Proliferation Medium Research Articles

Comparison of the Cost and Effect of Combined Conditioned Medium and Conventional Medium for Fallopian Tube Organoid Cultures

Fallopian tube epithelial cells (FTEC) are thought to be the cell of origin of high-grade serous ovarian carcinoma. FTEC organoids can be used as research models for the disease. Nevertheless, culturing organoids requires a medium supplemented with several expensive growth factors. We proposed that a combined conditioned medium based on the composition of the fallopian tubes, including epithelial, stromal, and endothelial cells could enhance FTEC organoid formation. We derived two primary culture cell lines from the fimbria portion of the fallopian tubes. The organoids were split into conventional or combined medium groups based on what medium they were grown in and compared. The number and size of the organoids were evaluated. Quantitative polymerase chain reaction (qPCR) and immunohistochemistry (IHC) were used to evaluate gene and protein expression (PAX8, FOXJ1, beta-catenin, and stemness genes). Enzyme-linked immunosorbent assay was used to measure Wnt3a and RSPO1 in both mediums. DKK1 and LiCl were added to the mediums to evaluate their influence on beta-catenin signaling. The growth factor in the combined medium was evaluated by the growth factor array. We found that the conventional medium was better for organoids regarding proliferation (number and size). In addition, WNT3A and RSPO1 concentrations were too low in the combined medium and needed to be added making the cost equivalent to the conventional medium. However, the organoid formation rate was 100% in both groups. Furthermore, the combined medium group had higher PAX8 and stemness gene expression (OLFM4, SSEA4, LGR5, B3GALT5) when compared with the conventional medium group. Wnt signaling was evident in the organoids grown in the conventional medium but not in the combined medium. PLGF, IGFBP6, VEGF, bFGF, and SCFR were found to be enriched in the combined medium. In conclusion, the combined medium could successfully culture organoids and enhance PAX8 and stemness gene expression. However, the conventional medium was a better medium for organoid proliferation. The expense of both mediums was comparable. The benefit of using a combined medium requires further exploration.

Investigation of In vitro Propagation Possibilities of Endemic Campanula phitosiana Yıldırım & Şentürk

Campanula phitosiana Yıldırım & Şentürk, is a local endemic to the Aydın Mountain range, which distribute across in Western Anatolia (both İzmir and Aydın). This species belongs to the Mediterranean basin floristic region. According to the International Union for Conservation of Nature’s criteria, C. phitosiana is classified as “Critically Endangered” (CR). The aim of the study is to develop in vitro regeneration protocol for critically endangered endemic Campanula phitosiana. To investigate the efficient medium and plant growth regulator combinations for callus initiation and shoot proliferation, petiole and leaves were used as explant and explants cultivated on MS medium including NAA (1-Naphthylacetic acid) (0.3 mg L-1), TDZ (Thidiazuron) (0.5, 1.0, 2.0, and 3.0 mg L-1), BA (6-Benzylaminopurine) (0.5, 1.0, 2.0, and 3.0 mg L-1) and Gibberellic Acid (GA3) (0.5, 1.0, 2.0, and 3.0 mg L-1). Explants subcultured 3 times. Experiments were conducted according to completely randomized design repeated with 5 replicates and each replicates including 5 explants. As a result of the experiment, callus initiation and shoot proliferation were investigated. Efficient callus initiation was observed petiole explants as 100% rate. Shoot proliferation was observed on MS medium including 0.3 mg L-1 NAA + 2.0 mg L-1 GA3.

Optimizing Somatic Embryogenesis Initiation, Maturation and Preculturing for Cryopreservation in Picea pungens

Picea pungens (Engelm.), known for its blue-green needles, has become a likable ornamental species in northeast China since 2000. Nonetheless, a lack of propagation methods that can maintain genetic fidelity and develop seedlings at a large scale prevents the further expansion of the species. Somatic embryogenesis (SE), paired with cryopreservation technologies, may provide a valid alternative. Picea pungens SE is not new, but its practical application has been limited due to low efficiencies in SE initiation and maturation as well as a lack of effective cryopreservation technology. In this study, experiments were carried out to overcome the limitations by modifying culture media. For initiation, the efficiency was enhanced by adjusting concentrations of 2.4-dichlorophenoxy acetic acid (2,4-D), 6-benzyl amino–purine (6-BA) or sucrose supplemented to the induction medium. The concentrations of 4.0 mg/L 2,4-D, 2 mg/L 6-BA, and 5 to 10 g/L sucrose were found optimal in maximizing initiation efficiency. For maturation, the efficiency, expressed as the number of mature somatic embryos per gram of fresh mass cultured (E/gFM), varied greatly with the choices of the basal medium and concentration of abscisic acid (ABA) of the maturation medium. Based on our results, the judicial choices were using the DCR medium as the basal medium and 10 mg/L ABA. The maturation efficiency could also be improved by adjusting the maturation medium’s osmotic pressure by manipulating the concentrations of carbohydrate and Gelrite and culture density. While the maturation medium, using sucrose as carbohydrate source or supplemented with a low (<8 g/L) Gelrite concentration, facilitated maturation, optimal selections were truly genotype-dependent. Our results also suggest that, while the optimal culture density varied with genotype, in general it is needless to culture more than 100 mg embryogenesis tissues per dish (size: 10 × 1.5 cm). Based on this study, the optimum pretreatment for embryogenesis tissue cryopreservation was culturing the tissues on the proliferation medium with 0.4 mol/L sorbitol for 24 h, followed by treatment with 5% Dimethyl sulfoxide. This study significantly improved the initiation (achieved a frequency of 0.56) and embryo maturation efficiencies (achieved 1030 E/gFM) and established an effective preculturing protocol for cryopreservation (recovered 1354 E/gFM) for the species. The protocols developed here, paired with the available ones for other SE steps in the literature, form a well-refined SE technology intended for commercial application to Picea pungens.

Cryopreservation of Abies alba embryogenic tissues by slow-freezing method

Embryogenic tissues of Abies alba Mill. were cryopreserved using the slow-freezing approach. Four cell lines were incubated for 24 h on a medium with 0.5 M sorbitol and pre-treated with 5% DMSO. Subsequently, the tissues were frozen at a cooling rate of 1 °C min-1 to -40 °C and transferred to liquid nitrogen for 72 hours. After thawing in a water bath at 40 °C, the tissues were cultivated on a proliferation medium. All tested lines recovered, but variations in regrowth frequencies across cell lines were noticed (91.66 to 100%). The recovered tissues showed similar features to the control 2 (non-pre-treated and non-cryopreserved tissues). In the accumulation of fresh and dry mass, no statistically significant differences were observed between cryopreserved cultures and control 2. The cryopreserved tissues produced cotyledonary somatic embryos capable of germination. Microscopic observations revealed considerable structural changes as a consequence of the cryopreservation procedure. The long vacuolated suspensor cells were disrupted, and mostly the meristematic cells of the embryonal region survived. The typical bipolar structure of early somatic embryos has been regained during the post-thaw period. Differences in cryotolerance across cell lines were also observed.

Effect of Growth Regulators on In Vitro Micropropagation of Stahlianthus thorelii Gagnep

Stahlianthus thorelii Gagnep is a plant belonging to the family Zingiberaceae, widely distributed in Asian countries like China, Thailand, India and Vietnam. In traditional oriental medicine, this plant is usually used to treat hemorrhage, heavy menstruation, poor digestion, rheumatism and bone/joint pain (tuberous roots). This research article presents the results of in vitro growth experiments on S.thorelii Gagnep using tubers as explants. The samples are grown in MS media enriched with BAP growth stimulant concentrations of 5.0 mg L−1 and a kinetin concentration of 4.0 mg L−1, yielding 5.55 ± 0.59 and 5.48 ± 0.87 shoots/explants, respectively. Once the plants reached a height of 3.0–4.0 cm, we inoculated 2.0–3.0 leaves with a MS rapid proliferation medium treated with BAP or NAA growth agents alone or in combination. The most shoots (7.54 ± 0.79 shoots/explants) were produced by the medium enhanced with 3.0 mg L−1 BAP and 0.5 mg L−1 NAA after 8 weeks of cultivation. The greatest root/shoot induction of 26.17 ± 1.5 was achieved with the medium that had been treated with 0.5 mg L−1 NAA and 0.5 mg L−1 IBA, which was prepared using the MS media that was administered alone or in combination with NAA and IBA for in vitro shoot rooting. Highest percentage of survival (100%) was observed when tissue cultured plantlets were acclimatized in soil:sand:compost (1:1:1).

TGF-β-Mediated Modulation of Cell-Cell Interactions in Postconfluent Maturing Corneal Endothelial Cells.

PurposeTransforming growth factor-beta (TGF-β) is known to influence many cell functions. In the corneal endothelium, TGF-β1 exerts contextual effects, promoting endothelial–mesenchymal transition in proliferating cells and enhancing barrier integrity in early confluent maturing cells. Herein, we studied how TGF-β isoforms participate in the formation of corneal endothelial intercellular junctions.MethodsCorneal endothelial cells (CECs) were cultured using a two-phase media approach. When CECs reached confluence, the proliferation medium was replaced with maturation medium, which was supplemented or not with TGF-β isoforms. The cell morphology (circularity index), intercellular junction protein expression, trans-endothelial electrical resistance (TEER), and permeability of 7-day postconfluent CECs were assessed. Gene transcription and signaling pathways that were activated following maturation in the presence of TGF-β2 were also studied. The beneficial effect of TGF-β2 on CEC maturation was evaluated using ex vivo corneas mounted on a corneal bioreactor.ResultsThe results showed increases in circularity index, membrane localization of junction-related proteins, and TEER when TGF-β isoforms were individually added during the maturation phase, and TGF-β2 was the most effective isoform. Gene profiling revealed an increase in extracellular matrix-related gene expression. In ex vivo cell adhesion experiments, CECs that were matured in the presence of TGF-β2 had a higher circularity index and cell density and exhibited cell membrane-localized junction-related protein expression at earlier time points.ConclusionsThese results suggest that TGF-β2 can strengthen cell–cell and cell–substrate adhesion, which accelerates barrier integrity establishment and thus enhances CEC functionality.

Effects of PPAR-γ and RXR-α on mouse meibomian gland epithelial cells during inflammation induced by latanoprost

The purpose of this study is to investigate the effects of latanoprost on the secretion of cytokines and chemokines from meibomian gland epithelial cells, and to evaluate the modulation of peroxisome proliferator-activated receptor γ (PPAR-γ) and retinoid X receptor α (RXR-α) during latanoprost-induced inflammation. Mouse meibomian gland epithelial cells were cultured in proliferation and differentiation medium, respectively. Cells were exposed to latanoprost, rosiglitazone (PPAR-γ agonist), or LG100268 (RXR-α agonist), respectively. The expression of IL−6, IL-1β, TNF-α, MMP-9, MCP-1, and CCL-5 were detected by real-time PCR and ELISA. The effect of latanoprost, rosiglitazone, LG100268, and inflammatory cytokines on the differentiation of meibocyte were evaluated by related gene expression and lipid staining. The expression of Keratin-1, 6, 17 protein was detected by western immunoblotting. The results showed that the above cytokines could be induced by latanoprost in meibomian gland epithelial cells. LG100268 and rosiglitazone could inhibit the production of IL-6 and TNF-α induced by latanoprost, respectively. Latanoprost suppressed the expression of differentiation-related mRNA through a positive feedback loop by enhancement of COX-2 expression via FP receptor-activated ERK signaling. The expression of Keratin-17 was upregulated by rosiglitazone and suppressed by LG100268. The application of IL-6 and TNF-α showed negative effects on lipid accumulation in meibomian gland epithelial cells. These results demonstrated that latanoprost could induce inflammation and suppress differentiation of mouse meibomian gland epithelial cells. The activation of PPAR-γ and RXR-α showed an anti-inflammatory effect, showing a potential role to antagonize the effect of latanoprost eyedrops on meibomian gland epithelial cells.

An Optimized Protocol for Indirect Organogenesis from Root Explants of Agapanthus praecox subsp. orientalis ‘Big Blue’

Agapanthus praecox has become a burgeoning variety in the flower market due to its high ornamental value with unique large blue-purple inflorescence. For rapid entering into the market, tissue culture technology or organogenesis has an attractive application over the conventional reproduction approach. In this study, a highly efficient protocol based on indirect organogenesis has been successfully established for A. praecox subsp. orientalis ‘Big Blue’. Two types of explants, root tips versus root segments, were compared for callus induction frequency in response to the induction culture media. The induction media contain Murashige and Skoog’s (MS) Basal Salt supplemented with various concentrations of picloram (PIC), 2,4-Dichlorophenoxyacetic acid (2,4-D), thidiazuron (TDZ), kinetin (KT) and naphthalene acetic acid (NAA). Of the two types of explants, root tips were found to be more effective for callus induction than root segments. Among the induction media tested, the highest callus induction rate (100.00%) was achieved when cultured on MS supplemented with 2.0 mg/L PIC, 1.5 mg/L KT and 0.1 mg/L NAA, which was probably accredited to higher endogenous phytohormone contents, especially of 3-indoleacetic (IAA). The optimal medium for callus proliferation was MS + 1.0 mg/L PIC + 1.0 mg/L 6-BA + 0.4 mg/L NAA, and the fresh weight increased by 72.74%. After being transferred onto the adventitious bud induction medium for 25 days, shoots were dedifferentiated from the surface of the flourishing callus, which then developed to the plantlet with roots in 90 days. The plantlets were transplanted in a greenhouse with a survival rate of 92.86%. This study innovatively established an indirect organogenesis tissue culture system of A. praecox with roots as explants, which provided a practical reference in its application.

Efficient In Vitro Sterilization and Propagation from Stem Segment Explants of Cnidoscolus aconitifolius (Mill.) I.M. Johnst, a Multipurpose Woody Plant.

Cnidoscolus aconitifolius (Mill.) I.M. Johnst is a multipurpose woody plant. In this study, an in vitro efficient propagation system of stem segment explants derived from field-grown C. aconitifolius plants was established for the first time. The sterilization effect, axillary bud initiation, and proliferation efficiency of stem segments were evaluated. The results showed that the sterilization time of 0.1% mercuric chloride, the concentration of Plant Preservative Mixture (PPM), the pretreatment method, and the sampling season had significant effects on the sterilization of stem segments (p < 0.05). The type of medium and plant growth regulators (PGRs) affected the initiation of axillary buds, and the proliferation efficiency was significantly affected by PGRs. The results showed that the best sterilization method for stem segment explants was as follows: a pretreatment by rinsing with running water for 120 min, soaking in 75% ethanol for 50 s, soaking in 0.1% mercuric chloride for 10 min, and medium supplemented with 3 mL/L PPM. When inoculated on the medium in spring, the contamination rate was as low as 25.56%. The optimal initiation medium for axillary buds in stem segments was half-strength Murashige and Skoog (1/2 MS) medium supplemented with 0.5 mg/L 6-benzyladenine (6-BA). The induction rate was as high as 93.33%, and the mean length of axillary buds was 2.47 cm. The optimal proliferation medium was 1/2 MS medium supplemented with 4.0 mg/L 6-BA and 0.2 mg/L indole-3-butyric acid (IBA). The induction rate was up to 80.00%, the total proliferation coefficient was 4.56, and the net proliferation coefficient was 5.69. The 1/2 MS medium supplemented with 0.1 mg/L 6-BA and 1.5 mg/L indole-3-acetic acid (IAA) was most conducive to the elongation of the adventitious shoot, and the adventitious shoot of approximately 1 cm reached 1.93 cm after culturing for 14 days. The best medium for adventitious shoot rooting was 1/2 MS medium supplemented with 0.1 mg/L α-naphthalene acetic acid (NAA), the highest rooting rate was 82.00%, and the survival rate of transplanting was over 90%.

Effect of Different Concentrations and Combinations of BAP and NAA on Micropropagation of Anthurium (Anthurium andraeanum Linn.) Red

Anthurium anderanum from Araneae family, is a plant with high commercial value. Tissue culture technique appears as an alternative way propagation to increase Anthurium production. The recent research intended to establish an efficient regeneration method for Anthurium andraeanum tissue culture. Young Anthurium plantlets including young shoots, petioles and leaves were applied for regeneration through in vitro indirect organogenesis method from the undefined plant tissues. Callus proliferation was induced by the use of Morishige and Skoog media along with 0.6 mg/l 2,4- Dichlorophenes acetic acid. Shoot regeneration medium consisted of MS salts with supplemented different combinations of BAP and NAA. The number of shoots per explants was significantly increased in 7 months of culture periods in all different combinations but there were found that 14 shoots/explant and 13.9 shoots/explant in NA+ BAP (0+3mg/l) and (0.2+0mg/l) respectively. The longest shoot length (17.3 cm) was found in NAA+BAP (0+0.5mg/l). Rooting was found automatically in shoot proliferation medium. Regenerated plantlets were potted in mixture of broken brick, coco husk and charcoal and successfully acclimatized.

Isolation of adult rat kidney derived stem cells and differentiation into podocyte-like cells

Studies have shown that adult stem cells can be isolated from different organs. However, no agreement has yet been reached on the identification of adult kidney stem cells. In this study, we have shown that the adult rat kidney contains a population of stem cells that are isolatable and, under suitable culture conditions, these stem cells can differentiate into podocyte. Six adult Wistar rats were used in the present study. Their kidneys were chopped and exposed to collagenase I. Then, with passing through 100 μm mesh, single cells in filtered fraction were cultured in a proliferation medium. After 4 passages, the cells were analyzed by flow cytometry, differentiation and gene expression. Morphological analyses revealed that isolated cells are spindle-shaped, and can differentiate into osteocytes and adipocytes. Flow cytometry demonstrated that these cells expressed cell surface markers CD44, CD90, CD133, c-kit, Pax- 2, Oct4, sca-1 and vimentin significantly. Also, the high expression of Wt-1 and Wnt-4 genes was seen in these cells. Moreover, expression of synaptopodin and podocalyxin genes showed differentiation of stem cells into podocyte cells. Our findings demonstrate that isolated cells from the kidney of adult rats are mesenchymal stem cells or progenitor cells and can differentiate to podocyte-like cells.

Somatic embryogenesis mediated micropropagation of polyembryonic cultivars of mango Vellaikolumban and Olour using nucellus tissue

Abstract Somatic embryogenesis mediated micropropagation from nucellus tissue was attempted in polyembryonic mango at the ICAR-Indian Institute of Horticultural Research, Bengaluru during 2016–2019. Studies were conducted to optimize the different stages of somatic embryogenesis namely induction, proliferation, conversion, maturation, germination of somatic embryos and ex vitro establishment of plantlets using nucellus tissue. For culture initiation, various developmental stages of fruits ranging from less than 20 days post-pollination to more than 60 days-post pollination of cultivars Vellaikolumban and Olour were utilized. Fruits of 30–40 days post-pollination in Vellaikolumban and 40–50 days post-pollination in Olour gave better percentage callusing with fewer days for callus initiation and callus formation. Embryonic calli developed on induction medium containing Rugini olive (RO) constituents, 6% sucrose, and 5 ppm each of 2, 4-Dichlorophenoxyacetic acid (2, 4-D) and Giberellic acid (GA3). Upon shifting to proliferation medium (RO with 6% sucrose, 5 ppm each of 2, 4-D and GA3, 400 mgL−1of glutamine and 20% v/v coconut water) pro-globular and globular embryos were formed within 4–5 weeks. Further development of somatic embryos through early heart, late heart and cotyledonary stages occurred in conversion medium (half strength B5 salts, full RO organics, 5 ppm of Indole-3-Acetic Acid (IAA), 20% coconut water and 200 mgL−1 of casein hydrolysate). Maturation of embryos was obtained by using 0.01 mg L−1of Abscisic acid (ABA) and 100 mg L−1of Polyethylene glycol (PEG). Germination of embryos with shoot and root initiation was observed in the presence of 5 ppm of zeatin. Our results show that somatic embryogenesis can be used as an alternative method to conventional propagation methods for rapid multiplication of uniform planting material in polyembryonic mango cultivars.

Rosmarinic acid production in cell suspension cultures of Ehretia asperula Zollinger &amp; Moritz

Plant cell cultures provide an alternative means for producing secondary compounds in food, cosmetic and pharmaceutical industries. Ehretia asperula Zollinger &amp; Moritzi is used as a traditional medicine for the treatment of liver detoxification, ulcers, tumors, inflammation and enhancing the body's resistance in Vietnam. The study was carried out to select suitable callus line for cell suspension cultures of E. asperula Zollinger &amp; Moritzi and investigate the effects of inoculum size, rotation speed and naphthalene acetic acid (NAA) on the proliferation of cell suspension cultures. In addition, the influence of light intensity on the growth and rosmarinic acid (RA) biosynthesis of cell suspension was also surveyed. After 4 weeks of culture, the white to pale yellow friable callus expanded significantly with a fresh weight (FW) of 0.788 g and a high RA content of 2.062 mg/g FW. An appropriate medium for cell proliferation was the liquid B5 medium, which contained 30 g/l glucose, 0.1 mg/l benzyl adenine (BA) and 0.4 mg/l NAA. The results also demonstrated that a 1:20 ratio (w/v) inoculum size, darkness and rotation speed of 90 rpm were the optimal conditions for the proliferation and RA accumulation to 188.217 mg/l in 4 weeks of culture. These findings showed that E. asperula Zollinger &amp; Moritzi cell suspension cultures could be a potential alternative approach for RA production in vitro.

Evaluating the role of nitric oxide in myogenesis in vitro

Skeletal muscle injury activates satellite cells to proliferate as myoblasts and migrate, differentiate and fuse with existing fibres at the site of injury. Nitric oxide (NO), a free radical produced by NO synthase, is elevated and supports healing after in vivo injury. NOS-independent elevation of NO levels in vitro is possible via donors such as molsidomine (SIN-1). We hypothesized that alterations in NO levels may directly influence myogenic processes critical for skeletal muscle wound healing. This study aimed to clarify the role of NO in myoblast proliferation, migration and differentiation. Baseline NO levels were established in vitro, whereafter NO levels were manipulated during myogenesis using l-NAME (NOS inhibitor) or SIN-1. Baseline NO levels generated by myoblasts in proliferation media did not change 1 h after stimulation. Addition of a pro-proliferative dose of HGF slightly elevated NO levels 1 h post-stimulation, whereas cell numbers assessed 24 h later increased significantly; l-NAME reduced the HGF-driven increase in NO and proliferation, reducing wound closure over 16 h. In differentiation media, NO levels increased significantly within 24 h, returning to baseline over several days. Regular addition of l-NAME to differentiating cells significantly reduced NO levels and fusion. SIN-1 increased NO levels in a dose-dependent manner, reaching maximal levels 16 h post-treatment. SIN-1, added at 0, 2 and 4 days, significantly increased myofiber area (26 ± 1.8% vs 18.6 ± 3.4% in control at 5 day, p < 0.0001), without affecting proliferation or migration. In conclusion, this study demonstrates that, during skeletal muscle regeneration, increased NO specifically stimulates myoblast differentiation.

Genomic Methylated Cytosine Level during the Dedifferentiation and Cellular Competence in Coffea arabica Lines: Insights about the Different In Vitro Responses

Coffea arabica genotypes present distinct responses in vitro, and somaclonal variation occurrence has been reported. Global cytosine methylation is one of the epigenetic mechanisms that influences the Coffea in vitro responses. We aimed to establish the indirect somatic embryogenesis in C. arabica ‘Catuaí Vermelho’, ‘Caturra’ and ‘Oeiras’, associate the distinct responses to the methylated cytosine genomic level, and check the ploidy stability. Leaf explants were cultured in callus induction and proliferation medium. The resulted calli were transferred to the regeneration medium, and the mature cotyledonary somatic embryos were transferred to the seedling medium. ‘Oeiras’ exhibited the highest number of responsive leaf explants, followed by ‘Caturra’ and ‘Catuaí Vermelho’. Global methylated cytosine level increased over time in the ‘Catuaí Vermelho’ and ‘Caturra’ friable calli, remaining constant in ‘Oeiras’. ‘Oeiras’ did not regenerate somatic embryos, while ‘Catuaí Vermelho’ exhibited the highest number. Somatic embryo regeneration was associated with the increase of the methylated cytosine level. However, the ‘Catuaí Vermelho’ embryogenic calli showed a lower methylated cytosine level than ‘Caturra’. Recovered plantlets exhibited the same 2C value and chromosome number to the explant donors. Therefore, cytosine hypermethylation occurred during C. arabica indirect somatic embryogenesis, influencing cell competence and somatic embryos regeneration.

DEVELOPMENT OF IN VITRO PLANT REGENERATION PROTOCOL OF BANANA (MUSA SP.) CV. GRAND NAINE USING SUCKER EXPLANT

Banana is an important fruit crop belongs to the family Musaceae. This has more demand for it multifarious uses like food, medicinal as well as industrial values. The present study was carried out to develop micropropagation protocol for large scale production of banana cv. Grand naine using sucker explant. Sucker explants were inoculated on Murashige and Skoog’s (1962) (MS) basal medium and MS basal medium supplemented with different types and concentrations and combination of plant growth regulators. Highest mean number of shoots (10.2) per explant having mean shoot length 5.2 cm was observed on MS medium supplemented with 4.0 mg/L BA, 2.0 mg/L Z, 1.0 mg/L NAA, and 3.0 mg/L ADS. For large scale production of shoot, in vitro regenerated shoots were harvested, cut into small pieces and inoculated on the optimum medium for multiple shoot proliferation. In this way, more than thousand numbers of in vitro shoots were regenerated from a single explant at six month of culture. In vitro regenerated shoots were excised and rooted on ½ MS medium supplemented with 1.0 mg/L IBA. Finally in vitro regenerated plants were acclimatized and subsequently transferred to field with zero mortality. This protocol helps to meet the demand of the farmers.

Embryogenic callus induction from immature zygotic embryos and genetic transformation of Larix kaempferi 3x Larix gmelinii 9.

To date, there are few reports of the successful genetic transformation of larch and other conifers, mainly because it is difficult to transform and integrate exogenous genes. In this study, hybrid larch Larix kaempferi 3x Larix gmelinii 9 cones were collected on June 27, July 1, July 4, July 7 and July 16, 2017. Embryogenic callus induction was studied using a combination of different plant growth regulators and concentrations. The results showed that July 1 was the best stage; the highest induction rate was 10.83%, which cultured in BM medium (Button medium, which formula was listed in S1 Table) with 1.0 mg/L 2,4-D (2,4-dichlorophenoxyacetic acid) and 0.2 mg/L KT(kinetin). When cultured on a proliferation medium for 12 days, proliferation was the fastest, reaching 323.08%, which could also maintain the freshness and vitality. The suitable pre-culture medium for somatic embryogenesis was 1/4 BM medium containing 10 g/L inositol and 60 g/L sucrose. The combination of 45 mg/L ABA (abscisic acid) and 75 g/L PEG4000 (Polyethyene glycol 4000) could promote the number of somatic embryos, and reached the maximum, 210 140 per 1 g FW. The genetic transformation was carried out by the Agrobacterium-mediated transformation method with embryogenic callus cultured for 12 days. The results showed the optimal OD600 of the infection solution(suspension of A. tumefaciens) was 0.5, co-culture time was 2 days, and screening concentration of Hyg (hygromycin B) was 4 mg/L. In this study, the transformation rate of resistance callus was 32.1%. It provides a reference for low genetic transformation efficiency of larch at present. This study could be beneficial for the innovation and breeding of larch by genetic engineering and provides a certain basis for rapid propagation of excellent larch germplasm resources and genetic engineering breeding of larch and other conifers.

In Vitro Rapid Multiplication of a Highly Valuable Ornamental Aquatic Plant Anubias Heterophylla

The present study focuses on the development of a micropropagation protocol for true to type plants of Anubias heterophylla, a valuable ornamental aquatic plant. Natural propagation of aquatic species is limited due to the production of a small number of plants with a long cultivation period, disease and requirement of a large space for propagation. Surface-disinfected shoot tip explants were cultured on MS medium containing 0.1 % (w/v) activated charcoal (AC) and supplemented with 6-benzylaminopurine (BAP) at different concentrations (0, 1.0, 3.0 and 5.0 mg/L) either singly or in combination with alpha Naphthalene acetic acid (NAA) and 2,4-Dichlorophenoxy acetic acid (2,4-D) (0.5 and 1.0 mg/L). Among different auxins and cytokinin used, the number of shoots initiated per explant varied significantly among the different auxin and cytokinin combinations. The highest number of shoots per explants (3.60 ± 0.24) was formed in the culture medium containing 1.0 mg/L NAA in combination with 1.0 mg/L BAP, followed by in the culture medium with 3.0 mg/L BAP (2.40 ± 0.24). In the culture medium with 1.0 mg/L NAA and 1.0 mg/L BAP, 100 % of the cultured explants regenerated new shoots within 60 days. The maximum number of roots (19.80 ± 0.20 roots) per explant was observed after 60 days of culture on MS medium contained 0.1 % (w/v) AC and supplemented with 1.0 mg/L NAA and 1.0 mg/L BAP. With a 100% survival rate, the regenerated plantlets were effectively acclimatized to submerged underwater environments. Plantlets exhibited phenotypic traits that were comparable to those of their donor plant. This protocol is suited for commercial Anubias heterophylla propagation in the aquarium industry. HIGHLIGHTS The development of a micropropagation protocol for true to type plants of Anubias heterophylla The initiation of aseptic cultures of Anubias heterophylla from shoot tip explants Optimization of culture medium for shoot proliferation Rooting micro shoots and hardening of rooted micro shoots

Characterization of a dual media system for culturing primary normal and Fuchs endothelial corneal dystrophy (FECD) endothelial cells.

Primary cultures of human corneal endothelial cells (HCECs) are an important model system for studying the pathophysiology of corneal endothelium. The purpose of this study was to identify and validate an optimal primary culture model of normal and Fuchs endothelial corneal dystrophy (FECD) endothelial cells by comparing cell morphology and marker expression under different media conditions to in vivo donor tissues. Primary and immortalized HCECs, isolated from normal and FECD donors, were cultured in proliferation media (Joyce, M4, Bartakova) alone or sequentially with maturation media (F99, Stabilization 1, M5). CD56, CD73 and CD166 expressions were quantified in confluent and matured cell lines by flow cytometry. HCECs that were allowed to proliferate in Joyce’s medium followed by maturation in low-mitogen containing media yielded cells with similar morphology to corneal endothelial tissues. Elevated expression of CD56 and CD166 and low expression of CD73 correlated with regular, hexagonal-like HCEC morphology. CD56:CD73 > 2.5 was most consistent with normal HCEC morphology and mimicked corneal endothelial tissue. Immortalization of normal HCECs by hTERT transduction showed morphology and CD56:CD73 ratios similar to parental cell lines. HCECs established from FECD donors showed reduced CD56:CD73 ratios compared to normal HCECs which coincided with reduced uniformity and regularity of cell monolayers. Overall, a dual media system with Joyce’s medium for proliferation and a low-mitogen media for maturation, provided normal cultures with regular, hexagonal-like cell morphologies consistent with corneal endothelial cells in vivo. CD56:CD73 expression ratio >2.5 was predictive of in vivo-like cellular morphology.

β1-adrenergic receptor but not β2 mediates osteogenic differentiation of bone marrow mesenchymal stem cells in normotensive and hypertensive rats

The sympathetic nervous system regulates bone remodeling via adrenergic receptors on the surface of bone cells. Herein, we evaluated the role of beta-adrenergic receptors (ADRBs) in osteoblastic differentiation of bone marrow mesenchymal stem cells (BMSCs) derived from normotensive (Wistar) and spontaneously hypertensive rats (SHRs). BMSCs were cultured in a proliferation medium or osteogenic medium (OM). Cells cultured in OM were treated with carvedilol (Cv) or nebivolol (Nb).In OM, cell proliferation was decreased in both strains. In Wistar rats, Cv increased BMSC proliferation and increased alkaline phosphatase (ALP) activity in OM. Both Cv and Nb decreased ALP activity. In addition, Cv and Nb reduced mineral deposition in Wistar rats. Moreover, NB decreased mineralization in SHRs, exhibiting superior efficacy. In OM, cells from Wistar rats and SHRs showed Adrb1 and Adrb2 expression. On day 7, Nb, but not Cv, reduced Adrb1 levels in BMSCs from Wistar rats. Nb inhibited Adrb2 in both strains, and Cv demonstrated superior efficacy. In BMSCs from Wistar rats, both antagonists inhibited Runx2, osterix, and β-catenin; in SHRs, Cv and Nb inhibited only osterix. Cv decreased osteopontin (Opn), osteocalcin (Ocn), and bone morphogenetic protein (Bmp2) in BMSCs from Wistar rats, inhibiting only Opn in SHRs. Nb effectively inhibited Ocn, bone sialoprotein, and Bmp2, but not Ocn, in BMSCs from Wistar rats, while suppressing Opn in BMSCs from SHRs. In addition, Nb inhibited p-p38 in BMSCs from Wistar rats; Cv inhibited p-p38 in BMSCs from SHRs. In Wistar rats, both antagonists inhibited p-ERK and reduced p-JNK; Cv reduced these expressions only in SHRs. In conclusion, ADRB1, but not ADRB2, could be involved in the osteogenic differentiation of BMSCs from Wistar rats and SHRs. The high ADRB1 expression might suppress the effect of ADRB2 on BMSCs.