Selection of Diazotrophic Bacterial Communities in Biological Sand Filter Mesocosms Used for the Treatment of Phenolic-Laden Wastewater

Agri effluents such as winery or olive mill wastewaters are characterized by high phenolic concentrations. These compounds are highly toxic and generally refractory to biodegradation. Biological sand filters (BSFs) represent inexpensive, environmentally friendly, and sustainable wastewater treatment systems which rely vastly on microbial catabolic processes. Using denaturing gradient gel electrophoresis and terminal-restriction fragment length polymorphism, this study aimed to assess the impact of increasing concentrations of synthetic phenolic-rich wastewater, ranging from 96 mg L^?1 gallic acid and 138 mg L^?1 vanillin (i.e., a total chemical oxygen demand (COD) of 234 mg L^?1) to 2,400 mg L^?1 gallic acid and 3,442 mg L^?1 vanillin (5,842 mg COD L^?1), on bacterial communities and the specific functional diazotrophic community from BSF mesocosms. This amendment procedure instigated efficient BSF phenolic removal, significant modifications of the bacterial communities, and notably led to the selection of a phenolic-resistant and less diverse diazotrophic community. This suggests that bioavailable N is crucial in the functioning of biological treatment processes involving microbial communities, and thus that functional alterations in the bacterial communities in BSFs ensure provision of sufficient bioavailable nitrogen for the degradation of wastewater with a high C/N ratio.     

Isolation and characterization of pyrene and benzo[a]pyrene-degrading Klebsiella pneumonia PL1 and its potential use in bioremediation

Polycyclic aromatic hydrocarbons (PAHs), which are hard to degrade, are the main pollutants in the environment. Degradation of PAHs in the environment is becoming more necessary and urgent. In the current study, strain PL1 with degradation capability of pyrene (PYR) and benzo[a]pyrene (BaP) was isolated from soil and identified as Klebsiella pneumoniae by morphological and physiological characteristics as well as 16S rDNA sequence. With the presence of 20 mg L^?1 PYR and 10 mg L^?1 BaP in solution, the strain PL1 could degrade 63.4 % of PYR and 55.8 % of BaP in 10 days, respectively. The order of biodegradation of strain PL1 was pH 7.0?>?pH 8.0?>?pH 10.0?>?pH 6.0?>?pH 5.0. Strain PL1 degradation ability varied in different soil. The half-life of PYR in soil was respectively 16.9, 24.9, and 88.9 days in paddy soil, red soil, and fluvo-aquic soil by PL1 degradation; however, the half-lives of BaP were respectively 9.5, 9.5, and 34.0 days in paddy soil, red soil, and fluvo-aquic soil by PL1 degradation. The results demonstrate that the degradation capability on PYR and BaP by PL1 in paddy soil was relatively good, and K. pneumoniae PL1 was the new degradation bacterium of PYR and BaP. K. pneumoniae PL1 has potential application in PAH bioremediation.     

Simultaneous production of propionic acid and vitamin B12 from corn stalk hydrolysates by Propionibacterium freudenreichii in an expanded bed adsorption bioreactor

Abstract

Vitamin B12 and propionic acid that were simultaneous produced by Propionibacterium freudenreichii are both favorable chemicals widely used in food preservatives, medicine, and nutrition. While the carbon source and propionic acid accumulation reflected fermentation efficiency. In this study, using corn stalk as a carbon source and fed-batch fermentation process in an expanded bed adsorption bioreactor was studied for efficient and economic biosynthesis of acid vitamin B12 and propionic. With liquid hot water pretreated corn stalk hydrolysates as carbon source, 28.65?mg L^?1 of vitamin B12 and 17.05?g L^?1 of propionic acid were attained at 168?h in batch fermentation. In order to optimize the fermentation outcomes, fed-batch fermentation was performed with hydrolyzed corn stalk in expanded bed adsorption bioreactor (EBAB), giving 47.6?mg L^?1 vitamin B12 and 91.4?g L^?1 of propionic acid at 258?h, which correspond to product yields of 0.37?mg g^?1 and 0.75?g g^?1, respectively. The present study provided a promising strategy for economically sustainable production of vitamin B12 and propionic acid by P. freudenreichii fermentation using biomass cornstalk as carbon source and expanded bed adsorption bioreactor.     

Biodegradation of the Pyrethroid Pesticide Esfenvalerate by Marine-Derived Fungi

Esfenvalerate biodegradation by marine-derived fungi is reported here. Esfenvalerate (S,S-fenvalerate) and its main metabolites [3-phenoxybenzaldehyde (PBAld), 3-phenoxybenzoic acid (PBAc), 3-phenoxybenzyl alcohol (PBAlc), and 2-(4-chlorophenyl)-3-methylbutyric acid (CLAc)] were quantitatively analyzed by a validated method in triplicate experiments. All the strains (Penicillium raistrickii CBMAI 931, Aspergillus sydowii CBMAI 935, Cladosporium sp. CBMAI 1237, Microsphaeropsis sp. CBMAI 1675, Acremonium sp. CBMAI 1676, Westerdykella sp. CBMAI 1679, and Cladosporium sp. CBMAI 1678) were able to degrade esfenvalerate, however, with different efficiencies. Initially, 100 mg L^?1 esfenvalerate (Sumidan 150SC) was added to each culture in 3 % malt liquid medium. Residual esfenvalerate (64.8–95.2 mg L^?1) and the concentrations of PBAc (0.5–7.4 mg L^?1), ClAc (0.1–7.5 mg L^?1), and PBAlc (0.2 mg L^?1) were determined after 14 days. In experiments after 7, 14, 21, and 28 days of biodegradation with the three most efficient strains, increasing concentrations of the toxic compounds PBAc (2.7–16.6 mg L^?1, after 28 days) and CLAc (6.6–13.4 mg L^?1, after 28 days) were observed. A biodegradation pathway was proposed, based on HPLC-ToF results. The biodegradation pathway includes PBAld, PBAc, PBAlc, ClAc, 2-hydroxy-2-(3-phenoxyphenyl)acetonitrile, 3-(hydroxyphenoxy)benzoic acid, and methyl 3-phenoxy benzoate. Marine-derived fungi were able to biodegrade esfenvalerate in a commercial formulation and showed their potential for future bioremediation studies in contaminated soils and water bodies.     

Bacterial community analysis in chlorpyrifos enrichment cultures via DGGE and use of bacterial consortium for CP biodegradation

The organophosphate pesticide chlorpyrifos (CP) has been used extensively since the 1960s for insect control. However, its toxic effects on mammals and persistence in environment necessitate its removal from contaminated sites, biodegradation studies of CP-degrading microbes are therefore of immense importance. Samples from a Pakistani agricultural soil with an extensive history of CP application were used to prepare enrichment cultures using CP as sole carbon source for bacterial community analysis and isolation of CP metabolizing bacteria. Bacterial community analysis (denaturing gradient gel electrophoresis) revealed that the dominant genera enriched under these conditions were Pseudomonas, Acinetobacter and Stenotrophomonas, along with lower numbers of Sphingomonas, Agrobacterium and Burkholderia. Furthermore, it revealed that members of Bacteroidetes, Firmicutes, α- and γ-Proteobacteria and Actinobacteria were present at initial steps of enrichment whereas β-Proteobacteria appeared in later steps and only Proteobacteria were selected by enrichment culturing. However, when CP-degrading strains were isolated from this enrichment culture, the most active organisms were strains of Acinetobacter calcoaceticus, Pseudomonas mendocina and Pseudomonas aeruginosa. These strains degraded 6–7.4 mg L^?1 day^?1 of CP when cultivated in mineral medium, while the consortium of all four strains degraded 9.2 mg L^?1 day^?1 of CP (100 mg L^?1). Addition of glucose as an additional C source increased the degradation capacity by 8–14 %. After inoculation of contaminated soil with CP (200 mg kg^?1) disappearance rates were 3.83–4.30 mg kg^?1 day^?1 for individual strains and 4.76 mg kg^?1 day^?1 for the consortium. These results indicate that these organisms are involved in the degradation of CP in soil and represent valuable candidates for in situ bioremediation of contaminated soils and waters.     

Growth Kinetics of Microbacterium lacticum and Nitrate-Dependent Degradation of Ethylbenzene under Anaerobic Conditions

A pure strain of Microbacterium lacticum DJ-1 capable of anaer-obic biodegradation of ethylbenzene was isolated from soil contaminated with gasoline. Growth of the strain and biodegradation of ethylbenzene in batch cultures led to stoichiometric reduction of nitrate. M. lacticum DJ-1 could degrade 100 mg L^?1 of ethylbenzene completely, with a maximum degradation rate of 15.02 ± 1.14 mg L^?1 day^?1. Increasing the initial concentration of ethy-lbenzene resulted in decreased degradative ability. The cell-specific growth rates on ethylbenzene conformed to the Haldane–Andrew model in the substrate level range of 10–150 mg L^?1. Kinetic parameters were determined by nonlinear regression on specific growth rates and various initial substrate concentrat-ions, and the values of the maximum specific growth rate, half saturation constant, and inhibition constant were 0.71 day^?1, 34.3 mg L^?1, and 183.5 mg L^?1, respectively. This is the first report of ethylbenzene biodegradation by a bacterium of Microbacterium lacticum under nitrate-reducing conditions.     

Biodegradation of 4-bromophenol by Arthrobacter chlorophenolicus A6 in batch shake flasks and in a continuously operated packed bed reactor

The present study investigated growth and biodegradation of 4-bromophenol (4-BP) by Arthrobacter chlorophenolicus A6 in batch shake flasks as well as in a continuously operated packed bed reactor (PBR). Batch growth kinetics of A. chlorophenolicus A6 in presence of 4-BP followed substrate inhibition kinetics with the estimated biokinetic parameters value of μ _max = 0.246 h^?1, K _i = 111 mg L^?1, K _s  = 30.77 mg L^?1 and K = 100 mg L^?1. In addition, variations in the observed and theoretical biomass yield coefficient and maintenance energy of the culture were investigated at different initial 4-BP concentration. Results indicates that the toxicity tolerance and the biomass yield of A. chlorophenolicus A6 towards 4-BP was found to be poor as the organism utilized the substrate mainly for its metabolic maintenance energy. Further, 4-BP biodegradation performance by the microorganism was evaluated in a continuously operated PBR by varying the influent concentration and hydraulic retention time in the ranges 400–1,200 mg L^?1 and 24–7.5 h, respectively. Complete removal of 4-BP was achieved in the PBR up to a loading rate of 2,276 mg L^?1 day^?1.     

CITRIC ACID PRODUCTION BY Candida SPECIES GROWN ON A SOY-BASED CRUDE GLYCEROL

Citric acid was produced by five species of the yeast Candida after growth on a medium containing soy biodiesel-based crude glycerol. After growth on a medium containing 10 g L^?1 or 60 g L^?1 crude glycerol for 168 hr at 30°C, Candida parapsilosis ATCC 7330 and C. guilliermondii ATCC 9058 produced the highest citric acid levels. On 10 g L^?1 or 60 g L^?1 crude glycerol for 168 hr at 30°C, the citric acid level produced by C. parapsilosis ATCC 7330 was 1.8 g L^?1 or 11.3 g L^?1, respectively, while C. guilliermondii ATCC 9058 produced citric acid concentrations of 3.0 g L^?1 or 10.4 g L^?1, respectively. Biomass production by C. guilliermondii ATCC 9058 on 10 g L^?1 or 60 g L^?1 crude glycerol for 168 hr at 30°C was highest at 1.2 g L^?1 or 6.9 g L^?1, respectively. The citric acid yields observed for C. guilliermondii ATCC 9058 after growth on 10 g L^?1 or 60 g L^?1 crude glycerol (0.35 g g^?1 or 0.21 g g^?1, respectively) were generally higher than for the other Candida species tested. When similar crude glycerol concentrations were present in the culture medium, citric acid yields observed for some of the Candida species utilized in this study were about the same or higher compared to citric acid yields by Yarrowia lipolytica strains. Based on the findings, it appeared that C. guilliermondii ATCC 9058 was the most effective species utilized, with its citric acid production being similar to what has been observed when citric acid-producing strains of Y. lipolytica were grown on crude glycerol under batch conditions that could be of significance to biobased citric acid production.     

Elicitor mediated enhancement of wedelolactone in cell suspension culture of <Emphasis Type="Italic">Eclipta alba</Emphasis> (L.) Hassk

The present research focused on enhancing the production of wedelolactone through cell suspension culture (CSC) in Eclipta alba (L.) Hassk. With an aim of attaining a sustainable CSC, various plant growth regulators, elicitors and agitation speed were examined. Nodal segments of in vitro propagated plantlets induced the maximum percentage (93.47?±?0.61%) of callus inoculated on Murashige and Skoog (MS) medium fortified with picloram (2 mg L^?1). The growth kinetics of CSC exhibited a sigmoid pattern with a lag phase (0–6 days), a log phase (6–18 days), a stationary phase (18–24 days) and then death phase thereafter. The highest biomass accumulation in CSC with 7.09?±?0.06 g 50 mL^?1 fresh weight, 1.52?±?0.02 g 50 mL^?1 dry cell weight, 1.34?±?0.01?×?10⁶ cell mL^?1 total cell count and 57.00?±?0.58% packed cell volume was obtained in the liquid MS medium supplemented with 1.5 mg L^?1 picloram plus 0.5 mg L^?1 kinetin at 120 rpm. High performance thin layer chromatography confirmed that yeast extract (biotic elicitor) at 150 mg L^?1 accumulated more CSC biomass with 1.22-fold increase in wedelolactone (288.97?±?1.94 µg g^?1 dry weight) content in comparison to the non-elicited CSC (237.78?±?0.04 µg g^?1 dry weight) after 120 h of incubation. Contrastingly, methyl jasmonate (abiotic elicitor) did not alter the biomass but increased the wedelolactone content (259.32?±?1.06 µg g^?1 dry weight) to an extent of 1.09-fold at 100 µM. Complete plantlet regeneration from CSC was possible on MS medium containing N⁶-benzyladenine (0.75 mg L^?1) and abscisic acid (0.5 mg L^?1). Thus, the establishment of protocol for CSC constitutes the bases for future biotechnological improvement studies in this crop.     

Phytotoxic analysis of coastal medicinal plants and quantification of phenolic compounds using HPLC

Abstract

Medicinal plants are rich sources of bioactive phytochemicals. Production of such chemicals usually increased under unfavorable conditions. This study investigated the phytotoxic potential of 105 medicinal plants distributed in arid/semiarid areas along Arabian Sea coast of Pakistan. Total polyphenols, flavonoids, tannins and composition of individual phenolic compounds were also determined in species which showed high phytotoxic potential. Sandwich method was used to determine the phytotoxicity of medicinal plants on the growth of lettuce seedlings. Radicle growth was inhibited more than the hypocotyl growth. In general, halophytes showed higher phytotoxic potential than non-halophytes. Capparis cartilaginea, Indigofera hochstetteri, Parkinsonia aculeata and Prosopis glandulosa showed highest degree of inhibition. Higher amount of total phenols (16.35–25.33?mg GAE g^?1), flavonoids (3.32-6.41?mg QE g^?1) and tannins (1.54–2.54?mg TAE g^?1) were found in these species. Pyrocatechol, quercetin, gallic, hydroxybenzoic and ferulic acids were detected as major phytotoxins, of which, gallic acid, pyrocatechol and quercetin were most abundant. These phytochemicals could be used for the production of natural, safe, healthy and eco-friendly agro-chemicals. Furthermore, these plants can be grown without encroaching agricultural lands, and can convert vast areas of arid/saline lands into economically viable resources, which also helps to halt climate change and desertification.     

Effect of different acclimation methods on the performance of microbial fuel cells using phenol as substrate

Single-chamber microbial fuel cells (MFCs) with air-cathode were constructed. MFCs were fed different feedstocks during their inoculation, their role on phenol degradation and MFC performance were investigated. The results showed that the MFC inoculated using glucose exhibited the highest power density (31.3 mW m^?2) when phenol was used as the sole substrate for MFC. The corresponding biodegradation kinetic constant was obtained at 0.035 h^?1, at an initial phenol concentration of 600 mg L^?1. Moreover, the phenol degradation rates in this MFC with closed circuit were 9.8–16.5 % higher than those in MFC with opened circuit. The cyclic voltammograms revealed a different electrochemical activity of the anode biofilms in the MFC, and this led to differences in performance of the MFCs with phenol as sole substrate. These results demonstrated that phenol degradation and power production are affected by current generation and type of acclimation.     

Kinetics of the aerobic co-metabolism of 1,1-dichloroethylene by Achromobacter sp.: a novel benzene-grown culture

Batch experiments were performed for the aerobic co-metabolism of 1,1-dichloroethylene (1,1-DCE) by Achromobacter sp., identified by gene sequencing of 16S rRNA and grown on benzene. Kinetic models were employed to simulate the co-metabolic degradation of 1,1-DCE, and relevant parameters were obtained by non-linear least squares regression. Benzene at 90 mg L^?1 non-competitively inhibited degradation of 1,1-DCE (from 125 to 1,200 μg L^?1). The maximum specific utilization (k_c) rate and the half-saturation constant (K_c) for 1,1-DCE were 54 ± 0.85 μg h^?1 and 220 ± 6.8 μg L^?1, respectively; the k_b and K_b for benzene were 13 ± 0.18 mg h^?1 and 28 ± 0.42 mg L^?1, respectively. This study provides a theoretical basis to predict the natural attenuation when benzene and 1,1-DCE occur as co-contaminants.     

High efficient somatic embryogenesis development from leaf cultures of <Emphasis Type="Italic">Citrullus colocynthis</Emphasis> (L.) Schrad for generating true type clones

We report an efficient somatic embryogenesis and plant regeneration system using leaf cultures of Citrullus colocynthis (L.) and assessed the effect of plant growth regulators on the regeneration process. Initially leaf explants were cultured on Murashige and Skoog medium supplemented with different concentrations of auxins viz., 2,4-dichlorophenoxyacetic acid, 1-naphthaleneacetic acid, gibberellic acid alone and along with combination of 6-benzylaminopurine. The different forms of calli such as compact, white friable, creamy friable, brownish nodular, green globular and green calli were induced from the leaf explants on MS medium containing different concentrations of auxins and gibberellins. Subsequently initial callus was subcultured at 1.5 mg L^?1 BAP + 1.0 mg L^?1 2,4-D which resulted in 25 % somatic embryos from 85 % nodular embryogenic nodular callus that is highest percentage. Similarly the lowest percentage of somatic embryos was recorded at 2.5 mg L^?1 BAP + 0.5 mg L^?1 NAA from 55 % embryogenic globular callus i.e., 16 %. High frequency of embryo development takes place at intermittent light when compared with continuous light in the individual subcultures. The cotyledonary embryos were developed into complete platelets on MS medium. In vitro regenerated plantlets were washed to remove the traces of agar and then transferred to sterile vermiculite and sand (2:1) containing pot.     

Enhancing of Phytoremediation Efficiency Using Indole-3-Acetic Acid (IAA)

In this study, a pot experiment using Solanum nigrum L. grown in cadmium-contaminated soil was conducted in a greenhouse. Indole-3-acetic acid (IAA) was applied at three different concentrations (1 mg L^?1, 10 mg L^?1, and 100 mg L^?1) to examine the effects on phytoremediation efficiency. According to the experimental results, IAA increased the shoot biomass of S. nigrum significantly, by 124% at the highest concentration used, and increased the Cd concentration in the shoot of S. nigrum by 16%. The Cd extraction amount from a single plant was increased by up to 158%, demonstrating potential practical application for remediation practice.     

Growth and nutrient removal of three macrophytes in response to concentrations and ratios of N and P

Wastewater from different sources shows great differences in concentrations and ratios of N and P. In order to choose suitable plant species to remove excess N and/or P from polluted waters, it is important to know the performances of these plants under different N and P concentrations. In this study, we investigated the growth and N and P removal rate of three macrophytes, Coix lacryma-jobi, Iris wilsonii, and Arundo donax under six N and P combination treatments. C. lacryma-jobi preferred higher N and P concentrations (16 mg N L^?1 and 3.2 mg P L^?1), and increasing N supply could increase its P removal rate. I. wilsonii exhibited a growth preference at a combination of moderate N and P concentrations (8 mg N L^?1 and 0.8 mg P L^?1). A. donax could grow well at all combinations of N and P and had significantly higher relative growth rate and N and P removal rates than the other two species. These results showed A. donax is a promising species to treat various polluted waters and the other two species can be used specifically to treat certain types of wastewater.     

A rapid and efficient in vitro shoot regeneration protocol using cotyledons of London plane tree (<Emphasis Type="Italic">Platanus acerifolia</Emphasis> Willd.)

A rapid, prolific and reproducible protocol for in vitro shoot regeneration from mature cotyledons of Platanus acerifolia has been developed. The influences of different plant growth regulator (PGR) combinations and donor seedling ages on shoot regeneration were investigated. The results showed that the application of BA in conjunction with NAA was the most effective PGR combination for the induction of shoot regeneration. When cotyledon explants of 5-day-old seedlings were incubated on MS basal medium supplemented with 4.0 mg L^?1 BA and 0.2 mg L^?1 NAA, 67.6?±?4.9% of the cotyledon segments produced adventitious shoots. These regenerated shoots were initially formed as stunted rosette cluster forms and were encouraged to elongate to produce distinct shoots by transfer onto MS medium containing 0.5 mg L^?1 BA and 0.05 mg L^?1 NAA; the resulting mean number of adventitious shoots per explant was 5.81?±?0.36. The elongated shoots were readily induced to root (i.e. 89.3% of shoots) by incubation on ½-strength MS medium supplemented with 0.1 mg L^?1 IBA. This is the first report of an efficient in vitro shoot regeneration protocol for P. acerifolia through direct organogenesis using cotyledon explants. Hence, this provides a more efficient basis for the Agrobacterium-mediated genetic transformation of Platanus than previously available.     

Effects of stationary phase elongation and initial nitrogen and phosphorus concentrations on the growth and lipid-producing potential of Chlorella sp. HQ

Higher lipid production and nutrient removal rates are the pursuing goals for synchronous biodiesel production and wastewater treatment technology. An oleaginous alga Chlorella sp. HQ was tested in five different synthetic water, and it was found to achieve the maximum biomass (0.27 g L^?1) and lipid yield (41.3 mg L^?1) in the synthetic secondary effluent. Next, the effects of the stationary phase elongation and initial nitrogen (N) and phosphorus (P) concentrations were investigated. The results show that the algal characteristics were affected apparently under different N concentrations but not P, which were verified by Logistic and Monod models. At the early stationary phase, the algal biomass, lipid and triacylglycerols (TAGs) yields, and P removal efficiency increased and reached up to 0.19 g L^?1, 46.7 mg L^?1, 14.3 mg L^?1, and 94.3 %, respectively, but N removal efficiency decreased from 86.2 to 26.8 % under different N concentrations. And the largest TAGs yield was only 6.4 mg L^?1 and N removal efficiency was above 71.1 % under different P concentrations. At the late stationary phase, the maximal biomass, lipid and TAGs yields, and P removal efficiencies primarily increased as the initial N and P concentrations increase and climbed up to 0.49 g L^?1, 99.2 mg L^?1, 54.0 mg L^?1, and 100.0 %, respectively. It is concluded that stationary phase elongation is of great importance and the optimal initial N/P ratio should be controlled between 8/1 and 20/1 to serve Chlorella sp. HQ for better biodiesel production and secondary effluent purification.     

Variability of phenolic composition and biological activities of two Tunisian halophyte species from contrasted regions

The present study consists in evaluating the inter- and intraspecific variability of phenolic contents and biological capacities of Limoniastrum monopetalum L. and L. guyonianum Boiss. extracts. Ultimately, they were subjected to HPLC for phenolic identification. Results showed a great variation of phenolic content as function of species and localities. In fact, L. guyonianum extracts (El Akarit) contained the highest polyphenol (57 mg GAE g^?1 DW), flavonoid (9.47 mg CE g^?1 DW) and condensed tannin contents (106.58 mg CE g^?1 DW). These amounts were accompanied by the greatest total antioxidant activity (128.53 mg GAE g^?1 DW), antiradical capacity (IC₅₀ = 4.68 μg/ml) and reducing power (EC₅₀ = 120 μg/ml). In addition, L. monopetalum and L. guyonianum extracts exhibited an important and variable antibacterial activity with a diameter of inhibition zone ranging from 6.00 to 14.83 mm. Furthermore, these extracts displayed considerable antifungal activity. L. monopetalum extracts (Enfidha) showed the strongest activity against Candida glabrata and C. krusei with a diameter exceeding 12 mm. The phytochemical investigation of these extracts confirmed the variability of phenolic composition, since the major phenolic compound varied as a function of species and locality. These findings suggest that these two halophytes may be a new source of natural antioxidants that are increasingly important for human consumption, as well as for agro-food, cosmetic and pharmaceutical industries.     

Conversion of oat (<Emphasis Type="Italic">Avena sativa</Emphasis> L.) haploid embryos into plants in relation to embryo developmental stage and regeneration media

Obtaining oat DH lines is only effective via wide crossing with maize. Seven hundred haploid embryos from 21 single F₁ progeny obtained from wide crosses with maize were isolated, divided into four groups according to their size (<0.5 mm, 0.5–0.9 mm, 1.0–1.4 mm, and ≥1.5 mm), and transferred into 190–2 regeneration medium with different growth regulators: 0.5 mg L^?1 kinetin (KIN) and 0.5 mg L^?1 1-naphthaleneacetic acid (NAA); 1 mg L^?1 zeatin (ZEA) and 0.5 mg L^?1 NAA; or 1 mg L^?1 dicamba (DIC), 1 mg L^?1 picloram (PIC), and 0.5 mg L^?1 kinetin (KIN). Among all isolated embryos, approximately 46.1% were between 1.0–1.4 mm, while the smallest group of embryos (7.1%) were those <0.5 mm. The ability of haploid embryos to germinate varied depending on oat genotypes and the size of embryos. Haploid embryos <0.5 mm were globular and did not germinate, whereas embryos ≥1.5 mm had clearly visible coleoptiles, radicles, and scutella, and were able to germinate. Germination of oat haploid embryos varied depending on growth regulators in the regeneration medium. Most haploid embryos germinated on medium with 0.5 mg L^?1 NAA and 0.5 mg L^?1 KIN, while the fewest germinated on medium with 1 mg L^?1 DIC, 1 mg L^?1 PIC, and 0.5 mg L^?1 KIN. One hundred thirty germinated haploid embryos converted into haploid plants. Fifty oat DH lines were obtained after colchicine treatment.