GENETIC ENGINEERING IN FLORICULTURE

Speaker : Zaman Mariya S. Course No. : MBB 591 Major Sub. : Plant BiotechnologyReg. No. : 04-1273-2010

ANAND AGRICULTURAL UNIVERSITY

Major Guide : Dr. G. C. Jadeja Minor Guide : Dr. J. G. Talati Date :15 /09/2012Time : 14:00 Hrs

wel come

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The Floriculture Industry……….Biotechnology In FloricultureCurrent Status Of Genetic Modification In FloricultureTransformation

Color ModificationVase life Flower Scent Modified Plant Structure and Architecture

Disease Resistance Case studiesConclusionFuture Prospects

CONTENTS

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THE FLORICULTURE INDUSTRY

• Floriculture is considered to include the cut flowers, potted plants, and ornamental bedding plants and garden plant industries (Chandler, 2003).

• The worldwide production value of flowers and floricultural plants is approx. 50 billion EURO.

• From the per capita consumption data provided by the Dutch flower council (Flower Council of Holland, 2007), it can be extrapolated that the global consumption value at consumer level is somewhere between 100 to 150 billion EURO.

• The main areas of production and consumption of floricultural products are in the United States and Europe, with a significant industry in Japan. The area of production in China is also increasing rapidly.

• The highest consumption per head is in the Netherlands, Luxembourg, Germany, Austria, and France.

Orchids

Arachnis

Aranda

Aranthera

Cattleya

Cymbidium

Dendrobium

Lycaste

Paphiodelphium

Miltonia

Odontoglossum

ChrysanthemumGerberaAntirrhinum RoseCarnation 

Cut flowers

Bulbs and cormsGladiolusTulipsLiliesTuberoseAmaryllisIris 

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World Flower Production and Consumption

Production Consumption

ChinaKenya

Columbia

JapanEurope

USA

$40 billion(Getu, 2009)

Market grow20% annually

Japan

EU

USA

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 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. At present, only consumer traits appear to provide a return capable of supporting what is still a relatively expensive molecular breeding tool.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

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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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Genetic modification can be used to transfer new

specific traits into the plantConventional 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.

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

Chandler and Brugliera, 20119

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Gene transformation

Bacterium mixed with plant cells

GENE identified and

isolated

Gene inserted into Ti plasmid

Gene replication

Gold particles coated with

DNA

Cells shot with gene gun and DNA incorporated into plant cell

chromosome

Ti plasmid moves into plant cell and

inserts DNA into plant chromosome

Cells screened for transgene

Transformed cells selected with

selectable marker

Transgenic plant regenerated from single

transformed cell

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Color and color patternsFlower color: most important trait, dictating

consumer attractionRole of color:• 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 compoundsColor pattern: Differential accumulation of

pigment(s)

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Pigments Compound Types Compound Examples Typical Colors

Porphyrins Chlorophyll Chlorophyll a & b Green Flavonoids Anthocyanins Pelargonidin, Cyanidin, Delphinidin,

PeonidinRed, Blue, Violet

Anthoxanthins Flavonols Kaempferol, Quercetin, Fisetin, Kaempferide, Morin, Myricetin,

Myricitrin, Rutin

Yellow

Flavones Apigenin, Biacalein, Chrysin, Diosmetin, Flavone, Luteolin

Yellow

Isoflavones Diadzin, Genistein, Enterodiol, Coumestrol, Biochanin

Flavonones Eriodictyol, Hesperidin, Naringin, Naringenin

Colorless Co-pigments

Flavans Biflavan, Catechin, Epicatechin Colorless Co-pigments

Carotenoids Carotenes Lycopene, α-carotene, β-carotene, γ-carotene

Yellow, Orange, Red

Xanthophylls Lutein, Cryptoxanthin, Zeaxanthin, Neoxanthin, Rhodoxanthin, Violaxanthin,

Canthaxanthin, Astaxanthin

Betalains Betacyanins Reddish To Violet

Betaxanthins Miraxanthin & PortulaxanthinYello

w To Orange

Major Pigments in Plants

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Site of colour accumulation

Chlorophylls and carotenoids are in

chloroplasts

Anthocyanins are in the vacuole

Factors on flower color perceptionpH of the vacuole:

pH of the vacuole is acidicSmall changes of pH have visible effects on flower color

Metal ions:Metal complexing has a blueing effect on flower color

Co-pigmentation:Co-pigmentation of anthocyanins with the colourless flavonols and flavones is an important factor influencing pigmentation.Co-pigmented flowers give a mauve colour, whereas in the absence of flavonols maroon flowers are formed.

Co-occurrence of anthocyanins and yellow pigments

Mixtures 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

low pH to high pH

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Protein (Enzyme) Red Pigment

Springob et al., 2003

Genes contain regulatory region and coding region

Regulatory region Coding region

The genes consist of DNA which is made up of four chemical letters

The chromosome is made up of genes

Chromosomes-23 pairs

A cell

Genes involved in pigment synthesis

Structural (enzyme) gene: Gene that codes for any RNA or protein product other than a regulatory protein. Enzyme Gen

eSpecies

CHS (Chalcone synthase) Chs Antirrhinum, Chrysanthemum, Orchid, Rosa, Dianthus

CHI (Chalcone-flavanone isomerase)

Chi Antirrhinum, Petunia, Eustoma, Dianthus

F3H (Flavone 3-hydroxylase) F3h Antirrhinum, Calistephus, Chrysanthemum, Dianthus, Orchid

F3’H (Flavonoid 3’ hydroxlase)

F3’h Antirrhinum, Dianthus, Petunia

F3’5’H (Flavonoid 3’,5’-hydroxlase)

F3’5’h

Calistephus, Eustoma, Petunia

FLS (Flavonol synthase) Fls Petunia, RosaFNS (Flavone synthase) FnsII Antirrhinum, GerberaDFR (Dihydroflavonol-4-reductase)

Dfr Antirrhinum, Calistephus, Gerbera, Orchid, Dianthus, Petunia

ANS (Anthocyanidin synthase)

Ans Antirrhinum, Calistephus, Petunia

GT (Glycosyltransferase) 3Gt Antirrhinum, Gentiana

Vainstein, 2004

Regulatory GenesStructural (Enzyme) Genes

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 Phmyb3, An2,

An4An1

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Genes involved in carotene pigment synthesis

(Vainstein, 2004)

Gene Enzyme Dxs Deoxyxylulose 5- phosphate

synthaseDxr Deoxyxylulose 5- phosphate

reducoisomeraseLpi LytB proteinGps Geranyl diphosphate synthaseFps Fernsyl diphosphate synthase

Ggps Geranyl geranyl diphosphate synthase

Psy Phytotene synthase Zds β-Carotene dessaturase

Lcy-b Lycopene β-cyclaseLcy-c Lycopene β-cyclaseNsy Neoxanthin synthaseCcs Capsanthin capsorubin synthasePtox Plastid terminal oxysidase

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Biosynthetic pathway

of

flavonoid

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Colour modification done by:

Over expression of structural genes

Use of sense or antisense enzyme construct

Inhibition of key biosynthetic enzyme

Addition of an enzyme in a particular biosynthetic step

FLORIGENE scientists  Isolated the blue gene in 1991 and patented in 1992.

Petunia gene  didn’t work in roses, so FLORIGENE scientists 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

moonique moonpearl moonvelvetmoonberry

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• 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.

Natural rose did not have delphinidin. pH of cell sap is 4.0-4.5 (acidic). Cell sap is govern by 7 genes and each gene

contributes 0.5 pH

Genetic engineering of blue Rose

Anthocyanidin

The price for a single blue rose is about $22 to $33. Source: The Japan Today The transgenic rose variety ‘‘Applause’’ was commercially released in Japan in 2009 (Tanaka et al., 2009)

Blue Gene Technology

www.suntory.com and www.florigene.com.au

In April of 2005, Suntory Ltd. and Florigene Ltd. announced the production of a blue rose by introducing three transformation constructs simultaneously into roses:

To make a blue roses:

(1)Turn off’ the

rose DFR gene

(2) Insert pansy gene to open

the ‘blue’ door

(3) Insert iris DFR gene

to make blue pigment23

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Genetic engineering for white flower 

Silencing anthocyanin biosynthetic pathway by

a) Transcriptional down-regulation b) By inactivating the key enzymes

CHS gene

F3’5’H gene

DFR gene

More sensitive to ranges of stresses

Alternative targets, better and increased fragrance

Zuker et al., 2002

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Gerbera 

(Courtney-Gutterson et al. 1994), 

Genetic engineering for white flower 

(Elomaa 1993),

Chrysanthemum Petunia 

(Krol et al. 1988),

Rose

(Gutterson 1995),

Carnation

(Gutterson 1995).

      Genetic engineering for red/ orange flowers

•Cyanidin and delphinidin derivatives occur but no pelargonidin derivatives in petunia.

•Petunia DFR cannot able to reduce dihydrokaempferol because it shows substrate specificity which lead to the synthesis of leucoanthocyanidins.

•Over expression of A1 gene + abundant substrate –opens a new pathway leading to the synthesis of novel brick red colour petunia.

Maize A1 gene encodes

dihydroquercetin 4 reductase

Meyer et al., 198726

Genetic engineering for yellow flowers

• Aurones are bright yellow flavonoids found in species such as snapdragon, dahlia etc..

• Aurone synthase, more specifically aureusidin synthase (AS), was purified from yellow snapdragon petals and the cDNA encoding the enzyme was cloned.

• A pale yellow petunia expressing a Lotus japonica PKR gene.

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•Chalcone and aurones contribute to yellow colours

• The most common chalcone, THC, is yellow but is spontaneously

isomerized to naringenin in vitro and rapidly isomerized in vivo by CHI.

• Discovery of chalcone 2′-glucosyltransferase (C2′GT) enzyme - stabilizes the chemically unstable chalcone.

• Recent activity: (Okuhara et al. 2004)

Lack of CHI activity + presence of UDP-glucose: THC 2’ glucosyltransferase (C2’GT) activity = yellow carnation

Plant species Original colours

Gene sources Methods Produced flower colors

References

Cyclamen persicum Purple  Endogenous F3’5’H Antisense  Red to pink  Boase et al. (2010)

Gentiana sp. Blue  Endogenous CHS Antisense  White  Nishihara et al. (2006)

Blue Endogenous F3’5’H  RNAi Lilac to purple blue Nakatsuka et al. (2008b)

Nierembergia sp. Violet Endogenous F3’5’H  Antisense  Pale blue Uyema et al. (20006)

Ostespermum hybrida

Magenta  Endogenous F3’5’H  RNAi reddish Seitz et al. (2007)

Gerbera hybrida DFR Over expression

Petunia hybrida  Blue  Mazus psonilum CHS Dominant negative Pale blue Hanumappa et al. (2007)

Red  Lotus japonicus PKR Over expression  Variegated red Shimada et al. (2006)

Rosa hybrida  Red to pale cyanic Viola sp. F3’5’H Over expression Bluish  Katsumoto et al. (2007)

Torenia hybrida Blue  Endogenous ANS RNAi Whitish to pale blue Nakamura et al. (2006)

Blue Endogenous ANS Antisense  Pale blue Nakamura et al. (2006)

Blue Antirrhinum majus AS 

Over expression yellow Ono et al. (2006)

Flower colour modifications by regulating flavonoid biosynthesis

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value of a cut flower=Post harvest longevitySenescence highly controlled process requires active gene expression & protein synthesis for programmed cell death.

Increased respiration and ethylene production induction of catabolic enzymes resulting in decreased proteins

Genetic engineering for longer vase life

Aging of petals

Inhibiting or by

blocking perception

of ethylene

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Florigene has developed carnation flowers with enhanced vase life using antisense RNA

technology. 

Down regulated petal ACC synthase

Control STS Transgenic

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A high level of resistance in osmotin, pr-1 and/or chitinase genes to a major carnation pathogen (fusarium oxysporum f. Sp. Dianthi ) was demonstrated in greenhouse tests. (Zuker et al., 2005)

Genetic Engineering for biotic stress 

Transformation of chrysanthemum cultivar 'shuho-no-chikara' was modified by delta-endotoxin gene cry1ab (mcbt) from  bacillus thuringiensis biological activity against lepidopteran insects into chrysanthemum.Protection of flower crops against coat protein viruses (William R. Woodson 1991)

Transgenic chrysanthemum showing resistance against chrysanthemum stunt viroid (csvd) and TSWV.

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Genetic engineering for improved shape, size

ABC MODELThe combination of three genes that give rise to the flower parts.A sepalsA + B petalsB + C stamensC carpels

• The ABC model (Coen and Meyerowitz 1991) and its modified version (Theißen 2001) are known to be applicable to a broad range of plants (Kim et al. 2005).

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Genetic engineering for improved shape, size

Constitutive expression of  Antirrhinum majus B genes DEF And GLO in

transgenic torenia resulted in the conversion of sepals to petals.

Constitutive expression of the C gene from Rosa rugosa in torenia resulted in a

carpeloid structure in place of sepals (Kitahara et al. 2004)

Studies on homeotic mutants have clarified many important aspects of

genetic control on flower development.

Deficiencies genes and AGAMOUS genes isolated from Antirrhinum majus

increased interest in novel flower shapes through molecular manipulation.

Transformant Wild type

 Genetic engineering

for floral scentmay enhance the value of cut flowers to consumers…

Fragrance numerous volatile aromatic organic substances present in the

flower. Such as,

hydrocarbons, alcohols,

aldehydes, ketones,

esters, ethers

Manipulation of fragrance in flowers chemicals contributing to the fragrance of roses, their pathways of synthesis and enzymes controlling these pathways to be identified.34

Genetic engineering to modify plant architecture

Control of plant height is of great importance in floriculture

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• A petunia plant transformed with Arabidopsis gai-1 (right).

• Genetic modification may replace chemical growth retardants in future.

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Limitation of Plant gene transformation

• Improvement of traits controlled by many gene.

• Improvement of traits controlled by gene that has not been identified.

• Requires high technology knowledge and equipment.

• Expensive cost.

• Government regulation.

Flower color modification of Petunia hybrida commercial varieties by metabolic engineering

o 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

37Japan

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Case studies

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

38Japan Tsuda et al., 2004

Vector constructionFigure 2. Schematic representation of the expression cassettes in T-DNA

of binary vectors constructed for colour modification.

39Japan

Generation of orange petunia expression of rose DFR gene + Suppression of F3H gene by antisense or RNAi method

Generation of red petunia expression of rose DFR gene

T-DNA copy number analysis of transgenic plants show that three most stable lines, two plants had a single copy insert, and the other one had two.

Contd…..

40Japan Tsuda et al., 2004

Modification of co-pigment by Suppressing flavonol biosynthesis resulted in darker and slightly redder colour.

A flower of a transgenic plant expressing rose FLS gene has a paler flower

Surfinia Violet Mini

transgenic petunia expressing torenia FNSII gene

Creeping character did not change

41Japan Tsuda et al., 2004

Engineering of the Rose Flavonoid Biosynthetic Pathway Successfully Generated Blue-Hued Flowers

Accumulating Delphinidin

Katsumoto et. al (2007)Japan

o Rosa hybrida lacks  violet  to blue  colour due  to  the absence of flavonoid  3’,5’-hydoxylase  (F3’5’H)  enzyme  which  produces delphinidin-based anthocyanins.

o Other  factors  such  as  co-pigments  and  vacuolar  pH  also  affect flower colour.

o Expression of the viola F3’5’H gene  accumulates (~95% high)  delphinidin  a novel bluish flower colour.

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• For more exclusive and dominant accumulation of delphinidin irrespective of the hosts, the endogenous dihydroflavonol 4-reductase (DFR) gene was down-regulated and overexpressed the Iris3hollandica DFR gene in addition to the viola F3’5’H gene in a rose cultivar.

• The resultant roses exclusively accumulated delphinidin in the petals, and the flowers had blue hues not achieved by hybridization breeding.

• Moreover, the ability for exclusive accumulation of delphinidin was inherited by the next generations.

43 Katsumoto et. al (2007)Japan

Biosynthetic pathway

Of

flavonoid

44

WKS77 WKS82 WKS100

WKS116 WKS124 WKS140

Rose Varieties transformed with pSPB130 and their flower colour changed are shown

Schematic representation of T-DNA region of binary vectors constructed for colour modification for the constitutive over expression of the viola F3’5’H BP40 gene and the torenia 5AT gene in rose.

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o Correlation of delphinidin content and petal colours in transgenic Lavande. o Pure red and blue have hue values of 360° and 290° in the hue angle.

46Japan Katsumoto et. al (2007)

Host flowerviolet-coloured

transgenic flower98% delphinidin

Flower and petal colour comparison

Transgenic roses exhibited paler flower colour

98% delphinidin Madam Violet Seiryu

The vector pSPB919 is to down-regulate the endogenous rose DFR gene using RNA interference (RNAi) and to over express the iris DFR and the viola F3’5’H genes.

• Northern blot analysis of LA/919-4-10.• The expected sizes of the transcripts of

viola F3’5’H BP40 (1.8 kb) and iris DFR (1.7 kb) genes & smaller size was detected for rose DFR mRNA (A).

• A rose DFR probe detected about 23 bp small sized RNA, which was supposed to be a degraded endogenous rose DFR transcript with RNAi (B).

• Delphinidin contents was confirmed in all transgenic (KmR) progeny of LA/919-4-10.

• The flowers of F1 and F2 progeny contained exclusively delphinidin.

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(si RNA)

Genetic engineering of novel flower colour by suppression of anthocyanin modification genes in gentian

Nakatsuka et.al (2009)Japan 49

(A)A binary vector, pSMABR-rolCpro-intF3’5’H:5/3’ATir(B)The typical flower of wild-type gentian cv. ‘Albireo’(C) 5/3’AT – suppressed transgenic gentian clone no.1.(D) 5/3’ATandF3’5’H double suppressed transgenic gentian clone no.15.

3

•Expressions of anthocyanin biosynthetic genes in transgenic gentian plants.

•The membranes were hybridised with DIG-labelled probes for GtCHS, GtF3’5’H and 5/3’AT, respectively.

•WT indicates untransformed wild-type gentian cv. ‘Albireo’. Ethidium bromide-stained ribosomal RNA bands (rRNA) are shown as a control.

•Accumulation of anthocyanidin and flavone derivatives in transgenic gentian plants.

(B) Flavone conc. in the petal of an untransformed control plant (WT) and transgenic plants were determined by measuring the absorbance at 330 nm using HPLC analysis with apigenin and luteolin as the standards.

• Data are the average ±SD of five replicate flowers.

(A) Anthocyanidin conc. in the petal of an untransformed control (WT) and transgenic plants were determined.

• Data are the average ±SD of five replicate flowers.

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RNA gel blot analysis

Green fluorescent flowers

Mercuri et. al (2001)Italy

GFP detection in Eustoma (Lisianthus) flower petals. (A)

GFP detection in Osteospermum ligules flower petals. (A)

GFP = Green Fluorescent Protein

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4

Block schemes of the gfp-expression constructs used in this work.

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GFP detection in leaves and roots of transgenic plants. (A, B) Leaf trichomes from Osteospermum transfected with vector alone. (C, D) Leaf trichomes from Osteospermum transfected with mGFP5. (E, F) Roots from Osteospermum transfected with vector alone (left) or mGFP5 (right). (A, C, E) were illuminated with white light; (B, D, F) were illuminated with UV light.

GFP detection in flower and leaf extracts

PAGE analysis of plant extracts

Western blot analysis

A B

C D

E F

(A) Phenotypes of transgenic Torenia flowers. • (Upper) Flower color under white light.• (Lower) Cellular fluorescence from adaxial side of petal in

each line.(B) Expression analysis of each transgenic line by RT-PCR/Southern blottingCoexpression of the Am4CGT and AmAS1 genes was sufficient for the accumulation of aureusidin 6-O-glucoside in transgenic flowers (Torenia hybrida).

54Japan Ono et. al (2006)

A

B

Yellow flowers generated by the expression

of aurone biosynthetic pathway

5

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Pathway Of Aurone

flavonoid Synthesis

Production of picotee-type flowers in Japanese gentian by CRES-T

A B

Wild type flower‐Solid colorsuppression of pigment production generates picotee type flower

Nakatsuka et al., 2011Japan

CRES-TChimeric repressor gene-silencing technology

56

6

• (CRES-T) is an efficient gene suppression system which worked successfully in Japanese gentian.

• A chimeric repressor of the anthocyanin biosynthetic regulator gene GtMYB3, under the control of the Arabidopsis actin2 promoter, was introduced into blue-flowered gentian.

• Of 12 transgenic lines, 2 exhibited a picotee flower phenotype with a lack of pigmentation in the lower part of the petal.

• HPLC analysis showed that the petals of these lines contained less anthocyanin and more flavone than the wild-type.

• The expressions of ‘late’ flavonoid biosynthetic genes, including F3H, F35H, DFR and ANS, were strongly suppressed in petals of these transgenic plants.

• In contrast, the ‘early’ flavonoid biosynthetic genes, such as CHS and FNSII, were not affected.

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• Expression of flavonoid biosynthetic genes in transgenic gentian plants.

• The expression levels of GtMYB3-SRDX and endogenous flavonoid biosynthetic genes were determined by semi-quantitative RT-PCR analysis in wild-type and GtMYB3-SRDX expressed transgenic gentian clone nos. 7 & 11

• Schematic representation of pSMABR-AtACT2pro-GtMYB3-SRDX.

• Bar herbicide bialaphos resistance gene as a selectable marker.• NOSp promoter of nopaline synthase (NOS) gene from A. tumefaciens.• rbcSt terminator of RuBisCO small subunit 2B gene from Arabidopsis.• NOSt terminator of NOS gene.• LB left border; RB right border.

semi-quantitative RT-PCR analysis

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Flavonoid analysis in the flowers of transgenic gentian plants by HPLC

A & D- wild type

B & E- transgenic gentian clones no. 7

C & F- transgenic gentian clones no. 11

AnthocyaninsFlavones

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Ethylene Insensitive Transgenic Petunias‐

Jones et al, 2005USA

Senescence was delayed by approx. 8 days

60

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Ethylene sensitive (MD)Ethylene-insensitive (35S:etr1-1)

Nine genes encoding putative cysteine proteases

Protein content (A) and protease activity (B) in wild-type Petunia (MD) & 35S:etr1-1

transgenic (etr-44568) petunia corollas during flower development.

PhCP2 to PhCP10

61 Jones et al, 2005USA

• In this study, 35S:etr1-1 transgenic petunias have been used to see how ethylene regulates flower senescence.

• To compare the senescence programmes in ethylene-sensitive (MD) and ethylene-insensitive (etr-44568) flowers, a comparative analysis was conducted of age-related changes in total protein, protease activity, and the expression of nine cysteine protease genes in the petals of MD and etr-44568 petunias.

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ETR1-1 - Makes plants ethylene insensitive

Long Lasting FlowersEtr1-1

Control Etr1-1Control Etr1-1

Generation of phenylpropanoid pathway-derived volatiles in transgenic plants

• Rose alcohol acetyltransferase produces phenylethyl acetate and benzyl acetate in petunia flowers.

• Esters are important volatiles contributing to the aroma of numerous flowers and fruits.

• Acetate esters such as geranyl acetate, phenylethyl acetate and benzyl acetate are generated as a result of the action of alcohol acetyltransferases (AATs).

• To study the function of rose alcohol acetyltransferase (RhAAT), they generated transgenic petunia plants expressing the rose gene under the control of a CaMV-35S promoter.

• Phenylethyl alcohol and benzyl alcohol produce the corresponding acetate esters, not generated by control flowers.

• The level of benzyl acetate is three times more than phenylethyl acetate in different transgenic lines of petunia.

USA Pichersky et al.,200663

8

Molecular analyses of transgenic petunia plants

expressing RhAAT

RNA blot

GC–MS analysis of volatile compounds

benzyl acetate

phenylethyl acetate

64USA Pichersky et al.,2006

Transgenic rose lines harboring an antimicrobial protein gene, Ace-AMP1, demonstrate enhanced

resistance to powdery mildew• An antimicrobial protein gene, Ace-

AMP1, was introduced into Rosa hybrida via Agrobacterium-mediated transformation.

• PCR analysis for both Ace-AMP1 and neomycin phosphotransferase (npt II) genes, showed that 62% of these plants were positive for both transgenes.

• These lines were further confirmed for stable integration of Ace-AMP1 and npt II genes by Southern blotting.

• Transcription of the Ace-AMP1 transgene in various transgenic rose lines was determined using Northern blotting.

USA Li et al., (2003)65

Fig. a) Powdery mildew development on leaflets & b) on whole leaves

Control

Control

Transgenic

Transgenic

9

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PCR analysis of transgenic plants of Rosa hybrida cv. Carefree Beauty

Tnos Ace-AMP1 Penh-35S Pnos Npt II Tg7

EcoR 1

The binary vector pFAJ3033. The Ace-AMP1 gene is driven by CaMV 35S with a duplicate enhancer region, and is terminated by (NOS).

Southern blot hybridization analysis of transgenic and control rose plants. A) Probed with a 0.35-kb fragment of the Ace-AMP1gene. B) Probed with a 0.7-kb fragment of npt II gene

Northern blot analysis whereby a 0.35-kb fragment of the Ace-AMP1 gene was used as a probe

A)

B)

Conclusions

• Genetic engineering overcomes almost all the limitations of traditional breeding approaches.

• Recent developments in plant molecular biology provide opportunities to use techniques of genetic engineering for improvement of flower crops for modify flower colour, improve vase life, floral morphology, scent and disease resistance.

• Spectral difference in flower colour is mainly determined by the ratio of different classes of pigments and other factors and knowledge of flower coloration at the biochemical and molecular level has made it possible to develop novel color. 67

68

• Recently genetic engineering has demonstrated the best examples such as ‘Moon’ series of transgenic carnations and transgenic blue rose marketed in North America, Australia and Japan.

• Ethylene sensitivity regulates the timing of flower senescence and incorporation of etr1-1 gene delayed senescence in petunia flowers.

• Recent advances in the isolation of scent biosynthetic genes have provided the basis and created the opportunity for the biotechnological manipulation of floral scent.

• Ace-AMP1 is a good candidate gene for genetic improvement of disease resistance in roses.

Conclusions

Future prospects and new avenues

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• New genes should be isolated that will have utility in floriculture, and new transformation methods for flower crops should be further optimized.

• A genetic map of rose, which is commercially the most valuable cut flower, has now been developed. Identification of quantitative trait locus (QTL) from this map, in conjunction with genetic modification, will assist breeders to improve productivity, disease and insect resistance.

• Information on expression of regulatory genes encoding transcription factors should be generated which have effects on flower colour, fragrance and disease resistance.

• The manipulation of colour in the yellow and orange range will become increasingly feasible as more is learnt about the carotenoid biosynthesis pathway.

• More research efforts are needed to modify flower shape and size.

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Thank you