A protocol for &lt;i&gt;Agrobacterium&lt;/i&gt;-mediated transformation of Japanese cedar, Sugi (&lt;i&gt;Cryptomeria japonica&lt;/i&gt; D. Don) using embryogenic tissue explants

et al. 2021

Sci Rep

Self Cite

Cryptomeria japonica (Japanese cedar or sugi) is one of the most important coniferous tree species in Japan and breeding programs for this species have been launched since 1950s. Genome editing technology can be used to shorten the breeding period. In this study, we performed targeted mutagenesis using the CRISPR/Cas9 system in C. japonica. First, the CRISPR/Cas9 system was tested using green fluorescent protein (GFP)-expressing transgenic embryogenic tissue lines. Knock-out efficiency of GFP ranged from 3.1 to 41.4% depending on U6 promoters and target sequences. The GFP knock-out region was mottled in many lines, indicating genome editing in individual cells. However, in 101 of 102 mutated individuals (> 99%) from 6 GFP knock-out lines, embryos had a single mutation pattern. Next, we knocked out the endogenous C. japonica magnesium chelatase subunit I (CjChlI) gene using two guide RNA targets. Green, pale green, and albino phenotypes were obtained in the gene-edited cell lines. Sequence analysis revealed random deletions, insertions, and replacements in the target region. Thus, targeted mutagenesis using the CRISPR/Cas9 system can be used to modify the C. japonica genome.

Section: Methodsmentioning

confidence: 99%

“…ET cell lines expressing GFP were generated through the Agrobacterium -mediated transformation of pZmUbi-GFP-Dt/HygR 26 , 34 . After screening with 5 mg/L hygromycin, lines with a strong GFP signal and a single-copy insertion (named N4) were selected through Southern blot analysis (Supplementary Fig.…”

Section: Methodsmentioning

confidence: 99%

CRISPR/Cas9-mediated targeted mutagenesis in Japanese cedar (Cryptomeria japonica D. Don)

Nanasato

Mikami

et al. 2021

Sci Rep

Self Cite

“…An embryogenic tissue (ET) cell line #13-8-12 was initiated using megagametophyte explants of C. japonica 23 . The ET was maintained in the dark at 25 °C in solid 1/2MD medium 24 and subcultured on fresh media after a 2-week interval.…”

Section: Plant Materialsmentioning

confidence: 99%

“…The detailed method for vector construction is described in Supplementary Information. Transformation of C. japonica ET cell lines expressing GFP were generated through the Agrobacterium-mediated transformation of pZmUbi-GFP-Dt/HygR 24,32 . After screening with 5 mg/L hygromycin, lines with a strong GFP signal with a single-copy line (named N4) were selected through Southern blot analysis ( Supplementary Fig.…”

Section: Vector Constructionmentioning

confidence: 99%

See 1 more Smart Citation

CRISPR/Cas9-mediated Targeted Mutagenesis in Japanese Cedar (Cryptomeria japonica D. Don)

Nanasato

Mikami

et al. 2021

Preprint

Self Cite

Cryptomeria japonica (Japanese cedar or sugi) is one of the most important coniferous tree species in Japan and breeding programs for this species have been launched since 1950s. Genome editing technology can be used to shorten the breeding period. In this study, we performed targeted mutagenesis using the CRISPR/Cas9 system in C. japonica. First, the CRISPR/Cas9 system was tested using green fluorescent protein (GFP)-expressing transgenic embryogenic tissue lines. Knock-out efficiency of GFP ranged from 3.1–41.4% depending on U6 promoters and target sequences. The GFP knock-out region was mottled in many lines, indicating genome editing in individual cells. However, in 102 of 103 mutated individuals (> 99%) from 6 GFP knock-out lines, embryos had a single mutation pattern. Next, we knocked out endogenous C. japonica magnesium chelatase subunit I (CjChlI) using two guide RNA targets. Green, pale green, and albino phenotypes were obtained in genome-edited cell lines. Sequence analysis revealed random deletions, insertions, and replacements in the target region. Thus, targeted mutagenesis using the CRISPR/Cas9 system can be used to modify the C. japonica genome.

CRISPR/Cas9-mediated disruption of CjACOS5 confers no-pollen formation on sugi trees (Cryptomeria japonica D. Don)

Nishiguchi

Endo

et al. 2023

Sci Rep

Self Cite

Sugi (Cryptomeria japonica D. Don) is an economically important coniferous tree in Japan. However, abundant sugi pollen grains are dispersed and transported by the wind each spring and cause a severe pollen allergy syndrome (Japanese cedar pollinosis). The use of pollen-free sugi that cannot produce pollen has been thought as a countermeasure to Japanese cedar pollinosis. The sugi CjACOS5 gene is an ortholog of Arabidopsis ACOS5 and rice OsACOS12, which encode an acyl-CoA synthetase that is involved in the synthesis of sporopollenin in pollen walls. To generate pollen-free sugi, we mutated CjACOS5 using the CRISPR/Cas9 system. As a result of sugi transformation mediated by Agrobacterium tumefaciens harboring the CjACOS5-targeted CRISPR/Cas9 vector, 1 bp-deleted homo biallelic mutant lines were obtained. Chimeric mutant lines harboring both mutant and wild-type CjACOS5 genes were also generated. The homo biallelic mutant lines had no-pollen in male strobili, whereas chimeric mutant lines had male strobili with or without pollen grains. Our results suggest that CjACOS5 is essential for the production of pollen in sugi and that its disruption is useful for the generation of pollen-free sugi. In addition to conventional transgenic technology, genome editing technology, including CRISPR/Cas9, can confer new traits on sugi.