CRISPR/Cas9-targeted mutagenesis of F3′H, DFR and LDOX, genes related to anthocyanin biosynthesis in black rice (Oryza sativa L.)
Altering a trait by CRISPR-Cas9-targeted mutagenesis offers great advantages in identifying gene function and crop improvement. In the present study, three genes (OsF3′H, OsDFR and OsLDOX) in the anthocyanin biosynthesis pathway were successfully edited on the Heugseonchal or Sinmyungheugchal variety using the CRISPR/Cas9 system. As a result, the ratio of the edited plants in the transformed early generation was 56.7%. These edited mutant lines were observed with the changes of seed color and anthocyanin content. All mutations were stably inherited to the T2 progeny. In addition, we could select edited homozygous mutant lines lacking the T-DNA already in the first offspring generation. Also the insertion of vector backbone sequences in f3′h-9, dfr-4 and ldox-16 lines was not detected in the whole genome resequencing. These results demonstrated that the CRISPR/Cas9 system can induce clearly gene-specific mutations with a high efficiency in rice and null plants selected from these mutants cannot be distinguished from non-GMO plants even under strict GMO regulation.
KeywordsBlack rice Anthocyanin OsLDOX OsF3′H OsDFR CRISPR/Cas9 Gene editing
This work was carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ01319302)” Rural Development Administration, Republic of Korea.
- Batschauer A, Rocholl M, Kaiser T, Nagatani A, Furuya M, Schäfer E (1996) Blue and UV-A light-regulated CHS expression in Arabidopsis independent of phytochrome A and phytochrome B. Plant J 9:63–69. https://doi.org/10.1046/j.1365-313X.1996.09010063.x CrossRefGoogle Scholar
- Lepiniec L, Debeaujon I, Routaboul JM, Baudry A, Pourcel L, Nesi N, Caboche M (2006) Genetics and biochemistry of seed flavonoids. Plant Biol 57:405–430. https://doi.org/10.1146/annurev.arplant.57.032905.105252 CrossRefGoogle Scholar
- Mazza G, Gao L (2005) Blue and purple grains. In: Grains S, for Food and Feed. E. Abdel-Aal and P. Wood, (eds) American Association of Cereal Chemists. St. Paul, MN, pp 313–350Google Scholar
- Nakornriab M, Sriseadka T, Wongpornchai S (2008) Quantification of carotenoid and flavonoid components in brans of some Thai black rice cultivars using supercritical fluid extraction and high-performance liquid chromatography-mass spectrometry. J Food Lipids 15:488–503. https://doi.org/10.1111/j.1745-4522.2008.00135.x CrossRefGoogle Scholar
- Schouten HJ, vande Geest H, Papadimitriou S, Bemer M, Schaart JG, Smulders MJ, Schijlen E, (2017) Re-sequencing transgenic plants revealed rearrangements at T-DNA inserts, and integration of a short T-DNA fragment, but no increase of small mutations elsewhere. Plant Cell Rep 36:493–504. https://doi.org/10.1007/s00299-017-2098-z CrossRefPubMedPubMedCentralGoogle Scholar
- Team RC (2015) R Foundation for Statistical Computing; Vienna, Austria: 2014. R: a language and environment for statistical computingGoogle Scholar
- Winkel BS (2004) Metabolic channeling in plants. Annu Rev Plant Biol 55:85–107. https://doi.org/10.1146/annurev.arplant.55.031903.141714 CrossRefPubMedGoogle Scholar