Proliferation Medium Research Articles

PLANT REGENERATION FROM SHOOT TIPS IN TRIPLOID LOQUAT

Triploid loquat (Eriobotrya japonica Lindl.) was regenerated for the first time using shoot tips as explants. Several experiments were carried out to investigate the effects of time of sampling and size of the explants, growth regulators on shoot tip sprouting, elongation, rooting and transplantation. Results showed that sampling in spring and shoot-tip size of 0.7 cm were appropriate, resulting in a survival rate of 77.5%. Appropriate initial culture medium was MS + 6-BA 1.0 mg L-1 + NAA 0.5 mg L-1 + NAA 1.5 mg L-1, in which the sprouting rate reached 91%. Suitable medium for shoot elongation was MS + 6-BA 0.5 mg L-1 + NAA 0.3 mg L-1 + GA3 0.3 mg L-1, which resulted in an average shoot height of 4.9 cm. The most suitable medium for shoot proliferation was MS + 6-BA 1.0 mg L-1 + NAA 0.3 mg L-1 + Tryptone 750 mg L-1, in which a high multiplication coefficient of 7.7 was obtained. The most suitable medium for rooting was ½ MS + NAA 0.25 mg L-1 + IBA 2.0 mg L-1, in which the rooting rate was 73.3%. The rooted plantlets, after hardening, survived at a rate of 93.67% in a transplantation substrate composed by organic fertilizers, garden soil, and sawdust (1:2:1).

TIPS to manipulate myogenesis: retention of myoblast differentiation capacity using microsphere culture.

Cell therapy is an emerging option for regenerating skeletal muscle. Improved delivery methods for anchorage-dependent myoblasts are likely to improve integration and function of transplanted muscle cells. Highly porous microspheres, produced using thermally induced phase separation (TIPS), have features ideally suited for minimally invasive cell delivery. The purpose of this study was to investigate, for the first time, the use of TIPS microspheres as highly porous microcarriers for manipulation of human skeletal muscle myoblasts (HSMM) under defined culture conditions. HSMM cells readily attached to the surface of poly (DL-lactide-co-glycolide) (PLGA) TIPS microcarriers, where they were induced to continue proliferating or to be driven towards differentiation whilst under static-dynamic culture conditions for 7 days. Switching from proliferation medium to differentiation medium for 7 days, resulted in increased protein expression of skeletal muscle cell contractile apparatus components, MyoD and skeletal muscle myosin heavy chain, compared with cells cultured on conventional culture plasticware for the same duration (p < 0.001). Growth of myoblasts on the surface of the microcarriers and their migration following simulated delivery, caused no change to the proliferative capacity of cells over 7 days. Results from this study demonstrate that TIPS microspheres provide an ideal vehicle for the expansion and delivery of myoblasts for therapeutic applications. Transplantation of myoblasts anchored to a substrate, rather than in suspension, will reduce the amount of ex vivo manipulation required during preparation of the product and allows cells to be delivered in a more natural state. This will improve the ability to control cell dosage and increase the likelihood of efficacy.

Effects of nanosilicon dioxide application on in vitro proliferation of apple rootstock

ABSTRACTAlthough silicon (Si) is the second most abundant element of the earth's crust and in soils, it is not listed among the essential elements for plants. However, the beneficial role of Si in stimulating the growth and development of many plant species has been recognized. This study investigated the effects of in vitro application of nanosilicon oxide on growth and proliferation of apple rootstock MM106 in tissue culture. The experiment included five levels nanosilicon oxide (0, 25, 50, 100, and 200 mg/L) added to Murashige and Skoog medium. The results showed that using nanosilicon increased in fresh and dry weights, length and number of branches, and chlorophyll in explants with the highest increase being at 100 mg/L. Growth suppression occurred at 200 mg/L. This investigation showed that 100 mg/L silicon oxide can be added to Murashige and Skoog medium for fast growth and proliferation of MM106 apple rootstock explants.

Effects of growth retardants on sprouting and development of apricot (Prunus armeniaca L.) and neem (Azarchta indica A. Juss.) nodal buds

As a first step to the establishment of an in vitro conservation protocol for apricot and neem, the effect of several growth retardants on the sprouting and development from nodal buds of both species and meristem development at the histological level was evaluated. Nodal section bud sprouting was affected by growth retardants, ranged from 4 to 100 % in apricot and from 10 to 100 % in neem, depending on the retardant and its concentration. Retardants paclobutrazol (PBZ), flurprimidol (FMD), diniconazole (DIN), and acetyl-salicylic acid (ASA) produced a drastic reduction of shoot length in apricot. PBZ, DIN and ASA decreased the number of leaves, whereas 1-Aminocyclopropane-1-carboxylic acid (ACC) at 10−5 µM improved the number of leaves. Length of neem sprouted buds as well as the number of leaves was affected by all of the growth retardants, with the exception of ACC that only affected the number of leaves. Triiodobenzoic acid (TIBA) at 10−5 µM improved neem shoot length. PBZ, FMD, and DIN produced several effects as very thin, long, and dark green colored leaves in shoots from both species. Once growth retardants were removed from the proliferation medium the recovery of apricot bud growth was good from PBZ and FMD at low and medium concentrations and from ACC and ASA at all concentrations tested. In neem, the recovery was good only from TIBA and ASA at low concentrations; TIBA at 10−5 µM produced an increment of shoot length higher than the control. Histological changes observed in the meristems of apricot and neem buds during growth retardation induced by FMD and ASA could be related to some extent with those observed in the dormant shoot apex.

Characterization of L‐Threonine transport in mouse embryonic stem cells

Stem cells are at the forefront of current regenerative and biomedical research. There exists an imperative need to understand the mechanisms that drive stem cell function in order to exploit their use as a therapeutic tool. Amino acids are potent inducers of signaling cascades that drive stem cell proliferation and differentiation. With a focus on mouse embryonic stem (mES) cells, L‐threonine is the only amino acid required in culture medium for mES cell proliferation. Current research associates this need for L‐threonine with threonine dehydrogenase (TDH), which catabolizes L‐threonine to glycine and acetyl‐CoA in mES cells. While this may be true for mES cells, human embryonic stem (hES) cells do not express a functional tdh gene. Additionally, the concentration of 3‐hydroxynorvaline (3‐HNV), a potent inhibitor of TDH, needed to inhibit mES cell proliferation is more than an order of magnitude less than the apparent Ki for TDH inhibition. Such findings suggest a different mechanism responsible for the relationship between L‐threonine and ES cell proliferation, which may be explained by Thr transport. To explore this novel idea, we characterized the L‐threonine transport systems in mES cells by conducting radiolabelling transport studies. We found that there is a Na+‐dependent and Na+‐independent component of transport, with the Na+‐dependent component predominating. We also found that Thr, Ser, Cys, Ala, 4‐OH‐Pro, Asn, 3‐HNV, and Met were the strongest inhibitors of the Na+‐dependent component of transport. Such findings are consistent with characteristics of the ASC transport system, suggesting that this system is responsible for majority of Thr transport in mES cells. We confirmed the presence of the ASC system transporters, ASCT1 and ASCT2, in mES cells using RT‐PCR. Project supported by Midwestern University.

Optimizing the concentrations of plant growth regulators for in vitro shoot cultures, callus induction and shoot regeneration from calluses of grapes

&lt;p style="text-align: justify;"&gt;&lt;strong&gt;Aim&lt;/strong&gt;: To optimize the concentrations of growth regulators in the media for the proficient micropropagation of grapevine (&lt;em&gt;Vitis vinifera &lt;/em&gt;L.) cv. King’s Ruby.&lt;/p&gt;&lt;p style="text-align: justify;"&gt;&lt;strong&gt;Methods and results&lt;/strong&gt;: Apical meristems of the grape cultivar were used to establish &lt;em&gt;in vitro&lt;/em&gt; shoot cultures. Nodal explants, each containing an axillary bud, taken from &lt;em&gt;in vitro&lt;/em&gt; grown shoots were inoculated in shoot proliferation medium, i.e., half strength Murashige and Skoog (MS) medium supplemented with benzyl aminopurine (BAP), kinetin, glycine and gibberellic acid (GA&lt;sub&gt;3&lt;/sub&gt;). A higher number of shoots (5.33) with greater shoot length (2.75 cm) was produced in the medium supplemented with 1.0 mg L&lt;sup&gt;-1&lt;/sup&gt; BAP and 0.1 mg L&lt;sup&gt;-1&lt;/sup&gt; GA&lt;sub&gt;3&lt;/sub&gt;. Calluses were induced from leaf explants taken from &lt;em&gt;in vitro&lt;/em&gt; grown shoots. Callus induction was greater (73.00%) on the medium containing 2.0 mg L&lt;sup&gt;-1&lt;/sup&gt; 2,4-dichlorophenoxyacetic acid (2,4-D), 0.3 mg L&lt;sup&gt;-1&lt;/sup&gt; BAP and 0.2 mg L&lt;sup&gt;-1&lt;/sup&gt; α-naphthaleneacetic acid (NAA). The maximum frequency of shoot regeneration (53.33%) was achieved on the medium supplemented with 1.5 mg L&lt;sup&gt;-1&lt;/sup&gt; BAP and 0.5 mg L&lt;sup&gt;-1&lt;/sup&gt; NAA, and the regenerated shoots successfully formed roots on growth regulator-free half strength MS medium.&lt;/p&gt;&lt;p style="text-align: justify;"&gt;&lt;strong&gt;Conclusion&lt;/strong&gt;: Optimizing the concentration of BAP and GA&lt;sub&gt;3&lt;/sub&gt; and omitting the glycine and kinetin in the culture medium increased the number and length of shoots. Similarly, for inducing the callus of the leaf explants, taken from &lt;em&gt;in vitro&lt;/em&gt; grown shoots, it is recommended to adjust the medium with the higher concentration of 2,4-D and lower concentrations of BAP. Moreover, the maximum number of shoots was regenerated on a medium supplemented with relatively high levels of both BAP and NAA (1.5 and 0.5 mg L&lt;sup&gt;-1&lt;/sup&gt;, respectively). Finally, we suggest the half strength MS medium that is free from growth regulators for the root formation of the regenerated shoots.&lt;/p&gt;&lt;p style="text-align: justify;"&gt;&lt;strong&gt;Significance and impact of the study&lt;/strong&gt;: Optimizing the concentration of growth regulators is crucial for the efficient micropropagation of a grape cultivar. Knowing the specific balance between the growth regulators is necessary to establish &lt;em&gt;in vitro&lt;/em&gt; shoot cultures, callus induction and shoot regeneration and, hence, to propagate disease-free true to type grape cultivars in a short time.&lt;/p&gt;

Role of NF-κB/survivin signal pathway on intima hyperplasia of rat carotid balloon injury restenosis model

To investigate the role of NF-κB/survivin signal pathway in the intima hyperplasia of rat carotid balloon injury restenosis model. NF-κB siRNA lentivirus vector (titer was 1 × 10⁸ TU/ml) was established. Carotid balloon injury restenosis model was made in 33 SD rats. The rats were divided into 4 groups according to different processing methods, including negative control (NC) group (n = 11), NF-κB siRNA group (n =11), NF-κB siRNA+YM155 (survivin inhibitor) (n = 11), the uninjured carotid artery served as the normal control group (n = 11). After 7 days, the carotid sample (n = 5 each group) were harvested to detect the NF-κB and survivin mRNA expression by RT-PCR.The carotid sample were harvested on 28 days (n = 6 each group) for HE staining and measuring intima hyperplasia. Immunohistochemical method was also used to detect the expression of intima proliferation cell nuclear antigen (PCNA) and media α-SM-actin. (1) After 7 days, NF-κB and survivin mRNA expression was significant higher in NC group than in normal control group (P<0.05), the NF-κB mRNA expression was significantly lower in NF-κB siRNA group than in NC group (P<0.05) and similar between NF-κB siRNA group and NF-κB siRNA+YM155 group. The survivin mRNA expression was significantly lower in NF-κB siRNA group compared to NC group (P<0.05) and significantly higher in NF-κB siRNA group than in NF-κB siRNA+YM155 group (P<0.05). (2) After 28 days, intima hyperplasia was observed in NC (0.13 ± 0.01), NF-κB siRNA (0.11 ± 0.01) and NF-κB siRNA+YM155 group (0.09 ± 0.01) mm² (P<0.05). Media area was similar among NC group, NF-κB siRNA group and NF-κB siRNA+YM155 group (P>0.05). I/M ratio was gradually reduced among NC group (1.55 ± 0.07), NF-κB siRNA group (0.92 ± 0.08), NF-κB siRNA+YM155 group (0.76 ± 0.06, all P<0.05). Similar results were found in the residual restenosis rate: NC group (58.71 ± 0.02) %, NF-κB siRNA group (32.13 ± 0.05) %, NF-κB siRNA+YM155 group (26.42 ± 0.03) % (all P<0.05) and expression of vascular smooth muscle cell PCNA: NC group (45.32 ± 7.21) %, NF-κB siRNA group (36.54 ± 6.42) %, NF-κB siRNA+YM155 group (28.57 ± 6.31) % (all P<0.05). On the contrary, the IOD of α-SM-actin in media increased gradually: NC group (0.055 ± 0.006), NF-κB siRNA group (0.072 ± 0.011), NF-κB siRNA+YM155 group (0.084 ± 0.008, all P<0.05). Inhibiting NF-κB expression can significant decrease intima hyperplasia in this model, and this effect may be mediated by inhibiting survivin and reducing the proliferation of vascular smooth muscle cells.

Micropropagation of centennial tertiary relict trees of Liquidambar orientalis Miller through meristematic nodules produced by cultures of primordial shoots

Key message This paper provides an important mi- cropropagation method that might be used for conser- vation and commercial production of tertiary relict tree Liquidambar orientalis. Abstract The Liquidambar orientalis Miller is an impor- tant tree as a tertiary relict endemic species in terms of plant biodiversity, and has economic value due to the balsam it produces. In the present study, an efficient micropropaga- tion method was developed for the L. orientalis. Initially, primordial shoot explants isolated from axillary buds were cultured on Murashige and Skoog and Woody Plant Medium (WPM) containing different plant growth regulators (PGRs). The highest number of shoots per ex- plant was obtained on WPM supplemented with 1.0 mg/L 6-benzylaminopurine (BAP) and 1.0 mg/L indole-3-butyric acid (IBA) (shoot proliferation medium: SPM). These shoots were then subcultured continuously on SPM for a period of 18 months. Meristematic nodule clusters were formed at the base of the cultured shoots in SPM, where multiple shoots developed from them. The number of shoots per explant was increased approximately 2.8-fold by ap- plying various strategies such as different explant type (single shoots and shoot clusters) and culture vessels (Glass Tube, 210-cc Glass culture jar, 400-cc Glass culture jar, and Vitro Vent). Shoot clusters cultured in ventilated Vitro Vent vessels on SPM gave best result for shoot propaga- tion. In vitro shoots rooted best on WPM containing 30 g/L sucrose, 120 mg/L ethylenediamine di-2-hydroxyphenyl acetate ferric (Fe-EDDHA) and 4.0 mg/L IBA. All plantlets were successfully acclimatized in a glasshouse and then plants were transferred to the field. This methodology has been adapted by a commercial producer.

IN VITRO SHOOT REGENERATION OF INDONESIAN BANANAS (Musa spp.) cv. AMBON KUNING AND RAJA BULU, PLANTLET ACCLIMATIZATIONAND FIELD PERFORMANCE

The aim of this study was to observe responses of two banana cultivars ‘Ambon’ and ‘Raja Bulu’ on different BA concentrations and effects of different media and fertilizer on survival and growth of plantlets. Sterilized explants were cultured on initiation medium (MS with 1mg L -1 BA) for 4 weeks, then subjected to media MS with 2.5, 5.0, or 7.5 mg L -1 BA. Numbers of shoot buds, shoots and propagules were recorded after 4 consecutive passages with 4 weeks intervals. Rooted plantlets were acclimatized in three different media, then treated with or without NPK (32:10:10) fertilizer solution once a week. After 2 months, the survival and growth of plantlets were recorded. Cultures of banana ‘Ambon Kuning’ showed higher regenerative capacity compared to ‘Raja Bulu’, producing higher numbers of shoot buds, shoots and propagules. The best medium for propagule proliferation of both banana cultivars was MS+5 mg L -1 BA, producing 40.7 propagules for ‘Ambon Kuning’, and 12.3 propagules for ‘Raja Bulu’ per explant. In all acclimatization media tested, 100% of plantlet survival was achieved. The best plantlet growth was found in sand: compost (1:1,v/v) with application of NPK solutions. The in vitro -derived plants were planted in the field and produced fruits of high quality.

Cation Exchange Properties of Zeolites in Hyper Alkaline Aqueous Media

Construction of multibarrier concrete based waste disposal sites and management of alkaline mine drainage water requires cation exchangers combining excellent sorption properties with a high stability and predictable performance in hyper alkaline media. Though highly selective organic cation exchange resins have been developed for most pollutants, they can serve as a growth medium for bacterial proliferation, impairing their long-term stability and introducing unpredictable parameters into the evolution of the system. Zeolites represent a family of inorganic cation exchangers, which naturally occur in hyper alkaline conditions and cannot serve as an electron donor or carbon source for microbial proliferation. Despite their successful application as industrial cation exchangers under near neutral conditions, their performance in hyper alkaline, saline water remains highly undocumented. Using Cs(+) as a benchmark element, this study aims to assess the long-term cation exchange performance of zeolites in concrete derived aqueous solutions. Comparison of their exchange properties in alkaline media with data obtained in near neutral solutions demonstrated that the cation exchange selectivity remains unaffected by the increased hydroxyl concentration; the cation exchange capacity did however show an unexpected increase in hyper alkaline media.

Improved secondary embryo production in Quercus alba and Q. rubra by activated charcoal, silver thiosulphate and sucrose: influence of embryogenic explant used for subculture

In somatic embryogenic systems of various oak species, including Quercus alba and Q. rubra, embryogenic lines generally proliferate through generation of nodular embryogenic structures (NS) as a form of repetitive embryogenesis, without embryo development progressing to the cotyledonary stage, or with a low proportion of embryo formation, resulting in the inability to achieve suitable material for plant recovery. In experiments we used previously initiated embryogenic lines derived from 7-year-old trees of Q. alba and Q. rubra. The aim of this work was to improve embryo proliferation and histodifferentiation of lines from these oak species to obtain well-developed embryos and plant regeneration. The effects of activated charcoal, silver thiosulphate (STS) and sucrose concentration on secondary embryo production were investigated. Overall, the best embryo proliferation medium consisted of basal Murashige and Skoog medium containing 0.4 % charcoal and 20 µM STS, and supplemented with 6 % sucrose for Q. alba and 3 % sucrose for Q. rubra. In these conditions, well-developed and singularised torpedo- and cotyledonary-shaped embryos were produced. The developmental stage of the embryogenic explant used for subculture significantly influenced embryo production and differentiation of new secondary embryos, with NS being the most effective explant despite being the smallest explant type in terms of size and fresh weight. Addition of STS in the germination medium also had a positive effect on germination response giving rise to approximately 39 % embryo conversion in Q. alba and 11 % in Q. rubra.

Establishment of a picloram-induced somatic embryogenesis system in Vitis vinifera cv. chardonnay and genetic transformation of a stilbene synthase gene from wild-growing Vitis species

To facilitate genetic improvement, an efficient system of embryogenic culture induction, maintenance and transformation in Vitis vinifera cv. Chardonnay was developed using picloram. Whole flowers produced the most embryogenic calluses on induction media containing Murashige and Skoog’s (MS) basal salts and 3.0 mg L−1 picloram with different concentrations of 2,4-dichlorophenoxyacetic acid and 6-benzyladenine. Proembryonic masses (PEM) cultured on maintenance and proliferation media (MPM) containing MS basal salts and picloram had the highest proliferation efficiency within 2 months. Additionally, the best PEM formation was found with MPM containing MS basal salts and 2.0 mg L−1 picloram. Then this PEM cultured on MPM were transferred to embryo germination medium containing MS basal salts, 0.2 mg L−1 kinetin and 0.1 mg L−1 2-naphthoxyacetic acid, the optimal medium for normal plantlets regeneration. A single copy of the stilbene synthase gene VpSTSgDNA2 from Chinese wild Vitis pseudoreticulata was transferred into regenerated Chardonnay via Agrobacterium tumefaciens-mediated transformation and confirmed by Southern blot analysis. The positive transgenic grapevine lines exhibited higher levels of stilbene and H2O2 than wild-type vines and could slightly reduced the growth of powdery mildew.

Effects of Abiotic Factors on Cell Biomass and Rosmarinic Acid Production in Cell Suspension Cultures of Orthosiphon stamineus Benth

Orthosiphon stamineus Benth. is a medicinal plant belongs to Lamiaceae family. Due to the high variations in rosmarinic acid produced from the field-derived plants, cell suspension culture techniques was previously suggested and practiced as the alternative for cell biomass and rosmarinic acid (RA) production. In the present study, the effects of medium pH, photoperiod, temperature and sucrose were evaluated on cell biomass and rosmarinic acid production using three different cell lines of O. stamineus. This study revealed that the slow and intermediate growing cell lines had similarities in terms of growth pattern. However, cells cultured in medium supplemented with lower sucrose concentration (30 g/L) were more productive in terms of cell biomass and RA in the intermediate and fast growing cell lines however slow growing cells cultured in proliferation medium with 45 g/l sucrose produced higher amount of RA. Medium pH in the range of 5.65-5.8 was optimal for both cell biomass and RA production. Photoperiod did not have a consistent effect on the three cell line’s biomass production but the alternation of darkness and illumination led to high RA production in the slow growing cell line. This study proved that abiotic factors including medium pH, photoperiod, temperature and carbohydrate source had impacts on cell biomass and rosmarinic acid production although in a diverse range among different growing cell lines

Pore size and LbL chitosan coating influence mesenchymal stem cell in vitro fibrosis and biomineralization in 3D porous poly(epsilon-caprolactone) scaffolds.

Poly(epsilon-caprolactone) (PCL) is a hydrophobic bioplastic under development for bone tissue engineering applications. Limited information is available on the role of internal geometry and cell-surface attachment on osseous integration potential. We tested the hypothesis that human bone marrow mesenchymal stem cells (MSCs) deposit more mineral inside porous 3D PCL scaffolds with fully interconnected 84 or 141 µm pores, when the surfaces are coated with chitosan via Layer-by-Layer (LbL)-deposited polyelectrolytes. Freshly trypsinized MSCs were seeded on PCL 3D cylinders using a novel static cold seeding method in 2% serum to optimally populate all depths of the scaffold discs, followed by 10 days of culture in proliferation medium and 21 additional days in osteogenic medium. MSCs were observed by SEM and histology to spread faster and to proliferate more on chitosan-coated pore surfaces. Most pores, with or without chitosan, became filled by collagen networks sparsely populated with fibroblast-like cells. After 21 days of culture in osteogenic medium, sporadic matrix mineralization was detected histologically and by micro-CT in highly cellular surface layers that enveloped all scaffolds and in cell aggregates in 141 µm pores near the edges. LbL-chitosan promoted punctate mineral deposition on the surfaces of 84 µm pores (p < 0.05 vs. PCL-only) but not the 141 µm pores. This study revealed that LbL-chitosan coatings are sufficient to promote MSC attachment to PCL but only enhance mineral formation in 84 µm pores, suggesting a potential inhibitory role for MSC-derived fibroblasts in osteoblast terminal differentiation.

188 DEVELOPMENT OF AN EFFECTIVE WHOLE-OVARY PERFUSION SYSTEM

The different structures of a mammalian ovary require complex 3-dimensional interactions to function properly. It is difficult to access the ovary in vivo and to study its physiology in vitro, it is necessary to dissect its different parts and culture them individually. Although informative, this approach prevents the understanding of the role played by their interactions. Perfusion systems are available for ovaries of laboratory animals while organs of larger species have been maintained in culture only for a few hours. This has prompted us to develop a system that can preserve the function of a whole sheep ovary for a few days ex vivo so that it is available for analysis in controlled conditions. Twenty-four sheep ovaries were collected at the local abattoir; 18 were assigned randomly to 3 experimental groups (media A, B, and C) and 6 were immediately fixed in 10% formaldehyde and used as fresh controls. Whole ovaries were cultured for up to 4 days using a semi-open perfusion system. Organs were perfused through the ovarian artery, at a flow rate of 1.5 mL min–1 with basal medium (M199, 25 mM HEPES, 2 mM l-glutamine and 100 µg mL–1 antibiotic-antimycotic solution) supplemented with 0.4% fatty acid free BSA (medium A); or 0.4% BSA heat shock fraction (medium B); or 10% FBS, 50 ng mL–1 IGF-1, and 50 mg bovine insulin (medium C). Ovaries were stimulated with FSH (Folltropin®-V, Bioniche Animal Health Inc., Belleville, Ontario, Canada) changing medium in a pulsatile manner (1 mg mL–1 for 2 h; 0.5 mg mL–1 for 2 h; 0 mg mL–1 for 20 h), with the same cycle repeated each day of culture. At every change, aliquots were collected for oestradiol (E2) and progesterone (P4) quantification. After culture, ovaries were examined for follicular morphology, cell proliferation, and apoptotic rate. Statistical analysis was performed using one-way ANOVA (SPSS 20, IBM, Armonk, NY, USA). In media A and B, all morphological parameters showed a small but significant decrease compared to fresh control, only after 3 days of culture. The different BSA in medium B did not affect follicle morphology but significantly increased cell proliferation (medium A, 28.59 ± 3.26%; medium B, 32.04 ± 2.67%) and decreased apoptosis (medium A, 32.51 ± 5.92%; medium B, 24.55 ± 2.55%). In both media, steroid concentration increased after FSH pulses (E2 range 1.95–10.50 pg mL–1; P4 range 0.34–3.08 ng mL–1), reaching levels similar to those measurable in peripheral plasma. The presence of FBS, IGF-1, and insulin in medium C allowed extension of the culture period to 4 days with a percentage of intact follicles comparable to that observed after 3 days in media A and B. Moreover, proliferation rates were comparable to fresh controls. Steroid pattern changed with P4 values dropping close to zero (range 0.03–1.18 ng mL–1) and E2 level (range 23.59–94.98 pg mL–1) increasing 10-fold, achieving a concentration similar to that measured in the ovarian vein around oestrous. Our data indicate that it is possible to support viability of large animal whole ovaries for up to 4 days, providing a physiologically relevant model for studying ovarian functions in vitro. Research was supported by AIRC IG 10376 and by the Carraresi Foundation.

Effect of different concentrations of plant growth regulators on micropropagation of Lantana camara

Lantana camara is a plant with numerous medicinal properties and belongs to Verbenaceae family. Too few studies on micropropagation of this plant exist in the literature. This study was designed and conducted to investigate the effective factors on in vitro shoot proliferation of L. camara. This experiment was conducted in Isfahan University of Technology, Isfahan, Iran. In this study, nodal segments of L. camara were used as explants and woody plant medium (WPM) as culture medium. The explants were placed in culture medium of WPM containing 6-benzyladenine (BA) and thidiazuron (TDZ) in various concentrations for shoot proliferation and MS medium containing different concentrations of indole-3-acetic acid (IAA) for rooting. After four weeks, different indices of shoot proliferation and rooting were investigated. The experiments’ data were analyzed by Statistical Analysis System (SAS) software and the means were compared using Duncan's multiple range test (DMRT) at 5% probability level. The results indicated that the highest shoot and internode length, fresh and dry weight and the maximum number of leaves were obtained in control treatment. The maximum number of shoots was observed in 8 mg/L BA and the highest fresh weight was in 4 mg/L BA. Cytokinins, particularly BA, in high concentrations cause apical dominance to be overcome through declining auxin effect, and proliferation was increased. Therefore, shoot and internode length was decreased with increasing BA concentration. The most number of roots was also obtained in treatment with 0.5 mg/l IAA. The increase in roots’ number occurs because of hydrolysis of foods and their transfer to the sprouts. Key words: Nodal segments, shoot proliferation, thidiazuron, woody plant medium.

Effect of Epoxomicin on 3D Neurosphere

Developmental neurotoxicity (DNT) entails the toxic effects imparted by many chemicals on brain during early childhood. Various chemicals would have their maximum effects on brains during early childhood. Some toxicants have confirmed to induce developmental toxic effects on CNS. Most of agents cannot be identified with certainty due to the defective nature of predictive toxicology models used. A novel method that can overcome most of the limitations of conventional techniques is the use of 3D neurospheres system (in-vitro system can recapitulate most of the changes during the period of brain development making it an ideal model for predicting neurotoxic effects). In the present study we verified the possible DNT of epoxomicin which is a potent anti-tumor agent isolated from Actinomycetes that is used as a selective and irreversible inhibitor of the 20S proteasome with anti-inflammatory activity. Methods Rat neural progenitor cells were isolated from rat embryos (E14) extracted from placental tissue. Cortices were aseptically dissected from brains of the fetuses and the tissues were triturated by repeated passage through a fire-polished constricted Pasteur pipette. The dispersed tissues were allowed to settle for 3 min. The supernatant was then transferred to a fresh tube and centrifuged at 1,000 g for 5 min. The pellet was placed in Hank’s balanced salt solution cultured as free-floating neurospheres in proliferation medium. Two doses of epoxomicin (1µM and 10µM) were used in cultured neurospheres for a period of 14 days. For proliferation analysis, spheres were cultured in proliferation medium. After 0,4,5,11 and 14 days, sphere size was determined by software analyses (CellProfiler, Broad Institute, www.cellprofiler.org) Diameter of each neurosphere was measured and exported as excel file to statistical analysis. For viability analysis, trypsin-EDTA solution were added to neurospheres for 3 min to dissociate them into single cells suspension, then viability was evaluated by the Trypan Blue exclusion test. Epoxomicin found to affect differentiation and viability of neurospheres, these effects were positively correlated to doses and progress of time. Results Epoxomicin found to affect proliferation and viability of neuropsheres. Conclusion The study confirms DNT of epoxomicin and suggests possible gross morphologic changes and the decrease in viability, it proposes possible focal lesions on exposure to epoxomicin in early childhood.

Antimicrobial activity of Melia azedarach fruit extracts for control of bacteria in inoculated in-vitro shoots of ‘MRS 2/5’ plum hybrid and calla lily and extract influence on the shoot cultures

The study aimed to investigate the antibacterial activity of Melia azedarach fruit extracts in plant shoot culture. Unripe fruits were collected in different periods and the extracts were obtained through homogenization in an ethanol (EtOH) solution (40 g fresh weight in 100 ml 50 % EtOH) followed by EtOH evaporation. Their activity was tested against isolates of several bacterial species (Bacillus spp., Pseudomonas spp.) of agronomic interest, and on shoots of ‘MRS 2/5’ (Prunus cerasifera x Prunus spinosa) and calla lily (Zantedeschia aethiopica) inoculated (bacterized) with Microbacterium oleivorans (Mo), Bacillus subtilis (A17), Sphingomonas meloni (Sm) and Bacillus nealsonii (Bn). Shoot number and fresh weight of explants of ‘MRS 2/5’ on the proliferation medium and shoot length on the rooting medium were reduced in cultures derived from shoots bacterized with Mo and A17. Moreover, rooting frequency and root number decreased in A17-bacterized shoots. In calla lily, the number of rooted shoots was strongly reduced also by Sm. The extracts had a bactericidal activity against Mo, A17, Bn and most other Bacillus spp. isolates within 2-year trials. This activity remained unchanged against most of the tested bacterial strains after autoclave-sterilization (20 min at 120 °C). The filter-sterilized (0.22-μm Millipore membrane) extract generally reduced shoot elongation, and caused the death of some ‘MRS 2/5’shoots at a 20 % level in the proliferation medium, while all calla lily shoots survived the treatment. It strongly reduced Mo and Bn spread in both plant genotypes, and was even bactericidal against Mo in’MRS 2/5’, without showing any negative effect on growth of survived shoots already at the second subculture on standard medium. The HPTLC analysis revealed the prevalence of polyphenols in the extracts from fruits collected in two successive periods; this was consistent with their antimicrobial activity.

English

Aseptic cultures of pineapple &lsquo;smooth cayenne&rsquo; were established from shoot tip explants. They were initiated on Murashige and Skoog (MS) basal medium with vitamins supplemented with various combinations of 6-benzylaminopurine (BAP) (0, 0.1, 0.2, 0.3 and 0.4 mg L-1) and Gelrite [1.0 g L-1&nbsp;(semi-liquid), 1.5 g L-1&nbsp;(semi-solid) and 2.0 g L-1&nbsp;(solid)]. Explants were transferred five weeks after&nbsp;in vitro&nbsp;initiation to semi-liquid medium containing higher concentrations of BAP (0, 0.5, 1.0, 1.5 and 2.0 mg L-1) for proliferation. Four sub-cultures were made at five weeks interval for twenty weeks in the proliferation medium. Established shoots were introduced into rooting medium containing either full (4.4 g L-1) or half (2.2 g L-1) strength MS basal medium with vitamins supplemented with 0, 0.5 mg L-1&nbsp;BAP alone or in combination with 0, 0.9 and 1.8 mg L-1&nbsp;&alpha;-naphthaleneacetic acid (NAA). Semi-liquid MS basal medium supplemented with 0.1 and 0.3 mg L-1&nbsp;BAP gave the best regeneration results, producing the highest average shoot length (34.6 mm) and average number of shoot buds (2.4) per explants. Significant difference (p&le;0.05) in average shoot number per explant was observed with semi-liquid MS basal medium supplemented with 1.5 mg L-1&nbsp;BAP, producing the highest average shoot number (6.1) per explant at 5 weeks and this increased to 167.7 after 20 weeks. The half-strength MS basal medium without growth regulators or with 0.9 mg L-1&nbsp;NAA gave the highest average root length (29.3 mm) and root number per shoot (7.9). The semi-liquid MS basal medium supplemented with low BAP (1.5 mg L-1) is a cost-effective method for&nbsp;in vitro&nbsp;propagation of pineapple. Half strength MS basal medium without hormones or with low NAA are also cost-effective methods for inducing roots in pineapple. &nbsp; Key words: Gelrite, growth regulators, medium type, pineapple, propagation.

Effect of MgCl2 and double concentration of Murashige and Skoog medium on in vitro plantlet and root cultures generation in halophytic grasswort Salicornia brachiata

An improved micropropagation protocol has been developed for halophytic grasswort Salicornia brachiata by using double concentration of Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium (DMS) supplemented with plant growth regulators. Best shoot bud induction (90 %) from nodal explants was observed on DMS supplemented with 3.0 mg L−1 6-benzyl aminopurine (BAP) and 0.5 mg L−1 Zeatin with >3 shoot buds. Shoot proliferation and elongation was achieved on DMS supplemented with 1.0 mg L−1 thidiazuron (TDZ) and 1.0 mg L−1 α-naphthalene acetic acid (NAA) with multiple shoot buds after 30 days of culture. Best callus response (78 %) was observed when stem explants were cultured on DMS supplemented with 2.0 mg L−1 2, 4-dichlorophenoxyacetic acid and 0.01 mg L−1 BAP. Regeneration from the callus was achieved when callus was cultured on DMS supplemented with 0.5 mg L−1 TDZ. These shoot buds were elongated on shoot proliferation and elongation medium. Elongated shoots (5 cm) could be rooted on DMS supplemented with 0.5–1.5 mg L−1 NAA, 0.5–1.5 mg L−1 indole-3-acetic acid and 0.5–1.5 mg L−1 indole-3-butyric acid. DMS medium supplemented with 0.5 mg L−1 NAA found to be best for rooting and the addition of 20 g L−1 magnesium chloride (MgCl2) to the medium resulted in highest percentage of rooting and not with the addition of sodium chloride (NaCl). Rooted plants could be established in soil with 55 % survival. In another set of study on root culture generation, many roots initiated from explants cultured on DMS supplemented with 2.0 mg L−1 NAA. Addition of 10 g L−1 MgCl2 was highly beneficial in stimulating root initiation and proliferation as compared to NaCl.