Changes of lignified-callus and wound-induced adventitious rooting in ancient Platycladus orientalis cuttings as affected by tree age

Stem cuttings of the endangered ancient Platycladus orientalis are a valuable genetic resource for cryopreservation and propagation, but their extremely hard adventitious root (AR) formation poses a challenge for regeneration. In this study, we investigated the causes of decreased AR formation during the initial stem (S1), callus expansion (S2), and AR formation (S3) stages in cuttings from 5-, 100-, and 700-year-old P. orientalis trees, and identified potential solutions to enhance AR formation. Proteomic analysis revealed an up-regulation of flavonoid and phenylpropanoid biosynthesis pathway-associated proteins, including chalcone synthase (CHS), chalcone isomerase (CHI), and flavonone-3-hydroxylase (F3H), as well as down-regulation of auxin transport-associated proteins, such as auxin transport protein (BIG) and auxin responsive (AIR12), in the S3 of 700-year-old donors cuttings. Subsequent biochemical analysis confirmed over-accumulation of flavonoids, phenolics, and lignin resulting in callus-lignification and inhibition of AR formation in 100- and 700-year-old donors. Notably, wounding of the lignified-callus significantly increased AR formation in cuttings of 100-year-old donors. The upregulation of expression of meristematic cells regulatory proteins (enolase (ENO) and elongation factor 1α (EF1α)), carbohydrate metabolism proteins (isoamylases (ISA)), and vitamin B6 biosynthesis proteins (pyridoxine synthase (PDX1) and pyridoxal kinase (PDXK)) after wounding, might promote AR formation in the lignified-callus with wounding of cuttings of 100-year-old donors. Overall, our findings reveal that callus-lignification inhibits AR formation, while wounding promotes it in ancient P. orientalis. This study provides a foundation for enhancing the rooting rate and shortening the rooting time in ancient P. orientalis, and potentially other difficult-to-root species.