Decoding a unique double flower pattern in herbaceous peony: insight into proliferate flower bud development

Background and Aims In herbaceous peony, the proliferate flower pattern exhibits a complex and two-layered floral structure with high ornamental value, offering new insights into flower development. The development of proliferate flowers is not well understood due to its complex structure. This study aimed to decode the developmental pattern of proliferate flowers through continuous observations of flower bud differentiation, focusing on the morphological and anatomical characteristics of pistils and stamens in proliferate flowers. Methods Floral differentiation in herbaceous peonies (including single flower variety, double flower variety and four proliferate flower varieties) was observed using a stereomicroscope, paraffin sectioning and free-hand sectioning. The qRT-PCR experiment was conducted to detect the expression of genes related to floral development, revealing key genes involved in proliferate flower development. Key Results In proliferate flowers, the development of lower and upper flowers occurs through two distinct stages. The lower flower sequentially generates bracts, sepals, petals, stamens and pistils. On the inner side of pistil primordia, a new flower primordium continuously differentiates to produce the upper flower, retaining carpels’ characteristics. The expression levels of classical ABCDE model genes (PlAP1, PlPl2, PlAP3, PlSEP1 and PlSEP3) were higher in proliferate flowers. In contrast, the expression of C-class gene PlAG was reduced in double flowers and was the lowest in proliferate flowers compared to single flowers. Conclusions The proliferate flower, a special type of double flower pattern, can be divided into upper and lower flowers. For lower flowers, the varying degrees of petaloid stamens are attributed to the diversity of proliferate flower cultivars, while the proliferation and directional mutation of pistils lead to the origination of upper flowers. For upper flowers, the differentiation of floral apical meristem includes petals, stamens and pistils. The limited expression of PlAG suggests its involvement in the induction of proliferate flowers.