Efficient propagation protocol and genetic similarity assessment of medicinal cannabis based on photoautotrophic micropropagation

Photoautotrophic micropropagation (PAM), which employs sugar-free media and ventilated culture vessels to enhance plantlet photoautotrophic capacity and overall quality, has substantial potential to increase propagation efficiency when integrated with an appropriate propagation protocol. In this study, we established a PAM propagation protocol for medicinal cannabis ( Cannabis sativa L.) using two genotypically different cultivars, ‘Charlotte’ and ‘Auto Charlotte’, and systematically evaluated the genetic similarity of plantlets across all successive batches via inter-simple sequence repeat (ISSR) markers as well as their performance after transplanting. Furthermore, a cultivar-specific logistic growth model was used to estimate annual yield and optimize the mother plant culture cycle for maximizing production. Results showed that under PAM conditions, mother plants sustained 11 batches of shoot tip harvest within a 98-d cycle. The propagation coefficients of ‘Charlotte’ and ‘Auto Charlotte’ reached 12.0 (95%CI: 11.0-13.0) and 11.3 (95%CI: 10.5-12.2), respectively. After 35 days of culture, plantlets from all batches exhibited uniform morphological and physiological traits, with rooting rates exceeding 70%. These plantlets could be directly transplanted without acclimatization, and minor growth differences between batches were eliminated by extending the culture cycle by one week. ISSR markers detected no obvious polymorphism between regenerated plantlets and their respective mother plants, with all similarity coefficients exceeding 0.90. Model simulations suggested that a 70-day culture cycle could maximize annual production, reaching a yield of 50,417–54,034 plants m −2 . In conclusion, this study established an efficient PAM propagation protocol with high genetic similarity for the two tested medicinal cannabis cultivars, thereby providing technical support for cannabis plantlet production.