(1) Tree peony (Paeonia suffruticosa Andrews) is a woody ornamental plant originating from China, and beloved by people worldwide. Non-structural carbohydrates (NSCs) play a crucial role in regulating the flowering quality of tree peonies in both field and potted conditions. However, the effects of NSCs accumulation and allocation in various organs during the vegetative growth stage on the flowering quality of tree peony under forcing culture remains unclear. (2) Two-year-old grafted seedlings of tree peony cv. 'Luoyanghong' were subjected to orthogonal treatments to investigate the role of NSCs accumulation in plants' developmental process. We measured leaf photosynthetic capacity, NSCs accumulation in the organs of seedlings, observed key ornamental characteristics of flowering quality under forcing culture conditions, and evaluated the qualities of seedlings and flowers using the seedling index (SI) and flowering index (FI), respectively. (3) There was a significant positive correlation between leaf photosynthetic capacity and NSCs accumulation in both the whole plant and roots of potted tree peony. Roots were identified as the primary organs for NSCs accumulation in potted tree peonies. Sufficient NSCs accumulation in the plant, particularly in the roots during the defoliation period, was essential not only for enhancing the seedling quality of potted tree peonies but also for improving the flowering quality under forcing culture conditions. Both the seedling index (SI) and flowering index (FI) exhibited a significant dose-response with increasing root NSCs accumulation at defoliation. The T3 group, which involved slight root pruning (by 25%), combined with a high-concentration rooting agent (750 mg·L-1) and Metarhizium anisopliae (20 million U·mL-1), resulted in the highest photosynthetic capacity, SI, FI and NSCs accumulation status (NSCAR), making it the optimal treatment combination. (4) This finding indicates that increasing NSCs accumulation in the roots of potted tree peonies is a crucial biological foundation for producing high-quality potted flowers under forcing culture conditions, which provide new insights into the importance of NSCs in tree peony flowering and may improve the production technology for high-quality potted tree peony flowers under forcing culture conditions.
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