| Title: | Site-directed mutagenesis by biolistic transformation efficiently generates inheritable mutations in a targeted locus in soybean somatic embryos and transgene-free descendants in the T-1 generation |
| Authors: | Adachi, Kohei Browse this author |
| Hirose, Aya Browse this author |
| Kanazashi, Yuhei Browse this author |
| Hibara, Miki Browse this author |
| Hirata, Toshiyuki Browse this author |
| Mikami, Masafumi Browse this author |
| Endo, Masaki Browse this author |
| Hirose, Sakiko Browse this author |
| Maruyama, Nobuyuki Browse this author |
| Ishimoto, Masao Browse this author |
| Abe, Jun Browse this author →KAKEN DB |
| Yamada, Tetsuya Browse this author →KAKEN DB |
| Keywords: | CRISPR |
| Cas9 |
| Glycine max |
| Transgenic embryo |
| Null-segregant |
| Nonsense mRNA decay |
| Issue Date: | Feb-2021 |
| Publisher: | Springer |
| Journal Title: | Transgenic Research |
| Volume: | 30 |
| Issue: | 1 |
| Start Page: | 77 |
| End Page: | 89 |
| Publisher DOI: | 10.1007/s11248-020-00229-4 |
| Abstract: | The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system is being rapidly developed for mutagenesis in higher plants. Ideally, foreign DNA introduced by this system is removed in the breeding of edible crops and vegetables. Here, we report an efficient generation of Cas9-free mutants lacking an allergenic gene, Gly m Bd 30K, using biolistic transformation and the CRISPR/Cas9 system. Five transgenic embryo lines were selected on the basis of hygromycin resistance. Cleaved amplified polymorphic sequence analysis detected only two different mutations in e all of the lines. These results indicate that mutations were induced in the target gene immediately after the delivery of the exogenous gene into the embryo cells. Soybean plantlets (T-0 plants) were regenerated from two of the transgenic embryo lines. The segregation pattern of the Cas9 gene in the T-1 generation, which included Cas9-free plants, revealed that a single copy number of transgene was integrated in both lines. Immunoblot analysis demonstrated that no Gly m Bd 30K protein accumulated in the Cas9-free plants. Gene expression analysis indicated that nonsense mRNA decay might have occurred in mature mutant seeds. Due to the efficient induction of inheritable mutations and the low integrated transgene copy number in the T-0 plants, we could remove foreign DNA easily by genetic segregation in the T-1 generation. Our results demonstrate that biolistic transformation of soybean embryos is useful for CRISPR/Cas9-mediated site-directed mutagenesis of soybean for human consumption. |
| Rights: | This is a post-peer-review, pre-copyedit version of an article published in Transgenic Research. The final authenticated version is available online at: http://dx.doi.org/10.1007/s11248-020-00229-4 |
| Type: | article (author version) |
| URI: | http://hdl.handle.net/2115/83986 |
| Appears in Collections: | 農学院・農学研究院 (Graduate School of Agriculture / Faculty of Agriculture) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)
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