Explant browning during callus induction of Juglans mandshurica might be caused by the cooperation of PAL, polyphenol and PPO

Juglans mandshurica is an important timber and economic tree species in Northeast China. Callus induction is a key step for propagation and genetic transformation of plant species. In this study, we performed callus induction using mature zygotic embryos of J. mandshurica under varying concentrations of 2,4-D and sucrose, and analyzed its physiological indicators at 10 d, 20 d, 30 d, and 40 d of culture. The results showed that 30 g·L⁻¹ sucrose combined with 2.26 μM 2,4-D are optimal for callus induction, with a callus induction rate of 88.89 %. Higher sucrose concentration inhibited callus induction and exacerbated explant browning. Highest activities (or content) of superoxide dismutase (SOD), phenylalanine ammonia lyase (PAL) and polyphenol content, were observed at 30 d of culture, with the values of 1208.841 U·g⁻¹ s, 53.429 U·h⁻¹·g⁻¹, and 1.740 U·g⁻¹, respectively; while at 40 d for peroxidase (POD), polyphenol oxidase (PPO) with the values of 55.686 U·g⁻¹ s, and 182.030 U·g⁻¹, respectively. However, the significant negative correlations (p < 0.05) indicated that elevated level of the physiological indicators (SOD, POD, PPO and polyphenol) are not conducive for callus induction. No significant correlation was detected between polyphenol content and explant browning, however, significant positive correlation between PAL and polyphenol (R = 0.75), polyphenol and PPO (R = 0.696), and PPO and explant browning (R = 0.82) were detected. These results pointed to the likelihood that explant browning might be attributed to a cooperation of PAL, polyphenol and PPO (where PAL synthesized polyphenol, and then polyphenol was oxidized by PPO and produced brown quinones), rather than to an increase in polyphenol content simply. These insights provided a theoretical foundation for developing an indirect somatic embryogenesis system via callus induction.