application of biotechnology for improvement of ornamental crops

transcript

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

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

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.

Biotechnology contd..

• 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

• 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)

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)

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?

• 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

CODING SEQUENCEINTRON poly A signalPROMOTER

Building the Transgenes

Plant Transgene

bacterial genes•antibiotic marker•replication origin

Plant Selectable Marker Gene

Plasmid DNA Construct

ON/OFF Switch Makes Protein stop sign

Introduction of recombineent vector into host:

Transfer of recombinent vector from E.coli into Agrobactrerium is usually achieved through conjugation.

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

• 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.

• 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

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

• Microinjection:

• DNA solution is injected directly inside the cell using capillary glass micropipetts

• 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.

• 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.

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.

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.

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

• 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

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 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)

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)

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

• 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).

Genetic engineering for flower color contd..

Phenyl alanine Trans cinnamic acid

P-coumaric acid

P-coumaroyl coA

Tetrahydroxy chalcone

Naringenin

Dihydrokaempferol

Kaempferol

Pelargonidin

Dihydroquercetin

Cyanidin

Dihydromyrecetin

Delphinidin 3glucoside

PAL C4H4CL

DQRF3’5’H

6deoxy chalconesCHS,CHR

F3’H

• 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),

Genetic engineering for white flower

Gerbera (Elomaa 1993),

Chrysanthemum (Courtney- Gutterson et al. 1994),

Rose (Gutterson 1995),

Carnation (Gutterson 1995).

• 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

3’ TAC ACC TCG TTC CTC 5’

Antisense gene

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 mRNA5’ AUG UGG AGC AAG GAG 3’

3’ UAC ACC UGC UUC CUC 5’

No translation

No enzyme

• 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 cntd..

• 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)

• 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.

Genetic engineering for red/ orange flowers

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)

• 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

• 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 yellow flowers contd..

• 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

• 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

• 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 ,

• 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

• 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

Development of blue Rose contd..

• 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

and then install the delphinidin gene – plus a new DFR gene to complete delphinidin synthesis.

Use of RNAi technology to switch off DFR gene in a red roseto block cyanidin pathway

In 2001

• 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)

• 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.

Co-supression

• 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.

• 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)

• 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 for floral scent

• 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.

Genetic engineering to modify plant architecture

Dwarf Bougainvilleas

• 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

• 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)

Genetic engineering for longer vase life contd..

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

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

Orchids

• Chrysanthemum• Gerbera• Anthurium • Rose• Carnation

Cut flowers

• Gladiolus• Tulips • LiliesGladiolus• Tulips • Lilies• Tuberose • Amaryllis• Iris• Tuberose • Amaryllis• Iris

Bulbs and corms

• 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

application of biotechnology for improvement of ornamental crops

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