In vitro propagation and biological activity of essential oil and methanol extracts of tansy (Tanacetum vulgare L.)

Rodu Tanacetum pripadaju mnoge aromatične, lekovite i ukrasne vrste bogate biološki aktivnim sekundarnim metabolitima. Tanacetum vulgare L. (syn. Chrysanthemum vulgare L., povratič, vratiželja, konopljika) je višegodišnja, zeljasta biljka, poreklom iz Evrope i centralne Azije koja se obično sreće duž puteva, pruga, pašnjaka i polja ali i priobalnih područja. U Srbiji je ova vrsta deo sinurbane (ruderalne) flore. Predmet istraživanja ove disertacije bila ja fitohemijska karakterizacija sastava i bioloških aktivnosti etarskog ulja i metanolnih ekstrakata ove vrste. In vitro kultura T. vulgare je uspešno uspostavljena iz semena biljaka sakupljenih u prirodi. Kultura izdanaka je uspešno održavana i multiplikovana na MS hranljivoj podlozi uz dodatak 6-benziaminopurina (BAP) a prilikom ožiljavanja BAP je zamenjen indolsirćetnom kiselinom (IBA). In vitro kultura korenova održavana je u tečnoj MS hranljivoj podlozi sa dodatkom IBA koja je uticala na povećanje biomase gajenih korenova tokom subkultura. Histoločkom analizom sekretornih struktura in vitro gajenih izdanaka utvrđeno je da su na listovima i stablu prisutne biserijatne glandularne trihome sa sekrecionim sadržajem u kojem su detektovani lipidi, terpeni i alkaloidi. Hemijska analiza metanolnih ekstrakata herbe i korena T. vulgare ukazala je na to da su ekstrakti biljaka gajenih in vitro kvalitativno siromašniji u odnosu na metanolne ekstrakte biljaka iz prirode ali su se ekstrakti biljaka in vitro odlikovali višestruko većom zastupljenošću pojedinih jedinjenja, pre svih 3,5-O-dikafeoilhininskom kiselinom. Najzastupljenija jedinjenja su fenolne kiseline iz grupe derivata cimetne kiseline i to: neohlorogena, 3,5-O-dikafeoilhininska i dikafeoilhininska kiselina. Pored fenolnih kiselina detektovano je i 17 flavonoida. Relativni udeo fenolnih kiselina i flavonoida se razlikovao u zavisnosti od dela biljke korišćenog za pripremanje matanolnog ekstrakta...Many aromatic, medicinal and ornamental species rich in biologically active secondary metabolites belong to the genus Tanacetum. Tanacetum vulgare L. (syn. Chrysanthemum vulgare L., tansy) is a perennial, herbaceous plant originating in Europe and Central Asia, commonly encountered along roads, stripes, pastures, fields, and coastal areas. In Serbia, this species is part of the ruderal flora. The research subject of this dissertation was the phytochemical characterization of the composition and biological activities of T. vulgare essential oil and methanol extracts. In vitro culture of T. vulgare was successfully established from seeds of plants harvested in nature. Shoot cultures have been successfully maintained and multiplied on the nutrient medium enriched with 6-benziaminopurine (BAP); for needs of rooting, BAP was replaced with indole buteric-acid (IBA). The root in vitro cultures were maintained in a liquid nutrient medium supplemented with IBA which influenced the increase in the biomass of cultivated roots during subcultures. Histological analysis of secretory structures of in vitro plantlets showed presence of biseriate glandular trichomes with detected lipids, terpenes and alkaloids as secretory content,, both on the leaves and the stalk. The chemical analysis of methanol extracts of T. vulgare herb and roots indicated that the extracts of in vitro cultivated plants were qualitatively poorer than methanol extracts of plants harvested in nature, but the extracts isolated from in vitro plants were characterized by several times higher presence of certain compounds, primarily 3,5-O-dicaffeoylquinic acid. The most common compounds were phenolic acids from the derivatives of cinnamic acid group, such as neohlorogenic, 3,5-O-dicaffeoylquinic,and dicaffeoylquinic acid. In addition, 17 flavonoids were detected. The relative proportion of phenolic acids and flavanoids differed depending on the part of the plant used to prepare the methanol extract..

Rooting and preventing shoot-tip necrosis of in vitro cultured horse chestnut shoots

Efficient bud regeneration was achieved from germinating horse chestnut (Aesculus hippocastanum L.) somatic embryos cultivated on 1-10 μM benzyladenine (BA). Adventitious buds were detached from the mother tissue and used to establish permanent shoot cultures on 0-20 μM BA. Secondary buds were regenerated from the shoot base of the explants. Bud multiplication was very poor (1.9) and shoot-tip necrosis was very high (100%) on plant growth regulator (PGR)-free medium. The highest multiplication was achieved on 5 and 10 μM BA (16.8 and 18.7, respectively), with no shoot-tip necrosis, while hyperhydration was rather frequent on shoots cultivated on BA above 5 μM. Individual shoots were elongated on medium with 1 μM BA and 500 mg/l polyvinylpyrrolidone (PVP MW40 000) for 4 weeks. However, it was necessary to reduce BA level below 1 μM for shoot rooting and that caused mass shoot-tip necrosis. As classical rooting methods failed, the basal part of each elongated shoot was first wounded by cutting with a sterile blade and then dipped into a 0, 5 or 10 mM indole-3-butyric acid solution for 1 min and cultivated on solid half-strength MS PGR-free medium with 0.02% activated charcoal for 2-3 weeks. To prevent shoot tip necrosis during this phase, a BA solution was applied directly on apical meristem. Shoot-tip necrosis was completely eliminated by weekly application of 10 μl of 1 μM BA. As soon as the root initials were observed, the shoots were transferred to MS medium supplemented with 500 mg/l PVP and 5 μM BA. The frequency of rooting was 23%, and further optimisation of root-inducing phase is needed

Somatic embryogenesis from stamen filaments of Aesculus flava Sol. and peroxidase activity during the transition from friable to embryogenic callus

Species of the genus Aesculus are among the most attractive ornamental woody plants. Conventional propagation methods of these species are either inefficient (stem cuttings) or unsuitable for clonal propagation (seeds). The aim of the present study was to develop an efficient protocol for clonal propagation of elite specimens of yellow buckeye (Aesculus flava) by somatic embryogenesis. For this purpose, stamen filaments of yellow buckeye were cultivated on media supplemented with 1, 5 or 10 μM 2,4-dichlorophenoxyacetic acid (2,4-D) combined with 0, 1, 5 or 10 μM 6-furfurylaminopurine (Kin), either under light or dark conditions, for 8 weeks, and then subcultivated on plant growth regulator (PGR)-free medium with 400 mg/l of glutamine. The highest somatic embryo (SE) initiation rates were achieved for the explants cultivated in darkness on medium containing 1 μM 2,4-D + 10 μM Kin during callus induction (CI) phase. Embryogenic calli (EC) were initiated from friable calli, starting from the 7th week of culture initiation, while SEs appeared two weeks later, following a week of subcultivation of the explants on PGR-free medium. EC and SEs were observed only in the explants grown in darkness during CI phase. Minimal duration of CI phase and darkness necessary for SE induction was four weeks, while the highest embryogenic response was achieved when each lasted for 8–10 weeks. Obtained SEs were efficiently multiplied on medium supplemented with 0.05 μM 2,4-D + 5 μM Kin by recurrent somatic embryogenesis. SEs at globular stage of development exhibited the highest capacity for secondary SE regeneration. High germination and conversion rates were attained in cotyledonary-stage SEs cultivated on medium with 0.05 μM 2,4-D + 5 μM Kin, but this phase needs to be further optimised, since the obtained plants failed to acclimatize to greenhouse conditions. During the transition of calli from friable to embryogenic state, total peroxidase (POX) activity significantly decreased, indicating their involvement in the acquisition of embryogenic capacity. The presented protocol is suitable for clonal propagation and genetic transformation of this ornamental species, and POX activity may be used as a marker for SE initiation

Induction of potato (Solanum tuberosum L.) defense genes by French marigold (Tagetes patula L.) essential oil

When attacked, plants activate defense mechanisms including volatile emission. Besides havinga pest repellent effect, volatiles can induce defense response in surrounding unharmed plants.French marigold (Tagetes patula L.) essential oil (EO) was isolated from the above-ground partsof plants collected from nature by hydrodistillation using Clevenger apparatus. GC/MS analysisrevealed the presence of 42 compounds, of which 97% (32 compounds) have been identified.The monoterpene fraction was dominant (76.9%) with terpinolene (32.4%) and limonene (14.7%)as the most abundant compounds. In order to investigate the potential of marigold EO to inducethe defense of potato, we analyzed 3 genes related to the induced resistance (IR): pathogenesisrelated PR-2 and PR-5 genes, and proteinase inhibitor Pin2. The potato plants were exposed to EOvolatiles during 4, 8 and 12 h. Two leaves of EO-treated or untreated control plants were collected,and plants were then additionally injured by mechanically wounding leaves across the leaf surface.After 24 h of wounding, another two leaves from each plant were collected and expressionof the IR related genes was analyzed by qPCR method. EO induced altered expression of PR genes,while Pin2 wasn’t affected by the treatment. Mechanical wounding increased the expression ofall genes after 4 and 8 h of EO treatment, with up to 4-fold change in PR-2. However, in plantsexposed to EO for 12 h before wounding, the level of transcription in PR-2 and Pin2 was similar tothat detected in untreated controls. These findings support the efforts for environmentally-safepest control in crops by using natural compounds.Uzelac B, editor. Book of abstracts. 3rd International Conference on Plant Biology (22nd SPPS Meeting); 2018 Jun 9-12; Belgrade, Serbia. Belgrade: Serbian Plant Physiology Society; Institute for Biological Research "Siniša Stanković"; Faculty of Biology; 2018. p. 58

Supplementary data for the article: Stupar, S.; Dragićević, M.; Tešević, V.; Stanković-Jeremić, J.; Maksimović, V.; Ćosić, T.; Devrnja, N.; Tubić, L.; Cingel, A.; Vinterhalter, B.; Ninković, S.; Savić, J. Transcriptome Profiling of the Potato Exposed to French Marigold Essential Oil with a Special Emphasis on Leaf Starch Metabolism and Defense against Colorado Potato Beetle. Plants 2021, 10 (1), 172. https://doi.org/10.3390/plants10010172.

Supplementary material for: [https://doi.org/10.3390/plants10010172]Related to published version: [https://cherry.chem.bg.ac.rs/handle/123456789/4499

Pollen morphology, viability, and germination of Prunus domestica cv. Pozegaca

The pollen morphology and exine ornamentation of an important autochthon plum cultivar (Prunus domestica cv. Pozegaca L), which has horticultural, medicinal and alimentary uses, was examined in detail using both light and scanning electron microscope (SEM). Acetocarmine and fluorescein diacetate were used to assess the pollen viability of four Pozegaca plum genotypes. Pollen viability varied from 67% in genotype Pdp1 to 99% in genotype Pdp4. Also, pollen nucleus status was determined, and binucleate mature pollens were detected. The effect of polyethylene glycol-PEG (10, 15 and 20%, w/v) on pollen germination and tube growth was evaluated. Overall, the inclusion of PEG in the medium improved both pollen germination and tube growth. Regardless of the PEG concentration genotype has an effect on germination and length of pollen tubes. Genotype has a pronounced effect on germination and length of pollen tubes, regardless of the PEG concentration used. Genotype Pdp4 had the highest germination percentage and the highest pollen tube length on all media when compared to the other three genotypes. The highest pollen germination (96%) and a tube length (822 mu m) of genotype PdP4 was attained in media with 20% PEG

Evaluation of tansy essential oil as a potential "green" alternative for gypsy moth control.

The development of "green" alternatives to chemical pesticides could play a crucial role in integrated pest management (IPM). Their use is considered either as a substitution for or in addition to hazardous synthetic products. We analysed the influence of three concentrations of tansy (Tanacetum vulgare L.) essential oil (EO), previously characterised by GC-MS, on the survival and moulting of the 2nd instar and the nutritional indices of the 4th instar gypsy moth (Lymantria dispar L.) larvae. In a residual contact toxicity assessment, the exposure to tansy EO caused low mortality (< 10%) while larval development was significantly slowed down, i.e., the percentage of larvae that moulted into the 3rd instar was reduced. On the other hand, when tansy EO was incorporated into the diet (digestive toxicity assay), high mortality and a lack of moulting after 120 h of eating were recorded for the highest applied concentration of EO. During 48 h of feeding on EO-supplemented food at concentrations of 0.5 and 1% (v/v), the relative growth rate (RGR) of the 4th instar larvae significantly decreased, which can be explained by a significant reduction of the relative consumption rate (RCR) and significantly or marginally significantly lower efficiency of conversion of ingested food into insect biomass (ECI). Although the RCR was also reduced with the lowest applied EO concentration (0.1%), the ECI was not affected which meant the RGR was as high as it was for the control larvae. ECI changes, when two higher EO concentrations were applied, were due to a reduction in the efficiency of conversion of digested food into biomass (ECD), while approximate digestibility was unaffected by the presence of EO in the food. Our results on the significant negative effects of tansy EO on gypsy moth larval survival, development time, and nutritional physiology suggest that it could be considered in future designs for botanical insecticides for gypsy moth control

Pollen morphology, viability, and germination of Prunus domestica cv. Pozegaca

The pollen morphology and exine ornamentation of an important autochthon plum cultivar (Prunus domestica cv. Pozegaca L), which has horticultural, medicinal and alimentary uses, was examined in detail using both light and scanning electron microscope (SEM). Acetocarmine and fluorescein diacetate were used to assess the pollen viability of four Pozegaca plum genotypes. Pollen viability varied from 67% in genotype Pdp1 to 99% in genotype Pdp4. Also, pollen nucleus status was determined, and binucleate mature pollens were detected. The effect of polyethylene glycol-PEG (10, 15 and 20%, w/v) on pollen germination and tube growth was evaluated. Overall, the inclusion of PEG in the medium improved both pollen germination and tube growth. Regardless of the PEG concentration genotype has an effect on germination and length of pollen tubes. Genotype has a pronounced effect on germination and length of pollen tubes, regardless of the PEG concentration used. Genotype Pdp4 had the highest germination percentage and the highest pollen tube length on all media when compared to the other three genotypes. The highest pollen germination (96%) and a tube length (822 mu m) of genotype PdP4 was attained in media with 20% PEG. (C) 2013 Elsevier B.V. All rights reserved.Ministry of Education, Science and Technological Development of the Republic of Serbia [173015

Pollen morphology, viability, and germination of Prunus domestica cv. Pozegaca

The pollen morphology and exine ornamentation of an important autochthon plum cultivar (Prunus domestica cv. Pozegaca L), which has horticultural, medicinal and alimentary uses, was examined in detail using both light and scanning electron microscope (SEM). Acetocarmine and fluorescein diacetate were used to assess the pollen viability of four Pozegaca plum genotypes. Pollen viability varied from 67% in genotype Pdp1 to 99% in genotype Pdp4. Also, pollen nucleus status was determined, and binucleate mature pollens were detected. The effect of polyethylene glycol-PEG (10, 15 and 20%, w/v) on pollen germination and tube growth was evaluated. Overall, the inclusion of PEG in the medium improved both pollen germination and tube growth. Regardless of the PEG concentration genotype has an effect on germination and length of pollen tubes. Genotype has a pronounced effect on germination and length of pollen tubes, regardless of the PEG concentration used. Genotype Pdp4 had the highest germination percentage and the highest pollen tube length on all media when compared to the other three genotypes. The highest pollen germination (96%) and a tube length (822 mu m) of genotype PdP4 was attained in media with 20% PEG. (C) 2013 Elsevier B.V. All rights reserved.Ministry of Education, Science and Technological Development of the Republic of Serbia [173015