Biochemical and molecular characterization of orange- and tangerine-colored rice calli

Ishihara, Atsushi; Ohishi, Kazuhiko; Yamada, Tetsuya; Shibata-Hatta, Mari; Arai-Kichise, Yuko; Watanabe, Satoru; Yoshikawa, Hideki; Wakasa, K.

doi:10.5511/plantbiotechnology.15.0526a

Cited by 7 publications

(6 citation statements)

References 44 publications

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“…In addition to δ-carotene, we also detected lycopene accumulation in the callus. Consistently, lycopene with red color is the upstream substrate of δ-carotene in the carotenoid biosynthesis pathway 23 . LCYβ disruption could induce not only δ-carotene accumulation but also that of upstream substrates.…”

Section: Targeted Mutagenesis Of Lycopene Cyclase Genes In Ricementioning

confidence: 94%

“…3a and b). A previous study implies that the expression level reduction of these genes results in rice with high lycopene content 23 , although no single gene knockouts of these genes have been reported so far. We designed three gRNAs for LCYβ or LCYε.…”

Section: Targeted Mutagenesis Of Lycopene Cyclase Genes In Ricementioning

confidence: 98%

“…We performed carotenoid extraction from the calli (0.5 g) and shoots (0.25 with biological triplicates following a previously reported procedure 23 . The acetone supernatants of the extracts were dried and the samples were kept at − 20°C.…”

Section: Carotenoid Identi Cation and Quanti Cationmentioning

confidence: 99%

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The sonication-assisted whisker method enables CRISPR-Cas9 ribonucleoprotein delivery to induce genome editing in rice

Sugano

Nakamura

Furubayashi

et al. 2023

Preprint

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CRISPR/Cas9-based genome editing represents an unprecedented potential for plant breeding. Unlike animal cells, plant cells contain a rigid cell wall, genome editing tool delivery into plant cells is thus challenging. In particular, the delivery of the Cas9-gRNA ribonucleoprotein (RNP) into plant cells is desired since the transgene insertion into the genome should be avoided for industrial applications in plants. In this study, we present a novel RNP delivery approach in rice. We applied the sonication-assisted whisker method, conventionally developed for DNA delivery in plants, for RNP delivery in rice. Combined with marker gene delivery, we successfully isolated LCYβgenome-edited lines generated by RNPs. The calli and regenerated shoot of the LCYβmutant showed abnormal carotenoid accumulation. In addition, we also detected, although at a low frequency, genome editing events in rice calli cells by RNP delivery using the sonication-assisted whisker method without any additiona. Therefore, the sonication-assisted whisker method could be an attractive way to create RNP-based genome-edited lines in plants.

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Section: Targeted Mutagenesis Of Lycopene Cyclase Genes In Ricementioning

confidence: 94%

Section: Targeted Mutagenesis Of Lycopene Cyclase Genes In Ricementioning

confidence: 98%

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The sonication-assisted whisker method enables CRISPR-Cas9 ribonucleoprotein delivery to induce genome editing in rice

Sugano

Nakamura

Furubayashi

et al. 2023

Preprint

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“…The obtained residue was dissolved in 500 μL of acetone and diluted 20 times. The absorption of the suspension was measured spectrophotometrically according to a previously reported method [ 47 ].…”

Section: Methodsmentioning

confidence: 99%

Biochemical Characterization of Orange-Colored Rice Calli Induced by Target Mutagenesis of OsOr Gene

Kim

et al. 2022

Plants

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We generated an orange-colored (OC) rice callus line by targeted mutagenesis of the orange gene (OsOr) using the CRISPR-Cas9 system. The OC line accumulated more lutein, β-carotene, and two β-carotene isomers compared to the WT callus line. We also analyzed the expression levels of carotenoid biosynthesis genes by qRT-PCR. Among the genes encoding carotenoid metabolic pathway enzymes, the number of transcripts of the PSY2, PSY3, PDS, ZDS and β-LCY genes were higher in the OC line than in the WT line. In contrast, transcription of the ε-LCY gene was downregulated in the OC line compared to the WT line. In addition, we detected increases in the transcript levels of two genes involved in carotenoid oxidation in the OC lines. The developed OC lines also showed increased tolerance to salt stress. Collectively, these findings indicate that targeted mutagenesis of the OsOr gene via CRISPR/Cas9-mediated genome editing results in β-carotene accumulation in rice calli. Accordingly, we believe that this type of genome-editing technology could represent an effective alternative approach for enhancing the β-carotene content of plants.

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“…Wakasa et al [ 20 ] reported the characteristics of two callus lines showing orange and tangerine colors by repeatedly subculturing anther culture-derived mutant calli. Ishihara et al [ 21 ] showed that the tangerine callus line is the LcyE gene (H523L), which carries a single nucleotide polymorphism responsible for His to Leu substitution at amino acid position 523, exhibiting a dramatically higher carotenoid content than WT calli. Here, we created HDR callus mutants ( LcyE sg2-HDR1 and LcyE sg2-HDR2) with golden SNP-carrying OsLcyE genes using the CRSPR/Cas9 and the geminiviral replicon system.…”

Section: Introductionmentioning

confidence: 99%

Genome Editing of Golden SNP-Carrying Lycopene Epsilon-Cyclase (LcyE) Gene Using the CRSPR-Cas9/HDR and Geminiviral Replicon System in Rice

Kim

et al. 2022

IJMS

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Lycopene epsilon-cyclase (LcyE) is a key enzyme in the carotenoid biosynthetic pathway of higher plants. Using the CRSPR/Cas9 and the geminiviral replicon, we optimized a method for targeted mutagenesis and golden SNP replacement of the LcyE gene in rice. We have exploited the geminiviral replicon amplification as a means to provide a large amount of donor template for the repair of a CRISPR-Cas-induced DNA double-strand break (DSB) in the target gene via homology-directed repair (HDR). Mutagenesis experiments performed on the Donggin variety achieved precise modification of the LcyE loci with an efficiency of up to 90%. In HDR experiments, our target was the LcyE allele (LcyE-H523L) derived from anther culture containing a golden SNP replacement. The phenotype of the homologous recombination (HR) mutant obtained through the geminiviral replicon-based template delivery system was tangerine color, and the frequency was 1.32% of the transformed calli. In addition, the total carotenoid content of the LcyEsg2-HDR1 and LcyEsg2-HDR2 lines was 6.8–9.6 times higher than that of the wild-type (WT) calli, respectively. The reactive oxygen species content was lower in the LcyEsg2-HDR1 and LcyEsg2-HDR2 lines. These results indicate that efficient HDR can be achieved in the golden SNP replacement using a single and modular configuration applicable to different rice targets and other crops. This work demonstrates the potential to replace all genes with elite alleles within one generation and greatly expands our ability to improve agriculturally important traits.

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Biochemical and molecular characterization of orange- and tangerine-colored rice calli

Cited by 7 publications

References 44 publications

The sonication-assisted whisker method enables CRISPR-Cas9 ribonucleoprotein delivery to induce genome editing in rice

The sonication-assisted whisker method enables CRISPR-Cas9 ribonucleoprotein delivery to induce genome editing in rice

Biochemical Characterization of Orange-Colored Rice Calli Induced by Target Mutagenesis of OsOr Gene

Genome Editing of Golden SNP-Carrying Lycopene Epsilon-Cyclase (LcyE) Gene Using the CRSPR-Cas9/HDR and Geminiviral Replicon System in Rice

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