Induction And Culture Of Meristematic Nodules In Paeonia Rockii

Tree peonies (Paeonia sect. Moutan) are famous ornamental plants in many parts of the world and have served as an important crude drug in Chinese traditional medicine for thousands of years. However, the propagation of tree peony plants by grafting and dividing takes so long time with a low propagation ratio that hardly satisfies the required demand for large scale commercial productions. Cross breeding by seedling also takes so many years for a plant to reach an effective production stage due to the slow development. Tissue culture could be a potential technique to solve these undesirable problems in tree peony propagation and breeding and may also be useful in the pharmaceutical ingredients production. An ideal protocol for the tissue culture of tree peony must have a high multiplication rate, low variation percentage, and automatic multiplication process, using vegetative tissues as explants because cultivars of tree peony are so highly hybridized through a long cultivation, but to date a reliable tissue culture protocol for tree peonies propagation is still unavailabe. Meristematic nodules are tissues with distinct status during plant in vitro development and thus provide an important regeneration pathway via organogenesis in many plant species, with a great value in large scale plant propagation programs. In this study, P. rockii cultivars were used as the main materials to examine the effect of basal media, the explant type and the inoculation method, and the class and concentration of plant growth regulators (PGRs) on the browning of explants, callus induction and subculturing. Subsequently, meristematic nodules (MN) were induced from P. rockii 'Gao Yuan Sheng Huo', and the aspects influencing MN initiation were tested to find the optimal treatments. The relationship among MN development, the media status, the media basal composition, the media pH fluctuation or the media PGRs content were also investigated. Finally, the main pharmaceutical ingredients of MN were analyzed to evaluate its value for drug production. Main results were as follows:1.The basal media, source of explants and the way of their inoculation significantly affected explant browning and callusing, according to the explant browning and callusing rate. Thin cell layers (TCLs) from petioles were selected as the ideal explants whereas SH medium was the best basal medium for P. rockii. Petiole TCLs cultured on SH medium almost did not change to brown, and the highest callusing rate reached 100%.2. Petiole TCLs and leaf explants had different levels of sensitivity to the concentration of 2,4-D. Maximum callusing rate was gained in 0.1mg/L 2,4-D containing media in petiole TCLs explants, while in leaf explants,0.5mg/L 2,4-D was needed.2,4-D could improve callus growth alone both in induction and in subculture, and higher concentrations had the similar effects to that of 0.1/0.5mg/L. BA inhibited callus formation in induction media, but improved callus growth in subcultures when it was not accompanied by 2,4-D. The effects of explant type and the PGRs on callus induction expended to the subculture of calli.3.MN of P. rockii was induced from callus. The callus induction phase of the whole process was termed as preinduction. MN initiation was comprehensively influenced by explant type, the time of their selection, the 2,4-D concentration in preinduction and the duration of preinduction. MN was obtained with petiole PGRs selected from mature'Gao Yuan Sheng Huo'plants 55 d after sprouting as explants, the SH medium containing 0.1-2.0 mg/L 2,4-D as the preinduction medium, the SH solid medium containing 0.5 mg/L 2,4-D and 4 mg/L BA as the induction medium, a 45 d duration of preinduction, and a 60 d duration of induction. The highest inducing rate of MN in this study was 85.7%.4. MN of P. rockii could be multiplied in liquid medium. The media status, the basal media composition and the media pH fluctuation were responsible to MN development. MN grew msinly to increase the size in solid medium, occasionally self-multiplied, while did self-multiplication in liquid medium. Inorganic salts took more responsibility of improving MN growth. Media pH changed as the MN cultured in them. The tendency and extent to which the media pH changed were affected by PGRs and initial media pH. But the initial media pH did not have distinct influence on MN development. Their development process contained 3 phases. Auxins and cytokinins acted in different phases. The state of culture medium (solidified or liquid) had even more significant influence on the development status of meristematic nodules, than that of PGRs. Organogenesis or somatic embryogenesis did not occurred under PGRs treatments in this study.5. The content of pharmaceutical ingredients such as paeoniflorin and paeonol in MN of P. rockii 'Gao Yuan Sheng Huo'was related to their development status, and was almostly the same as in the roots of t P. ostii'Feng Dan'and P.×suffruticosa, which are traditionally the original sources of Chinese drug Danpi. It means the MN should be a potential way to produce effective pharmaceutical ingredients in tree peonies, which will be very beneficial for protecting wild tree peony plants from being collected for medicine use in the habitats.Totally, the protocol of MN culture in P. rocki established in this study will provide a reliable foundation for the ideal tissue culture protocol for tree peony propagation as well as pharmaceutical ingredient production. Further investigations should be concentrated to inducting organogenesis from MN and its extending to more species and cultivars in Paeonia.