Paeonia ostii ‘Feng Dan’ plant regeneration through direct organogenesis and direct meristematic nodule culture

Abstract Tissue culture is preferred for solving the shortcoming of low efficiency in terms of conventional propagation ways in tree peony, an economically important woody plant in China with various purposes. However, callus differentiation is hard to obtain during in vitro regeneration. Meristematic nodule (MN) is a favorable way capable of overcoming this problem, but possesses a lengthy process. Direct organogenesis excluding the callus step is needed to simplify the procedure. This study firstly presented a protocol of direct organogenesis and direct MNs induction and differentiation using cotyledon explant for in vitro regeneration of P.ostii ‘Feng Dan’. The highest direct MNs induction rate (41.67%) and frequency of direct organogenesis (DO) (66.67%) was achieved under the following procedure. The explants were pretreated in dedifferentiation induction medium (DIM) [Murashige and Skoog (MS) medium with 2.27 µMthidiazuron (TDZ)+5.37 µM α-naphthylacetic acid (NAA)] for 10 days, and then the cotyledons without callus induced were transferred to differentiation medium (DM) [Woody plant medium (WPM) containing 2.02 µM N-(2-chloro-4-pyridyl)-N-phenylurea (CPPU)+2.27 µM TDZ and 4.04 µM CPPU+4.54 µM TDZ] respectively, with 6 subcultures, 90 days in total. The regenerated shoots rooted and transplanted successfully. Histological study confirmed the process of DO and direct MNs induction, and revealed that shoots and MNs were originated from increased division of meristematic cell under cortical tissue, as well as from actively divided meristematic cells around vascular center. Moreover, shoots regenerated through MNs differentiation were originated from the epidermal and subepidermal cells. This study is an innovation and supplement in the field of in vitro regeneration in tree peony, and will be conductive to clonal micropropagation, fundamental studies of developmental biology and genetic transformation.