CRISPR/Cas9-mediated generation of a green hypocotyl-marked male-sterile line for hybrid seed production in Solanum lycopersicum

Tomato ( Solanum lycopersicum ) is a self‑pollinating crop that exhibits significant heterosis. While F1 hybrid seeds offer high uniformity and strong stress resistance, the scarcity of male‑sterile materials means that production still relies on manual emasculation and pollination. This labor‑intensive process has become a major bottleneck for the tomato seed industry. CRISPR/Cas9 gene editing has emerged as a revolutionary molecular breeding technology, becoming a critical tool for generating novel crop germplasm. In this study, we employed CRISPR/Cas9 technology to create a male‑sterile line marked by a green hypocotyl via the simultaneous knockout of F3H ( flavanone 3‑hydroxylase ) and Ms10 35 ( male sterile 10 35 ), designated as CR‑ms10 35 /f3h . To evaluate its breeding potential, we used CR‑ms10 35 /f3h and the inbred line 19‑1 (WT) as maternal parents, crossed with paternal lines 1# and 8#, to analyze the performance of the resulting hybrids. Comparative analysis of F1 agronomic traits revealed no significant differences between hybrids derived from the gene‑edited sterile lines and those produced via manual emasculation; PCR assays confirmed the absence of transgenic elements (Cas9, NPTII) in the F1 hybrids. These results indicate that the CR‑ms10 35 /f3h line can be effectively applied to hybrid seed production without detectable affecting F1 generation performance. It was noted that incomplete co‑segregation between the green‑hypocotyl marker and male sterility was detected, with a recombination rate of approximately 5%. To enable more accurate screening, we generated a seed‑fluorescent transgenic line carrying the Ms10 35 gene and a DsRed2 marker as a candidate material for future artificial maintainer development, providing a promising approach for simplifying hybrid seed production in tomato and potentially other vegetable crops.