Application of Biotechnology for

improvement of ornamentals

Pavani. U
RHM/07-02
 Introduction
•Ornamental floriculture is becoming an important industry
•Ornamentals include a large variety of crop plants
Cut flowers,
Bulbs and corms
Flowering pot plants
Foliage plants
All the present day ornamental varieties and novelties are as
a result of extensive hybridization, induced mutation and
selection
 Biotechnology

Science of utilizing the properties and uses of

micro organisms or to exploit cells and the cell
constituents at different level for generating useful
products essential to life and human welfare

(B.D. Singh 2001)

 Biotechnology contd..
 Genetic engineering:

The technology of preparing recombinant DNA in vitro by

cutting up DNA molecules and splicing together fragments
from more than one organism

 Tissue culture:

Plant tissue culture is a practice used to propagate plants

under sterile conditions, often to produce clones of a plant.
 Genetic engineering

• Genetic engineering is a laboratory technique for gene

manipulation.

• Natural recombination of genes occurs through meiotic

crossing over.

• Genetic engineering brings about novel combination of

genes by using recombinent DNA technology which is not
possible through natural means.
 Genetic engineering

• Using r DNA technology - multiple copies of a desired gene -

all identical in a bacterial cell or any other host cell as inserted
DNA replicate along with host DNA - Gene cloning.

• Of late gene cloning in a computerized machine Thermocycler

by Polymerase Chain Reaction (PCR)
 Genetic engineering contd..
• Genetic engineering of plants is much easier than animals.

 there is natural transformation system for plants

(Agrobacterium)
 plant tissue can redifferentiate
 plant transformation and regeneration are relatively easy
for a variety of plants.

Agrobacterium tumefaciens can infect wounded plant

tissue, transferring a large plasmid, the Ti plasmid, to the
plant cell.
 Genetic engineering contd..
• Important methods in recombinent DNA technology are

Isolation of desired gene

Insertion of isolated gene into a suitable vector

Introduction of recombineent vector in to host

Selection of transformed host cells

(A.C.Dutta 2005)
 Genetic engineering contd..

Isolation of desired gene:

 Digestion of the cell wall by enzymatic action, dissolution of
the biological membranes by detergent losses, centrifugation
to isolate pure DNA.

 DNA cut into no. of fragments by restriction endonulcleases

- “molecular scissors” forming
i) Blunt (rarely) or flushends
ii) Cohesive or sticky ends (mostly)
 Genetic engineering contd..

• Insertion of isolated gene into a suitable vector:

• Desired fragment so obtained are inserted in to a suitable
vector to produce indefinite no.of copies of desired genes.

i) Plasmids
ii) λ phages
iii) cosmids
What is a plasmid?
 Genetic engineering contd..

• Most common - Ti plasmid of Agrobacterium tumefaciens

• The Ti plasmid - disarmed

• deleting the genes governing Auxin and cytokinin

production – replaced by pBR322 sequence.

• pBR322 – modified to produce an Intermediate Vector

• Intermediate vector must contain

• Origin of replication in E.coli

• pBR322 sequences in T-region of disarmed pTi

• T-DNA (with out borders) from pTi

• Selectable marker (for selecting plant cells with rT-DNA

• Kan - for selection of co-integrate vector in Agrobacterium

 Building the Transgenes
ON/OFF Switch Makes Protein stop sign

PROMOTER INTRON CODING SEQUENCE poly A signal

Plant Transgene

Plant Selectable
Marker Gene

Plasmid DNA
Construct
bacterial genes
•antibiotic marker
•replication origin
 Genetic engineering contd..

Introduction of recombineent vector into host:

Transfer of recombinent vector from E.coli into

Agrobactrerium is usually achieved through conjugation.
 Genetic engineering contd..
• Selection of transformed cells:

Recombinent DNA is placed in Agrobacteium – co

cultivated wiyh plant cells or tissues to be transformed.

The T-DNA would be integrated into plant genome and the

transgene would be expressed.

As a result transformed cells would become resistant to

kanamycin
 Genetic engineering contd..

• After 2 days leaf discs are transferred onto a regeneration

medium containing appropriate concentration of
Kanamycin and carbenicillin.

• Kanamycin allows only transformed plant cells to divide

and regenerate shoots in about 3-4 weeks, while
carbenicillin kills Agrobacterium cells.
 Genetic engineering contd..
• Direct gene transfer:

• Indirect i.e biological agent like Agrobacterium not

applicable for transforming monocots like orchids.

i) Electroporation:
• Exposing the cells to high voltage electrical pulse for very
breif periods
 • GenegunTM

www.tritechresearch .com
 Genetic engineering contd..
ii) Particle gun method:
(Klein et al., 1988)

• Ballistic or biolistic
• 1-2 µm of tungsten or gold
particles (microprojectiles)
coated with DNA to be used
for transformation are
accelerated to velocities
using pressurized Helium
gas
 Genetic engineering contd..

• Microinjection:

• DNA solution is injected

directly inside the cell
using capillary glass
micropipetts
 Genetic engineering contd..

• Genetic engineering can be used to create

genetically altogether a new plant of desired
nature

• It is possible to introduce genes from quite

unrelated organisms like bacteria, fugi, yeasts into
the plants to modify their traits.
 Genetic engineering contd..

• Novel plants with desirable characters created

through genetic engineering methods are called
“Transgenic plants”.

• Creation of transgenic plant utilises the genetic

engineerig technology through tissue culture
methods.
 Genetic engineering contd..

 How transgenic technology utilized in ornamentals?

For a modern and industrialized horticulture there is

always demand and necessity for new varieties.

To develop new varieties through genetic manipulation,

there are several plant breeding techniques.
 Genetic engineering contd..

However combining large parts of parental genomes in

rather uncontrolled fashion is hit- or- miss process to a
larger extent.

Genetic engineering on the other hand allows transfer of

very specific genes in to plants.
 Genetic engineering contd..
 This transgenic technology can be used to generate

Flower crops resistant to biotic and a biotic stresses

Flowers with new colors

Flowers with improved size, shape and floral scent

Flowers having long vase life

 Genetic Engineering for biotic stress

• Identification of genes that control general agronomic

traits – disease and insect resistance.

• Insect control protein genes from Bt – increased resistance

to lepidopteran larvae (Fischhoff et al., 1987 biotech :5)

• Expression of cowpea trypsin inhibitor gene in transgenic

tobacco – increased resistance to herbivorous insect pests
(Hilder et al., 1988 Nature: 330)
 Genetic Engineering for biotic stress

• The chrysanthemum cultivar 'Shuho-no-chikara' was

transformed with modified delta-endotoxin gene, modified
cry1Ab (mcbt) of Bacillus thuringiensis.

• which displays a specific biological activity against

lepidopteran insects into chrysanthemum.

(Shinoyama.H et al., Breeding science 53(4), 2003)

 Genetic Engineering for biotic stress contd..
Genetic engineering of plants to virus resistance:

• Coat protein mediated- resistance

• Expression of coat protein gene of Tobacco Mosaic Virus in

transgenic tomato – resistance to infection by TMV (Abel et al., 1986)

• Similar approach for alfalfa mosaic virus, cucumber and mosaic

virus (Tumer et al., 1987)

• Availability of cloned and sequenced plant viruses – use in

protection of flower crops (William R.Woodson 1991)
 Genetic Engineering for biotic stress contd..

• Transgenic chrysanthemum showing resistance against

chrysanthemum stunt viroid (CSVd) and TSWV.

• Double-stranded RNA-specific ribonuclease gene (pacl)

derived from Schizosaccharomyces pombe using an
Agrobacterium mediated transformation

(OgawaToshiya et al., Breeding science 55(1),2004)

Genetic Engineering for biotic stress contd..
Genetic engineering for fungal resistance:

• Limited success in area of fungal resistance through genetic

engineering.

• Chitinase – protein hydrolyses Chitin – component of fungal

cell wall – defense mechanism of plant.

• This enzyme has been shown to inhibit fungal growth in vitro

(Broekart et al., 1989 Science: 245)
Genetic Engineering for biotic stress contd..
Transgenic carnation with fungal resistance:

• To obtain fungal resistance, transgenic carnation with

osmotin, PR-1 and/or chitinase genes were generated.

• A high level of resistance in these transgenes to a major

carnation pathogen (Fusarium oxysporum f. sp. Dianthi)
was demonstrated in greenhouse tests.

(A.Zuker et al., 2005 Acta Horticulturae:560)

 Genetic engineering for flower color

Floriculture industry driven by availability of novel flower

crops.

Because of this desire for novel flower, tremendous interest

in genetic engineering to introduce genes for new flower
colors.

Particularly for rare shades of blue and purple

 Genetic engineering for flower color contd..

• Flavonoids are one of the main determinants of flower

colors.

• Flavonoid compounds are produced by the

phenylpropanoid pathway.

• Primary function of flavonoid pigments in flowers is to

attract insects and other animals which help in
crosspollination (Brouillard and Dangles 1993).

• Provide protection against U.Vradiation (Dixon et al.1995).

 Phenyl alanine Trans cinnamic P-coumaric acid
PAL acid C4H
4CL
C H R
,
CHS P-coumaroyl coA
6deoxy chalcones
CHS
Tetrahydroxy chalcone
CHI
Naringenin
FHT
F3’H
Dihydromyrecetin Dihydroquercetin Dihydrokaempferol
FLS
F3’5’H DQR
Kaempferol
DFR
Delphinidin Cyanidin
3glucoside Pelargonidin
 Genetic engineering for white flower
• Straight way – silecing anthocyanin biosynthetic pathway
by
a) transcriptional down-regulation or
b) by inactivating the key enzymes.

• Successful reduction of anthocyanin biosynthesis has been

reported in
Petunia (Krol et al. 1988),
 Gerbera (Elomaa 1993),

Chrysanthemum (Courtney-
Gutterson et al. 1994),
Rose (Gutterson 1995),

Carnation (Gutterson 1995).

 Genetic engineering for white flower contd..

• Reducing expression of endogenous pigment

biosynthesis:

• This has been accomplished in Petunia using

antisense RNA technique.
 Genetic engineering for white flower contd..

• It invloves insertion of a reverse orientation copy of the

endogenous gene ( encoding chalcone synthase)

• The expression of this inserted gene gives rise to

complementory mRNA or antisenese mRNA strand that
forms a duplex with the sense strand.

• This duplex likely is unstable and is not available for

translation
 Normal gene Antisense gene

3’ TAC ACC TCG TTC CTC 5’ 3’GAG GAA CGA GGT GTA 5’

5’ATG TGG AGC AAG GAG 3’ 5’ CTC CTT GCT CCA CAT 3’

mRNA Antisense mRNA

5’AUG UGG AGC AAG GAG 3’ 5’ CUC CUU GCU CCA CAU 3’

Double stranded mRNA

5’ AUG UGG AGC AAG GAG 3’

3’ UAC ACC UGC UUC CUC 5’

No translation

No enzyme
 Genetic engineering for white flower cntd..

• When the endogenous chalcone synthase gene was

completely inhibited , no coloration would be expected since
the substrate for this enzyme is colorless and gives white
colored flowers.

• Such plants were observed and unexpectedly novel

pigmentation patterns were also observed as a result of
antisense gene expression.
 Genetic engineering for white flower contd..

• CHS-silencing would lead to the abolishment of the entire

array of flavonoid compounds in plants.
• sometimes, this would cause the plants to be more sensitive
to environmental stresses and might lead to sterility in
some species.
• DFR and F3H – alternative silensing targets for producing
white flowers.
• When F3H is silenced in carnations transgenic plants were
obtained with reduced anthocyanins and increased
fragrance (Zuker et al., 2002)
 Genetic engineering for red/ orange flowers

• In petunia cyanidin and delphinidin derivatives but no

pelargonidin derivatives.

• Enzyme dihydroflavonol 4 reductase shows substrate

specificity - can’t reduce dihydrokaempferol – no
pelargonidin

• A1 gene from maize encodes dihydro quercetin 4

reductase- doesn’t show substrate specificity as does
petunia enzyme.
RL01 mutant petunia line - accumulates dihydrokaempferol -
no pigmentation

Insertion of Maize A1 gene as a chimeric constuct with ca MV

35s promoter (Schwarz –somner et al., 1987) encodes
dihydroquercetin 4 reductase.

Over expression of A1 gene + abundant substrate due to

petunia mutation – synthesis of novel brick red colored petunia.
(Meyer et al., 1987)
 Genetic engineering for yellow flowers

• Chalcones contribute to the yellow colors in Dianthus

caryophyllus (Forkman and Dangel meyer 1980).

• Silencing of CHI in petunia and Lisianthus aimed at

accumulating chalcones, did not produce yellow pigments
in flowers as expected (Van bockland et al., 1993).

• Later discovered - a chalcone 2′-glucosyltransferase

(C2′GT) enzyme - stabilizes the chemically unstable
chalcone and is necessary for producing chalcone-based
yellow pigments. Carnation C2′GT gene has been cloned
recently (Ishida et al. 2003, Okuhara et al. 2004) cloned.
 Genetic engineering for yellow flowers contd..
• Aurones are bright yellow flavonoids found in species such
as snapdragon, dahlia etc..

• Aurone synthases catalyze the hydroxylation and oxidative

cyclization of chalcone precursors.

• One of the aurone synthases, aureusidin synthase was

recently purified from yellow snapdragon petals
(Nakayama et al. 2000).

It belongs to the polyphenol oxidase enzymes, and could be

used for engineering yellow flowers.
 Genetic engineering for yellow flowers contd..

• In yellow varieties of some Asteraceae and Legumacea

plants such as cosmos and dahlia, 6′-deoxychalcones are
the main pigments (Davies and Schwinn 1997).

• The biosynthesis of 6′-deoxychalcones requires coordinate

activity of CHS with a chalcone reductase (CHS).

• Medicago truncatula CHR in a white petunia and obtained

pale yellow flower buds that accumulated the chalcones,
butein 3-O-glucoside and butein 4-O-glucoside.
 Genetic engineering for blue flowers

• The most economically significant flowers - Rose,

Chrysanthemum, and Carnations - no blue color - no
delphinidin - lack of F3′5′H in their flowers.

• Therefore, one can not produce a blue rose or blue

carnation by traditional breeding.
 Genetic engineering for blue flowers contd..

• Expression of a petunia F3′5′H in a carnation line that

accumulated cyanidin-based pigments resulted in very low
levels of delphinidin production and no dramatic effect on
flower color (Brugliera et al. 2000)

• It appears that the introduced petunia F3′5′H could not

efficiently compete with the endogenous carnation F3′H
and DFR enzymes
 Genetic engineering for blue flowers contd..

• Petunia cytochrome b5 gene + Petunia F3′5′H gene was

expressed in the same carnation line –
dramatic improvement in the level of delphinidin - shift in
the flower color from a pink and red to mauve and purple.

• Florigene's new lilac- and mauve-hued carnations

-'Moondust' and 'Moonglow', now dominate the North and
South American carnation cut-flower markets
 Genetic engineering for blue flowers contd..

• Florigene Ltd. and Suntory Ltd. Have successfully

developed a range of transgenic violet carnations by
introduction of a F3’5’H gene together with a petunia DFR
gene in to a DFR deficient white carnation (Fukui et al.
2003).

• In these petals, the engineered delphinidin is conjugated

by endogenous glucosyl transferase, and formed a complex
,
with co pigment such as apigenin 6-c glucosyl malonylester
under vascular pH 5.5
 Development of blue Rose

• Holy Grail of rose breeders since 1840.

• No blue rose - naturally – incapable of synthesizing

delphinidin

• Molecular geneticists with Florigene and Suntory achieved

by combining something old, something new, something
borrowed, and something blue.
 Development of blue Rose contd..

• The 'something blue' was the delphinidin gene cloned from

a pansy.

• The 'something borrowed' was an iris gene for an enzyme,

DFR, required to complete the delphinidin-synthesis
reaction.

• 'something new' was a man-made gene designed by

Suntory geneticists exploited a powerful new CSIRO-
developed technology - to switch off a rose gene
• Suntory's scientists created the 'silencer' gene to exploit a
cellular phenomenon called RNA interference (RNAi)

• Potentially the first commercial plant in the world to

exploit RNAi technology,
 The making of the blue rose

Early 20th century

Rose hybridists - range of novel floral hues.

In 1986

Calgene Pacific - major goal was to use gene technology

In 1991

Florigene's scientists cloned the delphinidin gene from a petunia

They had perfected techniques for genetically transforming roses

It enabled Florigene to create the first roses with delphinidin

 mid-1990s

Florigene had high level expression of delphinidin in an old

red variety, 'Cardinal'.

Combination of cyanidin and delphinidin - attractive dark

burgundy Rose – wasn’t blue – technically major advance

Need a white rose in which the DFR gene was inactivated.

But they were unable to identify a DFR-knockout white rose

Florigene researchers consulted Dr Waterhouse's team at CSIRO

 In 2001

Use of RNAi technology to switch off DFR gene in a red rose

to block cyanidin pathway

and then install the delphinidin gene – plus a new DFR gene
to complete delphinidin synthesis.
• Suntory's researchers had the same idea – they used RNAi
to create a synthetic gene to suppress the DFR gene in a
shapely pink rose .

• They cloned a new version of the delphinidin gene, from

pansy, and, on a hunch, teamed it with a DFR gene from
iris.
• The rose and iris genes are quite similar, and share much
of their DNA code,

• but RNAi is so exquisitely precise that they were able to

design a RNAi 'hairpin' gene targeting a DNA sequence
exclusive to the rose DFR gene,

• so the 'knockout' had no effect on the imported iris DFR

gene.
• The three-gene package (pansy delphinidin, iris DFR, anti-
rose DFR) package worked:

• Suntory's transgenic rose produced very high levels of

delphinidin in its petals, and a small residue of cyanidin.

• The new rose is an attractive shade of mauve, similar to

the current generation of mauve-lilac roses like 'Blue
Moon' and 'Vol de Nuit'.
• Blue shades should be achievable if Florigene and Suntory
researchers can make the rose's petals less acidic.

• Rose petals are moderately acidic, with a pH around 4.5,

while carnation petals are less so, with a pH of 5.5.

• Researchers ‘fished around’ for roses with high pH. But low
petal acidity trait - genetically limited

• Now using RNAi gene knockout technology to identify genes

influencing petal acidity.
 What is RNAi technology?

• Inhibition of gene expression with the aid of double-stranded

RNA (dsRNA) molecules is called RNA interference (RNAi)

• When antisense RNA (aRNA) is introduced into a cell - binds

to the already present sense RNA - inhibits gene expression

• If sense RNA is prepared and introduced into the cell?

• Since two strands of sense RNA do not bind to each other, it

is logical to think that nothing would happen with additional
sense RNA
 RNAi technology contd..

• The new sense RNA suppresses gene expression, similar to

aRNA

• sense RNA actually contain contaminating strands of

antisense RNA.

• Sense and antisense strands bind to each other, forming a

helix – supressor of its corresponding gene.

(www.agricola.org)
 Co-supression

• Researchers were trying to deepen the purple colour of the

flowers by injecting the gene responsible into the petunias
but were surprised at the result.

• Instead of a darker flower, the petunias were either

variegated (Figure 2) or completely white!

• It is now known that double stranded RNA is responsible

for this effect.
 Genetic engineering for improved shape, size

• Studies on homeotic mutants have clarified many

important aspects of genetic control of flower
development. (www.pubmed central)

• Deficiencies genes and agamous genes isolated from

Antirrhinum majus increased interest in novel flower
shapes through molecular manipulation
 Genetic engineering for improved shape, size

• The ABC model (Coen andMeyerowitz 1991) and its

modified version (Theißen 2001) are known to be
applicable to a broad range of plants (Kim et al. 2005).

• The ABC model proposed that three functionally different

genes, i.e., A, B, and C, specified the four-whorl structure
of flowers.

• Gene A is responsible for sepal development in the first

(outermost) whorl. Genes A and B together specify the
petals in the second whorl.
 Genetic engineering for improved shape, size

• Genes B and C determine the stamens in the third whorl,

and gene C alone specifies the carpels in the fourth whorl
(Coen and Meyerowitz 1991).

• Constitutive expression of Antirrhinum majus B genes DEF

and GLO in transgenic torenia resulted in the conversion
of sepals to petals (Dr. Takashi Handa, personal
communication)

• Constitutive expression of the C gene from Rosa rugosa in

torenia resulted in a carpeloid structure in place of sepals
(Kitahara et al. 2004, plant science:166)
 Genetic engineering for floral scent

• Genetically engineering floral scent may enhance the value

of cut flowers to consumers.

• Fragrance is a result of numerous volatile aromatic

organic substances present in the flower. These substances
include hydrocarbons, alcohols, aldehydes, ketones, esters,
ethers, (Flament et al., 1993).
 Genetic engineering for floral scent
• To be able to manipulate fragrance in flowers through
genetic engineering, the chemicals contributing to the
fragrance of roses, their pathways of synthesis and enzymes
controlling these pathways identified.

• Gene mapping is a means to locate such fragrance genes and

to identify the DNA markers associated with these genes.

• Metabolic engineering is a form of genetic engineering aimed

at changing the way living things metabolize, or rearrange
the nutrients they take in into different chemicals and thus
make useful fragrances.
 Genetic engineering to modify plant architecture

• Control of plant height is of great importance in

floriculture.

• Chrysanthemum cv. ‘Iridon’ engineered to express tobacco

phytochrome B1 gene under control of caMV35s

• Transgenic plants were shorter in structure, larger branch

angles than wild type. (Zheng et al., 2001)

• rolC -Transgenic carnation - exhibite increased axillary

bud break, more stem cuttings, increased flowering.
Dwarf Bougainvilleas
 Genetic engineering for longer vase life

• Post harvest longevity

determines value of a cut
flower.
• Senescence of a flower is
highly controlled process
requiring active gene
expression and protein
synthesis - amenable to
manipulation (Woodson
1987) programmed cell
death
 Genetic engineering for longer vase life

• Increased respiration and

ethylene production,
induction of catabolic
enzymes resulting in
decreased proteins.

• Biosynthesis of ethylene
is well characterized.
 Genetic engineering for longer vase life contd..

• Onset of increased ethylene production in aging petals

associated with
i) ACC synthase – converts S adenosyl -L- metheonine to
ACC
ii) EFE activities - oxidise ACC to Ethylene.

Senescence can be prevented either by inhibiting production

of ethylene or by blocking perception of ethylene.

Florigene has developed carnation flowers with enhanced

vase life using antisense RNA technology.
 Genetic engineering for longer vase life contd..

• Virus Induced Gene Silencing technology:

• A 447bp ACC oxidase of petunia was cloned from petunia

cDNA into TRV2-CHS vector to test simultaneous
silencing of ACO and CHS.

(C.Z.Jiang et al.,Acta Hort. 682, 2005)

 Micropropagation of ornamentals

Rapid clonal in vitro

propagation of plants from cells,
tissues or organs cultured
aseptically on defined media
contained in culture vessels
maintained under controlled
conditions of light and temperature.
 Micropropagation in ornamentals

• Orchids
• Cut flowers
• Bulbs and corms
• Flowering pot plants
• Foliage plants
 Orchids

• Arachnis
• Aranda
• Aranthera
• Cattleya
• Cymbidium
• Dendrobium
• Lycaste
• Paphiodelphium
• Miltonia
• Odontoglossum
 Cut flowers

• Chrysanthemum
• Gerbera
• Anthurium
• Rose
• Carnation
 Bulbs and corms

• Gladiolus
• Tulips
• LiliesGladiolus
• Tulips
• Lilies
• Tuberose
• Amaryllis
• Iris
• Tuberose
• Amaryllis
• Iris
• Micropropagation on a commercial scale
done via ….

• Meristem / shoot tip / axillary bud culture

(organogenesis)
• Somatic embryogenesis
• Thin cell layer technique
 Conclusion

 Recent developments in plant molecular biology – provide

opportunities to use techniques of genetic engineering for
improvement of flower crops.
 As horticulture scientists , we should not wait for
Developments to reach the stage of application