Proliferation Medium Research Articles

Transcriptomics and Functional Analysis of Graphene-Guided Osteogenic Differentiation of Mesenchymal Stem Cells.

To explore graphene's effects on the gene expression profile of mesenchymal stem cells, and to reveal the mechanisms of graphene-guided osteogenic differentiation. Human adipose-derived mesenchymal stem cells (hASCs) were cultured on single-layer graphene-coated titanium disks or titanium disks in proliferation medium (control) or osteoinduction medium for 7 days before RNA extraction. After library construction and RNA sequencing, identification of differentially expressed genes was performed through Limma package of R platform, with a cut-off value of log fold change (logFC) > = |1|. Pathway and Gene ontology (GO) analyses were conducted on DAVID Bioinformatics Resources 6.8 (NIAID/NIH). Network analyses were performed by the Ingenuity Pathways Analysis (IPA). Signalling pathway analysis revealed the top five pathways - cytokine-cytokine receptor interaction, neuroactive-ligand receptor interaction, calcium signalling pathway, PI3K-Akt signalling pathway and cell adhesion molecules. GO analyses demonstrated significant changes on cell adhesion, calcium signalling, and epigenetic regulation. IPA network analyses demonstrated that inflammation-related pathways were influenced by graphene, while the downstream factors of histone H3 and H4 were also altered especially under the existence of osteoinduction medium. Graphene promotes osteogenic differentiation of hASCs mainly by influencing cell adhesion, cytokine-cytokine receptor interactions, inflammatory responses, and potentially influences histone H3 and H4 through epigenetic regulation.

Changes in osmotic potential, protein content and proline accumulation in response to NaCl salinity and phosphorus in cucumber microshoots grown on proliferation medium

The aim of this work was to explore the contributions of phosphorus in mitigating the salinity impacts on osmotic potential, protein content and proline accumulation of cucumber grown in vitro on proliferation medium. Microshoots were grown on Murashige and Skoog (MS) proliferation media containing 1 mg/l kinetin and supplemented with a combination of NaCl at 0.0, 25, 50, 75, 100 mM with 0.5, 1.0 or 2.0 mM phosphorus using KH2PO4. Leaf osmotic potential was reduced with increasing NaCl concentration and within each of the 50, 75 and 100 mM NaCl concentrations, the incorporation of 2 mM KH2PO4 was needed to significantly increase leaf osmotic potential. Protein content of cucumber microshoots was shown to decrease with NaCl concentration, this reduction was lessened with increase in KH2PO4 level applied. In vitro-induced salinity caused profound proline accumulation in cucumber microshoots and combinations having 2 mM KH2PO4 showed lower proline accumulation especially at the highest two concentrations of NaCl.

In vitro differentiation of neural cells from human adipose tissue derived stromal cells.

Stem cells, including neural stem cells (NSCs), are endowed with self-renewal capability and hence hold great opportunity for the institution of replacement/protective therapy. We propose a method for in vitro generation of stromal cells from human adipose tissue and their differentiation into neural cells. Ten grams of donor adipose tissue was surgically resected from the abdominal wall of the human donor after the participants' informed consents. The resected adipose tissue was minced and incubated for 1 hour in the presence of an enzyme (collagenase-type I) at 370C followed by its centrifugation. After centrifugation, the supernatant and pellets were separated and cultured in a medium for proliferation at 370C with 5% CO2 for 9-10 days in separate tissue culture dishes for generation of mesenchymal stromal cells (MSC). At the end of the culture, MSC were harvested and analyzed. The harvested MSC were subjected for further culture for their differentiation into neural cells for 5-7 days using differentiation medium mainly comprising of neurobasal medium. At the end of the procedure, culture cells were isolated and studied for expression of transcriptional factor proteins: orthodenticle homolog-2 (OTX-2), beta-III-tubulin (β3-Tubulin), glial-fibrillary acid protein (GFAP) and synaptophysin-β2. In total, 50 neural cells-lines were generated. In vitro generated MSC differentiated neural cells' mean quantum was 5.4 ± 6.9 ml with the mean cell count being, 5.27 ± 2.65 × 103/μl. All of them showed the presence of OTX-2, β3-Tubulin, GFAP, synaptophysin-β2. Neural cells can be differentiated in vitro from MSC safely and effectively. In vitro generated neural cells represent a potential therapy for recovery from spinal cord injuries and neurodegenerative disease.

Optimization of In vitro Culture Conditions of Interleukin-10 Secreting B Regulatory Cells of Human Origin

AbstractAim: B-regulatory cells (B-regs), commonly characterized as CD19+/38hi/24hi, comprise of a major subset of IL-10 secreting B-cells. We report optimization of culture conditions for B-regs of human origin, obtained from in-vitro co-culture of donor adipose tissue derived mesenchymal stem cells (AD-MSC) and renal allograft recipient (RAR) peripheral blood mononuclear cells (PBMC) for potential cell therapy.Material and methodology: Five sets of 6-well plates were filled with proliferation media. Each plate was seeded with 5 × 104 ADMSC and 1 × 106 cells of responder and stimulator peripheral blood mononuclear cells (PBMC) and incubated at 37˚C, 5% CO2 after addition of lipopolysaccharide–E.coli-K12 strain (LPSEK) in varying concentrations. At each time point, one plate was withdrawn and contents centrifuged. IL-10 concentration of cell supernatant was estimated by quantitative ELISA. Pellet was resuspended to determine morphology, sterility, total count, viability and immunophenotype of cells.Results: The highest B-reg population (0.19×106 cells/ml) was noted at 24 hours with 400 ng/ml LPS-EK, which also correlated with optimum secretion of IL-10 (1.58 pg/ml), for the same time interval.Conclusion: The present study provides valuable information about the dynamics of IL-10 secretion by in vitro generated human B-regs, obtained from donor AD-MSC and recipient PBMC.

Effects of Different Light Qualities--LED on Apple Root-Stock JM7 Tissue Culture Seedling

Used apple root-stock JM7 stem-tips as material to study different LED influence of different stages of tissue culture. The results showed that in the culture medium (bud induction medium LS + 4.0mg/L 6-BA + 0.2 mg/L NAA, proliferation medium LS + 2.5mg/L 6-BA + 0.1mg/L NAA , rooting medium 1/2 LS + 0.5mg/L NAA + 1.0mg/L IAA), with 30g/L sugar and 9g/L agar, pH 5.8, autoclave 121°C, 20 minuets. At the same culture conditions, 21±1°C temperature, 12 hours of light, 12 hours of dark and same illumination time. The LED treatments were better than the fluorescent light of the germination; LED r/b ratio 5:5, was the best on germination effect, the height of seedling was 1.84cm and 10 leaves on 50 days of cultivation; LED r/b ratio 6:4, was the best on the proliferation stage and rooting effect stage, the height of seedling was 4.15cm on 45 days of proliferation and the main-root length was 5.97cm, and the height of seedling was 6.38cm on 45 days of rooting stage; followed by LED r/b 7:3 and 5:5. In conclusion LED treatments were advantageous to the germination, seedling differentiation and rooting of apple root-stock JM7 tissue culture, better than fluorescent lamp group. It could get more quantities and stronger plants in the same culture conditions, also saved the culture time. LED was cold light source, it could produce less excess heat and truly reflects the feature of power-saving effectively. So it was reduced of the tissue culture production in a certain extent.

Evaluation of different media for cell proliferation in mantle tissue culture of the green mussel, Perna viridis (Linnaeus, 1758)

The aim of the present study was to establish a suitable culture system for tissue explants from the mantle of the green mussel, Perna viridis. The experiments were conducted using healthy, live green mussels in the size range of 75 to 110 g collected from Pulicat Lake, Tamil Nadu. Three different culture media namely M199, Leibovitz L-15 and sterile seawater were used to assess the most suitable medium for growth, proliferation and viability of mantle epithelial cells. The effect of the addition of two supplements viz., 10% foetal calf serum (FCS) and 0.1% yeast extract to the culture media was also evaluated. After carefully isolating the pallial layer from the mantle tissue, 1-2 mm2 size explants were successfully cultured in 12-well plates at 25°C for up to 14 days. Cultures were monitored under light and phase contrast objectives in an inverted microscope. Cell counts were made and cell size was measured for each treatment. Cells were observed to migrate from the periphery of the explant within 24 h after initiation of cultures. The liberated cells were mostly round and were either granulocytes or hyalinocytes. Fibroblast-like cells were also observed. Our results showed that proliferation of epithelial cells from mantle tissue was maximum in seawater medium (7.4x104 cells ml-1), followed by L-15 medium (2.55x104 cells ml-1). Average cell size in seawater medium was 10.72 μm and that in L-15 and M199 media was 8.56 and 6.39 μm, respectively. Adherent cells were also more prominent and higher in number in seawater medium. Supplementation of culture media with 10% FCS and 0.1% yeast extract improved both cell proliferation and cell size in all the three culture media. Four concentrations of 0.1% yeast extract (@ 50 μl, 75 μl, 100 μl, 150 μl ml-1 medium) were tested in the present study and best results were obtained with 100 μl ml-1, with respect to both cell counts and size.

Application of whey protein isolate in bone regeneration: Effects on growth and osteogenic differentiation of bone-forming cells

Recently, milk-derived proteins have attracted attention for applications in the biomedical field such as tissue regeneration. Whey protein isolate (WPI), especially its main component β-lactoglobulin, can modulate immunity and acts as an antioxidant, antitumor, antiviral, and antibacterial agent. There are very few reports of the application of WPI in tissue engineering, especially in bone tissue engineering. In this study, we tested the influence of different concentrations of WPI on behavior of human osteoblast-like Saos-2 cells, human adipose tissue-derived stem cells (ASC), and human neonatal dermal fibroblasts (FIB). The positive effect on growth was apparent for Saos-2 cells and FIB but not for ASC. However, the expression of markers characteristic for early osteogenic cell differentiation [type-I collagen (COL1) and alkaline phosphatase (ALP)] as well as ALP activity, increased dose-dependently in ASC. Importantly, Saos-2 cells were able to deposit calcium in the presence of WPI, even in a proliferation medium without other supplements that support osteogenic cell differentiation. The results indicate that, depending on the cell type, WPI can act as an enhancer of cell proliferation and osteogenic differentiation. Therefore, enrichment of biomaterials for bone regeneration with WPI seems a promising approach, especially due to the low cost of WPI.

Simple and efficient protocols for the initiation and proliferation of embryogenic tissue of Douglas-fir

Key messageThis paper describes the micropropagation of Douglas-fir via somatic embryogenesis using media and methods available in the public domain.Douglas-fir is a conifer species with high and increasing interest for forest industries in both New Zealand and France. Delivery of the best trees to the forest from breeding programmes is currently constrained by an inability to effectively and reliably multiply selections through vegetative propagation. Somatic embryogenesis coupled with cryopreservation is essential biotechnology tool in conifers for scaling up variety design and production. The aim of this work was therefore to develop protocols for the initiation and proliferation of Douglas-fir embryogenic cell lines based on the modifications of previously published techniques and media available in the public domain, especially successful methods developed for P. radiata at Scion. Three years of initiation experiments have resulted in a simple and efficient protocol. Disinfection of whole cones (instead of seeds) was sufficient to prevent contamination. Immature zygotic embryos were excised from megagametophytes and placed onto a modified Litvay medium (Plant Cell Rep 4(6):325–328, 1985***). At optimal development stages of zygotic embryos, the average initiation percentage of embryogenic tissue was 69.3% when our most effective protocol was used. Proliferation of initiated cell lines on a Glitz formulation was challenging, with a high percentage of cell lines composed of a mixture of both embryonal masses and callus. 2,4-D at a lower concentration reduced the number of such mixed lines. Repeated liquid suspension and subculture of the embryogenic parts of the tissue was an efficient means to increase the number of embryogenic cell lines with sustained proliferation. Maltose added to the proliferation medium in place of sucrose improved fresh mass gain and consistently increased early somatic embryo patterning and growth. A sample of proliferating cell lines was successfully cryopreserved, thawed and somatic embryos were matured and germinated from these lines. The method may be of practical interest and provide new opportunities to realize increased genetic gain in this species through the clonal-assisted deployment of the best genetic material in both New Zealand and France.

Direct shoot regeneration and genetic fidelity analysis in finger millet using ISSR markers

Finger millet (Eleusine coracana (L.) Gaertn.), an economically important food crop is cultivated widely in the arid and semi-arid tropics of Africa and Asia. In the present study, an efficient micropropagation protocol has been established for finger millet genotypes CO 9, CO (Ra) 14 and GPU 28 using shoot apical meristems (SAMs). Shoot proliferation medium (SPM) containing Murashige and Skoog’s (MS) medium amended with 3.0 mg/l 6-benzylaminopurine produced the highest shoot regeneration frequency (86.60%) with an average of 26.45 ± 0.34 shoots per explant and 6.26 ± 0.38 cm shoot length in CO 9. An increase in the number of shoots per explant was observed when SAMs were repeatedly sub-cultured in SPM at 2 weeks interval for 8 weeks. Rooting of the regenerated shoots was achieved in full-strength MS medium containing indole-3-acetic acid (IAA) or indole-3-butyric acid. Rooting medium containing 0.25 mg/l IAA exhibited highest rooting frequency (100%) with an average root length of 4.44 ± 0.15 cm. In vitro rooted shoots transferred to the field conditions resulted in 100% survivability.Genetic fidelity of 3-month old mother plant and micropropagated plantlets was confirmed using 3′-anchored dinucleotide inter simple sequence repeats. A total of 115 amplicons generated for CO 9, CO (Ra) 14 and GPU 28 were monomorphic, revealing no variation among mother plant and micropropagated plantlets. Thus, SAMs could serve as a suitable explant for the mass multiplication of true-to-type plants and genetic transformation in finger millet.

Somatic Embryo from Basal Leaf Segments of Vanda tricolor Lindl. var. pallida

<p class="Els-Abstract-text">Somatic embryogenesis is one of techniques in plant micropropagation. The induction of somatic embryogenesis through callus phase was done on <em>Vanda tricolor</em> Lindl. var. pallida. This study aimed to find out the effect of naphtalene acetic acid (NAA) and benzyl amino purine (BAP) in inducing somatic embryogenesis via callus on the basal leaf segments of <em>Vanda tricolor</em> Lindl. var. pallida. The half-strength of Murashige and Skoog (½ MS) medium with 1 % sucrose, incorporated with (0.02 mg · L<sup>–1</sup>and 0.05 mg · L<sup>–1</sup>) NAA and also 0.01 mg · L<sup>–1</sup> BAP were used in this experiment. The best medium for embryogenic callus formation and proliferation was 0.05 mg · L<sup>–1</sup> NAA in combination with 0.01 mg · L<sup>–1</sup> BAP. The formation of somatic embryos occurred 30 d after the calluses were cultured on to ½ MS without the addition of plant growth regulator and subsequently formed shoots.</p>

505 A new tool for modelling stinging test in vitro: A comparative evaluation with in vivo results using a bacterial polysaccharide

Stinging test is an in vivo protocol that evaluates sensitive skin and tests formula or ingredients on soothing sensation. Stinging test is made using lactic acid (LA) at 10% placed on nasolabial fold right and left of the face of volunteers before addition of a product. To predict the soothing sensation of a product and to modelize effect on the skin, we developed culture models based on LA test and substance P (SP) release using human keratinocytes and PC12 cells. In order to predict a calming effect, we used a bacterial polysaccharide (bPS) present in Fucogel® in in vivo stinging test and our in vitro model. Cells were placed on a 96-wells plate. PC12 cells were differentiated using NGF for 3 days and keratinocytes were maintained one day in proliferation medium before application of LA or bPS or both. Firstly, the ideal concentration of LA (10%, 5%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.07%, 0.04% or 0%) was determined to release SP without cytotoxicity. Excluding toxic concentrations, the release of SP was significant from 0.2% of lactic acid for PC12 (pH6.2) cells and from 0.1% of lactic acid for keratinocytes (pH6.6). When pH was adjusted around 7.2, LA did not provoke SP release. In keratinocytes, 0.1% LA showed a cytosolic calcium entry. At these concentrations of LA, 0.1% of bPS showed a significant decrease of SP release on two cellular types and in co-culture without modified the pH of the medium. In vivo, stinging test using Fucogel® showed a decrease by 30% of prikling intensity versus placebo on 19 women from 21 to 69 years old. Our in vitro model is ethically interesting and is adapted for cosmetic ingredients screening because it does not use animal experimentation. Moreover, Fucogel® has an attractive soothing activity revealed by in vivo stinging test and our new in vitro stinging test based on SP release

An assessment of somatic embryogenesis and cryo-preservation methods with a wide range of Sitka spruce breeding material from the UK

A wide range of Sitka spruce breeding material (Picea sitchensis (Bong.) Carr.) was assessed for its ability to respond to somatic embryogenesis (SE) and cryo-preservation methods, with 765 cell lines being established from seeds taken from the UK’s breeding population for testing with the relevant methodologies. Cell lines were most readily established from immature seeds (63%) on a proliferation medium originally developed for white spruce (P. glauca) with agar as the gelling agent. However, it was also found that 48% of the zygotic embryos taken from freshly harvested mature seed were also able to establish cell lines on the same medium, as were 20% of seeds that had been stored at −20 °C for up to 8 years. Most of the cell lines tested (57%) produced significant numbers of somatic embryos, but the rate at which they could be germinated into plants capable of surviving the transfer to nursery conditions varied considerably from 67 to 8% between years for reasons that were not clear. The ability of the cell lines to survive cryo-preservation was also assessed, and out of 199 cell lines tested, 45 cell lines from 21 breeding families were successfully established in a cryo-storage facility. The best rates of recovery were obtained when Phytagel™ was used in the medium for growing the samples before and after they were cryo-preserved.

Mineral manipulation and antioxidative studies in carnation - Dianthus caryophyllus L.

The effect of ammonium nitrate (NH 4 NO 3 ) on shoot bud induction and proliferation from nodal explants of Dianthus caryophyllus L. was investigated. Shoot buds were induced on MS medium supplemented with 2.2µM BAP and 2.7µM NAA. The induced shoot buds were sub-cultured on medium with same hormonal composition for their proliferation. Both shoot bud induction and proliferation media were supplemented with different levels of NH 4 NO 3 ( 0, 5.15, 10.3, 20.61*, 41.20 mM ). NH 4 NO 3 highly influenced the shoot bud formation and their subsequent proliferation. In the present investigation, NH 4 NO 3 at one-fouth of MS level was found to be beneficial for controlling the hyperhydricity of regenerated shoots whereas the number of shoots was comparable to the control cultures. The MS or higher levels of NH 4 NO 3 were observed to suppress healthy morphogenesis and resulted in increased hyperhydricity. The physiological disorder of hyperhydricity, commonly observed in carnation micropropagation, was also controlled with 100 % efficiency by reducing NH 4 NO 3 at one-fouth of MS level. Antioxidant enzyme activity was minimum in normal and healthy shoots. The activity increased with increase in the number of hyperhydric shoots.

Red (660\u2009nm) or near-infrared (810\u2009nm) photobiomodulation stimulates, while blue (415\u2009nm), green (540\u2009nm) light inhibits proliferation in human adipose-derived stem cells

We previously showed that blue (415 nm) and green (540 nm) wavelengths were more effective in stimulating osteoblast differentiation of human adipose-derived stem cells (hASC), compared to red (660 nm) and near-infrared (NIR, 810 nm). Intracellular calcium was higher after blue/green, and could be inhibited by the ion channel blocker, capsazepine. In the present study we asked what was the effect of these four wavelengths on proliferation of the hASC? When cultured in proliferation medium there was a clear difference between blue/green which inhibited proliferation and red/NIR which stimulated proliferation, all at 3 J/cm2. Blue/green reduced cellular ATP, while red/NIR increased ATP in a biphasic manner. Blue/green produced a bigger increase in intracellular calcium and reactive oxygen species (ROS). Blue/green reduced mitochondrial membrane potential (MMP) and lowered intracellular pH, while red/NIR had the opposite effect. Transient receptor potential vanilloid 1 (TRPV1) ion channel was expressed in hADSC, and the TRPV1 ligand capsaicin (5uM) stimulated proliferation, which could be abrogated by capsazepine. The inhibition of proliferation caused by blue/green could also be abrogated by capsazepine, and by the antioxidant, N-acetylcysteine. The data suggest that blue/green light inhibits proliferation by activating TRPV1, and increasing calcium and ROS.

Propagation of mature Quercus ilex L. (holm oak) trees by somatic embryogenesis

Somatic embryogenesis from in vitro leaf and shoot apex explants excised from axillary shoot cultures established from two mature Quercus ilex trees has been developed. Somatic embryos (SE) were obtained from both explant types and genotypes evaluated, although embryogenic frequencies were influenced by the genotype, auxin concentration, and explant type. The explants were cultured on Murashige and Skoog salts and vitamins, supplemented with 500 mg L−1 casein hydrolysate (CH) and different concentrations of indole-3-acetic acid or α-naphthalene acetic acid (NAA) in combination with 2.22 µM 6-benzylaminopurine (BA). In both genotypes, shoot apex explants were more responsive than leaf explants. The best results were obtained with apex explants of clone Q3 (11%) cultured on medium with 21.48 µM NAA plus 2.22 µM BA. This combination was also effective for initiating SE from leaf explants, although the induction rates were lower (1–3%). Embryogenic lines were maintained by repetitive embryogenesis following culture of nodular embryogenic structures on Schenk and Hildebrand medium without plant growth regulators. Low embryo multiplication rates were obtained when torpedo or early cotyledonary SE were used as initial explant for embryo proliferation, or when glutamine or CH (500 mg L−1) was added to proliferation medium. For germination, cotyledonary-stage SE were isolated and stored at 4 °C for 2 months. After cold storage, SE were cultured on germination medium consisting of Gresshoff and Doy medium, supplemented with 0.44 μM BA and 20 μM silver thiosulphate. Under these conditions, plantlets were regenerated from 21 to 66.7% of the SE generated for both genotypes.

Cationic osteogenic peptide P15-CSP coatings promote 3-D osteogenesis in poly(epsilon-caprolactone) scaffolds of distinct pore size.

P15-CSP is a biomimetic cationic fusion peptide that stimulates osteogenesis and inhibits bacterial biofilm formation when coated on 2-D surfaces. This study tested the hypothesis that P15-CSP coatings enhance 3-D osteogenesis in a porous but otherwise hydrophobic poly-(ɛ-caprolactone) (PCL) scaffold. Scaffolds of 84 µm and 141 µm average pore size were coated or not with Layer-by-Layer polyelectrolytes followed by P15-CSP, seeded with adult primary human mesenchymal stem cells (MSCs), and cultured 10 days in proliferation medium, then 21 days in osteogenic medium. Atomic analyses showed that P15-CSP was successfully captured by LbL. After 2 days of culture, MSCs adhered and spread more on P15-CSP coated pores than PCL-only. At day 10, all constructs contained nonmineralized tissue. At day 31, all constructs became enveloped in a "skin" of tissue that, like 2-D cultures, underwent sporadic mineralization in areas of high cell density that extended into some 141 µm edge pores. By quantitative histomorphometry, 2.5-fold more tissue and biomineral accumulated in edge pores versus inner pores. P15-CSP specifically promoted tissue-scaffold integration, fourfold higher overall biomineralization, and more mineral deposits in the outer 84 µm and inner 141 µm pores than PCL-only (p < 0.05). 3-D Micro-CT revealed asymmetric mineral deposition consistent with histological calcium staining. This study provides proof-of-concept that P15-CSP coatings are osteoconductive in PCL pore surfaces with 3-D topography. Biomineralization deeper than 150 µm from the scaffold edge was optimally attained with the larger 141 µm peptide-coated pores. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2171-2181, 2017.

English

This study was conducted at Marine Science Center, Basrah University, Basrah, Iraq during the period of 2015 to 2016. In the present work, we used NaCl and polyethylene glycol (PEG) to induced abiotic stress in vitro. Results show that high shoot numbers (24) were produced on MS (solid) medium enriched with 0.8 g/L NaCl in addition to cytokinins while, inclusion of drought agent PEG to proliferation medium (solid and liquid) produced a minimum shoots of 5 and 11 respectively. Root growth and development were also investigated in this study. It was found that high root development was observed on MS (solid or liquid) medium free of hormone (control) and MS (solid or liquid) medium containing 0.8 g/L NaCl 26.67, 25, 21.67 and 18 respectively whereas, less root development was obtained on MS medium (solid or liquid) enriched with NaCl and 26 g/L PEG. In this study it was found that inclusion of the amino acid proline to the stressed media did not improve the growth of shoots or roots. Histological analysis of multiplied shoots under abiotic stress agents showed accumulation of insoluble starch granules in the parenchyma cells of the cortex layer and this may be a part of protective tolerate mechanism used by medicinal hyssop (Bacopa monnieri) against abiotic stress. Key words: Bacopa monnieri, medicinal hyssop, Brbin, micropropagation, salt stress, polyethylene glycol (PEG), proline.

In-vitro generation of interleukin-10 secreting B-regulatory cells from donor adipose tissue derived mesenchymal stem cells and recipient peripheral blood mononuclear cells for potential cell therapy

BackgroundInterleukin-10 secreting B-cells are a major subset of B-regulatory cells (B-regs), commonly recognized as CD19+/38hi/24hi/IL10+. They carry out immunomodulation by release of specific cytokines and/or cell-to-cell contact. We have generated B-regs in-vitro from donor adipose tissue derived mesenchymal stem cells (AD-MSC) and renal allograft recipient (RAR) peripheral blood mononuclear cells (PBMC) for potential cell therapy. Material and methodsMononuclear cells separated by density gradient centrifugation from 50 ml anti-coagulated blood of 15-RAR and respective donors were analysed for baseline B-regs using appropriate antibodies. Equal amount (20 × 106 cells/ml) of stimulator (irradiated at 7.45 Gy/min for 10 min) and responder (non-irradiated) cells were co-cultured with in-vitro generated AD-MSC (1 × 106 cells/ml) in proliferation medium containing lipopolysaccharide from E. coli K12 strain at 37 °C with 5% CO2. Cells were harvested on day-7 and analyzed for viability, sterility, quantity, morphology and phenotyping. In-vitro generated B-reg levels were compared with baseline B-regs. ResultsIn-vitro generated B-reg count increased to 16.75% from baseline count of 3.35%. ConclusionB-regs can be successfully generated in-vitro from donor AD-MSC and RAR PBMC for potential cell therapy.

Micropropagation of Pyracantha coccinea

Pyracantha coccinea is a thorny evergreen shrub native to southeast Europe to southeast Asia. It is a popular ornamental plant because of its showy bright red fruits and small white flowers. However, in vitro vegetative propagation of P. coccinea has not been studied. Nodal segments with one or two axillary buds (1 to 1.5 cm in length) were cut and disinfected in a solution of 0.1% (v/v) mercuric chloride (HgCl2) for 5 minutes, and proliferated on Murashige and Skoog (MS) basal medium supplemented with various concentrations 6-benzylaminopurine (6-BA). After 4 weeks, newly formed shoots were transferred to proliferation and rooting media containing various concentrations of indole-3-butyric acid (IBA). Establishment of axillary buds was significantly better with an establishing rate of 67% on basal MS medium augmented with 6.6 µm 6-BA. The best medium for proliferation of shoots was three-fourth basal MS supplemented with 1.5 µm IBA, with a proliferation rate of 3.4 axillary bud. The optimum rooting medium was one-fourth MS basal medium containing 93 µm IBA. Rooting of shoots was as much as 77%. Rooted plantlets were transferred to pots containing vermiculite:perlite:peat (6:1:2) and acclimatized to ambient greenhouse conditions with a 95% survival rate. This protocol can be used for in vitro propagation of P. coccinea.

Induksi dan Proliferasi Embriogenesis Somatik In Vitro pada Lima Genotipe Kedelai

ABSTRACT&lt;br /&gt;&lt;br /&gt;Somatic embryo induction medium was reported to be genotype dependent for soybean. This study was aimed to obtain the optimum medium for embryo somatic induction and proliferation, and to regenerate somatic embryo of five soybean genotypes. Five soybean genotypes (Tanggamus, Anjasmoro, Yellow Biloxi, CG-22-10, and SP-10-4) were used in this study. The research was divided into four steps: (1) embryogenic callus induction of five soybean genotypes, (2) embryogenic callus proliferation of five soybean genotypes, (3) optimation of embryo somatic induction on five soybean genotypes and (4) embryo somatic regeneration of five soybean genotypes. The induction experiment showed that based on number of embryogenic callus, the best somatic embryo-induction medium was 3% sucrose+ NAA 5 mg L-1+2,4-D 5 mg L-1+ Vitamin B5. Embryogenic callus number for each genotype tested was increased on proliferation media of 3% sukrosa + 2,4-D 5 mg L-1 + NAA 5 mg L-1+ Vit B5, and Yellow Biloxi gave the highest number of proliferated somatic embryos compared to other genotypes. Increasing number of globular somatic embryo of all genotypes was obtained from the optimation of somatic embryo induction media being used, and Tanggamus genotype gave the highest number of globular somatic embryo which followed by Yellow Biloxi genotype. Tanggamus and Yellow Biloxi genotypes were also successfully formed the four steps of somatic embryos (globular, heart, torpedo, and cotyledonary stages), but in regeneration medium of MS0 and media MS + sukrosa 10 g L-1 + GA3 2 mg L-1 + BAP 4 mg L-1 + Vit B5 only Tanggamus genotype was regenerated into plantlet. &lt;br /&gt;&lt;br /&gt;Keywords: 2,4-D, NAA, somatic embryos, induction, proliferation&lt;br /&gt;&lt;br /&gt;