Site-directed mutagenesis by biolistic transformation efficiently generates inheritable mutations in a targeted locus in soybean somatic embryos and transgene-free descendants in the T1 generation |
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Authors: | Adachi Kohei Hirose Aya Kanazashi Yuhei Hibara Miki Hirata Toshiyuki Mikami Masafumi Endo Masaki Hirose Sakiko Maruyama Nobuyuki Ishimoto Masao Abe Jun Yamada Tetsuya |
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Affiliation: | 1.Graduate School of Agriculture, Hokkaido University, Kita 9, Nishi 9, Kita-ku, Sapporo, Hokkaido, 060-8589, Japan ;2.Field Science Center Northern Biosphere, Hokkaido University, Kita 11, Nishi 10, Kita-ku, Sapporo, Hokkaido, 060-0811, Japan ;3.Plant Genome Engineering Research Unit, Institute of Agrobiological Sciences, National Agricultural and Food Research Organization, 1-2, Owashi, Tsukuba, Ibaraki, 305-8634, Japan ;4.Graduate School of Agriculture, Kyoto University, Uji, Kyoto, 611-0011, Japan ;5.Plant Genome Engineering Research Unit, Institute of Agrobiological Sciences, National Agricultural and Food Research Organization, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602, Japan ; |
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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 (T0 plants) were regenerated from two of the transgenic embryo lines. The segregation pattern of the Cas9 gene in the T1 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 T0 plants, we could remove foreign DNA easily by genetic segregation in the T1 generation. Our results demonstrate that biolistic transformation of soybean embryos is useful for CRISPR/Cas9-mediated site-directed mutagenesis of soybean for human consumption. |
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