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“GENETIC ENGINEERING FOR FLOWER
COLOUR MODIFICATION.”
PREPARED BY :
AVINASH GOWDA H
M.Sc.(Agri) (Plant Mol. Biol. & Biotec...
Introduction
Biotechnology In Floriculture
Flower and flower colour
Role of colour
Major plant pigments
Genetic impr...
• Floriculture is considered to include the cut flowers, potted
plants, and ornamental bedding plants and garden plant
ind...
 About 255 thousand hectares area is under cultivation, and
the production of flowers are estimated to be 17.54 million
t...
The global flower industry thrives on novelty.
Genetic engineering is providing a valuable means of
expanding the floricul...
Flower
Reproductive structure of a seed-bearing plant
Flower colour
Flower color is one of the most attractive
characteri...
ROLE OF COLOUR
Attraction of pollinators
Function in photosynthesis
In human health as antioxidants and precursors of
vita...
Why we need Modification in colour ?
Modification in flower colour of a variety with desirable
agronomic or consumer chara...
Chlorophylls and
carotenoids are in
chloroplast and
chromoplast
Flavonoids are
in the vacuole
9
9
Pigment
Class
Compound Types Compound Examples Typical Colours
Porphyrins Chlorophyll Chlorophyll a and b Green
Flavonoids...
Genes involved in pigment synthesis
1.structural (enzyme) genes
2.regulatory genes
Enzyme Gene Species
CHS Chs Antirrhinum...
Regulatory genes: Influence the type, intensity and pattern of flavonoid
accumulation but do not encode flavonoid enzyme.
...
Regulatory region Coding region
Protein (Enzyme)
Pigment
Genes contain regulatory
region and coding region
Springob et al....
Effects of regulatory genes on flower colour
modification
A complex of two transcriptional factor MYB and basic-
Helix-Lo...
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...
16
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Genetic Improvement of Flower Colour
 Genetic Improvement: involves changing the plant’s
genetic makeup
Making deliberat...
Conventional breeding
Hybridization:
=x
Traditional doner
Desired
gene
Commercial variety New variety
Many genes are
trans...
Inter-specific Hybridization
Studies on inter specific hybridization for transferring yellow
colour in Dianthus plumarius ...
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...
Ornamental plants are ideal
First officially released commercial mutant cultivars : Tulip (cv. ‘Faraday‘
from cv. ‘Fantasy...
Crop Cultivar Mutagen Parent Earlier colour Changed colour
Chrysanthemum
1. Agnisikha Gamma rays D-5 Magnolia purple Eryth...
Polyploidy
Natural origin or colchiploidy
Polyploidy can be obtained by colchicine treatment
24
24
Ex; The effect of induc...
Conventional Breeding
many gene and limited by genetic incompability
Plant biotechnology
single gene with no specific to p...
Transgenic
Technology
Resistant To
Biotic Stresses
26
26
Gene transfer methods
Indirect Direct
Most widely used
More economical
More efficient
Transformation success is 80-85%...
Gene transformation
28
28
Colour modification done by:
Over expression of structural genes
Inhibition of key biosynthetic enzyme
Addition of an enzy...
1. Chalcone synthase
Chalcone synthase (CHS) catalyze 3 molecules of malonyl-CoA
and 1 molecule of coumaroyl- CoA into 1 m...
2. Chalcone isomerase
 Chalcone isomerase (CHI) catalyzes yellow coloured chalcone
to colourless pigment naringenin. Can ...
3. Flavanone hydroxylase/ Flavonoid-3′hydroxylase/
Flavonoid-3′,5′-hydroxylase
 The hydroxylation in position 3 of the C ...
4. Dihydroflavonol-4-reductase (DFR)
The enzyme DFR catalyzes the reduction of
dihydroflavonols to leucoanthocyanidins.
E...
5. Anthocyanidin synthase
 ANS catalyzes leucoanthocyanidins into anthocyanidin
Dehydroxylation
 Application of transge...
6. Flavonoid 3-O-glucosyltransferase (3GT)
 3GT transfers the glucose moiety from UDP-glucose
to C-3 hydroxyl group of th...
7. Other enzymes
 In some sps. like snapdragon, cosmos and dahlia, chalcone -
aurones (yellow colour) produced by aureusi...
37
8. Transformation with multiple genes
Petunia & torenia carrying F3′5′H and DFR genes altered
flower colour of interest...
Colour modification through antisense RNA
technology
 Antisense RNA is a single stranded RNA that is
complementary to mRN...
Inhibition of gene expression by antisense RNA
39
39
Colour modification through RNAi mediated
gene silencing
40
“The proces by which the dsRNA silence gene
expression.”
Degra...
Difference between antisense technology
and RNAi
 The intended effect in both will same i.e., gene
silencing but the proc...
42
Ds RNA are chopped in to short
interfering RNA s (siRNA) by Dicer.
The siRNA –Dicer complex is recruits
to form an RN...
Land marks in RNAi discovery
 RNAi was firstly discovered and observe in transcriptional
inhibition by antisense RNA expr...
 This phenomenon was called co-suppression of gene
expression , since both the expression of the existing gene (the
initi...
Generation of variegated flowers by using
transposons
 Insertion or excision of transposons in flavonoid biosynthetic
or ...
Other factors affecting flower colouration
1 . Co-pigments
 Flavonols and flavones
 Copigments & anthocyanins complex st...
2. Vacuolar pH
 pH of vacuole : Acidic : stabilize anthocyanins
 Generally, in pH - reddening, and in pH - blueing effec...
3. Cell shape
 Accumulation of anthocyanin pigments is also affected by the
shape of the cells.
 In Snapdragon, if cells...
49
49
Targeted for suppression of three anthocyanin biosynthetic genes; chalcone
synthase (CHS), anthocyanidin synthase (ANS) an...
Result:
Suppressed CHS gene - Selected 20 line – 17 changed colour – 14
pure white & 3 pale-blue color
Suppressed ANS gene...
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 flav...
Steps:
 Down-regulation of the rose DFR gene and over-expression of the iris DFR gene
by RNAi technique
 The over-expres...
Steps:
1. Turn off the production of red pigment;
2. Open the ‘door’ to production of blue pigment; and then
3. Produce bl...
Violet/Blue Chrysanthemums
 Flavonoid analysis and precursor feeding experiments
 A selection of eight cultivars were su...
Aselectionofchrysanthemumcultivarshighlightingthosedeemedsuitablefortransformationto
achievebluecoloration 5757
Inflorescence color changes with the production of delphinidin-based anthocyanins
5858
Inflorescence color changes with the production of delphinidin-based anthocyanins
59
59
Redirection of flavonoid biosynthesis in
petunia
 Mitchell has white flowers due to the absence of
anthocyanin biosynthes...
61
Plant line Flvonols
(µmol/ g dw)
Chalcones
(µmol/ g dw)
Mitchel 132 0
CHR-MP 72 81
Introduction of the CHR cDNA into Mi...
62
Introduction of the CHR transgene into cyanic-flowered
Petunia lines
Plant line Chalcones (%) Flvonols (%) Anthocyanin ...
Flower colour alteration in lotus japonicus by modification
of the carotenoid pathway
 Colour modification is done by ove...
64
TLC analysis of wild type and transgenic type
64
65
CAROTENOID
CAROTENOID CONTENT (%)
WILD TYPE TRANSGENIC
Neoxanthin 12.6 4.5
Violoxanthin 27.5 66.8
Antheraxanthin 19.8 1...
Flower color modification of Petunia hybrida commercial
varieties by metabolic engineering
Flower colour changed from purp...
 Flowers of transgenic Surfinia Purple Mini plant harboring antisense DFR gene
 Expression of DFR gene change the expres...
Transgenic flowers harboring the sense
Hf1 F35H, AR–AT, and FLS genes
Suppression of the F3H gene by antisense and
express...
Flower colour modifications by regulating
flavonoid biosynthesis
69
69
Conclusion:
 Flower colour modification using molecular methods has now
become reality
 Flower colour is mainly determin...
Future thrust:
Species-specific genes in flavonoid biosynthetic pathway
Changing flower pigmentation by modification of ca...
Genetic engineering for flower colour modification
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Genetic engineering for flower colour modification

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Genetic engineering for flower colour modification

  1. 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. 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. 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. 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. 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. 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. 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. 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. 9. Chlorophylls and carotenoids are in chloroplast and chromoplast Flavonoids are in the vacuole 9 9
  10. 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. 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. 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. 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. 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. 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. 16 B i o s y n t h e ti c p a t h w a y o f fl a v o n o i d 16
  17. 17. 17 B i o s y n t h e ti c p a t h w a y o f c a r o t e n o i d s 17
  18. 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. 19. Conventional breeding Hybridization: =x Traditional doner Desired gene Commercial variety New variety Many genes are transferred Co dominance 19 19
  20. 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. 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. 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. 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. 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. 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. 26. Transgenic Technology Resistant To Biotic Stresses 26 26
  27. 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. 28. Gene transformation 28 28
  29. 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. 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. 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. 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. 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. 34. 5. Anthocyanidin synthase  ANS catalyzes leucoanthocyanidins into anthocyanidin Dehydroxylation  Application of transgenic ans to pigment modification is less reported 34 34
  35. 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. 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. 37 8. Transformation with multiple genes Petunia & torenia carrying F3′5′H and DFR genes altered flower colour of interest 37
  38. 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. 39. Inhibition of gene expression by antisense RNA 39 39
  40. 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. 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. 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. 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. 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. 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. 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. 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. 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 49
  50. 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. 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. 52 Anthocyanidin composition in the petals of transgenic gentian was measured by HPLC analysis. 52
  53. 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. 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. 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. 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. 57. Aselectionofchrysanthemumcultivarshighlightingthosedeemedsuitablefortransformationto achievebluecoloration 5757
  58. 58. Inflorescence color changes with the production of delphinidin-based anthocyanins 5858
  59. 59. Inflorescence color changes with the production of delphinidin-based anthocyanins 59 59
  60. 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. 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. 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. 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. 64 TLC analysis of wild type and transgenic type 64
  65. 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. 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. 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. 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. 69. Flower colour modifications by regulating flavonoid biosynthesis 69 69
  70. 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. 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

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