genetic transformation in flower crops

1. GENETIC TRANSFORMATION IN FLOWER CROPS
TAMIL NADU AGRICULTURAL UNIVERSITY
Jiji Allen. J

2. CONTENT
• Genetic engineering
• GM in flowers
• Genetic transformation for Colour modification
• Concept
• Research articles
• Genetic transformation for improved Vase life
• Concept
• Research articles
• Genetic transformation for improved Fragrance
• Concept
• Research articles
• Genetic transformation for pest and disesase resistance
• Concept
• Research articles
• Future thrust

4. Types of Gene Delivery
• Direct gene transfer:
• Electroporation method
• Microinjection method
• Microprojectile bombardment method
• Gene Gun Method
• Indirect gene transfer:
• Agrobacterium Mediated transfer

5. Color Modification
• Most widely reported type of genetic modification in floricultural
crops
• Flavonoids, carotenoids, and betalains are the three main
pigment classes that contribute to flower color
• Anthocyanins, a particular class of flavonoids, are major
constituents of orange, red, violet, and blue flowers.
• Flavonoids (flavones and flavonols)
• Presence of metal ions and vacuole pH also affects flower color,
giving an infinite variation

6. Pigment Compound Types Compound Examples Colours
Chlorophylls Chlorophyll Chlorophyll a and b Green
Carotenoids
Carotenes Lycopene, α-carotene, β-carotene,
γ-carotene etc.
Yellow, Orange,
Red
Xanthophylls Lutein,, Zeaxanthin, Neoxanthin,
Violaxanthin etc.
Flavonoids
Anthocyanins Cyanidin, Delphinidin, Malvidin,
Pelargonidin, Peonidin, Petunidin
Red, Blue, violet
Flavones
Flavone Luteolin, Apigenin, Tangeritin Yellow
Flavonol Quercetin, Kaempferol, Myricetin Yellow
Flavanone Hesperetin, Naringenin, Eriodicty
ol Homoeriodictyol
Colour less co-
pigments
Flavanonol Taxifolin , Dihydrokaempferol Colour less co-
pigments
Isoflavones Genistein, Daidzein, Glycitein

7. FlavonoidsCarotenoids
Modification in Pigments
Aurones Chalcones
Anthocyanins
Carnation
Peony
Cyclamen
Snapdragon
Dahlia
Statice
Zinnia

8. Flavonoid Biosynthesis Pathway
• Flavonoids - water-soluble - most common pigments in
flowers
• Yellow to red to violet to blue
• Flavonoids are classified into groups depending on their
structure
• Chalcones, Aurones, Anthocyanins, flavones, and flavonols
– major contributors
• flavones, and flavonols – colourless (co-pigments)
• Synthesized in cytosols - transported to vacuoles - acidic
conditions stabilize the colored anthocyanins

9. pH and Colour
1. Dark red
2. Dark pink
3. Pinkish red
4. Faint pink
5. Violet
6. Faint violet/blue
7. Faint green
8. Faint green
9. Bluish green
10. Green
11. Yellowish green
12. Yellow

11. Chalcones & Aurones

12. Anthocyanins
Flavones
Dihydroxy-Flavones
Anthocyanidins
F 3 H
DFR & AS
F 3 H – Flavon-3-hydroxylase
DFR – Di-hydroxy flavone
Reductase
AS – Anthocyanin synthase
Unstable
Glycosylation
Acylated
Methyl transferase

13. Blue
• In anthocyanidin number of hydroxyl group in B ring decides the
colour
• The one with highest number produces blue
• Pelargonidin – brick red or red
• Cyanidin – Red or Magenta
• Delphinidin - Violet
Morning glory
Peonidin
+
pH 7.7
Heavenlyblue
Corn flower
6 mol Cyanidin
+
Flavone
+
Fe3+
+
Mg
+
2Ca+

15. Enzymes that are essential for colour modification

17. Non-transgenic torenia - delphinidin-based anthocyanin
Down-regulation of anthocyanidin synthase
Down-regulation of flavonoid 3, 5-hydroxylase and flavonoid 3-
hydroxylase and expression of a heterologous dihydroflavonol
reductase
Expression of snapdragon teterahydroxychalcone 4-glucosyltransferase
and aureusidin synthase genes and downregulation of anthocyanin
pathway
TORENIA

18. Carotenoids
• Carotenoids are C-40 tetraterpenoids with polyene chains
that are lipid soluble
• 700 natural carotenoids have been identified
• They share the terpenoid biosynthetic pathway
• Contribute to the majority of yellow to orange hues in a
number of flowers, including rose and chrysanthemum

19. Dominant over yellow
Single dominant gene is
suggested to inhibit
carotenoid biosynthesis
Carotenoid cleavage
Dioxygenase (CCD)
inhibit carotenoid biosynthesis
Supression of CCD using
RNA i Tech

20. Engineering of the Rose Flavonoid Biosynthetic
Pathway Successfully Generated Blue-Hued Flowers
Accumulating Delphinidin
Yukihisa et al,.
Suntory Limited Research Center & Florigene
2007
1.

22. Criteria for selecting the host
variety
• They accumulated flavonols that
were expected to be co-pigment
• They had a higher vacuolar pH
• Ideally, they did not have F3’H
activity
• They accumulated pelargonidin
rather than cyanidin
• WKS77, WKS82, WKS100,
WKS116, WKS124 and WKS140
were selected for genetic
transformation

23. Viola
Iris
Toreina
Anthocyanin 5-
hydroxycinnamoyl
transferase (5AT)

24. Binary Vectors

29. Result
Down-regulation of the rose DFR gene and overexpression of
the iris DFR gene, as well as the overexpression of the viola F3’
5’ H gene is required to produce blue colour in rose and cv
Lavender can be used as a parent plant.

31. FINALLY ACHEIVED
• In June 2004, they publicly
announced the first success
of the development of blue
roses in the world
• They obtained the permits on
January 31, 2008
SUNTORY blue rose ”APPLAUSE”

32. Carnation
2.

34. History of blue carnation

35. Transforming the Snapdragon Aurone Biosynthetic Genes
into Petunia Alters Coloration Patterns in Transgenic
Flowers
Chen et al., 2015
Thaiwan
Antirrhinum majus cv. Ribbon Yellow
Petunia hybrid cv. Extra Blue
3.

36. AURONE BIOSYNTHETIC PATHWAY GENES
• 4'-O-glucosyltransferase (designated as SRY4'CGT)
• Aureusidin synthase (designated as SRYAS1)

37. 2',4',6', 4-tetrahydroxychalcone (THC)
THC 4'-O-glucoside (colorless)
4'-O-glucosyltransferase
2',4',6',3,4-pentahydroxychalcone
PHC 4'-O-glucoside (colorless)
Function of 4'-O-glucosyltransferase (designated as SRY4'CGT)
Function of aureusidin synthase (designated as SRYAS1)
THC 4'-O-glucoside (colorless) or PHC 4'-Oglucoside (colorless)
aureusidin 6-O-glucoside (yellow pigment) or bracteatin 6-O-
glucoside (yellow pigment)
aureusidin synthase

39. …..Result

40. Petunia plants carrying SRYAS1 transgene

41. Gene Expression in T1 Transgenic SRY4'CGT Plants

42. Violet/Blue Chrysanthemums—Metabolic
Engineering of the Anthocyanin Biosynthetic Pathway
Results in Novel Petal Colors
5.
Filippa et al,. 2013
Florigene Pvt Ltd
Australia

44. • The anthocyanins generally found in chrysanthemum petals
are cyanidin 3-mono or di malonyl glucosides
• Pelargonidin-based anthocyanins are rarely found in
chrysanthemum
• Cultivar selected - Improved Reagan

47. List of crops transformed for color modification

48. Vase life
• Retention of postharvest quality is essential in floricultural crops
• Premature senescence - reflected in leaf yellowing, leaf and/or
flower abscission in pot plants, or a short vase-life of cut flowers
• Ethylene - petal senescence
• With species that ripen or senesce in response to ethylene,
longevity can be extended by suppressing either the biosynthesis
of or tissue sensitivity to ethylene

49. Methionine
S-adenosylmethionine
SAM synthase
1- aminocyclopropane-1-carboxylic acid
ACC Synthase
Ethylene
ACC Oxidase
Ethylene biosynthesis
Inhibiting the
expression by
sense or anti-
sense
technique
• Separate receptors from petals and
leaves
• mutant etr1-1 gene from Arabidposis

50. Delayed flower senescence of Petunia hybrida plants
transformed with antisense broccoli ACC synthase and
ACC oxidase genes
Li-Chun Huang et al., 2007
Thaiwan

51. genes that encode ACC synthase
PhACS1 PhACS2
petunia
regulated in a tissue-specific
manner during flower senescence
increases ACC synthase
activity during petal
senescence
PhACS2 is specifically
expressed in pistils during
flower development

52. Three genes that encode ACC oxidase
PhACO1 PhACO3PhACO4
causing increased ACC oxidase
activity during petal senescence
regulated in a tissue-specific
manner during flower senescence
petunia
specifically expressed during pistil
development

53. BoACS1
Antisense orientation
Was fused to the CaMV 35S
promoter of binary vector
pBI121
resultant plasmid pCS2 was
transferred to A. tumefaciens strain
LBA4404.
Construction of antisense BoACS1 gene
Construction of antisense BoACO1 gene
BoACO1 Antisense orientation was also
fused to binary vector pBI121
transferred to A.
tumefaciens strain LBA4404
Transformed into petunia

54. Ethylene produced by transgenic plants
Longevity of flowers left undisturbed on
plants

55. Conclusion
• Most convincing clones were 2-28, 5-2, 5-26, and 5-30
• The antisense BoACO1 transformation was evidently more
effective than the antisense BoACS1
• Transformation with antisense constructs of broccoli genes
can result in reduced ethylene evolution in an unrelated
species, petunia, and the lower rates of ethylene evolution
are associated with extended flower longevity, especially of
excised flowers

56. Practical Lessons in the Commercialization of
Genetically Modified Plants - Long Vase-Life Carnation
S. Chandler
Florigene Pvt. Ltd.
Bundoora, VIC 3083
Australia

57. GENETIC MODIFICATION FOR IMPROVED VASE-LIFE
IN CARNATION
Suppression of ACC Synthase
Mutant etr1-1 gene from Arabidposis
Down-regulate the ethylene signal cascade
Promoter - carnation floral specific
MADS box gene, CMB2
the best ETR transgenics had a vase-life in water as
good as silver treated control flowers

59. Ethylene production in transgenic carnations

60. Flower Scent
• Floral scent is an important and fascinating character of
floricultural crops
• Many modern cut flower varieties have little or no scent.
• This is because there is a negative correlation between
postharvest vase life and fragrance
• Terpenoids - essential oils - ability to manipulate
• The type, concentration, and ratios of these compounds may also
be a beneficial application of gene-technology in floricultural
plants

61. Mechanism
• Terpenes, phenylpropanoids or fatty acid derivatives (Volatile
groups)
• They are secondary, or specialized, metabolism
• Produced only in specific plant lineages and function in specific
ecological roles unique to these lineages
• Primary and specialized metabolic pathways are not completely
separate
• In some cases a single reaction and a single enzyme will convert a
primary metabolite into a volatile compound, whereas in other
cases multiple steps are required

62. Rose,petunia,
etc.,

63. Trials conducted
Clarkia breweri
linalool synthase (LIS)
Geranyl diphosphate
(GPP)
(3S)-linalool,
sweet, pleasant fragrance
that is found in the flowers of
many species
Overexpressi
on of LIS
Petunia hybrida
and
Dianthus
caryophyllus
No monoterpene
But
linalool production in both leaves and flowers No effect

64. Reason
Petunia
linalool
into non-volatile linalyl b-D-glucoside
endogenous enzyme
Carnation
linalool
Cis and trans-linalyl oxides
Although these extra terpenes
constituted almost 10% of the total volatiles emitted from the
transgenic flowers, this increase in scent emission was still not enough
for most humans to detect a change in floral aroma in smell tests

65. • ClLIM, limonene synthase
• ClPIN, b-pinene synthase
• ClTER, Citrus limon g-terpinene synthase
• CbLIS, Clarkia breweri linalool synthase
• Dc F3’H, Dianthus caryophyllus F3’H
• MsLIM3H, Mentha spicata limonene-3-
hydroxylase
• PhBPBT, benzylalcohol/phenylethanol
benzoyltransferase
• PhBSMT, petunia benzoic acid/salicylic
acid carboxyl methyltransferase
• PhCFAT, coniferyl alcohol
acyltransferase
• PhODO1, ODORANT1 transcription
factor; PhPAAS, phenylacetaldehyde
synthase; RhAAT, Rosa hybrida alcohol
acetyltransferase
• Tobacco TERLIMPIN is a tobacco
transgenic line expressing ClTERM,
ClLIM and ClPIN

66. ODORANT1 Regulates Fragrance Biosynthesis in
Petunia Flowers
Verdonk, J.C. et al,. (2005)
P. hybrida cv Mitchell
ODORANT1 (ODO1)
Controls the synthesis of volatile benzenoids
Regulates the shikimate pathway in petals
toward benzenoid production
Downregulation of ODO1 does not
influence flower color

67. Petal-Specific Expression of ODO1
Correlates with Scent Emission.
(A) RNA gel blot analyses of ODO1 in Mitchell
petals harvested at 3-h intervals
(B) Bar graph depicting the emission of three
selected volatile benzenoids measured for 1 h
around the same time points (mean and SE, n
¼ 3).
(C) Organ- and tissue-specific expression. R,
roots; S, stems; L, leaves; Sp, sepals; PT, petal
tube; PL, petal limb; A, anthers; St, stigma.
(D) RNA gel blot analysis of ODO1 in Mitchell
(M) and W138 at 1800 h.

68. Gas chromatography time-of-flight mass spectrometry
chromatograms of volatiles emitted by Mitchell and RNAi line 3

69. PEST & DISEASE RESISTANCE
• Induced overexpression of particular antifungal proteins
may lead to enhanced pathogen resistance
rice
chitinase gene
CaMV 35S
promotor
Rose
reduced susceptibility to blackspot

70. Resistance against beet armyworms and cotton aphids
in caffeine-producing transgenic chrysanthemum
Yun et al., 2011, sweden
Chrysanthemum morifolium cv. Shinba
Agrobacterium tumefaciens strain LBA4404
three coffee Nmethyltransferases genes
(CaXMT1, CaMXMT1 and CaDXMT1
Eight kanamycin-resistant
transgenic shoots were obtained
All six lines successfully expressed
three transgenes and produced
caffeiene at ca. 3 mg per g fresh
weight

71. Detection of caffeine by HPLC in wild
type (1) and transgenic chrysanthemum
(2) in mature leaves.

72. Each caterpillar was transferred to
each leaf disc (diameter 15 mm)
prepared from wild type or
transgenic lines and then allowed
to feed for 24 h in the dark in a
climate chamber at 25°C with a
relative humidity of 60%
Second-instar caterpillars were
starved for 5 hours.

73. A choice test of aphids (C, D). After feeding for a week, each leaf detached from
whole plants of wild type or transgenic line C#1 was photographed
Caffeine-producing transgenic plants are commonly resistant
against a broad range of herbivore insects and pathogens

74. Future Thrust

77. Heterologous expression of the flavonoid 3’,5’-
hydroxylase gene of Vinca major alters flower color
in transgenic Petunia hybrida
Mori et al., 2004
‘Polo Red Target
4.