著者

Ikuko Kusaba Takahiro Nakao Hiroko Maita Shusei Sato Ryota Chijiiwa Emi Yamada Susumu Arima Mareshige Kojoma Kanji Ishimaru Ryo Akashi Akihiro Suzuki

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.38, no.1, pp.57-66, 2021-03-25 (Released:2021-03-25)

参考文献数

35

被引用文献数

3

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Licorice (Glycyrrhiza uralensis) is a medicinal plant that contains glycyrrhizin (GL), which has various pharmacological activities. Because licorice is a legume, it can establish a symbiotic relationship with nitrogen-fixing rhizobial bacteria. However, the effect of this symbiosis on GL production is unknown. Rhizobia were isolated from root nodules of Glycyrrhiza glabra, and a rhizobium that can form root nodules in G. uralensis was selected. Whole-genome analysis revealed a single circular chromosome of 6.7 Mbp. This rhizobium was classified as Mesorhizobium by phylogenetic analysis and was designated Mesorhizobium sp. J8. When G. uralensis plants grown from cuttings were inoculated with J8, root nodules formed. Shoot biomass and SPAD values of inoculated plants were significantly higher than those of uninoculated controls, and the GL content of the roots was 3.2 times that of controls. Because uninoculated plants from cuttings showed slight nodule formation, we grew plants from seeds in plant boxes filled with sterilized vermiculite, inoculated half of the seedlings with J8, and grew them with or without 100 µM KNO3. The SPAD values of inoculated plants were significantly higher than those of uninoculated plants. Furthermore, the expression level of the CYP88D6 gene, which is a marker of GL synthesis, was 2.5 times higher than in inoculated plants. These results indicate that rhizobial symbiosis promotes both biomass and GL production in G. uralensis.

著者

Mugito Kato Hajime Shiota

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.38, no.1, pp.31-36, 2021-03-25 (Released:2021-03-25)

参考文献数

33

被引用文献数

2

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Japanese honewort (Cryptotaenia japonica) is consumed as a traditional vegetable and has medicinal applications. In Japan, C. japonica is mainly produced using hydroponic culture systems; however, damping-off is often caused by the adherence of pathogens to its seeds. Therefore, the use of sterile artificial seeds in hydroponic culture is likely to be effective for preventing disease. In this study, we established methods for stress-induced somatic embryogenesis and artificial seed production in Japanese honewort. Shoot apex explants from seedlings were treated with 0.7 M sucrose as a hyperosmotic stress for 3 or 6 weeks, and then transferred to stress-free conditions. Somatic embryos were formed after culture in stress-free conditions for 7 weeks. Stress-treated shoot apex explants that formed somatic embryos were cultured in Murashige and Skoog liquid medium with shaking. After 2 weeks of culture, approximately 800 somatic embryos were formed from each explant. Somatic embryos were formed continuously during 37 weeks under the same culture conditions. Thus, somatic embryogenesis was effectively induced in Japanese honewort via hyperosmotic stress, and embryogenic competence was maintained under stress- and phytohormone-free conditions. The somatic embryos produced by liquid culture were used to produce artificial seeds by enveloping the embryos in whipped alginate gel to avoid hypoxic conditions. The artificial seeds had a high germination rate (72%). This system is suitable for the sterile, highly productive hydroponic culture of Japanese honewort.

著者

Masato Araragi Airi Ikeura Toshiki Uchiumi

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.38, no.1, pp.23-30, 2021-03-25 (Released:2021-03-25)

参考文献数

40

被引用文献数

2

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Many abiotic stresses induce the generation of nitric oxide (NO) in plant tissues, where it functions as a signal molecule in stress responses. Plants modulate NO by oxidizing it to NO3− with plant hemoglobin (GLB), because excess NO is toxic to cells. At least eight genes encoding GLB have been identified in soybean, in three clades: GLB1, GLB2, and GLB3. However, it is still unclear which GLB genes are responsible for NO regulation under abiotic stress in soybean. We exposed soybean roots to flooding, salt, and two NO donors—sodium pentacyanonitrosylferrate (III) dihydrate (SNP) and S-nitroso-N-acetyl-D,L-penicillamine (SNAP)—and analyzed expression of GLB genes. GmGLB1, one of two GLB1 genes of soybean, significantly responded to both SNP and SNAP, and its induction was almost completely repressed by a NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. GmGLB1 responded to flooding but not to salt, suggesting that it is responsible for NO regulation under NO-inducing abiotic stresses such as flooding. GmGLB3, one of two GLB3 genes of soybean, did not respond to NO donors at all but did respond to flooding, at a lower level than GmGLB1. These results suggest that flooding induces not only NO but also unknown factor(s) that induce GmGLB3 gene in soybean.

著者

Leila Riahi Marwa Snoussi Mériam Ben Romdhane Nejia Zoghlami

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.38, no.1, pp.17-22, 2021-03-25 (Released:2021-03-25)

参考文献数

29

被引用文献数

3

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Tunisian pearl millet (Pennisetum glaucum L.) landraces are still growing in contrasting agro-ecological environments and are considered potentially useful for national and international breeders. Despite its genetic potential, the cropping areas of this species are still limited and scattered which increases the risk of genetic erosion. The chloroplast DNA polymorphism and maternal lineages classification of forty nine pearl millet landraces representing seven populations covering the main distribution area of this crop in Tunisia were undertaken based on informative cpSSR molecular markers. A total of 21 alleles combining to 9 haplotypes were detected with a mean value of 3.5 alleles per locus and a haplotype genetic diversity (Hd) of 0.82. The number of chloroplast haplotypes per population ranged from 1 to 4 with an average of 1.28. The haplotypes median-joining network and UPGMA analyses revealed two probable ancestral maternal lineages with a differential pearl millet seed-exchange rate between the investigated areas. Northern and Central populations presented unique genetic backgrounds while historical farmers’ practices in the South-East area resulted in the isolation of their own local landraces. The genetic evidences strongly support at least two introduction origins of pearl millet in Tunisia, one in the North and the other in the South followed by distinct local dispersal histories. Complementary in-situ and ex-situ conservation strategies taking into account the conservation of the maternal lineage cytoplasmic diversity are required. The investigated chloroplast SSRs provide useful molecular markers which could be used in further genetic studies and breeding surveys of pearl millet genetic resources.

著者

Yuko Maki Hiroshi Soejima Toru Kitamura Tamizi Sugiyama Takeo Sato Masaaki K. Watahiki Junji Yamaguchi

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.38, no.1, pp.9-16, 2021-03-25 (Released:2021-03-25)

参考文献数

32

被引用文献数

9

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Bokashi fertilizer, an organic fertilizer made of plant residue, has been used in Japan not only to fertilize plants but to regulate their growth. Lactic acid bacteria have been found to play an important role in the fermentation process of Bokashi, but the relationship between these bacteria and plant growth activity has not been clarified. Using the adzuki rooting assay, this study identified 3-phenyllactic acid (PLA) produced by lactic acid bacteria as a root promoting compound in Bokashi. PLA showed synergistic effect with tryptophan, but no stem elongation activity. Lactic acid bacteria produced equal quantities of the L- and D-forms of PLA, which have similar root promoting activity. PLA did not significantly affect the amount of endogenous indole-3-acetic acid (IAA), although the chemical structure of PLA is highly similar to that of L-2-aminooxy-3-phenypropionic acid (L-AOPP), which inhibits IAA biosynthesis. These results indicate that the root promoting activity of PLA is not simply due to its increase in the amount of active auxin.

著者

Reira Suzuki Takashi Ueda Takuji Wada Masaki Ito Takashi Ishida Shinichiro Sawa

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.38, no.1, pp.1-8, 2021-03-25 (Released:2021-03-25)

参考文献数

45

被引用文献数

5

---

Root-knot nematodes (RKN; Meloidogyne incognita) are phytoparasitic nematodes that cause significant damage to crop plants worldwide. Recent studies have revealed that RKNs disrupt various physiological processes in host plant cells to induce gall formation. However, little is known about the molecular mechanisms of gall formation induced by nematodes. We have previously found that RNA expression levels of some of genes related to micro-RNA, cell division, membrane traffic, vascular formation, and meristem maintenance system were modified by nematode infection. Here we evaluated these genes importance during nematode infection by using Arabidopsis mutants and/or β-glucronidase (GUS) marker genes, particularly after inoculation with nematodes, to identify the genes involved in successful nematode infection. Our results provide new insights not only for the basic biology of plant–nematode interactions but also to improve nematode control in an agricultural setting.

著者

Sabrina Sultana Daiki Fujiwara Koh Aoki

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.38, no.1, pp.47-56, 2021-03-25 (Released:2021-03-25)

参考文献数

39

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Cuscuta campestris, a stem parasitic plant, commences its parasitic behavior by forming a specialized disk-like adhesive structure called a holdfast, which facilitates tight adhesion to the stem surface of the host plant. The morphology of epidermal cells in the holdfast is similar to that of the leaf trichome and root hairs of dicotyledonous plants. However, the regulatory network underlying the development of the holdfast has not been elucidated to date. In this study, we assessed the roles of epidermal cell-patterning genes in the development of a holdfast. Epidermal cell-patterning genes of C. campestris, including CcWER, CcGL3, CcTTG1, CcGL2, and CcJKD, were expressed slightly before the initiation of the outgrowth of stem epidermal cells. CcJKD-silencing repressed CcJKD, CcWER, CcGL3, CcTTG1, CcGL2; therefore, CcJKD is an upstream regulator of other epidermal cell-patterning genes. Unlike other genes, CcCPC was not upregulated after attachment to the host, and was not repressed by CcJKD-silencing. Protein interaction assays demonstrated that CcJKD interacted with CcTTG1 and CcCPC. Furthermore, CcJKD-silencing repressed the outgrowth of holdfast epidermal cells. Therefore, C. campestris invokes epidermal cell-patterning genes for the outgrowth of holdfast epidermal cells, and their regulatory mechanism is different from those for leaf trichome or root hairs.

著者

Nutwadee Chintakovid Rujira Tisarum Thapanee Samphumphuang Thanyaporn Sotesaritkul Suriyan Cha-um

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.38, no.1, pp.37-46, 2021-03-25 (Released:2021-03-25)

参考文献数

58

被引用文献数

3

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In vitro acclimatization has been validated as the successful key to harden the plantlets before transplanting to ex vitro conditions. In the present study, we investigated the potential of different sugar types (glucose, fructose, galactose, sucrose) in regulating morphological, physiological and biochemical strategies, survival percentage and growth performance, and rhizome traits of turmeric under iso-osmotic potential. Leaf greenness (SPAD value) in acclimatized plantlets (4% glucose; −1.355 MPa osmotic potential) of ‘ST018’ was retained and greater than in ‘PB009’ by 1.69-fold, leading to maintain high Fv/Fm (maximum quantum yield of PSII), ΦPSII (photon yield of PSII) and Pn (net photosynthetic rate) levels, and retained shoot height, leaf length, leaf width, shoot fresh weight and shoot dry weight after one month upon transplanting to ex vitro conditions. In addition, Pn, Ci (intracellular CO2), gs (stomatal conductance) and E (transpiration rate) in acclimatized plantlets (6% sucrose; −1.355 MPa osmotic potential) of ‘PB009’ were stabilized as physiological adapted strategies, regulating the shoot and root growth and fresh and dry weights of mini-rhizome. Interestingly, the accumulation of total curcuminoids in mini-rhizome derived from 6% sucrose acclimatized plantlets of ‘ST018’ was greater than in ‘PB009’ by 3.76-fold. The study concludes that in vitro acclimation of turmeric ‘PB009’ and ‘ST018’ using 6% sucrose and 4% glucose, respectively, promoted percent survival, physiological adaptations, and overall growth performances under greenhouse conditions.

著者

Michio Shibata

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.25, no.1, pp.3-8, 2008-03-01 (Released:2008-03-15)

参考文献数

19

被引用文献数

22 36

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

著者

Kumpei Shiragaki Rie Nakamura Shigeki Nomura Hai He Tetsuya Yamada Wataru Marubashi Masayuki Oda Takahiro Tezuka

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.37, no.3, pp.327-333, 2020-09-25 (Released:2020-09-25)

参考文献数

41

被引用文献数

11

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Hybrid lethality observed in hybrid seedlings between Nicotiana suaveolens and N. tabacum is characterized by browning, initially of the hypocotyls and eventually of entire seedlings. We investigated the mechanism underlying this browning of tissues. A phenylalanine ammonia-lyase (PAL) gene codes an enzyme involved in a pathway producing phenolic compounds related to the browning of plant tissues. The expression of PAL rapidly increased with the induction of hybrid lethality. Phenolic compounds were observed to be accumulated in whole parts of hybrid seedlings. Treatment of hybrid seedlings with L-2-aminooxy-3-phenylpropionic acid (AOPP), an inhibitor for PAL, suppressed browning and decreased the phenolic content of hybrid seedlings. Although programmed cell death (PCD) was involved in hybrid lethality, AOPP treatment also suppressed cell death and enhanced the growth of hybrid seedlings. These results indicated that PAL is involved in hybrid lethality, and phenolic compounds could be the cause of hybrid lethality-associated tissue browning.

著者

Takeshi Ara Kunihiro Suda Masayuki Amagai Kiyoshi Namai Hideyuki Suzuki Nozomu Sakurai Daisuke Shibata

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.37, no.3, pp.383-387, 2020-09-25 (Released:2020-09-25)

参考文献数

11

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Metabolome analysis of flavored vegetables, green spring onion (Allium fistulosum), Chinese chive (A. tuberosum), and their interspecies hybrid Negi-Nira chive, was conducted using liquid chromatography-Fourier transform ion cyclotron resonance-mass spectrometry, with ca. 2 ppm mass accuracy. Ion peaks in the chromatograms of four biological replicates of the vegetable leaves were processed using the alignment software PowerGet for metabolite comparison, from which we obtained the potential chemical formulae. In total, 860 ion peaks were reproducibly detected; of these, 506, 525, and 336 peaks were found in the hybrid, A. tuberosum, and A. fistulosum, respectively. There were 130 peaks specific to the hybrid; from these, 31 metabolites were annotated by searching compound databases. The sulfur-containing compounds and flavonoids were further analyzed using bioinformatics, to examine the sulfur metabolism of Allium volatiles and the flavonoid pathways in these species. In conclusion, our metabolome analysis of this interspecies hybrid and its parents provides a unique opportunity to elucidate their metabolic background.

著者

Yoko Kamiya Fumitaka Abe Masafumi Mikami Masaki Endo Kanako Kawaura

出版者

Japanese Society for Plant Cell and Molecular Biology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.37, no.2, pp.247-251, 2020-06-25 (Released:2020-06-25)

参考文献数

11

被引用文献数

12

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Genome editing using CRISPR/Cas9 is useful for common wheat because common wheat has allohexaploid nature and it can induce mutations simultaneously in three homoeologous genes. Although Agrobacterium-mediated transformation has advantages in genome editing, it still has low efficiency and requires relatively long time in wheat. Therefore, the use of guide RNAs (gRNAs) with efficient mutagenesis in vivo is one of the critical factors for producing genome-edited mutant lines in a short time. In this study, we targeted three genes in common wheat and established a rapid method for detection of mutations induced by the biolistic transient expression system. Biolistic transient expression of the gRNAs and Cas9 was achieved in immature wheat embryos. Mutations were detected a week later using PCR-RFLP and verified by the sequencing of genomic clones. We confirmed several types of mutations that occurred at different rates depending on the target sequences. Furthermore, frequencies of mutations tended to be higher at the targets that were edited at higher rates in the plants transformed by Agrobacterium. These results show that this method of rapid detection of edited mutations could be used for variety of applications, such as screening of target sequences or modified vectors for efficient CRISPR/Cas9 genome editing in wheat.

著者

Irene Ferreres Mirari Ortega Camilo López-Cristoffanini Salvador Nogués Xavier Serrat

出版者

Japanese Society for Plant Cell and Molecular Biology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.36, no.4, pp.269-273, 2019-12-25 (Released:2019-12-27)

参考文献数

22

被引用文献数

5

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Anther culture is a fast tool to obtain double haploid plant lines for breeding purposes. In rice, this procedure is commonly performed in two steps: i) induction of calli from anthers and ii) regeneration of plantlets from calli. It has been stated that genotype highly influences the anther culture efficiency, so the media used in each step should be optimized for each variety. In this study, we tested different media modifications of an efficient protocol optimized for a medium sized grain temperate japonica NRVC980385, used as a control, in a long grain temperate japonica rice variety (NRVC20120346), and two long grain tropical japonica varieties (303012 and 303013).We found that the addition of 150 mg l−1 colchicine to the induction medium worked best for all genotypes except for NRVC20120346, whose best induction was obtained with the colchicine-free medium. Referring to regeneration, increased gelling agent in the medium provided the best rates in NRVC980385, improving our former NRVC980385-optimized anther culture protocol. Sorbitol fortified regeneration medium worked the best in the case of the long grain varieties. The presence of colchicine in the induction medium was also related to a higher obtention of double haploid plantlets. This study highlights that genotype is a key factor in the performance of rice anther culture. It has set a first anther culture study on long grain japonica varieties and optimizes the anther culture protocol for temperate japonica medium grain NRVC980385 with the use of colchicine and other additives that increase osmotic stress.

著者

Hiroyuki Kajiura Kyoko Hiwasa-Tanase Hiroshi Ezura Kazuhito Fujiyama

出版者

Japanese Society for Plant Cell and Molecular Biology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.35, no.4, pp.375-379, 2018-12-25 (Released:2018-12-31)

参考文献数

17

被引用文献数

1 4

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Miraculin is a promising protein with taste-modifying properties. Focusing on the unique function and potential of miraculin, recombinant miraculin production has been explored with the use of heterologous expression systems, but the activities of recombinant miraculins were much lower than those of native miraculin, probably due to the difference in post-translational modification, especially N-glycosylation. For practical use therefore, the differences between N-glycan of recombinant miraculin compared to that of native miraculin should be minimized. Here, to establish the platform for functional miraculin production, we expressed miraculin in tomato plants with the same taste-modifying activity as native miraculin purified from miracle fruit, and we compared the N-glycan structures with those of native miraculin. Our N-glycan structural analysis using purified miraculin, followed by hydrazynolysis, 2-pyridylamine (PA)-labeling, high-performance liquid chromatography, and a liquid chromatography tandem-mass spectrometry analysis revealed that both the native and recombinant miraculins carried an M3 structure as a predominant structure and that most of the N-glycan structures on the miraculins were pauci-mannosidic structures with a smaller amount of plant-specific α1,3-fucosylated and/or β1,2-xylosylated N-glycans and without a Lewis a epitope. These results indicate that the N-glycoform of native miraculin from miracle fruit and recombinant miraculin expressed in tomato plants are almost identical to each other with similar ratios and that, therefore, plant-specific N-glycans are essential for showing the full taste-modifying activity of miraculin.

著者

Ryo Nakabayashi Tomoko Nishizawa Tetsuya Mori Hiroshi Sudo Isao Fujii Takashi Asano Kazuki Saito

出版者

Japanese Society for Plant Cell and Molecular Biology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

pp.19.1002a, (Released:2019-12-18)

参考文献数

6

被引用文献数

8

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Asparaptine, a conjugate of L-arginine and asparagusic acid, was found in green asparagus (Asparagus officinalis) using ultrahigh-resolution metabolomics for sulfur-containing metabolites (S-metabolites), called S-omics. Asparaptine has been shown to inhibit the activity of angiotensin-converting enzyme. Larger amounts of this S-metabolite are therefore required for further analysis; however, there are limitations that asparagus is a perennial plant and its spears, wherein asparaptine accumulates, can be mainly harvested at the spring to summer season. In order to overcome these, we prepared a callus and suspension cell line from green asparagus. Untargeted metabolome analysis using liquid chromatography-tandem mass spectrometry was performed in the materials as well as spears and three calluses derived from wild type Asparagus. The analysis demonstrated that the amount of asparaptine in the callus derived from the green asparagus was more than the others per mg dry weight. The suspension cell line treated with methyljasmonate showed the induction of asparaptine, suggesting that the asparaptine production is modifiable under appropriate culture conditions. The described materials can be utilized for the production of asparaptine and in integrated metabolomics to study the biosynthesis of this S-metabolite, which is currently unknown.

著者

Naoki Yamamoto Hiroyuki Kajiura Shinya Takeno Nobuaki Suzuki Yoshihisa Nakazawa

出版者

Japanese Society for Plant Cell and Molecular Biology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

pp.14.0609b, (Released:2014-08-23)

参考文献数

33

被引用文献数

1 3

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We established a DNA watermarking system for discriminating transgenic plants. The system contains an encryption algorithm based on a binary system, genetic transformation and a detection algorithm for encrypted DNA watermark sequences using a DNA dot plot. The encryption algorithm converted character strings into nucleic acid sequences through binary digits, and the sequence was designed to be resistant to transition mutations to decipher codes completely. Moreover, the encrypted sequences were capable of taking specific nucleotide sequences in using the algorithmic redundancy of the corresponding DNA. Genetic transformation enables labeling plant genomes with DNA watermarks. The detection algorithm allows finding traces of sequence changes in DNA watermarks, complementing the error protection function of the encryption algorithm. To validate the effectiveness of our DNA watermarking system, we introduced a DNA watermark to the tobacco genome and detected the DNA watermark in PCR products amplified from the genome. This indicates that DNA watermark technology is useful for introducing artificial genetic markers in plant organisms, in particular when several transgenic host plants and transgenes are used. The source codes of the Perl scripts are available in this report.

著者

Yuki Kanesaka Masaaki Okada Shogo Ito Tokitaka Oyama

出版者

Japanese Society for Plant Cell and Molecular Biology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

pp.19.0531a, (Released:2019-09-07)

参考文献数

27

被引用文献数

7

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The rapid assessment of gene function is crucial in biological research. The CRISPR/Cas9 system is widely used as a tool for targeted gene editing in many organisms including plants. Previously, we established a transient gene expression system for investigating cellular circadian rhythms in duckweed. In this system, circadian reporters and clock gene effectors—such as overexpressors, RNA interference (RNAi), and CRISPR/Cas9—were introduced into duckweed cells using a particle bombardment method. In the present study, we applied the CRISPR/Cas9 system at a single cell level to Arabidopsis thaliana, a model organism in plant biology. To evaluate the mutation induction efficiency of the system, we monitored single-cell bioluminescence after application of the CRISPR/Cas9 system targeting the ELF3 gene, which is essential for robust circadian rhythmicity. We evaluated the mutation induction efficiency by determining the proportion of cells with impaired circadian rhythms. Three single guide RNAs (sgRNAs) were designed, and the proportion of arrhythmic cells following their use ranged from 32 to 91%. A comparison of the mutation induction efficiencies of diploid and tetraploid Arabidopsis suggested that endoreduplication had a slight effect on efficiency. Taken together, our results demonstrate that the transiently introduced CRISPR/Cas9 system is useful for rapidly assessing the physiological function of target genes in Arabidopsis cells.

著者

Sumire Fujiwara Keiko Kigoshi Nobutaka Mitsuda Kaoru Suzuki Masaru Ohme-Takagi

出版者

Japanese Society for Plant Cell and Molecular Biology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

pp.14.0121a, (Released:2014-03-20)

参考文献数

58

被引用文献数

5 7

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Proper gene expression regulated by transcription factors is essential for plants to achieve proper growth and development. However, the biological functions of many transcription factors remain largely unknown. Furthermore, although there are transcription factors which possess a plant-specific repression domain(s), their biological functions and whether such transcription factors function as transcriptional repressors are unclear. Thus, aiming for searching clues to understand their functions, we generated transgenic plants in which a putative transcriptional repressor fused with a VP16 viral trans-activation domain was expressed constitutively. Several plants with strong morphological phenotypes such as leaf and flower development defects were isolated from those lines expressing potential transcriptional repressors with unknown functions, giving the clue to reveal the yet-to-be analyzed functions of each protein. Reversal of function of the well-known transcriptional and floral repressor SHORT VEGETATIVE PHASE by VP16 fusion was observed, exemplifying successful functional reversion by this system. Plants constitutively expressing VP16 fused WUSCHEL, which is known to function both as a transcriptional activator and repressor, showed both phenotypes reported in its overexpression and loss-of-function lines. Taken together, our data provide examples showing the efficacy of VP16 fusion to provide helpful information to uncover the unknown functions of potential transcriptional repressors. This technique could also be effective to produce “super plants” which obtained strong and useful traits for application by strongly activating genes which are usually silent.

著者

Anung Wahyudi Dinni Ariyani Gang Ma Ryosuke Inaba Chikako Fukasawa Ryohei Nakano Reiko Motohashi

出版者

Japanese Society for Plant Cell and Molecular Biology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.35, no.4, pp.303-312, 2018-12-25 (Released:2018-12-31)

参考文献数

29

被引用文献数

10

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In this study, two temperature-induced lipocalin genes SlTIL1 and SlTIL2, and a chloroplastic lipocalin gene SlCHL were isolated from ‘Micro-Tom’ tomato. The coding sequences of SlTIL1, SlTIL2 and SlCHL were 558, 558, and 1002 bp, respectively. By TargetP analysis, no characteristic transit peptides were predicted in the proteins of SlTIL1 and SlTIL2, while a chloroplastic transit peptide was predicted in the protein of SlCHL. The subcellular localization results indicated that SlTIL1 and SlTIL2 proteins were major localized in the plasma membrane, while SlCHL was localized in chloroplast. To understand the function of lipocalins, transgenic tomato over-expressed SlTIL1, SlTIL2 and SlCHL and their virus-induced gene silencing (VIGS) plants were generated. The phenotypes were significantly affected when the SlTIL1, SlTIL2 and SlCHL were over-expressed or silenced by VIGS, which suggested that the three lipocalins played important roles in regulating the growth and development of tomato. In addition, the level of ROS (O2− and H2O2) was low in SlTIL1, SlTIL2 and SlCHL over-expressed plants, while it was high in their silenced plants. The changes in the expression of SODs were consistent with the accumulations of ROS, which indicated that lipocalins might have an important role in abiotic oxidative stress tolerance in tomato plants. Especially SlTIL1 and SlTIL2 are localized around their membranes and protect them from ROS. The results will contribute to elucidating the functions of lipocalin in plants, and provide new strategies to improve the tolerance to abiotic stress in tomato plants.

著者

Shinzo Tsuda Yuko Fukui Noriko Nakamura Yukihisa Katsumoto Keiko Yonekura-Sakakibara Masako Fukuchi-Mizutani Kazuyuki Ohira Yukiko Ueyama Hideo Ohkawa Timothy A. Holton Takaaki Kusumi Yoshikazu Tanaka

出版者

日本植物細胞分子生物学会

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.21, no.5, pp.377-386, 2004 (Released:2005-06-03)

参考文献数

34

被引用文献数

35 76

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Petunia flower colors are mainly due to flavonoids. The flower color of commercial varieties of Petunia hybrida was successfully modified by the suppression of endogenous flavonoid biosynthetic genes, the expression of a hetelorogous flavonoid biosynthetic gene, and the combination of both. Flower color changed from purple to almost white or from purple to red by the suppression of the endogenous gene expression, from red to orange by the down-regulation of the flavonoid 3′-hydroxylase gene and the expression of the rose dihydroflavonol 4-reductase gene, and from violet to pale violet by the expression of the flavonol synthase or flavone synthase gene. These results clearly indicate the usefulness of metabolic engineering of the flavonoid biosynthetic pathway to modify flower color. Only a few of the transgenic petunia exhibited phenotypic stability. For commercialisation, it is necessary to generate many independent transgenic lines, select elite lines with stable phenotypes and maintain them in tissue culture.