High-efficiency callus induction from intact Camellia sinensis leaves via controlled incisions and its regulatory...

Tea (Camellia sinensis (L.) O. Kuntze) is a globally important economic crop, but its in vitro tissue culture is severely limited by polyphenol-induced explant browning and low callus induction efficiency. Conventional methods relying on fragmented leaf explants exacerbate these issues, and the regulatory mechanisms underlying genotypic differences in callus induction remain poorly understood.

We optimized a callus induction protocol using intact tea leaves with controlled transverse incisions, combining liquid-solid co-culture and staged dark/light cycles. The optimal protocol-5-8 transverse incisions on Huangjincha leaves, followed by 3-day liquid dark preculture, 12-day solid dark culture, and 15-day 16/8 h photoperiod-reduced the browning rate to 32.0 ± 0.9% and achieved a callus induction rate of 68.0 ± 2.2%, producing healthy, light-yellow, friable calli. Morphologically, Huangjincha (high induction efficiency) exhibited sparser epidermal wax, intact chloroplast ultrastructure, and larger stomatal area compared to Yunnandaye (low induction efficiency), which had dense wax aggregates and fragmented chloroplasts under in vitro conditions. Physiologically, Huangjincha maintained higher superoxide dismutase (SOD) and peroxidase (POD) activities, supporting effective reactive oxygen species (ROS) scavenging, while Yunnandaye showed elevated alternative antioxidant enzyme activities but persistent high browning. Transcriptomic analysis identified 27,520 differentially expressed genes (DEGs) between the two cultivars: Huangjincha upregulated genes related to cell cycle progression (CsCDK2, CsCYCD3) and ROS scavenging (CsSOD2, CsPOD12), whereas Yunnandaye enriched genes in polyphenol biosynthesis (CsCHS, CsC4H).

Our optimized protocol effectively mitigates key bottlenecks in tea tissue culture. The integrated "structure-physiology-transcriptome" analysis reveals the mechanistic basis of genotypic differences in callus induction, identifying actionable targets for improving tissue culture efficiency in recalcitrant tea varieties. This study provides a scalable tool for tea biotechnology and offers insights for optimizing tissue culture protocols in other polyphenol-rich crops.