Developing new germplasm of high-quality and early-maturing rice by editing Hd6 via CRISPR/Cas9

Heading date is a key agronomic trait that determines the seasonal and regional adaptability of rice varieties. Hd6 encodes the CK2α subunit, which regulates heading date in rice by phosphorylating florigen regulators. Loss-of-function mutants of Hd6 exhibit earlier heading under long-day conditions. To shorten the growth duration of the elite, tasty, late-season japonica cultivar Nanjing 46 (NJ46) and expand its cultivation range, the Hd6 gene in NJ46 was knocked out using CRISPR/Cas9- mediated gene editing. A target site was designed within the second exon of Hd6, and its specificity was validated by BLAST analysis. The expression vector pCAMBIA1305-Actin:Cas9-sgRNAHd6 was constructed and introduced into NJ46 callus via Agrobacterium-mediated transformation. Homozygous mutant lines without exogenous components were screened using PCR. The heading date, major agronomic traits, yield-related traits, and grain quality traits of these lines were evaluated in both central and northern Jiangsu and compared with those of local benchmark cultivars Nanjing 9108 (NJ9108) and Nanjing 518 (NJ518), respectively. In the T0 generation, 18 transgenic positive plants were obtained. In the T2 generation, three homozygous mutant lines with different mutation types and free of exogenous sequences were identified: NJ46-hd6ko-1 (TTGG deletion), NJ46-hd6ko-2 (25 bp deletion), and NJ46-hd6ko-3 (G deletion). Their heading dates were 21.1, 24.7, and 21.4 d earlier than that of NJ46, respectively. Among them, NJ46-hd6ko-3 showed similar heading date, theoretical yield, milled rice rate, amylose content, gel consistency, and taste value to NJ9108, but had a shorter plant height and a higher alkali spreading value, indicating superior overall performance and suitability for cultivation in the central Jiangsu region. NJ46-hd6ko-2 showed no significant differences in heading date, theoretical yield, or alkali spreading value compared to NJ518, but demonstrated significantly higher milled rice rate and taste value, along with a shorter plant height, suggesting enhanced overall traits and suitability for the northern Jiangsu region. By precisely editing the Hd6 gene in NJ46, we developed new elite, tasty japonica rice germplasms adapted to the central and northern Jiangsu ecological zones, thereby expanding the cultivation area for high-quality, palatable rice varieties.