Callus induction
Five days after cultured on CIM, there was no obvious difference in morpho-histological observation of the explants. The cotyledons turned white and became swollen (Fig. 2A) due to vigorously division of cortex parenchyma cells (Fig. 2D) after 10 days of culture. Meanwhile, meristematic cell mass with rapid cell division was observed locally around the area of the vascular bundle (Fig. 2E). After 15 days of culture, visible calli formed on the edge of the cotyledons (Fig. 2B). Sectional observation revealed that callus formation was unevenly along the explant tissue so that the explant not kept a regular shape (Fig. 2F). The original tissue structure and morphology of some parts of the explant disappeared, and a small proportion of calli expanded into clumps after 20 days (Fig. 2C). The proportion of clumpy calli increased, and gradually covering the entire explant after 25 days. There were two morphologically distinct types of calli after 30 days of induction: type I-yellow compact calli with densely arranged clumps (Fig. 3A) and type II-transparent watery loose calli (Fig. 3E).
Identification of ECs and NECs
It was observed through SEM that surface cells of type I callus showed globular shape and tightly packed structure (Fig. 3B), while type II callus exhibited disorganized, elongated and tubular cells (Fig. 3F). Cells of type I callus were small, isodiametric and densely arranged with large and clear nuclei under histological analysis (Fig. 3C). While type II callus were shown to be large, irregular, and highly vacuolated cells with abundance of intercellular space (Fig. 3G). Regarding cytological analysis,type I callus cells had large nucleus containing prominent nucleoli and chromatin that showed even distribution and little or no condensation, a few small scattered vacuoles, abundant mitochondria , and chloroplasts were degraded to plastids with starch grains accumulation in which the endometrial system was disassembled (Fig. 3D). In contrast, cells of type II callus showed a large vacuole occupying nearly the whole cytoplasmic space, the nucleus and other cytoplasmic organelles located in a narrow strip of cytoplasm between the cell wall and the large vacuole (Fig. 3H). Besides, no starch grain was observed. Type I callus cultured in CIM obtained regenerated shoots ultimately, while Type II callus gradually browning without differentiation. In summary, type I callus were identified as ECs.
MN induction and organogenesis
Morpho-histological study revealed a developmental sequence leading to the formation of MNs and shoot differentiation.
①Pre-nodular structures: The yellow callus turned green after subculture in DIM for 1-2 generations (Fig. 4A). Correspondingly, cells on the surface of the callus were organized into small units under SEM observation (Fig. 4B). The external layer of the callus were composed of small, isodiametric, densely stained meristematic cells which were organized into abundant meristematic cell masses in peripheral regions (Fig. 4C). During 3-4 generations, neo-formed tracheary elements developed inside of meristematic cell mass and organization centers (OCs) consisting of a central area of vascularization surrounded by meristematic cell layers with vigorous division were observed in histological analysis. Later, the OCs became autonomous and developed an epidermis-like layer (Fig. 4F). These visible small protuberances were termed as pre-nodular structures (Fig. 4D, E).
②MNs: After subculture for 5-6 generations, the pre-nodular structures greatly increased in diameter due to vigorous divisions taking place in meristematic cells surrounding vascularized centers, and rapidly formed conspicuous large protuberances (Fig. 4G, H) differentiated a more defined internal structure. Histological sections revealed that the large protuberance comprising of OCs, a cortical-like area of parenchymatous cells and an epidermal-like area (Fig. 4I). These typical features account for their classification as MNs. Smaller nucleus and larger vacuoles were observed under TEM, and less starch grains exhibited in the plastids, where containing moderately developed lamellar structures (Fig. 6A, B).
③Nodular clusters: After 7-8 generations, enlargement of nodules in size were accompanied by the formation of indentations created by differential expansion of multiple OCs,that appeared initially as small groove on the surface of the nodules, and then progressively deepened, yet nodule break-up was never observed. In the same way, smaller ‘daughter nodules’ were produced without detachment. Thus, several MNs displaying different levels of development were loosely attached to each other and developed into nodular clusters in appearance (Fig. 4A, B). During 7-10 generations, meristematic cells of OCs inside the nodules intensely divided and showed relative movement towards the nodules periphery (Fig. 4C), which resulted in the formation of primordia established vascular connection with the nodule (Fig. 4D, E, F). Smaller nucleus and larger vacuoles were also observed under TEM, nevertheless, plastids contained moderately developed lamellar structures without starch grain (Fig. 6C, D). There were lots of mitochondria and extensive rough endoplasmic reticulum (RER) adjacent to dictyosome that were active in producing vesicles (Fig. 6E).
④Shoots differentiation: After 11-12 generations, early stage of leaf clusters formed with development and elongation of primordia (Fig. 4G, H), and apical meristems establishing vascular connection with the nodule (Fig. 4I). Nodules with leaf clusters were not conducive to promoting shoot elongation when cultured in the same DIM (Fig. 7C), but new elongated shoots were able to successively developed after transferring to medium containing BA and GA3 (Fig. 7D), which had complete vascular system and axillary bud primordia (Fig. 7A, B). At this stage, chloroplasts developed a well-organized internal membrane system (Fig. 6F).
These regenerated shoots were developed into plantlets through rooting culture (Fig. 7E) and transplanted to culture chamber successfully (Fig. 7F), which supporting the usefulness of this in vitro regeneration protocol in tree peony.
The extracellular matrix (ECM)
SEM revealed the presence of a discontinuous amorphous secretions outside the callus surface,and varied in structure, which termed as ECM. Compared to ECs covered with numerous compact membranous layers, fibrillar structure and abundant granular mucilage-like secretions (Fig. 8A, B), cells of NECs provided a ‘‘peeling’’ appearance (Fig. 8C). Enlargement and multiplication of the surface cells of nodules resulted in the rupture at various sectors of membranous layer, consequently, transition from membranous layers to fibrillar structures were exhibited on the nodules surface (Fig. 8D, E). The structure of superficial cells become slightly elongated and regularly arranged leading to the formation of epidermis-like surface under SEM at stage of nodular clusters (Fig. 8F), and the ECM structures on the surface gradually decreased and presented fragments appearance (Fig. 8G). However, dense granular mucilage-like secretions were exposed on the top surface of the primordia regions exclusively (Fig. 8H). No ECM performed on the smooth surface of newly formed leaves (Fig. 8I).