breeding for pigments
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breeding for colour in ornamentalsTRANSCRIPT
Recent Advances in Genetic Improvement of Flower Colour
Speaker: Arvind Kumar Verma Ph.D. Scholar, IARI, New Delhi
Chairperson: Dr. S.S. Sindhu (Principal Scientist)
Colour and colour patternsFlower colour: most important trait, dictating consumer attraction
Role of colour:
Attraction of pollinators
Function in photosynthesis
In human health as antioxidants and precursors of vitamin A
Protecting tissue against photooxidative damage
Resistant to biotic and abiotic stress
Symbiotic plant-microbe interaction
Act as intermediary for other compounds
Colour pattern: Differential accumulation of pigment(s)
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, HesperidinNaringin, Naringenin
Colour less co-pigments
Flavans Biflavan, Catechin, Epicatechin,
Colour less 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
Chlorophyllsandcarotenoidsare inchloroplasts
Anthocyaninsare in thevacuole
Site of colour accumulation
Grotewold, 2006
LDOX/ANS= leucoanthocyanidin dioxygenase/anthocyanidin synthase.
Carotenoid biosynthesis pathway
GA3P: glyceraldehyde-3-phosphate, DXR: 1-deoxy-D-xylulose 5-phosphate reductoisomerase.DXS: 1-deoxy-D-xylulose 5-phosphate synthase. MEP: 2-C-methyl-D-erythritol 4-phosphate. IPP: isopentenyl diphosphate. IPI: isopentenyl diphosphate isomerase. GGPP: geranylgeranyl diphosphate. PSY: phytoene synthase(P), CrtB: phytoene synthase (B). PDS: phytoene desaturase (P). ZDS: γ-carotene desaturase (P). ZISO: γ-carotene isomerase (P). CrtISO: carotene isomerase (P). CrtI: phytoene desaturase/isomerase (P). LCY-b: β-cyclase (P). LCY-e: α-cyclase (P). BETA: chromoplast-specific beta-cyclase (P). CYP97B, CYP97C: cytochrome P450 carotene hydroxylases (P). CHY1, CHY2: non-heme carotene hydroxylases (P). CrtO: ketolase (algal/cyanobacterial). CrtW: ketolase (B). ZEP: zeaxanthin epoxidase (P).VDE: violaxanthin de-epoxidase (P). CaCCS: capsanthin/capsorubin synthase (P).
Grotewold, 2006
Factors on flower colour perceptionpH of the vacuole:pH of the vacuole is acidic
Small changes of pH have visible effects on flower colour
Some plants genes have been identified influencing pH
Metal ions:Metal complexing has a blueing effect on flower colour
Colour change from red to blue in hydrangea sepals is most likely due to complexing of Dp 3-glucoside with
aluminium ion and 3-caffeoylquinic acid
Co-pigmentation:Co-pigmentation of anthocyanins with flavones, flavonols and other compounds
Primula, where the contribution of flavonols to flower colour is genetically controlled (gene B). Co-
pigmented flowers (BB) give a mauve colour, whereas in the absence of flavonols (bb) maroon flowers are
formed
Co-occurrence of anthocyanins and yellow pigmentsMixtures of Ans and yellow flavonoids were found in the orange-yellow or orange-red flowers of antirrhinum and bronze flower colour of helichrysum
Tanaka et al., 2009
Regulatory region Coding region
Where? When?How Much?
Protein (Enzyme)
Red Pigment
Genes containregulatory region and
coding region
Springob et al., 2003
Genes involved in pigment synthesis1.structural (enzyme) genes2.regulatory genes
Enzyme Enzyme GeneGene Species Species
CHSCHS ChsChs Antirrhinum, Chrysanthemum, Orchid, Rosa, DianthusAntirrhinum, Chrysanthemum, Orchid, Rosa, Dianthus
CHICHI ChiChi Antirrhinum, Petunia, Eustoma, DianthusAntirrhinum, Petunia, Eustoma, Dianthus
F3HF3H F3hF3h Antirrhinum, Calistephus, Chrysanthemum, Dianthus, OrchidAntirrhinum, Calistephus, Chrysanthemum, Dianthus, Orchid
F3’HF3’H F3’hF3’h Antirrhinum, Dianthus, PetuniaAntirrhinum, Dianthus, Petunia
F3’5’HF3’5’H F3’5’hF3’5’h Calistephus, Eustoma, PetuniaCalistephus, Eustoma, Petunia
FLSFLS FlsFls Petunia, RosaPetunia, Rosa
FNSFNS FnsIIFnsII Antirrhinum, GerberaAntirrhinum, Gerbera
DFRDFR DfrDfr Antirrhinum, Calistephus, Gerbera, Orchid, Dianthus, PetuniaAntirrhinum, Calistephus, Gerbera, Orchid, Dianthus, Petunia
ANSANS AnsAns Antirrhinum, Calistephus, PetuniaAntirrhinum, Calistephus, Petunia
GTGT 3Gt3Gt Antirrhinum, GentianaAntirrhinum, Gentiana
GTSGTS Gts Gts PetuniaPetunia
1.structural (enzyme) genes: is a gene that codes for any RNA or protein product other than a regulatory protein.
Vainstein, 2004
Two classes of regulatory genes identifiedTF with MYB domainTF with MYC/bHLH motif
Infuence the type, intensity and pattern of flavonoid accumulation but donot encode flavonoid enzyme
Regulatory genes
PlantPlant GeneGene
Myb MycMyb Myc
Petunia Rosea,Mixta DeliaPetunia Rosea,Mixta Delia
Gerbera Gmyc IGerbera Gmyc I
Perilla Mybp IPerilla Mybp I
Petunia Phmyb3, An2, An4 An1Petunia Phmyb3, An2, An4 An1
(Vainstein, 2004)
GeneGene Enzyme Enzyme DxsDxs
DxrDxr
LpiLpi
GpsGps
FpsFps
GgpsGgps
PsyPsy
ZdsZds
Lcy-bLcy-b
Lcy-cLcy-c
NsyNsy
CcsCcs
Ptox Ptox
Dexoyxylulose 5-phosphate synthaseDexoyxylulose 5-phosphate synthase
Dexoyxylulose 5-phosphate reducoisomeraseDexoyxylulose 5-phosphate reducoisomerase
LytB proteinLytB protein
Geranyl diphosphate synthaseGeranyl diphosphate synthase
Fernsyl diphosphate synthaseFernsyl diphosphate synthase
Geranylgeranyl diphosphate synthaseGeranylgeranyl diphosphate synthase
Phytoene synthasePhytoene synthase
ββ-Carotene dessaturase-Carotene dessaturase
Lycopene bita-cyclaseLycopene bita-cyclase
Lycopene – Lycopene – ββcyclasecyclase
Neoxanthin synthaseNeoxanthin synthase
Capsanthin capsorubin synthaseCapsanthin capsorubin synthase
Plastid terminal oxysidasePlastid terminal oxysidase
Genes involved in carotene pigment synthesis
(Vainstein, 2004)
Genetic Improvement: involves changing the plant’s genetic makeup
Making deliberate crosses between two parents
Mutation
Polyploidy
Introducing genes of desired traits into recipient plant by
methods other than sexual crosses
Genetic Improvement of Flower Colour
Conventional breeding
Hybridization:=x
Traditional doner
Desired gene
Commercial variety New variety
Many genes are transferred
Dominance Co dominance
CASE STUDYStudies on inter specific hybridization for transferring yellow colour in Dianthus plumarius (2n=6x=90).
Gatt et al. (2005) Observation and result x =
Analysis of the flower pigments :Yellow flower colour of D. knappii resulted from flavone and flavonol glycosides.
Yellow carnations were chalcones.
Thus, the F1 hybrids with D. knappii were yellow because they contained the same pigments as D. knappii
but the hybrids with the carnations were pink due to their ability to convert chalcones through
dihydroflavones and then to anthocyanins.
Dianthus plumarius Yellow carnation
x =
D. knappiiDianthus plumarius
Many different genes are involved incontrolling 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:
a c
ed
b Two types of flower in a single branch
Datta et al., 2001
CASE STUDY
A quick method for establishment of solid mutant
Phenotypic expression in flower after mutationPhenotypic expression in flower after mutation
Ajay
Prasad et at., 2008Pusa AnmolA success story at IARI…………………
M1
M2
In vitro isolation, purification, rapid bulking and field establishment of a radio-mutant
Polyploidy
Auto polyploidy Allo polyploidy
Natural origin or colchiploidy
Mitotic doubling of chromosome number- colchiploidyFailure of meiotic reduction i.e. unreduced gamete- natural
CASE STUDYThe effect of induced polyploidy on the flavonols of Petunia ‘Mitchell' Griesbach and Kamo, 1996
increasing the relative concentration of the major metabolite quercetin-3-sophoroside and decreasing the relativeconcentration of the minor metabolite quercetin-3,7-diglucoside.
Analytical HPLC data for the flavonols of haploid, diploid and tetraploid cytotypes of Petunia
Polyploidy was inducedthrough in vitro colchicine treatment
Q3 = quercetin-3-glucoside, Q32 = quercetin-3-sophoroside, Q7 = quercetin-7-glucoside, Q3,7 = quercetin-3,7-diglucoside, 32,7 = quercetin-3-sophoroside-7-glucoside, Qc32,7 - quercetin-3-caffeoylsophoroside-7-glucoside.
Colour modification Over expression of structural genes Use of sense or antisense enzyme construct Inhibit production of key biosynthetic enzyme Add an enzyme of a particular biosynthetic step
Why transgenic crops are important ?
Limitations of conventional breeding for attaining the desirable traits
Development of organisms that express a “novel” trait: normally not
found in the species
Genetic engineering: Manipulation of plant genome through recombinant DNA technology to alter plant characteristics
Chandler and Brugliera, 2011
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
Gene transformation
Colour modification through antisense RNA technology
Inhibition of gene expression by antisense RNA
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 gene
Surfinia Purple Mini
CASE STUDY
Tsuda et al., 2004
Surfinia Pure White
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
Contd…..
C
Generating White colour
Down regulation of an anthocyanin biosynthesis structural gene (Using an antisense gentian CHS gene)
Petunia (Van der Krol et al., 1988), Gerbera (Elomaa et al., 1993), Chrysanthemum (Courtney-Gutterson et al., 1994), Rose (Gutterson, 1995), Carnation (Gutterson, 1995), Lisianthus (Deroles et al., 1998;Kato et al., 2001) Torenia (Aida et al., 2000a;)More recently, gentian (Nishihara et al. 2003)
Generating red to orange flowersIntroduction maize Dfr gene Petunia (Meyer et al.,1987)
Isolated the blue gene in 1991 and patented in 1992.
Petunia gene didn’t work in roses.
Transplanting genes is easy to say but hard to do in the lab, so they used their techniques on carnations- a much easier species to manipulate than roses.
In 1996, Florigene developed mauve-coloured carnation, FLORIGENE Moondust and it was the world's first genetically modified flower on sale
In 1997, developed second genetically-modified carnation, FLORIGENE Moonshadow with a richer and true purple colour.
Successfully developed a range of transgenic violet carnations by introduction of a F3′5′H gene together with a petunia DFR gene into a DFR-deficient white carnation
Blue carnation
Fukui et al., 2003
BLUE-VIOLET CARNATION Introduction of a F3’ 5’H gene together with a petunia DFR gene into DFR
deficient white carnation
FLORIGENE MOONSERIES
FLORIGENE Moonshadow
FLORIGENE Moonvista
FLORIGENE Moonlite
FLORIGENE Moonshade
FLORIGENE Moondust
FLORIGENE Moonaqua
FLORIGENE Moonique
FLORIGENE Moonpearl
FLORIGENE Moonvelvet
FLORIGENE Moonberry
Blue rose
Why a natural rose could not have the true blue colour?
"Flavonoid 3', 5'- hydroxylase" is one of the key enzymes involved in the flavonoid
biosynthesis for blue colour development deficient in rose
Did’t have Dp
pH of cell sap is 4.0-4.5
Cell sap is govern by 7 genes and each gene contributes 0.5 pH
Blue Gene Technology
In April of 2005, Suntory Ltd. and Florigen Ltd. announced the production of a blue
rose by introducing three transformation constructs simultaneously into roses:
www.suntory.com and www.florigene.com.au
Blue Gene Technology
www.suntory.com and www.florigene.com.au
The transgenic rose variety ‘‘Applause’’ was commercially released in Japan in 2009 (Tanaka et al., 2009)
Suntory selected APPLAUSE as the name for its blue rose as a symbol of congratulations for those whose dreams have come true, as well as of encouragement for those pursuing a dream, whatever it may be.
The price for a single blue rose is about $22 to $33. Source: The Japan Today
Flower colour modifications by regulating flavonoid biosynthesis
Conclusion:Classical breeding methods have been extensively used to develop cultivars with
flowers varying in both the colour and its intensity
Spectral difference in flower colour is mainly determined by the ratio of different classes of pigments and other factors such as vacuolar pH, co-pigmentation and metal ion complexation
Knowledge of flower colouration at the biochemical and molecular level has made it possible to developed novel colour
Genetic engineering overcome almost all the limitations of traditional breeding approaches
The expression of genes transferred across genera is not always predictable and so requires considerable trial to arrive at stable phenotype of commercial interest