MSM Medium Research Articles

Rooting technique of double haploids obtained in culture of microspore <i>in vitro</i> for European radish

Relevance. Doubled haploids (DH-plants) are excellent material for genetic research and breeding due to their complete homozygosity. The genus Raphanus from the Brassicaceae family is the toughest to produce doubled haploid plants through isolated microspore culture in vitro (IMC). The study of the causes of disturbed root formation and the development of elements of this stage of technology will significantly increase the effectiveness of the IMC technology for European radish.Methods. The study included three varieties from the collection of the Federal State Budgetary Scientific Institution Federal Scientific Vegetable Center (FSBSI FSVC): Teplichny Gribovsky, Rozovo-krasniy s belim konchikom and Rhodes. The experiments used a standard protocol for obtaining DH plants using IMC technology in a standard form and with a modification of the rooting stage. The solid MS medium (with agar 7g/L): MS without hormones, MS medium supplemented with IAA at concentrations of 0.5; 1 and 2 mg / L and liquid MSm medium supplemented with 0.1 mg / L kinetin were used for rooting of regenerated plants. All media were supplemented with 20 g/L sucrose. We used three types of techniques for transplanting plant explants onto a solid hormonefree MS medium: planting micro-shoots with their basal part immersed by 2-3 mm into the medium; planting in a well made in a nutrient medium using tweezers under sterile conditions; and landing on the surface of the medium without embedment.Results. In this work, we studied the features of the stage of rooting of regenerated European radish plants in vitro conditions. The transplant technique has been proven to be important for the successful establishment of radish micro-shoots. Plant explants must be planted strictly on the surface of a solid hormone-free nutrient medium MS, without embedment. The use of tubes with bridges made of filter paper and MSm liquid medium with the addition of 0.1 mg/L kinetin for the induction of root formation also showed high efficiency. For plants prone to the formation of root-like structures (RLS) with secondary tumors (ST), multiple dissection of abnormal formations with successive transplants is necessary. Modification at the rooting stage of micro-shoots growing has increased the percentage of successfully adapted DH plants in vivo conditions from 0-14% to 95-98%.

Microbial degradation, spectral analysis and toxicological assessment of malachite green by Streptomyces chrestomyceticus S20.

Malachite green (MG), a triphenylmethane dye is extensively used for coloring silk, aquaculture and textile industries, it has also has been reported toxic to life forms. This study aimed to investigate the biodegradation potential of MG by actinobacteria. The potent actinobacterial strain S20 used in this study was isolated from forest soil (Sabarimala, Kerala, India) and identified as Streptomyces chrestomyceticus based on phenotype and molecular features. Strain S20 degraded MG up to 59.65 ± 0.68% was studied in MSM medium and MG (300mgl-1) and degradation was increased (90-99%) by additions of 1% glucose and yeast extract into the medium at pH 7. The treated metabolites from MG by S20 characterized by FT-IR and GC-MS. The results showed MG has been degraded into nontoxic compounds evaluated by (1) phytotoxic assay on Vigna radiata, (2) microbial toxicity on Staphylococcus aureus, Bacillus subtilis, Micrococcus luteus, Streptococcus sp. and Escherichia coli, (3) cytotoxicity assay in a human cell line (MCF 7). The toxicity studies demonstrated that the byproducts from MG degradation by S. chrestomyceticus S20 were no toxic to plants and microbes and less toxic to human cells as compared to the parent MG. Perhaps this is the first work reported on biodegradation of MG by S. chrestomyceticus which could be a potential candidate for the removal of MG from various environments.

Purification, characterization and anticancer activities of exopolysaccharide produced by Rhodococcus erythropolis HX-2

In the present study, an exopolysaccharide (EPS) producer Rhodococcus erythropolis HX-2 was isolated from Xinjiang oil field, China. The HX-2 EPS (name HPS) production reached 8.957 g/L by RSM in MSM medium. The HPS was purified by ethanol precipitation and fractionation by DEAE-Cellulose and Sepharose column, the yield of HPS was 3.736 g/L. HPS composed by glucose, galactose, fucose, mannose and glucuronic acid. FT-IR spectroscopy indicated the presence of a large amount of hydroxyl groups. NMR spectroscopy indicated the existence of both α and β-configuration for sugar moieties present in HPS. The degradation temperature (255.4 °C) of the HPS was determined by thermogravimetric analysis (TGA). A reticular structure of HPS was observed by SEM and the AFM analysis of the HPS revealed straight chains line. Meanwhile, the WSI and WHC of HPS were 92.15 ± 3.05% and 189.45 ± 5.65%, respectively. Finally, In vitro anticancer activity purified EPS was evaluated on L929 normal cells, A549 cancer cells, SMMC-7721 liver cancer cells and Hela cervical cancer cell. HPS inhibited the growth of cancer cells in a certain concentration without damage to normal cells. These characteristics indicate that its potential application value in food, industry and pharmaceutical application.

Exploration on the bioreduction mechanism of Cr(Ⅵ) by a gram-positive bacterium: Pseudochrobactrum saccharolyticum W1

Bioremediation of chromium (Cr(Ⅵ)) contaminations has been widely reported, but the research on its removal mechanism is still scarce. Studies on Cr(Ⅵ) removal by strains affiliated to genus Pseudochobactrum revealed the Cr(Ⅵ) efficiency removal through the reduction of Cr(Ⅵ) to Cr(Ⅲ). However, the location of Cr(Ⅵ) reduction reaction and exact mechanism are still unspecified. In this work, a Gram-positive bacterial strain, Pseudochrobactrum saccharolyticum W1 (P. saccharolyticum W1) was isolated and tested to remove approximately 53.7% of Cr(Ⅵ) (initial concentration was 200 mg L−1) from the MSM medium. Analysis of SEM-EDS and TEM-EDS indicated that chromium-containing particles precipitated both on the cell surface and in the cytoplasm. Batch experiments indicated that the heat-treated bacterial cells almost had no ability to remove Cr(Ⅵ) from solution, while the resting cells could remove 62.0% of Cr(Ⅵ) at the initial concentration of 10 mg L−1. Additionally, at this concentration, 64.8% and 70.8% of Cr(Ⅵ) was reduced by cell envelope components and intracellular soluble substances after 6 h, respectively. These results suggested that the removal of Cr(Ⅵ) by P. saccharolyticum W1 was through direct reduction, which occurred on both cell envelop and cytoplasm. The results also showed that cytoplasm was the main site for Cr(Ⅵ) reduction compared to the cell envelop. Further analysis of FTIR and XPS verified that C–H, C–C, CO, C–OH and C–O–C groups of cells involved in correlation with chromium during Cr(Ⅵ) reduction. The study offered an insight into the Cr(VI) reduction mechanism of P. saccharolyticum W1.

Biosorption of Cr(VI) by Halomonas sp. DK4, a halotolerant bacterium isolated from chrome electroplating sludge.

This study evaluated Cr(VI) biosorption by a halotolerant gram-negative bacterium Halomonas sp. DK4 isolated from chrome electroplating sludge. The bacterium could withstand high concentrations of Cr(VI) exhibiting a minimal inhibitory concentration (MIC) of 250 mg/L. Plackett-Burman design confirmed glucose, KH2PO4, NaCl, inoculum size, and initial Cr(VI) concentration as significant variables influencing the Cr(VI) removal ability of the bacterium. The suspended culture of Halomonas sp. DK4 was able to remove 81% (100 mg/L) of Cr(VI) in optimized MSM medium from aqueous solutions within 48 h. The bacterium also removed 59% Cr(VI) in the presence of 15% NaCl concentration within 72 h. The main mechanism involved in Cr(VI) removal by Halomonas sp. DK4 was determined to be biosorption which was best explained using the Langmuir isotherm model, wherein the maximum adsorption of 150.7 mg/g was observed under equilibrium conditions. Kinetic studies reveal that chemisorption of Cr(VI) by Halomonas sp. DK4 was a rate-limiting process which followed pseudo-second-order kinetics (R2 = 0.99). Bacterial biomass exhibited maximum adsorption of 70.3% Cr(VI) at an initial concentration of 100 mg/L under optimal conditions. Fourier transform infrared spectroscopy (FTIR) analysis confirmed the presence of hydroxyl, carboxyl, amide, and phosphate groups on the bacterial surface which may be involved in Cr(VI) adsorption. Scanning electron microscopy coupled energy dispersive X-ray (SEM-EDX) analysis revealed morphological changes in the bacterial cell and accumulation of Cr(VI) on the cell surface. These results suggest the potential application of Halomonas sp. DK4 in the removal of Cr(VI) from saline chromium-containing industrial wastewaters.

A Practical and Efficient Method for the Micropropagation of Japanese Cherry (Prunus sp.)

This study was conducted to establish the procedure for in vitro propagation of Japanese cherry (Prunus sp.) to produce large quantity of plantlets and initial planting materials for climate adaptation research of this plant in Hanoi. Single nodal stems were used as the primary explants and initially produced shoots on MS medium supplemented with 1 mg L-1 BA. The highest shoot multiplication rate (9.57 times) was obtained on MS medium containing 1 mg L-1 BA and 0.25 mg L-1 a-NAA after 8 weeks of culture. 100% of the shoots produced roots with a mean of 10.10 roots per plant within 4 weeks on ½ MSM medium with 4 mg L-1 IBA. The survival rate of in vitro derived plantlets after a 6 to 7-week-period of rooting during acclimatization using a soil: coco peat: smoked rice husks (2:2:1, v/v/v) substrate was 100% and acclimatized plantlets showed good growth and development. This is the first report on a practical and efficient in vitro multiplication protocol for Japanese cherry in Vietnam, starting from shoot initiation to establishment of plants under greenhouse conditions.

Sugarcane molasses and vinasse as a substrate for polyhydroxyalkanoates (PHA) production

Los Polihidroxialcanoatos (PHAs) son biopolímeros de origen microbiano totalmente biodegradables con características físicoquímicas similares a los plásticos sintéticos convencionales, sin embargo, su producción actual es altamente costosa debido principalmente al sustrato o medio de cultivo. En esta investigación se evaluó como sustrato, una mezcla de melaza de caña y vinaza residuales de la industria licorera empleando la cepa referencia Ralstonia eutropha ATCC 17699 para la producción de PHAs. Los resultados obtenidos mostraron potencial en el uso de este medio de cultivo, alcanzando concentraciones de polímero de 3,17 g/L en medio MMS y de 2,71 g/L en el medio M/V con una relación melaza/vinaza de 25/75. El rendimiento de consumo de azucares (sacarosa, fructosa y glucosa) fue del 70,7% y una acumulación del biopolímero del 97,8% respecto a la biomasa producida. La caracterización del biopolímero presento una correlación con la muestra patrón de polihidroxibutirato - PHB del 99,25%.

Optimization of bio-surfactant production by Azo-rhizobium strain isolated from oil-contaminated soil

Bio-surfactants are amphiphilic molecules possessing both hydrophilic and hydrophobic moieties. They are surface active agents that are produced extracellularly or as a part of the cell membrane by bacteria, yeast, and fungi. In the present study, the screening and optimization of bio-surfactant production were carried out using oil-contaminated soil sample. The cultural, morphological and biochemical tests as well as 16s rRNA analysis identified the most efficient bio-surfactant producer as Azorhizobium strain. The preliminary screening of bio-surfactant production was done with the help of oil displacement test, drop collapse test and observed hemolysis on superimposed blood agar plates. Optimization studies revealed that 2% inoculum size of test strain (1.0 O.D 530 nm) can be exploited for bio-surfactant production using 2% coconut oil and ammonium nitrate with a C: N ratio of 20:1 in MSM medium (pH 8.5) and incubation conditions of 30 ˚C for 96 h. The crude yield of bio-surfactant produced was estimated as 2.5 g/L. Further purification of crude bio-surfactant was carried out using acid hydrolysis and rotary vacuum evaporator. The bio-surfactant thus obtained successfully reduced the surface tension of the medium from 59 mN/m to 38 mN/m with E24 60%. The characterization studies of the purified bio-surfactant carried out by FTIR analysis confirmed it to be lipopolysaccharide type of bio-surfactant. Thus our current study suggests useful application of bio-surfactant producing bacterial strain that may be helpful in the petroleum industry for the purpose of recovery of petroleum and other oils from oily sludge, cleaning of oil storage tanks and bioremediation of oil-contaminated sites.

STUDY OF BIOSURFACTANT PRODUCING BACTERIA AND PRELIMINARY CHARACTERIZATION OF BIOSURFACTNAT PRODUCED BY BACILLUS species ISOLATED FROM PETROLEUM CONTAMINATED SOIL

Chemically synthesized amphiphilic molecules like surfactants are used for variety of purposes, but they are costly and less or non-degradable and may be toxic once released in the environment. Microbially produced biosurfactants are better alternatives of chemical surfactants as they are stable as well as easily biodegradable and also due to structural and functional similarities with chemical surfactants. The aim of this study was to isolate and characterize biosurfactant producing bacteria from oil contaminated sample (garage soil, Anand, Gujarat). Thirty bacterial cultures were isolated on the basis of differences in their colony characters. To confirm and compare the efficiency of biosurfactant production, hemolytic activity and reduction in surface tension was analyzed. Biochemical and morphological tests identified bacterial isolate C5C (Gram positive rods arranged in short chains) as Bacillus species. Optimum pH and temperature for biosurfactant production were 5 and 30oC, respectively. It produced 3.12 g/l of biosurfactant in MSM medium with 1% diesel as carbon source. Biosurfactant produced by C5C contains lipid. No carbohydrates or amino acid component was detected on TLC plate.

Characteristics of metal-tolerant plant growth-promoting yeast (Cryptococcus sp. NSE1) and its influence on Cd hyperaccumulator Sedum plumbizincicola.

Plant growth-promoting yeasts are often over looked as a mechanism to improve phytoremediation of heavy metals. In this study, Cryptococcus sp. NSE1, a Cd-tolerant yeast with plant growth capabilities, was isolated from the rhizosphere of the heavy metal hyperaccumulator Sedum plumbizincicola. The yeast exhibited strong tolerance to a range of heavy metals including Cd, Cu, and Zn on plate assays. The adsorption rate Cd, Cu, Zn by NSE1 was 26.1, 13.2, and 25.2%, respectively. Irregular spines were formed on the surface of NSE1 when grown in MSM medium supplemented with 200mgL(-1) Cd. NSE1 was capable of utilizing 1-aminocyclopropane-1-carboxylate (ACC) as a sole nitrogen source and was capable of solubilization of inorganic phosphate at rates of 195.2mgL(-1). Field experiments demonstrated that NSE1 increased phytoremediation by increasing the biomass of Cd hyperaccumulator S. plumbizincicola (46%, p<0.05) during phytoremediation. Overall, Cd accumulation by S. plumbizincicola was increased from 19.6 to 31.1mgm(-2) though no difference in the concentration of Cd in the shoot biomass was observed between NSE1 and control. A Cd accumulation ratio of 38.0% for NSE1 and 17.2% for control was observed. The HCl-extractable Cd and CaCl2-extractable Cd concentration in the soil of the NSE1 treatment were reduced by 39.2 and 29.5%, respectively. Community-level physiology profiling, assessed using Biolog Eco plates, indicated functional changes to the rhizosphere community inoculated with NSE1 by average well color development (AWCD) and measurement of richness (diversity). Values of Shannon-Weiner index, Simpson index, and McIntosh index showed a slight but no significant increases. These results indicate that inoculation of NSE1 could increase the shoot biomass of S. plumbizincicola, enhance the Cd accumulation in S. plumbizincicola, and decrease the available heavy metal content in soils significantly without overall significant changes to the microbial community.

Ecofreindly, candle precursor (Paraffin wax) production by Iranian indigenous bacterium to reduce the indoor health risks

The potential indoor air impacts of burning candles have drawn increased attention in recent years. Burning paraffin waxes itself, regardless to wick and the essence, produce some allergens and even carcinogenic compounds. Further biorefining of petroleum paraffin wax with proper bacteria is one of the proposed resolutions to decrease the health threat. Therefore in this study Iranian heavy crude oil sample were screened for the best isolate to reach the target. Manual culture and plate count method and screening were done in MSM medium supplemented with paraffin wax as the sole source of carbon and energy in 40°C and 150rpm. The growth curve of the best isolate was depicted through spectrophotometry during 24 hours. Then the isolate was characterized by biochemical test and molecular identification using 16srRNA. The spore forming, biosurfactant producing bacterium was identified as Bacillus cereus. Gas chromatogaraphs revealed the efficient degradation of long chain paraffin wax with the selected Bacillus cereus into 15 to 25 carbon length n- alkanes. The high physicochemical endurance of this bacterium including salinity tolerance from 0.5to 15 percent, pH from 4 to 10 and temperature growth range from 40 to 65°C, makes it a decent choice for industrial and environmental approaches.

Exploration of cellulolytic potential of Termite gut flora for sustainable development

Cellulose is the most abundant organic polymer on Earth. Cellulose is an important structural component of the primary cell wall of green plants. Cellulose degrading bacteria from termite gut flora were isolated, screened and their characterization was studied in relation to cellulase activity. Out of the 16 isolates the three strains, showed higher production of cellulase using CMC and Blotting paper compared to filter paper as sole carbon source. CDB-W utilized CMC in higher level while CDB-Y utilized blotting paper in higher level. The maximum growth was recorded at pH 7.0 and at temperature 30°C. Among three isolates, the CDB- W strain showed higher enzyme activity as compared to other strains. Among carbon sources, maximum growth was observed in fructose amended MSM medium followed by Xylose, Glucose and Starch. Ammonium nitrate and potassium nitrate were good nitrogen sources for growth of CDB. The cellulolytic activities of these organisms may be utilized for various industrial and biotechnological applications for sustainable development.

Biosurfactant production by Serratia rubidaea SNAU02 isolated from hydrocarbon contaminated soil and its physico-chemical characterization

The aim of the study was to characterize and optimize the growth media for biosurfactant production from Serratia rubidaea SNAU02 isolated from hydrocarbon-contaminated soil from Cuddalore district, Tamilnadu, India. The biosurfactant produced by S. rubidaea SNAU02, was able to reduce the surface tension to 34.4 mN m(-1) in MSM medium. The biosurfactant was characterized by FT-IR and GC-MS analysis. The GC-MS analysis shows that dirhamnolipid was detected in abundance as predominant congener than monorhamnolipid. The response surface methodology (RSM) -central composite design (CCD) was performed to optimize the media for biosurfactant production. The maximum emulsification index was obtained under the optimal condition of 29.31 g L(-1) mannitol; 2.06 g L(-1) yeast extract, medium pH 6.97 and 5.69 g L(-1) NaCl. The biosurfactant produced by S. rubidaea recovered 92% of used engine oil adsorbed to a sand sample, suggested the potential application in microbial enhanced oil recovery and bioremediation.

Vacuum distillation residue upgrading by an indigenous Bacillus cereus

BackgroundBiological processing of heavy fractions of crude oils offers less severe process conditions and higher selectivity for refining. Biochemical Processes are expected to be low demand energy processes and certainly ecofriendly.ResultsA strain of biosurfactant producing bacterium was isolated from an oil contaminated soil at Tehran refinery distillation unit. Based on selected phenotypic and genotypic characteristic including morphology, biochemical proprety, and 16 SrRNA sequencing identified as a novel strain of Bacillus cereus (JQ178332). This bacterium endures a wide range of pH, salinity and temperature. This specific strain utilizes both paraffin and anthracene as samples of aliphatic and polycyclic aromatic hydrocarbons. The ability of this bacterium to acquire all its energy and chemical requirements from Vacuum Distillation Residue (VR), as a net sample of problematic hydrocarbons in refineries, was studied. SARA test ASTM D4124-01 revealed 65.5% decrease in asphaltenic, 22.1% in aliphatics and 30.3% in Aromatics content of the VR in MSM medium. Further results with 0.9% saline showed 55% decrease in asphaltene content and 2.1% Aromatics respectively.ConclusionRemarkable abilities of this microorganism propose its application in an ecofriendly technology to upgrade heavy crude oils.

Introducción y multiplicación in vitrodel cultivo de ajo variedad Morado Barranquino

We determine the effect of modified culture medium using different growth regulators concentrations (auxins / cytokinins) for regeneration and propagation of in vitro garlic plants (Allium sativumL.) var. ‘Morado Barrran-quino’. In the introduction stage meristem tips were cultured in test tubes up to regeneration. It was determined that medium MSM + IBA 0.6 mg/L was most suitable for the meristem tip differentiation and development. 13 weeks old microplants obtained on introduction stage were used for multiplication, obtaining the formation of a cluster after each subcultive. The best multiplication ratio was obtained in MMA medium + IAA 2.5 mg/L + KIN 5.0 mg/L and MSM medium + 2iP 1.5 mg/L with 4.71 and 4.79 respectively.

The Optimization of Alcohol Dehydrogenase Production from Pseudomonas aeruginosa NW

The locally isolated Pseudomonas aeruginosa NW was tested for their ability to produce alcohol dehydrogenase (ADH) enzyme by growing the bacteria on three different aromatic hydrocarbons: naphthalene, xylene and toluene. Following five days incubation the bacterium showed a good ability to utilize xylene, limited ability for toluene utilization and failed to degrade naphthalene. The ADH activity was assayed following each day of incubation on xylene as sole source of carbon and energy and it was found that a maximum ADH volume activity was 2.86 and 4.11 U/ml following the third and fourth day of incubation, respectively. The production of ADH was optimized under different conditions involving: temperature, pH, xylene concentration and incubation period. Results indicated that the optimal conditions for ADH production were by incubating the bacterium in MSM medium containing 1% xylene, pH: 8 for four days and incubation at 37ºC.

23. Comparison of vitrification-based techniques in the efficacy of cryopreservation of Passiflora suberosa L. and P. foetida L. shoot tips

Passiflora are used as ornamentals and in popular medicine. Passiflora suberosa is commonly used to treat hypertension, diabetes, skin diseases and as a sedative, whereas P. foetida shows analgesic and antibacterial activities. The goal of this study was the development of in vitro conservation strategies for long-term storage for both species through cryopreservation using vitrification and encapsulation-vitrification techniques. The efficacy of vitrification and encapsulation-vitrification protocols was compared, and several parameters were evaluated, including pregrowth on medium with high sucrose concentrations, type of vitrification solution (PVS2 and PVS3), exposure time to vitrification solutions, and recovery conditions. Shoot tips (0.3 cm) excised from in vitro-grown plants were precultured for 24 h on solid 1/2 MSM medium supplemented with different sucrose concentrations (0.3, 0.5 or 0.7 M), at 25 °C. Explants precultured on medium supplemented with 0.3 M sucrose were incubated in the cryoprotectant solutions PVS2 or PVS3 for different periods (0–240 min) at room temperature, and immersed into liquid nitrogen (LN) for 72 h. In the encapsulation-vitrification protocol, precultured shoot tips were encapsulated in calcium alginate beads, followed by incubation in liquid 1/2 MSM medium containing 0.75 M sucrose for 24 h, on a rotary shaker (100 rpm) at 25 ± 2°C. After this period, beads were maintained in cryovials containing PVS2 or PVS3 for 30, 60, 120 or 180 min, before immersion into LN. The materials were rapidly thawed in a warm water bath (38-40 °C) for 2 minutes and transferred to the recovery medium (1/2 MSM or MSM supplemented with 0.44, 2.2 or 4.4 μM BA). Alternatively, explants were kept in the dark for 30 or 60 days before transfer to the presence of light. Plant recovery from the shoot tips before and after cooling in LN was evaluated by assessing the percentage of shoot formation. The shoots were cultured on 1/2 MSM medium for 60–90 days for root induction, and whole plants were transferred to ex vitro conditions. The highest recovery rates for both P. suberosa and P. foetida (28% and 60%) were obtained with the encapsulation-vitrification protocol, after a pre-treatment with 0.3 M sucrose, followed by exposure to PVS2 for 60 or 120 min, respectively, and post-freezing incubation in the absence of light for 30 days on MSM medium supplemented with 0.44 μM BA. These results suggest that alginate beads provided additional protection, reducing the toxicity of the cryoprotectant solution. In addition, they demonstrate that different species from genus Passiflora display distinct responses to cryopreservation treatments.

72. Cryopreservation of Passiflora pohlii – Vitrification and encapsulation-vitrification

Passiflora pohlii is a wild tropical species of the genus Passiflora , it is native to Brazil and found in Cerrado, Atlantic Forest and Pantanal. This species has a potential agronomic interest due to its tolerance to soil-borne pathogens of Phytophthora sp, which causes damage to passionfruit culture. P. pohlii occurs in regions that are impacted by human actions, with the possibility of disappearance of many genotypes that could be used in breeding. Considering these facts, the establishment of different conservation strategies for this species is of great importance. The goal of this work was to develop cryopreservation protocols for P. polhii using vitrification-based techniques. Nodal segments (0.3 cm) excised from in vitro -grown plants were precultured for 24 h on solid MSM (Monteiro et al. In vitro Cell Dev Biol – Plant 36, 527–531) medium supplemented with 0.7 M sucrose, at 25 °C. For vitrification, the precultured explants were placed into 2.0 ml cryovials and incubated in the cryoprotectant solution PVS2 for different periods (0, 30, 60 or 120 min) at room temperature. After this, the cryovials were plunged into liquid nitrogen (LN 2 ) and stored for 72 h. For encapsulation-vitrification, the precultured explants were suspended in calcium-free MS medium supplemented with 3% sodium alginate. The mixture, including the nodal segments, was dispensed with a sterile pipette into MS medium containing 100 mM CaCl 2 , and held for 20 min at 25 °C. Beads, each containing one explant, were precultured in liquid 1/2 MSM medium containing 0.75 M sucrose for 24 h on a rotary shaker (100 rpm) at 25° ± 2 °C, and then placed into 2 ml cryovials containing PVS2 for 0, 30, 60 or 120 min. After these periods, cryovials were immersed into liquid nitrogen (LN 2 ) and stored for 72 h. Thereafter, the cryovials were removed from LN 2 and rapidly re-warmed in a water bath (40 °C) for 2 min, and transferred to the MSM supplemented with 31 μM BA. The explants were maintained in the dark for 30 or 60 days in a growth chamber at 30° ± 2 °C. After these periods, the explants were exposed to total irradiance (46 μ mol m −2 s −1 ). When using the vitrification technique, the highest recovery rate (40%) was obtained after exposure to PVS2 for 60 or 120 min. On the other hand, a very low survival rate was attained when explants were submitted to the encapsulation-vitrification protocol. In summary, in the present study we established a protocol for long-term conservation of P. pohlii through cryopreservation. We demonstrated that the vitrification protocol resulted in higher recovery rates when compared to the results obtained with the encapsulation-vitrification technique. In addition, the high multiplication capacity of nodal segments was not affected by cryostorage.

Innovative protocol for “ex vitro rooting” on olive micropropagation

Abstract The commercial micropropagation of olive trees is currently limited by the production cost. An ex vitro method for olive microshoot rooting could reduce both the production cost per plant and the propagation time. In this study a successful ex vitro rooting protocol tested on seven olive cultivars is reported. The explants of cv. Maurino were collected from fifth, sixth, and seventh proliferative subcultures carried out on MSM medium, while for the other cultivars the explants were collected from only seventh proliferative subculture. Continuous light during the rooting phase was a prerequisite for the success of the ex vitro protocol. The best source of microshoots for a high rooting percentage was the seventh proliferative subculture. Cvs. Coratina, Maremmano, Maurino, Picholine, and S. Francesco showed high rooting percentages with a range of 62–76%; whereas for cvs. Correggiolo and Frantoio the experimental conditions need to be optimised. Up to 90% of the rooted microplants survived, and continuous growth of shoots was subsequently observed. The proposed protocol can be easily applied to several different olive cultivars to produce microplants by commercial laboratories. The approach makes olive micropropagation in the nursery industry both possible and profitable.

Effects of plant growth regulators and elicitors on production of secondary metabolites in shoot cultures of Hypericum hirsutum and Hypericum maculatum

We investigated the effects of plant growth regulators [6-benzyladenine (BA), kinetin (Kin), 6-γ,γ-dimethylallylaminopurine (2iP), thidiazuron (TDZ) and α-naphthaleneacetic acid (NAA)], modified Murashige and Skoog (MS) medium containing 10 mM NH4 + and 5 mM NO3 − and supplemented with 2iP, BA, Kin and NAA (MSM medium), and two elicitors [jasmonic acid (JA), and salicylic acid (SA)], on plant growth and accumulation of hypericins (hypericin and pseudohypericin) and hyperforin in shoot cultures of Hypericum hirsutum and H. maculatum. Our data suggested that culture of shoots on MS medium supplemented with BA (0.4 mg l−1) or Kin (0.4 mg l−1) enhanced production of hypericins in H. maculatum and hyperforin in H. hirsutum. Hypericins and hyperforin concentrations decreased in both species when TDZ (0.4 mg l−1) was added to the MS medium. Also, TDZ induced hyperhydric malformations and necrosis of regenerated shoots. Cultivation of H. maculatum on MSM medium resulted in approximately twofold increased production of hypericins compared to controls, and the growth of H. hirsutum shoots on the same medium led to a 6.16-fold increase in hyperforin production. Of the two elicitors, SA was more effective in stimulating the accumulation of hypericins. At 50 μM, SA enhanced the production of hypericin (7.98-fold) and pseudohypericin (13.58-fold) in H. hirsutum, and, at 200 μM, enhanced the production of hypericin (2.2-fold) and pseudohypericin (3.94-fold) in H. maculatum.