Sterilization protocols and the effect of plant growth regulators on callus induction and secondary metabolites production in in vitro cultures Melia azedarach L.

Melia azedarach L. is a valuable source of antioxidants and secondary metabolites. This study is a first extensive report about the effect of different serialization protocols and plant growth regulators (PGRs) on explant disinfection efficiency, callus induction and secondary metabolites production and accumulation in callus cultures of M. azedarach L. In this regard, the effect of plant growth regulators on callus induction and secondary metabolites production were examined. In addition, different sterilization agents were evaluated for disinfection of chinaberry leaf explants. The results showed that the lowest percentage of explant contamination and browning with the highest percentage of callus induction and callus growth obtained with explants pretreated with benomyl (2 g/L) for 2 h and sterilized with 7% H2O2 for 10 min and NaOCl 2% (without pH adjustment) for 12 min. Although adjusting the pH of NaOCl to pH = 7 and 10 significantly reduced the microbial contamination and increased the percentage of contamination-free cultures of M. azedarach L., adversely influenced the explant viability and callus induction and growth. The highest percentage of callus induction obtained on the MS medium containing 3 mg/L NAA/2,4-D and 1 or 3 mg/L Kin/BAP, and the highest callus yield (1804.833 mg/explant) belonged to the MS medium supplemented with 5 mg/L 2,4-D and 5 mg/L Kin. The callus cultures grown on the MS medium supplemented with 3 mg/L NAA and 1 mg/L Kin produced the highest amount of Quercetin (2.06 mg/g fresh weight), Rutin (5.56 mg/g fresh weight) and Kaempferol (1.84 mg/g fresh weight).

Interaction of causative agents of sapronoses with the land plant Lithospermum erythrorhizon

Background. A significant role in the ecology of the sapronotic pathogens Yersinia pseudotuberculosis and Listeria monocytogenes and in the epidemiology of the infections they cause is played by land plants used for food. These microorganisms are often found on plant substrates, they multiply on various vegetable and root crops. In this regard, it is relevant to study the viability and biological activity of Y. pseudotuberculosis and L. monocytogenes in contact with various land plants, including those that are not eaten, but are used in medicine.Aim. Study of the interaction of sapronotic pathogens Y. pseudotuberculosis and L. monocytogenes with callus cultures of the land plant Lithospermum erythrorhizon Siebold et Zucc.Materials and methods. The studies included strains of Y. pseudotuberculosis 512 serotype 1b, pYV+, 82MD+ and L. monocytogenes NCTC (4b) 10527 from the Collection of Somov Institute of Epidemiology and Microbiology, and cell culture from the roots of red-root gromwell Lithospermum erythrorhizon line VC-39 (from the Collection of FSC of the East Asia Terrestrial Biodiversity FEB RAS).Before the study, Y. pseudotuberculosis and L . monocytogenes were cultured 18–20 hours on nutrient agar pH 7.1–7.2. A working dilution of microorganisms was prepared (106 micobial cells per 1 ml) and applied at a dose of 100 μl to the surface of plant calli. Material samples were taken in dynamics after 3 and 14 days and prepared for scanning electron microscopy.Results. Y. pseudotuberculosis and L. monocytogenes formed biofilms on the surface of plant cells within 3 days after the start of the experiment. It was noted that Y. pseudotuberculosis destroyed the components of the plant cell membrane.Conclusion. New data obtained during the study expand the understanding of environments and forms of habitation, as well as the potential for pathogenicity of sapronotic pathogens in the environment.

Expression of Heat Shock Protein HSP90 in Genomic-DNA of Chickpea (Cicer arietinum L.) Callus by Heat Shock Treatment

This study was able to detect of the expression activity of heat shock proteins HSP90 and heat transcription factors HSFs for the first time in callus cultures of chickpea, Cicer arietinum L., that exposed to abiotic shocks, grown on MS medium supplemented with 1.0 mg L-1 naphthalene acetic acid (NAA) and 2.0 mg L-1 benzyl adenine (BA). Heat shock proteins HSPs were constructed for increase of withstand long-term physical shocks, and production of resistant to heat chickpeas plants, this shock was enhancement of tolerance of chickpea callus to abiotic stresses (high - temperatures). Results enhanced the ability of chickpea callus to abiotic stresses bearing and induce of HSF genes to heat shock proteins HSP90 production quickly to removing denatured proteins, avoid apoptosis, thus, supporting tolerance to the sudden action of these shocks. Expression activity of heat shock genes and transcription factors by determined based on polymerase chain reaction qPCR, that explained the gene activity increasing at shocks intensity increased

Optimization of Biomass Accumulation and Production of Phenolic Compounds in Callus Cultures of Rhodiola rosea L. Using Design of Experiments

Rhodiola rosea L. is a valuable medicinal plant with adaptogenic, neuroprotective, antitumor, cardioprotective, and antidepressant effects. In this study, design of experiments methodology was employed to analyze and optimize the interacting effects of mineral compounds (concentration of NO3− and the ratio of NH4+ to K+) and two plant growth regulators [total 6-benzylaminopurine (BAP) and α-naphthylacetic acid (NAA) concentration and the ratio of BAP to NAA] on the growth and the production of total phenolic compounds (TPCs) in R. rosea calluses. The overall effect of the model was highly significant (p < 0.0001), indicating that NH4+, K+, NO3−, BAP, and NAA significantly affected growth. The best callus growth (703%) and the highest production of TPCs (75.17 mg/g) were achieved at an NH4+/K+ ratio of 0.33 and BAP/NAA of 0.33, provided that the concentration of plant growth regulators was 30 μM and that of NO3− was ≤40 mM. According to high-performance liquid chromatography analyses of aerial parts (leaves and stems), in vitro seedlings and callus cultures of R. rosea contain no detectable rosarin, rosavin, rosin, and cinnamyl alcohol. This is the first report on the creation of an experiment for the significant improvement of biomass accumulation and TPC production in callus cultures of R. rosea.

Profiling of Polyphenolic Compounds of Leontopodium alpinum Cass. Callus Cultures Using UPLC/IM-HRMS and Screening of In Vitro Effects

Leontopodium alpinum Cass. (edelweiss) is recognized as a frequent constituent of anti-aging skin care products, providing increased antioxidant and anti-inflammatory defense. Considering the growing demand and the protected status of edelweiss in many countries, alternative methods of production have been developed, one of them being callus culturing. This study reports the phytochemical composition of a methanolic extract of L. alpinum callus cultures, characterized by liquid chromatography coupled to ion-mobility high resolution mass spectrometry (UPLC/IM-HRMS). The methanolic extract exhibited strong free radical scavenging activity (122.19 ± 7.28 mg AAE/g dw), while the quantitative evaluation revealed that four major constituents (phenylpropanoid derivatives) represent 57.13% (m/m) of the extract. Consequently, a screening of antiproliferative effects was performed on ten cancer cell lines, representative of prostate, colon, lung and breast cancer, showing inhibition of colony formation in all cases. These results provide a comprehensive phytochemical characterization of L. alpinum callus cultures using advanced IM-HRMS, while the in vitro explorations confirmed the potent antioxidant properties of edelweiss which are worth exploring further in cancer prevention.

Producing Medicinal Phytochemicals from Phyllanthus acuminatus in Plant - Cell Suspension Cultures

Pharmaceutical use is not feasible for important medicinal compounds derived from certain plant materials, including Phyllanthus acuminatus roots, due to their low natural abundance. New technologies in non-traditional biomass generation are needed to produce these remarkable natural compounds. Therefore, this article describes a methodology for establishing Phyllanthus acuminatus plant-cell suspensions from callus cultures: An evaluation on inoculum concentration and agitation speed displayed significant changes in plant cell growth kinetics. It was determined that treatment with 2 g of inoculum in 25 mL of medium and 100 rpm agitation creates the best conditions for generating thick cell suspensions. Likewise, treatment with 2 g of inoculum and 120 rpm agitation produces the best conditions for establishing fine cell suspensions. Phytochemical comparison through high-resolution mass spectrometry of P. acuminatus roots and plant cell suspension extracts confirmed presence in the plant cell culture of multiple phyllantostatins of pharmaceutical interest. Here, we demonstrate that Phyllanthus acuminatus can be cultured in plant cell suspensions to produce secondary metabolites of medical interest – technology that could be scaled up for implementation in industrial bioprocesses.

Analysis of Phenolic Compounds for the Determination of Grafts (in) Compatibility Using In Vitro Callus Cultures of Sato-Zakura Cherries

The aim of this study was to prove that under in vitro conditions, the adhesiveness of the callus between rootstock and scion, the development of callus cells at the points of fusion, and the presence of phenolic components are closely related to the level of (in) compatibility of the grafting combinations between Sato-zakura cherry cultivars (‘Amanogawa’, ‘Kanzan’, and ‘Kiku-shidare-zakura’) and commercial rootstocks. Prunus avium, Prunus ‘Colt’, Prunus mahaleb and Prunus serrulata were used as compatible and Prunus serotina and Pyrus communis ‘Pyrodwarf’ were used as two potentially incompatible rootstocks. The results indicated the significant manifestations of the early signs of the incompatibility on the callus junction. Phenols, as well as tissue senescence, were very precisely localized by toluidine blue and alcian blue as well as safranin staining, which can indicate the early signs of the callus incompatibility in some grafting unions. In the callus unions of Prunus avium with ‘Amanogawa’ and ‘Kiku-shidare-zakura’ the results of chemical analyses indicated that the existence of several flavonols, flavones and phenol acids could be involved in the incompatibility process in grafted combination. The detection of flavonol astragalin in the unions can be a biomarker of compatibility between scion and the rootstock, while some polyphenols, such as neochlorogenic acid, sinapic acid, ellagic acid, caffeic acid, baicalein, naringenin, apigenin and luteolin can be used as the indicators of graft incompatibility. p-coumaric acid and ferulic acid could be used for detection of delayed incompatibility.

Flavonoids in in vitro cultures of Astragalus hamosus

Astragalus hamosus contains valuable biologically active compounds, incl. flavonoids. The possibility for in vitro cultivation of the species as a source of important flavonoids was studied. Shoot and callus cultures were established and successfully cultivated on different nutrition media, complemented or not with growth regulators. An ultra-high performance liquid chromatography – high-resolution electrospray ionisation mass spectrometry (UHPLC-HRESIMS) qualitative and quantitative analysis of non-purified methanol extracts of these cultures was performed. It was found that the cultures produced rutin in comparable quantity. Interestingly, both shoots and callus cultures accumulated the rare triglycosides alcesefoliside and mauritianin. The quantity of mauritianin, biosynthesized in shoots, was significantly higher to that in callus cultures. Alcesefoliside, was in lower quantity, compared to mauritianin. In addition, callus cultures produced alcesefoliside trice as the shoots, besides their lower level of differentiation. These findings could serve as initial research to establish the value of in vitro cultures from A. hamosus as an alternative mean of production of pharmaceutically important flavonol glycosides.

The possibility to enhance flavonoids production in Rubia tinctorum L. callus cultures

Production of flavonoids in madder callus culture (Rubia tinctorum L.) was dependent on culture conditions and culture media composition. The content of flavonoids increased in calli maintained on media supplemented with NAA (4 mg.l-1) or NAA:BAP (4 mg.l-1 a 1 mg.l-1) in 16 h photoperiod. Flavonoids represented 2.08 – 2.25 % of callus dry mass. The presence of Cd(NO3)2 (3.1 or 31.0 mg.l-1 concentrations) negatively influenced callus growth, but enhaced the percentage of dry mass in callus cells. During 42 days of culture an increase of cadmium accumulation and even of flavonoids has been observed. The most considerable influence of CdCl2 or Cd(NO3)2 on flavonoids content has been shown in short-term experiments after 48 h of callus culture. More distinct influence has been observed under the treatment with CdCl2 (0.005 mg.l-1) in comparison with Cd(NO3)2.

Polyphenol contents and antioxidant activity of carob callus cultures

Carob (Ceratonia siliqua L.) is an important Mediterranean plant species with worldwide commercial and medicinal uses. The establishment of a callus culture protocol as an alternative system to produce polyphenols of chemical and pharmaceutical interest was made in the present study for the first time in carob. Explant type and the light regime are two important factors that influence morphogenic responses and biochemical production. Maximal callus induction (100 %) and biomass accumulation were obtained in cotyledon explants under both tested light regimes (16-hour photoperiod and darkness). However, leaf callus produced a higher amount of polyphenols (TPC) and flavonoids (TFC) but a lower amount of total condensed tannins (TCT) as compared to cotyledon callus. Light treatment has significantly increased TCT content but decreased the antioxidant activity in carob callus cultures. Strong and positive correlations were obtained between TPC and TFC and the antioxidant activities with correlation coefficients ranging between 0.68 and 0.98. The obtained results indicate that calli of C. siliqua have the potential for enhanced production of phenolic compounds with antioxidant activity that is favored by culture under dark condition.