Induction, Genesis And Development Of Meristematic Nodules And Their Cytohistological Observations In Tree Peonies

Tree peonies (Paeonia sect. Moutan) are Chinese traditional flowers. However, low propagation rate and time-consuming operation by traditional propagation methods are the main obstacles hindering the development of tree peonies. And that is why tissue culture is becoming an issue in breeding and propagation of this species. Since about half a century when the in vitro culture of tree peonies was firstly reported, there is still a long way to form a protocol in the production. Meristematic nodules (MN) represent a morphogenetic pathway of importance equal to that of embryogenesis. And MN culture leads many recalcitrant woody plants amenable to in vitro regeneration. Therefore, it is reasonable to say MN culture as the most valuable and promising way to solve the challenges of tree peonies with low propagation rate. In this study, thin cell layers (TCLs) from petiole were used as materials to examine the influence of cultivars, plant growth regulators (PGRs) and other factors on the pre-induction callus formation, MN induction, multiplication and differentiation. Meanwhile, histological analysis was used to examine the induction, genesis and development of MN and determine their anatomical characteristics. The process is realized basically from MN to adventitious buds or leaflets. The induction conditions were defined and can be used as reference for the in vitro protocol for tree peonies propagation. Main results were as follows:1. Callus formation was the premise of MN formation. The result suggested that cultivar, developmental stage and position of explant significantly affected callus formation. Among the seven tested cultivars,'Golden Era'performed best and produced the higher induction rate and amount of compact callus. Explants taken between flowering and 1 week after blossom fall showed higher callus induction rate and negligible browning. Petiole layers explants taken from the basal or top petioles showed lower browning rate and higher callus induction rate. While, the explants taken from middle petioles showed serious browning problem. The optimum formulation for callus formation was SH medium containing 1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D). Higher concentration of 2,4-D leaded to vitrification or browning. Meanwhile thidiazuron (TDZ) promoted callus formation, and the effect was more noticeable when TDZ was combined with 2,4-D.1 mg/L 2,4-D and 1 mg/L TDZ produced the higher formation rate and good quality callus of'Golden Era'.2. The genesis and development of tree peonies callus were divided into 0-4 five grades. The cytohistological observation suggested that callus formation underwent three stages, namely activation, division and formation. During activation, fascicular cambium cells were most notable and started the division process. Cells kept division during the second stage division and led to the formation of callus in cambial area firstly and then in the region of cortex parenchyma cells. These callus tissues formed a compact cell cluster and the callus cells were characterized by dense cytoplasm, nucleus in center and small in volume. However, callus proliferated at a slower rate during formation stage, only the peripheral callus cells which facing the medium kept division.3. Tree peonies MN differentiated from the callus internally. There were two kinds of structures in the center of tree peonies MN. Some of tracheids organized as vascular centers and some specialized parenchyma cells could also act as centers, and then these centers were surrounded by loosely arranged, plastid-dense cell layers. This way, MN formed. The formation of tree peonies MN were regulated by cultivar, pre-formation of position of explants, PGRs, nitrogen form and sucrose concentration. Callus of'Golden Era','Bartzella'and'High Noon'proliferated into nodule structures and the explants from the central part of petiole produced higher induction rate of MN than from the basal or top petioles. The optimum formulation for MN was SH medium containing 0.2 mg/L2,4-D and 6 mg/L N6-benzyl adenine (BA) for both of'Golden Era'and'Bartzella'. While the ratio of NO3-/NH4+ was 2.4 and sucrose concentration was 50 g/L in the medium,'Golden Era'produced highes MN induction rate (78.0%). And when the ratio of NO3-/NH4+ was 9.5 and sucrose concentration was 30 g/L in the medium, 'Bartzella'produced its highes MN induction rate (55.6%).4. Proliferation was an important part of the MN protocol, a-naphthalene acetic acid (NAA) and TDZ influenced the MN multiplication according to cultivar. Maximum MN multiplication rate was gained in 0.5 mg/LNAA and 1 mg/L TDZ for'Golden Era'(83.3%) and 8 mg/L BA and 5 mg/L TDZ for'Bartzella'(91.7%). The MN mainly increased their number when cultured in liquid medium. And the best liquid culture period was 30 days for'Golden Era'.5. Differentiation was the key of the MN protocol. The results suggested that the differentiation of 'Golden Era'MN has experienced two steps. The first step related to transfer the MN to SH medium containing 0.25 mg/L NAA+0.5 mg/L TDZ or 0.5 mg/L TDZ under light condition for 45 days. The second step, the SH medium containing 25 g/L sucrose+2 g/L activated carbon were useful. Then 16% rate of adventitious buds and leaflets were observed. Among these factors, lower concentration of PGRs and light condition are major factors for differentiation. In addition, fewer subculture time and long subculture cycle were more easily differentiated. Epidermic cell divisions preceded shoot formation of MN and the communication-vascular bundles between buds and the MN was observed.