Most of the economically important ornamental plants are cut flowers, which are produced by vegetative propagation. For many years, new varieties of ornamental plants have been produced by cross-hybridization and mutation breeding techniques, separately or in combination. Similar to mutation breeding, genetic transformation would also be a useful way of making a one-point improvement of a trait in original cultivars bred by cross-hybridization. Mutation breeding can change a dominant trait to a recessive one mostly. In other words, genetic transformation produces an “additive” one-point improvement, whereas mutation breeding produces a “subtractive” one-point improvement. Furthermore, genetic transformation can modify target traits by direct incorporation of related genes. Genetic transformation methods will be used in the near future as standard breeding tools in combination with traditional breeding methods.

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A broad spectrum of living modified organisms (LMOs) have been developed and commercial production of them have already been started. It is the global requirement to conduct environmental biosafety after the implementation of Cartagena Protocol on Biosafety. Environmental biosafety assessment is of cardinal importance even in confined field trials and at the deliberate release to an environment for practical uses. It is necessary to collect various categories of data prior to fields; not only about the morphological and physiological traits of the LMOs in some confined experimental facilities, but also about characteristics of host organisms in the natural habitat and field for intended releases. Environmental release of LMOs is possible only after collected information is examined systematically and comprehensively in a scientifically-sound manner, and target end-points of such uses are well-identified. Environmental biosafety assessments principally require case-by-case evaluation. Gene-flow, allelopathy and invasiveness are three key aspects in GM plants. In Japan, many crop species do not have their wild relatives due to their historical introduction from overseas. Furthermore, crops grow properly only under agricultural practices, and invasive weediness of these plants is very likely low. In contrast, floricultural plants and trees have related native species located around the fields that can cross with the transgenic counterparts in many cases. In this review, we examine the environmental biosafety assessment of LMO floricultural plants and trees enumerating the example of field trials in Japan, and describe concepts that should be noted for the commercial cultivation and environmental release of these species.

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Insect-resistant transgenic chrysanthemum plants expressing a modified cry1Ab gene of Bacillus thuringiensis were produced, and an environmental risk assessment of these plants was undertaken in preparation for release their into the field. These plants were examined for molecular profiles, morphological and growth characteristics, cross compatibility, production of allelopathic substances, and influence on soil microbes. The results showed that the insect-resistance trait of the GM chrysanthemum plants is stably integrated in the nuclear genome, and the expression of the mcbt gene did not cause significant differences in the morphological characteristics, the production of allelopathic substances, or the effect on soil microorganisms compared to non-GM chrysanthemums. However, because these plants have cross-compatibility with wild related species, they will be forbidden to be cultured in the open field under the Cartagena Protocol on Biosafety. Therefore, we are attempting to introduce a male and/or female sterility trait to GM chrysanthemums with insect resistance to reinforce their biosafety for the practical uses.

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Cross-pollination is an effective method of breeding flowering plants to produce novel variation. However, this strategy is often protracted or demands repeated crossing for several generations to obtain desired traits. Recently, gene-modification technologies have been utilized in plant breeding for manipulation of floral traits. Many homeotic genes that regulate flower development have been shown to encode transcription factors. In this review we describe the utility of transcription factors and a novel gene-silencing technology, the CRES-T system, to effectively manipulate floral traits. When AGAMOUS (AG) and APETALA3 (AP3) were subjected to this system in Arabidopsis, ag-like and ap3-like phenotypes, respectively, were induced with high efficiency as a dominant trait. Plant transcription factors are conserved between different species to some extent and consequently the chimeric repressor derived from Arabidopsis can be applied to other species without any modification. By applying this system, new floral color and shape phenotypes were obtained in Torenia fournieri and Ipomoea nil. Since the CRES-T system is able to overcome the problem of gene redundancy, polyploid plants may be also manipulated with this system. In addition, with the CRES-T system modification of the flower morphology of plants for which limited genome sequence information is available can be expected.

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Manipulation of horticultural plants' traits using genetic engineering has been a challenge because of gene redundancy and limited information concerning genome or other factors necessary for successful engineering. Recently we have developed a powerful tool with potential to overcome these difficulties, a novel gene silencing technology targeting transcription factor, which is designated Chimeric REpressor gene-Silencing Technology (CRES-T). Using this system, we are now analyzing biological functions of transcription factors in Arabidopsis and trying to manipulate morphological traits of various floricultural plants. To provide these information for genetic engineering of horticultural plants, we have developed the ‘FioreDB’ database in a web-based interface (http://www.cres-t.org/fiore/public_db/), which stores phenotypic information induced by various chimeric repressors in Arabidopsis and six floricultural plants, namely torenia, chrysanthemum, gentian, cyclamen, eustoma, morning glory. Users can find gene constructs that induce their preferred phenotype in Arabidopsis using simple searches, and can browse induced phenotypes in floricultural plants. Most phenotypic information has photo data. FioreDB is continually updated by addition of new data derived from the CRES-T analyses. FioreDB will help to improve traits of horticultural plants using the CRES-T system.

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The homeotic protein AGAMOUS (AG) terminates the floral meristem and promotes the development of stamens and carpels in Arabidopsis. Disruption of its function or expression of the chimeric AG repressor (AGSRDX) results in redundant petals, known as a double flower phenotype. To investigate whether this morphological change in Arabidopsis is applicable to ornamental flowers to increase their horticultural value, we introduced AGSRDX into torenia (Torenia fournieri Lind.) plants. Transgenic torenia plants expressing AGSRDX showed no redundancy in petal number, although they exhibited serration in petal margins, anthocyanin accumulation and morphological change in the stigma surface, and formation of extra vascular bundles in petals and styles. Anatomical observation of petals and styles revealed that these phenotypes are highly similar to those of forchlorfenuron (CPPU)-treated torenia plants especially in the derangement of vascular bundles. Phenotypes similar to AGSRDX transgenic torenia plants were also observed when the chimeric repressors for torenia C-function genes TfFAR or TfPLE1, homologs of Antirrhinum FARINELLI and PLENA respectively, were expressed. These results suggest that the morphological changes in AGSRDX transgenic torenia plants are induced by the disruption of C-function. These novel phenotypes might be caused by the modification of cytokinin-dependent regulation in vascular bundle formation and ectopic expression of the chimeric repressors in all whorls by the cauliflower mosaic virus (CaMV) 35S promoter.

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Creation of new flower shapes is a major breeding target for ornamental plants. The ABC model on the flower structure is known to be applicable to a broad range of plants. Suppression of the C gene would produce a double flower phenotype with loss of floral determinacy. The flower shape of chrysanthemum (Chrysanthemum morifolium) was modified by suppressing the chrysanthemum-AGAMOUS (CAG) gene, which might be a C gene, with an antisense transgene. We obtained 103 transgenic plants; however, only a single line (951-2) showed modified flower shape. The pistil of each ray floret in line 951-2 was transformed to several corolla-like tissues (secondary corolla) and a pistil-like tissue. Southern blot analysis showed multiple-copy integration of the transgene into the 951-2 genome. The amount of CAG mRNA was reduced in line 951-2 compared to the wild-type. On the ray florets, the cell shapes of the adaxial surfaces of the corolla seemed to be almost the same between the proper corolla and the secondary corolla. The stigma was poorly developed and plain structured both on the ray and disk florets. We observed abnormally wide filaments in 951-2. The structure of the surface cells of the abnormal filaments transformed to corolla-like cells. In this study, we demonstrate that suppression of the CAG gene converted the stamen and pistil into corolla-like tissues.

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RNA interference (RNAi) is an efficient and powerful technique for gene silencing compared with antisense and sense suppression. Here we report adaptation of RNAi technology to modify flower colors in gentian, targeted for suppression of three anthocyanin biosynthetic genes; chalcone synthase (CHS), anthocyanidin synthase (ANS) and flavonoid 3′,5′-hydroxylase (F3′5′H). The petals of transgenic gentian plants with a suppressed CHS gene exhibited pure white to pale-blue color, while those with a suppressed ANS gene showed only pale-blue. The suppression of the F3′5′H gene decreased delphinidin derivatives and increased cyanidin derivatives, and led to magenta flower colors. Northern blot analyses confirmed that all transgenic gentian plants showing typical phenotypes had strongly suppressed transcriptions of the targeted genes, corresponding with a change in anthocyanin accumulation and composition in their petals. Some rolCpro-CHSir transgenic gentians exhibited bicolor phenotypes with reduced anthocyanin accumulation along the vascular bundles. These data demonstrated that the suppression of anthocyanin biosynthetic genes by RNAi was successfully applied to gentian plants to change flower color, and this could be useful for designing novel flower color and patterns. Transgenic gentian plants produced in this study might be utilized as elite materials in the breeding of gentian plants in the near future.

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We investigated the efficiency of the 5′-untranslated region (UTR) of the tobacco alcohol dehydrogenase gene (NtADH-5′UTR) as a translational enhancer in chrysanthemum (Chrysanthemum morifolium) and torenia (Torenia fournieri). Three constructs were introduced: control, “ADH−,” cauliflower mosaic virus 35S RNA (CaMV 35S) promoter::β-glucuronidase (GUS); NtADH-5′UTR with a spacer, “ADH+S,” CaMV 35S promoter::NtADH-5′UTR::25-bp spacer::GUS; and NtADH-5′UTR with no spacer, “ADH+,” CaMV 35S promoter::NtADH-5′UTR::GUS. The highest GUS activity in ADH+S and ADH+ for chrysanthemum was about 45 and 190 times, respectively, and for torenia was 12 and 22 times, respectively, than that for the ADH− plants. NtADH-5′UTR enhanced translational efficiency in both species. With the lowest translational efficiency set to 1, the relative translational efficiencies were 1 to 15 (ADH−), 47 to 568 (ADH+S), and 267 to 1360 (ADH+) for chrysanthemum, and 1 to 3 (ADH−), 47 to 114 (ADH+S), and 85 to 226 (ADH+) for torenia. NtADH-5′UTR would facilitate practical breeding and fundamental genetic research in chrysanthemum and torenia.

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Cyclamen persicum is one of the most important pot plants in the world. We developed an effective method for somatic embryo-mediated plant regeneration and genetic transformation using Agrobacterium, with somatic embryos as the source of plant material. Numerous somatic embryos were formed from the leaf segments of Cyclamen cv. “Fragrance Mini” 16 weeks after initiation on a 2,4-D- and kinetin-containing medium. The maximum regeneration frequency from the somatic embryos was achieved using a regeneration medium containing 0.1 mg l⁻¹ benzylaminopurine, 0.01 mg l⁻¹ α-naphthaleneacetic acid, and 0.2 mg l⁻¹ gibberellin. After co-cultivating the somatic embryos with Agrobacterium strain LBA4404 (the plasmid vector pIG121Hm) that harbors genes for hygromycin phosphotransferase, neomycin phosphotransferase, and β-glucuronidase, hygromycin-resistant clones were selected on a medium containing 5 mg l⁻¹ hygromycin. Hygromycin-resistant plantlets were regenerated from 14 hygromycin-resistant embryos. Using this transformation system, 74 independent plants were obtained from 1-ml packed cell volume, approximately 2,000 embryos.

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To shorten the time required for breeding and optimize the risk-cost/benefit ratio of genetically modified ornamental plants, we applied heavy-ion beam irradiation to wild-type and genetically modified torenia (Torenia fournieri Lind. CV. ‘Crown Violet’ plants in which petal color and pattern had been modified by controlling two anthocyanin biosynthesis-related genes encoding chalcone synthase (CHS) and dihydroflavonol-4-reductase (DFR). Ion beams of ¹²C⁶⁺ and ²⁰Ne¹⁰⁺ were applied to 11,500 leaf disks from wild type and five transgenic lines, and over 3,200 regenerated flowering plants were then investigated for visible phenotypes. The mutation rate after whole irradiation averaged 10.4%, and the maximum rate in the initial screening was 44.2% (²⁰Ne, 30 Gy). Mutant phenotypes were observed mainly in flowers and showed wide variation in color and shape. Mutation efficiencies for petal color and coloration pattern were higher in transgenic plants than in wild-type plants, while those for petal shape and corolla divergence were almost equivalent in the two plant groups. Mutation spectrums in petal color in transformant-based mutants were obviously wider than those in wild-type plants. Among these mutants, a class B gene-deficient mutant was investigated as a model case for further study to facilitate the control of flower phenotype. Expression of the TfGLO gene was found to be repressed in this line, probably due to dysfunctioning of the upstream signaling. We propose that the combination of genetic engineering and ion beam irradiation greatly facilitates improvement of agrobiological and commercial traits within a short period. We also discuss characteristic changes observed at high frequency in torenia flowers and the mutant-based approach to the identification of useful genes.

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Sterile mutants of Verbena×hybrida were isolated at high frequency from nodal cultures of developed plants irradiated with heavy-ion beams. Sixty four in vitro-cultured nodes of fertile cultivars ‘Temari Sakura’ (FS), ‘Temari Coral Pink’ (FC) and ‘Temari White’ (FW) were irradiated with 1 to 10Gy of ¹⁴N-ion beam (1890MeV). Lateral shoot development of FS, FC and FW was not affected by irradiation with up to 10Gy. After open-pollination, shoots with inflorescence forming unenlarged ovaries were selected and propagated several times by cutting. Shoots were grown to flowering and the selection process for isolating stable sterile mutants was carried out by the same method. Finally, one mutant out of 104 FS lateral shoots (5Gy), one mutant out of 115 FC shoots (5Gy) and 3 mutants out of 108 FC shoots (10Gy) were successfully isolated. With the exception of sterility all these mutants showed normal morphology. Two sterile mutants SS and SC, which were isolated from 5Gy-irradiated FS and FC, respectively, were characterized by their flowering habits. These two mutants grew well, had a larger number of inflorescences, and a better longevity compared with their parental cultivars. These results show that heavy-ion beam irradiation is an excellent tool for isolating sterile mutants without alterations in others important traits at a high frequency. In addition, the characterization of SS and SC indicated that they have different sterile phenotypes: male and female gametes of SS are non-functional, in contrast, SC exhibits self-incompatibility, which results in mutants unable to produce seeds.

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We previously used heavy-ion beam irradiation to generate a self-incompatible mutant of Verbena×hybrida ‘Temari Coral Pink’ (SC), which exhibits a late-acting self-incompatibility system. In the present study, the behavior of pollen tubes and seed productivity after self-pollination were comparatively investigated in SC and wild species Verbena peruviana (VP), one of the parental species of Verbena×hybrida. Although reciprocal cross-pollination between VP and SC produced seeds with high frequencies, namely 57.1% for SC×VP and 59.7% for VP×SC, self-pollinated VP flowers produced no seeds. In the latter, almost all of the pollen germinated on the stigma, but further growth of the pollen tube was inhibited at the upper part of the style. These observations of pollen tube behavior may indicate that the SI system of VP was different from that of SC. VP may possess a homomorphic gametophytic SI system, which is characterized by the inhibition of pollen tube growth at style. We propose a set of SC and VP as a novel model plants for genetic analysis of the SI mechanism in the Verbenaceae family.

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The biological effects of heavy-ion beam irradiation on cultured tissues of cyclamen were investigated by establishing an irradiation-mediated mutation breeding protocol for producing a new variety of cyclamen. Initially, a callus, a somatic embryo, and a plantlet were irradiated with ¹²C⁶⁺ ion beam at doses of 10, 20, 40, 60, and 80 Gy. Distinct mutants were not obtained in plants regenerated from the irradiated cultured materials. Next, we used the tuber of cyclamen as a target for irradiation. Irradiation (8–16 Gy) of 8–15 mm diameter tubers produced male sterility, change in petal colour, and petal form. Mutation induction by heavy-ion beam irradiation to the tuber is useful for changing flower characteristics of cyclamen.

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Ion-beam mutagenesis is a highly effective way to rapidly create new cultivars. To optimize conditions for heavy ion mutagenesis, we irradiated tobacco (Nicotiana tabacum L.) tissues at various developmental stages with heavy ion beams of various doses and examined the effects of irradiation by monitoring plant growth and mutation induction. The effects differed among irradiated tissues. Sensitivity to heavy ion-beam irradiation increased in the following order: dry seeds, imbibed seeds, and culture tissues. We isolated three white flower mutants. One, BWF1, was found to be a novel mutant, in which the synthesis of proanthocyanidin was up-regulated. The others may have a mutation in some regulatory genes involved in the flavonoid biosynthetic pathway. These results suggest that the developmental state of plant tissues is critical for efficient plant mutagenesis, and that the broad spectrum of mutations may be induced by heavy ion-beam irradiation at molecular level.

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We irradiated Arabidopsis thaliana with several kinds of heavy-ion beams to investigate the linear energy transfer (LET)-dependent effects of heavy-ion beam irradiation. First, dry seeds were irradiated with C, N, Ne, Ar, or Fe ions at doses ranging from 5 to 400 Gy to compare the flowering and mutation rates among the ion species. The sensitivity of the flowering and mutation rates to irradiation differed markedly among the ion species. Of the ion species, N (30 keV μm⁻¹) was the most effective at inducing albino plants. Second, we examined the effects of LET on mutation induction. The LET of C, N, Ne, and Ar ion beams was controlled at 30–640 keV μm⁻¹. Regardless of ion species, irradiation with the same LET value resulted in the same flowering rate and mutation rate. Thus, the LET of ion beams seems to be an important factor affecting mutagenesis. We found a 440-bp deletion in the hy (elongated hypocotyl) mutant that was isolated from M₂ progeny. These fundamental data on LET-dependence can be used to develop advanced technologies for plant mutagenesis.

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Biological samples are irradiated with heavy-ion beams at a heavy-ion beam irradiation facility in RIKEN to perform mutation breeding. The energies of the heavy-ion beams are sufficiently high to irradiate biological samples of macroscopic thickness in air. A uniform dose distribution is a key to a systematic study of the effect of the heavy-ion beams, and thus to the improvement of mutation efficiency. We selected a sufficiently high beam energy to avoid the Bragg peak to realize a uniform dose distribution along the beam path. The outline of the beam line is presented. The linear energy transfers (LETs) of the heavy-ion beams are selected using a range shifter and an energy adjuster to investigate the LET dependence of the irradiation effect. More than five hundred samples are automatically sent to the beam position using an automatic sample changer, which put the heavy-ion beam induced mutation breeding to practical use. The structure and function of the automatic irradiation system are also presented.

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