“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.comMob: +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

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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

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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

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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

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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

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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

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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

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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

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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

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“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

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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

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CASE STUDIES

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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

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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

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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

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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

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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

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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