Modification of Flower Colour

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This ppt is about different approaches to be implies to modify the flower colour with emphasis on flavonoid synthesis pathway modification.

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Indian Agricultural Research Institute, New Delhi DIFFERENT APPROACHES FOR MODIFICATION OF FLOWER COLOUR Student: Abhay Kumar Gaurav 20307 M. Sc. 1 st Year Seminar: FLA 691

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Indian Agricultural Research Institute, New Delhi Introduction Flower : Reproductive structure of a seed-bearing plant Flower colour : Determines the market value in ornamental plants The demand varies with trend, season and occasions Role of colour: Function in photosynthesis Attraction of pollinators Protecting tissue against photo-oxidative damage Seed dispersal Act as intermediary for other compounds

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Indian Agricultural Research Institute, New Delhi Flower Colour Plant compounds that are perceived by humans to have colour - ‘ pigments ’ Flower colour : Flavonoids , Carotenoids , Betalains The flavonoids : Most common Betalains are the least abundant ( Forkmann , 1991) Ex: Amaranthus , Bougainvillea, Celosia, Gomphrena , Iresine etc. Colour pattern: Differential accumulation of pigment(s)

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Indian Agricultural Research Institute, New Delhi Pigment Compound Types Compound Examples Typical 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 , Eriodictyol , Homoeriodictyol Colour less co- pigments Flavanonol Taxifolin , Dihydrokaempferol Colour less co- pigments Isoflavones Genistein , Daidzein , Glycitein Betalains Betacyanins Red - Violet Betaxanthins Yellow -Orange Major plant pigments and their occurrence

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Indian Agricultural Research Institute, New Delhi Chlorophylls & Carotenoids (Fat soluble) Plastids Anthocyanins & Betalains (Water soluble) vacuole Site of colour accumulation

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Indian Agricultural Research Institute, New Delhi 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

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Indian Agricultural Research Institute, New Delhi Approaches to change flower colour: Conventional Hybridization Inter-specific Hybridization Mutation Genetic Engineering of flower colour Via Flavonoid biosynthesis pathway Via Carotenoid biosynthesis pathway Via Betalain biosynthesis pathway Tinting

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Indian Agricultural Research Institute, New Delhi Conventional Hybridization: = x Traditional donor Desired gene Commercial variety New variety Many genes are transferred Incomplete dominance Ex: Antirrhinum majus Mirabilis jalapa Co dominance: Ex. Rhododendron

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Indian Agricultural Research Institute, New Delhi Inter-specific Hybridization : By this method also new colour has been developed which were not present in that species. Ex. Garden Pink, Summer Lilac etc . Ex: Inter specific hybridization for transferring yellow colour in Dianthus plumarius – Garden pink (2n=6x=90) Gatt et al . (1998) Observation and result × = x = Dianthus plumarius Yellow carnation D. knappii Dianthus plumarius

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Indian Agricultural Research Institute, New Delhi Outlines of experiment: To transfer yellow flower colour from yellow-flowered carnations (2n.2x.30) and D. knappii (2n.2x.30) to a white-flowered cultivar of the garden pink, D. plumarius (2n.6x.90) D. plumarius × D. knappii - F1 pale cream-yellow D. plumarius × Carnation - F1 Pink Analysis of the flower pigments Yellow colour of D. knappii - Flavone and flavonol . Yellow carnations - chalcones The F1 hybrids with D. knappii were yellow because they contained the same pigments as D. knappii but the F1 hybrids with the carnations were pink due to their ability to convert chalcones through to dihydroflavones and then to anthocyanins

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Indian Agricultural Research Institute, New Delhi Mutation: 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: Chrysanthemum, Bougainvillea, Rose etc. Datta et al. , 2001 Phenotypic expression in flower after mutation

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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 Indian Agricultural Research Institute, New Delhi

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Ajay Prasad et al., 2008 Pusa Anmol A success story at IARI …… M1 M2 In vitro isolation, purification, rapid bulking and field establishment of a radio-mutant Indian Agricultural Research Institute, New Delhi Chrysanthemum variety from IARI through Mutation induced by Gamma rays Pusa Anmol (Ajay) 2. Pusa Centenary (Thai Chen Queen) 3. Pusa Kesari (Thai Chen Queen) 4. Pusa Arunodaya (Thai Chen Queen)

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Indian Agricultural Research Institute, New Delhi Genetic Engineering of flower colour : Genetic engineering focused on metabolic engineering of the flavonoid biosynthesis pathway. Reason being flavonoids are major contributors to flower colour Different approaches are : Over-expressing or silencing the structural gene expression in flavonoid biosynthetic pathway Effects of regulatory genes on flower colour modification Generation of variegated flowers by using transposons Other factors affecting flower colouration Co-pigments Vacuolar pH Cell shape

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Indian Agricultural Research Institute, New Delhi BIOSYNTHESIS OF FLAVONOIDS: Flavonoids are the most common pigment in flower tissue Major classes of flavonoids contributing colours: anthocyanins , flavonols , chalcones and aurones . Anthocyanins - largest class Three common anthocyanins are pelargonidin -, cyanidin -, and delphinidin -based pigments Flavonoids are derived from phenyl- propanoid pathway with precursor amino acid phenyl- alanine Phenyl- alanine

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Indian Agricultural Research Institute, New Delhi Fig: Flavonoid Bio-Synthetic Pathway Dihydroflavonols Leucoanthocynidin Anthocynidins Anthocyanins

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Indian Agricultural Research Institute, New Delhi 1.Over-expressing or silencing the structural gene expression in flavonoid biosynthetic pathway. : 1.1 Chalcone synthase Chalcone synthase (CHS) catalyze 3 molecules of malonyl-CoA and 1 molecule of coumaroyl - CoA into 1 molecule of chalcone Over-expression of sense or antisense chs constructs: To modify flower colour in Petunia, Torenia , chrysanthemum, lisianthus etc. Generating white flowers by Genetic modification, can be accomplished by transforming chimerical chs construct into plants

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Indian Agricultural Research Institute, New Delhi

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Indian Agricultural Research Institute, New Delhi 1.2. 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 Yellow flowers - chi mutants of aster and carnation ( Schijlen et al. 2004)

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Indian Agricultural Research Institute, New Delhi 1.3 . 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) In Petunia and Antirrhinum - Mutation in f3h locus caused a loss of F3H activity - white flowers ( Schijlen et al. 2004) Petunia, torenia , carnation etc. with novel flower colours - transgenic approach Dihydroflavonols

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Indian Agricultural Research Institute, New Delhi 1.4. Dihydroflavonol-4-reductase (DFR) The enzyme DFR catalyzes the reduction of dihydroflavonols to leucoanthocyanidins 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) Leucoanthocynidin Dihydroflavonols

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Indian Agricultural Research Institute, New Delhi 1.5. Anthocyanidin synthase ANS catalyzes leucoanthocyanidins into anthocyanidin Application of transgenic ans to pigment modification is less reported Leucoanthocynidin Anthocynidins

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Indian Agricultural Research Institute, New Delhi 1.6. 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 Overexpression of snapdragon 3GT cDNA in lisianthus - novel anthocyanins . Anthocynidins

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Indian Agricultural Research Institute, New Delhi 1.7. Other enzymes In some sps . like snapdragon, cosmos and dahlia, chalcone - aurones (yellow colour) by aureusidin synthase (AS) Chalcone reductase (CHR) co-acts with chalcone synthase (CHS), catalyzing 1X coumaroyl-CoA and 3X malonyl-CoA to produce iso-liquiritigenin (yellow in colour) – a precursor of 5-deoxy-isoflavonoids 1.8. Transformation with multiple genes Petunia & torenia carrying F3′5′H and DFR genes altered flower colour of interest

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Indian Agricultural Research Institute, New Delhi 2. 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 Studied in Maize, snapdragon, Petunia, Arabidopsis etc. Ex: maize leaf colour ( Lc ) gene - bHLH , maize colourless (C1) gene – MYB By transforming maize Lc gene into Petunia -- vegetative and floral tissues in transgenic plants had increased pigmentation (Bradley et al. 1998)

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Indian Agricultural Research Institute, New Delhi 3. 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 transpogon results in white sectors of a coloured background, and excision of such a transposon results in coloured sectors on a white background The sizes of sectors : Depend on the timing of insertion and excision Morning glory and Petunia etc.

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Indian Agricultural Research Institute, New Delhi 4. Other factors affecting flower colouration : 4.1 Co-pigments Flavonols and flavones Copigments & anthocyanins complex stabilizes and determine the colour The enzyme flavonol synthase (FLS) [ Also flavone synthase (FNS)] converts dihydroflavonols into flavonols Gene coding for enzyme is cloned from Petroselinum , Chrysanthenum , Dahlia and Gerbera Flavonols and flavones share common precursors with anthocyanins , so their down regulation often reduces anthocyanin level.

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Indian Agricultural Research Institute, New Delhi 4.2. Vacuolar pH pH of vacuole : Acidic : stabilize anthocyanins Generally, in pH - reddening, and in pH - blueing effect In Petunia, loci (ph1 to ph7) identified. Mutated- blueing of the flower. Morning glory ( Ipomea tricolor ) Strong reddish purple buds change to light blue open flowers - 4 hrs- by the PURPLE (PR) protein transports Na + into and H + out of the vacuole, resulting in the increased vacuolar pH (6.5 to 7.5)

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Indian Agricultural Research Institute, New Delhi 4.3 Cell shape 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

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Indian Agricultural Research Institute, New Delhi Tinting The process of artificial colouring Practiced in white or shades of white flower Common flower: Carnation, Orchid, Rose, Chrysanthemum etc. Common dyes: Bromocresol red/green, Eosin yellow, ammonium purpurate , phenol red and other food colourant (Conc. 0.1-0.2%)

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Indian Agricultural Research Institute, New Delhi Gentian: Gentiana triflora , Blue flowers Case study-1

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Indian Agricultural Research Institute, New Delhi RNA interference ( RNAi ) technique: Efficient tool for gene silencing. Most popular methods to down-regulate gene expression in higher plants. Higher frequency and phenotypic stability. RNAi technology: Targeted for suppression of three anthocyanin biosynthetic genes; chalcone synthase ( CHS), anthocyanidin synthase (ANS) and flavonoid 3’,5’-hydroxylase ( F3’5’H) in Gentia .

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Indian Agricultural Research Institute, New Delhi Vector construction gentian transformation: 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

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Indian Agricultural Research Institute, New Delhi Expression level analysis: RNA gel blot tech Pigment analysis: HPLC 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) rolCpro-CHSir C) rolCpro-CHSir D) rolCpro-ANSir F) rolCpro-F35Hir

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Indian Agricultural Research Institute, New Delhi Case study-2 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 Co-pigments and the vacuolar pH (3.69 to 5.78).

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Indian Agricultural Research Institute, New Delhi Steps: The over-expression of a viola F3’5’H – efficient accumulation of delphinidin and color changes to blue hue Down-regulation of the rose DFR gene and over-expression of the iris DFR gene by RNAi technique Efficient and exclusive delphinidin production and a bluer flower colour Lavande , and transgenic blue colour roses

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Indian Agricultural Research Institute, New Delhi In April 2005, Suntory Ltd. and Florigene Ltd . announced the production of a blue rose. The blue rose – developed using CSIRO’s hairpin RNAi Transgenic rose variety ‘‘ Applause ’’ was commercially released in Japan (2009)

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Indian Agricultural Research Institute, New Delhi Steps: Turn off the production of red pigment; 2. Open the ‘door’ to production of blue pigment; and then 3. Produce blue pigment.

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Indian Agricultural Research Institute, New Delhi Conclusions: 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 Metabolic cross-stock between different pathway makes the production of transgenic challenging Spatial and temporal expression of colour is need to be understand

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Indian Agricultural Research Institute, New Delhi Future thrust: Function, expression, regulation and interaction of the structural genes and regulatory genes Transport mechanism of pigments Species-specific genes in flavonoid biosynthetic pathway Changing flower pigmentation by modification of carotenoids and betalain biosynthetic pathway

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