著者

Seung-won Choi Kie Kumaishi Reiko Motohashi Harumi Enoki Wiluk Chacuttayapong Tadashi Takamizo Hiroaki Saika Masaki Endo Tetsuya Yamada Aya Hirose Nobuya Koizuka Seisuke Kimura Yaichi Kawakatsu Hiroyuki Koga Emi Ito Ken Shirasu Yasunori Ichihashi

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.39, no.3, pp.323-327, 2022-09-25 (Released:2022-09-25)

参考文献数

28

---

Agrobacterium-mediated transformation is a key innovation for plant breeding, and routinely used in basic researches and applied biology. However, the transformation efficiency is often the limiting factor of this technique. In this study, we discovered that oxicam-type nonsteroidal anti-inflammatory drugs, including tenoxicam (TNX), increase the efficiency of Agrobacterium-mediated transient transformation. TNX treatment increased the transformation efficiency of Agrobacterium-mediated transformation of Arabidopsis thaliana mature leaves by agroinfiltration. The increase of efficiency by TNX treatment was not observed in dde2/ein2/pad4/sid2 quadruple mutant, indicating that TNX inhibits the immune system mediated by jasmonic acid, ethylene, and salicylic acid against to Agrobacterium. We also found that TNX-treatment is applicable for the transient expression and subcellular localization analysis of fluorescent-tagged proteins in Arabidopsis leaf cells. In addition, we found that TNX increases the efficiency of Agrobacterium-mediated transient transformation of Jatropha. Given that treatment with oxicam compounds is a simple and cost effective method, our findings will provide a new option to overcome limitations associated with Agrobacterium-mediated transformation of various plant species.

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

---

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.