Human bone marrow colony formation in diffusion chambers. Normal values.

In situ hybridization techniques have previously employed a series of manipulations to effect denaturation of chromosomal DNA and reannealing of DNA-RNA hybrids. This report presents a new protocol which combines the denaturation and reannealing processes. DNA is heated in a solution of 50% formamide, 50% 4 × SSC containing the RNA to be hybridized. After l h at 70 °C the preparation is slowly cooled to 37 °C over a period of 6 h and incubated at 37 °C for an additional 10 h. This technique eliminates the possibility of premature reannealing of the DNA while employing hybridization conditions which, in vitro, lead to accurate base pairing.     

Target concentration dependence of DNA melting temperature on oligonucleotide microarrays

The design of microarrays is currently based on studies focusing on DNA hybridization reaction in bulk solution. However, the presence of a surface to which the probe strand is attached can make the solution‐based approximations invalid, resulting in sub‐optimum hybridization conditions. To determine the effect of surfaces on DNA duplex formation, the authors studied the dependence of DNA melting temperature (T_m) on target concentration. An automated system was developed to capture the melting profiles of a 25‐mer perfect‐match probe–target pair initially hybridized at 23°C. Target concentrations ranged from 0.0165 to 15 nM with different probe amounts (0.03–0.82 pmol on a surface area of 10¹⁸ Ų), a constant probe density (5 × 10¹² molecules/cm²) and spacer length (15 dT). The authors found that T_m for duplexes anchored to a surface is lower than in‐solution, and this difference increases with increasing target concentration. In a representative set, a target concentration increase from 0.5 to 15 nM with 0.82 pmol of probe on the surface resulted in a T_m decrease of 6°C when compared with a 4°C increase in solution. At very low target concentrations, a multi‐melting process was observed in low temperature domains of the curves. This was attributed to the presence of truncated or mismatch probes. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Successful development of a shed-microspore culture protocol for doubled haploid production in Indonesian hot pepper (Capsicum annuum L.)

Various systems of anther and microspore cultures were studied to establish an efficient doubled haploid production method for Indonesian hot pepper (Capsicum annuum L.). A shed-microspore culture protocol was developed which outperformed all the previously reported methods of haploid production in pepper. The critical factors of the protocol are: selection of flower buds with more than 50% late unicellular microspores, a 1 day 4°C pretreatment of the buds, followed by culture of the anthers in double-layer medium system for 1 week at 9°C and thereafter at 28°C in continuous darkness. The medium contained Nitsch components and 2% maltose, with 1% activated charcoal in the solid under layer and 2.5 μM zeatin and 5 μM indole-3-acetic acid in the liquid upper layer. All the ten genotypes of hot pepper tested, responded to this protocol. The best genotypes produced four to seven plants per original flower bud. This protocol can be used as a potential tool for producing doubled haploid plants for hot pepper breeding.     

Polymer supported synthesis of aminooxyalkylated oligonucleotides,and some applications in the fabrication of microarrays

A new protocol has been described for solid phase preparation of 3′- and 5′-aminooxylalkylated oligonucleotides using commercially available reagents. This involves attachment of linker 4 either with an LCAA-CPG support via succinoylation followed by synthesis (3′-aminooxyalkylated oligomers) or formation of its phosphoramidite 6 followed by coupling with desired oligomer (for generating 5′-aminooxyalkylated oligomers). Both the routes produced modified oligonucleotides in sufficiently high yields and purity (on HPLC) via conventional oligonucleotide synthesis on an automated synthesizer and deprotection step using aqueous ammonia (16 h, 60 °C). Aminooxyalkylated oligonucleotides were used to construct microarrays on glass surface (biochips). The performance of the biochips was evaluated by immobilizing modified oligonucleotides on epoxylated glass microslides under different sets of conditions with respect to pH, temperature and time. Further, the constructed microarrays were successfully used for detection of nucleotide mismatches and bacterial typhoid.     

A simple wheat haploid and doubled haploid production system using anther culture

Summary Wheat (Triticum aestivum L.) haploids and doubled haploids have been used in breeding programs and genetic studies. Wheat haploids and doubled haploids via anther culture are usually produced by a multiple step culture procedure. We improved a wheat haploid and doubled haploid production system via anther culture in which plants are produced from microspore-derived embryos using one medium and one culture environment. In the improved protocol, tillers of donor plants were pretreated at 4°C for 1–2 wk before anthers were plated on a modified 85D12 basal medium with phenylacetic acid (PAA) and zeatin and cultured at 30°C with a 12-h daylength (43 μEs⁻¹m⁻²) in an incubator. Microspore-derived embryos developed in 2–3 wk and the plants were produced 3–4 wk after anther plating. In the improved system, as much as 53% of the anthers of Pavon 76 were responsive with multiple embryos. For plant regeneration, as many as 22 green and 25 albino plants were produced from 100 anthers. Sixty-five green plants were grown to maturity and 32 (49%) plants were fertile and produced seeds (indicating spontaneous chromosome doubling) while 33 plants did not produce seed. Of five Nebraska breeding lines tested using the protocol, NE96675 was very responsive and the other lines less so, indicating that the protocol is genotype-dependent.     

In vitro conservation of enset under slow-growth conditions

Studies on in vitro storage of enset under slow-growth conditions were carried out to develop an efficient protocol for conservation of the genetic diversity of the crop. The response to different growth retardation treatments was examined using three enset clones collected from southwestern Ethiopia. In vitro cultures could be effectively maintained for 6 months at 15 °C and 18 °C on MS medium supplemented with 10 μM BAP, in the presence of mannitol at concentrations of 0, 1 or 2% as a growth retardant. Shoots were subsequently recovered and multiplied on MS medium supplemented with 10 and 20 μM BAP at 25 °C and rooted shoots were successfully transferred to the greenhouse. Incubation at the lower temperature (15 °C) and the presence of mannitol in the culture medium had a significantly positive effect on maintenance, measured by the number of recovered shoots after storage. This revised version was published online in June 2006 with corrections to the Cover Date.     

The effect of root temperature on growth of curled parsley

Summary Curled parsley was grown at root-zone temperature (RZT) of 18, 21, 24, 27 and 36°C at air temperature (AT) of 18 and 21°C. Maximum growth was obtained at 18°C AT and 24°C RZT, but there were no significant differences between 18 and 27°C RZT. Shoot and root growth were severely inhibited at 36°C constant RZT. The growth was also retarded when RZT rose to 36°C for 30 minutes per day, even when compared to a RZT of constant 27°C. This indicates that a short exposure to RZT above 30°C retards growth. A relatively low daily average RZT did not compensate for the damage caused by a short daily high temperature exposure. Optimum temperature for curled parsley seems to be about 21°C. Report No. 316.     

Effect of Temperatures on Dormancy and Germination in Three Species in the Lamiaceae Occurring in Northern Wetlands

In the temperate region temperature is the main factor influencing the germination period of plant species. The purpose of this study was to examine effects of constant and fluctuating temperatures on dormancy and germination under laboratory and field conditions in the three wetland species Lycopus europaeus, Mentha aquatica and Stachys palustris. The results should give indications if the temperature-dependent regulation of dormancy and germination is phylogenetically constrained. Tests for germination requirements showed a minimum temperature for germination of 9 °C in Mentha and 12 °C in Lycopus and Stachys, and a maximum temperature of 33 °C for Lycopus and 36 °C for Mentha and Stachys. Fluctuating temperatures promoted germination in all three species but the amplitude required for high germination (>50%) differed: it was 8 °C in Mentha, 10 °C in Stachys and 14 °C in Lycopus (mean temperature 22 °C). The effect of temperatures on the level of dormancy was examined in the laboratory by imbibing seeds at temperatures between 3 °C and 18 °C for periods between 2 and 28 weeks, as well as by a 30-month burial period, followed by germination tests at various temperatures, in light and darkness. In the laboratory only low temperatures (≤12 °C) relieved primary dormancy in seeds of Lycopus, while in Mentha and Stachys also higher temperatures lead to an increase of germination. Dormancy was only induced in Lycopus seeds after prolonged imbibition at 12 °C in the laboratory. Buried seeds of all species exhibited annual dormancy cycles with lower germination in summer and higher germination from autumn to spring. Exhumed seeds, however, showed considerable differences in periods of germination success. Dormancy was relieved when ambient temperatures were below 12 °C. Ambient temperatures that caused an induction of dormancy varied depending on species and test condition, but even low temperatures (8 °C) were effective. At high test temperatures (25 °C) in light, exhumed seeds of all three species showed high germination throughout the year. The three species showed various differences in the effects of temperatures on dormancy and germination. Similarities in dormancy and germination found among the species are in common with other spring-germinating species occurring in wetlands, so it seems that the temperature dependent regulation of dormancy and germination are related to habitat and not to phylogenetic relatedness.     

A novel tissue array technique for high-throughput tissue microarray analysis — microarray groups

Tissue microarrays are ordered arrays of hundreds to thousands of tissue cores in a single paraffin block. We invented a novel method to make a high-throughput microarray group. Conventional smaller tissue microarrays were made first and then sectioned. Separate paraffin films were arrayed orderly onto a regular-sized glass slide to form a larger microarray group. Sections were not floated in a water bath but, rather, were cut singly using conventional microtome, arrayed orderly onto the glass slide with forceps instead of using a tape-based tissue transfer system, and then unfolded with warm water (46° C) using a micropipette. This not only lowers the difficulty in sectioning but the overall tissue disks can be included in the same section. A microarray group of 2,534 small disks (theoretically, 2,560 disks can be made; 26 fell off during the procedure), the most up to now, was successfully made and may be used in immunohistochemistry, mRNA in situ hybridization, and flourescent in situ hybridization.     

Formulation of stable <Emphasis Type="Italic">Bacillus subtilis</Emphasis> AH18 against temperature fluctuation with highly heat-resistant endospores and micropore inorganic carriers

To survive the commercial market and to achieve the desired effect of beneficial organisms, the strains in microbial products must be cost-effectively formulated to remain dormant and hence survive through high and low temperatures of the environment during transportation and storage. Dormancy and stability of Bacillus subtilis AH18 was achieved by producing endospores with enhanced heat resistance and using inorganic carriers. Heat stability assays, at 90°C for 1 h, showed that spores produced under a sublethal temperature of 57°C was 100 times more heat-resistant than the ones produced by food depletion at the growing temperature of 37°C. When these highly heat-resistant endospores were formulated with inorganic carriers of natural and synthetic zeolite or kaolin clay minerals having substantial amount of micropores, the dormancy of the endospores was maintained for 6 months at 15–25°C. Meanwhile, macroporous perlite carriers with average pore diameter larger than 3.7 μm stimulated the germination of the spores and rapid proliferation of the bacteria. These results indicated that a B. subtilis AH18 product that can remain dormant and survive through environmental temperature fluctuation can be formulated by producing heat-stressed endospores and incorporating inorganic carriers with micropores in the formulation step.     

Direct determination of the influence of extreme temperature on transposition and structural mutation rates of Drosophila melanogaster mobile elements

Two sets of mutation accumulation lines, one reared at 28°C and the other at 24°C, were compared for their transposition and rearrangement rates of eleven transposable element families. The changes affecting mobile elements were analysed by the Southern technique and in situ hybridization. No differences were found between treated and control lines. The role of the host genotype in transposition control and the significance of structural mutations in transposable element dynamics are discussed.     

An Activated GOPS‐poly‐L‐Lysine‐ Coated Glass Surface for the Immobilization of 60mer Oligonucleotides

To explore a method for enhancing the immobilization and hybridization efficiency of oligonucleotides on DNA microarrays, conventional protocols of poly‐L‐lysine coating were modified by means of surface chemistry, namely, the slides were prepared by the covalently coupling of poly‐L‐lysine to a glycidoxy‐modified glass surface. The modified slides were then used to print microarrays for the detection of the SARS coronavirus by means of 60mer oligonucleotide probes. The characteristics of the modified slides concerning immobilization efficiency, hybridization dynamics, and probe stripping cycles were determined. The improved surface exhibited high immobilization efficiency, a good quality uniformity, and satisfactory hybridization dynamics. The spotting concentration of 10 μmol/L can meet the requirements of detection; the spots were approximately 170 nm in diameter; the mean fluorescence intensity of the SARS spots were between 3.2 × 10⁴ and 5.0 × 10⁴ after hybridization. Furthermore, the microarrays prepared by this method demonstrated more resistance to consecutive probe stripping cycles. The activated GOPS‐PLL slide could undergo hybridization stripping cycles for at least three cycles, and the highest loss in fluorescence intensity was found to be only 11.9 % after the third hybridization. The modified slides using the above‐mentioned method were superior to those slides treated with conventional approaches, which theoretically agrees with the fact that modification by surface chemistry attaches the DNA covalently firmly to the slides. This protocol may have great promise in the future for application in large‐scale manufacture.     

Temperatures lethal for citrus blackfly parasites,Encarsia opulenta andE. Smithi [Hym.: Aphelinidae]

Seasonally acclimatized adult and immature parasites of the citrus blackfly (CBF),Aleurocanthus woglumi Ashby, were exposed to high or low temperature extremes for 3 h periods. Death of all summer adults ofEncarsia opulenta Silvestri andE. smithi Silvestri occurred between 35° and 40°C. Within CBF hosts,E. opulenta were not able to emerge when temperatures reached between 45° and 50°C. In winter experiments adults of bothEncarsia species succumbed between −5° and −10°C. In a comparison of the 2 seasonal tests, a higher percentage ofE. smithi adults were able to survive both higher and lower temperatures thanE. opulenta, but the main interspecific difference was the ability ofE. opulenta within CBF to survive −10° to − 15°C whileE. smithi did not. Limited data forAmitus hesperidum Silvestri [Hym.: Platygasteridae] indicated that the immatures survived better at low, and not as well at high, temperatures as either species ofEncarsia. Florida Agricultural Experiment Station Journal Series # 5549.     

The influence of temperature and nitrogen source on growth and nitrogen uptake of two polar-desert species,Saxifraga caespitosa and Cerastium alpinum

Polar-desert plants experience low average air temperatures during their short growing season (4–8 °C mean July temperature). In addition, low availability of inorganic nitrogen in the soil may also limit plant growth. Our goals were to elucidate which N sources can be acquired by polar-desert plants, and how growth and N-uptake are affected by low growth temperatures. We compared rates of N-uptake and increases in mass and leaf area of two polar-desert species (Cerastium alpinum L. and Saxifraga caespitosa L.) over a period of 3 weeks when grown at two temperatures (6 °C vs. 15 °C) and supplied with either glycine, NH₄ ⁺ or NO₃ ⁻. At 15 °C, plants at least doubled their leaf area, whereas there was no change in leaf area at 6 °C. Measured mean N-uptake rates varied between 0.5 nmol g⁻¹ root DM s⁻¹ on glycine at 15 °C and 7.5 nmol g⁻¹ root DM s⁻¹ on NH₄ ⁺ at 15 °C. Uptake rates based upon increases in mass and tissue N concentrations showed that plants had a lower N-uptake rate at 6 °C, regardless of N source or species. We conclude that these polar-desert plants can use all three N sources to increase their leaf area and support flowering when grown at 15 °C. Based upon short-term (8 h) uptake experiments, we also conclude that the short-term capacity to take up inorganic or organic N is not reduced by low temperature (6 °C). However, net N-uptake integrated over a three-week period is severely reduced at 6 °C. This revised version was published online in June 2006 with corrections to the Cover Date.     

Pilot plant scale extraction of alginates from Macrocystis pyrifera. 2. Studies on extraction conditions andmethods of separating the alkaline-insoluble residue

The effect of temperature (70, 80, 90 ^°C) and time (1–9 h) during the alkaline extraction step on alginate yield and quality were studied. The alginate yield increased with time and maximum yield was obtained after 3.5 h treatment, ranging from19.4% at 70 ^°C to 21.9% at 90 ^°C. The viscosity of the alginate produced was inversely correlated with the temperature and time. At70 ^°C the slope of the curve was almost zero(753 to 923 mPa s); at 90 ^°C the viscosity loss was 154 mPa s per hour during the first two hours, reducing from 523 to 86 mPa s after 5 h; 80 ^°C yielded values between those for 70 ^°C and90 ^°C. The best conditions for alkaline extraction were using pH 10 at 80 ^°C for two hours. The curves obtained gave useful information for controlling the viscosity of the alginate during production. It was found that viscosity of the paste formed during alkaline extraction (`process viscosity') was the best parameter to determine there action rate during extraction. Alginate yield increased during filtration time from 17.6% to 23.7%after 55 min at 70 ^°C. In this step the viscosity of the alginate obtained remained almost constant (522–610 mPa s), indicating no degradation of the products during filtration. The best dilution to filter the alginate extract was obtained at 45 mPa s. Diatomaceous earth (Celite) and expanded lava(Perlite) were tested as filter aids. Expanded lava was the best filter aid, using 1 kg per kilogram of alginate produced. Three methods were studied to separate the alkaline-insoluble residues after extraction: filtration, centrifugation, flocculation, and combinations of them. The best system found was filtration with flocculant in a rotary vacuum filter, with a knife advance of 0.1 mm every 3.5 seconds and drum rotation of 2 rpm, yielding an average filtration flow rate of 10.5 L min^-1. This revised version was published online in August 2006 with corrections to the Cover Date.     

Salinity and Temperature Effects on Germination for Three Salt-resistant Euhalophytes, Halostachys caspica, Kalidium foliatum and Halocnemum strobilaceum

The effects of three salinities (0, 100 and 500 mM NaCl) and four constant temperatures (10, 20, 30 and 35 °C) on seed germination of Halostachys caspica (M. B.) C. A. Mey., Kalidium foliatum (Pall.) Mop. and Halocnemum strobilaceum (Pall.) Bieb. were investigated. After seeds were treated with different concentrations of NaCl at constant temperatures of 10–35 °C for 16 days, ungerminated seeds were transferred to distilled water for 10 days to investigate the total germination; after this time, the ungerminated seeds from the 10 and 20 °C treatments were then moved to 35 °C for another 5 days to determine the final germination. The three plant species in the present experiment are salt-resistant euhalophytes growing in high saline soils in the Zhungur Basin in Xinjiang, a northwest province of China.Compared with germination under control conditions, germination percentages of all three species were not affected by 100 mM NaCl at 10–35 °C, while severely inhibited by 500 mM NaCl; germination percentages were very low at 10 °C up to 100 mM NaCl for all species; the optimum temperature for germination of H. caspica and K. foliatum was 20–30 °C, while 35 °C for H. strobilaceum, up to 100 mM NaCl; seeds did not suffer ion toxicity for all species, as evidenced by the high total germination after ungerminated seeds pretreated with 500 mM NaCl were transferred to distilled water at constant temperatures of 10–35 °C for 10 days, and the high final germination after the ungerminated seeds from the 10 and 20 °C treatments were subsequently moved to 35 °C for another 5 days; Halostachys caspica had greater sensitivity to increasing temperatures from 10 and 20 °C to 35 °C compared with the other two species.     

Plant regeneration from microspore-derived embryos ofBrassica Napus: Effect of embryo age,culture temperature,osmotic pressure,and abscisic acid

Summary Microscope cultures ofBrassica napus cv. Topas undergo high frequency embryogenesis in vitro; however, the majority of microspore-derived embryos do not develop directly into plants but usually undergo abnormal development including the formation of secondary embryos on the hypocotyls. The present studies show that older embryos or embryos cultured at higher temperature (25° C) were more likely to undergo secondary embryogenesis whereas embryos cultured at 20° C or pretreated at 5° to 10° C for 28 days developed more readily into normal plants. Compared with embryos cultured at 25° C, those cultured at 20° C gave a threefold increase in normal plant production. Pretreatments at cooler temperatures (5° to 10° C) resulted in an additional two-to threefold increase in the recovery of normal plants. Higher osmoticum during pretreatment improved embryo survival at low temperatures but generally inhibited normal plant development. Abscisic acid was ineffective or deleterious.     

Triploidization in the streaked prochilod Prochilodus lineatus inferred by flow cytometry,blood smears and karyological approaches

The streaked prochilod Prochilodus lineatus is an important fish species from the Neotropical region. In this study, a protocol of triploidization was established by temperature shock. Fertilized eggs were shocked at 2 min post-fertilization at 0, 38, 40 and 42°C. At 0°C, the embryos were maintained for 30 min, while the rest for 2 min. Heat shocked embryos and control (during all experiment) were incubated at 27°C. The ploidy status was confirmed by flow cytometry, erythrocyte diameter, conventional cytogenetics (Giemsa staining), chromosome banding and fluorescence in situ hybridization using 5S and 18S rDNA probes. Heat-shock at 40°C produced 96.7% of triploids and such a procedure did not reduce the fertilization, hatching rate and the percentage of normal embryos. The use of chromosome banding and FISH gave rise to effective procedure to identify triploids. The data obtained here is innovative for the species and bring new information for basic and applied studies.     

Effect of Chilling on DNA Endoreplication in Root Cortex Cells and Root Hairs of Soybean Seedlings

Relative nuclear DNA contents in cortex parenchyma cells in root segments of 3- and 7-d-old soybean seedlings grown at 25 °C and in plants grown for 3 d at 25 °C, and then for 4 d at 10 °C, were determined with cytophotometry. Measurements revealed that in each variant the cortex cell nuclei with DNA content between 2C and 8C were in all the examined segments and nuclei with 8C – 16C DNA appeared in higher parts of roots. However, in chilled plant cells the number of 8C – 16C DNA nuclei was very low. Therefore, chilling inhibited endoreplication in comparison with plants grown at 25 °C for 7 d, and even reduced endopolyploidy level as compared to the initial seedlings, i.e. 3-d-old plants. DNA contents in root hairs grown at 25 °C (control) and in root hairs emerged at 10 °C were also determined. In controls 4C – 8C DNA nuclei predominated while in chilled plants an additional population of 2C – 4C DNA appeared. Thus a reduction of DNA synthesis was brought about by low temperature. The occurrence of an intermediate DNA contents besides those with full endoreplication cycles suggests the possibility of differential DNA replication. This suggestion seems to be supported by the lack of ³H-thymidine incorporation into root hair nuclei at the examined developmental stage both in control and chilled root hairs. The same number, but larger, chromocentric lumps in polyploid cortex cell nuclei of higher root zones, in comparison to meristematic nuclei, suggests that endoreduplication process occurred. This revised version was published online in July 2006 with corrections to the Cover Date.