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Abstract Background Betalains are reddish and yellow pigments that accumulate in a few plant species of the order Caryophyllales. These pigments have antioxidant and medicinal properties and can be used as functional foods. They also enhance resistance to stress or disease in crops. Several plant species belonging to other orders have been genetically engineered to express betalain pigments. Betalains can also be used for flower color modification in ornamental plants, as they confer vivid colors, like red and yellow. To date, betalain engineering to modify the color of Torenia fournieri—or wishbone flower—a popular ornamental plant, has not been attempted. Results We report the production of purple-reddish-flowered torenia plants from the purple torenia cultivar “Crown Violet.” Three betalain-biosynthetic genes encoding CYP76AD1, dihydroxyphenylalanine (DOPA) 4,5-dioxygenase (DOD), and cyclo-DOPA 5-O-glucosyltransferase (5GT) were constitutively ectopically expressed under the cauliflower mosaic virus (CaMV) 35S promoter, and their expression was confirmed by quantitative real-time PCR (qRT-PCR) analysis. The color traits, measured by spectrophotometric colorimeter and spectral absorbance of fresh petal extracts, revealed a successful flower color modification from purple to reddish. Red pigmentation was also observed in whole plants. LC-DAD-MS and HPLC analyses confirmed that the additional accumulated pigments were betacyanins—mainly betanin (betanidin 5-O-glucoside) and, to a lesser extent, isobetanin (isobetanidin 5-O-glucoside). The five endogenous anthocyanins in torenia flower petals were also detected. Conclusions This study demonstrates the possibility of foreign betacyanin accumulation in addition to native pigments in torenia, a popular garden bedding plant. To our knowledge, this is the first report presenting engineered expression of betalain pigments in the family Linderniaceae. Genetic engineering of betalains would be valuable in increasing the flower color variation in future breeding programs for torenia.

Background: Betalains are reddish and yellow pigments that accumulate in a few plant species of the order Caryophyllales. These pigments have antioxidant and medicinal properties and can be used as functional foods. They also enhance resistance to stress or disease in crops. Several plant species belonging to other orders have been genetically engineered to express betalain pigments. Betalains can also be used for flower color modification in ornamental plants, as they confer vivid colors, like red and yellow. To date, betalain engineering to modify the color of Torenia fournieri—or wishbone flower—a popular ornamental plant, has not been attempted. Results: We report the production of purple-reddish-flowered torenia plants from the purple torenia cultivar "Crown Violet." Three betalain-biosynthetic genes encoding CYP76AD1, dihydroxyphenylalanine (DOPA) 4,5-dioxygenase (DOD), and cyclo-DOPA 5-O-glucosyltransferase (5GT) were constitutively ectopically expressed under the cauliflower mosaic virus (CaMV) 35S promoter, and their expression was confirmed by quantitative real-time PCR (qRT-PCR) analysis. The color traits, measured by spectrophotometric colorimeter and spectral absorbance of fresh petal extracts, revealed a successful flower color modification from purple to reddish. Red pigmentation was also observed in whole plants. LC-DAD-MS and HPLC analyses confirmed that the additional accumulated pigments were betacyanins—mainly betanin (betanidin 5-O-glucoside) and, to a lesser extent, isobetanin (isobetanidin 5-O-glucoside). The five endogenous anthocyanins in torenia flower petals were also detected. Conclusions: This study demonstrates the possibility of foreign betacyanin accumulation in addition to native pigments in torenia, a popular garden bedding plant. To our knowledge, this is the first report presenting engineered expression of betalain pigments in the family Linderniaceae. Genetic engineering of betalains would be valuable in increasing the flower color variation in future breeding programs for torenia. [ABSTRACT FROM AUTHOR]

The expression pattern of an interested gene at a cellular level provides strong evidence for its functions. RNA in situ hybridization has been proved to be a powerful tool in detecting the spatial–temporal expression pattern of a gene in various organisms. However, classical RNA in situ hybridization (ISH) technique is time-consuming and requires sophisticated sectioning skills. Therefore, we developed a method for whole-mount in situ hybridization (WISH) on ovules of Torenia fournieri, which is a model species in the study of plant reproduction. T. fournieri possesses ovules with protruding embryo sacs, making it easy to be observed and imaged through simple manipulation. To determine the effect of classical ISH and our newly established WISH, we detected the expression of a D-class gene, TfSTK3, using both methods. The expression patterns of TfSTK3 are similar in classical ISH and WISH, confirming reliability of the WISH method. Compared with WISH, classical ISH always leads to distorted embryo sacs, hence difficult to distinguish signals within the female gametophyte. To understand whether our WISH protocol also works well in detecting genes expressed within embryo sacs, we further examined the expression of a synergid-enriched candidate, TfPMEI1, and clearly observed specific signals within two synergid cells. To summarize, our WISH technique allows to visualize gene expression patterns in ovules of T. fournieri within one week and will benefit the field of plant reproduction in the future. [ABSTRACT FROM AUTHOR]

There are significant societal repercussions from our excessive use of plastic products derived from petroleum. In response to the increasing environmental implications of plastic wastes, biodegradable materials have been proven to be an effective means of mitigating environmental issues. Therefore, protein- and polysaccharide-based polymers have gained widespread attention recently. In our study, for increasing the strength of a biopolymer (Starch), we used ZnO dispersed nanoparticles (NPs), which resulted in the enhancement of other functional properties of the polymer. The synthesized NPs were characterized using SEM, XRD, and Zeta potential values. The preparation techniques are completely green, with no hazardous chemicals employed. The floral extract employed in this study is Torenia fournieri (TFE), which is prepared using a mixture of ethanol and water and possesses diverse bioactive features and pH-sensitive characteristics. The prepared films were characterized using SEM, XRD, FTIR, contact angle and TGA. The incorporation of TFE and ZnO (SEZ) NPs was found to increase the overall nature of the control film. The results obtained from this study confirmed that the developed material is suitable for wound healing and can also be used as a smart packaging material. [ABSTRACT FROM AUTHOR]

Plants can exert remarkable capacity for cell reprogramming even from differentiated cells. This ability allows plants to regenerate tissues/organs and even individuals in nature and in vitro. In recent decades, Arabidopsis research has uncovered molecular mechanisms of plant regeneration; however, our understanding of how plant cells retain both differentiated status and developmental plasticity is still obscure. In this review, we first provide a brief outlook of the representative modes of plant regeneration and key factors revealed by Arabidopsis research. We then re-examine historical tissue culture systems that enable us to investigate the molecular details of cell reprogramming in differentiated cells and discuss the different approaches, specifically highlighting our recent progress in shoot regeneration from the epidermal cell of Torenia fournieri. [ABSTRACT FROM AUTHOR]

Torenia fournieri (T. fournieri) is one of the most widely used horticultural flowers and is considered a potential model plant for the genetic investigation of ornamental traits. In this study, we optimized an efficient protocol for high efficiency preparation and transformation of T. fournieri protoplast. The transformation rate reached ~75% when a 35S:GFP construct was used for the transformation. Using this system, we characterized the subcellular localization of several TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) transcription factors (TFs), and found a distinct localization pattern between the CIN and CYC classes of TCP TFs. Furthermore, we also demonstrated the feasibility of the expression of dual luciferase assay system in T. fournieri protoplasts for the measurement of the activity of cis-regulatory elements. Taken together, a well-optimized transient expression system in T. fournieri protoplasts would be crucial for rapid exploration of the gene function or cis-regulatory elements. [ABSTRACT FROM AUTHOR]

Torenia fournieri belongs to the genus Torenia in the family Linderniaceae. The complete chloroplast genome of T. fournieri was sequenced and analyzed by Illumina sequencing in this study. The full length of the complete chloroplast genome is 153,938 bp, containing a pair of inverted repeat regions of 24,805 bp (IRa and IRb) separated by a large single copy region (LSC) of 85,498 bp and a small single copy region (SSC) of 18,830 bp. The T. fournieri chloroplast genome encodes 131 genes, comprising 87 protein-coding genes, 36 tRNA genes, 8 rRNA genes, without pseudogene. Phylogenetic analysis showed that T. fournieri was closely related to T. benthamiana and T. concolor within the genus Torenia in family Linderniaceae.

Teosinte branched1/cycloidea/proliferating cell factor (TCP) transcription factors (TFs) are essential for regulating plant developmental processes, which is still largely unknown in Torenia fournieri (T. fournieri), a widely used horticultural flower. In this study, we used a de novo transcriptome assembly method to predict the TCP transcription factors in T. fournieri. In total, 15 out of 21 predicted T. fournieri TCPs (TfTCPs) were isolated and verified with Sanger sequencing. Phylogenetic analysis showed that these 15 TfTCPs could be classified into two major classes. Most of these TfTCPs were expressed in floral buds, flowers, or leaves, suggesting an important role in developmental regulation in these tissues. Moreover, TfTCP8 and TfTCP13, the homologues of the Arabidopsis thaliana TCP5‐like transcription factor, were able to bind to the conserved Class II TCP binding motifs and are localized to the nucleus, indicating that TfTCP8 and TfTCP13 act as transcriptional regulators. In agreement with the overexpression phenotype of AtTCP5, ectopic expression of TfTCP8 and TfTCP13 resulted in narrow leaves and the small petal phenotype in Arabidopsis, suggesting that these two TfTCPs potentially regulate leaf or flower shape in T. fournieri. [ABSTRACT FROM AUTHOR]

Torenia fournieri (T. fournieri) is one of the most widely used horticultural flowers and is considered a potential model plant for the genetic investigation of ornamental traits. In this study, we optimized an efficient protocol for high efficiency preparation and transformation of T. fournieri protoplast. The transformation rate reached ~75% when a 35S:GFP construct was used for the transformation. Using this system, we characterized the subcellular localization of several TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) transcription factors (TFs), and found a distinct localization pattern between the CIN and CYC classes of TCP TFs. Furthermore, we also demonstrated the feasibility of the expression of dual luciferase assay system in T. fournieri protoplasts for the measurement of the activity of cis-regulatory elements. Taken together, a well-optimized transient expression system in T. fournieri protoplasts would be crucial for rapid exploration of the gene function or cis-regulatory elements.

Abstract Background CRISPR/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.). Results We 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. Conclusions These 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.

Abstract Background ALOG (Arabidopsis LSH1 and Oryza G1) family with a conserved domain widely exists in plants. A handful of ALOG members have been functionally characterized, suggesting their roles as key developmental regulators. However, the evolutionary scenario of this gene family during the diversification of plant species remains largely unclear. Methods Here, we isolated seven ALOG genes from Torenia fournieri and phylogenetically analyzed them with different ALOG members from representative plants in major taxonomic clades. We further examined their gene expression patterns by RT-PCR, and regarding the protein subcellular localization, we co-expressed the candidates with a nuclear marker. Finally, we explored the functional diversification of two ALOG members, TfALOG1 in euALOG1 and TfALOG2 in euALOG4 sub-clades by obtaining the transgenic T. fournieri plants. Results The ALOG gene family can be divided into different lineages, indicating that extensive duplication events occurred within eudicots, grasses and bryophytes, respectively. In T. fournieri, seven TfALOG genes from four sub-clades exhibit distinct expression patterns. TfALOG1–6 YFP-fused proteins were accumulated in the nuclear region, while TfALOG7-YFP was localized both in nuclear and cytoplasm, suggesting potentially functional diversification. In the 35S:TfALOG1 transgenic lines, normal development of petal epidermal cells was disrupted, accompanied with changes in the expression of MIXTA-like genes. In 35S:TfALOG2 transgenic lines, the leaf mesophyll cells development was abnormal, favoring functional differences between the two homologous proteins. Unfortunately, we failed to observe any phenotypical changes in the TfALOG1 knock-out mutants, which might be due to functional redundancy as the case in Arabidopsis. Conclusion Our results unraveled the evolutionary history of ALOG gene family, supporting the idea that changes occurred in the cis regulatory and/or nonconserved coding regions of ALOG genes may result in new functions during the establishment of plant architecture.

Torenia fournieri belongs to the genus Torenia in the family Linderniaceae. The complete chloroplast genome of T. fournieri was sequenced and analyzed by Illumina sequencing in this study. The full length of the complete chloroplast genome is 153,938 bp, containing a pair of inverted repeat regions of 24,805 bp (IRa and IRb) separated by a large single copy region (LSC) of 85,498 bp and a small single copy region (SSC) of 18,830 bp. The T. fournieri chloroplast genome encodes 131 genes, comprising 87 protein-coding genes, 36 tRNA genes, 8 rRNA genes, without pseudogene. Phylogenetic analysis showed that T. fournieri was closely related to T. benthamiana and T. concolor within the genus Torenia in family Linderniaceae. [ABSTRACT FROM AUTHOR]

Flowers of honey plants (Torenia) face various abiotic stressors, including rain, that can damage pollens and dilute nectar. Many Torenia species are thought to have evolved a modified corolla base termed the corolla neck to prevent raindrops from contacting the nectar. Although this hypothesis was postulated long ago, direct validation is lacking. Here, we have evaluated Torenia fournieri, the corolla tube of which differentiates into distinct regions: a conical tube above that connects to an inflated base through a constriction. This constriction and inflated base are collectively referred to as the corolla neck. Using transcriptomic sequencing and genome-editing approaches, we have characterized an ALOG gene, TfALOG3, that is involved in formation of the corolla neck. TfALOG3 was found expressed in the epidermis of the corolla neck. Cells in the corolla bottom differentiated and expanded in wild-type T. fournieri, whereas such cells in TfALOG3 loss-of-function mutants failed to develop into a corolla neck. Water easily contacted the nectary in the absence of the corolla neck. Taken together, our study unveils a novel gene that controls corolla tube differentiation and demonstrates a hypothetical property of the corolla neck. [ABSTRACT FROM AUTHOR]

Summary: Changes in the amount of mitochondrial DNA (mtDNA) have never been investigated in plant zygotes or early plant embryos due to the difficulty in isolating these cells, although such changes have been investigated in mammalian embryos. Using the single‐cell quantitative real‐time polymerase chain reaction (PCR) and laser confocal microscopy, we surveyed the changes in mtDNA levels during early embryogenesis in Torenia fournieri and Arabidopsis thaliana. In contrast with the amount of mtDNA in early mammalian embryos, which does not change, we found that mtDNA doubling occurred during zygotic development in T. fournieri and during two‐cell proembryo development in A. thaliana. These findings reveal that mtDNA doubling occurs during early embryogenesis in T. fournieri and A. thaliana, indicating that the dynamics of mtDNA in early plant embryos differs from that in early mammalian embryos. [ABSTRACT FROM AUTHOR]

The diverse pigmentation patterns of flower corollas probably result from pollinator-mediated selection. Previous studies demonstrated that R2R3- MYB factors may have been recruited in the regulation of corolla pigmentation. However, how R2R3-MYBs became so diverse in their regulation of different pigmentation patterns remains unclear., Here, we studied a Lamiales species, Torenia fournieri, which has elaborate zygomorphic flowers with dorsal-ventral asymmetries in corolla pigmentation. We found recent gene duplication events in CYCLOIDEA-like ( CYC-like) and RADIALIS-like ( RAD-like) genes, and functionally analyzed three dorsal-specific expression factors: Tf CYC1, Tf CYC2, and Tf RAD1., We found that the CYC- RAD module coordinates petal shape and corolla pigmentation, as ectopic expression of Tf CYC2 or Tf RAD1 disrupted the asymmetric corolla pigmentation pattern and produced strongly dorsalized flowers. Dorsal petal identity was lost when Tf CYC2 was down-regulated or when Tf RAD1 was knocked out. In T. fournieri, the diversified CYC and RAD genes have evolved regulatory loops, and Tf CYC2 binds directly to the regulatory regions of an R2R3- MYB factor gene, Tf MYB1, which might lead to its asymmetric expression and ultimately establish the asymmetric pigmentation pattern., These findings support the existence of a regulatory module that integrates dorsal-ventral patterning and asymmetric corolla pigmentation in T. fournieri. [ABSTRACT FROM AUTHOR]

A tissue culture system was established from young leaves of in vitro grown seedling of Torenia fournieri Lind. Leafsegments were cultured on Murashige and Skoog medium (MS) medium supplemented with combinations of NAA and BA. Shoot organogenesis was observed in all growth regulators containing medium. High frequency regeneration was obtained from leaves cultured on MS medium supplemented with 0.05 mg/l NAA plus 3 mg/l BA. When individual isolated shoots were transferred to MS medium devoid of growth regulators, complete plantlets were obtained. Adventitious shoots were also regenerated from callus that was derived from leaf explants and a high frequency of shoot organogenesis was found on MSmedium containing 0.1 mg/l NAA plus 3 mg/l BA.

Torenia fournieri, a popular potted flower crop, is frequently used as a model plant in genetic transformation studies, and transgenic plants expressing various foreign genes have been obtained in this plant. A safe and long-term conservation of the transgenic plants is necessary. In this study, shoot tips of transgenic plants of T. fournieri were successfully cryopreserved by droplet-vitrification. Growth patterns, and morphologies of leaves and flowers were identical between the plantlets recovered from cryopreservation and the in vitro stock cultures (control). No polymorphic bands and no alternations at ploidy level assessed by ISSR and FCM, respectively, were found in the transgenic plantlets recovered from cryopreservation in comparison with the in vitro stock cultures. The transgene Cry 1Ab analyzed by PCR was maintained, and its expression levels measured by qRT-PCR did not change in the cryo-derived transgenic plantlets. Therefore, cryopreservation can be considered a promising strategy for safe and long-term conservation of the transgenes. [ABSTRACT FROM AUTHOR]

We developed two types of artificial platforms, T-junction and crossroad microchannel devices, and obtained guidance response ratio of pollen tubes to the female tissue as 56–57%. The crossroad device was also able to collect the attracted pollen tubes with high purity, which is useful for future omics analysis. [ABSTRACT FROM AUTHOR]

We developed a fertilization method in which pollen tubes entered into embryo sacs without any need to contact surrounding female sporophytic cells by using Torenia fournieri (Torenia) plants under the condition of hindering movement of the virus from a stigma, which is the first infection site leading to systemic infection. When RBDV-infected Torenia pollen grains were used for the developed fertilization method, the virus was transmitted to the seeds by pollen tubes germinating from them. On the other hand, no seeds were infected with the virus when Torenia plants were pollinated with healthy Torenia pollen grains in combination with RBDV-infected raspberry pollen grains, which caused the virus infection in the stigma by penetration of their pollen tubes arrested in its style. Our results indicate that vertical transmission of RBDV by pollen occurs in the transport of the virus into embryo sacs by pollen tubes reaching the embryo sacs. [ABSTRACT FROM AUTHOR]

The female gametophytes of many flowering plants contain one egg cell, one central cell, two synergid cells and three antipodal cells with respective morphological characteristics and functions. These cells are formed by cellularization of a multinuclear female gametophyte. However, the dynamics and mechanisms of female gametophyte development remain largely unknown due to the lack of a system to visualize directly and manipulate female gametophytes in living material. Here, we established an in vitro ovule culture system to examine female gametophyte development in Torenia fournieri, a unique plant species with a protruding female gametophyte. The four-nucleate female gametophyte became eight nucleate by the final (third) mitosis and successively cellularized and matured to attract a pollen tube. The duration of final mitosis was 28 ± 6.5 min, and cellularization was completed in 54 ± 20 min after the end ofthe third mitosis. Fusion of polar nuclei in the central cell occurred in 13.1 ± 1.1 h, and onset of expression of LURE2, a pollen tube attractant gene, was visualized by a green fluorescent protein reporter 10.7 ± 2.3 h after cellularization. Laser disruption analysis demonstrated that the egg and central cells were required for synergid cells to acquire the pollen tube attraction function. Moreover, aberrant nuclear positioning and down-regulation of LURE2 were observed in one of the two synergid cells after disrupting an immature egg cell, suggesting that cell specification was affected. Our system provides insights into the precise dynamics and mechanisms of female gametophyte development in T. fournieri. [ABSTRACT FROM AUTHOR]

Highlights: [•] We describe a new pollen tube growth assay using a microchannel device. [•] The microchannel restricts the growth of the pollen tube to a single focal plane. [•] The growth of each pollen tube can be observed under a microscope and quantified. [•] This method might enable exploring the effects of signaling molecules on pollen tube. [Copyright &y& Elsevier]

Background Torenia (Torenia fournieri Lind.) is a model plant increasingly exploited in studies in various disciplines, including plant engineering, biochemistry, physiology, and ecology. Additionally, cultivars with different flower colors have been bred and made commercially available. Flower color in torenia is mainly attributed to the accumulation of anthocyanins, but the molecular mechanisms inducing flower color mutations in torenia have not been well elucidated. In this study, we therefore attempted to identify the cause of white coloration in torenia by comparing the white-flowered cultivar Crown White (CrW) with Crown Violet (CrV), a violet-flowered variety. Results In an expression analysis, no flavanone 3-hydroxylase (TfF3H) transcript accumulation was detected in CrW petals. Sequence analyses revealed that a novel long terminal repeat (LTR)- type retrotransposable element, designated as TORE1 (Torenia retrotransposon 1), is inserted into the 5'-upstream region of the TfF3H gene in CrW. A transient expression assay using torenia F3H promoters with or without TORE1 insertion showed that the TORE1 insertion substantially suppressed F3H promoter activity, suggesting that this insertion is responsible for the absence of F3H transcripts in white petals. Furthermore, a transformation experiment demonstrated that the introduction of a foreign gentian F3H cDNA, GtF3H, into CrW was able to recover pink-flower pigmentation, indicating that F3H deficiency is indeed the cause of the colorless flower phenotype in CrW. Detailed sequence analysis also identified deletion mutations in flavonoid 3'-hydroxylase (TfF3'H) and flavonoid 3',5'- hydroxylase (TfF3'H) genes, but these were not directly responsible for white coloration in this cultivar. Conclusions Taken together, a novel retrotransposable element, TORE1, inserted into the F3H 5'-upstream region is the cause of deficient F3H transcripts in white-flowered torenia, thereby leading to reduced petal anthocyanin levels. This is the first report of a retrotransposable element involved in flower color mutation in the genus Torenia. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background and Aims During sexual reproduction in higher angiosperms, the pollen tubes are directed to the ovules in the pistil to deliver sperm cells. This pollen tube attraction is highly species specific, and a group of small secreted proteins, TfCRPs, are necessary for this process in Torenia fournieri. Methods A candidate pollen tube attractant protein in Torenia concolor, a related species of T. fournieri, was isolated and the attractant abilities between them were compared. Key Results TcCRP1, an orthologous gene of TfCRP1 from T. concolor, is expressed predominantly in the synergid cell. The gene product attracted pollen tubes in a concentration-dependent manner, but attracted fewer pollen tubes from the other species. Conclusions The results indicated that this class of CRP proteins is a common pollen tube attractant in Torenia species. The sequence diversity of these proteins is important for species-specific pollen tube attraction. [ABSTRACT FROM PUBLISHER]

A tissue culture system was established from young leaves of in vitro grown seedling of Torenia fournieri Lind. Leaf segments were cultured on Murashige and Skoog medium (MS) medium supplemented with combinations of NAA and BA. Shoot organogenesis was observed in all growth regulators containing medium. High frequency regeneration was obtained from leaves cultured on MS medium supplemented with 0.05 mg/l NAA plus 3 mg/l BA. When individual isolated shoots were transferred to MS medium devoid of growth regulators, complete plantlets were obtained. Adventitious shoots were also regenerated from callus that was derived from leaf explants and a high frequency of shoot organogenesis was found on MS medium containing 0.1 mg/l NAA plus 3 mg/l BA. [ABSTRACT FROM AUTHOR]

Phosphoinositide-specific phospholipase C (PI-PLC) is an important enzyme, which is a key player involved in eukaryotic signal transduction pathways. In plants, it plays a key role in growth and development as well as environmental stress. However, little is known about its roles in signal transduction during sexual reproduction process. In this study, we cloned and characterized a gene of full-length PI-PLC from ovules of Torenia fournieri, designated as TfPLC1. It was 2,171 bp in length, including an open reading frame encoding a polypeptide of 583 amino acids with molecular mass of 66.02 kDa. The amino acid sequence deduced from the cDNA sequence shows 40–76% similarity to other plant PI-PLCs and contains the characteristic X, Y and C2 domains. Northern blot analysis demonstrated it was predominantly expressed in ovules and flowers. Furthermore, TfPLC1 promoter::GUS transgenic analysis indicated it specifically expressed in ovule, stigma and mature pollen grain. Immunohistochemical staining showed that, in mature stigma, TfPLC1 protein was principally localized in the cells of stigmatic receptive surface. Together, our data suggest that TfPLC1 may play an important role in plant sexual reproduction. [ABSTRACT FROM AUTHOR]

The effects of several hormones on pollen tube growth were compared in Torenia fournieri and it was found that IAA was the most effective, stimulating pollen tube growth and causing the shank part of pollen tubes to be slender and straighter. The role of IAA was investigated by studying the changes in ultrastructure and PM H+-ATPase distribution in the pollen tubes and the modification of the tube wall. Using the fluorescent marker FM4-64, together with transmission electron microscopy, it was shown that secretory vesicles and mitochondria increased in IAA-treated tubes. Immunolocalization and fluorescence labelling, together with Fourier-transform infrared analysis, detected that IAA enhanced the level of PM H+-ATPase and the synthesis of pectins, and reduced the cellulose density in pollen tubes. Importantly, to observe the orientation of cellulose microfibrils in pollen tubes in situ, atomic force microscopy was used to examine the ‘intact’ tube wall. Atomic force microscopy images showed that cellulose microfibrils were parallel to each other in the subapical region of IAA-treated tubes, but disorganized in control tubes. All results provided new insights into the functions of cellulose microfibrils in pollen tube growth and direction, and revealed that the IAA-induced changes of pollen tubes were attributed to the increase in secretory vesicles, mitochondria, and PM H+-ATPase, and the modification of pectin and cellulose microfibrils in the tube wall. [ABSTRACT FROM PUBLISHER]

A full-length gene encoding phosphoinositide-specific phospholipase C (PI-PLC), named TfPLC2, was cloned from Torenia fournieri by RT-PCR and RACE, using the designed degenerate primers. The TfPLC2 gene is a 2087-bp-long sequence including one open reading frame encoding a polypeptide of 601 amino acids. Northern blot analysis showed that the TfPLC2 gene was expressed predominantly in leaves and stems and at very low levels in other tissues. The expression of TfPLC2 was strongly induced under the conditions of high salt or ABA treatment. [ABSTRACT FROM AUTHOR]

The two sperm cells of Torenia fournieri are dimorphic. The dimorphic character suggests that they might be preferentially involved in fertilization during in vivo fusion with the egg cell and central cell. To probe the mechanism of preferential fertilization, it is necessary to use the most current molecular techniques. For this purpose, populations of >1000 individuals of the two dimorphic sperm cells, Sua (unassociated with the vegetative nucleus) and Svn (associated with the vegetative nucleus) were isolated from pollen tubes that had grown out of the cut ends of the styles. The two sperm cells released from pollen tubes remained attached to one another. When the two attached sperm cells were transferred into a solution containing 0.01% cellulose, 0.01% pectinase, and 5% mannitol, the connection between the two cells disappeared, and they were easily separated using a micromanipulator. The collection of these two individual populations containing over a thousand cells will permit research on gametic recognition at the molecular level. [ABSTRACT FROM AUTHOR]

Anthocyanin synthesis and chlorophyll degradation in regenerated torenia ( Torenia fournieri Linden ex Fourn.) shoots induced by osmotic stress with 7% sucrose were examined to identify the genes regulating the underlying molecular mechanism. To achieve this, suppression subtractive hybridization was performed to enrich the cDNAs of genes induced in anthocyanin-synthesizing and chlorophyll-degrading regenerated shoots. The nucleotide sequences of 1,388 random cDNAs were determined, and these were used in the preparation of cDNA microarrays for high-throughput screening. From 1,056 cDNAs analyzed in the microarrays, 116 nonredundant genes were identified, which were up regulated by 7% sucrose to induce anthocyanin synthesis and chlorophyll degradation in regenerated shoots. Of these, eight genes were selected and RNAi transformants prepared, six of which exhibited anthocyanin synthesis inhibition and/or chlorophyll degradation in their leaf discs. Notably, the RNAi transformants of the glucose 6-phosphate/phosphate translocator gene displayed inhibition both of anthocyanin synthesis and chlorophyll degradation in both leaf discs and regenerated shoots. There was also less accumulation of anthocyanin in the petals, and flowering time was shortened. The genes we identified as being up-regulated in the regenerated torenia shoots may help further elucidate the molecular mechanism underlying the induction of anthocyanin synthesis and chlorophyll degradation. [ABSTRACT FROM AUTHOR]

To elucidate the mechanism of anthocyanin synthesis induction concomitant with chlorophyll degradation, we established a system in which anthocyanin synthesis and degradation of chlorophyll in regenerated torenia ( Torenia fournieri) shoots was induced on medium containing 7% sucrose. Here, we studied the effect of several plant-growth regulators on anthocyanin synthesis and the degradation of chlorophyll in the torenia shoot regenerating system. Exogenous abscisic acid (ABA) could induce anthocyanin synthesis and chlorophyll senescence in regenerating torenia shoots on the medium containing a low concentration of sucrose (1.5%). We determined the changes in the amount of endogenous ABA in the regenerated shoots during the process of anthocyanin synthesis on the medium containing 7% sucrose. It was revealed that the 7% sucrose treatment elevated endogenous ABA levels before the induction of anthocyanin synthesis and chlorophyll degradation. However, while retransfer to the 1.5% sucrose medium resulted in a gradual decrease in the ABA level and a failure of induction of anthocyanin synthesis, normal shoot regeneration. These results suggest that changes in the amount of endogenous ABA may play an important role in the induction of anthocyanin synthesis and chlorophyll degradation in regenerated torenia shoots. [ABSTRACT FROM AUTHOR]

A system in which anthocyanin synthesis can be induced under defined conditions was established in regenerated torenia shoots. Leaf discs prepared from torenia plantlets grown under sterile conditions were placed on solidified half-strength MS medium containing 3% sucrose and 4.4×10-6 M benzyladenine (BA) and cultured under 16 h light/8 h dark (standard light) conditions for 10 days, then in the dark for a further 10 days. The discs were transferred to medium containing 7% sucrose without BA and cultured under standard light conditions. Six days after transfer, anthocyanin synthesis started in the regenerated shoots, and thereafter, anthocyanin accumulation increased while chlorophyll content decreased. Experiments in which either the timing of illumination was altered or shoots were retransferred to medium containing 1.5% sucrose or other sugars as well as sucrose indicated that both osmotic stress and light are required to induce anthocyanin synthesis. Once anthocyanin synthesis was induced in the torenia shoots 6 days after transfer, the shoots were fated to the synthesis of anthocyanins and the degradation of chlorophylls, and could not revert to the developmental pathway of shoot regeneration. This system may provide a good model for the investigation of the mechanisms underlying the induction of anthocyanin synthesis. [ABSTRACT FROM AUTHOR]

Changes in actin organization in the living egg apparatus of Torenia fournieri from anthesis to post-fertilization have been investigated using microinjection and confocal microscopy. Our results revealed that the actin cytoskeleton displays dramatic changes in the egg apparatus and appears to coordinate the events of synergid degeneration, pollen tube arrival and gametic fusion during fertilization. Synergid degeneration occurs after anthesis and is accompanied by actin fragmentation and degradation. The actin cytoskeleton becomes organized with numerous aggregates in the chalazal end of the degenerating synergid, and some of the actin infiltrates into the intercellular gap between synergids, egg and central cell, forming a distinct actin band. An actin cap is present near the filiform apparatus after anthesis and disappears after pollen tube arrival. In the egg cell, actin filaments initially organize into a network and after pollination become fragmented into numerous patches in the cortex. These structures, along with the actin in the degenerating synergid and intercellular spaces form two distinct actin coronas during fertilization. The actin coronas vanish after gametic fusion. This is the first report of changes in actin organization in the living egg apparatus. The reorganization of the actin cytoskeleton in the egg apparatus and the presence of the actin coronas during fertilization suggest these events may be a necessary prelude to reception of the pollen tube and fusion of the male and female gametes. [ABSTRACT FROM AUTHOR]

During the course of a fluorescence microscopic investigation on the extra-ovular micropylar portion of the embryo sacs of Torenia fournieri Lind. ( Scrophulariaceae) a callosic wall was found which surrounded it almost completely until the time of anthesis. In addition, the walls of young synergids and the filiform apparatus also showed callosic fluorescence. Treatments with PAS reaction revealed a PAS-positive substance filling up the locular cavity. Our attempts to induce fluorochromasia by employing fluorescein diacetate failed, indicating the low permeability of the callosic wall around the embryo sac. It is assumed that the callose wall around the embryo sac isolates the latter from the contents of the locular cavity whereas the callose in the synergid walls may represent an intermediate stage in the maturation of these walls; the filiform apparatus is mainly composed of callose. [ABSTRACT FROM AUTHOR]

Background: CRISPR/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.). Results: We 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. Conclusions: These 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. [ABSTRACT FROM AUTHOR]