Plant regeneration through two pathways of direct organogenesis in Paeonia ostii ‘Feng Dan’

Tissue culture is preferred for solving the shortcomings of low efficiency of conventional propagation in tree peony (Paeonia sect. Moutan), an economically important woody plant in China with various uses. Paeonia ostii ‘Feng Dan’ is one of the varieties with strong adaptability and high oil productivity. However, in vitro regeneration via organogenesis is hard to obtain. For instance, the indirect organogenesis from callus is a lengthy process, and the direct organogenesis is lacked. This study presents a protocol of direct organogenesis using cotyledon explant for in vitro regeneration of P. ostii ‘Feng Dan’, and includes two pathways. The explants need pretreated in the optimal dedifferentiation induction medium (DIM) [Murashige and Skoog (MS) medium with 2.27 µM thidiazuron (TDZ) + 5.37 µM α-naphthylacetic acid (NAA)] for 10 days, and then the cotyledons without callus induced were transferred to differentiation medium (DM) with 6 subcultures, 90 days in total. The best DM for the frequency of leaf clusters induction (66.67%) was Woody plant medium (WPM) containing 4.04 µM N-(2-chloro-4-pyridyl)-N-phenylurea (CPPU) + 4.54 µM TDZ, and for the frequency of MNs induction (41.67%) was WPM containing 2.02 µM CPPU + 2.27 µM TDZ. For two pathways, mean number of regenerated shoots (≈ 4 shoots per explant), the frequency of root induction (≈ 50%) and the frequency of survival (≈ 40%) were similar. Histological studies confirmed the process of direct organogenesis, and revealed that shoots and MNs were originated from the increased division of meristematic cells under cortical tissue, as well as from actively dividing meristematic cells around the vascular center. Moreover, shoots regenerated through MNs differentiation were originated from the epidermal and subepidermal cells. This study is supplements in the field of in vitro regeneration in tree peony, and is an improvement to current protocols of clonal micropropagation, at the same time facilitating fundamental studies of developmental biology and genetic transformation.