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The creation of herbicide-resistant rice germplasm is essential for breeding new herbicide-resistant rice varieties. By using CRISPR/Cas9 gene editing technology, the acetyl-CoA carboxylase OsACC1 gene was edited with the sterile line An 9S as the material, and a two-line sterile rice germplasm with herbicide resistance was created.
By using CRISPR/Cas9 technology, a single-base mutation vector, ePE2-ACC-I1879V, was constructed with acetyl-CoA carboxylase OsACC1 as the target gene. With An 9S as the transformation material, transgenic plants were obtained by Agrobacterium-mediated transformation. The genotypes and phenotypes were identified by sequencing the mutation sites of transgenic plants, conducting herbicide spraying tests and agronomic traits analysis.
Twenty-four ACC1I1879V mutant plants were obtained as confirmed by molecular detection in T0 generation; A total of 96 homozygous mutant plants were obtained from 24 lines in the T1 generation through segregation and screening, all of which had herbicide resistance. High-throughput sequencing showed that the three tested lines (NS512, NS513, NS514) contained no exogenous transgenic elements. Furthermore, the survival rate after spraying with 4% haloxyfop-P-methyl was 100%. Compared with the control An 9S, the NS512 line had increased plant height and wider flag leaf; the NS513 line had increased plant height and longer and wider flag leaf; and the NS514 line had a narrower flag leaf; the fertility of the three tested lines remained unchanged, and there was no significant difference in the number of effective panicles per plant and the panicle length. The transgenic safety evaluation indicated that the OsACC1 protein shares no sequence similarity with any known toxic proteins, antinutritional factors or allergens. Therefore, it is considered safe for human consumption.
Using CRISPR/Cas9 gene editing technology, we obtained homozygous mutant lines that are resistant to ACCase inhibitor herbicides. These lines are genetically stable, free of transgenic components, exhibit unaltered fertility, and are safe, providing fundamental materials for herbicide-resistant breeding in rice.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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