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The potentiality of PGPR and biocontrol bacteria in agriculture is steadily increased as it offers an attractive way to replace the use of chemical fertilizers, pesticides and other supplements. This study aimed to investigate the effects and potential mechanism of inoculation with two Bacillus velezensis strains on the growth and quality improvement of Anoectochilus roxburghii (Wall.) Lindl. (MRH) and Anoectochilus formosanus (Wall.) Lindl. (YYB). The control treatment was non-inoculated (CK) and the coculture treatments were inoculated with Bacillus velezensis D2WM (D2), Bacillus velezensis ZJ-11 (ZJ-11), Bacillus velezensis D2WM and Bacillus velezensis ZJ-11 (D2+ZJ-11). The fresh weight, plant length, amount of active compounds, as well as chlorophyll content were significantly increased under D2, ZJ-11, and D2+ZJ-11 treatments compared with MRH and YYB plants in CK. In particular, D2+ZJ-11 treatment resulted in the greatest growth promotion. In MRH and YYB plants, the fresh weight increased by 82.6 % and 106.6 %, the kinsenoside content increased by 9.33 % and 21.65 % per gram, and the flavonoid content increased by 44.70 % and 21.07 % per gram, respectively. D2WM and ZJ-11 were capable of secreting siderophore, phytase, indole-3-acetic acid (IAA), and zeatin to promote plant nutrient assimilation and growth. Moreover, when D2WM and ZJ-11 were both inoculated with Anoectochilus roxburghii, the rhizosphere soil enzyme activities of urease, phosphatase, and invertase were significantly higher than control, thus helped to provide more nutrients to Anoectochilus. Further, the analysis of microbial community diversity indicated the increase of abundance of beneficial microorganisms, such as nitrogen-fixing bacteria, Basidiomycota and Ascomycota both of which are known to fuel their plant partners with mineral nutrients from the soil. Additionally, the abundance of pathogens belonging to Xanthobacteraceae as well as Cladophialophora and Penicillium were reduced. Hence, coculture with beneficial microorganisms can improve the growth and quality of Anoectochilus plants via promoting nutrient assimilation and regulating rhizosphere microbial community. This technique is of great application value in promoting the efficient cultivation and production of high-quality plant materials.

Maca (Lepidium meyenii Walp.) is a traditional medicine and nutritional supplement from South America with many pharmacological effects, such as enhancing female and male fertility, improving sexual dysfunction, preventing osteoporosis and relieving menopausal syndrome. Until now, the active principles behind most of these effects have not been clarified, severely hindering the exploitation and application of maca products. In this study, the effective anti-osteoporotic components of maca are uncovered. Through virtual screening against estrogen receptor and verification of pharmacological activity on osteoblasts, we found that N-benzyl-palmitamide is an active constituent of maca in preventing osteoporosis. As well, N-benzyl-palmitamide promotes osteoblast proliferation, differentiation and mineralization, plus it benefits bone formation through enhanced expression of osteogenesis-related genes, such as bone morphogenetic protein-2, core binding factor alpha 1, type 1 collagen and alkaline phosphatase. The effects of N-benzyl-palmitamide on enhanced bone formation are very likely through the estrogen receptor pathway, as N-benzyl-palmitamide also increases the expression of ERα and ERβ genes. Clarifying single-component biological activity will greatly enable exploitation and application of maca as a medication and health supplement to prevent osteoporosis.

Sucrose metabolism generates material and energy foundations and regulates the biosynthesis of secondary metabolites in plant cell cultures. However, this regulation mechanism is unclear. The neutral/alkaline invertase (NINV) gene is among the key enzymes in sucrose hydrolysis. Thus, the complete open reading frame (ORF) of this gene was obtained from Taxus chinensis cells. This gene was also successfully expressed in Escherichia coli . The expressed protein was verified through the function of sucrose cleavage. Furthermore, the transcriptional levels of the NINV gene can respond to the sucrose concentration changes and relate to taxane biosynthesis in T. chinensis cells. Subsequently, we developed the gene expression vector and introduced it into T. chinensis suspension cells to confirm the role of the NINV gene in taxane biosynthesis. Results indicated that this gene was overexpressed and that it significantly enhanced the expression of the taxadiene synthase (TAS) gene, as well as the biosynthesis of the seven detected individual taxanes. Thus, NINV-mediated sucrose metabolism can promote the biosynthesis of secondary metabolites. This study presents new concepts and approaches to increase the production of secondary metabolites in plant cell cultures.

Yields of paclitaxel decreased with repeated subculturing of Taxus media cells. We used minimal growth conservation and manipulation of genome methylation to sustain paclitaxel production by Taxus media cell cultures. The subculture period of Taxus cells can be prolonged to 180 d by incubating them at a low temperature (5 °C). Paclitaxel levels increased in the cells after conservation and during the first recovery subculture cycle, and then decreased during the subsequent recovery subculture cycle. Analysis of genetic variations in these cultures using amplified fragment-length polymorphism (AFLP) technology identified only two polymorphic bands associated with the second and sixth recovery cycle cultures. However, the results of high-performance liquid chromatography indicated that DNA methylation increased during the course of repeated subculturing. A decrease in DNA methylation level caused by treatment with 5-Aza-2′-deoxycytidine coincided with an increase in paclitaxel levels. Simultaneous exposure to both methyl jasmonate and 5-Aza-2′-deoxycytidine increased paclitaxel levels to 320.43 μg g −1 (dry weight), which is more than six times the paclitaxel content before conservation. To our knowledge, this is the first report about improving paclitaxel production by ensuring sustainable use of Taxus cells.

For a better understanding of the simulation, optimization, and process control in cell cultures, good kinetic models are necessary for large scale plant cell culture. In this paper, the systematic kinetics of taxol production by Taxus media cell suspension cultures in a stirred 15-L bioreactor under substrate-sufficient conditions and the absence of inducer intervention were studied. A kinetic model of cell growth was established by logistic equation, and kinetic unstructured models of substrate consumption, product synthesis and rheological behavior were constituted, which incorporated energy spilling. These models were verified by comparing the simulation results with those obtained experimentally. These results showed that energy spilling was a key factor that must be considered in constructing unstructured kinetic models of Taxus media cell suspension cultures in a stirred bioreactor under substrate-sufficient conditions. Besides, an optimized operation measure of decreasing energy spilling was proposed. An increase of 17.64% in cell biomass and 14.88% in taxol concentration were obtained when the strategy of limiting added carbon several times was experimentally implemented in a 15-L bioreactor. Results demonstrated that these established models should be helpful in the process prediction and operation optimization to guide the production and amplification of Taxus media cell suspension cultures in a bioreactor.

The multi-arm star polymer (ESOPLA) was obtained by ring-opening polymerization of dl -lactide using multifunctional epoxidized soybean oil (ESO) as an initiator in the presence of a stannous actuate (SnOct 2 ) catalyst. Gel permeation chromatography with multi-angle laser light scattering (GPC-MALLS), FTIR, 1 H NMR, thermal analysis and in vitro degradation were used to qualitatively characterize the synthesized polymers. The results revealed that ESO plays an important role in increasing the molecular weight, polymerization rate and monomer conversion rate. Degradation analysis demonstrated that the decrease in molecular weight and the weight loss ratio of the star-shaped ESOPLA were lower than that of linear poly( dl -lactide) (PDLLA). The surface topography of pre- and post-degradation materials was characterized by scanning electron microscopy (SEM). These SEM images showed that the linear PDLLA films underwent water erosion more readily than the star-shaped polymer films.