Transgenic Torenia Research Articles

Downregulation of a Phi class glutathione S-transferase gene in transgenic torenia yielded pale flower color.

The members of glutathione S-transferase (GST) belonging to the Phi class of the GST family are known to play a role in anthocyanin transport to the vacuole. We isolated a GST orthologue from the torenia petal cDNA library. Transgenic plants transcribing GST double stranded RNA were generated from a torenia cultivar having blue flowers. These plants exhibited a range of flower colors, from blue to almost white. Quantitative RT-PCR confirmed the downregulation of the GST transcript, accompanied by a decrease in anthocyanin levels in the petals of the transgenic plants, whereas flavone levels remained unchanged. These results suggest that GST is involved in anthocyanin transport in torenia petals, and that anthocyanins and flavones are likely transported to the vacuole through different mechanisms.

Overexpression of Rice BSR2 Confers Disease Resistance and Induces Enlarged Flowers in Torenia fournieri Lind

Plant pathogens evade basal defense systems and attack different organs and tissues of plants. Genetic engineering of plants with genes that confer resistance against pathogens is very effective in pathogen control. Conventional breeding for disease resistance in ornamental crops is difficult and lagging relative to that in non-ornamental crops due to an inadequate number of disease-resistant genes. Therefore, genetic engineering of these plants with defense-conferring genes is a practical approach. We used rice BSR2 encoding CYP78A15 for developing transgenic Torenia fournieri Lind. lines. The overexpression of BSR2 conferred resistance against two devastating fungal pathogens, Rhizoctonia solani and Botrytis cinerea. In addition, BSR2 overexpression resulted in enlarged flowers with enlarged floral organs. Histological observation of the petal cells suggested that the enlargement in the floral organs could be due to the elongation and expansion of the cells. Therefore, the overexpression of BSR2 confers broad-spectrum disease resistance and induces the production of enlarged flowers simultaneously. Therefore, this could be an effective strategy for developing ornamental crops that are disease-resistant and economically more valuable.

Sucrose Enhances Anthocyanin Accumulation in Torenia by Promoting Expression of Anthocyanin Biosynthesis Genes

We examined the effects of different sucrose concentrations (3%, 5%, and 7%) on anthocyanin accumulation and plant growth in wild type (WT) and transgenic (T2) torenia cultivar “Kauai Rose” overexpressing the anthocyanin regulatory transcription factors B-Peru + mPAP1 or RsMYB1. Sucrose increased anthocyanin production in both WT and transgenic plants, with higher anthocyanin production in transgenic plants compared to WT plants. Higher sucrose concentrations increased production of anthocyanin in transgenic and WT plants, with increased anthocyanin production associated with increased expression of anthocyanin biosynthesis genes. Higher sucrose concentrations reduced growth of WT and transgenic plants. Our results indicate that sucrose enhances anthocyanin production in torenia by regulating anthocyanin biosynthesis genes.

Production of multi-petaled Torenia fournieri flowers by functional disruption of two class-C MADS-box genes.

Simultaneous knockdown or knockout of Torenia fournieri PLENA (TfPLE) and FALINELLI (TfFAR) genes with RNAi or genome-editing technologies generated a multi-petal phenotype in torenia. The MADS-box gene AGAMOUS (AG) is well known to play important roles in the development of stamens and carpels in Arabidopsis. Mutations in AG cause the morphological transformation of stamens and carpels into petaloid organs. In contrast, torenia (Torenia fournieri Lind.) has two types of class-C MADS-box genes, PLENA (PLE) and FALINELLI (FAR); however, their functions were previously undetermined. To examine the function of TfPLE and TfFAR in torenia, we used RNAi to knockdown expression of these two genes. TfPLE and TfFAR double-knockdown transgenic torenia plants had morphologically altered stamens and carpels that developed into petaloid organs. TfPLE knockdown transgenic plants also exhibited morphological transformations that included shortened styles, enlarged ovaries, and absent stigmata. Furthermore, simultaneous disruption of TfPLE and TfFAR genes by CRISPR/Cas9-mediated genome editing also resulted in the conversion of stamens and carpels into petaloid organs as was observed in the double-knockdown transgenic plants mediated by RNAi. In addition, the carpels of one TfPLE knockout mutant had the same morphological abnormalities as TfPLE knockdown transgenic plants. TfFAR knockdown genome-edited mutants had no morphological changes in their floral organs. These results clearly show that TfPLE and TfFAR cooperatively play important roles in the development of stamens and carpels. Simultaneous disruption of TfPLE and TfFAR functions caused a multi-petal phenotype, which is expected to be a highly valuable commercial floral trait in horticultural flowers.

Application of the CRISPR/Cas9 system for modification of flower color in Torenia fournieri

BackgroundCRISPR/Cas9 technology is one of the most powerful and useful tools for genome editing in various living organisms. In higher plants, the system has been widely exploited not only for basic research, such as gene functional analysis, but also for applied research such as crop breeding. Although the CRISPR/Cas9 system has been used to induce mutations in genes involved in various plant developmental processes, few studies have been performed to modify the color of ornamental flowers. We therefore attempted to use this system to modify flower color in the model plant torenia (Torenia fournieri L.).ResultsWe attempted to induce mutations in the torenia flavanone 3-hydroxylase (F3H) gene, which encodes a key enzyme involved in flavonoid biosynthesis. Application of the CRISPR/Cas9 system successfully generated pale blue (almost white) flowers at a high frequency (ca. 80% of regenerated lines) in transgenic torenia T0 plants. Sequence analysis of PCR amplicons by Sanger and next-generation sequencing revealed the occurrence of mutations such as base substitutions and insertions/deletions in the F3H target sequence, thus indicating that the obtained phenotype was induced by the targeted mutagenesis of the endogenous F3H gene.ConclusionsThese results clearly demonstrate that flower color modification by genome editing with the CRISPR/Cas9 system is easily and efficiently achievable. Our findings further indicate that this system may be useful for future research on flower pigmentation and/or functional analyses of additional genes in torenia.

Generation of brilliant green fluorescent petunia plants by using a new and potent fluorescent protein transgene

The application of fluorescent proteins in ornamental plants has lagged behind despite the recent development of powerful genetic tools. Although we previously generated transgenic torenia plants expressing green fluorescent protein from marine plankton (CpYGFP), in which bright fluorescence was easily visible at the whole plant level, the maximum excitation of this protein within the visible light spectrum required the use of a coloured emission filter to eliminate exciting light. Here, to overcome this limitation, we generated transgenic petunia plants expressing eYGFPuv, a CpYGFP derivative exhibiting bright fluorescence under invisible ultraviolet (UV) light excitation, with a novel combination of transcriptional terminator plus translational enhancer. As expected, all transgenic plants exhibited brilliant green fluorescence easily visible to the naked eye without an emission filter. In addition, fluorescence expressed in transgenic petunia flowers was stable during long-term vegetative propagation. Finally, we visually and quantitatively confirmed that transgenic petunia flowers resist to long-term exposure of UV without any damages such as fluorescence decay and withering. Thus, our whole-plant fluorescence imaging tool, that does not require high sensitive imaging equipment or special imaging conditions for observation, might be useful not only for basic plant research but also for ornamental purposes as a novel flower property.

Construction of Flower-specific Chimeric Promoters and Analysis of Their Activities in Transgenic Torenia

Flower-specific promoters can enable transgenic enhancement of valuable ornamental traits, including flower shape and color. However, the identification of strong, tissue-specific promoters remains a limiting factor. To obtain enhanced flower-specific promoters, we constructed four chimeric promoters (p35S-PCHS-Ω, p35S-LCHS-Ω, pOCS-PCHS-Ω and pOCS-LCHS-Ω) combining the 35S or OCS enhancer fused to a 302 bp CHSA core promoter fragment from petunia (PCHS) or a 307 bp CHS core promoter fragment from lily (LCHS), and also containing an omega element (Ω). Each promoter was fused to the β-glucuronidase (GUS) reporter gene, and we examined the levels and tissue specificity of GUS expression in transgenic Torenia fournieri. p35S-PCHS-Ω and p35S-LCHS-Ω drove strong, constitutive GUS expression in all tissues, especially in colored corollas (p35S-PCHS-Ω) or in colored corollas and roots (p35S-LCHS-Ω). pOCS-PCHS-Ω drove stronger GUS expression in colored corollas than in other tissues but expression was weaker than that of p35S-PCHS-Ω. pOCS-LCHS-Ω drove GUS in colored corollas but also in roots. Among the four chimeric promoters, pOCS-PCHS-Ω exhibited stronger activity only in colored corollas, making it useful for transgenic enhancement of floral traits, such as expressing ‘blue genes’ in lily to produce new lines with blue flowers.

Erratum: Acquired immunity of transgenic torenia plants overexpressing agmatine coumaroyltransferase to pathogens and herbivore pests

Erratum: Acquired immunity of transgenic torenia plants overexpressing agmatine coumaroyltransferase to pathogens and herbivore pests

Enhancement of phosphate absorption by garden plants by genetic engineering: a new tool for phytoremediation.

Although phosphorus is an essential factor for proper plant growth in natural environments, an excess of phosphate in water sources causes serious pollution. In this paper we describe transgenic plants which hyperaccumulate inorganic phosphate (Pi) and which may be used to reduce environmental water pollution by phytoremediation. AtPHR1, a transcription factor for a key regulator of the Pi starvation response in Arabidopsis thaliana, was overexpressed in the ornamental garden plants Torenia, Petunia, and Verbena. The transgenic plants showed hyperaccumulation of Pi in leaves and accelerated Pi absorption rates from hydroponic solutions. Large-scale hydroponic experiments indicated that the enhanced ability to absorb Pi in transgenic torenia (AtPHR1) was comparable to water hyacinth a plant that though is used for phytoremediation causes overgrowth problems.

Acquired immunity of transgenic torenia plants overexpressing agmatine coumaroyltransferase to pathogens and herbivore pests.

We investigated the ability of transgenic torenia (Scrophulariaceae) plants to resist fungi and arthropod herbivores. Torenia hybrida cv. Summerwave Blue was manipulated to produce Arabidopsis agmatine coumaroyltransferase (AtACT). This catalyses the last step in the biosynthesis of hydroxycinnamic acid amides (HCAAs) which function in defence. Transgenic plants accumulated substantial HCAAs, predominantly p-coumaroylagmatine, and the HCAAs were isomerized from the trans-form to the cis-form in planta. The transgenic line, accumulated the highest amount of endogenous HCAAs (CouAgm at 32.2 µM and total HCAAs at 47.5 µM) and this line was resistant to the necrotrophic fungus, Botrytis cinerea. There was no resistance, however, in their wild-type progenitors or in other transgenic lines accumulating low HCAA amounts. In contrast, the transformants were not significantly resistant to three representative herbivores, Frankliniella occidentalis, Aphis gossypii, and Tetranychus ludeni.

Arabidopsis chimeric TCP3 repressor produces novel floral traits in Torenia fournieri and Chrysanthemum morifolium

Chimeric REpressor gene-Silencing Technology (CRES-T) is a powerful gene-silencing tool to analyze the function of Arabidopsis transcription factors. To investigate whether CRES-T is also applicable to horticultural plants inadequate for genetic engineering because of their limited molecular biological characterization and polyploidy, we applied CRES-T to torenia and the hexaploid chrysanthemum and produced their transgenic plants expressing the chimeric repressor derived from the Arabidopsis TEOSINTE BRANCHED1, CYCLOIDEA, and PCF family transcription factor 3 (TCP3) fused with a plant-specific transcriptional repression domain named SRDX, consisting of 12 amino acids originated from the EAR-motif (TCP3-SRDX). Transgenic torenia and chrysanthemum expressing TCP3-SRDX exhibited fringed leaves and short pistils, while those expressing TCP3 fused with either the mutated repression domain (TCP3-mSRDX) or the overexpressor of TCP3 (TCP3-ox) did not exhibit phenotypic changes. In addition to fringed leaves, TCP3-SRDX transgenic torenia plants exhibited petals with fringed margins, distinctive color patterns, and reduced anthocyanin accumulation. In TCP3-SRDX transgenic chrysanthemum plants, floral organ development was suppressed as compared with the wild type. These results indicate that the Arabidopsis-derived TCP3-SRDX induced morphological changes in transgenic torenia and chrysanthemum although the observed phenotypes partially differ from each other. CRES-T may function in various plant species including polyploid species and modify their biological characteristics.

Utilization of a floral organ-expressing AP1 promoter for generation of new floral traits in Torenia fournieri Lind

To establish an efficient way to create novel floral traits in horticultural flowers, we have introduced many chimeric repressors of Arabidopsis transcription factors into torenia. Among them, we found a transgenic torenia exhibiting unopened flower buds and glossy dark green leaves with curled margins as a consequence of overexpression of Arabidopsis MYB24 with a transcriptional repression domain (MYB24-SRDX). Petals inside the flower buds exhibited a distinct coloration pattern. To bring out this favorable petal trait without inducing the unfavorable phenotypes due to the constitutive expression of chimeric repressors by the cauliflower mosaic virus 35S (35S) promoter, we tested the ability of a floral organ-specific Arabidopsis APETALA1 (AP1) promoter, which was found to be active in both petals and flower buds of torenia. As expected, AP1 pro:MYB24-SRDX transgenic torenias resulted in the opening of flowers and a normal leaf phenotype. Furthermore, these AP1 pro:MYB24-SRDX torenias exhibited wavy petals with a characteristic configuration. This is a good example of the utilization of a floral organ-specific promoter for creating distinct flower phenotypes without causing unfavorable morphological and physiological changes in other organs.

Functional divergence within class B MADS-box genes TfGLO and TfDEF in Torenia fournieri Lind

Homeotic class B genes GLOBOSA (GLO)/PISTILLATA (PI) and DEFICIENS (DEF)/APETALA3 (AP3) are involved in the development of petals and stamens in Arabidopsis. However, functions of these genes in the development of floral organs in torenia are less well known. Here, we demonstrate the unique floral phenotypes of transgenic torenia formed due to the modification of class B genes, TfGLO and TfDEF. TfGLO-overexpressing plants showed purple-stained sepals that accumulated anthocyanins in a manner similar to that of petals. TfGLO-suppressed plants showed serrated petals and TfDEF-suppressed plants showed partially decolorized petals. In TfGLO-overexpressing plants, cell shapes on the surfaces of sepals were altered to petal-like cell shapes. Furthermore, TfGLO- and TfDEF-suppressed plants partially had sepal-like cells on the surfaces of their petals. We isolated putative class B gene-regulated genes and examined their expression in transgenic plants. Three xyloglucan endo-1,4-beta-d-glucanase genes were up-regulated in TfGLO- and TfDEF-overexpressing plants and down-regulated in TfGLO- and TfDEF-suppressed plants. In addition, 10 anthocyanin biosynthesis-related genes, including anthocyanin synthase and chalcone isomerase, were up-regulated in TfGLO-overexpressing plants and down-regulated in TfGLO-suppressed plants. The expression patterns of these 10 genes in TfDEF transgenic plants were diverse and classified into several groups. HPLC analysis indicated that sepals of TfGLO-overexpressing plants accumulate the same type of anthocyanins and flavones as wild-type plants. The difference in phenotypes and expression patterns of the 10 anthocyanin biosynthesis-related genes between TfGLO and TfDEF transgenic plants indicated that TfGLO and TfDEF have partial functional divergence, while they basically work synergistically in torenia.Electronic supplementary materialThe online version of this article (doi:10.1007/s00438-010-0574-z) contains supplementary material, which is available to authorized users.

Generation of pink flower varieties from blue Torenia hybrida by redirecting the flavonoid biosynthetic pathway from delphinidin to pelargonidin

Transgenic torenia plants with various pink petal tones were obtained from blue or violet cultivars by combining downregulation of endogenous flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′, 5′-hydroxylase (F3′5′H) genes with expression of a heterologous gene. Knockdown of the F3′H and F3′5′H genes in a blue torenia cultivar resulted in pale pink lines by decreasing the cyanidin and delphinidin levels and increasing the pelargonidin-based anthocyanin levels. Additional expression of rose dihydroflavonol 4-reductase (DFR) gene elevated the level of pelargonidin and yielded darker pink petals. Expression of pelargonidin DFR gene instead of the rose DFR gene increased the level of pelargonidin and darkened the petal color. Introducing the two genetic constructs containing a DFR gene into a violet torenia cultivar, which had more anthocyanins and a darker color than the blue one, further increased the level of pelargonidin and pink color intensity. These results reveal that selection of a suitable gene source and host greatly affects the phenotypes of the resultant transgenic plants.

Improvement of Torenia fournieri salinity tolerance by expression of Arabidopsis AtNHX5.

In this paper, transgenic torenia plants expressing the AtNHX5 gene from Arabidopsis in sense and antisense orientations were produced to examine the potential role of AtNHX5 in plant salt tolerance and development. We found that torenia plants overexpressing AtNHX5 showed markedly enhanced tolerance to salt stress compared with both wild-type and antisense AtNHX5 transgenic plants upon salt stress. Measurements of ion levels indicated that Na+ and K+ contents were all higher in AtNHX5 overexpressing shoots than in those of both wild-type and antisense AtNHX5 shoots treated with 50 mm NaCl. This indicated that overexpression of AtNHX5 could improve the salt tolerance of transgenic torenia via accumulation of both Na+ and K+ in shoots, in which overall ion homeostasis and osmotic adjustment was changed to sustain the increase in shoot salt tolerance. Further, we found that overexpression of AtNHX5 in torenia significantly improved the shoot regeneration frequency in leaf explants and increased the plantlet survival rate when transferring the regenerated plants to soil. In addition, the AtNHX5 expressing plants produced flowers earlier than both wild-type and the antisense AtNHX5 plants. Taken together, the results indicated that AtNHX5 functions not only in plant salt tolerance but also in plant growth and development.

High-efficiency improvement of transgenic torenia flowers by ion beam irradiation

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 12 C 6� and 20 Ne 10� 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% ( 20 Ne, 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.

Chimeric AGAMOUS repressor induces serrated petal phenotype in Torenia fournieri similar to that induced by cytokinin application

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.

Genetic transformation of Torenia fournieri using the PMI/mannose selection system

Transgenic torenia plants were obtained using the selectable marker gene phosphomannose isomerase (manA), which encodes the enzyme phosphomannose isomerase (PMI) to enable selection of transformed cells on media containing mannose. We found that shoot organogenesis in torenia leaf explants was effectively suppressed on medium supplemented with mannose, which indicated that torenia cells had little or no PMI activity and could not utilize mannose as a carbon source. Leaf pieces from in vitro-germinated plants were inoculated with Agrobacterium tumefaciens EHA105 containing the binary vector pKPJ with both hpt and ManA genes, and subsequently selected on shoot induction (SI) medium (half strength MS basal + 4.4 μM BA + 0.5 μM NAA) supplemented with 20 g l−1 mannose and 5 g l−1 sucrose as carbon sources. Transformed plants were confirmed by PCR and Southern blot. The transgene expression was evaluated using Northern blot and the chlorophenol red assay. The transformation efficiency ranged from 7% to 10%, which is 1–3% higher than that obtained by selection with hygromycin. This system provides an efficient manner for selecting transgenic flower plants without using antibiotics or herbicides.

RNAi suppression of the anthocyanidin synthase gene in Torenia hybrida yields white flowers with higher frequency and better stability than antisense and sense suppression

Post-transcriptional gene silencing, such as antisense suppression, sense suppression (or cosuppression), and RNAi, is often used to down-regulate a target gene in transgenic plants. Novel flower color is industrially important; furthermore, flower color is a convenient tool to monitor the stability of such silencing. Previously, we obtained white torenia through sense suppression of chalcone synthase or dihydroflavonol 4-reductase (Suzuki et al. 2000). However, their phenotypes were not stable. In this study, we suppressed the anthocyanidin synthase gene using three methods in transgenic torenia. About half of the transgenic torenia plants gave white flowers by RNAi suppression of the gene, while antisense and sense suppression yielded a few and no white flowers, respectively. The white flower color obtained by RNAi has been stable for three years in a greenhouse. This study shows the usefulness of RNAi to suppress a target gene.

Molecular and biochemical characterization of torenia flavonoid 3′-hydroxylase and flavone synthase II and modification of flower color by modulating the expression of these genes

Cytochrome P450 (P450) enzymes play important roles in the biosynthesis of flavonoids that determine flower color. Three P450s, flavonoid 3′-hydroxylase (F3′H), flavonoid 3′,5′-hydroxylase (F3′,5′H) and flavone synthase II (FNSII), are involved in torenia flavonoid biosynthesis. In this study, we isolated a full-length cDNA of F3′H from a torenia petal cDNA library. The deduced amino acid sequence of torenia F3′H has 82 and 80% identity to those of Arabidopsis and petunia F3′Hs, respectively. Phylogenetic analysis showed that F3′H and F3′,5′H genes diverted before speciation of higher plants during evolution. Expression of torenia F3′H cDNA in yeast demonstrated that torenia F3′H catalyzed hydroxylation at the 3′ position of naringenin, dihydrokaempferol, kaempferol and apigenin. Km values for these compounds were 0.83, 3.95, 2.96 and 21.5 μM, respectively. Northern analysis showed that the accumulation of anthocyanins and flavones was transcriptionally regulated and that the transcription of the FNSII gene was differently regulated from F3′H and F3′,5′H genes. The torenia, whose F3′,5′H expression had been suppressed, was further transformed with the F3′H gene driven by a constitutive promoter. Some of the transgenic torenia plants had an elevated amount of cyanidin-type anthocyanins and thus redder flower color. Co-suppression of the FNSII gene in the torenia successfully decreased the amount of flavones and increased the amount of flavanones, and yielded paler flower color.