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7 Pags.- 2 Figs.- 2 Tabls. The definitive version is available at: http://www.actahort.org/index.htm ; Pistachio grafting often shows a high degree of uncertainty, with variable and inconsistent results with commonly used rootstocks, producing irregular orchards and frequent regrafting. Besides, the size of the buds of pistachio cultivars far exceeds that of the juvenile rootstock terebinth (Pistacia terebinthus L.), widely used in Spain, making pistachio grafting more difficult. Here, we have applied in vitro techniques to obtain reduced-size cultivars that match to the rootstock size. By reducing bud/scion of pistachio cultivars we can thus graft them on terebinth seedlings few weeks after germination. The female cultivars 'Larnaka', 'Kerman' and 'Sirora', as well as the male cultivar 'Peters' and the selection AD15 have been successfully cultivated in vitro. Grafting was performed on juvenile terebinth plants grown in small containers using a) micropropagated and acclimatized pistachio plants or b) in vitro grown shoot-tips as scion source. We have got up to 68% successful grafts with scions derived from in vitro techniques. Scions resumed growth few days after grafting, and time required to obtain new plants was significantly shortened. ; This work was partly funded by the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria-FEDER RTA2014-00056-C02 Project and by Group A-43 Excellence grant by the Government of Aragon. ; Peer reviewed

In vitro production of naturally-occurring medicinally-important secondary metabolites from plants using callus or cell suspension culture has become an industrially promising project. In vitro production of secondary metabolites has many advantages: i) year-round availability of plant material for the production of functional phytomolecules; ii) better avenues for processing and isolation; iii) the possibility of accentuation of chemical reactions leading to other useful secondary metabolites under in vitro conditions; and iv) elimination of potential political and geographical boundaries against crop production. (-)-Colchicine is one of the most important and well-studied natural compounds. It occurs mainly in plant species belonging to the genus Colchicum. (-)-Colchicine is still in use today as a pharmaceutical agent and as a laboratory tool. Because of the difficulties in seed germination, young corms are used in the propagation of different Colchicum species. (-)-Colchicine was produced via tissue culture from calluses and cell suspensions of different Colchicum and Gloriosa species.

Open Access Article ; Banana Bunchy Top Disease (BBTD) caused by the Banana Bunchy Top Virus (BBTV) is one of the most important banana diseases in the Democratic Republic of Congo. This study focused on the production of BBTV-free plantain seedlings from infected banana plants. A total of 10 suckers from the French plantain Litete (Musa AAB) and the False Horn plantain Libanga Likale (Musa AAB) with advanced BBTD symptoms were collected. Meristematic apices excised from those suckers were cultured in vitro and subcultured five times. The presence of BBTV was evaluated by the Triple-Antibody Sandwich Enzyme-linked Immunosorbent Assay (TAS-ELISA). The BBTV was confirmed in all suckers prior to in vitro culture but 73.3% of Litete plantlets and 66.6% of Libanga Likale plantlets regenerated from meristematic tissues were virus-free. This indicates that in vitro culture is a simple tool to generate BBTV-free plantains. ; Peer Review

It was established that for in vitro rhyzogenesis of apple rootstock 106-13 it is better to use medium with 1/2 MS macro- and microsalts supplemented with IBA in concentration of 0.5 mg/l without use of etiolation (the yield of rooted microplants was 90.0 % with a coefficient of root system development – 1.14 ± 0.18). For in vitro rhyzogenesis of apple rootstock 54-118, it is better to use medium with 1/3 MS macro and microsalts, supplemented with IBA in concentration of 1.0 mg/l without etiolation (the yield of rooted microplants was 85.35 % with a coefficient of root system development – 0.53 ± 0.12). The positive effect of etiolation on root system growth depended on cultivar. For rootstock 54-118, the use of etiolation stimulated the growth of roots on medium with 1/3 MS macro- and microsalts combined with IBA in concentration of 1.0 mg/l (until the end of subculture). Use of etiolation inhibited the root system growth of rootstock 106-13 microplants until the end of subculture both on medium with 1/2 MS macro- and microsalts and on medium with 1/3 MS macro- and microsalts, but it stimulated root formation of rootstock 106-13. However, use of 7 days etiolation at stage of in vitro rhyzogenesis of rootstocks 106-13 and 54-118 is not expedient, because it does not stimulate the increase in the number of rooted microplants at the end of subculture.

Rasva- ja hammaskudos ovat mielenkiintoisia ja runsaita kantasolujen lähteitä, joilla on terapeuttisia sovellusmahdollisuuksia lääketieteen eri osa-alueilla kudosvaurioiden korjauksessa. Mesenkymaalisia kantasoluja esiintyy monissa eri kudoksissa, kuten esimerkiksi luuytimessä, rasvakudoksessa ja hampaan kudoksissa. Näillä kantasoluilla on kyky uusiutua kantasoluina ja erilaistua useiksi eri solutyypeiksi kudoksen signaalien ohjaamina. Toisin kuin luuytimen kantasolut, joiden esiintymistiheys luuytimessä on alhainen, rasvakudoksen kantasoluja saadaan eristettyä suurissa määrin joko rasvaimunäytteestä tai rasvakudosnäytteestä ja niitä voidaan vaivattomasti lisätä laboratoriossa. Myös hampaan kantasolupopulaatioita saadaan eristettyä runsaasti viisaudenhampaasta. Lisäksi viisaudenhampaat ovat ihmiselle useimmiten tarpeettomia, ja ne voidaan poistaa suhteellisen kivuttomasti. Rasvakudoksen ja hampaan kantasoluilla on samantyyppisiä ominaisuuksia kuin luuytimen kantasoluilla; ne erilaistuvat laboratorio-olosuhteissa vähintään luusolujen, rustosolujen ja rasvasolujen suuntaan. Lisäksi rasvakudoksen kantasolujen on todettu alentavan immunologista vastetta, estävän vakavaa käänteishyljintäreaktiota solu- ja eläinmalleissa, ja niiden on todettu olevan geneettisesti vakaampia pitkäaikaisviljelyssä kuin luuytimen kantasolujen. Ennen kuin soluja voidaan hyödyntää kliinisissä kantasoluhoidoissa, niitä täytyy lisätä laboratoriossa. Tällä hetkellä yleisesti käytössä olevat soluviljelyreagenssit sisältävät eläinperäisiä ainesosia, joista turvallisuussyistä on päästävä eroon, sillä ne voivat kliinisessä käytössä aiheuttaa vakavan immuunivasteen soluterapiapotilaassa. Muihin eläinperäisten ainesosien tuomiin riskeihin kuuluu viruksien ja bakteerien aiheuttamat infektiot, prionit ja toistaiseksi tunnistamattomat eläintaudit. Tässä työssä tutkittiin aikuisten eri kudoksista peräisin olevien kantasolujen uusiutumis- ja erilaistumiskykyä laboratorio-olosuhteissa. Tutkimuksissa käytettiin viisaudenhampaan kantasolupopulaatioita, kuten hammaspulpan, -follikkelin ja juurikalvon kantasoluja sekä rasvakudoksen kantasoluja. Lisäksi rasvakudoksen kantasolujen kasvatusolosuhteita kehitettiin vastaamaan Euroopan unionin edellyttämien hyvien tuotantotapojen (eng. Good Manufacturing Practice, GMP) vaatimuksia. Viimeisessä osatyössä arvioitiin rasvakudoksen kantasolujen kliinistä soveltuvuutta kokeellisessa kudosteknologisessa luunkudoksen rekonstruktiossa. Tulokset osoittivat hampaan kantasolupopulaatioilla olevan mesenkymaalisille kantasoluille ominaisia piirteitä ja kyky erilaistua rusto- ja luusolujen suuntaan. Siten ne soveltuisivat käytettäviksi kovakudossovelluksissa kallon ja kasvojen alueella. Rasvakudoksen kantasolut ilmensivät eri kasvatusolosuhteissa mesenkymaalisille kantasoluille tyypillisiä pintamolekyylejä ja ne voitiin erilaistaa luu-, rusto- ja rasvasolujen suuntaan. Yhtäläisyyksistä huolimatta eri kasvatusolosuhteissa viljellyissä rasvakudoksen kantasoluissa esiintyi olennaisia eroja solusyklin etenemisen ja solujen erilaistumisen keskeisten geenien ilmentymisessä. Rasvakudoksen kantasoluja hyödyntäviä kliinisiä soluterapiakokeita on paraikaa käynnissä, mikä edellyttää keskittymistä laboratorio-olosuhteissa lisättyjen solujen turvallisuuteen, menetelmien toistettavuuteen ja laatuun. Tämä työ osoittaa, että eri kudoksista lähtöisin olevilla kantasoluilla on samantapaiset ominaispiirteet, ja niitä voidaan hyödyntää kantasoluterapiassa. Tulokset osoittavat myös kasvatusolosuhteilla olevan huomattava vaikutus soluihin geenitasolla; muokkaamalla kasvatusolosuhteita voidaan vaikuttaa kliiniseen tarkoitukseen tähtäävän solutuotteen laatuun. Lisäksi solukasvatusolosuhteiden huolellinen arviointi erilaisilla, kuitenkin tarkoin määrätyillä analyysimenetelmillä on ensiarvoisen tärkeää, jotta solujen soveltuvuus voidaan määrittää. Täydentäviä prekliinisiä turvallisuus- ja tehokkuustutkimuksia tarvitaan vielä ennen kuin aikuisten kantasolujen kaikki hyödyntämismahdollisuudet voidaan toteuttaa. ; Adipose and dental tissues are attractive and abundant sources of stem cells with therapeutic applicability in diverse medical fields for the repair and regeneration of acute and chronically damaged tissues. Mesenchymal stem cells (MSCs) are found in a variety of tissues including bone marrow, adipose tissue and dental tissues, and reside within the tissue in a hierarchical arrangement maintaining their proliferative potential and differentiation stage. Importantly, unlike the human bone marrow stromal/stem stem cells (BMSCs) that are present at low frequency in the bone marrow, adipose stem cells (ASCs) can be retrieved in high number from either liposuction aspirates or subcutaneous adipose tissue fragments and can easily be expanded in vitro. Also, the dental stem cell sources (DSCs) can easily be retrieved in high numbers from third molars representing an appealing source of MSCs owing to the fact that third molars are an expendable tissue that can be removed with minima! l surgical stress. ASCs and DSCs display properties similar to those observed in BMSCs and, upon induction, undergo at least osteogenic, chondrogenic and adipogenic differentiation in vitro. Furthermore, ASCs have been shown to be immunoprivileged, prevent severe graft-versus-host disease in vitro and in vivo and to be more genetically stable in long-term culture than BMSCs. They have also proven applicability in other functions, such as providing hematopoietic support and gene transfer. Nevertheless, expansion of ASCs and DSCs is necessary prior to performing clinical studies. Currently, standard in vitro cell expansion techniques utilize reagents of animal origin as part of the cultivation procedures. Due to critical safety issues, use of animal-derived reagents in clinical applications is not a suitable option, since severe immune response may develop in the recipient. Other possible risks for the recipient related to the introduction of animal-derived reagents include viral or bacterial infections, prions, and unidentified zoonoses. In this study, the renewal and differentiation capacity of stem cells from different adult tissues were evaluated in vitro. Stem cells from impacted third molars such as dental pulp stem cells, dental follicle cells and periodontal ligament stem cells, and also stem cells from adipose tissue were investigated. Furthermore, for ASCs, the culture conditions were developed and optimized to meet the GMP (Good Manufacturing Practice) standards set by the European Union. In the final part of the study, the clinical applicability of adipose stem cells was evaluated in a bone tissue engineering case study. The results show, that the cells from the dental pulp, follicle, and periodontal ligament exhibit mesenchymal stem cell-like characteristics and show capacities for chondrogenic and osteogenic differentiation thus suggesting usability in hard tissue engineering applications in the craniofacial area. Furthermore, albeit ASCs showed comparable surface marker expression profiles and multipotential differentiation capacity in different culture conditions demonstrating characteristics of mesenchymal stem cells, fundamental differences in genes central for cell cycle progression and cell differentiation were detected. Clinical stem cell therapy studies using ASCs are under way, which call for a strong focus on the safety, reproducibility and quality of transplanted in vitro expanded stem cells. The results from the study suggests that cells from various adult tissue sources may be utilized for clinical cell therapy, but that the choice of culturing supplements has considerable effects on the cells at gene level. Thus, the culturing conditions may have significant implications on the cell product aimed for clinical therapy. The results show that proper characterization of culture conditions with different, yet specific, analyzing methods is necessary for cells intended for clinical stem cell therapy. Nevertheless, additional pre-clinical safety and efficacy studies will be needed before the promise of the adult stem cells can be fully realized.

In vitro culture emerges as a sustainable way to produce bioactives for further applicability in the food industry. Herein, vegetative parts of Fragaria vesca L. (wild strawberry) obtained by in vitro culture were analyzed regarding nutritional and phytochemical compounds, as well as antioxidant activity. These samples proved to have higher content of protein, polyunsaturated fatty acids, soluble sugars, organic acids (including ascorbic acid) and tocopherols (mainly a-tocopherol) than wild grown F. vesca, as well as containing additional phenolic compounds. The antioxidant activity of hydromethanolic extracts could be correlated with the content of different phenolic groups and other compounds (sugars and organic acids). It was demonstrated that in vitro culture could enhance nutritional and bioactive compounds of Fragaria vesca L. plants, providing a very interesting biotechnological tool for potential food applications. ; The authors are grateful to Fundação para a Ciência e a Tecnologia (FCT, Portugal) for financial support to CIMO (strategic project UID/AGR/00690/2013) and to REQUIMTE (national funds and co-financed by FEDER, under the Partnership Agreement PT2020) and to POCI-01-0145-FEDER-006984 (LA LSRE-LCM) funded by ERDF through POCI-COMPETE2020 and FCT. L. Barros and M.I. Dias thank FCT for their grants (SFRH/BPD/107855/2015 and SFRH/BD/84485/2012, respectively). The GIP-USAL is financially supported by the Spanish Government through the project AGL2015-64522-C2-2-R. ; info:eu-repo/semantics/publishedVersion

Crude extract of the heartwood of Auxemma oncocalyx (A. oncocalyx) and its main component i.e., Oncocalyxone A (onco A), have elevated antioxidant and anti-tumoral activity, but studies on the action of these drugs regarding folliculogenesis are lacking. The aim of this study was to evaluate the effect of A. oncocalyx and onco A on the in vitro culture of isolated secondary follicles and on the in vitro maturation of oocytes from caprine antral follicles grown in vivo. Isolated secondary follicles were randomly distributed in six groups; the non-cultured control was immediately fixed upon isolation. The remaining follicles were cultured for 7 days in ?-MEM+ alone (control) or supplemented with DMSO, doxorrubicin, A. oncocalyx or onco A. After culture, follicles were evaluated for antrum formation, growth rate, apoptosis (TUNEL) and cellular proliferation (PCNA), as well as gene expression of Bcl2 and Bax. Additionally, cumulus oocyte complexes (COCs) were aspirated and allocated into five treatments for in vitro maturation: control, cultured only in maturation base medium (TCM 199+); or supplemented with DMSO; DXR; A. oncocalyx or onco A. After in vitro maturation, oocyte chromatin configuration and viability were assessed. After 7 days of culture, there was a reduction (P < 0.05) in the percentage of morphologically intact follicles, antrum formation, growth rate and number of PCNA positive granulosa cells in DXR treatment compared to the other treatments. In the DXR treatment a higher percentage (P < 0.05) of TUNEL positive follicles and higher (P < 0.05) relative BAX:BCL2 mRNA ratio's were observed. After in vitro maturation of the COCs DXR, A. oncocalyx and onco A treatments had a greater (P < 0.05) percentage of abnormal oocytes and a lower (P < 0.05) percentage of viable oocytes as compared with the control group. However, only DXR and onco A treatments increased (P < 0.05) the percentage of alive oocytes with abnormal chromatin configuration. There were no differences in maturation rates between the control group and DXR, A. oncocalyx and onco A treatments. In conclusion, under our culture conditions, A. oncocalyx and onco A do not exhibit a toxic effect on isolated secondary follicles and on maturation rates of COCs recovered from antral follicles, however, these drugs negatively affected the COCs viability. Thus, the use of culture biotechnologies as an in vitro secondary follicle culture and in vitro oocyte maturation toxicity testing are appropriated methods to evaluate the possible effects of drugs in folliculogesis.

Soil salinization is an important abiotic factor negatively affecting plant growth, development and productivity. Fast-growing poplar and willow trees are important plants for bioenergy production demonstrating varying degrees of adaptation to different habitats. The study of salt resistance in different clones of poplars and willows will reveal genotypes that can be planted in saline soils for producing biomass for the bioenergy industry. Therefore, the aim of the study was to investigate the effects of salt stress on poplar plants of clone 'INRA 353-38' (Populus tremula × P. tremuloides) and willow clone 'Zhytomyrska – 1' (Salix sp.) under in vitro culture. For this purpose the plants were cultivated on MS nutrient medium with the addition of sodium chloride in concentrations 25 mM, 50 mM and 100 mM. The control plants were grown on the sodium chloridefree medium. The plant status (with a 4-score scale), the intensity of their growth (by shoot length) and rooting capacity (by the number of roots) were assessed on the 10th and the 30th day of cultivation. The results obtained indicate a high level of sensitivity to sodium chloride of both studied clones under in vitro cultivation. But the willow 'Zhytomyrska – 1' had a higher sensitivity to salt stress comparing to hybrid polar 'ІNRA 353-38' since growth parameters of willow were significantly decreased even under the concentration of sodium chloride 50 mM, and in the case of short term influence (10 days) of the highest concentration of sodium chloride (100 mM) all willow plants terminated their growth and quickly died. The growth parameters of hybrid poplar were declined within a month, mainly under the highest concentration of sodium chloride, but even under such conditions some part of the shoots were able to survive.

This study was conducted to evaluate the effects of the acidified extract of M. oleifera leaves as a supplement into the base medium for in vitro culture of sheep isolated secondary follicles. Follicles were isolated and cultured for 12 days in α-MEM+(supplemented with bovine serum albumin, insulin, glutamine, hypoxanthine, transferrin, selenium, and ascorbic acid) with or without 0.1; 0.2 or 0.4 mg/ml of the acidified extract of M. oleifera. Follicle morphology, antral cavity formation, follicular and oocyte diameter, glutathione (GSH) concentration, mitochondrial activity and meiotic resumption were evaluated. After 12 days of culture, there was no significant difference among treatments in relation to follicular morphology, antral cavity formation, diameter and mitochondrial activity. Nevertheless, oocytes from follicles cultured in α-MEM+ showed greater GSH concentration than media containing M. oleifera extract. Furthermore, the concentration of 0.4 mg/ml M. oleifera extract significantly increased the percentage of fully grown oocyte (≥ 110 µm) when compared to the other treatments. In conclusion, the concentration of 0.4 mg/ml M. oleifera extract as a supplement of the culture medium, maintained the survival, and increased the percentage of fully grown oocytes.

The article contains the results of study of the influence of added to culture medium silver-containing chitosan- based nanocomposites (Chitosan-Ag) at a dilution of 1:500 and 1:1000 (the mass ratio of the components is 50:1 for Chit- Ag 50:1 and 100:1 for Chit-Ag 100:1) on the development of potato microshoots and microclones with a formed root system. Potato microshoots cultivated for 4 weeks on nutrient medium modified with nanocomposites were characterized by slow development and the absence of rhizogenesis, which indicates the toxic effect of the studied nanocomposite concentrations. When replacing the standard nutrient medium with nanocomposites modified for a potato microclone with developed roots, the Chit-Ag 50:1 reduced the rate of growth and development of microclones compared to control and pure chitosan. The Chit- Ag 100:1 nanocomposite had no influence on the microclone growth compared to the control, but reduced the root biomass compared to chitosan. The preservation of photosynthetic pigments and proline concentrations with decreasing the hydrogen peroxide level indicates the absence of the toxic effect of silver-containing chitosan-based nanocomposites on the formed potato microclones. The chitosan concentration increase in the nanocomposite composition helps us to reduce the toxic effect due to the formation of a dense stabilizing shell that delays the silver ion generation.

AbstractAs a proinflammatory cytokine, tumor necrosis factor-α (TNF-α) is central to the female reproductive tract and affects various phases of follicular development and uterine cycles. High levels of TNF-α play a vital role in the pathogenesis of polycystic ovary syndrome (PCOS) in patients. Clinicians know that dexamethasone can inhibit the induction of androgen by suppressing the adrenal glands which improves the status of the endocrine system in PCOS patients. We hypothesize that dexamethasone has much more functionality and can exert a therapeutic effect by antagonizing TNF-α. We added TNF-α to the follicular culture medium to simulate the high TNF-α levels observed in the endocrine environment of PCOS patients. Dexamethasone was added to the medium to determine if it could counteract the inhibitory effect of TNF-α on follicular growth and 17β-estradiol (E2) secretion. Follicular diameter, E2 concentration, follicle survival, antral-like cavity formation, and ovulation were measured to assess the effects of dexamethasone. In our work, TNF-α inhibited in vitro follicular growth and E2 secretion in a dose-dependent manner. Based on the results of the present research, we concluded that the addition of dexamethasone partially counteracts the repressive effect of TNF-α on follicle growth and E2 secretion during in vitro culture of the preantral follicles of mice. Thus, the findings in this paper suggest that dexamethasone may act as a therapy by counteracting the effects of TNF-α in PCOS patients. These results provide a new foundation for exploring the treatment of PCOS patients with dexamethasone.