Development of High-quality Aromatic japonica Rice Lines by Using CRISPR/Cas9 Technology

doi:10.16819/j.1001-7216.2026.250108

Abstract

Abstract:

【Objective】 Fragrance is an important trait of rice eating quality. In this study, the fragrance gene Badh2 in rice was edited using CRISPR/Cas9 technology to create a high-quality aromatic japonica rice line. 【Method】 Two target sequences located in the third and fourth exons of Badh2 were selected as target sites for constructing the binary expression vector pHUE411-Badh2-gRNA. The high-quality japonica rice line Tai 20-29 was used as the transformation material. 【Result】 Twenty T0 transgenic plants were obtained after transformation. Through propagation, PCR, and sequencing verification, six homozygous mutants with different base alterations in the third and fourth exons of Badh2 were identified. Except for a slightly lower seed setting rate and a higher grain number per panicle, the six mutants(bh62, bh64, bh65, bh67, bh68, and bh74) showed similar agronomic traits such as panicle length, grain length, grain width, and 1000-grain weight compared with the wild-type Tai 20-29. The mutants showed a significantly lower seed setting rate and a higher grain number per panicle compared to Tai 20-29. The 2-AP content in brown rice of mutants bh62, bh64, bh65, and the wild type was determined by GC-MS. The results showed that the 2-AP content in the mutants increased significantly, reaching 24.27, 33.89, and 38.96 mg/kg, respectively, which was much higher than that in Tai 20-29. 【Conclusion】 We successfully generated a high-quality aromatic japonica rice line by editing the Badh2 gene using CRISPR/Cas9 technology.

Key words: rice, hygromycin, CRISPR/Cas9, fragrance gene, 2-acetyle-1-pyrroline (2-AP)

摘要：

【目的】香味是水稻重要的食味品质性状。本研究选择优质粳稻品系台20-29为研究对象，利用CRISPR/Cas9技术对其香味基因Badh2进行编辑，以期获得具有香味的优质粳稻品系。【方法】选择香味基因Badh2第3、第4外显子上的2段序列作靶位点，构建双元表达载体pHUE411-Badh2-gRNA并转入台20-29获得转基因植株。【结果】20株T₀植株经过加代、潮霉素标记检测、测序比对，共计获得22个T₁代无载体纯合突变株，包括6种突变类型，每种突变体在第3、第4外显子上存在不同程度的碱基变化。6个香味突变体bh62、bh64、bh65、bh67、bh68、bh74在穗长、粒长、粒宽、千粒重等方面与野生型台20-29基本一致，结实率有所下降，每穗粒数稍有提升，稻米品质没有明显变化。利用气相色谱-质谱技术检测突变体bh62、bh64、bh65的糙米中香味物质2-AP含量，结果表明糙米中2-AP含量较台20-29极显著提高，分别达到24.27、33.89、38.96 mg/kg。【结论】对水稻香味基因Badh2进行编辑可以高效获得具有香味的水稻优质品系。

关键词: 水稻, 潮霉素, CRISPR/Cas9, 香味基因, 2-乙酰-1-吡咯啉

DUAN Min, XIE Liujie, YUE Yani, HUANG Shanjun. Development of High-quality Aromatic japonica Rice Lines by Using CRISPR/Cas9 Technology[J]. Chinese Journal OF Rice Science, 2026, 40(2): 235-243.

段敏, 谢留杰, 岳雅妮, 黄善军. 基于CRISPR/Cas9技术创制优质香味粳稻品系[J]. 中国水稻科学, 2026, 40(2): 235-243.

Figures/Tables 7

Fig. 1. Schematic diagram of Badh2 and target sites Red letters represent protospacer adjacent motif (PAM) sequence; Black boxes represent exons.

Table 1. Primers used in this study

引物名称 Name of prime	寡核苷酸序列（5’-3’） Oligonucleotide sequence (5’-3’)
HYG-F	GCGTCTGCTGCTCCATACAA
HYG-R	TGACATTGGGGAGTTTAGCG
Bad-IF	TAGCCTTCCAGTCTAAATGACA
Bad-IR	CAACTACACCGATAGGCTCTT

Fig. 2. Identification result of hygromycin in T1 generation transgenic plants (partial) in the second test

Fig. 3. Comparison of target sequences between six aromatic mutants and wild type Red lines display protospacer adjacent motif (PAM) sequence. Arrows show the location of mutant bases.

Fig. 4. Agronomic traits of six aromatic mutants and wild type Values are shown as mean±SD. Sample sizes n=5 for plant height, panicle number per plant and 1000-grain weight, n=15 for the rest factors. **and * indicate significant difference between mutants and the wild type at 0.01 and 0.05, respectively.

Fig. 5. Phenotypic comparison during mature stage between the mutant bh62 and its wild type Tai 20-29 A, Plant comparison, bar=20 cm; B, Panicle comparison, bar=5 cm.

Fig. 6. Rice phenotype (A) and 2-AP content of brown rice (B) in mutants and the wild type Bar=1 cm. Values are shown as mean±SD, n=3. **represents P <0.01 compared with wild type.

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