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

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

Kanako Kawaura Asuka Saeki Takehiro Masumura Shigeto Morita Yasunari Ogihara

出版者

日本遺伝学会

雑誌

Genes & Genetic Systems (ISSN:13417568)

巻号頁・発行日

vol.86, no.4, pp.249-255, 2011 (Released:2011-12-29)

参考文献数

23

被引用文献数

3 8 2

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The plant chondriome confers a complex nature. The atp4 gene (formerly called orf25) of Aegilops crassa (CR) harbors the promoter sequence of the rps7 gene from common wheat (Triticum aestivum cv. Chinese Spring, CS). The rps7 gene of CR has the promoter sequence of CS atp6. The atp6 gene of CR contains an unknown sequence inside of its coding region. Since repeat sequences have been found around the breaking points, these structural alterations are most likely generated through homologous recombination. In this study, PCR analysis was performed to detect structural alterations in each of three lines: euplasmic lines of Ae. crassa, Chinese Spring, and alloplasmic Chinese Spring wheat with the cytoplasm of Ae. crassa ((cr)-CS). We found that each of these lines contained both genotypes, although mitochondrial genotypes of CR in Chinese Spring wheat and CS genotypes in Ae. crassa were still retained as minor fractions (less than 10%). On the other hand, CS mitochondrial gene frequencies in ((cr)-CS) were shown to be ca. 30%. SNP analysis after DNA sequencing of these genes indicated that minor types of all three mitochondrial genes in alloplasmic wheat contained the mitochondrial gene types from pollens. Since the frequencies of paternal mitochondrial gene types in F1 were about 20%, successive backcrossing increased the frequencies of paternal mitochondrial gene types to around 30% in alloplasmic wheat. Expression profiles of these mitochondrial genes were quantitatively analyzed by RT-PCR. Transcripts of paternal mitochondrial gene types were scarcely found. This suggests that minor fractions including paternal mitochondrial gene types are maintained and silenced in the descendants.