSp  promoter of nopaline synthase (NOS) gene
from A. tumefaciens.
• rbcSt  terminator of RuBisCO small subunit 2B gene
from Arabidopsis.
• NOSt  terminator of NOS gene.
• LB  left border; RB  right border.
semi-quantitative RT-PCR analysis
58

59. Flavonoid analysis in the flowers of transgenic gentian plants by HPLC
A & D- wild type
B & E- transgenic
gentian clones no. 7
C & F- transgenic
gentian clones no. 11
Anthocyanins
Flavones
59

60. Ethylene‐Insensitive Transgenic Petunias
Jones et al, 2005USA
Senescence was delayed by approx. 8 days
60
7
Ethylene sensitive
(MD)
Ethylene-insensitive
(35S:etr1-1)

61. Nine genes encoding putative cysteine proteases 
Protein content (A) and protease activity
(B) in wild-type Petunia (MD) & 35S:etr1-
1 transgenic (etr-44568) petunia corollas
during flower development.
PhCP2 to PhCP10
61 Jones et al, 2005USA
• In this study, 35S:etr1-1 transgenic
petunias have been used to see
how ethylene regulates flower
senescence.
• To compare the senescence
programmes in ethylene-sensitive
(MD) and ethylene-insensitive (etr-
44568) flowers, a comparative
analysis was conducted of age-
related changes in total protein,
protease activity, and the expression
of nine cysteine protease genes in
the petals of MD and etr-44568
petunias.

62. ETR1-1 - Makes plants ethylene insensitive
Long Lasting Flowers
Etr1-1
62
Control Etr1-1Control Etr1-1

63. Generation of phenylpropanoid pathway-derived
volatiles in transgenic plants
• Rose alcohol acetyltransferase produces phenylethyl acetate and
benzyl acetate in petunia flowers.
• Esters are important volatiles contributing to the aroma of numerous
flowers and fruits.
• Acetate esters such as geranyl acetate, phenylethyl acetate and
benzyl acetate are generated as a result of the action of alcohol
acetyltransferases (AATs).
• To study the function of rose alcohol acetyltransferase (RhAAT), they
generated transgenic petunia plants expressing the rose gene under
the control of a CaMV-35S promoter.
• Phenylethyl alcohol and benzyl alcohol produce the corresponding
acetate esters, not generated by control flowers.
• The level of benzyl acetate is three times more than phenylethyl
acetate in different transgenic lines of petunia.
USA Pichersky et al.,200663
8

64. Molecular analyses of
transgenic petunia plants
expressing RhAAT
RNA blot
GC–MS analysis of volatile compounds
benzyl acetate
phenylethyl acetate
64
USA Pichersky et al.,2006

65. Transgenic rose lines harboring an antimicrobial
protein gene, Ace-AMP1, demonstrate enhanced
resistance to powdery mildew
• An antimicrobial protein gene, Ace-AMP1,
was introduced into Rosa hybrida via
Agrobacterium-mediated transformation.
• PCR analysis for both Ace-AMP1 and
neomycin phosphotransferase (npt II)
genes, showed that 62% of these plants
were positive for both transgenes.
• These lines were further confirmed for
stable integration of Ace-AMP1 and npt II
genes by Southern blotting.
• Transcription of the Ace-AMP1 transgene
in various transgenic rose lines was
determined using Northern blotting.
USA Li et al., (2003)65
Fig. a) Powdery mildew development
on leaflets & b) on whole leaves
Control
Control
Transgenic
Transgenic
9

66. 66
PCR analysis of transgenic plants of
Rosa hybrida cv. Carefree Beauty
Tnos Ace-AMP1 Penh-35S Pnos Npt II Tg7
EcoR 1
The binary vector pFAJ3033. The Ace-AMP1 gene is
driven by CaMV 35S with a duplicate enhancer
region, and is terminated by (NOS).
Southern blot hybridization
analysis of transgenic and control
rose plants. A) Probed with a 0.35-
kb fragment of the Ace-AMP1gene.
B) Probed with a 0.7-kb fragment
of npt II gene
Northern blot analysis whereby a 0.35-kb fragment
of the Ace-AMP1 gene was used as a probe
A)
B)

67. Conclusions
• Genetic engineering overcomes almost all the limitations
of traditional breeding approaches.
• Recent developments in plant molecular biology provide
opportunities to use techniques of genetic engineering
for improvement of flower crops for modify flower colour,
improve vase life, floral morphology, scent and disease
resistance.
• Spectral difference in flower colour is mainly determined
by the ratio of different classes of pigments and other
factors and knowledge of flower coloration at the
biochemical and molecular level has made it possible to
develop novel color.
67

68. • Recently genetic engineering has demonstrated the best
examples such as ‘Moon’ series of transgenic carnations
and transgenic blue rose marketed in North America,
Australia and Japan.
• Ethylene sensitivity regulates the timing of flower
senescence and incorporation of etr1-1 gene delayed
senescence in petunia flowers.
• Recent advances in the isolation of scent biosynthetic
genes have provided the basis and created the
opportunity for the biotechnological manipulation of floral
scent.
• Ace-AMP1 is a good candidate gene for genetic
improvement of disease resistance in roses. 68
Conclusions

69. Future prospects and new avenues
69
• New genes should be isolated that will have utility in
floriculture, and new transformation methods for flower
crops should be further optimized.
• A genetic map of rose, which is commercially the most
valuable cut flower, has now been developed.
Identification of quantitative trait locus (QTL) from this
map, in conjunction with genetic modification, will assist
breeders to improve productivity, disease and insect
resistance.
• Information on expression of regulatory genes encoding
transcription factors should be generated which have
effects on flower colour, fragrance and disease resistance.
• The manipulation of colour in the yellow and orange range
will become increasingly feasible as more is learnt about
the carotenoid biosynthesis pathway.
• More research efforts are needed to modify flower shape

70. 70
Thank you