Genetic engineering for flower colour modification
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Transcript of Genetic engineering for flower colour modification
“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: [email protected]: +91 9067840639
IntroductionBiotechnology In FloricultureFlower and flower colourRole of colourMajor plant pigmentsGenetic improvement of flower colour
Making deliberate crosses between two parents Mutation PolyploidyGenetic Engineering of flower colour
Over-expressing or silencing the structural gene expression in flavonoid biosynthetic pathway.
Colour modification through antisense RNA / RNAi technology
Case studiesConclusionFuture Prospects
CONTENTS
2
• 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
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
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
FlowerReproductive 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
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
Why we need Modification in colour ?
Modification in flower colour of a variety with desirable agronomic or consumer characteristicsEx: 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
Site of colour accumulation
Chlorophylls and carotenoids are in
chloroplast and chromoplast
Flavonoids are in the vacuole
9 9
Pigment Class
Compound Types Compound Examples Typical ColoursPorphyrins Chlorophyll Chlorophyll a and b GreenFlavonoids 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, HesperidinNaringin, 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
Genes involved in pigment synthesis1.structural (enzyme) genes2.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 1111
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 IPerilla MybpIPetunia An2, An4 An1
1212
Regulatory region Coding region
Protein (Enzyme)
Pigment
Genes contain regulatory region and coding region
Springob et al., 2003
1313
Influence the type, intensity and pattern
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.
1414
Gene Enzyme
DxsDxrLpiGpsFpsGgpsPsyZdsLcy-bLcy-cNsyCcsPtox
Deoxy xylulose 5-phosphate synthaseDeoxy xylulose 5-phosphate reducoisomeraseLytB proteinGeranyl diphosphate synthaseFernsyl diphosphate synthaseGeranylgeranyl diphosphate synthasePhytoene synthaseβ-Carotene dessaturaseLycopene β-cyclaseLycopene β-cyclaseNeoxanthin synthaseCapsanthin capsorubin synthasePlastid terminal oxysidase
Genes involved in carotene pigment synthesis
(Vainstein, 2004)1515
16
Biosynthetic pathway
of
flavonoid
16
17
Biosynthetic pathway
of
carotenoids
17
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
Conventional breeding
Hybridization:=x
Traditional doner
Desired gene
Commercial variety New variety
Many genes are transferred
Co dominance1919
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
2020
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 earlystep in the synthetic pathway will not happen.
A x B C D E G
H I J L
Mutation:
2121
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
2222
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
Gladiolus1. Shobha Gamma rays Wild Rose Roseine purple Shell pink
2. Tambari Gamma rays Oscar Single Altered flower colour
Source: http://mvgs.iaea.org 2323
Polyploidy
Natural origin or colchiploidy
Polyploidy can be obtained by colchicine treatment
2424
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 relativeconcentration of the minor metabolite quercetin-3,7-diglucoside.
Polyploidy was inducedthrough in vitro colchicine treatment
Griesbach and Kamo, 1996
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.
Genetic modification can be used to transfer new specific traits into the plant
Genetic engineering
2525
26
Transgenic Technology
New
Colo
urs
Long
Vase
Life
Resistant To Biotic Stresses
Resistant To Abiotic Stresses
Improved Size
Impr
oved
Flo
ral S
cent
Improved
Shape
26
Gene transfer methods
Indirect Direct
Most widely usedMore economicalMore efficientTransformation 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 2727
Gene transformation
2828
29
Colour modification done by:
Over expression of structural genes
Inhibition of key biosynthetic enzyme
Use of sense or antisense enzyme construct 29
1. Chalcone synthaseChalcone 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
3030
2. Chalcone isomeraseChalcone 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)
3131
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).
3232
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).
3333
5. Anthocyanidin synthase ANS catalyzes leucoanthocyanidins into anthocyanidin
Dehydroxylation
Application of transgenic ans to pigment modification is less
reported
3434
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.
3535
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.
3636
37
8. Transformation with multiple genes
Petunia & torenia carrying F3′5′H and DFR genes altered
flower colour of interest
37
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
3838
Inhibition of gene expression by antisense RNA
3939
Colour modification through RNAi mediated gene silencing
40
“The proces by which the dsRNA silence gene expression.”
Degradation of mRNA or translation inhibition
40
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.
4141
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
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
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
4444
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.
4545
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.
4646
2. Vacuolar pH pH of vacuole : Acidic : stabilize anthocyanins Generally, in pH - reddening, and in pH - blueing effectEx: 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)
4747
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
4848
49
CASE STUDIES
49
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 5050
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
5151
52
Anthocyanidin composition in the petals of transgenic gentian was measured by HPLC analysis. 52
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 5353
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
5454
Steps:1. Turn off the production of red pigment; 2. Open the ‘door’ to production of blue pigment; and then3. Produce blue pigment. 55
55
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 5656
A selection of chrysanthemum cultivars highlighting those deemed suitable for transformation to achieve blue coloration 5757
Inflorescence color changes with the production of delphinidin-based anthocyanins5858
Inflorescence color changes with the production of delphinidin-based anthocyanins
5959
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 6060
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
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
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
63Suzuki et al., (2007)Japan 63
64
TLC analysis of wild type and transgenic type
64
65
CAROTENOIDCAROTENOID 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
Flower color modification of Petunia hybrida commercialvarieties by metabolic engineering
Flower colour changed from purple to almost white by the down-regulation of the CHS geneSurfinia 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.
6666Japan
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
6767
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
Flower colour modifications by regulating flavonoid biosynthesis
6969
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.
7070
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
7171