著者

Bera Subhankar Katsushi Yamaguchi Shuji Shigenobu Koh Aoki

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

pp.21.0121a, (Released:2021-06-01)

参考文献数

41

被引用文献数

8

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Parasitic plants exchange various types of RNAs with their host plants, including mRNA, and small non-coding RNA. Among small non-coding RNAs, miRNA production is known to be induced at the haustorial interface. The induced miRNAs transfer to the host plant and activate secondary siRNA production to silence target genes in the host. In addition to interfacial transfer, long-distance movement of the small RNAs has also been known to mediate signaling and regulate biological processes. In this study, we tested the long-distance movement of trans-species small RNAs in a parasitic-plant complex. Small RNA-Seq was performed using a complex of a stem parasitic plant, Cuscuta campestris, and a host, Arabidopsis thaliana. In the host plant’s parasitized stem, genes involved in the production of secondary siRNA, AtSGS3 and AtRDR6, were upregulated, and 22-nt small RNA was enriched concomitantly, suggesting the activation of secondary siRNA production. Stem-loop RT-PCR and subsequent sequencing experimentally confirmed the mobility of the small RNAs. Trans-species mobile small RNAs were detected in the parasitic interface and also in distant organs. To clarify the mode of long-distance translocation, we examined whether C. campestris-derived small RNA moves long distances in A. thaliana sgs3 and rdr6 mutants or not. Mobility of C. campestris-derived small RNA in sgs3 and rdr6 mutants suggested the occurrence of direct long-distance transport without secondary siRNA production in the recipient plant.

著者

Azusa Ono Kyoko Hiwasa-Tanase Satoko Nonaka Hiroshi Ezura

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.161-165, 2021-03-25 (Released:2021-03-25)

参考文献数

21

被引用文献数

2

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The taste-modifying protein miraculin (MIR) has received increasing interest as a new low-calorie sweetener. In our previous study using the tomato variety ‘Micro-Tom,’ it was shown that in transgenic tomatoes in which MIR was expressed by using the cauliflower mosaic virus 35S promoter (p35S) and a heat shock protein terminator (tHSP) cassette (p35S-MIR-tHSP), higher levels of miraculin accumulated than when MIR was driven by the nopaline synthase terminator (tNOS) cassette (p35S-MIR-tNOS). ‘Micro-Tom’ is a dwarf tomato used for research and shows a low yield. To achieve high productivity of MIR, it is essential to improve the MIR accumulation potential by using high-yielding cultivars. In this study, we evaluate whether the high MIR accumulation trait mediated by the tHSP appears even when fruit size increases. A line in which the p35S-MIR-tHSP cassette was introduced into a high-yielding variety was bred by backcrossing. The line homozygous for MIR showed higher accumulation of MIR than the heterozygous line. Despite large differences in fruit size, the MIR level in the backcross line was similar to that in the p35S-MIR-tHSP line (background ‘Micro-Tom’). It was approximately 3.1 times and 4.0 times higher than those in miracle fruits and the p35S-MIR-tNOS tomato line 5B (‘Moneymaker’ background, which exhibits the highest miraculin productivity achieved thus far), respectively. These results demonstrate that the high MIR accumulation trait mediated by the tHSP appears even when fruit size is increased.

著者

Mariko Ohnuma Kosuke Ito Karin Hamada Ami Takeuchi Kenji Asano Takahiro Noda Akira Watanabe Akiko Hokura Hiroshi Teramura Fuminori Takahashi Hiromi Mutsuro-Aoki Koji Tamura Hiroaki Shimada

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.40, no.3, pp.219-227, 2023-09-25 (Released:2023-09-25)

参考文献数

34

被引用文献数

2

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Glucose chains in starch are phosphorylated and contribute to structural stabilization. Phosphate groups contained in starch also play a role in retaining moisture. α-Glucan water dikinase 1 (GWD1) is involved in the phosphorylation of glucose chains in starch. In this study, we generated potato mutants of the GWD1 gene using the CRISPR/dMac3-Cas9 system. Observation of the phenotypes of the GWD1-deficient mutants revealed their physiological roles in tuber starch formation. The 4-allele mutants showed growth retardation and a delay in tuber formation. A significant decrease in phosphorus content was detected in the tuber starch of the gwd1 mutant. This mutant starch showed a higher amylose content than the wild-type starch, whereas its gelatinization temperature was slightly lower than that of the WT starch. The peak viscosity of the mutant starch was lower than that of the WT starch. These observations revealed that the starch of the gwd1 mutants had peculiar and unique properties compared to those of WT, sbe3 and gbss1 mutant starches. The amount of tissue-released water due to freeze–thawing treatment was determined on tubers of the gwd1 mutant and compared with those of WT and the other mutants. Significantly less water loss was found in the gwd1, sbe3 and gbss1 mutant tubers than in the WT tubers. Our results indicate that the GWD1 gene is not only important for potato growth, but also largely effective for the traits of tuber starch.

著者

Mitsuko Kishi-Kaboshi Fumitaka Abe Yoko Kamiya Kanako Kawaura Hiroshi Hisano Kazuhiro Sato

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.40, no.3, pp.237-245, 2023-09-25 (Released:2023-09-25)

参考文献数

36

被引用文献数

1

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Genome editing is a promising method for simultaneously mutagenizing homoeologs in the three subgenomes of wheat (Triticum aestivum L.). However, the mutation rate via genome editing must be improved in order to analyze gene function and to quickly modify agronomic traits in wheat. Here, we examined the Cas9-induced mutation rates in wheat plants using two promoters for single guide RNA (sgRNA) expression and applying heat treatment during Agrobacterium tumefaciens-mediated transformation. Using the TaU6 promoter instead of the OsU6 promoter from rice (Oryza sativa L.) to drive sgRNA expression greatly improved the Cas9-induced mutation rate. Moreover, a heat treatment of 30°C for 1 day during tissue culture increased the Cas9-induced mutation rate and the variety of mutations obtained compared to tissue culture at the normal temperature (25°C). The same heat treatment did not affect the regeneration rates of transgenic plants but tended to increase the number of transgene integration sites in each transgenic plant. These results lay the foundation for improving the Cas9-induced mutation rate in wheat to enhance research on gene function and crop improvement.

著者

Naoyuki Umemoto Shuhei Yasumoto Muneo Yamazaki Kenji Asano Kotaro Akai Hyoung Jae Lee Ryota Akiyama Masaharu Mizutani Yozo Nagira Kazuki Saito Toshiya Muranaka

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.40, no.3, pp.211-218, 2023-09-25 (Released:2023-09-25)

参考文献数

19

被引用文献数

1

---

Genome editing is highly useful for crop improvement. The method of expressing genome-editing enzymes using a transient expression system in Agrobacterium, called agrobacterial mutagenesis, is a shortcut used in genome-editing technology to improve elite varieties of vegetatively propagated crops, including potato. However, with this method, edited individuals cannot be selected. The transient expression of regeneration-promoting genes can result in shoot regeneration from plantlets, while the constitutive expression of most regeneration-promoting genes does not result in normally regenerated shoots. Here, we report that we could obtain genome-edited potatoes by positive selection. These regenerated shoots were obtained via a method that combined a regeneration-promoting gene with the transient expression of a genome-editing enzyme gene. Moreover, we confirmed that the genome-edited potatoes obtained using this method did not contain the sequence of the binary vector used in Agrobacterium. Our data have been submitted to the Japanese regulatory authority, the Ministry of Education, Culture, Sports, Science and Technology (MEXT), and we are in the process of conducting field tests for further research on these potatoes. Our work presents a powerful method for regarding regeneration and acquisition of genome-edited crops through transient expression of regeneration-promoting gene.

著者

Masaki Odahara Most Tanziman Ara Remi Nakagawa Yoko Horii Shougo Ishio Shinjiro Ogita Keiji Numata

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.40, no.4, pp.263-271, 2023-12-25 (Released:2023-12-25)

参考文献数

34

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The plastid is a promising target for the production of valuable biomolecules via genetic engineering. We recently developed a plastid-specific gene delivery system for leaves or seedlings using KH-AtOEP34, a functional peptide composed of the polycationic DNA-binding peptide KH and the Arabidopsis thaliana plastid-targeting peptide OEP34. Here, we established a liquid culture system for inducing multiple shoots in the model plants A. thaliana and Nicotiana benthamiana and the crop plant strawberry (Fragaria×ananassa) and tested the use of these plant materials for peptide-mediated gene delivery to plastids. Our liquid culture system efficiently induced multiple shoots that were enriched in meristems. Using these meristems, we performed KH-AtOEP34-mediated gene delivery to plastids and tested the delivery and integration of a cassette composed of the spectinomycin resistance gene aadA, the GFP reporter gene, and sequences homologous to plastid DNA. Genotyping PCR revealed the integration of the cassette DNA into plastid DNA several days after delivery in all three plants. Confocal laser scanning microscopy and immunoblotting confirmed the presence of plasmid-derived GFP in the plastids of meristems, indicating that the plasmid DNA was successfully integrated into plastid DNA and that the cassette was expressed. These results suggest the meristems developed in our liquid culture system are applicable to peptide-mediated delivery of exogeneous DNA into plastids. The multiple shoots generated in our liquid novel culture system represent promising materials for in planta peptide-mediated plastid transformation in combination with spectinomycin selection.

著者

Jaechol Sim Yuhei Kanazashi Tetsuya Yamada

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.40, no.3, pp.247-254, 2023-09-25 (Released:2023-09-25)

参考文献数

24

被引用文献数

2

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In general, plant organ size is determined using cell number and expansion. In our previous study, we generated soybean (Glycine max) mutants of the PEAPOD (PPD) genes GmPPD1 and GmPPD2 using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 system. Some of these mutants exhibited extremely abnormal phenotypes, such as twisted pods and limited seeds. These phenotypes were attributed to the frameshift mutation in both GmPPD loci. In this study, the physiological and molecular biological properties of mutant plants with two knocked-out GmPPD loci (ppd-KO) were characterized. The ppd-KO mutant exhibited a delayed growth phase from the time of development of the unifoliolate leaves to that of first trifoliolate leaves and a stay-green phenotype, which were not observed in the other mutants of soybean or ppd mutants of other plant species. Gene expression analysis revealed considerably decreased expression of SPIRAL1-LIKE 5 (GmSP1L5), mainly causing the twisted pod phenotype observed in the ppd-KO mutant. The relationship between PPD and SP1L5 has not been previously reported, and in this study, we showed that that loss of PPD functioning affects SP1L5 expression in soybean. In this study, we revealed that the decrease in PPD function contributed to organ enlargement and that complete knockout of PPD has a negative effect on soybean organogenesis.

著者

Jaechol Sim Chikako Kuwabara Shota Sugano Kohei Adachi Tetsuya Yamada

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.40, no.3, pp.193-200, 2023-09-25 (Released:2023-09-25)

参考文献数

59

被引用文献数

1

---

Genetic improvement of soybean seed traits is important for developing new varieties that meet the demand for soybean as a food, forage crop, and industrial products. A large number of soybean genome sequences are currently publicly available. This genome sequence information provides a significant opportunity to design genomic approaches to improve soybean traits. Genome editing represents a major advancement in biotechnology. The production of soybean mutants through genome editing is commonly achieved with either an Agrobacterium-mediated or biolistic transformation platform, which have been optimized for various soybean genotypes. Currently, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system, which represents a major advance in genome editing, is used to improve soybean traits, such as fatty acid composition, protein content and composition, flavor, digestibility, size, and seed-coat color. In this review, we summarize the recent advances in the improvement of soybean seed traits through genome editing. We also discuss the characteristics of genome editing using the CRISPR/Cas9 system with transformation platforms.

著者

Naoki Yokotani Yoshinori Hasegawa Yusuke Kouzai Hideki Hirakawa Sachiko Isobe

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.40, no.4, pp.273-282, 2023-12-25 (Released:2023-12-25)

参考文献数

51

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Salicylic acid (SA) is known to be involved in the immunity against Clavibacter michiganensis ssp. michiganensis (Cmm) that causes bacterial canker in tomato. To identify the candidate genes associated with SA-inducible Cmm resistance, transcriptome analysis was conducted via RNA sequencing in tomato plants treated with SA. SA treatment upregulated various defense-associated genes, such as PR and GST genes, in tomato cotyledons. A comparison of SA- and Cmm-responsive genes revealed that both SA treatment and Cmm infection commonly upregulated a large number of genes. Gene Ontology (GO) analysis indicated that the GO terms associated with plant immunity were over-represented in both SA- and Cmm-induced genes. The genes commonly downregulated by both SA treatment and Cmm infection were associated with the cell cycle and may be involved in growth and immunity trade-off through cell division. After SA treatment, several proteins that were predicted to play a role in immune signaling, such as resistance gene analogs, Ca2+ sensors, and WRKY transcription factors, were transcriptionally upregulated. The W-box element, which was targeted by WRKYs, was over-represented in the promoter regions of genes upregulated by both SA treatment and Cmm infection, supporting the speculation that WRKYs are important for the SA-mediated immunity against Cmm. Prediction of protein–protein interactions suggested that genes encoding receptor-like kinases and EF-hand proteins play an important role in immune signaling. Thus, various candidate genes involved in SA-inducible Cmm resistance were identified.

著者

Keito Mineta Junya Hirota Kesuke Yamada Takashi Itoh Poyu Chen Hidekazu Iwakawa Hirotomo Takatsuka Yuji Nomoto Masaki Ito

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.40, no.4, pp.353-359, 2023-12-25 (Released:2023-12-25)

参考文献数

20

---

Although it is well known that hierarchical transcriptional networks are essential for various aspects of plant development and environmental response, little has been investigated about whether and how they also regulate the plant cell cycle. Recent studies on cell cycle regulation in Arabidopsis thaliana identified SCARECROW-LIKE28 (SCL28), a GRAS-type transcription factor, that constitutes a hierarchical transcriptional pathway comprised of MYB3R, SCL28 and SIAMESE-RELATED (SMR). In this pathway, MYB3R family proteins regulate the G2/M-specific transcription of the SCL28 gene, of which products, in turn, positively regulate the transcription of SMR genes encoding a group of plant-specific inhibitor proteins of cyclin-dependent kinases. However, this pathway with a role in cell cycle inhibition is solely demonstrated in A. thaliana, thus leaving open the question of whether and to what extent this pathway is evolutionarily conserved in plants. In this study, we conducted differential display RT-PCR on synchronized Nicotiana tabacum (tobacco) BY-2 cells and identified several M-phase-specific cDNA clones, one of which turned out to be a tobacco ortholog of SCL28 and was designated NtSCL28. We showed that NtSCL28 is expressed specifically during G2/M and early G1 in the synchronized cultures of BY-2 cells. NtSCL28 contains MYB3R-binding promoter elements, so-called mitosis-specific activator elements, and is upregulated by a hyperactive form of NtmybA2, one of the MYB3R proteins from tobacco. Our study indicated that a part of the hierarchical pathway identified in A. thaliana is equally operating in tobacco cells, suggesting the conservation of this pathway across different families in evolution of angiosperm.

著者

Yong-Gen Yin Atsuko Sanuki Yukihisa Goto Nobuo Suzui Naoki Kawachi Chiaki Matsukura

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.40, no.4, pp.345-351, 2023-12-25 (Released:2023-12-25)

参考文献数

32

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In early developing tomato (Solanum lycopersicum L.) fruit, starch accumulates at high levels and is used by various primary metabolites in ripening fruits. ADP-glucose pyrophosphorylase is responsible for the first key step of starch biosynthesis. Although it has been reported that AgpL1 and AgpS1 isoforms are mainly expressed in early developing fruit, their regulatory mechanism has not been elucidated. The present study investigated the transcriptional response of AgpL1 and AgpS1 to various metabolizable sugars, nonmetabolizable sugar analogues, hexokinase inhibitors and proline by an experimental system using half-cut fruits. AgpL1 was upregulated in response to sucrose and constituted hexoses such glucose, whereas the AgpS1 gene almost did not exhibit a prominent sugar response. Further analyses revealed that other disaccharides such maltose and trehalose did not show a remarkable effect on both AgpL1 and AgpS1 expressions. These results indicate that there are two distinct regulatory mechanisms, namely, sugar metabolism-dependent and -independent, for the regulation of AGPase gene expression. Interestingly, the ADP treatment, a hexokinase inhibitors, cancelled the sugar response of AgpL1, indicating that hexokinase-mediated sugar signaling should be involved in the sugar response of AgpL1. These results suggest that sugar-dependent (AgpL1) and sugar-independent (AgpS1) pathways coordinatively regulate starch biosynthesis in immature tomato fruit.

著者

Yoshimi Nakano Maki Kawai Moeca Arai Sumire Fujiwara

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.40, no.4, pp.337-344, 2023-12-25 (Released:2023-12-25)

参考文献数

26

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Correct flower organ formation at the right timing is one of the most important strategies for plants to achieve reproductive success. Ectopic overexpression of LATE FLOWERING (LATE) is known to induce late flowering, partly through suppressing expression of the florigen-encoding gene FLOWERING LOCUS T (FT) in Arabidopsis. LATE is one of the C2H2 zinc finger transcription factors, and it has a canonical transcriptional repression domain called the ethylene-responsive element-binding factor-associated amphiphilic repression (EAR) motif at the end of its C terminus. Therefore, LATE is considered a transcriptional repressor, but its molecular function remains unclear. Our genome-edited late mutants exhibited no distinct phenotype, even in flowering, indicating the presence of redundancy from other factors. To reveal the molecular function of LATE and factors working with it, we investigated its transcriptional activity and interactions with other proteins. Transactivation activity assay showed that LATE possesses transcriptional repression ability, which appears to be attributable to both the EAR motif and other sequences. Yeast two-hybrid assay showed the EAR motif-mediated interaction of LATE with TOPLESS, a transcriptional corepressor. Moreover, LATE could also interact with CRABS CLAW (CRC), one of the most important regulators of floral meristem determinacy, through sequences in LATE other than the EAR motif. Our findings demonstrated the possibility that LATE can form a transcriptional repression complex with CRC for floral meristem determinacy.

著者

Emi Iida Kazunori Kuriyama Midori Tabara Atsushi Takeda Nobuhiro Suzuki Hiromitsu Moriyama Toshiyuki Fukuhara

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.40, no.4, pp.289-299, 2023-12-25 (Released:2023-12-25)

参考文献数

29

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Agrobacterium tumefaciens (Rhizobium radiobacter) is used for the transient expression of foreign genes by the agroinfiltration method, but the introduction of foreign genes often induces transcriptional and/or post-transcriptional gene silencing (TGS and/or PTGS). In this study, we characterized the structural features of T-DNA that induce TGS during agroinfiltration. When A. tumefaciens cells harboring an empty T-DNA plasmid containing the cauliflower mosaic virus (CaMV) 35S promoter were infiltrated into the leaves of Nicotiana benthamiana line 16c with a GFP gene over-expressed under the control of the same promoter, no small interfering RNAs (siRNAs) were derived from the GFP sequence. However, siRNAs derived from the CaMV 35S promoter were detected, indicating that TGS against the GFP gene was induced. When the GFP gene was inserted into the T-DNA plasmid, PTGS against the GFP gene was induced whereas TGS against the CaMV 35S promoter was suppressed. We also showed the importance of terminator sequences in T-DNA for gene silencing. Therefore, depending on the combination of promoter, terminator and coding sequences on T-DNA and the host nuclear genome, either or both TGS and/or PTGS could be induced by agroinfiltration. Furthermore, we showed the possible involvement of three siRNA-producing Dicers (DCL2, DCL3 and DCL4) in the induction of TGS by the co-agroinfiltration method. Especially, DCL2 was probably the most important among them in the initial step of TGS induction. These results are valuable for controlling gene expression by agroinfiltration.

著者

Ryszard Zamorski Kei’ichi Baba Takahiro Noda Rimpei Sawada Kana Miyata Takao Itoh Hanae Kaku Naoto Shibuya

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.40, no.4, pp.321-336, 2023-12-25 (Released:2023-12-25)

参考文献数

85

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Plant cell wall plays important roles in the regulation of plant growth/development and affects the quality of plant-derived food and industrial materials. On the other hand, genetic variability of cell wall structure within a plant species has not been well understood. Here we show that the endosperm cell walls, including both starchy endosperm and aleurone layer, of rice grains with various genetic backgrounds are clearly classified into two groups depending on the presence/absence of β-1,4-linked glucomannan. All-or-none distribution of the glucomannan accumulation among rice varieties is very different from the varietal differences of arabinoxylan content in wheat and barley, which showed continuous distributions. Immunoelectron microscopic observation suggested that the glucomannan was synthesized in the early stage of endosperm development, but the synthesis was down-regulated during the secondary thickening process associated with the differentiation of aleurone layer. Significant amount of glucomannan in the cell walls of the glucomannan-positive varieties, i.e., 10% or more of the starchy endosperm cell walls, and its close association with the cellulose microfibril suggested possible effects on the physicochemical/biochemical properties of these cell walls. Comparative genomic analysis indicated the presence of striking differences between OsCslA12 genes of glucomannan-positive and negative rice varieties, Kitaake and Nipponbare, which seems to explain the all-or-none glucomannan cell wall trait in the rice varieties. Identification of the gene responsible for the glucomannan accumulation could lead the way to clarify the effect of the accumulation of glucomannan on the agronomic traits of rice by using genetic approaches.

著者

Hiroaki Kisaka Dong Poh Chin Tetsuya Miwa Hiroto Hirano Sato Uchiyama Masahiro Mii Mayu Iyo

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.40, no.4, pp.311-320, 2023-12-25 (Released:2023-12-25)

参考文献数

35

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The biosynthetic pathway of Catharanthus roseus vinca alkaloids has a long research history, including not only identification of metabolic intermediates but also the mechanisms of inter-cellular transport and accumulation of biosynthesized components. Vinca alkaloids pathway begins with strictosidine, which is biosynthesized by condensing tryptamine from the tryptophan pathway and secologanin from the isoprenoid pathway. Therefore, increasing the supply of precursor tryptophan may enhance vinca alkaloid content or their metabolic intermediates. Many reports on the genetic modification of C. roseus use cultured cells or hairy roots, but few reports cover the production of transgenic plants. In this study, we first investigated a method for stably producing transgenic plants of C. roseus, then, using this technique, we modified the tryptophan metabolism system to produce transgenic plants with increased tryptophan content. Transformed plants were obtained by infecting cotyledons two weeks after sowing with Agrobacterium strain A13 containing a plant expression vector, then selecting with 1/2 B5 medium supplemented with 50 mg l−1 kanamycin and 20 mg l−1 meropenem. Sixty-eight regenerated plants were obtained from 4,200 cotyledons infected with Agrobacterium, after which genomic PCR analysis using NPTII-specific primers confirmed gene presence in 24 plants with a transformation rate of 0.6%. Furthermore, we performed transformation into C. roseus using an expression vector to join trpE8 and aroG4 genes, which are feedback-resistant mutant genes derived from Escherichia coli. The resulting transformed plants showed exactly the same morphology as the wild-type, albeit with a marked increase in tryptophan and alkaloids content, especially catharanthine in leaves.

著者

Masahiro Nishihara Toshiya Muranaka

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.40, no.3, pp.181-184, 2023-09-25 (Released:2023-09-25)

参考文献数

30

著者

Hiroaki Kusano Ami Takeuchi Hiroaki Shimada

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.40, no.3, pp.201-209, 2023-09-25 (Released:2023-09-25)

参考文献数

29

被引用文献数

3

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Potato (Solanum tuberosum L.) has a tetraploid genome. To make a mutant lacking a specific gene function, it is necessary to introduce mutations into all four gene alleles. To achieve this goal, we developed a powerful genome editing tool, CRISPR/dMac3-Cas9, which installed the translation enhancer dMac3 that greatly increased the translation of the downstream open reading frame. The CRISPR/dMac3-Cas9 system employing three guide RNAs (gRNAs) greatly elevated the frequency of the generation rate of mutation. This system enabled to create the 4-allele mutants of granule-bound starch synthase (GBSS) and starch branching enzyme (SBE). These mutants indicated functionally defective features, suggesting that we succeeded in efficient genome editing of the potato tetraploid genome. Here, we show the effect of the number of gRNAs for efficient mutagenesis of the target gene using the mutants of the GBSS1 gene. CRISPR/dMac3-Cas9 employing three gRNA genes achieved a higher mutation efficiency than the CRISPR/dMac3-Cas9 with two gRNAs, suggesting being influenced by the dose effect of the number of gRNAs at the target region. The alleles of the SBE3 gene contained SNPs that caused sequence differences in the gRNAs but these gRNAs functioned efficiently. However, many rearrangement events and large deletions were induced. These results support the importance of accurate binding of gRNA to the target sequence, which may lead to a hint to avoid the unexpected mutation on the off-target sites.

著者

Muxiu Tan Fengming Liu Yueying Xie Qiaocheng Mo Fenghua Shi

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.40, no.2, pp.167-174, 2023-06-25 (Released:2023-06-25)

参考文献数

34

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In this study, the transformed system mediated by Agrobacterium tumefaciens of Gynostemma pentaphyllum was constructed by using the phosphomannose-isomerase (PMI) gene as a marker. To investigate the cefotaxime sodium salt (Cef) concentration of bacteriostatic medium and the appropriate mannose concentration in the selectable medium, explants of the stems with buds were cultured in a basic medium supplemented with different Cef and mannose concentrations, respectively. After these were optimized, 288 explants were transformed according the protocol described above to verify their availability by using the polymerase chain reaction (PCR), reverse transcription-PCR and chlorophenol red. The results showed that the appropriate Cef concentration for bacteriostatic culture and mannose concentration for selectable culture were 150 mg l−1 and 3 g l−1 for stem with buds, respectively. According to the PCR results, the transformation frequency of stems with buds was 20.49% with a regeneration rate of 29.16%. In future, the CPR assay could be the auxiliary method of choice as it is moderately accurate, but it has good maneuverability and is cost effective for large-scale use.

著者

Mitsuko Kishi-Kaboshi Ayako Nishizawa-Yokoi Ichiro Mitsuhara Seiichi Toki Katsutomo Sasaki

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.40, no.2, pp.157-165, 2023-06-25 (Released:2023-06-25)

参考文献数

24

被引用文献数

1

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Chrysanthemum morifolium is one of the most popular ornamental plants in the world. However, as C. morifolium is a segmental hexaploid, self-incompatible, and has a sizable heterologous genome, it is difficult to modify its trait systematically. Genome editing technology is one of the attractive methods for modifying traits systematically. For the commercial use of genetically modified C. morifolium, rigorous stabilization of its quality is essential. This trait stability can be achieved by avoiding further genome modification after suitable trait modification by genome editing. Since C. morifolium is a vegetatively propagated plant, an approach for removing genome editing tools is required. In this study, we attempted to use the piggyBac transposon system to remove specific DNA sequences from the C. morifolium genome. Using the luminescence as a visible marker, we demonstrated that inoculation of Agrobacterium harboring hyperactive piggyBac transposase removes inserted 2.6 kb DNA, which harbors piggyBac recognition sequences, from the modified Eluc sequence.

著者

Xiaolan Li Huan Hu Qunli Ren Miao Wang Yimei Du Yuqi He Qian Wang

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.40, no.2, pp.145-155, 2023-06-25 (Released:2023-06-25)

参考文献数

70

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Dendrobium officinale usually lives on rock or tree, but their endophyte diversity has not yet been fully revealed? In this study, high-throughput sequencing technology was used to investigate the endophyte diversity of the roots of D. officinale lived on tree (Group 1–3, arboreal type) and rock (Group 4, lithophytic type). The results showed that their composition of endophytic fungi and bacteria were similar at phylum level, while their relative abundance were different. Their taxa composition and abundance of endophytes differed significantly among groups at the genus level. Alpha diversity of endophytic fungi of lithophytic type was higher than those from arboreal type, while there was no advantage in endophytic bacteria. Beta diversity revealed that the endophytic fungi tended to cluster in each group, but the endophytic bacteria were dispersed among the groups. LEfSe analysis found that the numbers of predicted endophyte biomarkers of lithophytic type were more than arboreal types at genus level, and the biomarkers varied among groups. Microbial network analysis revealed similarities and differences in the taxa composition and abundance of shared and special endophytes in each group. These results suggested that the root endophytes of lithophytic and arboreal D. officinale differed in diversity.