Explants In Medium Research Articles

Study on Tissue Culture and Rapid Propagation Techniques of Different Explants of Codonopsis convolvulacea Kurz

The tissue culture technology of Dioscorea bulbifera L. is of great significance for the effective protection and utilization of its germplasm resources. This paper reviews the research progress of tissue culture technology of Dioscorea bulbifera L. with different explants. It introduces the historical development of tissue culture technology of Dioscorea bulbifera L., including early exploration and gradual improvement of modern technology. The paper elaborates various methods and strategies of tissue culture technology of Dioscorea bulbifera L. at present, including research progress in aspects such as explant selection, medium formulation, and application of growth regulators. It also looks forward to the future development direction and potential applications of tissue culture technology of Dioscorea bulbifera L., and discusses its application prospects in germplasm resource preservation, new variety breeding, and improvement of disease and pest resistance. This paper is of important theoretical and practical significance for promoting the research and application of tissue culture technology of Dioscorea bulbifera L.

Human placenta releases extracellular vesicles carrying corticotrophin releasing hormone mRNA into the maternal blood

The human placenta releases diverse extracellular vesicles (EVs), including microvesicles (100–1000 nm) and exosomes (30–150 nm), into the maternal blood for feto-maternal communication. Exosomes and microvesicles contribute to normal pregnancy physiology and major pregnancy pathologies. Differences in miRNA expressions and protein content in placental exosomes have been reported in complicated pregnancies. During human pregnancy, Corticotropin-Releasing Hormone (CRH) is produced and released by the placenta into the maternal blood. CRH is involved in regulating gestational length and the initiation of labour. CRH mRNA levels in the maternal plasma rise with gestation. High levels of CRH mRNA are reported to be associated with preeclamptic and preterm pregnancies. However, the underlying mechanism of placental CRH mRNA secretion remains to be elucidated. We hypothesise that the placenta releases CRH mRNA packaged within extracellular vesicles (EVs) into the maternal blood. In this study, placental EVs (microvesicles and exosomes) were isolated from human term healthy placentas via villus washes and from explant culture media by differential centrifugation and purified by density gradient ultracentrifugation using a continuous sucrose gradient (0.25–2.5 M). Western blotting using placenta- and exosome-specific markers and electron microscopy confirmed exosomes and microvesicles in the placental wash and explant media samples. Real-time quantitative RT-PCR data detected CRH mRNA in placenta-derived EVs from placental washes and explants. We also sorted placenta-secreted EVs in maternal plasma samples (≥37 weeks) by high-resolution flow cytometry using a fluorescent-labelled PLAP antibody. CRH mRNA was demonstrated in placental EVs obtained from maternal blood plasma. We therefore show that human placental EVs carry CRH mRNA into the maternal blood. Our study implies that measuring CRH mRNA in placental EVs in the maternal plasma could beused for monitoring pregnancy.

Development of individual elements of a protocol for sustainable growth and propagation of garden strawberries (Fragaria ananassa Duch.) under aseptic conditions

Garden strawberry (Fragaria ananassa Duch.) is one of the most valuable fruits the demand for which in the food market is consistently high. One of the limiting factors for achieving consistently high-quality strawberry yields is the presence of diseases caused by bacteria, phytoplasmas, viruses, and viroids. In order to intensify the technology of garden strawberries cultivation, the problem of production in significant volumes of genetically constant material free from pathogens is essential. Biotechnological methods are currently relevant technologies that allow mass production of planting material with high phytosanitary and genetic quality. The purpose of the study is to update the protocol for microclonal propagation of garden strawberries to obtain virus-free planting material. The research was conducted in the micropropagation laboratory of LLC «Blahodatne» (ТеvittaTM) Cherkasy region, Ukraine using the «Alba» and «Present» strawberry cultivars. A series of experiments were conducted according to the «step by step» principle on two types of explants: buds and meristems. The determinants for obtaining aseptic cultures from bud and meristem explants were investigated. The trophic influence was studied in media with different mineral content (at the multiplication stage) and sucrose concentrations during rhizogenesis. Among the phytohormonal determinants during the multiplication stage, the best combination among those investigated was the use of substances with cytokinin activity consisting of BAP at 0.2 mg/l and kinetin at 0.8 mg/l. The addition of 0.1 ml/l of «Gibb plus preparation» (GK4 + GK7) was effective for the reproduction rate increasing. Growing of donor explants in media with BAP at 0.2 mg/l, kinetin at 0.3 mg/l, and adenine at 0.5 mg/l, compared to the control (BAP at 1.0 mg/l) improved rhizogenesis in regenerants. The highest root formation rates were observed in the variant with 4 % of sucrose (40 g/l). Key words: propagation; microclonal propagation; aseptic culture; trophic and hormonal determination.

Rosmarinic Acid Production from Origanum dictamnus L. Root Liquid Cultures In Vitro.

In the present work Origanum dictamnus L. was studied as a suitable in vitro adventitious root culture system for the production of important bioactive molecules, such as rosmarinic acid (RA). Callus culture was initiated from leaf, petiole and root explants on solid MS medium supplemented with either 5 μM NAA + 5 μM kinetin (ODK3) or 5 μM NAA + 0.5 μM kinetin (ODK4). New roots formed from leaf, petiole and root calluses were aseptically transferred into Erlenmeyer flasks containing 100 mL liquid medium and shaken at 120 rpm in the dark. The liquid medium used was the MS supplemented either with 35 μM IBA + 2.5 μM kinetin (ODY1) or 5 μM NAA + 0.5 μM kinetin (ODY2). Biomass production parameters, RA content (%) and yield index (YI) were recorded for each treatment explant type, medium composition and incubation period. Results showed, in every case, the production of RA in vitro. Between the two liquid media (ODY1, ODY2) and the different culture periods, the ODY1 medium and the longest 200-day-culture period were more effective for RA and biomass production, regardless of the initial explant type used. The combination of ODK4-ODY1 resulted in higher RA (5.1% and 4.7%), fresh biomass production (19.0 g and 11.6 g), mean YI (93.7 mg and 51.4 mg) and YI per explant (3.75 mg and 2.06 mg) for roots derived from leaf calluses and root calluses, respectively. However, the solid ODK3 (200 days)-liquid ODY1 (40 days) transition treatment was more beneficial for roots derived from petiole calluses leading to an 18.8-fold increase in fresh biomass growth rate. RA accumulation and YIs were also significantly influenced by explant type, with the highest value produced from root petiole calluses (6.6% RA dry weight, 115.3 mg mean YI and 4.61 mg YI per explant) after 240 days.

In Vitro Floral Emergence and Improved Formation of Saffron Daughter Corms

In vitro cormogenesis is a potential tool for improving saffron production under controlled conditions. In this study, the effects of explant type, culture type, and medium supplements on saffron daughter corm formation in vitro were assessed. Saffron flowers emerged 30 days after culture, and the sizes of in-vitro- and ex-vitro-produced flowers and stigmas were similar. In vitro daughter corm formation and the saffron life cycle was completed after 10 and 14 weeks of culture, respectively. Using in vitro intact corms was more effective for corm production than using apical buds. Compared with apical bud explants, mother corm explants produced more corms with a higher fresh weight and diameter. Compared with solid culture, liquid cultures using bioreactors provided corms with a higher fresh weight and diameter, regardless of explant type. An ebb and flow system provided the highest cormlet fresh weight and diameter but the fewest cormlets, whereas an immersion system provided more cormlets with a smaller size. Saffron apical buds cultured with salicylic acid at 75 mg L−1 or glutamine at 600 mg L−1 exhibited the highest cormlet diameter and fresh weight. These findings will improve the process of in vitro cormogenesis and the production of saffron under controlled conditions.

Clodronate disodium is neither cytotoxic nor cytoprotective to normal and recombinant equine interleukin-1β-treated joint tissues in vitro.

To determine the effects of clodronate disodium (CLO) on control and recombinant equine interleukin-1β (IL-1β)-treated equine joint tissues. In vitro experimental study. Cartilage explants, chondrocytes, and synoviocytes (n=3 horses). Monolayer cultures of chondrocytes and synoviocytes from three horses were subjected to: control media (CON), 5ng/ml CLO (C/low), 50 ng/ml CLO (C/med), 100 ng/ml CLO (C/high), with and without IL-1β, and 10ng/ml IL-1β (IL) alone for 72 hours. Cartilage explants from three horses were subjected to CON, IL, C/low, and C/med with and without IL-1β for 72 hours. Culture media was analyzed for prostaglandin-E2 (PGE2 ), interleukin-6 (IL-6), and nitric oxide (NO). Explant media was analyzed for glycosaminoglycan (GAG) content and NO. At 72 hours, explant and monolayer culture viability were assessed, and explant GAG content was measured. IL-1β treatment resulted in higher media concentrations of GAG, NO, PGE2 , and IL-6 compared to the CON treatment (p < .05), demonstrating a catabolic effect of IL-1β on explants and monolayer cultures. CLO treatments did not increase media concentrations of GAG, NO, PGE2 , or IL-6 compared to CON, indicating no cytotoxic effect. Nevertheless, CLO treatments administered to IL-1β-treated monolayer cultures and explants did not significantly reduce the inflammatory response regardless of concentration. CLO did not demonstrate cytotoxic nor cytoprotective effects in normal and IL-1β-stimulated chondrocytes, synoviocytes or explants in culture. This study does not support the use of CLO as an anti-inflammatory treatment. Further research is necessary to confirm any anti-inflammatory effects of CLO on joint tissues.

Effect of explant type, culture medium, and BAP concentration on in vitro shoot development in highbush blueberry (Vaccinium corymbosum L.) cultivars

Effect of explant type, culture medium, and BAP concentration on in vitro shoot development in highbush blueberry (Vaccinium corymbosum L.) cultivars

Effect of Explant Origin and Medium Plant Growth Regulators on In Vitro Shoot Proliferation and Rooting of Salvia tomentosa, a Native Sage of the Northeastern Mediterranean Basin

Salvia tomentosa is a perennial sage of the maquis vegetation, strongly aromatic, with medicinal and ornamental value. Aiming to facilitate the promotion of the species in the floriculture sector, its micropropagation was studied in terms of the effect of explant origin and medium plant growth regulators on in vitro cultures establishment, shoot proliferation and rooting. Shoot tip or single node explants excised from either in vitro grown seedlings or greenhouse plants grown from cuttings of mature plants were cultured on MS medium supplemented with 6-benzyladenine (BA) at concentrations 0.0–3.2 mg L−1 in combination with 0.0, 0.01 and 0.1 mg L−1 1-naphthaleneacetic acid (NAA). Infections were the biggest problem for establishing cultures from greenhouse plants, while explants from in vitro seedlings showed hyperhydricity, especially the nodal ones. In the multiplication stage, high BA concentrations reduced explant response to produce shoots as well as the length of shoots produced, and although they increased shoot number per responding explant, a large number of shoots were hyperhydrated especially in explants of seedling origin. In general, the highest multiplication indexes, relevant to normal shoots, were given by the hormone-free medium or one with low BA concentration, and by shoot tip explants originated from greenhouse adult plants. The highest rooting rates (70–78%) were achieved either at full or half strength hormone-free or with 0.1 mg L−1 indole-3-butyric acid (IBA) medium. Increasing IBA resulted in an increase in root number, but a decrease in rooting rate and root length. Ex vitro acclimatization was highly successful (92–96%) in peat-perlite (1:1 v/v) substrate. The micropropagation protocol developed will contribute to the promotion of S. tomentosa in the floriculture and pharmaceutical industry and will be a tool for breeding programs targeting high value products.

Assessment of somaclonal variation occurrence in micropropagated Mansonia altissimia (A. Chev.) A. Chev. using molecular marker

Plant tissue culture that offers limited space and short time for clonal propagation of plant species can sometimes be discouraging if true to type clones are not generated. This study investigated direct shoot induction and genetic stability of Mansonia altissimia using molecular markers in order to ascertain that the in vitro propagated plantlets were true to type. Nodal cutting was used as explant, and Murashige and Skoog (MS) medium was supplemented with 6-benzyl aminopurine (BAP) (µM) + naphthaleneacetic acid (NAA) (µM), BAP (µM) + Gibberellic acid (GA3) (µM), Kinetin (KIN) (µM) + NAA (µM), KIN (µM) + GA3 (µM), thidiazuron (TDZ) (µM) + NAA (µM), TDZ (µM) + GA3 (µM), for direct micropropagation. PCR amplification of wild plant and direct micropropagation of Mansonia altissimia were done using 15 RAPD and 6 ISSR primers. The highest shoot height (4.77 cm) and number of leaves (5.33) were obtained from 2.5 µM BAP + 3.0 µM GA3. Moreover, the polymerase chain reaction amplification results showed 100% monomorphic and 1.0 level of similarity coefficients, which indicates that there is no occurrence of somaclonal variation.

Mechanism of anti-inflammatory effects of rifampicin in an ex vivo culture system of hidradenitis suppurativa.

Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease of the hair follicles leading to painful lesions, associated with increased levels of pro-inflammatory cytokines. Numerous guidelines recommend antibiotics like clindamycin and rifampicin in combination, as first-line systemic therapy in moderate-to-severe forms of inflammation. HS has been proposed to be mainly an auto-inflammatory disease associated with but not initially provoked by bacteria. Therefore, it has to be assumed that the pro-inflammatory milieu previously observed in HS skin is not solely dampened by the bacteriostatic inhibition of DNA-dependent RNA polymerase. To further clarify the mechanism of anti-inflammatory effects of rifampicin, ex vivo explants of lesional HS from 8 HS patients were treated with rifampicin, and its effect on cytokine production, immune cells as well as the expression of Toll-like receptor 2 (TLR2) were investigated. Analysis of cell culture medium of rifampicin-treated HS explants revealed an anti-inflammatory effect of rifampicin that significantly inhibiting interleukin (IL)-1β, IL-6, IL-8, IL-10 and tumour necrosis factor (TNF)-α production. Immunohistochemistry of the rifampicin-treated explants suggested a tendency for it to reduce the expression of TLR2 while not affecting the number of immune cells.

Preservation of Quercus Robur and Quercus Petraea Genetic Resources through In Vitro Culture

In this paper, the effect of various factors (clone, initial explant, sterilization method, and culture medium) on the in vitro multiplication of pedunculate and sessile oak species was tested. No influence of clone was found and a 44-83% survival rate was found for different types of explants, the best result being that of using mature acorns germinated in vitro. The most efficient sterilization method was: mercuric chloride 0.2 mg/l, ascorbic acid 1 mg/l, for 30 min., with or without antibiotic. The best results were obtained with GD medium complemented with 0.5 mg/l BAP and 0.1 mg/l IBA, which allowed obtaining of a high rooting percentage. The regenerated plants were transferred in vivo, in a climate controlled greenhouse.

Effects of orexin A on PTGS2, PTGES, CBR1 and PGFS mRNA transcript abundances and prostaglandin E2 and F2α concentrations in culture medium of pig uterine explants collected during early gestation and the estrogenic cycle

In this study, aims were to evaluate orexin A (OXA) effects on mRNA abundance of important enzymes involved in prostaglandin production, such as cyclooxygenase 2 (PTGS2), microsomal PGE2 synthase-1 (PTGES), PGF2α synthase (PGFS) and carbonyl reductase 1 (CBR1), as well as prostaglandin E2 (PGE2) and F2α (PGF2α) culture medium concentrations for endometrial and myometrial explants. Tissues were collected from gilts during specific phases of the estrogenic cycle or early gestational period. There were greater concentrations of PGE2 with OXA treatments of endometrial tissues collected on days 12–13 and 27–28, as well as PGF2α on days 10–11 of the gestational period. The PGF2α concentrations were less in tissues collected on days 27–28 of the gestational period. The OXA treatments resulted in lesser concentrations of PGE2 from myometrial tissues collected on days 10–11 and greater PGF2α on days 10–11 of the gestational period and 10–11 of the estrogenic cycle. Effects of OXA may occur due to actions at PTGS2, PTGES, PGFS and CBR1 genes because mRNA abundances for proteins encoded by these genes were affected by OXA. Results indicate there is an OXA effect on mRNA abundances and prostaglandin culture medium concentrations of uterine tissue collected at different stages of the gestational period or estrogenic cycle using different doses of OXA. It, therefore, is concluded OXA may affect de novo synthesis and secretion of PGE2 and PGF2α in the uterus of pigs.

Metal Akümülatör Bitki olan Brassica nigra L.’nin in vitro Kallus İndüksiyonu ve Sürgün Gelişimi

This study established an efficient in vitro callus formation and plant regeneration protocol for a prevıously reported Cu accumulator, Brassica nigra, black mustard collected from Diyarbakir (Station site). Node explants from 10-day old mature plants were used for callus formation and shoot regeneration. The highest callus formation frequency (100%) was observed on Murasige Skoog (MS) medium containing 0.1 mg/L Benzylaminopurine (BAP) + 0.5 mg/L Naphthylacetic acid (NAA) (MS 2), 0.6 mg/L BAP + 0.2 mg/L NAA (MS 7), the highest shoot regeneration frequency (100%) was achieved on MS medium containing 0.6 mg/L BAP + 0.05 mg/L Indole butyric acid (IBA) (MS 8), 0.2 mg/L IBA + 0.2 mg/L NAA (MS 10) and the highest number of shoots per explant (3,25) was obtained on MS medium supplemented 0.6 mg/L BAP + 0.05 mg/L IBA (MS 8). After root, stem, and leaf formation from explants in MS medium, these plants were transferred to soil and grown in the plant growth room for one month. A dependable and effective shoot regeneration procedure was developed, laying the groundwork for genetic transformation in Brassica nigra.

Reserpine improves Enterobacteriaceae resistance in chicken intestine via neuro-immunometabolic signaling and MEK1/2 activation

Salmonella enterica persist in the chicken gut by suppressing inflammatory responses via expansion of intestinal regulatory T cells (Tregs). In humans, T cell activation is controlled by neurochemical signaling in Tregs; however, whether similar neuroimmunological signaling occurs in chickens is currently unknown. In this study, we explore the role of the neuroimmunological axis in intestinal Salmonella resistance using the drug reserpine, which disrupts intracellular storage of catecholamines like norepinephrine. Following reserpine treatment, norepinephrine release was increased in both ceca explant media and Tregs. Similarly, Salmonella killing was greater in reserpine-treated explants, and oral reserpine treatment reduced the level of intestinal Salmonella Typhimurium and other Enterobacteriaceae in vivo. These antimicrobial responses were linked to an increase in antimicrobial peptide and IL-2 gene expression as well as a decrease in CTLA-4 gene expression. Globally, reserpine treatment led to phosphorylative changes in epidermal growth factor receptor (EGFR), mammalian target of rapamycin (mTOR), and the mitogen-associated protein kinase 2(MEK2). Exogenous norepinephrine treatment alone increased Salmonella resistance, and reserpine-induced antimicrobial responses were blocked using beta-adrenergic receptor inhibitors, suggesting norepinephrine signaling is crucial in this mechanism. Furthermore, EGF treatment reversed reserpine-induced antimicrobial responses, whereas mTOR inhibition increased antimicrobial activities, confirming the roles of metabolic signaling in these responses. Finally, MEK1/2 inhibition suppressed reserpine, norepinephrine, and mTOR-induced antimicrobial responses. Overall, this study demonstrates a central role for MEK1/2 activity in reserpine induced neuro-immunometabolic signaling and subsequent antimicrobial responses in the chicken intestine, providing a means of reducing bacterial colonization in chickens to improve food safety.

Plant Regeneration from Leaf Explants of the Medicinal Herb Wedelia chinensis

Wedelia chinensis, belonging to the Asteraceae family, has been used in folk medicine in East and South Asia for the treatment of common inflammatory diseases and protection against liver toxicity. Previously, in vitro propagation through different tissue explants has been reported, including through nodal segments, axillary buds, and shoot tips, whereas leaf segments failed to proliferate. Here, we report on the in vitro propagation of W. chinensis by culturing young leaf explants in MS medium supplemented with 0.5 mg/L α-naphthaleneacetic acid (NAA), 0.75 mg/L thidiazuron (TDZ), 1 mg/L gibberellic acid (GA3), 3.75 mg/L adenine, 3% sucrose, and 0.8% agar at pH 5.8. Calli were observed in all explants derived from the youngest top two leaves, and the average percentage of shoot regeneration was 23% from three independent experiments. Then, several shoots were excised, transferred onto MS basal medium supplemented with 3% sucrose and 0.8% agar at pH 5.8, and cultured in a growth chamber for 1 to 2 months. Roots were easily induced. Finally, plantlets carrying shoots and roots were transferred into soil, and all of them grew healthily in a greenhouse. No morphological variation was observed between the regenerated plantlets and the donor wild-type plants. In addition, we also established root cultures of W. chinensis in culture medium (MS medium, 3 mg/L NAA, 3% sucrose, pH 5.8) with or without 0.8% agar. To the best of our knowledge, this is the first paper reporting plant regeneration from leaf explants in the herbal plant W. chinensis.

Activation of the Adipose Tissue NLRP3 Inflammasome Pathway in Cancer Cachexia.

BackgroundCachexia is a paraneoplastic syndrome that accompanies and compromises cancer treatment, especially in advanced stages, affecting the metabolism and function of several organs. The adipose tissue is the first to respond to the presence of the tumor, contributing to the secretion of factors which drive the systemic inflammation, a hallmark of the syndrome. While inflammation is a defensive innate response, the control mechanisms have been reported to be disrupted in cachexia. On the other hand, little is known about the role of NLRP3 inflammasome in this scenario, a multiprotein complex involved in caspase-1 activation and the processing of the cytokines IL-1β and IL-18.Aimbased on the evidence from our previous study with a rodent model of cachexia, we examined the activation of the NLRP3 inflammasome pathway in two adipose tissue depots obtained from patients with colorectal cancer and compared with that another inflammatory pathway, NF-κB.ResultsFor CC we found opposite modulation in ScAT and PtAT for the gene expression of TLR4, Caspase-1 (cachectic group) and for NF-κB p50, NF-κB p65, IL-1β. CD36, expression was decreased in both depots while that of NLRP3 and IL-18 was higher in both tissues, as compared with controls and weight stable patients (WSC). Caspase-1 basal protein levels in the ScAT culture supernatant were higher in WSC and (weight stable patients) CC, when compared to controls. Basal ScAT explant culture medium IL-1β and IL-18 protein content in ScAT supernatant was decreased in the WSC and CC as compared to CTL explants.ConclusionsThe results demonstrate heterogeneous responses in the activation of genes of the NLRP3 inflammasome pathway in the adipose tissue of patients with cancer cachexia, rendering this pathway a potential target for therapy aiming at decreasing chronic inflammation in cancer.

Influence of light on the accumulation of anthocyanins in callus culture of Vaccinium corymbosum L. cv. Sunt Blue Giant

Blueberry is considered as a health-promoting food with a high content of anthocyanins. The extraction of anthocyanins from callus has been used to solve some problems of anthocyanins production, being more effective for commercial application and high production of anthocyanins. One of the most important aims of our study is to enhance anthocyanin synthesis in callus cultures. The results showed that callus induction from the highbush blueberry V. corymbosum L. cv. Sunt Blue Giant depends on the type of explant (leaf, root, and stem segments) and culture medium. Maximum callus induction was established on Woody Plant nutrient Medium (WPM) containing 2.0 mg/L ɑ–naphthalene acetic acid (NAA) in combination with 1.0 mg/L 6-benzylaminopurine/benzyl adenine (BAP) from all used explants and the maximum fresh weight (for leaf, 1.03; root, 1.0; and stem, 0.8 g/jar) was recorded. After four subcultures on the same medium composition, we studied the effect of different light types on the accumulation of anthocyanins in callus culture. The highest levels of anthocyanin content were observed in callus from root origin (5.7 fold) and leaf origin (4.8 fold) grown under red light comparing with callus grown in dark as a control. The anthocyanins composition were evaluated using high-performance liquid chromatography, under the influence of red light the qualitative composition of anthocyanins significantly expanded in comparison with the dark condition. In addition, the red light increased the qualitative composition of anthocyanins in callus obtained from leaf origin largely than in callus obtained from root origin. From callus cultures obtained from root and leaf origin growing under red light were isolated ten anthocyanins and three proanthocyanidins. The present study could suggest an important protocol for improving the production of anthocyanins from callus culture of V. corymbosum L. cv. Sunt Blue Giant.

4 The effects of clodronate disodium on normal and inflamed equine cartilage in vitro

Bisphosphonates (BP) are a group of drugs which modulate osteoclastic resorption and alter bone turnover. BP are only approved for use in navicular syndrome in adult horses, however, these drugs are being used off-label for their potential anti-inflammatory and pain-relieving effects. When the BP, clodronate disodium (CLO), is administered intramuscularly at the labeled dose, synovial fluid concentrations are detectable within 2 h of administration. There are currently no published studies evaluating the effects of CLO on equine joint tissues. This study evaluated the effects of CLO on normal and inflamed equine cartilage explants in vitro. Cartilage explants were collected from the femorotibial joints of 3 horses which underwent euthanasia for reasons other than stifle osteoarthritis. Only visually normal cartilage was selected for biopsy. The harvested cartilage from each femorotibial joint was subdivided into 24-well plates. Cartilage explants were subjected to 6 different treatments with 8 replicates each, after a 24 h equilibration period: Control (Con), CLO 5 ng/ml (C/lo), CLO 50 ng/ml (C/hi), rEqIL-1β 10 ng/ml (IL), C/lo plus IL (ILC/lo) and C/hi concentration plus IL (ILC/hi). Explant culture media was collected every 24 h and stored at −20°C until analysis of sulfated glycosaminoglycan (GAG) content and nitric oxide (NO) content using a Dimethylmethylene Blue (DMMB) assay and a Greiss reaction, respectively. At the conclusion of the experiment (96 h), cartilage explants were papain-digested and GAG were quantified using a DMMB assay. Data were analyzed using PROC MIXED procedure (SAS 9.4), where horses were considered a random effect, and treatment and day were fixed effects. For all analyses, values of P < 0.05 were considered significant. At d 1, d 2, and d 3, the IL treatment group had a significantly higher concentration of GAG within the media compared with the Con group ( P < 0.05), indicating the expected catabolic effect of IL-1β on the explants. Treatments with clodronate (C/lo, C/hi) alone did not result in an increase in GAG or NO within the media compared with the control indicating CLO did not have a cytotoxic effect. However, the addition of CLO to the IL-1β treated explants (ILC/lo, ILC/hi) did not result in a significant decrease in GAG or NO release into the media when compared with rEqIL-1β (IL) alone. No differences were observed between treatments when GAG content of cartilage explants was measured following papain-digestion. Our in vitro study suggests that CLO does not result in cartilage toxicity. However, no anti-inflammatory or chondroprotective effects were observed.

Direct and Indirect Somatic Embryogenesis Induction in Camellia oleifera Abel.

Camellia oleifera Abel. is an important woody oil species; however, the shortage of rapid and industrialized seedling culture is a large constraint on the development of the tea oil industry. Somatic embryogenesis (SE) is one of the main powerful biotechnological tools for plant mass regeneration, but the largely unknown SE in C. oleifera limits the scale production of clonal plants. In this study, we described a high-efficiency SE system via direct and indirect pathways in C. oleifera and investigated the effect of genotype, explant age and phytohormones on SE. In the direct pathway, somatic embryos were highly induced from immature seeds 220 days after full blossom, and the development of embryoids was achieved with a combination of 0.19 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.05 mg/L thidiazuron (TDZ). In the indirect pathway, embryogenic calli were induced from the same explants in medium containing 1.5 mg/L 2,4-D, while 0.75 mg/L 2,4-D treatment led to high proliferation rates for embryogenic calli. The addition of 0.19 mg/L 2,4-D alone stimulated the production of globular embryos while causing a 75% loss of the induction rate in the heart embryo stage. Upon transfer of the globular embryos to phytohormone-free medium, an optimal induction rate of 62.37% from globular embryos to cotyledonary embryos was obtained. These data suggest that the subsequent differentiation process after the globular embryo stage in ISE is more similar to an endogenous phytohormones-driven process. Mature embryos germinated to produce intact plantlets on half-strength MS basal medium with a regeneration rate of 63.67%. Histological analysis confirmed the vascular bundle isolation of embryoids from the mother tissue. We further studied the different varieties and found that there were no significant genotype differences for SE induction efficiency in C. oleifera. Thus, we established a high-efficiency induction system for direct and indirect somatic embryogenesis (ISE) in C. oleifera and regenerated intact plantlets via SE, not organogenesis. ISE has a more complicated induction and regulatory mechanism than direct somatic embryogenesis. The improved protocol of SE would benefit mass propagation and genetic manipulation in C. oleifera.

In-vitro propagation of Pluchea lanceolata (DC) C.B.Clarke a potent antiarthritic medicinal herb through axillary bud

Pluchea lanceolata, commonly known as Rasana, is highly valued for its anti-inflammatory, antiarthritic, and analgesic properties and is extensively employed in treating rheumatoid arthritis, cough, neurological diseases, edema, sciatica, psoriasis, bronchitis, dyspepsia, and piles. Shrinking populations and insufficient variation due to rootstock propagation methods warrant efficient mass multiplication and conservation protocols. In the present study, explant (axillaries bud was cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of cytokinins and auxin (PGRs) individually, as well as in combination. The highest mean shoot number (2.06 ± 1.43) and second highest mean shoot length (2.62 ± 1.49 cm) were obtained in MS media enriched with 4 mg/L Benzyl amino purine (BAP) + 1mg/L Naphthalene acetic acid (NAA). Whereas, the best shoots multiplication (2.62 ± 1.41) and shoots elongation (5.88 ± 2.44) responses were displayed when MS medium supplemented with 0.25 mg/L (BAP) +0.50 mg/L Kinetin (KIN) were used.