Establishment of an Efficient Rooting System for Paeonia lactiflora Shoots Derived from Tissue‐Cultured Zygotic Embryos: Effects of Indole‐3‐Butyric Acid, Coumarin, and Light

Paeonia lactiflora Pall. is a globally recognized ornamental and medicinal plant of immense economic value. However, conventional cross‐breeding and germplasm innovation are severely hampered by deep seed double dormancy and a protracted life cycle. While in vitro culture of zygotic embryos offers a strategy to accelerate breeding, the widespread application of this technique is strictly bottlenecked by the recalcitrant rooting of embryo‐derived shoots and subsequent low ex vitro survival rates. To overcome this, the present study established a sequentially optimized in vitro rooting and acclimatization system for P. lactiflora embryo‐derived shoots. Initially, testing of light/dark regimes revealed that 7 days of complete darkness followed by 28 days of light was most conducive to root development. Based on this optimal physical environment, chemical formulations were systematically refined. The optimal adventitious root induction occurred on a modified half‐strength Murashige and Skoog basal medium (with double Ca 2+ concentration) co‐supplemented with 0.5 mg/L putrescine, 1.0 mg/L indole‐3‐butyric acid, and 5.0 mg/L coumarin. This specific combination resulted in a rooting percentage of 75%, with an average of 8.3 roots per shoot. Furthermore, substituting conventional agar with a highly porous mixed substrate (perlite and vermiculite, 2:1, v/v) during the rooting phase significantly improved root architecture, vigor, and subsequent ex vitro survival. These optimized protocols offer a robust regeneration platform, providing vital technical support for accelerating breeding cycles, facilitating embryo rescue, and advancing genetic transformation in herbaceous peony.