CRISPR/Cas9-mediated mutagenesis of the dihydroflavonol-4-reductase-B (DFR-B) locus in the Japanese morning glory Ipomoea (Pharbitis) nil

Kenta Watanabe,Michiyuki Ono,Seiichi Toki,Kimiyo Sage-Ono,Anna Kobayashi,Masaki Endo

doi:10.1038/s41598-017-10715-1

Abstract

CRISPR/Cas9 technology is a versatile tool for targeted mutagenesis in many organisms, including plants. However, this technique has not been applied to the Japanese morning glory (Ipomoea [Pharbitis] nil), a traditional garden plant chosen for the National BioResource Project in Japan. We selected dihydroflavonol-4-reductase-B (DFR-B) of I. nil, encoding an anthocyanin biosynthesis enzyme, as the target gene, and changes in the stem colour were observed during the early stages of plant tissue culture by Rhizobium [Agrobacterium]-mediated transformation. Twenty-four of the 32 (75%) transgenic plants bore anthocyanin-less white flowers with bi-allelic mutations at the Cas9 cleavage site in DFR-B, exhibiting a single base insertion or deletions of more than two bases. Thus, these results demonstrate that CRISPR/Cas9 technology enables the exploration of gene functions in this model horticultural plant. To our knowledge, this report is the first concerning flower colour changes in higher plants using CRISPR/Cas9 technology.

Highlights

The Japanese morning glory, Ipomoea nil or Pharbitis nil, is one of two traditional horticultural model plants selected for the National BioResource Project in Japan by the Agency for Medical Research and Development (AMED)[5]
One-third of the stable transgenic plants were bi-allelic mutants at the DFR-B locus in one generation. This high efficiency indicates that the CRISPR/Cas[9] system is a highly applicable technique for next-generation breeding in I. nil and other horticultural plants
We successfully visually selected targeted mutagenesis in the early stage during transformation, as the loss of function of the target gene DFR-B prevents the synthesis of stem pigmentation with anthocyanin

Summary

Introduction

The Japanese morning glory, Ipomoea nil or Pharbitis nil, is one of two traditional horticultural model plants selected for the National BioResource Project in Japan by the Agency for Medical Research and Development (AMED)[5]. Since initiating the National BioResource Project (NBRP) in Japan[5], libraries of expression sequence tags (ESTs) and bacterial artificial chromosomes (BACs) have been constructed[6], and genetic and molecular markers have been prepared[27] These resources make I. nil an ideal model plant for basic and horticultural studies in physiology and molecular biology. In I. nil, these three genes are structurally normal, but I. nil DFR-B (InDFR-B) is the genetically dominant gene responsible for pigmentation in the stems and flowers, as several spontaneous mutants of InDFR-B have shown the null phenotype[30] It remains unknown whether the targeted mutagenesis of InDFR-B located between InDFR-A and InDFR-C causes the null phenotype. The PAM is essential for the binding of CRISPR/Cas[9] to the DNA target[31, 32]

Methods

Results

Conclusion