Effects of long-term successive subculture on phenotype, organogenesis capacity, and genetic stability in pear

To study the effect of long-term successive subculture on pear somaclonal variation, so as to provide a theoretical basis for the control and utilization of variation in vitro culture and reproduction. Here, we compared newly established (3‑month) tissue‑cultured plantlets of the dwarfing rootstock ‘Qingzhen D1’ ( Pyrus communis × P. bretschneideri ) with those subcultured for five years, using an integrated approach that combined phenotypic and anatomical evaluation, endogenous hormone quantification, flow cytometry (FCM), whole‑genome resequencing (WGS), and functional validation of candidate mutations. Long-term subcultured plantlets exhibited notable phenotypic changes, with approximately 20% of individuals developing lobed leaves; the shoot proliferation coefficient increased from 2.7 to 5.4, and the rooting rate rose from 75% to 100%. Plant growth regulator and endogenous hormone quantification revealed elevated 6-benzylaminopurine (6-BA), indole-3-butyric acid (IBA), and zeatin (ZT), and decreased indole-3-acetic acid (IAA) in long-term subcultured plantlets. FCM confirmed that no ploidy variation occurred during subculture. WGS identified 452,432 SNPs and 44,892 Indels, yielding a mutation frequency of approximately 5.0 × 10⁻⁴ per base pair. Based on the KEGG enrichment results and the observation that long-term subcultured plantlets exhibited elevated endogenous ZT levels, we selected the cytokinin oxidase/dehydrogenase ( CKX ) gene ( pycom07g02010 ) located on chromosome 7 for functional validation. A non-synonymous SNP was identified in this gene, corresponding to a T‑to‑C transition on the coding strand, which results in a leucine-to-proline substitution at position 34 (L34P). Transient expression in tobacco demonstrated that the L34P variant partially impaired the ability of CKX to degrade ZT. Overall, long-term successive subculture altered morphological and physiological traits linked to shifted growth regulator and endogenous hormone levels, while maintaining relatively stable genetic integrity. These findings suggest a mechanistic link between somaclonal variation and cytokinin homeostasis, providing a scientific basis for optimizing pear micropropagation strategies.