Current Status of Genetically Modified Ornamentals

John Hammond USDA-ARS. United States National Arboretuni Floral and Nursery Plants Research Unit Beltsville MD 20705 USA

Keywords: Virus resistance, disease resistance. trarisgenic plants, flower color

Abstract Biotechnology offers new means of improving ornamental crops by the addition of desirable traits to existing horticulturally-adapted cultivars. Traditional plant breeding involves the exchange of many hundreds or thousands of genes between related species to create novel combinations, and improved genotypes must be selected from thousands of segregating progeny over multiple c ycles of selection. Introgression of a single desirable new character, such as disease resistance, from an unadapted relative of a crop species may require multiple backcross generations to regain the horticultural or ornamental qualities of the original cultivar, often resulting in reduced intensity of expression of the back-crossed character. In the case of many ornamental species, related germplasm with the desired disease resistance has not been identified. This is particularly true in the case of viruses, where few sources of resistance are known among ornamentals and related species. However, it has been demonstrated that effective resistance against many plant viruses can be obtained, in model systems and in some field crops, by introduction of virus-derived sequences or various anti-viral genes. Viral infection is a significant problem in many ornamentals, and especially floral crops, because so many ornamentals are vegetatively propagated. Virus infection frequently reduces floral quality and value as well as y ield. Transformation of crop species with one or more gene constructs can lead to addition of desirable traits, typically with minimal effects on other desirable characteristics. Transformation systems have currently been developed for over 30 species of herbaceous ornamentals and several woody species, and efforts continue in man y laboratories around the world. Several groups are working to introduce virus resistance into various ornamentals, including lilies, gladiolus, chrysanthemums, and various orchids. Other traits of interest include plant architecture, floral longevity, flower color, and resistance to other environmental stresses including drought, pests, and fungal and bacterial diseases. A few genetically modified ornamental crops have been commercialized, notably carnations with altered flower color, and there is future expectation of the introduction of effective virus resistance into commercial floral crops.

INTRODUCTION The ability to introduce additional traits into horticulturally-adapted genotypes is important in all crop types. and perhaps especially so in ornamentals. Whereas there is a broad genetic base available for most agronomic crops, with extensive collections of germplasm from the centers of diversity, ornamentals are frequently derived from a far more restricted gene pool. Apart from a few major crops such as chrysanthemum, roses, and petunia, relatively ftw professional plant breeders work on each of the very large number of crops that make up the ornamentals industry. Some of the variety present in the form and color of ornamentals has arisen from somatic mutations. or sports, of existing varieties, that are often propagated vegetatively. In some of the major ornamental crops radiation breeding or other means of inducing mutations are used as an adjunct to traditional breeding, especially to produce series of plants that dilThr in color but otherwise have uniform production characteristics. It is probable that some resistance has been lost in the selection tbr ornamental

Proc. XI h IS on Virus Diseases in Ornamentals Ed. C.A. (hang Acta tIort. 722, ISIIS 2006 117 h1k traits because many of the crops that we currently grow have been selected almost entirely for their ornamental qualities, with little regard to potential resistance to pests or diseases. Mass production, particularly in facilities where many other crops are also grown, often exposes ornamentals to pathogens or pests that are not present in their native habitat. When susceptibility to pests or diseases becomes apparent following large-scale production of a new crop, or as a consequence of the co-production of different crops in protected structures that sometimes offer an ideal environment for pests and diseases, there are few places to turn for genes to combat the problems. Frequently little is known about the genetics of the genus, and in those relatively rare instances where resistance can be identified in related gennplasm or wild species, transfer of the resistance is often very difficult: the problems of maintaining ornamental qualities while introgressing disease resistance from a wild relative may be almost insurmountable. Indeed, any traditional hybridization involves the interaction between at least several thousand different genes to create novel genotypes: hundreds or thousands of segregating progeny must he grown to the point at which desirable phenotypes can be selected from the population. Typically multiple cycles of selection are required, and introgression of a single desirable new character from an unadapted relative of the crop species (such as disease resistance) may require multiple backcross generations to recover the horticultural or ornamental qualities of the original cultivar. In many cases the desirable character is also expressed at reduced intensity due to pteiotropic effects. Additional problems involving wide hybridization, resulting in low fertility or the necessity to use techniques such as embryo rescue, are also frequently encountered. The advent of transformation, with the ability to add a single gene, or a small number of genes in a relatively controlled manner, was recognized as an opportunity to introduce additional traits into ornamentals that had already been highly selected for aesthetic qualities. If a sufficient number of transformants are examined, there is a good probability of identifying a line that has the introduced gene expressed at an appropriate level, without interference with plant growth or other traits. There is also the possibility to add traits that do not exist in closely-related germplasm, and that could not therefore be introduced even by an extensive traditional breeding program. Examples of such traits include genes affecting [lower pigmentation (mainly genes in the anthocyanin pathway) or longevity (mainly ethylene insensitivity), plant architecture (e.g. stature or branching patterns), resistance to environmental stresses (including heat and drought), and resistance to insects, bacteria, fungi, and viruses. Genes from different organisms can be combined, if necessary for example transgenic "golden rice" utilizes two genes from narcissus under the control of an endosperm-specific promoter, and one gene from the bacteria Erwin/a wedovera expressed from the cauliflower mosaic virus 35S promoter (Ye et al., 2000). In other instances synthetic peptides can confer resistance to bacterial, fungal, or viral diseases (e.g. Jaynes. 1993: Rudolph et al., 2003).

TRANSFORMATION SYSTEMS FOR ORNAMENTALS Transgenic ornamentals of various species have been produced by different systems, including Agrobacterium-mediated transformation (e.g. chrysanthemum. Courtney-Gutterson et al., 1994), biolistic or particle bombardment (e.g. gladiolus, Kamo et al., 1995a.b), and direct electrophoresis of DNA into meristems (e.g. orchids, Griesbach, 1994). Electroporation or microinection of protoplasts, silicon carbide fibers. pollen treatments, or addition of DNA directly to pollen tubes in an excised style have all been used to obtain transgcnic plants, but not necessarily ornamentals (see Deroles et al.. 2002; and references therein). Agrobac/eriurn or biolistic bombardment are most commonly used, and can be combined (Hansen et al., 1997: Zuker et al., 1999). While Agrohacteuiutn-mediated transformation was initially largely restricted to dicotyledonous species, various modifications have resulted in successful transformation of an increasing number of monocotyledonous species (e.g. lily: Iloshi et al.. 2004). In theory almost any plant species can he transformed. Production of transgenic plants requires a successful transformation event at the cellular event, expression of a

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marker gene allowing favorable growth of transformed cells among a population of non- transfomed cells and selection of putative transformants, and successful regeneration from the transformed cells into whole plants (Robinson and Firoozabady, 1993). One of the methods of introducing DNA into plant cells can probably he adapted to get DNA into the nucleus of cells of the target species that have the potential to form a meristeni. The second requirement is to he able to regenerate the transformed cell back into a whole plant. With most transformation methods this requires an efficient tissue culture system, but using the direct electrophoresis of DNA into meristems on a living plant (e.g. Griesbach. 1994 Burchi et al., 1995 Vik et al., 2001). it is possible to avoid tissue culture altogether. Shoots arising from the treated meristem may he chimeric, but secondary shoots derived solely from transgenic tissues can be isolated in some instances, excised and rooted to yield non-chimeric plants. To date over 30 species of transformed ornamentals have been reported, including most of the major crops such as chrysanthemums, carnations, several species of orchids. and roses (see Deroles et al., 2002).

COMMERCIAL PRODUCTION OF IRANSGENIC ORNAMENTALS At this time the only commercially available transgenic ornamentals appear to he the "Moon" series of carnations offered for sale by Florigene in Australia. Canada, and the USA. There are six eultivars 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. Florigene has arrangements with two major carnation breeders, and additional types are expected to reach the market before long. The "blue" coloration of the transgenic carnation is due to expression of a heterologous flavonoicl 3,5-hydroxylase (F3,51-1) 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 Moneymaker, although the proportion of cuttings with pale pink (lowers was higher in Florida than in California trials (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 Florigenc), F3,5H and dihydroflavonol 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: see also Aida et al., 2000a,b; Suzuki et al., 2000). Meyer et al. (1987. 1992) produced and field-tested an orange-flowered petunia

119 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 tisianthus with a snapdragon anthocyanin pathway gene. while Deroles and co-workers have introduced a number of genes related to (lower color (Deroles ci at., 2002 and references therein). Zuker et at. (2001) have reported carnation transformed with roiC, resulting in altered plant form, and with osmotin, PR-1 and/or chitinase for resistance to Fusariu,n oxvsporwn. Takatsu ci a! (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 roK (Pcllegrineschi ci at.. 1994). Bi et at. (1999) transformed scented geranium with an anti-microbial protein from onion to yield increased resistance to Botrrti.s cinerea and bacteria. Rcnou et al. (2000) reported that a chimeric cecropin gene conferred resistance to Xanthoinonas in transgenie Pelargoniwn horiorum and P. peltatuni worthy of field testing. Chen and Keuhnle (1996) produced anthurium plants expressing the antibacterial peptide attacin, white Liau et at. (2003a) showed that Onciclium orchids expressing sweet pepper ferredoxin-like protein were resistant to bacterial soft rot caused by Erwinia caJoto vera. Roses have been transk)rmed with a rice chitinase gene to increase resistance to blackspot caused by Diplocarpon ro,sae (Marchant et al., 1998), and with cecropin B to increase bacterial resistance and thus enhance vase life (Derks ci al., 1995). Expression of the anti-microbial peptide Ace-AMP I conferred enhanced resistance to powdery mildew of rose caused by Sphaerothcca pannosa (Li et al.. 2003). A rolA,B.0 rose rootstock rooted more efficiently, and produced more flowers on non-transgenic scions grafted to the rootstock (Van der SaIm et al., 1998). VIRUS AND VtROID RESISTANCE IN ORNAMENTALS A number of ornamentals expressing virus-derived constructs expected to confer disease resistance have been produced to date. A greater number of constructs of relevance to viruses affecting ornamental plants have been tested in model systems, and shown to confer some resistance (see below). It is beyond the scope of this overview to review the mechanisms of resistance conferred by different constructs, but in many instances resistance is apparently conferred by post-translational gene silencing (e.g. Goldbach et al., 2003). Given that methods of transforming ornamentals are becoming more efficient, we can expect to see many more reports of virus-resistant transgenic ornamentals over the next few years. Prior reports of virus-resistant transgenic ornamentals include chrysanthemum, Gerhera. and Osteospermum expressing tospovinis genes for resistance. Yepes et al. (1995) used hiolistic transformation to introduce the Tomato sj)otfedl ni/I virus (TSWV) N-gene into leaf and stem explants of four cultivars of chrysanthemum. Stem segments yielded the greatest regeneration efficiency, and a total of 82 transgenic lines were produced. In later workthey compared biolistic and Agrohacterium-mediated transformation methods with three cultivars (Yepes et at.. 999). The easily regenerated cultivar "lridon" was used in both studies, but in the second study they transformed plants with the N-gene of either TS\VV. Impatiens necrotic spot virus (INSV), or Groundnut ring.spot virus (G RSV), data on resistance were not presented. Sherman ci al. (1998) used Agrohacteriuin-mediated transformation to introduce a full-length NT-gene (N 4 ), a translationally truncated N-gene (Nt), or an antisense N-gene (N-) of a dahlia TSWV isolate into chrysanthemum CV. Polaris. They demonstrated that one Nt and two N- lines were fully resistant to thrips-transmutted infection by a virulent chrysanthemum isolate of TSWV. Several N+ lines were infected but showed significantly reduced symptoms

120 W, compared to controls (Sherman et al., 1998). Korbin et at. (2002) used the TSWV N-gene to transform four cultivars of Gerhera. whereas Allavena et al. (2000) transformed Osteosperinum. and in each case resistance to mechanical inoculation with TSWV was demonstrated. Osteosperinum has also been transformed with several constructs derived from Lettuce mosaic virus with the intent of obtaining resistance (Mörhel et al.,2002). Other crops transformed to obtain resistance to various viruses include geranium, gladiolus, hyacinth, lily. ornithogalum, and various orchids. Berthomé et al. (2000) have transformed geranium (Pelargonium x hoitoruin) with the coat protein (CP) gene of Pc/argon/urn tlowerbreak virus (PFBV), with rat 2-5 ol igoadenylate synthetase (2-5A), and the dsRNA-specific ribonuclease Pad no resistance assays were reported. Kamo and co-workers have transformed gladiolus with the CP gene or antisense RNA of Bean yellow mosaic virus (BYMV). the CP gene or defective replicase gene of Cucumber mosaic virus (CMV), and anti-CMV single chain antibodies (Kamo, 1997: Kai-no et al.. 1997: K.K. Kamo, H.T. Ilsu. and R.L. Jordan, personal communication). Some BYMV CP and antisense RNA lines showed a seasons delay in detectability of infection, hut not the apparent immunity to infection or recovery from infection observed with similar constructs in ,Vicooana hentharniana (Kamo et al., submitted: Hammond and Kamo. 1995a.h). Langeveld et al. (cited in Dcroles et al.. 2002) obtained hyacinth plantlets from transformations with the h yacinth mosaic virus (p gene, but analysis of virus resistance has not yet been reported. Similarly Lipsky et al. (2002) report transformation of lily with a delCciivc CMV replicase gene. and Dc Villiers et at. (2000) attempted transformation of Ornithogalurn with the ()rnirhoga/um mosaic virus (OrMV) CP gene. Ishida et al., (2002) transformed chrysanthemum and tobacco with the Pad dsRNA-specific nuclease, and 2-5A/rihonuclease L (RNase L), and have shown that Pac I-expressing chrysanthemum are resistant to Chrysanthemum stunt viroid (CSVd). Pad I and 2-5A/RNase L tobacco are resistant to TMV, CMV, and Potato virus Y(PVY), and Pad I -transgenic potato are resistant to Potato spindle tuber iiroid (PSTVd). At this meeting Clarke et al. (2004) report initial attempts to produce transgenic poinsettia with resistance to Poinsettia mosaic virus (PnMV): Mitiouchkina et al. (2004) describe transformation of chrysanthemum with three constructs derived from the CP gene of Chrvsanihenuirn viril.s B (CVB); and Borth et al. (2004) describe characterization of transgenic resistance to Cvmbidium mosaic virus (CymMV) in Dendrohium orchids and N. henthamiana. RESISTANCE IN MODEl, PLANTS TO VIRUSES OF RELEVANCE TO ORNAMENTALS Tobacco (N. henthamiana) transformed with the CP gene of CymMV was shown to be resistant to mechanical infection (the only means of transmission relevant for CyniMV) by Chia et al. (1992). CymMV infects many other orchid species, and transformation has been reported for Cvmhidium (Yang et al.. 1999), Oocidiu,n (Liau et at., 2003a,h), Ca/ant/ic (Grieshach. 1994 Knapp et at., 2000): Brassia and Doritaenop.sis (Knapp et al., 2000): and Pha/aenopsis (Chan et al., cited m Deroles et al., 2002) in addition to Dendrobium. Rubino and Russo (1995) showed that the replicase gene of another orchid-infecting virus. Cymbidiurn ring,spot virus (CymRSV) conferred effective resistance to CymRSV in N. henthamiana, while Kollar et al. (1993) showed that defective interfering RNA also conferred effective resistance to CymRSV. Various tospovirus-derived constructs have been shown to confer resistance to one or more tospovirLises (e.g. Pang et al., 1992. 1993), including a composite construct of partial TSWV N-protein fused to the CP gene of Turnip mosaic virus (potyvirus), which conferred resistance to both viruses (Jan et al,. 2000). A non-viral approach utilized by Rudolph et al. (2003) recently demonstrated effective resistance to multiple tospoviruses conferred by a dominant interfering peptide. or "aptamer" that interacts with the viral N gene (nudleocapsid) protein. As several crops are susceptible to multiple tospoviruses, and as new tospovirus variants can arise by reassortment or recombination, the aptarner approach may well be one of the more effective approaches.

121 I Other viruses commonly affecting multiple ornamentals are CMV, Arahis mosaic virus (ArNIV; nepovirus), CVB (carlavirus), Broad bean will virus (BBWV; fabavirus), Tobacco streak virus (ISV; ilarvirus), TMV and Thmaio mosaic virus (ToMV) tobamoviruses, and many potyviruses. Transgenic resistance against CMV has been demonstrated in tobacco, tomato and cucurhitaceous plants expressing CP, movement protein, or replicase genes (e.g. Nakajima, et at., 1993; Gonsalves et at., 1992; Anderson et at., 1992; Wintermantet and Zaitlin, 2000), dsRNA (Kalantidis et al., 2002), or satellite RNA (Harrison ci at., 1987; Cillo et at, 2004). Hsu et at. (2004) have shown that anti- CMV single chain antibodies (ScFv) expressed in Ni henthamiana conferred significant resistance to mechanical inoculation with CMV. Lipsky et at. (2002) have reported transformation of lily, and Kai-no and co-workers (K.K. Kamo and H-T. Hsu, personal communication) have transformed gladiolus with a defective CMV replicase gene and separately with anti-CM V ScFv. Plant-expressed antibodies against Artichoke mottled crinkle virus Beet necrotic ye//ow vein virus, and TMV have also been shown to protect plants against infection by the respective viruses (Taviadoraki et al., 1993; Fecker et at., 1997; Zimmermann et al., 1998). A delay in infection, and reduction in the number of plants infected, was reported for N. henthamiana transformed with the CP gene of ArMV (Spielmann et at., 2000). Mitiouchkina et al. (2004) report trans formation of chrysanthemum with different forms of the CVB CP gene. Resistance to tobamoviruses has been reported from expression of truncated repticase, defective movement protein, or CF transgenes (e.g. Golemboski et at., 1990; Cooper et at.. 1995; Lapidot et at., 1993; Powell-Abel et at., 1986). To date no effective resistance has been reported against TSV or BBWV. Resistance against a large number of potyviruses has been reported, including BYMV (Hammond and Kai-no, t995a,h), TuMV (Jan et al., 1999, 2000), and Plum pox virus (e.g. Scorza ci al., 2001) which threatens ornamental as well as fruit-bearing Prunus. Both the CP and a truncated replicase protein of Tomato yellow lea! curl virus (TYLCV; begomovirus) conferred resistance to TYLCV in N. henthamiana and tomato (Kunik et at., 1994; Noris et at.. 1996), suggesting that TYLCV resistance could be engineered into lisianthus. General resistance against viruses and viroids may be possible by expression of dsRNA-specific ribonucteases such as the yeast Pact, or the mammalian interferon- induced 2.5-oligoadenytate synthetase (2-5A)/RNase L system. Ogawa et at. (1996) showed that tobacco plants expressing 2-5A and RNase L were extremely resistant to CMV; however, PVY-infected plants died within 20 days of inoculation, even though PVY infection could not be detected beyond the inoculated leaf, in contrast. Ishida et at. (2002) produced transgenic Pacl and 2-5A/RNase L tobacco plants resistant to TMV, CMV and PVY. Pact potato resistant to PVY and PSTVd. and Pact chrysanthemum resistant to CSVd. This suggests that it may he possible to produce plants with effective multiple virus resistance.

CONCLUSIONS Effective virus resistance has been demonstrated against a large number of plant viruses in a number of transgenic crops. and new means of obtaining resistance are stilt being developed. As more is understood about the mechanisms inducing viral resistance. it is probable that it will he possible to engineer effective antiviral constructs against any given virus. Similarly, it appears that obstacles to transformation of ornamental crops will likely be overcome in the foreseeable future. It is currently possible to produce nuclear stocks of virus-free plants, and to minimize re-infection by careful control of propagation and production facilities to exclude vectors. However, the nature of the industry, with continuous introduction of new plant species and production of many different crops within a single facility. makes complete exclusion of both pathogens and vectors very difficult. The potential for increased quality and productivity that would result from transgenic virus-resistant plants are therefore appealing, especially in an industry in which aesthetic quality is so important. Virus resistance introduced to existing cultivars would

122 also he available to breed new cultivars by traditional breeding methods, with the added advantage that molecular marker techniques for identification of the transgene would he readily available to aid in selection. Against this must be balanced consumer sensitivities and perceptions relating to genetically modified organisms, as well as concerns about worker safety and pesticide usage to control vector and disease spread in current production practices. Perhaps the most critical question for those seeking to develop virus-resistant transgenic ornamentals is whether such crops will be favorably received by the consumer. As only one company currently has commercial product available, which is not yet offered for sale in the European market, it is far more difficult to predict the long term acceptability of transgenic ornamentals in the market place than it is to foresee the technical ability to produce such plants. 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.

ACKNOWLEDGEMENTS Thanks are due to Roger Lawson, John Stommel. Rob Griesbach, Kathryn Kamo, Ramon Jordan. and I lei-ti Hsu for critical review and useful suggestions.

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