Plating Efficiency Research Articles

Radiation-induced bystander effect in non-irradiated glioblastoma spheroid cells.

Radiation-induced bystander effects (RIBEs) are detected in cells that are not irradiated but receive signals from treated cells. The present study explored these bystander effects in a U87MG multicellular tumour spheroid model. A medium transfer technique was employed to induce the bystander effect, and colony formation assay was used to evaluate the effect. Relative changes in expression of BAX, BCL2, JNK and ERK genes were analysed using RT-PCR to investigate the RIBE mechanism. A significant decrease in plating efficiency was observed for both bystander and irradiated cells. The survival fraction was calculated for bystander cells to be 69.48% and for irradiated cells to be 34.68%. There was no change in pro-apoptotic BAX relative expression, but anti-apoptotic BCL2 showed downregulation in both irradiated and bystander cells. Pro-apoptotic JNK in bystander samples and ERK in irradiated samples were upregulated. The clonogenic survival data suggests that there was a classic RIBE in U87MG spheroids exposed to 4 Gy of X-rays, using a medium transfer technique. Changes in the expression of pro- and anti-apoptotic genes indicate involvement of both intrinsic apoptotic and MAPK pathways in inducing these effects.

Fundamental Studies of Iron Redox Flow Battery Electrochemistry in a Deep Eutectic Solvent

Current redox flow battery (RFB) technologies are limited by low energy and power densities; thus, a recent focus has been on non-aqueous electrolytes. Although organic solvents allow for increased voltage windows, they possess a sensitivity to moisture, low active species solubilities, high flammability and toxicity, and limited availability. Another class of non-aqueous solvents, ionic liquids (IL) or more specifically, deep eutectic solvents (DES) offer promise for future non-aqueous RFBs. These “designer solvents” can be created using a number of combinations of bulky asymmetric cations and weakly coordinating anions, allowing for the ability to tailor different solvent properties, presenting an opportunity to generate optimized solvents. DES are advantageous in that they have large electrochemical windows similar to organic solvents, and they have low volatilities, high thermal stabilities, high polarities, and high conductivities without the need for a supporting electrolyte [1-4]. They offer a “greener” alternative to volatile organic solvents and are non-toxic and nonflammable [5, 6]. Two different deep eutectic systems, reline (1:2 molar ratio choline chloride and urea) and ethaline (1:2 molar ratio choline chloride and ethylene glycol), have been investigated as well as an iron eutectic (1:3 molar ratio iron chloride and ethylene glycol). Wide potential windows of 2.6V and 2.0V are achieved using reline and ethaline respectively (compared to 1.23V for aqueous). Electrolyte properties such as conductivities and viscosities as a function of temperature are reported. Iron plating efficiencies are reported; higher coulombic efficiencies compared to the aqueous system are attributed to elimination of the side reaction of hydrogen evolution (due to the expanded voltage window). Characterization of the electrokinetics, diffusion coefficients, and solubility of the iron chemistry at 50°C have been determined using voltammetric measurements and electrochemical impedance spectroscopy at platinum, glassy carbon, and carbon fiber microelectrodes. Iron was chosen as proof of concept chemistry; higher potential couples are currently under investigation. [1] S.-H. Wu, Thermochimica Acta, vol. 544, pp. 1-5, 2012. [2] G. A. Baker, S. N. Baker, S. Pandey, and F. V. Bright, Analyst, p. 800, 2005. [3] T. Welton, Chem Rev., p. 2071, 1999. [4] S. A. Forsyth, J. M. Pringle, and D. R. MacFarlane, Aust J. Chem, vol. 57, 2004. [5] M. J. Earle and R. K. Seddon, Pure Appl. Chem., vol. 72, p. 1391, 2000. [6] K. N. Marsh, A. Deev, A. C.-T. Wu, T. E., and A. Klamt, Korean J Chem. Eng. , vol. 19, 2002. Figure 1

Using Copolymers As Suppressors in a Copper Plating Bath for through-Hole Filling

Recently, a copper electroplating technique, namely butterfly technique (BFT) [1], was developed to achieve a void-free filling of a through hole (TH) of a printed circuit board (PCB). However, the BFT for TH filling caused an issue of thick copper layer on the PCB surface, which increases the process cost due to a requirement of post copper etching process. Literature has mentioned that copolymers have strong suppression on copper electroplating than poly(ethylene glycol) (PEG). The strong suppressor can minimize surface copper thickness to achieve excellent filling performance. In this work, we employ a copper filling formula to perform the TH filling. Several copolymers were employed as suppressors to replace PEG. The result shows that the surface copper thickness is decreased by using the copolymer. It can not only enhance the copper filling performance but also increase plating efficiency. Reference W. P. Dow, H. H. Chen, M. Y. Yen, W. H. Chen, K. H. Hsu, P. Y. Chuang, H. Ishizuka, N. Sakagawa, R. Kimizuka, “Through-Hole Filling by Copper Electroplating”, Journal of The Electrochemical Society, 2008, 155, D750-D757. Figure 1

Prediction and Supplementation of Reducing Agent to Improve the Coating Efficiency and Wear Behavior of Electroless Ni-P Plating

Prediction and Supplementation of Reducing Agent to Improve the Coating Efficiency and Wear Behavior of Electroless Ni-P Plating

Phage T4‐induced DNA breaks activate a tRNA repair‐defying anticodon nuclease

The natural role of the conserved bacterial anticodon nuclease (ACNase) RloC is not known, but traits that set it apart from the homologous phage T4-excluding ACNase PrrC could provide relevant clues. PrrC is silenced by a genetically linked DNA restriction-modification (RM) protein and turned on by a phage-encoded DNA restriction inhibitor. In contrast, RloC is rarely linked to an RM protein, and its ACNase is regulated by an internal switch responsive to double-stranded DNA breaks. Moreover, PrrC nicks the tRNA substrate, whereas RloC excises the wobble nucleotide. These distinctions suggested that (i) T4 and related phage that degrade their host DNA will activate RloC and (ii) the tRNA species consequently disrupted will not be restored by phage tRNA repair enzymes that counteract PrrC. Consistent with these predictions we show that Acinetobacter baylyi RloC expressed in Escherichia coli is activated by wild-type phage T4 but not by a mutant impaired in host DNA degradation. Moreover, host and T4 tRNA species disrupted by the activated ACNase were not restored by T4's tRNA repair system. Nonetheless, T4's plating efficiency was inefficiently impaired by AbaRloC, presumably due to a decoy function of the phage encoded tRNA target, the absence of which exacerbated the restriction.

Radiosensitivity of Patient-Derived Glioma Stem Cell 3-Dimensional Cultures to Photon, Proton, and Carbon Irradiation

To investigate the radiosensitivity of primary glioma stem cell (GSC) cultures with different CD133 status in a 3-dimensional (3D) model after photon versus proton versus carbon irradiation. Human primary GSC spheroid cultures were established from tumor specimens of six consented glioblastoma patients. Human U87MG was used as a classical glioblastoma radioresistant cell line. Cell suspensions were generated by mechanical dissociation of GSC spheroids and embedded in a semi-solid 3D matrix before irradiation. Spheroid-like colonies were manually counted by microscopy. Cells were also recovered and quantified by fluorescence. CD133 expression and DNA damage were evaluated by flow cytometry. The fraction of CD133(+) cells varied between 0.014% and 96% in the six GSC cultures and showed a nonsignificant correlation with plating efficiency and survival fractions. The 4 most photon-radioresistant GSC cultures were NCH644, NCH421k, NCH441, and NCH636. Clonogenic survival for proton irradiation revealed relative biologic effectiveness (RBE) in the range of 0.7-1.20. However, carbon irradiation rendered the photon-resistant GSC cultures sensitive, with average RBE of 1.87-3.44. This effect was partly attributed to impaired capability of GSC to repair carbon ion-induced DNA double-strand breaks as determined by residual DNA repair foci. Interestingly, radiosensitivity of U87 cells was comparable to GSC cultures using clonogenic survival as the standard readout. Carbon irradiation is effective in GSC eradication with similar RBE ranges approximately 2-3 as compared with non-stem GSC cultures (U87). Our data strongly suggest further exploration of GSC using classic radiobiology endpoints such as the here-used 3D clonogenic survival assay and integration of additional GSC-specific markers.

Optimum Design of Copper Stabilizer on Coated Conductors

Metallic copper is the obvious engineering stabilizing partner for the second-generation coated conductor (CC) wire. The copper shunt on the conductor gives a number of electric, thermal, and mechanical advantages. Nonvacuum deposition of copper by electrochemical plating is now an established CC technology, both single sided as well as surrounded. Cu plating on RABITS Ag/YBCO/Ni5%W and IBAD Ag/YBCO/YSZ/Hastelloy conductors are reported in the applied cell potentials, currents, and Coulombs. The diffusion-limited plating current in copper sulfate and alkaline electrolyte is investigated in view of the largest rate of mass transfer. The electrochemical deposition opens a further window of design and conductor engineering. Beneficial Cu plating with Ag cap layer is performed, and J c is evaluated. All parameters are transferred into the reel technique to improve the efficiency of copper plating. This paper describes the optimum and merits of Cu shunt parameters in geometry, thickness, and wire robustness.

Irradiación subletal de fibroblastos murinos 3T3 con rayos X para su utilización como capa celular alimentadora

Keratinocytes, the main cells of the epidermis, require mitotically inactive but live feeder-layer cells for their survival and proliferation. We evaluated four X-ray irradiation doses on 3T3 murine fibroblasts for feeder-layer production. Each dose effect was evaluated by cell counting and cellular viability determination (MTT), cell adhesion (plating efficiency) and cell migration (scratch wound healing). Mitotic arrest was suggested by nuclear staining (GIEMSA). We determined that an 80 Gy X-ray dose generated enough cell damage to cause mitotic arrest yet keeping metabolically active cells up to 25 days.

Evaluation of the Performance of an Iron-Chloride Redox Flow Battery for Large Scale Energy Storage

Highly efficient and inexpensive batteries are essential for the successful integration of renewable energy sources like solar and wind into the electricity grid [1, 2]. The present study focuses on the evaluation of the performance of an iron-chloride redox flow battery for such large scale energy storage applications. An iron-chloride redox flow battery is based on inexpensive and globally-abundant materials – iron and chloride. The all-iron redox flow battery was first reported by Hruska and Savinell in 1981[3]. While the economics and eco-friendly nature of this battery system is very promising, successful commercialization of the iron-chloride redox flow battery has been hindered by several technical challenges such as low charging efficiency of the negative electrode and poor cycle life. During discharge of the iron chloride flow battery, iron (III) chloride is converted to iron (II) chloride in the positive electrode and iron is oxidized to iron (II) chloride at the negative electrode (Eq. 1, 2). The reverse reactions happen during battery charging. Positive electrode: 2FeCl3 + 2e- 2FeCl2 + 2Cl- Eo= 0.77V (1) Negative electrode: Fe + 2Cl- FeCl2 + 2e- Eo= -0.44V (2) Overall cell reaction: Fe + 2 FeCl3 3FeCl2 Ecell= 1.21V (3) Negative electrode side reaction: 2H+ + 2e- H2 Eo= 0V (4) The reversible potential of the Fe/Fe2+ couple is more negative compared to the reversible potential for hydrogen evolution (Eq. 4). As a result, in addition to the reduction of Fe2+ ions to metallic iron, hydrogen evolution also takes place on the negative electrode during battery charging. This hydrogen evolution results in very low charging efficiency of the negative electrode. The amount of hydrogen evolved on the negative electrode during charging is dependent upon the electrolyte pH, charging current and the presence of any electrolyte additives. The pH of the electrolyte increases due to the evolution of hydrogen and results in the precipitation of Fe2+ and Fe3+ions in the electrolyte. Operating at low charging efficiencies is therefore highly undesirable for stable long-term operation. The faradaic efficiency of iron deposition in the presence of various additives was studied at a glassy carbon rotating disc electrode with a platinum wire counter electrode and a silver/silver chloride reference electrode. The baseline electrolyte consisted of 3M ferrous chloride (FeCl2.4H2O) and 2M ammonium chloride (NH4Cl). The ammonium chloride served as a supporting electrolyte. Full-cell studies were performed in an in-house designed flow cell with graphite electrodes and an anion exchange membrane that separated the positive and negative electrode compartments. In addition, reference electrodes were placed in the positive and negative electrode compartments to facilitate the characterization of the polarization characteristics of the individual electrodes in the flow cell [4]. The faradaic efficiency for iron deposition in the presence of different electrolyte additives at various current densities is shown in Figure 1. It is observed that the “round-trip” plating/stripping efficiency with the baseline electrolyte was less than 40%. In the presence of ascorbic acid or citric acid as buffering agents, the electrolyte could be stabilized at pH = 2 without the precipitation of Fe2+ and Fe3+ions. The round-trip plating efficiency with an electrolyte pH of 2 and ascorbic acid additive was about 92% (Figure 1). The charging efficiency of the negative electrode of an iron-chloride redox flow battery under different operating conditions is shown in Figure 2. The effect of charging current density, electrolyte pH, electrolyte flow rate and electrolyte additives on the charging efficiency and cycling performance of the iron-chloride flow battery will be discussed. Acknowledgement: The authors acknowledge the financial support for this research from US Army RDECOM CERDEC CP&I. We also thank Donald Wiggins and Mike Cowan of the USC Dornsife Machine Shop for support with the fabrication of the flow cell.

Establishment and characterization of a continuous cell line from thymus of striped snakehead, Channa striatus (Bloch 1793).

The establishment and characterization of a continuous cell line from the thymus of air-breathing fish Channa striatus are described. The cell line, designated C. striatus thymus (CST), has been subcultured over 71 times and shows optimal growth at 28°C in Leibovitz's-15 (L-15) medium supplemented with 20% fetal bovine serum. The CST cells exhibited low plating efficiency which improved with increase in seeding density. The karyotype analysis revealed that CST cells have a normal diploid karyotype with 2n = 40. Partial amplification and sequencing of two mitochondrial genes, viz. 16S ribosomal RNA (rRNA) and cytochrome oxidase I, confirmed that the cell line originated from C. striatus. CST cells were successfully transfected indicating their potential application for expression of recombinant proteins. In immunocytochemical staining, CST cells showed characteristics of epithelial cells. These cells were sensitive to extracellular products of Vibrio cholerae MTCC 3904 as well as to heavy metal mercuric chloride. The CST cell line would be a useful tool in functional genomic studies such as RNA interference and gene knockout as well as for cytotoxicity studies.

Direct confirmation of quiescence of CD34+CD38- leukemia stem cell populations using single cell culture, their molecular signature and clinicopathological implications.

BackgroundThe proliferating activity of a single leukemia stem cell and the molecular mechanisms for their quiescent property remain unknown, and also their prognostic value remains a matter of debate. Therefore, this study aimed to demonstrate the quiescence property and molecular signature of leukemia stem cell and their clinicopathological implications.MethodsSingle cell sorting and culture were performed in the various sets of hematopoietic stem cells including CD34+CD38- acute myeloid leukemia (AML) cell population (ASCs) from a total of 60 patients with AML, and 11 healthy controls. Their quiescence related-molecular signatures and clinicopathological parameters were evaluated in AML patients.ResultsSingle cell plating efficiency of ASCs was significantly lower (8.6%) than those of normal hematopoietic stem cells i.e.: cord blood, 79.0%; peripheral blood, 45.3%; and bone marrow stem cell, 31.1%. Members of the TGFβ super-family signaling pathway were most significantly decreased; as well as members of the Wnt, Notch, pluripotency maintenance and hedgehog pathways, compared with non ASC populations. mtDNA copy number of ASCs was significantly lower than that of corresponding other cell populations. However, our data couldn’t support the prognostic value of the ASCs in AML.ConclusionsASCs showed remarkable lower plating efficiency and slower dividing properties at the single cell level. This quiescence is represented as a marked decrease in the mtDNA copy number and also linked with down-regulation of genes in various molecular pathways.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1233-x) contains supplementary material, which is available to authorized users.

48 Expression pattern and significance of homeodomain only protein (HOPX) in head and neck cancer

Background The homeodomain only protein, HOPX (also known as HOP, NECC1, LAGY or OB1) was initially identified as a gene essential for cardiac development. HOPX is unusual because although it forms a classical homeodomain fold it lacks DNA-binding capacity due to the absence of several key residues known to mediate critical contacts with DNA that are conserved among other homeodomain proteins. The aim of this study is to investigate the expression pattern and role of HOPX in head and neck cancer. Methods Immunohistochemistry, Reverse Transcriptase-PCR and QPCR were performed to study the expression pattern of HOPX in head and neck carcinoma tissues and cell line. In addition, MTT, Transwell migration, Annexin V/PI staining for apoptosis and colony forming assay have been used to investigate the effect of forced HOPX expression in head and neck carcinoma cells. Results HOPX expression is markedly reduced in head and neck carcinoma tissues and cell line when compared with normal epithelial cells. Interestingly, forced expression of HOPX reduces plating efficiency, migration and sensitizes carcinoma cell to UV induced apoptosis. Conclusion Our results demonstrate that the loss of HOPX leads to more aggressive phenotype and insists the need to investigate the precise role of HOPX in homeostasis and tumor progressive for therapeutic implication.

Isolation of phages for phage therapy: a comparison of spot tests and efficiency of plating analyses for determination of host range and efficacy.

Phage therapy, treating bacterial infections with bacteriophages, could be a future alternative to antibiotic treatment of bacterial infections. There are, however, several problems to be solved, mainly associated to the biology of phages, the interaction between phages and their bacterial hosts, but also to the vast variation of pathogenic bacteria which implies that large numbers of different phages are going to be needed. All of these phages must under present regulation of medical products undergo extensive clinical testing before they can be applied. It will consequently be of great economic importance that effective and versatile phages are selected and collected into phage libraries, i.e., the selection must be carried out in a way that it results in highly virulent phages with broad host ranges. We have isolated phages using the Escherichia coli reference (ECOR) collection and compared two methods, spot testing and efficiency of plating (EOP), which are frequently used to identify phages suitable for phage therapy. The analyses of the differences between the two methods show that spot tests often overestimate both the overall virulence and the host range and that the results are not correlated to the results of EOP assays. The conclusion is that single dilution spot tests cannot be used for identification and selection of phages to a phage library and should be replaced by EOP assays. The difference between the two methods can be caused by many factors. We have analysed if the differences and lack of correlation could be caused by lysis from without, bacteriocins in the phage lysate, or by the presence of prophages harbouring genes coding for phage resistance systems in the genomes of the bacteria in the ECOR collection.

Autotetraploid plant regeneration by indirect somatic embryogenesis from leaf mesophyll protoplasts of diploid Gentiana decumbens L.f.

Somaclonal variation, often manifested as the increased ploidy of plants observed following in vitro culture, can be advantageous in ornamental species or those used for secondary metabolite production. Polyploidy occurs especially when plantlets are produced by protoplast and callus cultures. Plants were regenerated from green leaf mesophyll protoplasts of diploid Gentiana decumbens L.f. through somatic embryogenesis. A yield of more than 9 × 105 protoplasts per gram of fresh weight was achieved by incubating fully expanded young leaves in an enzyme mixture containing 1.0% (w/v) cellulase and 0.5% (w/v) macerozyme. Protoplasts, cultured in agarose beads using a modified Murashige and Skoog medium, divided and formed microcalli, with the highest plating efficiency obtained on medium containing 2.0 mg l−1 1-naphthaleneacetic acid and 0.1 mg l−1 thidiazuron. Callus proliferation was also promoted by including thidiazuron in agar-solidified medium, while somatic embryogenesis was induced from microcalli on medium supplemented with 1.0 mg l−1 kinetin, 0.5 mg l−1 gibberellic acid, and 80 mg l−1 adenine sulfate. Flow cytometric analysis and chromosome counting revealed that all regenerants were tetraploid.

Effect of Pd concentration on electroless dense Pd-PSS membrane fabrication

This work addresses the effect of palladium solution concentration on combinatorial plating characteristics of surfactant and sonication coupled electroless plating baths for the fabrication of dense palladium films on porous stainless steel substrates. All plating experiments were carried out using palladium solution concentration of 0·005 and 0·01M plating baths with cetyltrimethylammonium bromide surfactant at four critical micelle concentrations and loading ratio of 203 cm2 L−1. The evaluated combinatorial plating characteristics include selective conversion, plating efficiency, plating rate, Pd film thickness and per cent pore densification. The enhancement in palladium solution concentration was found to be insignificant to provide better combinatorial plating characteristics, and the plating bath consisting of 0·005M Pd solution concentration has provided 99·98% per cent pore densification with a Pd film thickness of 8·81 μm and plating efficiency of 80·69%.

Rate enhanced electroless fabrication of nickel ceramic composite membranes

Elaborating upon the role of surfactant and sonication, this work addresses the combinatorial plating characteristics for the fabrication of nickel ceramic composite membranes. Specific novelty corresponds to controlled addition of hydrazine hydrate as a reducing agent to nickel electroless plating bath. The experimental investigations evaluated the optimality of solution concentrations of cetyltrimethylammonium bromide (CTAB) and sodium dodecyl sulphate surfactants during coupled sonication and surfactant based electroless plating. Among both surfactants, CTAB surfactant with a solution concentration of 4 CMC provided maximum combinations of pore densification (73·4%), metal film thickness (8·4 μm) and plating efficiency (89·9%). In all, it was observed that with CTAB surfactant the pore densification and plating efficiencies increased with increasing surfactant solution concentration from 1–4 CMC and were maximum at 4 CMC which drastically decreased at 6 CMC respectively probably due to surfactant adsorption on the substrate surface.

Dental Plating Efficiency of Metal Base Dental Alloys by Thermal Diffusivity Study

The need for reliable data on thermophysical properties of materials has increased continuously in the last period. The existing data, except for some pure elements or alloys and compounds, often unreliable, and in many cases the need for accurate and acceptable data, can only be achieved by measuring a given property. This paper focuses on elements of the used test methods associated with the description of the newly introduced parameter (effectiveness of dental coating). The selected method was the “pulse method” for determining thermal diffusivity (the method allows simultaneous determination of thermal diffusivity, specific heat capacity and thermal conductivity) directly applicable in the study of the thermal compatibility of metal-ceramic systems in dental applications.

Characterization of Two Temperate Lactobacillus paracasei Bacteriophages: Morphology, Kinetics and Adsorption

Background/Aims: Adsorption and kinetic parameters, latent period, burst size and burst time, are characteristics of phage/host systems and can be affected by several environmental factors. As only few studies have focused on temperate dairy phages, we characterized these parameters on temperate Lactobacillus paracasei phages Φ iLp84 and Φ iLp1308, infective for probiotic strains. Methods: Phages were characterized by transmission electron microscopy and genomic DNA restriction. Adsorption under different environmental conditions, phage kinetics and efficiency of plating (EOP) were determined using the double-layer titration method. Results: Phages Φ iLp84 and Φ iLp1308 belong to the Siphoviridae family and have genome sizes of 38 and 34 kbp, respectively. Adsorption was affected by calcium concentration, pH, temperature and host viability, and reached a limit at very high multiplicity of infection. Latency, burst time and burst size were of 85 min, 131 min and 46 for Φ iLp84, and 51 min, 92 min and 28 for Φ iLp1308, respectively, at 37°C. A clear influence of temperature on phage kinetics was observed. Regarding EOP, Φ iLp84 produced plaques on only 1 out of 8 strains tested. Conclusion: Noticeable differences in adsorption, kinetics and EOP were found for two morphologically identical temperate L. paracasei phages of similar origin.

Plant regeneration from protoplasts of Gentiana macrophylla Pall. using agar-pool culture

An efficient protocol for plant regeneration was developed from protoplasts of Gentiana macrophylla Pall. through somatic embryogenesis. Viable protoplasts were isolated from cell suspensions derived from young seedling leaves in an enzyme solution containing 2 % Cellulase Onozuka R-10, 0.5 % Macerozyme R-10, 0.5 % Hemicellulase, and 0.4 M sorbitol with a yield of 6.2 × 106 protoplasts g−1 fresh weight. Liquid, solid–liquid double layer (sLD) and agar-pool (aPL) culture systems were used for protoplast culture. The frequency of protoplast cell divisions and colony formations in aPL culture were significant (p < 0.05) higher than those in liquid and sLD cultures. The highest division frequency and plating efficiency were 37.7 % and 16.5 %, respectively, in aPL culture supplemented with 2 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-d) and 0.5 mg l−1 6-benzylaminopurine (BA). Protoplast-derived microcalli obtained from aPL culture system were transferred to solid MS medium with a reduced concentration of 2,4-d (0.5 mg l−1) to promote the formation of embryogenic calli. Somatic embryos developed into plantlets on MS medium supplemented with 2 mg l−1 BA at a rate of 51.3 %. RAPD analysis of G. macrophylla revealed a low variation among regenerants.

Tri-parental protoplast fusion ofBrassicaspecies to produce somatic hybrids with high genetic and phenotypic variability

Plating efficiencies and the plant regeneration frequencies of bi-parental fusion of Brassica campestris pekinensis with Ogura cytoplasmic male sterility (CMS), B. oleracea L. var. italica with Ogura CMS, and Brassica juncea var. crispifolia were compared for illustrating the advantages of tri-parental somatic hybridization. The results showed that in the triparental fusion combination, 73 plants regenerated from 712 calli and the plant regeneration frequency was 10.3%, three or four times that obtained with the 2 fusion combinations. These hybrids were classified into 12 types based on morphology, and most showed intermediate characteristics between 2 or more of the parental species. The somatic hybiridity were confirmed by flow cytometry, genomic in situ hybridization (GISH) and PCR analysis, indicating that these regenerated plants were all true hybrids. Most of the progenies with normal pollen showed varied number of seed set after backcrossing with B. juncea. The high variability in the hybrids obtained illustrated that somatic hybridization may be useful in broadening existing Brassica gene pools and obtaining material for breeding.