In vitro regeneration and clonal fidelity assessment on induction and differentiation of protocorm-like body from Pleione bulbocodioides (Franch.)

Key message The present work reports the DNA hereditary stability character and optimum medium for induction and differentiation of PLBs from Pleione bulbocodioides (Franch.) Rolfe (PB), which will be useful for the efficient micropropagation popularization for PB. In clinical treatment, Pleione bulbocodioides (Franch.) Rolfe (PB) has been widely used as an anti-tumor drug. However, low productivity impeded its extensive application. The lack of endosperm in the seeds of PB led to a low natural germination rate, causing inefficient production and long reproductive cycle. Improving production and retaining clonal fidelity are the challenges in its efficient micropropagation. This study is to optimize medium and evaluate clonal fidelity on induction and differentiation of Protocorm-like Body (PLB) from PB. The effects of plant growth regulators (PGR) and natural additives on the formation and proliferation of protocorms from seeds and the induction and proliferation of PLBs were investigated. The ultra-structural were performed via a scanning electron microscope (SEM) and transmission electron microscope (TEM) and clonal fidelity assessment was conducted using histochemical assays, DNA electrophoresis analysis and Inter-simple sequence repeat (ISSR) molecular marker analysis. The optimum medium for inducing protocorm was that the Murashige and Skoog (MS) medium supplemented with 2.0 mg/L 2,4-D, 2.0 mg/L KT (Kinetin), 2.0 mg/L AgNO3, and 50 mL/L coconut water in the orthogonal experiments and validation experiments, and for inducing PLBs was that MS medium with 0.3 mg/L indole-3-butyric acid (IBA), 0.3 mg/L KT, 100 mg/L potato extract, and 1 g/L activated charcoal. The SEM and TEM showed the ultra-structural of PB had no change from protocorm to PLBs, and the DNA electrophoresis showed the total DNA did not change in different stages of PB by induction and differentiation and ISSR analysis revealed no DNA polymorphisms included bulb, PLBS, and the differentiated tissue of PLBs. In conclusion, the DNA hereditary character and ultra-structural of PB was retained using our optimum medium induction and differentiation of PLBs from PB, which will be useful for the efficient micropropagation popularization for PB.

Automated summary

This study optimized the medium for induction and differentiation of PLBs from Pleione bulbocodioides, maintaining the DNA hereditary stability and ultrastructural integrity. The optimal medium formulations were identified for inducing protocorms and PLBs, leading to no DNA polymorphisms during the process. This finding is beneficial for efficient micropropagation of Pleione bulbocodioides.