Liquid MS Research Articles

In vitro propagation and conservation of Cattleya tigrina A. Rich

ABSTRACT: Cattleya tigrina A. Rich has been suffering heavy losses in its natural habitat and it is now included in the list of plants that are vulnerable to extinction. The development of in vitro propagation and conservation methodologies, as well as acclimatization, are considered important for species at the risk of extinction, as they promote the multiplication and conservation of the species, hence avoiding the loss of their genetic variability. The present study established the protocol of micropropagation and the in vitro conservation of C. tigrina. For the in vitro propagation, the study tested two volumes of the MS medium and two medium consistencies (stationary liquid and semi-solid). For acclimatization, the substrate mixtures containing pine bark, charcoal, vermiculite, and coconut coir were analyzed. For the in vitro conservation, different concentrations of the salts were tested in the MS medium, together with the osmotic regulators (sucrose, mannitol, and sorbitol), and at two temperatures (18 and 25 °C). The results obtained inferred that the semi-solid medium was superior to the stationary liquid medium in the variables of survival and the presence of roots, while the liquid medium was superior to the semi-solid medium in the number of shoots. For acclimatization, pine bark was the substrate where the plants developed an improved height, with sprouting, and rooting. The conservation was satisfactory and the plants remained viable for a period of 730 days, with the MS medium with 25% of the salts, and at temperatures of 18 ºC or 25 ºC. The plants were propagated in the stationary liquid MS medium (10 mL) and the semi-solid medium (25 mL), while they were acclimatized in pine bark and preserved in the MS medium with 25% of the salts (18 ºC or 25 ºC).

Identification of synthetic cannabinoid methyl 2-{[1-(cyclohexylmethyl)-1H-indol-3-yl] formamido}-3-methylbutanoate using modern mass spectrometry and nuclear magnetic resonance techniques

Abstract The samples of plant material suspected to contain new psychoactive substances are very often the subject of chemical-toxicological analyses. Gas chromatography-mass spectrometry (MS), liquid chromatography-quadrupole time-of-flight-MS, and liquid chromatography-tandem MS were applied with the aim to identify synthetic cannabinoid, methyl 2-{[1-(cyclohexylmethyl)-1H-indol-3-yl] formamido}-3-methylbutanoate (MMB-CHMICA) without the analytical standard, which is very often the case when a new drug arrives. The structure of compound was also confirmed by one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy and conformational analysis. After identification, methanolic extract of plant material containing MMB-CHMICA was successfully used for developing a multiple reaction monitoring method on liquid chromatography-tandem MS instrument. The optimization procedure is shown in detail. The complete fragmentation pattern and also the optimization of the extraction procedure of MMB-CHMICA from plant material were shown. The obtained data are useful for forensic, toxicological, and clinical purposes.

Rapid in-vitro micropropagation of Bamboo (Dendrocalamus strictus) and its genetic fidelity testing using ISSR markers

Bamboo is a versatile, arborescent, perennial and non-wood forest tree with tremendous commercial importance. For mass scale propagation of bamboo, the micropropagation is an effective way for producing elite, infection free and true-to-type planting material. Here, the nodal explants of Dendrocalamus strictus used to develop an effective protocol for micropropagation based on tissue culture technique. In this study, the sterilization treatment of 70% ethanol + Tween 20 + Bavistin + Hgcl2 + PPM was successfully controlled the contamination up to 90 % as compared to other treatments. The shoots were initiated from nodal segments in MS medium supplemented with BAP (4 mg/l) and PPM (500µl/l). Shoot multiplication was found best with BAP (4 mg/l) and kinetin (2 mg/l) by using liquid MS medium. Whereas, rooting in solid MS medium has shown good results when supplemented with NAA (4mg/l). Healthy and disease-free plants were obtained after hardening under greenhouse conditions. Genetic fidelity testing by using ISSR markers reported that there was no variation in plantlets developed through micropropagation.

Growth response of diploid and tetraploid taro (Colocasia esculenta (L.) Schott) shoot culture to drought stress using polyethylene glycol

Taro genetic improvement through polyploidy induction is expected to be tolerant to abiotic stress. Several studies have shown that polyploidy plants have a higher adaptability to dry environments. In vitro selection technique for evaluation of plant tolerance to drought stress can be done by applying polyethylene glycol (PEG) as a selection agent. The aim of the research was to investigate the growth response of diploid and tetraploid taro shoot culture to drought stress using PEG. The experiment was conducted using a completely randomized design with two factors. The first factor was the concentration of PEG at 0, 5, 10, 15 and 20%. The second factor was Bentul taro clones which were 1 diploid clone and 2 tetraploid clones (clones 4.6.3 and 5.4.4). PEG was added to liquid MS medium containing 2 mg/l BAP. Observations of growth variables were carried out every week until the six weeks of culture. Fresh and dry weights, mortality percentage and proline content were determined at six weeks of culture. The results showed that the addition of PEG in liquid medium significantly affected the number of leaves, petiole length, number of roots, fresh and dry weights, as well as shoot mortality percentage. Differences in clones significantly affected the number of leaves, fresh and dry weights. The two factors tested (PEG and clones), gave an interaction on the number of leaves, fresh and dry weights. The proline content in all diploid and tetraploid clones showed an increase with increasing PEG concentration. The LC50 value in diploid clones was 9.82%, in tetraploid clones 4.6.3 and 5.4.4 were 14.14 and 15.45%, respectively. The results showed that PEG at 10% and 15% could be used for in vitro selection to drought stress both for diploid and tetraploid taro.

Kriopreservasi Tunas in Vitro Pepaya ‘Sukma’ Dengan Perlakuan Prakultur, Loading, Dan Dehidrasi Dengan Pvs2 Dan Modifikasinya

Papaya has high genetic variability because it is an open-pollinated plant and has genotype and phenotypeare that are easily changed due to environment changes. Cryopreservation is a storing method of germplasm in liquid nitrogen (-196 oC) which can maintain the genotype and phenotype of germplasm. The experiment aimed to obtain the best preculture, loading, and dehydration for cryopreservation of papaya ‘Sukma’ in vitro shoots. For preculture, we planted shoots on MS media with 0.3 M and 0.4 M sucrose for 1, 2, and 3 days. In the loading treatment, we immersed shoots in loading solution (liquid MS+1.2M glycerol+0.4M sucrose) for 0, 10, 20, and 30 minutes. For dehydration, we immersed shoots in cryoprotectant (PVS2 and its modification) for 5, 10, and 15 minutes. Then, shoots were immersed in liquid nitrogen. The results showed thatshoots had the best survival rate while they had been precultured on MS medium with 0.3 M sucrose for 3 days. The best loading treatment time was 20–30 minutes. The best dehydration treatment was obtained by modification of PVS2 for 10 minutes. The shoots have not been able to recovery after cryopreservation, so it can be concluded that cryopreservation of in vitro papaya ‘Sukma’ shoots has not been successful.

In vitro Micropropagation of Duckweed (Lemna minor L) Plant with Temporary Immersion System Bioreactors

Which is very rich in protein in plant Lemna minor L. abundant in Turkey ecologically plays a very important role in protecting the elimination of water pollution and aquaculture environment balance. In this study, in vitro propagation of this plant with Temporary Immersion System Bioreactor and determination of the effects of used plant growth regulators on the protein content of the plant were aimed. With this objective different variant of media with and without sucrose, varying pH and concentrations of BAP, kinetin, TDZ in medium were analyzed. Experiments for micropropagation were performed for 8 hours in the dark and 16 hours in white fluorescent light (150 µ mol photons m-2s-1) under photoperiod and at 24 ± 1 ° C. The highest plant growth was observed at pH 7.23 in sugar free liquid MS medium containing 0.2 mg/L BAP. 50.44 plants per explant were recorded in this medium. In addition, the maximum number of plants per explant in liquid MS medium containing 0.05 mg/L kinetin was calculated as 57,823 and the maximum number of plants per explant in liquid MS medium containing 0.6 mg/L TDZ was calculated as 50.74. As a result of nitrogen determination studies with Kjeldahl method, the protein value of the plant was determined as 25.5%. As a result of hormone application, it was seen that protein content in plant increased to 29.18% with 0.5 mg/L BAP. It was concluded that the aim of the study were fulfilled and positive effects of Temporary Immersion System Bioreactors on plant multiplication were found.

Concentrations and distribution of chlorinated paraffins in Belgian foods

This study reports on concentrations of short- and medium-chain chlorinated paraffins (SCCPs and MCCPs, respectively) in a wide range of food samples (n = 211) purchased in Belgium during 2020. Samples were analysed by gas chromatography-mass spectrometry (GC-MS) and quantified using chlorine content calibration. ∑SCCPs were present above LOQ in 25% of samples with an overall range of <LOQ to 58 ng/g wet weight (ww), while ∑MCCPs were identified in 66% of samples ranging from <LOQ to 250 ng/g ww. ∑MCCP concentrations were greater than those of ∑SCCPs in all 48 samples in which both groups were detected with an average ∑MCCP/∑SCCP ratio of 5.8 (ranging from 1.3 to 81). In general, the greatest CP concentrations were observed in foods classified as animal and vegetable fats and oils and sugar and confectionary for both SCCPs and MCCPs. Significant correlations between lipid content in food samples and CP levels illustrated the role of lipids in accumulating CPs within foodstuffs, while industrial processing, food packaging and environmental conditions are each likely to contribute to overall CP loads. Selected samples (n = 20) were further analysed by liquid chromatography-high resolution MS (LC-HRMS) to investigate homologue profiles and the occurrence of long-chain CPs (LCCPs). LCCPs were detected in 35% of the 20 subset samples while the HRMS results for SCCPs and MCCPs matched closely with those obtained by GC-MS. This study reveals the widespread occurrence of SCCPs and MCCPs in Belgian food and indicates that LCCPs may represent a substantial contribution to overall CP levels in foodstuffs.

The Effects of 2,4-Dichlorophenoxyacetic Acid and α-Naphthaleneacetic Acid on Biomass Increment, Rhizogenesis and Somatic Embryogenesis of Suspension-cultured Dinh Lang Cells [Polyscias fruticosa (L.) Harms

Background: Dinh Lang [Polyscias fruticosa (L.) Harms] is a medicinal plant widely grown in Vietnam, with proven note-worthy health benefits. However, Dinh Lang’s amounts of triterpenoid saponins could not meet the need of the pharmaceutical industry. Thus, this study’s purpose is to figure out the optimal condition for raising Dinh Lang’s cell biomass, rhizogenesis and somatic embryogenesis to provide materials for bioactive compound productions. Methods: Different 2,4-dichlorophenoxyacetic acid and α-naphthaleneacetic acid concentrations (0.5, 1.0, 1.5 and 2.0 mg/L) were examined to determine the best amount of each plant growth regulator for raising cells’ biomass, rhizogenesis and somatic embryogenesis. In each treatment, two grams of eight-week-old calli were cultured in 50 mL of liquid MS medium. Result: It is demonstrated by the results that liquid MS medium containing 1.5 mg/L α-naphthaleneacetic acid has the capacity of producing the highest numbers of somatic embryos (489 embryos per flask) and rooted cells (259.5 cells per flask), while the fresh weight of cells cultured in the medium given 1.5 mg/L 2,4-dichlorophenoxyacetic acid reached its peak of 5.7 g.

Development of high frequency cost-effective micropropagation protocol for Juncus rigidus using liquid culture medium and extraction of cellulose from their in vitro shoots - An important rush

Juncus rigidus is important halophyte with high cellulose content, but no studies has been made on micropropagation of the plant and cellulose extraction from in vitro shoots. To optimize an effective system for shoot multiplication, explants were grown in agar medium, liquid static culture, and bioreactor. Best shoot bud induction was obtained in liquid MS medium supplemented with2.27 μM thidiazuron (TDZ) over 0.46–4.65 μM Kinetin (KN) and 2.22–6.66 μM 6-benzyl aminopurine (BAP). Maximum number of shoot bud (36 ± 1.3) were achieved in static liquid MS medium supplemented with 2.22 μM BAP and 0.57 μM indole acetic acid (IAA). Upon subsequent subcultures >80 shoot buds were achieved in static liquid MS medium supplemented with 1.11 μM BAP, 0.57 μM IAA, and 0.46 μMKN within 30 days of culture. Being halophytic plant NaCl did not have any significant effect on shoot proliferation. Elongated shoots cultured in MS medium supplemented with 0.44 μM BAP, and 0.57 μM IAA developed 97% rooting in filter paper boat culture vessel. Sapling production cost in liquid culture medium was 90% less as compared to solid medium. Cellulose was extracted from the in vitro shoots and 17.64% cellulose achieved from liquid medium cultured shoots. For the first time micro-shoots are tested for in vitro cellulose production. • Maximum shoot bud (36 ± 1.2) induction was achieved in static liquid medium. • More than 80 shoot buds generated in static liquid MS medium supplemented with BAP, IAA, and KN. • 33.4 multiplication rate was recorded in static liquid medium. • 17.64 % cellulose extracted form in vitro shoots of J. rigidus. • In vitro plant based cellulose synthesis is important with future industrial application and bioengineering.

Rhodococcus comparative genomics reveals a phylogenomic-dependent non-ribosomal peptide synthetase distribution: insights into biosynthetic gene cluster connection to an orphan metabolite.

Natural products (NPs) are synthesized by biosynthetic gene clusters (BGCs), whose genes are involved in producing one or a family of chemically related metabolites. Advances in comparative genomics have been favourable for exploiting huge amounts of data and discovering previously unknown BGCs. Nonetheless, studying distribution patterns of novel BGCs and elucidating the biosynthesis of orphan metabolites remains a challenge. To fill this knowledge gap, our study developed a pipeline for high-quality comparative genomics for the actinomycete genus Rhodococcus, which is metabolically versatile, yet understudied in terms of NPs, leading to a total of 110 genomes, 1891 BGCs and 717 non-ribosomal peptide synthetases (NRPSs). Phylogenomic inferences showed four major clades retrieved from strains of several ecological habitats. BiG-SCAPE sequence similarity BGC networking revealed 44 unidentified gene cluster families (GCFs) for NRPS, which presented a phylogenomic-dependent evolution pattern, supporting the hypothesis of vertical gene transfer. As a proof of concept, we analysed in-depth one of our marine strains, Rhodococcus sp. H-CA8f, which revealed a unique BGC distribution within its phylogenomic clade, involved in producing a chloramphenicol-related compound. While this BGC is part of the most abundant and widely distributed NRPS GCF, corason analysis unveiled major differences regarding its genetic context, co-occurrence patterns and modularity. This BGC is composed of three sections, two well-conserved right/left arms flanking a very variable middle section, composed of nrps genes. The presence of two non-canonical domains in H-CA8f’s BGC may contribute to adding chemical diversity to this family of NPs. Liquid chromatography-high resolution MS and dereplication efforts retrieved a set of related orphan metabolites, the corynecins, which to our knowledge are reported here for the first time in Rhodococcus. Overall, our data provide insights to connect BGC uniqueness with orphan metabolites, by revealing key comparative genomic features supported by models of BGC distribution along phylogeny.

Regeneration of Hemidesmus indicus (L.) R. Br. using in vitro nodes: an alternative method for efficient multiplication of shoots

In vivo nodes of Hemidesmus indicus (L.) R. Br. induced healthy multiple shoots with branching in our earlier studies and thus in the present study, potency of in vitro nodes to regenerate shoots was evaluated. In vitro nodes were excised from eight-week-old shoots and placed in Murashige and Skoog’s (MS) medium fortified with sucrose (3%) and different concentrations of 6-benzyladenine (BA) and kinetin (Kn). After eight weeks, optimum of 5.42 ± 0.36 shoots with 100% response were regenerated in medium supplemented with BA (10 µM) and Kn (5 µM). These healthy shoots were placed in full, half and quarter strengths of liquid MS medium fortified with sucrose (1%) and α-naphthaleneacetic acid (NAA, 1-25 µM) for rooting. Among all the strengths of MS medium, full strength MS medium having 8 µM NAA formed maximum of 3.42 ± 0.55 roots (91.67% response) within four weeks. The protocol is in continuation with earlier study and it was confirmed that a single in vivo nodal explant can regenerate around 385 healthy elongated shoots within 4 months, which will help in mass-propagation of the species.

Development of low cost micropropagation protocol for Nardostachys jatamansi: A critically endangered medicinal herb of Himalayas

The study reports a low-cost micropropagation protocol for Nardostachys jatamansi, a high value, critically endangered Himalayan herb. Shoot-bud bearing rhizome segments of the plant were made to sprout and elongate on 0.8% agar-gelled Murashige and Skoog's medium containing 3% sucrose. The sprouted shoots were then multiplied on agar-gelled as well as liquid MS medium containing various concentrations and combinations of kinetin (Kn) and 1-phenyl-3- (1,2,3-thiadiazol-5-yl) urea (thidiazuron or TDZ). Maximum number of shoots developed per explant after 30 days on 100 ml agar gelled (6.7 shoots) and 30 ml liquid (6 shoots) MS medium containing 0.5 mg l−1 Kn and 0.75 mg l−1 TDZ. Although rooting of in vitro shoots was attempted on full as well as half-strength MS medium containing different auxins, rooting was achieved in 100% shoots (3.07 roots/ shoot) after 30 days on half-strength MS medium supplemented with 1.0 mg l−1 indole-3- acetic acid (IAA). Different potting mixtures were used to acclimatize the micropropagated plantlets under polyhouse condition, but a maximum of 83% survival was recorded in sand and sand plus soil. The genetic fidelity of the in vitro plantlets was also assessed using Start Codon Targeted Polymorphism (SCoT) and Directed Amplification of Minisatellite DNA (DAMD) markers, and a total of 8.03% polymorphism was recorded. Eventually, the cost of micropropagation was reduced by 6.4 times during shoot multiplication and further by half during rooting. The present micropropagation method developed for N. jatamansi can have potential applications in medicinal plant-based industry.

Flow Field Effect on the Performance of Direct Formic Acid Membraneless Fuel Cells: A Numerical Study

The performance of both air-breathing and air-feeding direct formic acid membraneless fuel cells (DFAMFCs) possessing different flow fields were numerically investigated in this study at given concentration and flow rate for both fuel and electrolyte. Single serpentine, stepwise broadening serpentine, multi-serpentine and parallel channel were tested as liquid flow field, while single serpentine, stepwise broadening serpentine, multi-serpentine and pin channel were tested as air flow field. The channel width was either 0.8 mm or 1.3 mm. The simulation results showed that the air-breathing DFAMFC having identical flow field for both fuel and electrolyte yielded highest cell output. The air-breathing DFAMFC having SBS liquid flow field yielded a maximum power density of 10.5 mW/cm2, while the air-breathing DFAMFC having S(1.3) liquid flow field produced an open circuit voltage of 1.0 V owing to few formic acid penetration into the cathode. Concerning the air-feeding DFAMFCs, the DFAMFC having SBS liquid flow field and MS(0.8) air flow field yielded highest peak power density, 12 mW/cm2, at an airflow rate of 500 sccm. Considering the power generated by the DFAMFCs together with the power consumed by the air pump, DFAMFC having SBS liquid flow field and Pin(0.8) air flow field could be the preferred design.

DETEKSI VARIASI SOMAKLONAL PLANLET Jatropha curcas Linn. HASIL REGENERASI EMBRIO SOMATIK DENGAN MARKA MOLEKULAR ISSR

Physic nut (Jatropha curcas Linn.) has the potential as a source of sustainable biofuels. Somatic embryo proliferation of J. curcas may cause somaclonal variations. This research aimed to investigate somaclonal variations of J. curcas somatic embryo derived-plantlet using ISSR markers. Somatic embryos of J. curcas at the globular phase were cultured on liquid MS medium supplemented with 0, 0.5, 1.0, 1.5, and 2.0 mg L-1 of 2,4-D. Parameter observed were embryos weight, embryos volume, colour, and size of embryos. After proliferation, the embryos were cultured on a germination medium until the cotyledonary phase. The results showed that proliferation of J. curcas somatic embryos was optimal, with the highest weight and volume, at MS medium added with 1 mg L-1 2,4-D.

Selection of vascular wilt resistance cumin callus to culture filtrate of Fusarium equiseti and regeneration of plants

A method is presented for cellular selection of Cumin plant resistant to Fusarium wilt. In vitro selection of resistant cells using culture filtrates of Fusarium equiseti is attempted which could lead to the development of new Cumin cultivars resistant to Fusarium wilt. In vitro selection of resistant cells using culture filtrates of Fusarium equiseti was attempted in order to obtain cellular selection of Cumin plant resistant to Fusarium wilt. MS basal medium with 1.0 μM 2, 4 D and 10 μM Kinetin was found most suitable for high callus induction. Cell suspension cultures were derived from these calli by transfer to liquid MS medium with 1.0 μM 2, 4 D and 10 μM Kinetin. These cells growing in suspension culture were plated on MS medium gelled with agar and containing 1–10% dilutions of Fusarium equiseti wilt toxin. After 3–4 weeks incubation, Survival calli (Clumps) were selected from 8% CF and put into 5–50% CF of Fusarium equiseti wilt toxin. In next phase resistant calli were obtained from 30% CF. Shoot and root proliferation was induced in these wilt toxin resistant calli in presence of culture filtrate (30%), to maintain the selection pressure to the regenerating shoots and plantlets obtained after rooting. The plants regenerated were hardened in coco-peat. Again the selection pressure was maintained on the plants growing further by irrigating them with nutrients mixed with culture filtrate till maturity. Thus the plants were continuously exposed to the fungal toxin to establish Fusarium equiseti wilt resistant selections.

In-vitro development of Nauclea diderrichii (de Willd. &amp; Th. Dur) Merrin liquid-M Smedia supplemented with benzyl amino purine (BAP) and naphthalene acetic acid (NAA)

The growth of plantlets in Temporary Immersion Bioreactor system (TIBs) relies on initial successful liquid phase transition process. The response of N. diderrichii explants was assessed in liquid-M Smedia with a view to mass produce its seedlings using TIBs. Seven treatments consisting (A) 0.0/0.0, (B) 0.0/0.1, (C) 0.1/0.0, (D) 0.2/0.1, (E) 0.3/0.2, (F) 0.4/0.3 and (G) 0.5/0.4mg/lBAP/NAA combinations were studied. Each group consist of seven replicates and group A without Growth Regulators (GR) serves as control. The results at 4 Weeks after Inoculation (WAI) showed that effects of the growth regulators were significant on shoot length and number of adventitious shoots while number of roots and leaves were closely related. Treatment E produced highest number of adventitious shoots (3.6) which was higher than 0.9 shoots from treatment G and closely related to others. Maximum number of leaves (16.6) was produced by treatment F followed by E (15.7) while the least (12) was obtained in treatment A. The highest number of roots (4.9) was obtained from treatments B, followed by E (4.3) with the lowest being recorded in C (2.43). Liquid MS medium supplemented with 0.3/0.2mg/lBAP/NAA shows some promise for plantlets generation for the purpose of multiple shoot production of N. diderrichii in TIBs.

In vitro tuberization in potato varieties of different ripe time

The formation of potato microtubers in vitro is discrete, and in order to increase its efficiency, it is important to ensure a regular supply of nutrients in a sterile vessel during plant ontogenesis. The aim of the research was to study the effect of phytohormones kinetin and IAA on the formation of microtubers in 20 potato varieties of different ripen time using two compositions of liquid media MS with 2% and 8% sucrose in the process of plant ontogenesis. The biomaterial was grown in plastic containers, the design of which allows the exchange of the nutrient medium in different phases of plant growth. The ontogeny of plants in tissue culture was significantly influenced by the ripening period of varieties. The early varieties were characterized by more intensive growth, their multiplication factor was 1.1-1.7 times higher than that of the middle early and mid-season varieties. The introduction of 0.4 mg/l kinetin into the nutrient medium had a positive effect on the formation of microtubers. For early varieties, the number of formed microtubers per plant increased by 1.2-1.8 pcs. compared to a hormone-free environment. The excess in later varieties was 0.8-0.9 pcs. per plant. The use of IAA without the addition of kinetin did not increase the number of microtubers. Complex application of phytoharmones promoted the growth of the fractional composition.

Meta-topolin and liquid medium enhanced in vitro regeneration in Scaevola taccada (Gaertn.) Roxb

Scaevola taccada (Gaertn.) Roxb. is a hemi-sclerophyllous littoral shrub of the family Goodeniaceae. It is a salt-tolerant plant and used in soil reclamation and coastal landscaping programs to control beach erosion. The present article reports a reproducible in vitro regeneration system using meta-topolin (mT) through liquid medium employing nodal segment cultures. The highest number of axillary shoots (5.8 ± 0.29) was differentiated on Murashige and Skoog’s (MS) medium containing meta-topolin (1.5 mg L−1). The amplification of shoots (49.8 ± 0.37) and fresh growth was achieved by recurrent subcultures on liquid MS medium supplemented with 0.25 mg L−1 mT and 0.15 mg L−1 indole-3-acetic acid (IAA) in the presence of additives (50 mg L−1 ascorbic acid + 25 mg L−1 citric acid, adenine sulphate and l-arginine). Rooting and acclimatization was simultaneously achieved using concurrent ex vitro rooting and acclimatization (CEVRA) technique by pulse treating the shoot base with 300 mg L−1 IBA for 5 min. The in vitro developed shoots represented a maximum percentage (100%) of rooting in autoclaved soilrite in a greenhouse. After 2 mo, the plantlets were established in the field with cent percentage survival success. The genetic fidelity analysis using inter simple sequence repeats (ISSR) DNA molecular markers developed maximum of 55 monomorphic bands and revealed the genetic homogeneity of in vitro regenerated plantlets with the mother plant.

Aeration Volume and Inoculum Density Using in Bioreactor to Optimized Biomass Production and Secondary Metabolites in Gynura procumbens (Lour.) Merr. Adventitious Roots Culture

Phenolic compounds and flavonoids are secondary metabolite compositions of the Gynura procumbens plant. Limited amounts of these secondary metabolites in nature limit their potential as raw material for pharmaceutical drugs. Hence, the optimization of adventitious root culture in vitro should be used as an alternative to producing these compounds. Under optimal culture conditions, the target secondary metabolites can be significantly increased. In this work, we determined the optimum aeration volume and inoculum density on growth and secondary metabolite production of Gynura procumbens adventitious roots in a laboratory-scale bioreactor. The adventitious roots were cultured in a 2 L liquid MS in a 3 L capacity Balloon-Type Bubble Bioreactor (BTBB). The 3 g/L adventitious roots were cultured in 4 variations of aeration volume treatment. The best aeration treatment is 0.15 vvm were used in 4 variations of the inoculum density treatment. The highest biomass (75.38 ± 0.95 g/L), total phenolic contents production (27.98 mg/DW), and flavonoid production rates (256.24 mg/DW) from adventitious roots culture in the BTBB has produced from a treatment combination aeration volume of 0.15 vvm and inoculum density 3 g/L. Accumulation of malondialdehyde and proline in roots in that treatment was lower than other treatments. The four flavonoid compounds that known used as drugs pharmaceutical compound as myricetin, catechin, kaempferol, and quercetin was successfully determined under that treatment. These optimal conditions of aeration volume and inoculum density could be used to mass-produce the desired compounds from Gynura procumbens adventitious root on a pilot scale for industries.

A Rat Model of Orthopedic Implant-Associated Infection for Identification of Staphylococcal Biofilm Proteins.

Secreted bacterial proteins are difficult to identify directly from an infection site due to a limited amount of bacteria and presence of a large quantity of host proteins. Here we describe a rat model of orthopedic implant that allows us to harvest bacterial biofilm materials sufficient for identification of bacterial proteins in the biofilm matrix by liquid chromatography-tandem MS (GeLC-MS/MS) analysis.