MSM Medium Research Articles

Isolation and Characterization of Indigenous Bacteria with Purifying Potential in Solid Palm Oil Extraction Sludge Generated by SOCAPALM-Mbambou

The oil palm industry contributes significantly to the economic development of producing countries such as Cameroon. Unfortunately, the exploitation of palm oil constitutes a source of environmental pollution due to the production of enormous quantities of waste during its extraction process, including solid sludge generating greenhouse gases which contribute to global warming climatic. All this leads to the search for alternatives which consists of isolating and characterizing indigenous bacteria with biodegradation capacities in sludge from palm oil extraction. The pH and bacterial counts were determined by the potentiometric method and the decimal dilution technique, respectively. The isolated bacteria were identified by their cultural, cellular and biochemical characteristics. In addition, the identification of Gram- bacteria was further explored by the API 20 E gallery. The palm oil biodegradability test was carried out on M2 medium supplemented with 2% palm oil. The solid sludge biodegradability test was carried out on liquid MSM medium supplemented with 4% sludge stock solution. The results showed that the sludge sample had a slightly alkaline pH of 7.3. A bacterial load of around 10<sup>9</sup> CFU/g of soil was counted. Thirty-one bacterial strains were isolated and purified, including 12 <i>Bacillus sp</i>, 10 <i>Pseudomonas sp</i>, 8 <i>Proteus mirabilis</i> and 1 <i>Klebsiella pneumoniae</i>. All isolates tested for their ability to degrade palm oil or solid sludge grew in culture media with palm oil or solid sludge as the sole source of carbon and energy but with a difference in load. Thus, isolates BI2, BI5, BI31, BI10 and BI 9 showed the highest degradation capacities. These isolates could be used to constitute consortia of microorganisms that can be used in the treatment of waste generated by palm oil extraction.

Isolation, characterization and identification of Klebsiella sp. dor_27f isolated from textile effluents

This research study aimed to isolate and characterize a new bacterial strain from textile effluents. To do this, bacteria were cultured using the MSM medium, where a colony was isolated through six successive pickings. It then underwent a DNA extraction process using the phenol-chloroform-isoamyl alcohol methodology, and an electrophoresis was carried out to confirm the extraction. In addition, the isolated colony was identified as Klebsiella sp. by sequencing the 16S rRNA gene using bioinformatics tools. To observe its biotechnological potential, the bacterial strain was grown in an MSM broth enriched with Golden Yellow K2R azoic dye at a concentration of 50 mg/L, showing a percentage of decolorization of 74 % after 72 hours at 37 °C, indicating the potential of the isolated colony for the development of bio-remediation processes for effluents containing azoic dyes. Keywords: Bacterial strain, Klebsiella sp., DNA extraction, decolorization, effluents.

Bioremediation of Imidacloprid by the Local Isolate Psychrobacter celer

Pesticide biodegradation can be accomplished by the technique of bioremediation, which makes use of microorganisms’ ability to degrade pesticide residues. This study aimed to separate and identify imidacloprid-biodegradable from botanical fields soil of greenhouses in the Plant Protection Directorate /Ministry of Agriculture in Baghdad, which has been using imidacloprid pesticides for many years. Using high-performance liquid chromatography, residual imidacloprid concentrations in MSM medium at a concentration of 25 mg/L after 21 days were measured to identify the best degrading bacterial isolates. Isolate No.37 the best bacterial isolate was able to degrade 63% of imidacloprid. was identified as Psychrobacter celer according to morphological, cultural and biochemical tests. Moreover, genetic analysis for the 16S rRNA gene and given a new accession number (OP672320.1) in the GenBank of NCBI, from this study that the soil previously contaminated for long periods of time with imidacloprid can be treated by degrading the imidacloprid residues in it by isolated bacteria Psychrobacter celer.

Development of the bacterial consortia for the degradation of benzo[a]pyrene, pyrene from hydrocarbons waste

The environment is heavily populated with polycyclic aromatic hydrocarbons (PAHs), which are dangerous to human health. Degradation and cleaning of PAH chemicals from water and soil regions are crucial due to their chemical and biological impacts and persistent nature. In this study, we found that a very efficient bacterial consortium A-LOBP-19A+LOP-9 (99.62%) for benzo[a]pyrene up to 1000ppm and B-LOP-9 +GWP-2 (93.8%) for pyrene up to 2000ppm concentration degradation and it was done in MSM medium with isolated bacterial strains and incubated at 37° C for 50 days and 30 days respectively. This consortium consisting of the Mycobacterium vaanbaalenii GWP-2 (ON715011), Staphylococcus aureus LOP-9(ON715121), and Stutzerimonas stutzeri (LOBP-19A) OP389146, and these have capabilities of mentioned PAHs. The HPLC analysis suggested that both benzo[a]pyrene and pyrene degraded through peaks by both consortia. Degraded metabolites were identified by GC-MS and reported the presence of Phthalic acid, Naphthalene, 1,4-benzodicarboxylic acid, Butoxyacetic acid, Benzeneacetic acid and benzo [a]pyrene-1,6-dione. Thus, the study demonstrated efficient bacterial community enhancement for PAHs (benzo[a]pyrene, pyrene) decomposition, and these can be further explored for the cleanup of hydrocarbons pollution.

In Vitro Propagation and Genetic Uniformity Assessment of Manglietiastrum sinicum: A Critically Endangered Magnoliaceae Species.

Manglietiastrum sinicum Y.W. Law is a critically endangered species with great ornamental and commercial value, which urgently requires protection. We tested different combinations of basal media and plant growth regulators to determine (i) the optimal conditions for bud induction and proliferation of explants and (ii) optimal rooting conditions. RAPD- and ISSR-PCR were used to assess the genetic fidelity of regenerated plantlets. Murashige and Skoog medium (MS) supplemented with 0.5 mg/L 6-benzyladenine (BA) and 0.05 mg/L indole-3-butyric acid (IBA) is the optimal medium for bud induction (100% induction). MSM medium (a special basal medium for M. sinicum) was more suitable for the efficient proliferation and rooting of M. sinicum. Maximum bud proliferation rate (446.20%) was obtained on MSM, with 0.4 mg/L BA, 0.5 mg/L kinetin, and 0.06 mg/L IBA, while maximum root induction rate (88.89%) was obtained on MSM supplemented with 0.4 mg/L 1-naphthylacetic acid and 1.0 mg/L IBA with a 7-day initial darkness treatment. The rooted plantlets were transferred to a substrate containing peat soil, perlite, coconut chaff, and bark (volume ratio 2:1:1:1), with a resulting survival rate of 92.2%. RAPD and ISSR markers confirmed the genetic uniformity and stability of regenerated plants.

Protocol for in vitro regeneration and rapid mass multiplication of apetalous male sterile lines of marigold

The apetalous form of male sterility, controlled by a single recessive gene (msms), is widely used for F1 hybridseed production in marigold (Tagetes spp.). Maintenance of this form of male sterility through conventionalmethods is a highly labour-intensive and costly task. Hence, we have developed a viable in vitro regenerationprotocol for rapid multiplication in three apetalous male sterile lines of marigold, MS-5, MS-7, and MS-8. Theeffect of Thidiazuron (TDZ) was assessed on in vitro establishment of apetalous male sterile lines using shoottipexplants. The modified MSm medium supplemented with 1.5 mg/l TDZ and 0.2 mg/l NAA was found suitablefor culture establishment from shoot tip explants. We have also assessed the efficiency of two cytokinins,TDZ and Kinetin (KIN), for rapid multiplication of male sterile lines of marigold. The culture media, genotypesand their interactions significantly influenced the shoot proliferation of marigold. Our findings have revealedthe superiority of kinetin over TDZ in inducing quality shoots during in vitro proliferation phase. The highestproliferation of quality shoots was obtained on modified MSm medium enriched with 0.5 mg/l kinetin and 0.1mg/l NAA. Half-strength MS medium supplemented with 0.5 mg/l of IBA was found effective for the inductionof quality roots in all the tested genotypes of marigold. Among the three male sterile lines of marigold, MS-5exhibited better in vitro establishment, proliferation and rooting abilities over the MS-7 and MS-8 lines. Thedeveloped protocol can be efficiently utilized for rapid in vitro mass multiplication of apetalous male sterilelines of marigold.

Degradation of poly(butylene adipate-co-terephthalate) films by Thermobifida fusca FXJ-1 isolated from compost.

In recent years, Poly(butylene adipate-co-terephthalate) (PBAT) films were wildly used due to its biodegradable properties. However, there are few reports of strains that can high efficiently degrade PBAT. Thermobifida fusca FXJ-1, a thermophilic actinomycete, was screened and identified from compost. FXJ-1 can efficiently degrade PBAT at 55°C in MSM medium. The degradation rates of the pure PBAT film (PF), PBAT film used for mulching on agricultural fields (PAF), and PBAT-PLA-ST film (PPSF) were 82.87±1.01%, 87.83±2.00% and 52.53±0.54%, respectively, after nine days of incubation in MSM medium. Cracking areas were monitored uniformly distributed on the surfaces of three kinds of PBAT-based films after treatment with FXJ-1 using scanning electron microscopy. The LC-MS results showed that PBAT might be degraded into adipic acid, terephthalic acid, butylene adipate, butylene terephthalate and butylene adipate-co-terephthalate, and these products are involved in the cleavage of ester bonds. We also found that amylase produced by FXJ-1 played an important role in the degradation of PPSF. FXJ-1 also showed an efficient PBAT-based films degradation ability in simulating compost environment, which implied its potential application in PBAT and starch-based film degradation by industrial composting.

Biodegradation of imidacloprid in liquid media by an isolated soil bacteria Cytobacillus firmus strain VG5

Imidacloprid (IMI), a neonicotinoid-class synthetic organic insecticide has the potential to have a negative influence on ecosystems and human health, and it has been identified as an emerging pollutant in all parts of the world in recent years. Microbial degradation is an efficient, clean and environmentally acceptable technique over physical and chemical methods for the bioremediation of organic contaminants like IMI. Our aim was to evaluate the ability of soil isolate Cytobacillus firmus strain VG5 to degrade IMI in MSM medium with glucose as a co-substrate. In liquid media, degradation was initially confirmed by nitrate reduction test. VG5 reduced IMI into guanidine with the removal of NO2- group and showed up to 87% degradation in 7 days. Spots with different Rf values on TLC sheet were identified by HPLC and GC-MS/ MS techniques as imidacloprid guanidine and 6-chloronicotinic acid based on respective standard spectra and m/z ratio. Strain VG5 showed potential to reduce pesticide pollution and further studies should be conducted to understand the degradation mechanism of IMI in soil under in- situ conditions.

High yield production of lipid and carotenoids in a newly isolated Rhodotorula mucilaginosa by adapting process optimization approach

Rhodotorula sp. is a robust microbe, which can synthesize a wide range of products such as lipids and carotenoids. In the present study, we have attempted to enhance the production of lipids and carotenoids from a newly isolated Rhodotorula mucilaginosa using a process optimization approach. Among the carbon source screened, glucose showed enhanced lipid and carotenoid production of 46.19% (w/w) and 36.2 mg/g, respectively. The low C/N has influenced lipid production by 43.5% and the high C/N ratio favored carotenoid production (41.06 µg/g). The sodium acetate with 1.5% (w/v) concentration showed a lipid content of 48.28% and carotenoid of 41.16 mg/g. Further with the optimum levels of RPM (180), pH (6), and temperature (30 °C), the maximum lipid and carotenoid were found to be 51.89%, and 43.34 µg/g, respectively. In a batch bioreactor, MSM medium with sodium acetate showed a maximum lipid content of 54%. The lipid and carotenoid were successfully characterized by laser Raman spectroscopy, a label-free method and the FAME analysis clearly showed the high content of palmitic acid (C16:0) 21.05 (% w/w), oleic acid (C18:1) 56.18 (%w/w) and linoleic acid (C18:2) 12.25 (% w/w), which is suitable for biodiesel preparation.

Evaluation of production of lipopeptide biosurfactants and surfactin micelles by native Bacillus of Iran, for a broader application range

AbstractSurfactin is one of the most important lipopeptide biosurfactants obtained by biocatalysts of Bacillus subtilis. The aim of this study was to isolate surfactin‐producing bacilli from native soils of the country (Iran), investigate their properties, convert surfactin to surfactin micelles, determine the properties of surfactin micelles and investigate the effect of starch‐coated Fe0 and Fe3+ nanoparticles on the production of surfactin from B. subtilis. To do so, 20 bacilli were isolated from the native soil sample by heat shock method and genome sequenced by SrRNA16 method. The samples with strong β‐hemolysis activity were screened as surfactant‐producing strains. Two species of 61 and 63 (B. subtilis subspecies. Inaquosorum) were selected and examined by quantitative and qualitative screening tests such as hemolysis activity, surfactin production, droplet aggregation, emulsifying activity, and surface tension reduction in MSM medium containing Fe0 and Fe3+ nanoparticles. Surfactin was converted to surfactin micelles by sonication and confirmed by SEM. The antimicrobial and emulsifying activity and surface tension reduction of surfactin micelles were investigated. According to the results, the surface tension reduction of surfactin micelles was greater than that of surfactin. The strain 61 (99.7%) culture in 5 L bioreactors containing Fe3+ nanoparticles produced more surfactin than culture of the same strain without nanoparticles. This study presents an efficient method to increase the biosynthesis of microbial metabolites.

Microbial degradation of low density polyethylene by Exiguobacterium sp. strain LM-IK2 isolated from plastic dumped soil.

Low-Density Polyethylene (LDPE) is one of the significant environmental pollutants as it is resistant to natural degradation. In this study, we reported the LDPE-degrading bacterial straini.e., Exiguobacterium sp. strain LM-IK2 isolated from plastic dumped soil which shows potential degradation capability. The percent weight loss of LDPE was calculated as -5.70 ± 0.7 after 90days of incubation in a carbon-free MSM medium. The Field Emission Scanning Electron Microscopy (FE-SEM) analysis shows that LDPE films show slight surface disruption after treatment with bacteria. The Fourier Transform Infrared Spectroscopy (FTIR) revealed the chemical changes in LDPE films e.g., formation and reduction of typical carbonyl peaks after incubation with bacteria. The X-Ray Diffraction (XRD) analysis displayed an increase in percent crystallinity, with a slight change in total carbon content. Genetic analysis showed the presence of Laccase (167bp) and Alkane Hydroxylase (330bp) genes that are responsible for LDPE degradation.Thus, Exiguobacterium sp. strain LM-IK2 has the potential to degrade LDPE and could be further explored to improve its efficiency in the bioremediation of LDPE.

Statistical Approach to Optimize Production of Biosurfactant by Achromobacter Xylos

This study aimed to optimize medium composition biosurfactant production of achromobacter xylos using response surface quadratic model . Lipoprotein and lipopeptides are used in many industries such as petroleum refining, pharmaceutical, mining, agriculture and bioprocess industries. The point of this assessment was to pull out and portray the biosurfactant passing on restriction of microorganisms from oil corrupted soil and considering their advancement energy at various temperatures and pH. The separation and growth study was directed in MSM medium using lamp fuel oil as sole carbon hotspot for bacterial turn of events. Confined strains were found to be Gram positive bacillus and in general Gram's positive minuscule life forms can convey lipopeptides type biosurfactants. The ideal conditions for achromobacteria xylos growth were discovered to be at pH seven (7) and temperature 30oC. Central composite design (CCD) was utilized to pick the following medium components (MgSO4, NaNO3, CaCl2, (NH4)2SO4, FeSO4, and KH2PO4). Central composite arrangement (CCD) of RSM was utilized to analyze the four parts at five stages, and biosurfactant fixation was evaluated as reaction. Backslide coefficients were directed by backslide examination, and the quadratic model condition was settled. R2 an impetus for bio-surfactant was endeavored to be 0.7527, showing that the quadratic model was basic with the exploratory outcomes. Confirmation of the numerical model was driven by playing out the assessment with the normal overhauled values, and bio-surfactant production was found to be 10.53 g/L. Underwriting of the normal quadratic model was 97.3% exact with the test results facilitated under the ideal conditions. CaCl2, (NH4)2SO4, FeSO4, and KH2PO4 were perceived as successful portions for bio-surfactant delivering 98% of achromobacter xylos microorganism.

Poly-cis-isoprene Degradation by Nocardia sp. BSTN01 Isolated from Industrial Waste.

The natural and synthetic rubber (NR and SR) products are made up of poly-cis-isoprene which are estimated as one of the major solid-wastes and need to be cleared through bacterial bioremediation. The present research reports isolation and characterization of a gram-positive, non-spore forming, filamentous actinomycete Nocardia sp. BSTN01 from the waste of a rubber processing industry. We found NR- and SR-dependent growth of BSTN01 over a period of time. BSTN01 has been found to degrade NR by 55.3% and SR by 45.9% in 6weeks. We have found an increase in the total protein of BSTN01 cells up to 623.6 and 573.9µg/ml for NR and SR, respectively, after 6weeks of growth in rubber-supplemented MSM medium. Scanning electron microscopy revealed adhesive growth of BSTN01 on the surface of NR and SR. Formation of aldehyde groups due to the degradation was indicated by Schiff's test and confirmed by FTIR-ATR analysis. The genome sequence of BSTN01 revealed the gene responsible for rubber degradation. The presence of lcp gene and structural analysis of the latex clearing protein further confirmed the reliability. Studies on quantification of rubber degradation capability by the isolated strain prove it to be an efficient degrader of NR and SR. This study revealed the genome sequence and structural analysis of the proteins responsible for degradation of rubber. A new fast-growing Nocardia strain can degrade both NR and SR with higher efficiency and have future potential for rubber solid-waste management either alone or in consortia.

Isolation and characterization of a polyurethane-degrading bacterium

Biodegradation of polyurethane (PUR) pollutants by microorganisms has received widespread attention currently. Identification of microorganisms capable of efficiently degrading PUR plastics is a key point. In this study, a strain P10 capable of degrading PUR was isolated from the plastic wastes, and identified as a bacterium belonging to the genus of Brevibacillus based on colony morphology and 16S rDNA phylogenetic analysis. Brevibacillus sp. P10 was capable of degrading 71.4% of waterborne polyurethane (Impranil DLN) after 6 days growth in MSM medium with DLN as a sole carbon source. In addition, strain P10 can use commercial PUR foam as the sole carbon source for growth. Brevibacillus sp. P10 can degrade 50 mg PUR foam after 6 days growth in MSM medium supplemented with 5% (V/V) LB after optimization of degradation conditions. This indicates that Brevibacillus sp. P10 has potential to be used in biodegradation of PUR waste.

Characterization of a novel beta-cypermethrin-degrading strain of Lactobacillus pentosus 3-27 and its effects on bioremediation and the bacterial community of contaminated alfalfa silage

In this study, a novel beta-cypermethrin (beta-cyp)-degrading strain Lactobacillus pentosus 3–27 (LP3–27) was screened from beta-cyp-contaminated silage. The strain could degrade 96% of beta-cyp (50 mg/L) in MSM medium after 4 d of culture, while the strain lost its degradation ability when the beta-cyp concentration reached 250 mg/L. The effects of LP 3–27 on fermentation, bacterial community, and bioremediation of contaminated alfalfa silage at two dry matter (DM) contents were studied. The results showed that inoculation with LP3–27 not only degraded beta-cyp, but also improved the fermentation quality of alfalfa silage after 60 d of ensiling. Meanwhile, L. pentosus dominated the bacterial community during ensiling in LP3–27 inoculated silages, whereas Pediococcus acidilactici was the dominant species in the control silage. LP3–27 inoculation also simplified the bacterial interaction networks of ensiled alfalfa. Beta-cyp degradation was positively correlated with L. pentosus in LP- inoculated silages, which confirmed the function of beta-cyp degradation by L. pentosus. In addition, higher beta-cyp degradation was observed in silage with 35% versus 43% DM. In summary, strain LP3–27 could be used as a candidate inoculum for bioremediation of beta-cyp-contaminated silage and to produce safe silage for animal production.

Investigating the potential of bioremediation in aged oil-polluted hypersaline soils in the south oilfields of Iran.

To date, studies for bioremediation of oil-polluted hypersaline soils have been neglected or limited to specific spots. Hence, in this study, ten samples of oil field soils in the Khuzestan province of Iran were collected to evaluate bioremediation's feasibility. These samples were analyzed for their physicochemical properties as well as the most probable number of total and hydrocarbon-degrading bacteria. Thirty-nine hydrocarbon-degrading bacteria were isolated from these soils over a 1-month incubation in an MSM medium enriched with diesel oil as the sole source of carbon. As revealed by 16S-rRNA analysis, the identified strains belonged to the genera Ochrobactrum, Microbacterium, and Bacillus with a high frequency of Ochrobactrum species. Additionally, by using degenerate primers, the third group of alkB gene was detected in Ochrobactrum and Microbacterium isolates through the touchdown nested PCR method for the first time. Ochrobactrum species possessing the alkB gene showed the highest population, and therefore, the highest adaptation to harsh environmental conditions. Most isolates showed outstanding results in the ability to grow with crude and diesel oil and tolerate high salt percentages, biosurfactant production, and emulsification activity, which are considered the most effective factors in bioremediation of such environments. Considering the soil analysis, limiting factors in bioremediation like available phosphorous, and the abundance of bacteria with remediation traits in these soils, these extremely polluted environments can be refined.

The Influence of Nutrient Media Modification on the Morphological Parameters in Raspberry (Rubus idaeus L.) Micropropagation in the Liquid and Semi-solid Media

Within the nursery sector, a global interest in functional fruits imposes a permanent need to improve the tissue culture technology. A modern bioreactor immersion system is currently a valid solution for the micropropagation of many cultures. At the Faculty of Agrobiotechnical Sciences Osijek (FAZOS, Croatia), a research was conducted with the aim of improving the raspberry (Rubus idaeus L., cv. HimboTop®) micropropagation protocol. The treatments implied the modifications of the MS and DKW nutrient medium (DKW – standard; DKWm – substitution of FeEDTA with the FeEDDHA; MSm – macroelement modification, 3 x CaCl2, MgSO4, and KH2PO4 + FeEDDHA), differently depending on the aggregate condition (semi‐solid and liquid SETISTM). Subsequent to 15 (SETISTM) and 30 cultivation days (semi-solid medium), respectively, a morphological parameter analysis and a multiplication success evaluation were commenced. In all treatments (DKW, DKWm, and MSm), the observed parameters (shoot height, number of leaves, leaf width and length) were significantly better in an interaction with the liquid medium. Comparing the models, a liquid medium (SETISTM) resulted in a significantly higher explant multiplication (4.02) (a semi‐solid medium - 2.74). The best reproduction quality, implying a better plant vigor with a reduced vitrification, was obtained on the MSm medium in the SETISTM. A concentration optimization of individual mesocomponents (i.e., of the macroelements) and their chemical forms (FeEDDHA) may contribute to their improved adoption and consequently to the stress suppression due to explant transplantation. Selecting the modern technical solutions (TIS bioreactors - SETISTM) and continuously improving the protocols, we may obtain the realistically sustainable models, suitable for a massive clonal reproduction.

Burkholderia thailandensis E264 as a promising safe rhamnolipids\u2019 producer towards a sustainable valorization of grape marcs and olive mill pomace

Within the circular economy framework, our study aims to assess the rhamnolipid production from winery and olive oil residues as low-cost carbon sources by nonpathogenic strains. After evaluating various agricultural residues from those two sectors, Burkholderia thailandensis E264 was found to use the raw soluble fraction of nonfermented (white) grape marcs (NF), as the sole carbon and energy source, and simultaneously, reducing the surface tension to around 35 mN/m. Interestingly, this strain showed a rhamnolipid production up to 1070 mg/L (13.37 mg/g of NF), with a higher purity, on those grape marcs, predominately Rha-Rha C14-C14, in MSM medium. On olive oil residues, the rhamnolipid yield of using olive mill pomace (OMP) at 2% (w/v) was around 300 mg/L (15 mg/g of OMP) with a similar CMC of 500 mg/L. To the best of our knowledge, our study indicated for the first time that a nonpathogenic bacterium is able to produce long-chain rhamnolipids in MSM medium supplemented with winery residues, as sole carbon and energy source.Key points• Winery and olive oil residues are used for producing long-chain rhamnolipids (RLs).• Both higher RL yields and purity were obtained on nonfermented grape marcs as substrates.• Long-chain RLs revealed stabilities over a wide range of pH, temperatures, and salinities

Haploid Embryogenesis in Isolated Microspore Culture of Carrots (Daucus carota L.).

The process of embryogenesis in isolated microspore culture was studied in eight carrot accessions of different origin. The ½NLN-13 medium supplemented with 0.2 mg/L 2,4D and 0.2mg/L kinetin was used to induce embryogenesis. The temperature treatment was performed at 5–6 °C for three days, followed by cultivation at 25 °C in darkness. As was shown, the first embryogenesis was only observed in microspores at the late vacuolated stage when the nucleus moved from the center to one pole following the long cell axis. Depending on the nucleus position, the microspore can divide into two equal or two different sized cells. Following divisions occurred either in one of these cells or in two. However, microspores that divided into two unequal cells were morphologically different form bi-cellular pollen grain. Embryogenic divisions in bi-cellular pollen grains were not observed. First divisions began by the third day of cultivation, and continued until the globular embryoid stage that was well-seen after the fourth week of cultivation. The already-formed embryoids can develop the secondary embryoids on their surface. Depending on the genotype, up to 1000 secondary embryoids can be produced from one embryoid in the liquid MSm medium supplemented with 0.1 mg/L of kinetin for regeneration. All carrot accessions studied were split into three groups: responsive genotypes, weakly responsive genotypes, and reluctant genotypes. The highest yield was 53 initial embryoids per a 6 cm diameter petri dish. Thus, the Nantskaya 4 cultivar totally produced 256 initial embryoids, out of which 94 developed into green plantlets and 162 into albino plantlets, whereas 97 initial embryoids with 45 albino plantlets formed from them were obtained from Chantenay cultivar.

Efficiency of shoot regeneration and micropropagation of purple passion fruit (Passiflora edulis Sims.) via internodal longitudinal thin cell layer culture

The thin cell layer culture technique (TCL) has been used for the effective tissue culture of several dozen plants with commercial importance such as field crops (rice, cereals), horticultural commodities (fruits, vegetables, ornamental plants), medicinal plants and herbs (Panax vietnamensis Ha et Grushv.), and even forestry trees (Pinus sp.), and woody fruit plants (Citrus spp., apple). In the present, TCL was used to evaluate the efficiency of shoot regeneration and propagation for purple passion fruit (Passiflora edulis Sims.). The internodes were cut longitudinally (lTCL) and was used as an initial material. The results showed that the shoot regeneration rate and number of shoots from internode-lTCL depended on position of internodes (1, 2, 3, 4 and 5) and the plant growth regulator (BA and NAA). After 8 weeks of culture, internode-lTCL derived from 3rd internode of P. edulis cultured on MS medium supplemented with 1.5 mg/L BA in combination with 1.0 mg/LNAA gave the highest shoot regeneration rate (83.33%), and number of shoots (3.00 shoots/explant). These shoots were cultured on modified MS (MSM) medium supplemented with 1.0 mg/L BA showed higher efficiency of shoot multiplication (3.56 shoots/explant and 6.67 cm height) than the other BA treatments. In addition, the highest rooting rate was 76.67% when cultured on MSM medium containing 2.0 mg/L IBA. The survival rate of plantlets was 83.33% when transferred into greenhouse condition after 10 weeks. The results of this study were the initial success in establishing an effective in vitro regeneration and propagation of Passiflora edulis Sims. through internode-lTCL.