The development of subnear-isogenic lines (sub-NILs) and near-isogenic lines (NILs) populations significantly impacts the study of exotic germplasm and enhances our understanding of the genetic basis of adaptation and abiotic stress tolerance. Cold stress poses a major challenge to tomato crop production, necessitating the development of cold-tolerant varieties. This study aimed to develop a novel sub-NIL population by crossing the cold-sensitive LA4024 line with the tolerant introgression line (IL) LA3969 (introgression segment between CT180 to TG394 on chromosome 12) to reduce introgression segment size. To detect the major chromosomal segment of the Solanum habrochaites genome responsible for cold tolerance, 200 sub-NIL seedlings, along with the two parent lines, LA4024 and LA3969, were assessed under cold stress conditions using several physiological indicators. Also, CAPS marker analysis identified FUM122 as a novel cold-tolerant line, exhibiting a reduced introgression segment associated with cold tolerance, flanked by SGN-E358810 to SGN-E359201 markers. Under cold stress, we examined physiological responses through several analyses, including measurements of malondialdehyde (MDA) levels, photosystem II (PSII) efficiency, proline content, total pigment and peroxidase (POD) activity. The phenotypic performance of FUM122 during cold stress was similar to that of LA3969 and demonstrated greater cold tolerance compared to LA4024. Additionally, gene expression analysis revealed that RD26, Hsp90-1 and F-box genes were upregulated in FUM122 during cold stress, supporting phenotyping analysis findings and suggesting their role in cold tolerance. Developing FUM122 with reduced introgression region size provides a valuable resource for breeding cold-tolerant tomato varieties and insights into the genetic mechanisms underlying cold stress tolerance.

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