Genetic engineering for flower colour modification

1. “GENETIC ENGINEERING FOR FLOWER
COLOUR MODIFICATION.”
PREPARED BY :
AVINASH GOWDA H
M.Sc.(Agri) (Plant Mol. Biol. & Biotech.)
Dept. of Biotechnology
Junagadh Agricultural University
Junagadh Gujarat
Email: avinashgowda.gh@gmail.com
Mob: +91 9067840639

2. Introduction
Biotechnology In Floriculture
Flower and flower colour
Role of colour
Major plant pigments
Genetic improvement of flower colour
Making deliberate crosses between two parents
Mutation
Polyploidy
Genetic Engineering of flower colour
Over-expressing or silencing the structural gene
expression in flavonoid biosynthetic pathway.
 Colour modification through antisense RNA / RNAi
technology
Case studies
Conclusion
Future Prospects 2

3. • Floriculture is considered to include the cut flowers, potted
plants, and ornamental bedding plants and garden plant
industries.
 Commercial floriculture is becoming important from the
export angle.
 commercial floriculture has higher potential per unit area
than most of the field crops.
• Government of India has identified floriculture as a sunrise
industry and accorded it 100% export oriented status.
 Indian floriculture industry has been shifting from
traditional flowers to cut flowers for export purposes
Introduction
3

4.  About 255 thousand hectares area is under cultivation, and
the production of flowers are estimated to be 17.54 million
tonnes loose flowers and 543 million tonnes cut flowers.
 The country has exported 22,947.23 MT of floriculture
products to the world for the worth of Rs. 460.75 crores in
2014-15.
• The main areas of production and consumption of
floricultural products are in the United States and Europe,
• The highest consumption per head is in the Netherlands,
followed by Germany, Austria, and France.
4

5. 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.
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
5

6. Flower
Reproductive structure of a seed-bearing plant
Flower colour
Flower color is one of the most attractive
characteristics in ornamental plants.
Determines the market value in ornamental plants
The demand varies with trend, season and occasions
6

7. ROLE OF COLOUR
Attraction of pollinators
Function in photosynthesis
In human health as antioxidants and precursors of
vitamin A
Seed dispersal
Protecting tissue against photooxidative damage
Resistant to biotic and abiotic stress
Symbiotic plant-microbe interaction
Act as intermediary for other compounds
7

8. Why we need Modification in colour ?
Modification in flower colour of a variety with desirable
agronomic or consumer characteristics
Ex: A white carnation from preferable red-flowering variety
A flower colour not occurring naturally in a particular crop
Ex: Blue colour in rose, carnation, orchids
Change in trend for colour season to season, year to year
High price for Novel colour.
Ex: The price for a single blue rose is about $22 to $33
8

9. Chlorophylls and
carotenoids are in
chloroplast and
chromoplast
Flavonoids are
in the vacuole
9
9

10. Pigment
Class
Compound Types Compound Examples Typical Colours
Porphyrins Chlorophyll Chlorophyll a and b Green
Flavonoids Anthocyanins Pelargonidin, Cyanidin,
Delphinidin, Peonidin
Petunidin, Malvidin
Red, Blue, violet
Anthoxanthins Flavonols Kaempferol, Quercetin, Fisetin,
Kaempferide, Morin, Myricetin,
Myricitrin, Rutin
Yellow
Flavones Apigenin, Biacalein, Chrysin,
Diosmetin, Flavone, Luteolin
Yellow
Isoflavonones Diadzin, Genistein, Enterodiol,
Coumestrol, Biochanin
Flavonones Eriodictyol, Hesperidin
Naringin, Naringenin
Colourless co pigments
Flavans Biflavan, Catechin, Epicatechin, Colourless co pigments
Carotenoids Carotenes Lycopene, α-carotene, β-carotene,
γ-carotene
Yellow, Orange, Red
Xanthophylls Lutein, Cryptoxanthin,
Zeaxanthin, Neoxanthin,
Rhodoxanthin, Violaxanthin,
Canthaxanthin, Astaxanthin,
Major Pigments in Plants
Betalains Betacyanins Reddish to Violet
Betaxanthins miraxanthin and portulaxanthin Yellow to Orange
Red
Colourless
1010

11. Genes involved in pigment synthesis
1.structural (enzyme) genes
2.regulatory genes
Enzyme Gene Species
CHS Chs Antirrhinum, Chrysanthemum, Orchid, Rosa, Dianthus
CHI Chi Antirrhinum, Petunia, Eustoma, Dianthus
F3H F3h Antirrhinum, Calistephus, Chrysanthemum, Dianthus, Orchid
F3’H F3’h Antirrhinum, Dianthus, Petunia
F3’5’H F3’5’h Calistephus, Eustoma, Petunia
FLS Fls Petunia, Rosa
FNS FnsII Antirrhinum, Gerbera
DFR Dfr Antirrhinum, Calistephus, Gerbera, Orchid, Dianthus, Petunia
ANS Ans Antirrhinum, Calistephus, Petunia
GT 3Gt Antirrhinum, Gentiana
GTS Gts Petunia
1.structural (enzyme) genes:Is a gene that codes for any RNA or protein
product other than a regulatory protein.
Vainstein, 2004 11
11

12. 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 An2, An4 An1
12
12

13. Regulatory region Coding region
Protein (Enzyme)
Pigment
Genes contain regulatory
region and coding region
Springob et al., 2003
13
13
Influence the type, intensity and pattern

14. Effects of regulatory genes on flower colour
modification
A complex of two transcriptional factor MYB and basic-
Helix-Loop-Helix (bHLH) and WD40 activates the
flavonoid biosynthesis genes.
These DNA binding proteins interact with promoter
regions of the target genes and regulate the initiation
rate of mRNA synthesis.
14
14

15. Gene Enzyme
Dxs
Dxr
Lpi
Gps
Fps
Ggps
Psy
Zds
Lcy-b
Lcy-c
Nsy
Ccs
Ptox
Deoxy xylulose 5-phosphate synthase
Deoxy xylulose 5-phosphate reducoisomerase
LytB protein
Geranyl diphosphate synthase
Fernsyl diphosphate synthase
Geranylgeranyl diphosphate synthase
Phytoene synthase
β-Carotene dessaturase
Lycopene β-cyclase
Lycopene β-cyclase
Neoxanthin synthase
Capsanthin capsorubin synthase
Plastid terminal oxysidase
Genes involved in carotene pigment synthesis
(Vainstein, 2004) 15
15

16. 16
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17. 17
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17

18. Genetic Improvement of Flower Colour
 Genetic Improvement: involves changing the plant’s
genetic makeup
Making deliberate crosses between two parents
 Conventional Hybridization
 Inter-specific Hybridization
Mutation
Polyploidy
Genetic Engineering of flower colour
18

19. Conventional breeding
Hybridization:
=x
Traditional doner
Desired
gene
Commercial variety New variety
Many genes are
transferred
Co dominance
19
19

20. Inter-specific Hybridization
Studies on inter specific hybridization for transferring yellow
colour in Dianthus plumarius (2n=6x=90).
Gatt et al. (2005)
x =
.
Dianthus plumarius D. knappii
20
20

21. Many different genes are involved in controlling the
synthesis of the pigments. In a multi-step process.
A B C D E G
H I J L
If a single enzyme is not present and early
step in the synthetic pathway will not happen.
A x B C D E G
H I J L
Mutation:
21
21

22. Ornamental plants are ideal
First officially released commercial mutant cultivars : Tulip (cv. ‘Faraday‘
from cv. ‘Fantasy by irradiation) expressing an altered flower colour in 1936
(Broertjes and van Harten 1988)
Approx 55% of the mutant cultivar changes in flower colour
Successfully achieved in Chrysanthemum, Bougainvillea, Rose etc.
Datta et al., 2001
Phenotypic
expression
in flower
after mutation
22
22

23. Crop Cultivar Mutagen Parent Earlier colour Changed colour
Chrysanthemum
1. Agnisikha Gamma rays D-5 Magnolia purple Erythrite red
2. Alankar Gamma rays D-5 Magnolia purple Spanish orange
3. Batik Gamma rays Flirt Red
Yellow stripes on red
background
4. Tulika Gamma rays M-24 Purple
5. Surekha Yellow Gamma rays Surekha Ruby red Yellow
6. Raktima Gamma rays Shyamal Purple crimson
Bougainvillea
1. Mahara variegata Gamma rays Mahara green leaves Variegated leaves
2. Jaya Gamma rays Jayalakshmi - Purple bracts
3. Suvarna Gamma rays Ceylon Single Altered flower colour
Rose
1.Abhisarika Gamma rays Kiss of fire Normal Striped
2. Curio Gamma rays Imperator - Cherry red
3. Light Pink Prize
Gamma rays First Prize
Light red and deep
pink
Light Pink
4.Sharada Gamma rays
Queen
Elizabeth
Carmine rose Light pink
5.Madhosh H.T EMS Gulzar -
Mauve coloured
stripes against deep
red base
Gladiolus
1. Shobha Gamma rays Wild Rose Roseine purple Shell pink
2. Tambari Gamma rays Oscar Single Altered flower colour
Source: http://mvgs.iaea.org 2323

24. Polyploidy
Natural origin or colchiploidy
Polyploidy can be obtained by colchicine treatment
24
24
Ex; The effect of induced polyploidy on the flavonols of Petunia
‘Mitchell'
Increasing the relative concentration
of the major metabolite
quercetin-3-sophoroside
and decreasing the relative
concentration of the minor
metabolite quercetin-3,7-diglucoside.
Polyploidy was induced
through in vitro colchicine
treatment
Griesbach and Kamo, 1996

25. 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.
Geneticmodificationcanbeusedtotransfernewspecifictraitsintotheplant
Genetic engineering
25
25

26. Transgenic
Technology
Resistant To
Biotic Stresses
26
26

27. 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
 Electroporotic uptake
Chandler and Brugliera, 2011 27
27

28. Gene transformation
28
28

29. Colour modification done by:
Over expression of structural genes
Inhibition of key biosynthetic enzyme
Addition of an enzyme in a particular
biosynthetic step
Use of sense or antisense enzyme construct
29
29

30. 1. Chalcone synthase
Chalcone synthase (CHS) catalyze 3 molecules of malonyl-CoA
and 1 molecule of coumaroyl- CoA into 1 molecule of chalcone
Ex: Over-expression of sense or antisense chs constructs to
modify flower colour in Petunia, Torenia, chrysanthemum,
lisianthus etc.
Over-expressing or silencing the structural gene
expression in flavonoid biosynthetic pathway
30
30

31. 2. Chalcone isomerase
 Chalcone isomerase (CHI) catalyzes yellow coloured chalcone
to colourless pigment naringenin. Can also occur spontaneously
 Most plants do not accumulate chalcones
 Some mutant plants accumulate chalcones mutation in the chi
locus
Ex: Yellow flowers - chi mutants of aster and carnation
(Schijlen et al. 2004)
31
31

32. 3. Flavanone hydroxylase/ Flavonoid-3′hydroxylase/
Flavonoid-3′,5′-hydroxylase
 The hydroxylation in position 3 of the C ring in flavanones,
results in dihydrokaempferol by flavanone-3-hydroxylase (F3H).
Ex: In Petunia and Antirrhinum - Mutation in f3h locus caused a
loss of F3H activity - white flowers (Schijlen et al. 2004).
32
32

33. 4. Dihydroflavonol-4-reductase (DFR)
The enzyme DFR catalyzes the reduction of
dihydroflavonols to leucoanthocyanidins.
Ex: Transgenic carnation plants carrying sense dfr and
sense F3′5′H from Petunia produced violet flowers as
compared to the wild-type white flowers (Forkmann
and Martens 2001).
33
33

34. 5. Anthocyanidin synthase
 ANS catalyzes leucoanthocyanidins into anthocyanidin
Dehydroxylation
 Application of transgenic ans to pigment modification is less
reported
34
34

35. 6. Flavonoid 3-O-glucosyltransferase (3GT)
 3GT transfers the glucose moiety from UDP-glucose
to C-3 hydroxyl group of the anthocyanidin - coloured
pigments of anthocyanidin 3-O-glucosides.
 3GT – stabilized anthocynidins for accumulation in
vacuole.
Ex: Overexpression of snapdragon 3GT cDNA in
lisianthus - novel anthocyanins.
35
35

36. 7. Other enzymes
 In some sps. like snapdragon, cosmos and dahlia, chalcone -
aurones (yellow colour) produced by aureusidin synthase
(AS).
Chalcone reductase (CHR) co-acts with chalcone synthase
(CHS) and catalyzing 1 coumaroyl-CoA and 3 malonyl-CoA
to produce iso-liquiritigenin (yellow in colour), this is a
precursor of 5-deoxy-isoflavonoids.
36
36

37. 37
8. Transformation with multiple genes
Petunia & torenia carrying F3′5′H and DFR genes altered
flower colour of interest
37

38. Colour modification through antisense RNA
technology
 Antisense RNA is a single stranded RNA that is
complementary to mRNA strand transcribed
within a cell.
 They are introduced in a cell to inhibit the
translation machinery by base pairing with the
sense RNA activating RnaseH, to develop
perticular novel transgenic.
mRNA sequence AUGAAACCCGUG
Antisence RNA UACUUUGGGCAC
38
38

39. Inhibition of gene expression by antisense RNA
39
39

40. Colour modification through RNAi mediated
gene silencing
40
“The proces by which the dsRNA silence gene
expression.”
Degradation of mRNA or translation inhibition
40

41. Difference between antisense technology
and RNAi
 The intended effect in both will same i.e., gene
silencing but the processing is little but different.
 Antisense technology degrades RNA by enzymes
RNaseH while RNAi employed the enzyme
DICER to degrade the mRNA.
 RNAi are twice larger than the antisense
oligonucleotides.
41
41

42. 42
Ds RNA are chopped in to short
interfering RNA s (siRNA) by Dicer.
The siRNA –Dicer complex is recruits
to form an RNA Induced Silencing
Complex (RISC).
The siRNA unwinds .
The unwond siRNA base pairs with
complementory mRNA , thus guiding
the RNAi machinery to the target
mRNA.
The target mRNA is effectively cleaved
and subsequently degraded. Resulting
in gene silencing.
Mechanism of RNAi
42

43. Land marks in RNAi discovery
 RNAi was firstly discovered and observe in transcriptional
inhibition by antisense RNA expressed in transgenic plants and
more directly by reports of unexpected outcomes in experiments
performed in 1990s (Jorgensen et al.,).
 In an attempt to produce more intense purple coloured Petunias,
researchers introduced additional copies of a transgene encoding
chalcone synthase . But were surprised at the result that instead
of a darker flower, the Petunias were variegated.
43
Upon injection of the transgene responsible for purple colorings in
Petunias, the flowers became variegated.
43

44.  This phenomenon was called co-suppression of gene
expression , since both the expression of the existing gene (the
initial purple colour) and the introduced gene/transgene (to
deepen the purple) were suppressed.
 It was subsequently shown that suppression of gene activity
could take place at the transcriptional level (transcriptional
gene silencing, TGS) or at the post-transcriptional level (post-
transcriptional gene silencing, PTGS
44
44

45. Generation of variegated flowers by using
transposons
 Insertion or excision of transposons in flavonoid biosynthetic
or regulatory genes produces a mosaic or variegated phenotype
 Insertion of a transposon results in white sectors of a coloured
background.
 Excision of transposon results in coloured sectors on a white
background
 The sizes of sectors depend on the timing of insertion and
excision
Ex: Morning glory and Petunia etc.
45
45

46. Other factors affecting flower colouration
1 . Co-pigments
 Flavonols and flavones
 Copigments & anthocyanins complex stabilizes and determine
the colour
 The enzyme flavonol synthase (FLS) and flavone synthase
(FNS) converts dihydroflavonols into flavonols
 Flavonols and flavones share common precursors with
anthocyanins, so their down regulation often reduces
anthocyanin level.
46
46

47. 2. Vacuolar pH
 pH of vacuole : Acidic : stabilize anthocyanins
 Generally, in pH - reddening, and in pH - blueing effect
Ex: 1.In Petunia, identified. Mutated- blueing of the
flower. (pH1 to pH7)
Ex: 2. Morning glory (Ipomea tricolor)
Strong reddish purple buds change to light blue when flower
opens due to purple protein transports Na+ into and H+
out of the vacuole, resulting in the increased vacuolar pH
(6.5-7.5)
47
47

48. 3. Cell shape
 Accumulation of anthocyanin pigments is also affected by the
shape of the cells.
 In Snapdragon, if cells of the inner epidermis are conical- the
properties of higher light absorption and a velvet sheen
 The fainter colour from a flattening of epidermal cells
48
48

49. 49
49

50. Targeted for suppression of three anthocyanin biosynthetic genes; chalcone
synthase (CHS), anthocyanidin synthase (ANS) and flavonoid 3’,5’-
hydroxylase (F3’5’H) in Gentia.
 Approx 500 bp fragments of gentian CHS, F35H and ANS genes
connected with the first intron of the caster bean catalase gene in inverted
orientation and driven by the rolC promoter
 Vectors have herbicide resistance (bar) gene as marker
 A. tumefaciens harboring vector inoculated into targeted plant
 Expression level analysis: RNA gel blot tech
Pigment analysis: HPLC
Flower color modification of gentian plants by
RNAi-mediated gene silencing
Nakatsuka et al., (2008)Japan 50
50

51. Result:
Suppressed CHS gene - Selected 20 line – 17 changed colour – 14
pure white & 3 pale-blue color
Suppressed ANS gene – Most line pale-blue, no white
Suppression of the F3’5’H gene - Decreased delphinidin derivatives
and increased cyanidin derivatives, and led to magenta flower colors
A) Wild-type B) Suppresed Suppression of the ANS gene
F3’5’H gene
51
51

52. 52
Anthocyanidin composition in the petals of transgenic gentian was measured by
HPLC analysis. 52

53.  Rosa hybrida lacks violet to blue flower.
 Due to absence of delphinidin-based anthocyanins
 Roses do not possess flavonoid 3’,5’-hydoxylase
(F3’5’H) For delphinidin biosynthesis
Engineering for Blue Rose
Katsumoto et al.,(2007)Australia 53
53

54. Steps:
 Down-regulation of the rose DFR gene and over-expression of the iris DFR gene
by RNAi technique
 The over-expression of a F3’5’H – efficient accumulation of delphinidin and
colour changes to blue.
 Efficient and exclusive delphinidin production and a bluer flower colour
54
54

55. Steps:
1. Turn off the production of red pigment;
2. Open the ‘door’ to production of blue pigment; and then
3. Produce blue pigment. 55
55

56. Violet/Blue Chrysanthemums
 Flavonoid analysis and precursor feeding experiments
 A selection of eight cultivars were successfully
transformed with F3’5’H genes under the control of
different promoters.
 A pansy F3’5’H gene under the control of a chalcone
synthase promoter fragment from rose resulted in the
effective diversion of the anthocyanin pathway to produce
delphinidin in transgenic chrysanthemum flower petals.
The resultant petal color was bluish.
Bruglier et al.,(2013)Australia 56
56

57. Aselectionofchrysanthemumcultivarshighlightingthosedeemedsuitablefortransformationto
achievebluecoloration 5757

58. Inflorescence color changes with the production of delphinidin-based anthocyanins
5858

59. Inflorescence color changes with the production of delphinidin-based anthocyanins
59
59

60. Redirection of flavonoid biosynthesis in
petunia
 Mitchell has white flowers due to the absence of
anthocyanin biosynthesis in petal limbs and pollen.
 A binary vector, pLN64, was constructed in which the
Medicago CHR7 cDNA (Ballance and Dixon, 1995)
was placed.
 pLN64 was used in Agrobacterium mediated
transformation to produce transgenic plants of the
Petunia line Mitchell and cyanic-flowered
(anthocyanin-producing) Petunia lines.
Davies et al., (1998)New Zealand 60
60

61. 61
Plant line Flvonols
(µmol/ g dw)
Chalcones
(µmol/ g dw)
Mitchel 132 0
CHR-MP 72 81
Introduction of the CHR cDNA into Mitchell Petunia
61

62. 62
Introduction of the CHR transgene into cyanic-flowered
Petunia lines
Plant line Chalcones (%) Flvonols (%) Anthocyanin (%)
Cyanic lines 27.4 42 30.6
Transgeneic line 62.5 20.5 17
62

63. Flower colour alteration in lotus japonicus by modification
of the carotenoid pathway
 Colour modification is done by over expression of crtW gene
 Gene was isolated from marine bacteria Agrobacterium aurantiacum
 Flower of petel color changed light yellow to deep yellow
 TLC was conducted to analyse percent accumilation of carotene
63
Suzuki et al., (2007)
Japan
63

64. 64
TLC analysis of wild type and transgenic type
64

65. 65
CAROTENOID
CAROTENOID CONTENT (%)
WILD TYPE TRANSGENIC
Neoxanthin 12.6 4.5
Violoxanthin 27.5 66.8
Antheraxanthin 19.8 11.3
Lutein 11.3 19.5
Zeaxanthin 10.2 8.1
β-carotenoid 14.4 20.5
Ketocarotenoid 0 23.2
Other 4.2 6.1
Total 21 36
65

66. Flower color modification of Petunia hybrida commercial
varieties by metabolic engineering
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
The flower color of commercial varieties of Petunia hybrida was
successfully modified by the suppression of endogenous flavonoid
biosynthetic genes, the expression of a heterologous flavonoid
biosynthetic gene, and the combination of both.
66
66Japan

67.  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
C
67
67

68. Transgenic flowers harboring the sense
Hf1 F35H, AR–AT, and FLS genes
Suppression of the F3H gene by antisense and
expression of the rose DFR gene.
Transgenic petunia expressing torenia
FNSII gene
Transgenic plant harboring the sense Hf1
F35H gene
68

69. Flower colour modifications by regulating
flavonoid biosynthesis
69
69

70. Conclusion:
 Flower colour modification using molecular methods has now
become reality
 Flower colour is mainly determined by the ratio of different pigments
and other factors such as vascular pH, co-pigments and metal ions.
 Knowledge at the biochemical and molecular level has made it
possible to develop novel colour which are otherwise absent in
nature.
 Transgenic floricultural crops, only carnation and rose -
commercialized, indicating development of commercial crops by GE
is still very challenging.
70
70

71. Future thrust:
Species-specific genes in flavonoid biosynthetic pathway
Changing flower pigmentation by modification of carotenoids
and betalain biosynthetic pathway.
Production of colour in a scented flowers.
Function, expression, regulation and interaction of the structural
genes and regulatory genes
Transport mechanism of pigments
71
71