Establishment of foreign-gene-free genome editing technology in Japanese gentian

• Project/Area Number: 19K06038
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 39030:Horticultural science-related
• Research Institution: Iwate Biotechnology Research Center
• Principal Investigator: Takahashi Shigekazu 公益財団法人岩手生物工学研究センター, 園芸資源研究部, 研究員 (10600033)
• Project Period (FY): 2019-04-01 – 2022-03-31
• Project Status: Completed (Fiscal Year 2021)
• Budget Amount: ¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000); Fiscal Year 2021: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2020: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000); Fiscal Year 2019: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
• Keywords: CRISPR/Cas9 / Gentian / Genome editing / ゲノム編集 / リンドウ / 外来遺伝子フリー / 園芸作物 / アグロバクテリウム / タバコ

Outline of Research at the Start

ゲノム編集は様々な作物で適用が進んでおり、リンドウでもCRISPR/Cas9系を利用してアグロバクテリウム法により編集体を作出する系が確立されている。実用を考えた場合、外来遺伝子を有しない編集体の作出が有効と考えられるが、リンドウはヘテロシス性が高く、育成に時間がかかる多年生植物であるため、交配によるnull-segregantの作出は困難である。そこで本研究では、Cas9とguide RNAを一過的に発現させることでリンドウのゲノム編集を可能とする、外来遺伝子組み込みフリーの系の確立と、その効率的な選抜法の開発を目指す。

Outline of Final Research Achievements

CRISPR / Cas9, one of the genome editing technologies, is widely used in the world because it is easy to operate. Generally, genome-edited plants carry foreign genes, however it is required genome edited plants with no foreign gene for practical use. Furthermore, in vegetative propagation plants and horticultural crops with high heterosis, it is important to obtain foreign gene-free genome edited lines in the present generation. Therefore, we performed the Agrobacterium method using Japanese gentian and tobacco as materials under various conditions, and tried to obtain foreign gene-free genome edited lines in the present generation. As a result, several signals for genome editing were detected in the shoots in the early stage of redifferentiation, however these signals disappeared with growth, and it was not possible to obtain genome edited plants with no foreign gene in the present generation.

Academic Significance and Societal Importance of the Research Achievements

本研究により当代での外来遺伝子フリーのゲノム編集体の取得において問題となる複数の課題をあぶり出すことができた。本知見をフィードバックすることで、今後の当該分野の研究の進展が期待される。