Establishment of an efficient tissue culture system for Paeonia ostii by combining vernalization and etiolation pretreatment with optimized culture conditions.

Background: Paeonia ostii, an economically important oil-producing peony cultivar, faces challenges in large-scale cultivation due to low propagation rates and long cultivation cycles. This study aimed to optimize tissue culture protocols for P. ostii 'Fengdan No. 3' by evaluating vernalization and etiolation pretreatments on single-node and leaf explants.

Results: Vernalization and etiolation treatments significantly enhanced in vitro regeneration of P. ostii, resulting in improved organogenic responses and reduced browning. Optimal sterilization and culture conditions were established for both single-node and leaf explants. For single-node explants, NN69 medium delivered the highest shoot induction rate (66.7%) with moderate browning. Supplementation with 0.1 mg·L⁻¹ indole-3-butyric acid (IBA) and 0.2 mg·L⁻¹ N-(2-chloro-4-pyridyl)-N'-phenylurea (CPPU) further enhanced shoot multiplication (4.5-fold) without hyperhydricity. The addition of white-red light increased shoot elongation to 2.27 cm. For leaf explants, callus induction reached 67.8% under 0.3 mg·L⁻¹ IBA and 0.9 mg·L⁻¹ CPPU, while shoot induction peaked at 54.4% with 0.2 mg·L⁻¹ IBA and 0.2 mg·L⁻¹ CPPU, without browning. The incorporation of 0.2 mg·L⁻¹ IBA and 3 mg·L⁻¹ CaCl₂ in the rooting medium promoted rapid adventitious root formation (60%) with robust, non-browning roots systems.

Conclusion: This study established an effective tissue culture platform for P. ostii by integrating vernalization-etiolation pretreatment with optimized culture conditions. This platform addresses the limitations of conventional propagation methods and offers a foundation for large-scale clonal propagation and future genetic improvement of this valuable species.