ASSAM AGRICULTURAL UNIVERSITY Credit Seminar of Post-Graduate Student M.Sc. (Agri.

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Name of the Student Roll No. Programme of study Major Discipline Supporting discipline Major Advisor : Ruby Sharma : 11-AMJ-54 : M.Sc (Agri.) : Horticulture : Agril. Statistics and Agril. Biotechnology : Dr. Sunil Bora Professor Deptt. of Horticulture AAU, Jorhat-13

A. Title of the seminar: Genetic transformation in ornamental plants. B. Contents: 1. 2. 3. 4. 5. 6. 7. 8. Introduction Significance of genetic transformation in ornamental plants Methods of genetic transformation in ornamental plants History and present status of genetic transformation in ornamental plants Use / importance of genetic transformation in ornamental plants Barriers of genetic transformation Future prospects Conclusion

Introduction: The genetic manipulation process is dependent on the ability of plant tissue to produce totipotent cells that can be regenerated into a completely viable plant. The use of

genetic engineering to incorporate useful genes into elite breeding lines is dependent on the development of tissue culture techniques. The genetic manipulation of plants has been an ongoing science since prehistoric times, when early farmers along the Euphrates began carefully selecting and maintaining seed from their best crops to plant for the next season. With the advent of recombinant DNA technology in the 1970s, the genetic manipulation of plants entered a new age. Genes and traits previously unavailable through traditional breeding became available through DNA recombination, and with greater specificity than ever before. Genes from sexually incompatible plants, or from animals, bacteria or insects can now be introduced into plants. What is genetic transformation? The ability to move foreign DNA into an organism and thereby alter its genetic makeup is the central theme to both basic and applied aspects of genetic transformation. Genes derived from unrelated species & even other kingdoms, e.g. bacteria, fungi, plants, animals, that would otherwise be inaccessible to an organism, can be combined using genetic transformation techniques. Process of plant transformation: Genetic transformation process normally involves 1. Uptake of naked DNA (gene of interest)by competent cells 2. Penetration into the nucleus & 3. Subsequent integration into the chromosome for proper expression to have the desired gene product Incorporation& integration of foreign DNA into the genetic elements of the host cell organelles (i.e. chloroplast, mitochondria) is another approach for foreign DNA delivery. Requirements for plant transformation: 1. Cell culture and plant regeneration system 2. Cloned DNA to be introduced a. Selectable marker gene b. Promoter, coding region 3. Method of delivery of DNA into the cell 4. Proof of transformation of plant

Formula: Tissue culture + DNA delivery and integration = transgenic plants Why Genetic transformation: Biochemical and physiological characterization of gene, protein or cell function Generate new plant strains for desired trait

This method is faster and allows for transgenic (genes from different species/organisms) plants to be created.

For two reasons, selection of the parental variety for transformation is very important: Genetic characters of background variety: The transformed variety must have an excellent genetic background, with good speed, productivity, and disease resistance. Without these characteristics, it will be expensive to grow and/or susceptible to damage during distribution. Cost: The cost of transforming these varieties for a producer trait, such as disease resistance would, therefore, be prohibitively expensive. A long-term view has to be taken and the choice of variety for transformation determined by its suitability and status in long-term breeding programs. This strategy applies to other crops too (Morandini and Salamini, 2003). Why we need genetic transformation in ornamentals or significance of genetic transformation in ornamentals: In ornamental horticulture, creation of novel traits through broadening the genetic variability is always necessary because of the constant demands of something new in cultivars and crops. Under these circumstances, interspecific hybridization has successfully been used in ornamental plants to produce novel cultivars with useful traits of both parents and to incorporate desirable traits of one species to another. However, interspecific hybridization is always encountered with the difficulty to overcome the sexual barriers lying between the target two species even though embryo rescue techniques are applied. Moreover, conventional breeding techniques including interspecific hybridization are lack of appropriate germplasm for achieving the breeding objectives such as conferring disease and pest resistances and creating novel flower colors. To achieve these breeding objectives, it has been expected to apply genetic transformation technologies in these horticultural crops. The majority of transformation protocols used in ornamental species is based on gene transfer mediated by Agrobacterium and particle bombardment (biolistic process). There are some compelling reasons to make transgenic ornamentals. Mol et al. (1995) discuss how traits such as flower color, plant size, plant architecture, flower vase life and fragrance are suitable targets for transgenic ornamental plant improvement. Genetic Engineering vs. Traditional Breeding: Ornamental horticulture, and particularly floriculture, is well suited to the application of genetic engineering technology. One reason is that the end product is not a food. Not only does this remove possible obstacles to commercialization as a result of some consumer attitudes towards consumption of genetically modified organisms, but reduces the need to undergo food safety studies, thereby reducing the cost of commercialization. Genetic engineering allows the introduction of genes from outside the gene pool, and is precise, because a gene or genes targeted for a specific trait can be introduced. Biotechnology also shortens the time frame for new variety development. Where phenotypic novelty is very important, as it is in ornamental horticulture, this is critical.

Advantage of genetic transformation: Genetic transformation can improve traits derived from one or several kinds of genes. Thus, the technique would be useful in producing a one-point improvement of traits in original cultivars bred by cross-hybridization in a manner similar to the mutation breeding method. The advantage of genetic transformation compared with the traditional breeding method is shown in Figure A. As most mutations involve a change from dominance to recessiveness, mutation breeding has to start from plant material in which the target genes are known to be present in a recessive condition. For example, the direction of flower color mutations in chrysanthemum is known (Machin and Scope 1978). Pink is dominant and yellow is recessive; therefore, pink-flowered cultivars are thought to be useful as mutation breeding materials. White-flowered cultivars frequently give rise to yellow-flowered mutants, but the reverse mutation has rarely been observed. Therefore, mutation breeding is subtractive one-point improvement. On the other hand, genetic transformation can produce changes from dominance to recessiveness and vice versa. Genetic transformation is thus additive one-point improvement, which is a great advantage. Genetic transformation is superior to mutation breeding because the target trait can be modified directly by incorporating related genes. In mutation breeding, the desirable traits can be obtained only when the genes related to the targeted traits are altered accidentally. Interspecific hybridization has also contributed much to the production of a broad range of genetic variation in cultivated plants. It has been successfully performed using style and ovary manipulation, or ovary, ovule, and embryo culture. However, interspecific hybridization introduces unexpected traits along with desirable ones. Because only one gene or several genes are introduced directly in genetic transformation, the possibility of incorporating unexpected traits is much lower than in interspecific hybridization. Furthermore, genetic transformation is a method with essentially infinite possibilities because the necessary resources are available in every living being on earth.

Figure A. Combinations of breeding methods for vegetatively propagated ornamental plants. Common Plant Transformation Methods: The gene transfer method and the gene expression vectors to be used must be compatible with the plant genotype and the tissue to be treated. Several gene transfer methods were used: Vector mediated Agrobacterium mediated

 Virus mediated Vectorless or Direct gene transfer methods Chemical methods: PEG DEAE-dextran Calcium phosphate Physical methods : Electroporation Particle gun delivery Lipofection Microinjection Pollen transformation Laser induced Fibre mediated

Vector mediated : 1. Agrobacterium mediated: In the laboratory, bacteria are co-cultured or inoculated with plant tissue and the bacteria transfer part of their DNA into plant cells. the keys: make a segment of DNA that contains a selectable marker and a gene of interest to look like a T-DNA get this T-DNA into an Agrobacterium cell so that it can be mobilized by the vir genes produce and find transformed plant cells that can be regenerated into normal, fertile plants

2. Virus mediated: Virus-based vectors requires the ability to modify plant viruses to express foreign genes. Vector less or Direct gene transfer methods: In the direct gene transfer methods, the foreign gene of interest is delivered into the host plant cell without the help of a vector. The methods used for direct gene transfer in plants are: Chemical methods: 1. PEG: In this method protoplasts are isolated and a particular concentration of protoplast suspension is taken in a tube followed by addition of plasmid DNA (donor or carrier). To this 40% PEG 4000(w/v) dissolved in mannitol and calcium nitrate solution is slowly added because of high viscosity, and this mixture is incubated for few minutes (5 min.). Among the most important parameters that affect the efficiency of PEG-mediated gene transfer are the concentration of calcium and magnesium ions in the incubation mixture, and the presence of carrier DNA. The

linearized dsDNA are more efficiently expressed and integrated in the genome than the supercoiled forms. The advantage of the method is that the form of DNA applied to the protoplast is controlled entirely by the experimenter and not by intermediate biological vector. Main disadvantage is that the system requires a protoplast. 2. DEAE-dextran: Transformation of cells with DNA complexed to the high molecular weight diethyl amino ethyl (DEAE) dextran is used to obtain efficient transient expression. The efficiency increase when 80% DMSO shock is given. But this technique does not produce stable transformants. 3. Calcium phosphate: DNA when mixed with calcium chloride solution isotomic phosphate buffer DNA-CaPO4 precipitate. The precipitate is allowed to react with actively dividing cells for several hours, washed and then incubated in the fresh medium. Giving them a physiological shock with DMSO can increase the efficiency of transformation to a certain extent. Relative success depends on high DNA concentration and its apparent protection in the precipitate. Physical methods: 1. Electroporation : It involves a pulse of high voltage applied to protoplasts/cells/ tissues to make transient (temporary) pores in the plasma membrane which facilitates the uptake of foreign DNA. The cells are placed in a solution containing DNA and subjected to electrical shocks to cause holes in the membranes. The foreign DNA fragments enter through the holes into the cytoplasm and then to nucleus. 2. Particle gun/Particle bombardment : In this method, the foreign DNA containing the genes to be transferred is coated onto the surface of minute gold or tungsten particles (1-3 micrometers) and bombarded onto the target tissue or cells using a particle gun (also called as gene gun/shot gun/microprojectile gun).The microprojectile bombardment method was initially named as biolistics by its inventor Sanford (1988). Two types of plant tissue are commonly used for particle bombardment- Primary explants and the proliferating embryonic tissues. 3. Lipofection: - Liposomes are circular lipid molecules with an aqueous interior that can carry nucleic acids. Liposomes encapsulate the DNA fragments and then adher to the cell membranes and fuse with them to transfer DNA fragments. Thus, the DNA enters the cell and then to the nucleus. Lipofection is a very efficient technique used to transfer genes in bacterial, animal and plant cells. 4. Microinjection: where the DNA is directly injected into plant protoplasts or cells (specifically into the nucleus or cytoplasm) using fine tipped (0.5 - 1.0 micrometer diameter) glass needle or micropipette. This method of gene transfer is used to introduce DNA into large cells such as oocytes, eggs, and the cells of early embryo. 5. Pollen transformation: In 1976, attempts were made to use pollen to take up foreign DNA and then cross fertilize a related species. Glauca pollen were incubated in DNA isolated from langsdorfii. The DNA treated pollen were used to pollinate emasculated glauca plant. 6. Laser induced: The laser pokes precise tiny holes in the cells allowing plasmid DNA to be taken up. Not yet successful in transforming plants. 7. Fibre mediated: Use silicon carbide fibers to punch holes through cultured plant cells. Silicon carbide fibers and cultured plant cells are added to a tube and vortexed

vigorously. The mechanical force generated by the vortex drives the fibres into the cell. The most used methods of DNA delivery for ornamental plants have been Agrobacterium-mediated transformation and microprojectile bombardment. Alternative methods of DNA delivery, such as infiltration (whole plant transformation), silicon carbide fiber-mediated transformation, electroporation of cells and tissues, electrophoresis of embryos, microinjection, transformation via pollen-tube pathway and liposome mediated transformation have seen little use with ornamentals (RakoczyTrojanowska, 2002; Zucker et al., 1998). AGROBACTERIUM-MEDIATED TRANSFORMATION: This transformation method takes advantage of the natural ability of the soil microorganism Agrobacterium tumefaciens to transfer DNA to plant cells. During transformation, a specific segment of the vector, the T-DNA, is transferred to the plant cell and is inserted into the plants nuclear genome. The T-DNA region can be substituted with engineered DNA sequences coding for selectable markers and/or genes of interest. DNA transfer functions are mediated by a set of virulence genes located separately from the TDNA region. Parts of the vir gene set sense the existence of wounded plant tissue and phenolic compounds and activate other vir genes. These vir genes replicate and process the DNA to be transferred and facilitate its movement to the plant cell nucleus. Important considerations: Strain Specificity: Agrobacterium tumefaciens mediated transformation had been successful with a broad range of dicotyledonous plants and few monocotyledons. There are differences in the susceptibility between species and even between cultivars and genotypes of the species. The gene expression vectors to be used must be compatible with the plant genotype and the tissue to be treated. Bacterial Density: Concentration of bacterial cells in the induction medium is another important factor to be considered for efficient transformation. Very low density of bacterial population could lead to ineffective transformation, whereas very high density may lead to necrosis and death of the explant. Some species are very sensitive to bacterial infection and hence very low density of bacterial population is used. Genes Transformed: A variety of gene expression vectors with selectable marker or reporter genes have been used in ornamentals. Chia et al. (1994) transformed Dendrobium with the firefly luciferase gene, luc. The product of this gene produces light upon reaction with luciferin, which can be detected with a camera photo multiplier. Co-cultivation: The explant chosen, in its most receptive stage, is exposed to the Agrobacterium culture in the induction media at an optimum bacterial density. Both the composition of the induction media and the time of induction play a key role in the efficiency of transformation.

Addition of Phenolic Compounds: Agrobacterium tumefaciens respond to certain phenolic compounds such as acetosyringone and hydroxyl acetosyringone which are excreted by wounded plants. These small molecules act to induce the activity of virulence (vir) genes that are encoded on the plasmid. The vir genes are located on 35Kb region of the plasmid that lies outside the TDNA region. When A. tumefaciens get attached to a plant cell, and the vir genes are induced, then T-DNA (which contains the gene of interest) is transferred to plant cell. Elimination of Bacteria after Co-cultivation: Complete elimination of bacteria from the explant after co-cultivation is very essential; otherwise it will interfere with the growth and organogenesis of the explant. Overgrowth of bacteria causes death of the explant and disrupts the experiment. Elimination of bacteria from the explant is done by the use of antibiotics. The antibiotic chosen should be such that it efficiently kills the bacteria at the same time it does not affect the growth and organogenesis of the explants. The most commonly used antibiotics for this purpose are carbenicillin and cefotaxime. However, their effect on the explant has to be studied before choosing any one of them as they are also reported to have detrimental effect on some species. Selection of Transformed Cells: Screening of un-transformed cells or selection of transformed cells is an important aspect of transformation work. The selection agent must be toxic to plant cells, though not so toxic that the products from the dying, non tranformant cells kill adjacent transformed cells. Thus the most effective toxins are those which either inhibit growth of untransformed cells or slowly kill the untransformed cells. Optimal selection pressure will use the lowest level of toxin needed to kill the untransformed tissues. Confirmatory experiments: Some reporter gene products can be detected in intact plant tissues. The most popular of these systems is the E.coli b-D-glucuronidase (GUS) gene. The GUS activity in transformed plant tissues can be localized by observing the blue colour that is formed after hydrolysis of the uncoloured substrate 5- bromo4-chloro3-indolyl b-D-glucuronic acid. PCR analysis is also used to analyse the presence of transgene in the genome of the transformed plant. Agrobacterium transformation has several advantages for plant transformation, including its simplicity and low cost, precise transfer and integration of DNA with defined ends, linked transfer of marker and trait genes, a high frequency of stable transformation with single-copy insertions, a low incidence of gene silencing and the ability to transfer large T-DNA pieces (Velethambi et al., 2003). The vast majority of ornamental plant transformations reported in the literature were made using Agrobacterium (approximately 80%), with a much smaller percentage made by microprojectile bombardment. Agrobacterium tumefaciens strains used for ornamental plants are binary vector systems where the recombinant T-DNA and the vir region reside on separate plasmids. The most commonly used Agrobacterium tumefaciens for ornamental plant transformation has been LB4404, comprised of an octopine-type vir helper plasmid, a disarmed Ti plasmid and a binary vector plasmid carrying the trait gene(s) (Hoekema et al., 1983). The L, L-succinamopine-type EHA 101 and EHA 105 (Hood et al., 1993), also with vir helper plasmids and supervirulent genes have also seen wide use in ornamental species. Other binary vectors seeing common use are AGLO and GV3101.

While Agrobacterium has historically been considered a poor transformation method for monocot plants, the development of improved supervirulent vectors and vir helper plasmids has largely overcome this limitation. The monocot ornamental genera including Agapanthus, Alstroemeria, Anthurium, Dendrobium, Iris, Lilium, Muscari, Oncidium, and Phalenopsis have been successfully transformed using Agrobacterium tumefaciens.

MICROPROJECTILE BOMBARDMENT: This transformation method is also known as biolistics, particle bombardment or particle acceleration. The equipment used for microprojectile bombardment is often referred to as a gene gun. It gets this name because heavy, DNA-coated particles are shot into target plant tissues, directly through cell walls and membranes (Klein et al., 1987; Sanford, 1988). Gold or tungsten particles between 1 and 4 M are surface coated with DNA by precipitation with spermidine. The particles are accelerated at target cells using compressed gas, electrical discharge or gun powder. The PDS 1000/He Particle Delivery System has been commonly used with ornamental species. It uses high-pressure helium, released by a rupture disk, and a partial vacuum to propel a macro-carrier coated with micro-carrier particles toward target cells. A partial vacuum is needed to allow the microscopic micro-carriers to travel straight to the target and maintain velocity. The macrocarrier is stopped by a screen and the micro-carriers detach and continue their progress into target cells. The accelerated particles pass through the cell wall and cell membrane and land in various parts of the cell. The DNA dissociates from the micro-carrier and moves into the nucleus where it is integrated into the genome of the plant (Yamashita et al., 1991).

The advantages of microprojectile bombardment are the ability to transform plants not infected by Agrobacterium, specialized vectors are not needed, multiple DNA fragments/plasmids can be simultaneously co-bombarded, elimination of false positive reporter gene expression seen with Agrobacterium, and transformation can be applied to plants where high efficiency regeneration systems are lacking and organelle transformation is a possibility (Veluthambi et al., 2003). On the other hand, biolistics requires specialized and expensive equipment, has a low efficiency of transformation relative to Agrobacterium and needs adjustments to be made to the firing protocol for each plant species or tissue type (Sanford, 1988). Perhaps the biggest drawback to the gene gun is high copy number and rearrangement of transgenes, often leading to gene silencing or genomic rearrangements (Hansen and Wright, 1999; Veluthambi et al., 2003). Despite the limitations of microprojectile bombardment, it has been used successfully to transform several ornamental monocot genera including Alstroemeria, Gladiolus, Lilium, and several orchid genera. In addition, the dicot genera Chrysanthemum, Dianthus, Euphorbia, Eustoma, Gentiana, Liquidambar, Pelargonium, Petunia, Rhododendron and Rosa have been transformed by microprojectile bombardment. Due to patent issues and potential future commercialization of transgenic plants, microprojectile bombardment was utilized instead of Agrobacterium for transformation of Rhododendron (Knapp et al., 2001). With Dianthus, microprojectile bombardment was used in combination with Agrobacterium to enhance transformation efficiency (Zucker et al., 2000; Lavy et al., 2002; Zucker et al., 1999).

Selection of transformed cells from untransformed cells:

The selection of transformed plant cells from untransformed cells is an important step in the plant genetic engineering. For this, a marker gene (e.g. for antibiotic resistance)

is introduced into the plant along with the transgene followed by the selection of an appropriate selection medium (containing the antibiotic). The segregation and stability of the transgene integration and expression in the subsequent generations can be studied by genetic and molecular analyses (Northern, Southern, Western blot, PCR).

Transformation protocols of ornamental plants:

Looking at transformation protocols of ornamental plants (as e.g. listed in Brand (2006)), it is clear that there are some similarities in the way that the experiments have been set up. The majority of the experiments are conducted on dicotyledonous plants; probably because the majority of the floricultural plants are belonging to this group. The preferred tissue varies from protocol to protocol, but stem and leaf explants are often used in adult tissue and hypocotyls or cotyledons from seedling tissue. From the orchids the protocorm like bodies are the preferred choice of explants. The choice of transformation technique is in most protocols Agrobacterium tumefaciens mediated transformation followed by particle bombardment as second choice. The mostly used vectors are AGL0, LBA4404 and EHA101, but others are used as well. Kanamycin (nptII) resistance as selection marker is very common, even though it can cause implications, if the product has to be registered for commercial use, especially in European countries because regulations are very strict to avoid the spread of bacterial resistance. The majority of the transformation systems are using the CaMV 35S promoter for dicots and the maize ubiquitin 1 promoter for monocots to obtain a high level of transgene expression. For protocol development the marker gene uidA coding for -glucuronidase (GUS) are often inserted without any gene of interest. When a gene of interest has been inserted, the focus from the researchers has been on traits such as flower colour, plant size and architecture, vase life, and disease resistance (Brand 2006; Chandler & Tanaka 2007). In newer reviews programming of flowering time has also started to show up (Chandler & Brugliera 2011).

History of ornamental genetic transformation:

Genetic transformation in higher plants was first reported by Zambryski et al. (1983). Meyer et al. (1987) reported that the introduced dihydroflavonol 4-reductase gene derived from Zea mays changed the color of petunia flowers to red, which was the first reported instance of flower color alteration by genetic transformation. Flower color is one of the most successfully improved target traits using the gene transfer technique. In 1997, the first genetically modified blue carnation was introduced in the market (Tanaka et al. 2005).

Use and importance of genetic transformation in ornamentals:

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, fungi, 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 utilizes the genetic engineering technology through tissue culture methods. 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: Flowers with new colors. Flower crops resistant to biotic and abiotic stresses. Flowers having long vase life. Control flowering time. Flowers with improved size and shape. Flowers with improved floral scent.

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. The modification of flower colour has been the most widely applied tool of genetic modification in flower species and is where the bulk of research on GM flower crops is focussed. Several reviews provide comprehensive assessments of the state of the art for modification of flower colour by genetic modification (Tanaka and Brugliera 2006; Tanaka and Ohmiya 2008; Nishihara and Nakatsuka 2010). Flower colour modification is an obvious application of the technology as flower colour is one of the most important traits for flower breeders and in many species the whole spectrum of flower colours is not available. This is because in some cases specific varieties are male sterile and so cannot be used in conventional breeding programs. In other cases, certain coloured anthocyanins are not present in the species, due to lack of the necessary genes (Tanaka and Brugliera 2006; Nakatsuka et al. 2007). Flavonoids, carotenoids and betalains are the major pigment classes found in flowers, with flavonoids the most abundant and diverse type. 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.V radiation (Dixon et al.1995). Some of the earliest reported research on genetically modified plants concerned the alteration of flower colour in Petunia hybrida (petunia) via manipulation of the flavonoid pathway. Since then, the molecular basis of the biosynthesis of the flavonoids including anthocyanins (pigments conferring pink, red, violet and blue hues to flowers), flavones and flavonols (colourless co-pigments that enhance colour and contribute to blueing of anthocyanins) and aurones (yellow coloured flower pigments) has been elucidated (Davies and Schwinn 2007). The cytochrome P450 enzymes flavonoid 30,50-hydroxylase and flavonoid 30-hydroxylase are predominantly responsible for the type of anthocyanin produced in a petal. The genes encoding both these enzymes have been isolated and have been used to transform several flowering plant species (Tanaka et al. 2009; Nishihara and Nakatsuka 2010). The genetic manipulation methods used to create potential new flower colours include creation of new flavonoid biosynthesis pathways by introducing genes encoding enzymes that are absent, redirection of pathways by over-expression of genes and/or suppression of pathways by down-

regulation of genes. An example of pathway creation is the genetic manipulation of flower colour in carnation, chrysanthemum and rose. These are all major cut-flower species and all lack flavonoid 30,50-hydroxylase, the key gene necessary for the biosynthesis of blue delphinidin-derived pigments. Purple or blue varieties are thus not able to be conventionally bred.

Fig: 1. Photographs of flowers from naturally occurring and transgenic plants. A. Scarlet verbena(1) and violet petunia (2) contain pelargonidin and delphinidin based anthocyanins. The yellow colour of many flowers including rose (3) and chrysanthemum is derived from carotenoids. Marigold flowers (4,5) contain carotenoids, as well as unusual yellow 6-hydroxy flavonol and cyaniding-based anthocyanins. Yellow marigold (4) contains carotenoids and 6hydroxy flavonol and orange marigold varieties (5) contain all the three types of pigment. B. Trangenic carnation. The Florigene Moonseries of transgenic carnation produce delphinidin based anthocyanins which can not be produced by carnation. Currently spray

(1,2) and standard (3,4) varieties are produced commercially. The difference in colour intensity between the different varieties due to variation in the amount of anthocyanin. C. Flowers from transgenic rose. Flowers from a transgenic rose producing delphinidin in the petals are shown to the right in panel 1 compared to flowers from the untransformed parent (1, left). Transgenic rose (flowers are shown in panel 2) are now under field trial in Japan, Colombia, USA, in order to meet the requirements for deregulation of genetically modified organisms. D. Flowers from non-transgenic torenia host flower mainly accumulate delphinidin based anthocyanin (1). Downregulation of anthocyanin synthase gene yielded white and partly white varieties (2). Downregulation of flavonoid 3,5-hydroxylase and flavonoid 3hydroxylase and expression of a heterologous dihydroflavonol reductase activity yielded transgenic plants with pink flowers (3) accumulating pelargonidin anthocyanins. Expression of snapdragon tetrahydroxychalcone 4-glucosyltransferase and aureusidin synthase genes and downregulation of anthocyanin pathway lead to aureusidin accumulation and a yellow flower colour (4) Fig: Alterations in flower colour. From Chandler and Tanaka (2007).

Genetically modified, purple coloured carnations androses accumulating delphinidinbased anthocyanins have been produced by introduction of the flavonoid 30,50-hydroxylase gene from petunia or pansy. The Moon series GM carnations have been available commercially for the past 15 years (Tanaka et al. 2009), and a genetically modified variety of rose was released in 2009 (Katsumoto et al. 2007). In some species the endogenous anthocyanin pathway may be functionally intact genetically but is directed towards certain anthocyanins. By adding genes on the flavonoid biosynthesis pathway from species whose enzymes have different substrate affinities to endogenous ones, the ratio of anthocyanins can be adjusted, creating flower colours not possible by breeding (Nakaysuka et al. 2007; Seitz et al. 2007;Ueyama et al. 2006). There are examples of manipulation of flowers colour through down-regulation of endogenous biosynthesis pathways via RNAi, co-suppression and antisense- mediated silencing (Tanaka and Brugliera 2006). Recent examples of generation of novel flower colour using suppression technologies include those in gentian (Nakatsuka et al. 2010) and torenia (Tanaka et al. 2010).

Abiotic stress resistance:

Under the intensive management conditions of commercial cut-flower and pot plant production, abiotic stress is not a problem, particularly in a greenhouse situation. In outdoor production areas, severe frost may sometimes damage crops. However, in the hands of a consumer or in a landscape situation, where watering may not be as rigorously controlled, drought-tolerance may be a desirable trait, in order to reduce plant losses. As drought tolerant genes are being developed for the major crops, the application of these genes to floricultural crops is also being evaluated (Potera, 2007).

Disease Resistance:
The most important factor for ornamental products for retail sales is the visual quality, especially for cut flowers and potted plants. Visible disease symptoms have a major impact on quality and consumer perception, and plant diseases can cause significant losses in the production of ornamental crops. At the same time diseases are difficult to control, and as new plant species are introduced to new areas of the World as ornamentals, new diseases become a problem for the growers. Over the years, much research has been done in increased disease resistance using transgenes, but the challenge is to find resistance genes that are efficient enough to justify the costs of making the transgenic plant commercially viable. The use of resistant cultivars is the most commonly used strategy for control of disease in many agricultural crop species. Traditional breeding strategies require that sources of disease resistance genes can be identified within the primary and secondary gene pool. This is a difficult task in ornamentals, because of the large diversity of floral and nursery crop species that are susceptible to an equally large group of different pathogens, and because many genera of ornamental plants do not have a gene pool that includes the necessary disease resistance genes. In addition, ornamental plant cultivars have a limited life span, before new and more appealing cultivars are introduced making traditional breeding for disease resistance even more difficult. Finally, backcrossing through multiple generations may be required, which may lead to reduced resistance compared with the source material (Hammond 2006). This can be inhibited further by the fact that many ornamentals are vegetative propagated, because they cannot produce viable offspring. It is important to bear in mind the diversity of pathogens, when attempting to find the right strategy to develop genetically modified disease resistant plants. The organisms are highly taxonomically diverse, and the major groups include cellular pathogens such as bacteria, fungi, and the algal oomycetes as well as molecular pathogens such as viruses. They are physiologically very different from each other, and therefore no single gene product can be expected to have a direct toxic effect on all types of pathogens.Consequently, enhanced disease resistance has been achieved using different strategies depending on the targeted pathogen. Fungal and viral diseases are a significant cost to producers of floricultural products and flower breeders continually try to improve pathogen resistance. Fusarium, Botrytis, rusts and black spot tolerance are major targets as these diseases are prolific and difficult to control. Genetic modification strategies to improve the resistance of floricultural species to pathogens have been reviewed recently by Hammond et al. (2006). As yet, there are no GM flowers varieties engineered to resist pathogens on the market. As specific loci are identified which are linked to pathogen resistance, marker assisted breeding may prove a useful source of genes for genetic modification in the future (Debener and Linde 2009). An example is the locus for black spot resistance in rose (Whitaker et al. 2010). Using a hairpin RNA gene silencing strategy, transgenic poinsettia resistant to Poinsettia mosaic virus has been developed (Clarke et al. 2008). As yet there are no GM virus tolerant flower or pot plant varieties on the market

The chrysanthemum cultivar 'Shuho-no-chikara' was transformed with modified deltaendotoxin 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) Collinge et al (2008) described a model that simplifies the different strategies used in research towards the creation of transgenic disease resistant plants (Figure 8). The most straight forward approach is to add genes encoding for antimicrobial proteins or peptides that originate from plants or other organisms either alone or in combination and express them constitutively (Figure 8; 1a). This approach includes new antimicrobial secondary metabolites, detoxification of the pathogen or destruction of pathogen signals. A variant concerns the use of pathogen-inducible promoters to regulate these anti-microbial factors (Figure 8; 1b). Another strategy concerns the manipulation and sharpening of the natural defence in the plant (Figure 8; 2a and 2b). This can be done by altering how the pathogen is detected (Figure 8; 2a) or by changing downstream regulatory pathways including transcription factors (Figure 8; 2b). In a third strategy, called pathogen mimicry (Figure 8; 3), the plant is primed to recognise a specific pathogen. This is also known as pathogen derived resistance or genetic vaccination (Collinge et al. 2008).

Figure 8: A simplified model of defence illustrating successful transgenic strategies. Strategy one concerns direct interference with pathogenicity or inhibition of pathogen physiology. Thus 1a involves constituative expression of antimicrobial factors and 1b involves pathogen induced expression of one or more genes in the transgenic plant. Strategy 2 concerns the regulation of the natural induced host defences. 2a concerns altering recognision of the pathogen (eg R-genes) and 2b concerns downstream regulatory pathways (e.g. SAR) and includes transcription factors. Strategy 3 is pathogen mimicry: the manipulation of the plant to prime recognision of a specific pathogen through pathogen derived gene sequences (genetic vaccination). (Collinge et al. 2008) 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) 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)

Insect resistance:
Control of specific insect pests is one of the primary costs for producers of floricultural products as well as being a nuisance to consumers and landscape gardeners. Thrips, spider mites and aphids are particularly serious pests, and can have a devastating impact on both yield of crops and the potential for export, because quarantine inspectors typically reject or fumigate product in which even a low frequency of insects are identified. There is a limited usefulness for the Bt toxin strategy for insect control in floriculture, as the Bt toxin is ineffective on the most common pests. In the future it is likely other genes conferring insect tolerance (directly or indirectly) will become available (Gatehouse 2008).

Genetic engineering for longer vase life:

Post harvest longevity determines value of a cut flower. Leaf and flower senescence is mainly regulated by the levels of ethylene and cytokinin in the plant. Senescence of a flower is highly controlled process requiring active gene expression and protein synthesis amenable to manipulation (Woodson 1987) programmed cell death, Increased respiration and ethylene production, induction of catabolic enzymes resulting in decreased proteins. Production of ethylene in plants is a 3 step process (Figure 6) (Yang & Hoffman 1984). First, Methionine is converted to S-Adenosyl Methionine (AdoMet). Then, 1aminocyclopropane-1-carboxylic acid (ACC) synthase facilitates the conversion of AdoMet to ACC. Finally, ACC oxidase transforms ACC to ethylene using oxygen. Senescence can be prevented either by inhibiting production of ethylene or by blocking perception of ethylene. It is possible to control the ethylene synthesis by altering either the ACC synthase or the ACC oxidase step. Florigene has developed carnation flowers with enhanced vase life using antisense RNA technology. In Torenia fournieri Aida and coworkers (2008) inserted a fragment of the ACC oxidase in sense and/or antisense direction and found that lines from both types displayed a significant greater longevity than the wild-type plants.

Figure 6: The ethylene biosynthesis pathway and the Yang cycle (Kieber & Gepstein 2010). However, the flowers remain sensitive to external ethylene (Chandler 2007; Stead et al. 2006). Another way to reduce ethylene sensitivity is to insert mutated ethylene receptors in the plants. Overexpression of mutant ethylene receptor genes has been shown to delay senescence and overcome the sensitivity to exogenous ethylene thereby improving vase life (Milbus et al. 2009; Raffeiner et al. 2009; Sriskandarajah et al. 2007). In carnation both technologies have been used and the subsequent longer vase life phenotypes have been proven to be stable in field trials and inter-continental transport studies. However, GM carnation with modified vase life has not been commercialised (Chandler 2007), due to the high cost of regulatory approval. Even though five ethylene receptors have been found, ethylene insensitivity is achieved if the binding site of one receptor in changed (Hall et al. 1999). Ethylene insensitivity has been obtained in e.g. Kalancho, Campanula, Rhipsalidopsis, Oncidium and Odontoglossum by insertion of the mutated ethylene receptor etr1-1 from Arabidopsis (Mibus et al. 2009). It is important that the insensitive ethylene receptor is limited to the flowers, because ethylene affects various processes in the plant including disease sensitivity and regulation of vegetative propagation (Mibus et al. 2009). Narumi et al (2005) constructed a mutated ethylene receptor in chrysanthemum, where the mutated mDG-ERS1 receptor, similar to etr1-4 from Arabidopsis, reduced leaf

senescence occurring prior to flower senescence. The transformed lines showed reduced ethylene sensitivity, and the leaves stayed green longer than the wild-type plants. It is worth noting that not all plant species have flowers that are sensitive towards ethylene, and for these species the described strategies cannot be applied. An example is Gladiolus, on which exogenous ethylene and ethylene inhibitors have no effects on the petal senescence process, and therefore other strategies have to be chosen to improve longevity. Milbus et al. (2009) have shown enhanced ethylene resistance in a number of important pot plants, but noted the need to ensure expression of the transgene is strictly confined to the floral organs, as ethylene is an important endogenous hormone involved in disease sensitivity and regulating vegetative propagation. This is a significant issue as many floricultural crops are vegetatively propagated (Shibata 2008).

Flowering time:
In cut flowers, speed to flowering has a significant effect on production costs, as it determines the productivity of a defined area of greenhouse space. In pot plants, the ability to speed flowering time, or predictably control flowering time, is vitally important to ensure that the plants are floriferous at the point of sale during special occasions. Though some control of flowering can be achieved by manipulation of photoperiod and/or use of plant growth regulators, the possibility of using genetically modified plants which will grow under day-neutral climates or can be induced to flower is one possible use of genetic modification technology (Franklin and Whitelam 2006; Tremblay and Colasanti 2006; Flachowsky et al. 2009). Two types of genes, meristem identity genes and floral organ identity genes, regulate floral development (Coen & Meyerowitz 1991). The first type of genes encodes transcription factors that are necessary in the positive regulation of the initial induction of organ identity genes. Examples from Arabidopsis of this type of genes are: APETALA1 (AP1), and LEAFY (LFY) and SUPPRESSOR OF CONSTANS 1 (SOC1) also known as AGAMOUSLIKE20 (AGL20). These meristem identity genes are critical in the genetic pathway and must be activated to establish floral meristem identity. The second type of genes determines where the different organs within a plant are formed. Most plant homeotic genes belong to a class of related sequences known as MADS box genes and many of these determine floral organ identity. In Arabidopsis five genes have been specified that encode homeotic transcription factors: APETALA1 (AP1), APETALA2 (AP2), APETALA3 (AP3), AGAMOUS (AG) and PISTILLATA (PI). The homeotic genes are divided into three categories (the ABC model of floral development) (Coen & Meyerowitz 1991). Type A activity is encoded by AP1 and AP2 and controls organ identity in the sepals and petals. Loss of type A activity causes formation of carpels instead of sepals in the first whorl, and of stamens instead of petals in the second whorl. Type B activity encoded by AP3 and PI controls organ determination in the second and third whorls. Loss of type B activity results in the formation of sepals instead of petals in the second whorl and carpels instead of stamens in the third whorl. Type C activity encoded by AG controls events in the third and fourth whorls. Loss of type C activity results in the

formation of petals instead of stamens in the third whorl. Without type C activity the fourth whorl, which normally is a carpel, can be replaced with a new flower. Shulga and co-workers (2008) transformed chrysanthemum with three AP1 homologue genes HAM75 and HAM92, from sunflower and CDM111 from chrysanthemum. Chrysanthemum flowers normally under short day conditions and little is known about the genetic regulation of flowering in chrysanthemum. In Arabidopsis thaliana, CONSTANS (CO) and FLOWERING LOCUS T (FT) are involved in day length regulation of flowering. They up-regulate the floral meristem identity genes LEAFY (LFY) and APETALA1 (AP1). Constitutive over-expression of the compositae AP1homologs in cut chrysanthemum under long-day conditions did not affect plant development. However, under short day conditions transgenic plants started bud initiation two weeks earlier than wild type chrysanthemum. Also the flower bud development was faster resulting in a 5 week reduction in production time. Thiruvengadam and Yang (2009) transformed Eustoma grandiflorum by insertion of two MADS box genes from lily and orchid. The genes were under control of constitutive promoters and the results showed that the second whorl of petals was converted into sepallike structures, and a third whorl of stamen formation was observed. Plants expressing both genes at the same time flowered significantly earlier than the wildtype. Using genetic modification, Natkasuka et al. (2009) reduced the time to flowering of ornamental gentian from one year to four months by insertion of the Arabidopsis FLOWERING LOCUS T (FT) gene, which encodes for a major component protein of the flowering hormone Florigen.

Genetic engineering for improved shape, size:

Other important factors that contribute to the general appearance of ornamentals are the size and architecture of the plants. Control of plant height is done in the commercial production of many potted plant cultures, where the distribution and retail still require a compact growth. This is done by combinations of pruning, in order to break the apical dominance, chemical growth retardants, climate control and other strategies like reduced water or nutrient availability and mechanical stress. To achieve more compact growth several studies have focused on limiting the growth using different transgenic strategies. In a recent study in Kalancho (Ltken et al. 2010) the short internode (shi) mutant gene from Arabidopsis under the control of both a 35S promoter and the gene s own promoter. Dwarfing phenotypes with reduced height and diameter as well as more compact inflorescences were achieved.

Another strategy to reduce plant height is the use of root loci genes ( rol genes) from Agrobacterium rhizogenes. The bacterium inserts genes into the plant, which alter the endogenous hormone levels and thereby reduce the growth of the plant. Examples of ornamentals transformed with rol genes are Antirrhinum majus (Handa 1992a), Datura sp.(Giovannini et al. 1997), Eustoma grandiflorum (Giovannini et al. 1996; Handa 1992b; Handa et al. 1995), Gentiana sp. (Hosokawa et al. 1997; Momcilovic et al. 1997; Suginuma & Akihama 1995; Vinterhalter et al. 1999), Kalancho (Christensen et al. 2008) and Pelargonium sp. (Pellegrineschi & DavolioMariani 1996; Saxena et al. 2007). Besides altering plant height, insertion of rol genes can increase or reduce the time to flowering, increase the number of flowers, reduce the flower size, change the flower shape and reduce the fertility and seed set as well as change the leaf morphology and colour (Christensen & Mller 2009). A study in carnation has shown that the insertion of PttKN1 (Populus tremula X Tremuoides knotted 1), a member of the KNOX gene family (Meng et al. 2009), could alter plant morphology. KNOX genes are differentially required for meristem development and reduce cell expansion and differentiation associated with organogenesis (Scofield & Murray 2006). Meng (2009) observed modification in phyllotaxis, with leaf position changing from opposite to whorled, modifications in the stem changing from round to thicker and flatter as well as dwarfism . Alteration of the gibberellin pathway has been reviewed by Bhattacharya et al (2010). They concluded that gibberellin modifications either induced dwarfism or increased plant height. Dwarfism has been achieved mainly using the PcGA2ox1 gene encoding a GA2 oxidase from runner bean (Thomas et al. 1999). GA2 ox catalyses the degradation of GA1 and GA4, and thereby reduces the active form of Giberillic Acid (GA) (Figure 7). Insertion have produced dwarfed plants of Solanum malanocearasum, Solanum nigrum, Petunia sp and Nicotiana sylvestris, T. aestivum and O. sativa (Bhattacharya et al. 2010; Dijkstra et al. 2008; Hedden & Phillips 2000; Sakamoto et al. 2003).The genes have a multifunctional activity and have been identified in several plant species (Lee & Zeevaart 2002; Lester et al. 1999; Martin et al. 1999; Sakamoto et al. 2003; Thomas et al. 1999; Ubeda-Tomas et al. 2006). Ectopic expression of many different GA2 oxidase genes results in dwarfism. Dwarfism can be induced by other GA manipulating strategies as well. Examples of this is antisense insertion of GA20 (Figure 7) (Bulley et al. 2005; Coles et al. 1999), interference of micro-RNA (RNAi), double stranded RNA against specific GA biosynthesis genes (Fire 1999; Sharp 2001) and the introduction of GA insensitive (GAI) genes (Yasumura et al. 2007) are other ways to achieve reduced height. Experiments to increase the length of the plant using GA alteration has been achieved by overexpression of GA20 oxidase from various plant species, while overexpression with GA3 oxidase results in a significant but less dramatic effect (Chiang et al. 1995; Itoh et al. 1999; Lange 1997; Martin et al. 1997; Toyomasu et al. 1998).

Figure 7: A portion of the GA biosynthesis pathway showing the metabolic steps that can be blocked by mutants or altered by genetic modification (Sponsel & Davies 2010)

Genetic engineering for floral scent:

Genetically engineering floral scent may enhance the value of cut flowers to consumers. Floral scent is due to volatiles that are primarily derivatives of terpenoids, phenylpropanoids/benzenoids or fatty acids (Pichersky and Dudareva 2007; Zvi et al. 2008). The biochemical pathways involved in the production of plant volatiles are complex (van Schie et al. 2006; Yu and Utsumi 2009) but some genes encoding enzymes of the pathways have been isolated and characterised (Dudareva et al. 2006; Colquhoun et al. 2010; Guterman et al. 2006; Dudareva and Pichersky 2008). Metabolic engineering of floral scent via overexpression of heterologous structural or regulatory genes or by down regulation of existing pathways has been used to elucidate biochemical pathways (Pichersky and Dudareva 2007; Dudareva and Pichersky 2008; Colquhoun et al. 2010; Guterman et al. 2006; Zvi et al. 2008). However, we are unaware of any reports where flower fragrance has been restored or manipulated to commercially significant levels. One aspect of modification of fragrance that must be taken into account is the possibility of altered plant insect interaction, as many insect pests are attracted to or deterred from plants by volatile chemicals produced by the plants (Dudareva et al. 2006). Plant volatiles play a role in insect resistance (Dudareva and Pichersky 2008) and in attracting predatory mites. In some flowers, including rose (much admired for the fragrance of its flowers in garden varieties) natural fragrance has been lost in modern cut flower varieties. This is because fragrance is correlated to poor vase life and breeders have largely eliminated fragrance during selection

for longer vase life. The re-introduction of fragrance into some of the more widely grown cut flowers would be a very useful commercial achievement.

Current status of ornamental genetic transformation:

Transgenic plants were first produced more than 20 years ago from protoplastderived calluses and leaf discs co-cultivated with Agrobacterium tumefaciens (Horsch et al., 1984, 1985). Other gene transfer methods have also been developed, including: (1) Microprojectile bombardment or biolistic transformation (Fitch et al., 1990; Vasil et al., 1992; Marchant et al., 1998a); (2) Infiltration with A. tumefaciens or in-planta transformation (Bechtold et al., 1993; Trieu et al., 2000); (3) Sonication-assisted Agrobacterium-mediated transformation (Trick and Finer, 1997; Zaragoza et al., 2004); (4) Electrophoresis (Griesbach, 1994); (5) Agrobacterium rhizogenes (Berthomieu and Jouanin, 1992); and (6) Direct DNA uptake mediated by electroporation or polyethylene glycol treatment (Shillito, 1999). The majority of transgenic dicot floral crops have been transformed by cocultivation with A. tumefaciens. Microprojectile bombardment was used either as means of gene delivery (Zuker et al., 1995) or as a wounding method to increase Agrobacterium mediated transformation in carnation (Zuker et al., 1999). Microprojectile bombardment and A. tumefaciens have also both been used as gene delivery methods to transform several monocot floral crops successfully. More than 20 floral crops have now been transformed. Recently, the green fluorescent protein gene (GFP) has been employed as a useful marker for developing transformation systems in several crops (Elliott et al., 1999; Huber et al., 2002) including floral crops (Tamura et al., 2003; Kim et al., 2004). GFP can be visualized using a fluorescence microscope allowing detection, isolation, and tracking of transformation events. Lately, five novel fluorescent proteins have been evaluated and shown to be valuable reporting tools for transformation in both dicots and monocots (Wenck et al., 2003).

Application of Genetic Modification to Ornamental Horticulture: Cut Flowers: Already, varieties of carnation are commercially available that have a modified flower color. The primary focus on color modification is important in cut flower because flower color is such an important driver of new variety development. Strategies for color modification by manipulating genes in the anthocyanin biosynthesis pathway have been reviewed elsewhere (Tanaka et al., 1998, 2005; Mol et al., 1999). Other important traits in cut flowers are vase life and perfume, both of which are potentially amenable to genetic modification. The plant hormone ethylene is involved in senescence in many flowers and fruits and vase life can be extended by either blocking ethylene biosynthesis (Savin et al., 1995) or ethylene reception (Bovy et al., 1999). As a result of breeding, fragrance has been lost in many modern floral varieties. S-Linalool is found in the scents of many flowers and the gene coding for S-linalool synthase has now been isolated from Clarkia breweri. This

gene and other genes derived from the same species are being used to modify scent in carnation and lisianthus (Lewinsohn et al., 2003). Several genes involved in scent formation in roses have also been identified and characterized (Guterman et al., 2002; Lewinsohn et al., 2003). From the producers perspective, disease and insect control are major cost inputs, so any increased resistance to the most common pests, particularly virus, aphids, mites, and thrips would be very valuable. Pot and Bedding Plants: Novelty is also an important driver of new variety development in the pot and bedding plant industries. Color modification, enhancement of flower life, and manipulation of plant and flower form are all attractive traits from a marketing point of view. Herbicide resistance in bedding plants can be expected to significantly reduce the cost of weeding in a landscape environment. From a growers perspective, any genetic improvements that would reduce the cost of chemical application to combat diseases and pests would be of significant benefit. Research on modification of hormone biosynthesis or metabolism and on expression of the floral meristem identity gene has been undertaken (Meeks-Wagner, 1996; Zheng et al., 1999; Theissen, 2000). These results may one day be directed towards manipulating the speed to flower, or the day-length requirements for flowering. In some pot plants, chemicals are used routinely to dwarf plants. By manipulation of hormone metabolism, this could instead be achieved by genetic modification (Baker et al., 2002; Petty et al., 2003). Delayed leaf senescence has been achieved in tobacco and petunia by manipulation of cytokinin synthesis (Clark et al., 2003).

COMMERCIAL PRODUCTION OF TRANSGENIC ORNAMENTALS: At this time the only commercially available transgenic ornamentals appear to be the "Moon" series of carnations offered for sale by Florigene in Australia. Canada, and the USA. There are six cultivars with different intensity of blue/purple pigmentation ranging from pastel shades to almost black; the Florigene web site (www.flori(Yene.com) lists twenty outlets in the USA, and states that the flowers are grown under contract in Ecuador and Colombia for the North American market. At an earlier time these flowers were also offered in the Japanese market, but that is no longer apparent from the company web site. The "blue" coloration of the transgenic carnation is due to expression of a heterologous flavonoid 3',5'-hydroxylase(F3',5'1-1) gene (Fukui et al., 2003). Florigene, together with chrysanthemum breeders Fides, also produced transgenic chrysanthemums expressing sense or antisense copies of the Chs gene encoding chalcone synthase (CHS), which were field tested in California and Florida. These plants were white or light pink instead of pink like the parental cultivar 'Money maker', although the proportion of cuttings with pale pink (Courtney-Gutterson et al.,1994). One line was named 'Floriant', and was apparently intended as a test case for the approval procedure for genetically modified ornamentals by the Dutch government, but may not have had high commercial prospects because of the large number of white chrysanthemum cultivars already available (Bijman, 1994). OTHER FUCTIONAL TRANSCENIC ORNAMENTALS: Over 30 genera of ornamentals have been transformed and regenerated, but many of these were transformed only with marker genes such as GUS or selectable markers such

as antibiotic or herbicide resistance (reviewed by Deroleset al., 2002). There are as yet relatively few reports of ornamentals transformed with genes that contribute to ornamental traits (mainly flower color), or pest and disease resistance. Most of these reports are also restricted to laboratory and greenhouse evaluations. The web site of the Japanese Ministry of Agriculture. Forestry and Fisheries, Biotechnology Safety Division (www.s.affrc.go.jp/docs/sentan/eguide/edevelp.htm).reports that several ornamental crops have been field-tested in Japan, including carnation expressing ACC synthase (for ethylene insensitivity and longer vase life; Suntory and Florigene), F3',5'H and dihydro-flavonol reductase (DFR) (flower color; Florigene and Suntory); viroid-resistant chrysanthemum (Ishida et al.. 2002); TM V-resistant petunia (Suntory); and torenia expressing DFR and CHS (flower color; Florigene and Suntory; Aida et al., 2000a,b; Suzuki et al., 2000). Meyer et al. (1987. 1992) produced and field-tested an orange-flowered petunia resulting from expression of a maize DFR gene. Bradley ciat. (1998) produced red-leaved petunia expressing the maize Lc regulatory gene, which have been field-evaluated for potential commercial production (Deroles et al.. 2002). Plant form (Winefield ci al..1999), ethylene sensitivity (Gubrium et at., 2000). and fungal resistance (Esposito et al..2000) are among other traits that have been examined in petunia. Gerbera have been transformed with a heterologous gene encoding CHS (Elomaa et at., 1993, 1996).Markham (1996) transformed lisianthus with a snapdragon anthocyanin pathway gene, while Deroles and coworkers have introduced a number of genes related to flower color (Deroles et al., 2002). Zuker et at. (2001) have reported carnation transformed with rolC, resulting in altered plant form, and with osmotin, PR-1 and/or chitinase for resistance to Fusarium, oxysporum. Takatsu et al (1999) generated chrysanthemum with a rice chitinase gene for resistance to Botrvtis, now being evaluated in the field (Deroles et al., 2002). Improved stature and increased fragrance was reported in lemon-scented geranium transformed with rolC (Pellegrineschi et al.. 1994). Bi et at. (1999) transformed scented geranium with an anti-microbial protein from onion to yield increased resistance to Botrytis cinerea and bacteria. Renou et al. (2000) reported that a chimeric cecropin gene conferred resistance to Xanthomonas in transgenic Pelargonium hortorum and P. peltatum worthy of field testing. Chen and Keuhnle (1996) produced anthurium plants expressing the antibacterial peptide attacin, white Liau et at. (2003a) showed that Oncidium orchids expressing sweet pepper ferredoxin-like protein were resistant to bacterial soft rot caused by Erwinia carotovera. Roses have been transformed with a rice chitinase gene to increase resistance to black spot caused by Diplocarpon rosae (Marchant et al., 1998), and with cecropin B to increase bacterial resistance and thus enhance vase life (Derks et al., 1995). Expression of the anti-microbial peptide Ace-AMP 1 conferred enhanced resistance to powdery mildew of rose caused by Sphaerotheca pannosa (Li et al.. 2003). A rolA,B,C rose rootstock rooted more efficiently, and produced more flowers on non-transgenic scions grafted to the rootstock (Van der SaIm et al., 1998). Table: Ornamental species for which trangenic plants have been generated (revised from Bajaj 2001; Deroles et al. 2002; and Chandler and Tanaka 2007) Species Agapanthus sp. Gladiolus grandiflorus Species Ornithogalum spp. Delphinium spp.

Alstroemeria spp. Hemerocallis sp. Antirrhinum majus Ipomea nil Anthurium andraeanum Iris germanica Begonia spp. Kalanchoe spp. Calanthe sp. Lavatera sp. Campanula spp. Lentopodium alpinum Cattleya spp. Linum spp. Chrysanthemum morifolium Lilium spp. Cyclamen persicum Lobelia erinus Cymbidium spp. Nierembergia scoparia Datura spp.

Osteospermum spp. Dendrobium spp. Pelargonium spp. Dianthus caryophyllus Petunia hybrida Doritaenopsis hybrids Phalaenopsis spp. Eschscholzia californica Rhododendron spp. Euphorbia pulcherrima Rosa spp. Eustoma grandiflorum Rudbeckia hirta Forsythia intermedia Saintpaulia ionantha Gentiana spp. Torenia fournieri Geranium spp. Verbena hybrida Gerbera hybrid Verticordia grandis

Flower colour: Recently, genes involved in metal ion uptake (Colangelo and Guerinot 2006) ion storage (Shoji et al. 2010) and the regulation of vacuole pH (Verweij et al. 2008) have been identified. As both metal ion-anthocyanin complexes and the vacuolar pH environment are known to be factors affecting petal colour, utilisation of these genes for further modification of flower colour is a strategy being employed by several groups. The manipulation of colour in the yellow and orange range will become increasingly feasible as more is learnt about the carotenoid biosynthesis pathway (Cazzonelli and Pogson 2010; Tanaka and Ohmiya 2008). Only flower colour altered GM flowers varieties have been released to date, and only in carnation and rose.

Table . Breeding goals and ornamental genera involved in programs using genetic transformation. (adapted from Cadic, 1999 and completed with year 2000 data ) Breeding goals Flower (or vegetative parts) color Morphology (whole plant or organs) Longevity (whole plant or flowers) Ornamental genera Dendranthema, Dianthus, Eustoma, Forsythia, Gerbera, Orchidaceae, Pelargonium, Rosa, Torenia Begonia, Datura, Dendranthema, Erica, Eustoma, Gentiana, Gerbera, Lilium, Limonium, Osteospermum, Rosa Begonia, Dianthus, Rosa, Torenia

Virus resistance Bacteria resistance Fungi resistance Insect resistance Nematod resistance Herbicide resistance Frost resistance Rooting ability

Dendranthema, Gladiolus, Orchidaceae, Osteospermum, Pelargonium Anthurium, Pelargonium Agrostis, Dendranthema, Rosa, Pelargonium Dendranthema Tagetes, Rudbeckia Agrostis Dendranthema Dianthus, Rosa

Barriers to genetically engineered ornamentals:

Genetic engineering is a technique well suited to the generation of new commercial varieties of ornamentals. It provides a precise and predictable method to modify color, habit, flower, form shelf life and many other valuable traits. Yet, despite the commercial success of genetic engineering in the development of new commodity crops, genetic engineering has not yet been broadly adopted as a tool by ornamental breeders. Despite the large number of published examples and issued permits for GE ornamentals, GE violet carnations, represents the only example of a commercial product (Tanaka 2005). Part of the reason for the failure to commercialize a broader selection of GE ornamentals may be a lack of technical success. However, there are most likely several nontechnical factors underlying the delay in commercializing GE ornamentals. These include product development costs, intellectual property costs, regulatory costs and public perception.

Product development costs:

The most significant limitation to fully using genetic engineering for ornamental plant improvement is the cost associated with the technology. These costs include time, facilities, personnel, and expenses for developing the enabling technology for transforming new plants (e.g., regeneration methods, gene discovery and isolation, etc.), licensing of existing technology, securing regulatory approval, and the eventual development and evaluation of transgenic plants. Discovery and characterization of a novel trait gene is a cost- and labor intensive pursuit lasting several years. Product development costs include transformation of commercial germ-plasm with a previously characterized gene expression cassette as well as all related preliminary greenhouse testing. Costs can be measured in relation to labor, materials, supplies, equipment and facilities. With a defined trait gene-expression cassette, and a reliable transformation system, a novel transgenic line of petunia can be developed in six to nine months. In conclusion, although product development costs can represent a significant incremental increase in costs relative to classical breeding, they probably do not necessarily represent a significant cost increase when compared to other laboratory based non-transgenic techniques such as tissue culture, embryo rescue or mutagenesis.

The regulatory process:

Transgenic plant research is perhaps one of the most highly regulated areas of genetic research. At the international level, GE plants are covered by the Cartagena Protocol on Biosafety, as adopted by signatories to the Convention on Biodiversity in 2000 (SCBD 2000). For release and commercialization there are three agencies involved in review and approval of GE plants. The USDA-APHIS is responsible under the Plant Protection Act (USDA 2000), for reviewing the plant pest potential of a GE crop. The EPA is, under the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA), responsible for reviewing and registering their pesticidal properties. The Food and Drug Administration (FDA) is involved in voluntary consultation on the safety of GE foods, which generally would not apply to ornamental plants (FDA 1992). A unified website that provides a single point of information access for all three agencies has been developed (NBII 2007). This web site provides an up to date summary of registrations and reviews by each agency, as well as access on the laws and regulations enforced by each agency.

Intellectual property challenges:

The challenge to obtain freedom to operate (FTO) among an increasingly complex web of utility patents covering traits genes, selectable markers and transformation methods has become a major barrier to the development and commercialization of GE ornamental plants and other specialty crops. The barrier can be measured in terms of costs and risks that a developer might encounter in terms of attempting to obtain FTO. These includes costs related to FTO analysis, license negotiation, license fees and royalties, as well as risks related to undisclosed and unpublished patent applications, the refusal of a patent owner to license the technology, the uncertainty of patent ownership as well as the cost and risks of legal disputes.

Public perception:
It is not so easy to know and predict consumers tastes as they are changing following most of the time an unpredictable way where fashion may be a strong driving force. To avoid this situation, one can try to forecast consumers' tastes through surveys. Public perception of genetic engineering, is very much dependent on the crop and the country in which the product is to be commercialized. There has traditionally been much more public and media opposition to the commercialization of GM plants in Europe and Japan than in the United States. GE carnations are the only example of a commercially available GE ornamental. These are now sold in more than ten countries around the world, including Japan, the United States, and several European countries. Market acceptance has been generally good, and it does not appear that wholesalers or retailers have experienced significant opposition to these products.

Future Prospects for Genetically Modified Ornamental Plants:

Ornamental horticulture is a very important economic aspect of horticulture, and floriculture is in turn a dominant sector of ornamental horticulture, one feature of floriculture, which covers both cut flowers and pot plants, is that certain crops dominate sales. This is obviously an important consideration in any program aimed towards the development of genetically modified ornamental products. A second feature is that the cut flower industry is a global industry. Countries with high altitude, equatorial, or tropical

latitudes favorable to flower production have developed large industries around the airfreight export of flowers. New genes will be isolated that will have utility in floriculture, and transformation methods for flower crops will be further optimised. As a result, ways for tackling some of the more qualitative traits using genetic modification, such as productivity, may be on the horizon. A genetic map of rose, which is commercially the most valuable cut flower, has now been developed (Debener and Linde 2009). Identification of quantitative trait locus (QTL) from this map, in conjunction with genetic modification, may assist breeders to improve productivity, disease resistance and insect resistance. Another potential avenue of interest is the use of regulatory genes, such as those encoding transcription factors (Century et al. 2008). Expression of these genes can have effects on flower colour (Yamagishi et al. 2010), fragrance (Zvi et al. 2008) and disease resistance (Pandy and Somssich 2009). Addition of transcription factors such as MADS box genes may be used to change the colour of vegetative plant parts of floricultural species (Han et al. 2009) or to change time to flowering (Shulga et al. 2009) and flower form (Thiruvengadam and Yang 2009). The development of new and superior plants helps to stimulate excitement and demand, improve competitiveness and profitability, and ultimately deliver superior and more diverse products to consumers. Genetic engineering has numerous theoretical advantages over other plant improvement methods. For example, specific traits can be inserted or modified in superior cultivars without altering other desirable traits. Conventional breeding can be used to similarly modify existing traits. However, seven to ten years are typically required to develop a new plant with the novel trait. In addition, conventional breeding is limited by the existing gene pool within a given species. Mutation breeding is useful for introducing traits that do not already exist within a gene pool, but the process is completely random necessitating the evaluation of large numbers of progeny. There are, however, a growing number of unpatented alternative technologies that may make it more feasible for researchers to develop genetically engineered minor crops including ornamentals. In spite of these stated challenges to genetically engineering ornamentals, numerous projects are underway within both public and private institutions. Although the majority of these projects are focused on herbaceous ornamental crops, some include woody ornamental species as well. Herbaceous crops, especially those grown for cut-flowers, are an obvious starting point for ornamental genetic engineering for several reasons. Herbaceous ornamental plant species are sold in much greater numbers and are often exported worldwide unlike most woody ornamental plants. This makes the potential short-term return on investments much more likely. Also, systems for regenerating many herbaceous plants from single cells, a process that is currently essential for most transformation systems, are already in place. Numerous ornamental traits have been targeted for modification through genetic engineering including cut-flower vase life, extra flower petals, rose flower fragrance, flower color in forsythia and numerous herbaceous plants, and enhanced plant architecture. Efforts to engineer sterility in some woody ornamental plants have been reported. Objectives of these efforts include preventing the spread of invasive woody species, elimination of fruit litter on streets, parking lots and sidewalks and insect and disease resistance. Significance to Industry: Large numbers of new ornamental plants are introduced each year serving to maintain or even bolster enthusiasm among consumers. Sources of new plants

are numerous each having its own distinct advantages. Augmenting current plant improvement methods with genetic engineering technologies should facilitate developing traits that would be difficult or impossible to capture using more traditional methods. Insect and disease resistance, increased environmental stress tolerance, adventitious root formation in recalcitrant species, reduced invasiveness, reduced pollen and allergen production, and improved floral and foliage characteristics are a few possible targets in ornamentals. As this technology continues to develop and becomes more affordable, it is inevitable that genetic engineering will impact new ornamental plant development.

Conclusion:
Transformation of ornamental plants has progressed dramatically in the last decade, to the point where it is realistic to expect that almost any species can be transformed if sufficient time and resources are directed at the task. Still, most transformed ornamentals contain only single genes that are coupled to a selectable marker. As with other groups of plants, transferring multiple foreign genes, such as those for a biosynthetic pathway, remains a challenge. Two ways to achieve this are: (1) to introduce several genes in a single step, or (2) cross different transgenic plants containing individual genes. The second option is generally not possible with most ornamental species due to their high degree of heterozygosity. Crossing phenotypically superior single transgene plants would undoubtedly unravel combinations of desired ornamental traits that took years of traditional breeding to assemble in one genotype. It can therefore be concluded that there was an immense potential in the improvement of ornamentals through transgenic technologies. Once the transformation protocol is standardized, then further improvement of transformation efficiency could be made possible by adopting certain techniques like addition of Vir gene inducing compounds (stimulants), use of various promoters, alternate selection agents or positive selection agents, etc. With the optimised conditions of explant selection, co-cultivation, infection, incubation and screening procedures the genes could be effectively engineered to produce desirable traits. So, by using these improved transformation procedures, genes for modifying the floral architecture, precocious flowering, improved flower colour, size, fragrance, extended vase life and viral resistance could be engineered into ornamentals. It is necessary for the scientists producing transgenie plants to provide convincing evidence to potential growers and consumers on the benefits of the technology. Nurserymen must be convinced by grower trials that show the superiority of transgenic crops resulting from the introduced traits, which will increase the value of the crop. For example, increased vase life due to ethylene insensitivity or reduced bacterial damage is a trait that has benefits for both growers and consumers. The potential for disease-resistant plants to result in improved quality and lowered application of pesticides is another area where scientists must clearly demonstrate the advantages to both growers and consumers in order to gain commercial acceptance.