Ion beams have been used as a mutagen to improve the efficiency of plant mutation breeding. Mutation breeding is sometimes perceived as a random process. In this review, we describe our recent progress in developing a more efficient mutagenesis technique using ion beam irradiation combined with sucrose pretreatment or subsequent re-irradiation. To shorten the time required for breeding new cultivars of cyclamen, we identified anthocyanin biosynthesis genes and examined the effectiveness of PCR screening of irradiated deletion-mutant candidates at early growth stages. We believe this research is a step toward more efficient and controlled mutation breeding using ion beams.
The fragrant cyclamens ‘Uruwashi-no-kaori’ (UR), ‘Kaori-no-mai’ (KM), ‘Kokou-no-kaori’ (KO) were bred from amphidiploids that were derived by chromosome doubling of the hybrid among Cyclamen persicum ‘Strauss’, ‘Pure White’ and fragrant wild species of C. purpurascens. Amphidiploid of GBCP was produced by chromosome doubling of the hybrid between C. persicum ‘Golden Boy’ and fragrant wild species of C. purpurascens. In order to create novel flower colors, mutation breeding with carbon–ion-beam irradiation was carried out using amphidiploids of UR, KM and KO as well as dihaploids of UR, KM and GBCP. Immature petals or etiolated petioles of the amphidiploids and dihaploids were irradiated, and plant tissue culture techniques were used to produce material for additional investigations. Furthermore, the stepwise improvement of flower color was achieved by irradiating ‘ion3’, which had been derived from dihaploid of UR by ion-beam irradiation. The mutants thus obtained were evaluated for breeding new cultivars of fragrant cyclamens. The tissues were then analyzed in terms of their flavonoid pigment content and their related genes.
We have developed a systematic and directed method to create novel glittering mutants in carnation (Dianthus caryophyllus) by combining the advantages of ion-beam breeding and genomic information. The method is a series of steps that include: (i) establishing a basic strategy to select appropriate genotypes for specific breeding aims using genomic information, (ii) identifying factors that induce anthocyanic vacuolar inclusions (AVIs), (iii) using ion-beam irradiation consecutively to modify pigment glycosylation and/or acylation, (iv) tuning shading of color, and (v) selecting stable mutants with markers. During the course of this work, we identified a factor that causes AVIs and analyzed the content of anthocyanins and related compounds in the flowers of these mutants. Applying the method, we have created two highly novel carnations with the most glittering color ever reported.
We analyzed flower color mutants of cyclamen (Cyclamen spp.) and carnation (Dianthus caryophyllus) obtained by ion-beam irradiation with an idea that a comprehensive analysis of anthocyanin and its biosynthetically related compounds, such as flavonols and cinnamic acid derivatives, is necessary in order to understand flower color expression mechanism. In this review, we discuss mechanisms for flower color mutation and deduce the following ideas: anthocyanin and its biosynthetically related compounds are cooperatively and compensatively regulated; multiple factors are often concerned in the expression of the same color phenotypes; and changes in chemical structure of a pigment induces new properties that generate novel phenotypes.
Two cDNAs with homology to glutathione S-transferase (GST) were isolated from the carnation (Dianthus caryophyllus); these cDNAs are termed here DcGSTF1 and DcGSTF2. Phylogenetic analysis suggested that both DcGSTF1 and DcGSTF2 belonged to the Phi class of GSTs. DcGSTF2 showed high levels of transcription at late stages of petal development when anthocyanin biosynthesis is most active. Sequencing of DcGSTF2 indicated that it consisted of three exons and two introns. A truncated DcGSTF2 gene, resulting from the insertion of a CACTA-type transposable element, was found in the genome of a mutable flower line bearing deep pink sectors on pale pink petals. A full length DcGSTF2 gene driven by a continuous expression promoter was introduced into the epidermal cells of carnations with pale pink petals. The transformed cells were deep pink. These results suggest that the DcGSTF2 gene is responsible for flower color intensity in carnations.
Thirteen glutamate receptor-like genes have recently been identified in tomato plants; however, their functions have not been fully elucidated. We overexpressed SlGLR1.1 and SlGLR3.5 in Arabidopsis and found that transgenic plants showed symptoms such as curled and deformed leaves, dwarf stature and retarded growth resembling those of Ca2+ deficiency. The results revealed that the levels of Ca2+ in aerial tissues did not differ between wild-type and transgenic plants, suggesting that overexpression of SlGLR1.1 and SlGLR3.5 did not affect Ca2+ uptake. Transgenic lines were hypersensitive to K+ and Na+ ionic stresses, which was rescued by addition of Ca2+ to the growth medium. Ectopic expression of SlGLR1.1 and SlGLR3.5 resulted in reduced efficiency of Ca2+ utilization, suggesting that these genes may play a role in calcium assimilation in tomato plants by controlling ionic transport across the membrane.
Meristems (length 100, 200, 300 µm) were excised from infected Potato plants of Binella and Burren cultivars and cultured on solidified MS medium containing 30 g l－1 sucrose, 5 mg l－1 Ascorbic acid, 5 mg l－1 Pyridoxine, 5 mg l－1 Nicotinic acid, 5 mg l－1 Thiamine, 200 mg l－1 Inositole, 2 mg l－1 GA3, and 0.2 mg l－1 kinetin. Virus status of in vitro plantlets was determined by double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA). Results showed that the highest rate of virus-free plants was obtained by using explants 100 µm in length. The rate of PVY elimination was improved after treatment by thermotherapy at approximately 37±1°C for 40 days, (81%) in Binella and (75%) in Burren. Chemotherapy was undertaken with (10–20–30) mg l－1 of ribavirin (RBV), The highest percentage of virus free plantlets 87% in Binella and 82% in Burren were obtained from the ribavirin's concentration (20 mg l－1) combined with meristem-tips (100 µm) in length. Severe growth abnormalities were observed on medium containing high concentration of RBV (30 mg l－1). Finally, the highest percentage virus free plantlets of PVY (93% in Binella and 87% in Burren) were obtained from meristem-tips 100 µm in length excised after electric treatments (15 mA/10 min).
The Q gene has played substantial roles in wheat domestication. As it pleiotropically governs domestication-related traits, such as free threshing, glume shape and tenacity, rachis fragility, spike length, plant height, and flowering time, wheat is cultivated in widespread adaptation. The Q gene located on the A genome encodes the APETALA2-like transcription factor WAP2AQ. The allelic mutation from q (WAP2Aq) to Q took place in the polyploidy wheats, and the B and D genomes of bread (hexaploid) wheat conferred its homoeoalleles (WAP2B and WAP2D, respectively). Although WAP2Aq and WAP2D revealed allelic phenotypes against WAP2AQ, their functions remain to be clarified. We overexpressed full-length cDNAs of WAP2AQ, WAP2Aq, and WAP2D in the ap2 mutant line of Arabidopsis. WAP2AQ fully recovered their flower organs similar to the wild type, WAP2D showed less recovery, and WAP2Aq rescued the least mutant flower phenotype. Use of a yeast two-hybrid system showed that WAP2AQ formed the most homodimers, WAP2Aq formed the next highest, and WAP2D formed the least. The sequence comparisons between the three transcription factors and with AP2 of Arabidopsis revealed that WAP2Aq confers two single protein substitutions, I329-to-V substitution and K108-to-E in the nuclear translocation signal, WAP2D harbors SNPs of I329-to-L similar to Arabidopsis, and other 6 substitutions. These data support the idea that a critical point mutation at the functional domain and structure alteration(s) resulting from sequence diversifications caused functional differences in the genes. Mutant lines of Arabidopsis can become a powerful tool for analyzing foreign gene functions as in the case of wheat.
Many abiotic and biotic stresses can reduce plant growth and development. Low temperature is one of the most harmful abiotic stresses, particularly for plants that are tropical or subtropical in origin. To elucidate the molecular mechanisms underlying the cold-stress response, components involved in the signal transduction of cold stress have been characterized. In this study, we characterized a basic helix–loop–helix (bHLH) transcription factor encoding gene, SlICE1, from tomato (Solanum lycopersicum), which shows similarity with Arabidopsis ICE1. The expression of SlICE1 was observed in younger leaves, flowers, and green and red fruits. To characterize the function of SlICE1, overexpressing tomato lines were produced. SlICE1-overexpressing tomatoes exhibited chilling tolerance, and SlICE1 enhanced the expression of cold-responsive genes, such as SlCBF1 and SlDRCi7, as well as accumulation of ascorbic acid. The SlICE1 protein was degraded after cold treatment. These results indicate that SlICE1 enhances cold tolerance in tomatoes.
Antioxidants and antioxidant activity confer important protective effects in plants against the effects of free radicals, which are generated by biotic and abiotic stresses, such as cold. In another study, we identified SlICE1 as a basic helix–loop–helix transcription factor to improve cold tolerance. Here, we demonstrate that SlICE1 plays an important role in the accumulation of antioxidants and in the regulation of antioxidant activity in tomato Solanum lycopersicum. Overexpression of SlICE1 in tomatoes enhanced the accumulation of antioxidants, such as β-carotene, lycopene, and ascorbic acid, as well as antioxidant activity, measured as the scavenging of 2,2-diphenyl-1-picrylhydazyl (DPPH) free radicals and O2－ radicals. Furthermore, the sugar content in SlICE1-overexpressing tomatoes red fruits was higher than that in wild-type red fruits. Metabolite profiling analysis performed by capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) revealed that several amino acids and amines were more highly accumulated in SlICE1-overexpressing tomato red fruits compared to those in wild-type tomatoes. These results suggest that SlICE1 plays a role in the regulation of antioxidant activity through the accumulation of several antioxidants.
Traditional methods used to study strawberry ripening-related gene function are time-consuming, and require at least 15 months from initiating the transformation experiment until the first ripe fruits are available for analysis. To accelerate data acquisition during gene function studies, we explored a transient assay method that employs an Agrobacterium-mediated RNAi (AmRNAi) technique in post-harvest strawberry fruit, Fragaria×ananassa (Fa) cv. Sachinoka, a Japanese cultivar. Our results showed that artificial white light induced strong expression of Fa′chalcone synthase (Fa′CHS), Fa′chalcone isomerase (Fa′CHI), and Fa′flavonoid 3′-hydroxylase orthologues (Fa′F3′H) in post-harvest fruit. Fa′CHS and Fa′F3′H function was subsequently examined by performing AmRNAi with post-harvest fruit. Although reduction of light-induced Fa′F3′H expression by AmRNAi resulted in no significant change in anthocyanin content, reduction of Fa′CHS significantly decreased anthocyanin levels, and up-regulated Fa′F3′H levels. Our results are consistent with previous data indicating that while CHS is required for anthocyanin accumulation during late stage strawberry fruit maturation, Fa′F3′H is not required. The novel system described here enabled gene function data to be available within 10 days of initiating the incubation period following infiltration. Therefore, we conclude our system is a valuable tool to elucidate the molecular mechanisms underlying light-induced ripening of strawberry fruit.
The ubiquitin/26S proteasome system (UPS) plays a central role in the degradation of short-lived regulatory proteins that control many cellular events. In this study, the Arabidopsis knockout mutant rpt2a, which contains a defect in the AtRPT2a subunit of the 26S proteasome regulatory particle, showed hypersensitivity to sugars as well as enlarged leaves. When the role of RPT2a in sugar response was examined in further detail it was found that putatively only the AtRPT2a gene of 19S proteasome was markedly transcriptionally promoted by sugar application. Notably, poly-ubiquitinated proteins degraded by the UPS accumulated significantly in rpt2a mutant under 6% sucrose conditions compared to wild type. In addition, the AtRPT2a gene in gin2, a glucose insensitive mutant with a defective glucose-sensing hexokinase, was not upregulated by sugar application, indicating that AtRPT2a is involved in hexokinase-dependent sugar response. Taken together, the above findings indicate that AtRPT2a plays an essential role in the maintenance of proteasome-dependent proteolysis activity in response to sugars.
Disease resistance potential of a synthetic fungal chitinase (NIC) gene was evaluated in transgenic Petunia hybrid employing Agrobacterium tumefaciens-mediated genetic transformation. The NIC gene (1271-bp in length) was synthesized to encode the same amino acid sequence (except 25Ser to 25Arg) as that of chitinase 1 gene (chi1) from Rhizopus oligosporus. As 18% of codons in the NIC gene were changed from fungal type (AT-rich) to plant type (GC-rich), the nucleotide sequence of NIC had 82% homology with the chi1 gene. Petunia hybrida ‘Danty Lady’ was transformed with A. tumefaciens EHA 101 harboring a binary vector plasmid containing NIC (gene of interest) and nptII (selection marker) genes. Putative transgenic plants were produced on MS medium containing kanamycin monosulphate as a selective chemical. PCR analysis revealed that the NIC and nptII genes are integrated into the genome of transgenic plants. Integrations of the transgenes were indicated by Southern blot analysis of genomic DNA from the transgenic plants. Expression of NIC gene at the mRNA level was confirmed by reverse-transcription-polymerase chain reaction (RT-PCR). Western blot analysis detected the accumulation of NIC protein in the leaves of transgenic plants. The transgenic plants exhibited enhanced resistance against Botrytis cinerea (grey mold) as indicated by inhibition of the fungal growth in detached leaves and by the total protein extract. From these results it could be concluded that the NIC gene was successfully integrated into the genome of transgenic petunia plants and produced the NIC protein. Expression of the synthetic chitinase, NIC gene, probably, conferred enhance resistance to B. cinerea in transgenic petunia.
We isolated and characterized Ralstonia solanacearum-responsive genes (RsRGs) related to the hypersensitive response (HR) by virus-induced gene silencing using Nicotiana benthamiana and the potato virus X vector system. We focused on RsRGM10, because induction of HR was delayed in RsRGM10-silenced plants challenged with incompatible R. solanacearum 8107, which induces the HR in, N. benthamiana. The amino acid sequence deduced from the full-length RsRGM10 cDNA showed approximately 90% identity with rolB overexpressed 1 (ROX1) from N. tabacum, and contained an asparagine-rich sequence. We designated this protein NbARP (N. benthamiana asparagine-rich protein). Expression of HR-related hin1 and onset of the oxidative burst were delayed in NbARP-silenced plants, and induction of myelin basic kinase activity was reduced. Growth of R. solanacearum 8107 was accelerated in NbARP-silenced plants. Induction of the HR by Pseudomonas cichorii and Pseudomonas syringae pv. syringae was also delayed in NbARP-silenced plants. Silencing of NbARP reduced induction of HR-cell death by Agrobacterium tumefaciens-mediated transient expression of HR elicitors and a constitutively active form of mitogen activated protein kinase kinase. The population of R. solanacearum 8107 was decreased in NbARP-overexpressing plants. These results suggest that NbARP is closely related to the HR.
The soybean is relatively sensitive to disturbances arising from flooding before its emergence from the soil. When young soybean seedlings at an early stage are transferred to flooding anaerobic conditions, alcohol dehydrogenase (Adh) mRNA and Adh protein increase temporarily in the root tips, where active cell division demands high energy production. Since there is little information on the significance of the up-regulation of Adh for the tolerance of soybeans to flooding stress, we examined the response to flooding in transgenic soybean lines in which the soybean Adh (GmAdh2) gene was introduced under the control of a constitutive promoter. Acquired transgenic soybean seeds from one out of 14 transgenic lines were subjected to flooding stress. Growth inhibition of soybean seedlings caused by flooding stress was reduced in soybeans with the GmAdh2 transgene. Protein analysis and enzyme assay at the early stage of growth of the soybean seedlings confirmed that Adh expressions and activities in transgenic soybeans were increased compared to control soybeans. These results indicated that the introduced GmAdh2 gene might have induced some change in glycolysis and alcohol fermentation, and improved the germination of transgenic soybeans under flooding stress.
Plastid transformation in higher plants has been established in tobacco using particle bombardment, and the protocol has been used as a model in other plant species; however, as target materials, tobacco leaves may be partially exposed to unexpected abiotic stresses―drought stress during post-bombardment culture and damage due to cutting before selection culture. Plastid transformation efficiency may be increased by modification of leaf treatment to lessen such damage. In the modified protocol, tobacco leaves were cut into pieces (5×5 mm) and placed on the culture medium plate (approximately fifty pieces per plate) for one day before bombardment. After bombardment, these pieces were transferred onto the non-selection medium and cultured for three days; they were then transferred onto spectinomycin selection culture medium. A transformation vector containing an aminoglycoside 3′-adenylyltransferase gene and a green fluorescence protein gene were used for plastid transformation. Approximately four independent plastid transformants per bombarded plate were obtained on average using the modified protocol; the transformation efficiency was 1.6 times higher than that in a control experiment using the standard protocol. Modified leaf treatment improved the efficiency and stability of plastid transformation. This finding should aid plastid transformant production in tobacco and other plant species.
Leaf explants formed embryogenic calluses at a frequency of 53.9% when cultured on B5 media supplemented with 0.1 mg l－1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.01 mg l－1 6-benzyladenine (BA) for 6 weeks. Upon transfer onto media with 5 mg l－1 abscisic acid, embryogenic calluses yielded somatic embryos at 73%. Somatic embryos developed into plantlets on media without plant growth regulators at 90%. Embryogenic calluses proliferated and maintained embryogenic capacity when subcultured on media with 0.1 mg l－1 2,4-D and 0.01 mg l－1 BA at 4-week intervals. This culture system is an effective means for clonal propagation and genetic manipulation of soapberry because it ensures taproot development required for tree stability.
Gentians are medicinal plants whose roots and rhizomes have been used as natural medicines for gastrointestinal problems. The active components are bitter compounds known as secoiridoid glycosides such as gentiopicrin and swertiamarin. Previously, procedures for the in vitro clonal culture of gentian plantlets were established. In this study, the effect of different wavelengths of light emitted by LED on plantlet growth and the concentrations of active components was investigated. We found that plantlet growth was promoted after irradiation with red light. Because a similar trend was observed in all gentian cultivars tested, the improved effect of red light on plantlet growth seemed to be a common characteristic of Gentiana species. We also found that both leaves and roots of the plantlets contained gentiopicrin and swertiamarin. Gentiana triflora ‘Maciry’ had the highest concentration of both compounds compared with several gentian lines and cultivars. Furthermore, the concentrations of roots were increased by far-red and blue light irradiation. These results suggest that modification of light quality by LED is an effective strategy for improving the growth and concentration of active components in gentian plantlets that can be used as a novel ingredient in natural medicine.
For higher expression of a foreign gene in plant cells, it is important to optimize nucleotide sequences corresponding to 5′-untranslated region (5′-UTR), because it usually has great impacts on the expression of the gene mainly at the translational level. In this study, with an aim to find useful 5′-UTRs, thirty nine 5′-UTRs derived from Arabidopsis thaliana genes were tested by transient expression of firefly luciferase (Fluc), and that of A. thaliana arabinogalactan-protein 21 (AtAGP21) gene was selected for further analyses. Its activity was either equaling or surpassing that of known translational enhancer, A. thaliana alchol dehydrogenase (AtADH) 5′-UTR in dicotyledons, and was further improved by the optimizing sequence context of the initiating codon (－3 to －1 of AUG). Finally, we also found that the modified AtAGP21 5′-UTR was useful in recombinant expression of horseradish peroxidase (HRP) in tobacco cultured cells, and the yield was as much as 23 mg l－1 culture medium in seven days.