Transformation of Brassica napus and Brassica oleracea Using Agrobacterium tumefaciens and the Expression of the bar and neo Genes in the Transgenic Plants

An efficient and largely genotype-independent transformation method for Brassica napus and Brassica oleracea was established based on neo or bar as selectable marker genes. Hypocotyl explants of Brassica napus and Brassica oleracea cultivars were infected with Agrobacterium strains containing chimeric neo and bar genes. The use of AgNO₃ was a prerequisite for efficient shoot regeneration under selective conditions. Vitrification was avoided by decreasing the water potential of the medium, by decreasing the relative humidity in the tissue culture vessel, and by lowering the cytokinin concentration. In this way, rooted transformed shoots were obtained with a 30% efficiency in 9 to 12 weeks. Southern blottings and genetic analysis of S1-progeny showed that the transformants contained on average between one and three copies of the chimeric genes. A wide range of expression levels of the chimeric genes was observed among independent transformants. Up to 25% of the transformants showed no detectable phosphinotricin acetyltransferase or neomycin phosphotransferase II enzyme activities although Southern blottings demonstrated that these plants were indeed transformed.     

Procedures for the Generation of Mature Chlamydomonas reinhardtii Zygotes for Molecular and Biochemical Analyses

Zygotes represent an important stage in the sexual cycle of the unicellular green alga Chlamydomonas reinhardtii. To study zygote germination at a molecular level, a protocol was elaborated for the generation of zygotes in large quantities and a method was developed for the extraction from zygotes of RNA that could be translated in vitro.     

The role of endogenous estrogens in the maturation process of the bovine placenta

The plasma estrogen and progesterone concentrations of 19 pregnant cows (average duration of pregnancy 266.0 +/- 2.3 d at the start of the study) were determined daily from Day 6 pre partum to Day 1 post partum. Parturition was induced in all cows by administration of 10 mg i.m. flumethasone. Values were centered around the delivery date (Day 0) following either induced normal calving (n = 3) or surgical delivery (n = 16). In animals showing spontaneous expulsion of the fetal membranes (Group 1, n = 6) the average total estrogen concentration increased significantly from Day 6 until Day 1 before parturition (1329.2 +/- 317.9 to 3719.8 +/- 951.2 pg/ml in total estrogens). A marked decrease was observed on Day 1 post partum (459.4 +/- 344.2 pg/ml). In comparison with Group 1, animals showing either a delayed or partial expulsion of the fetal membranes, or in which the placenta could be withdrawn 16 h after calving (Group 2, n = 5), had consistently lower total estrogen concentrations between Day 6 (595.4 +/- 174.8 pg/ml) and Day 1 (1884.3 +/- 565.1 pg/ml) before parturition. The estrogen values of the cows with retained placenta (Group 3, n = 8) from Days 6 to 0 pre partum were significantly lower than those of Group 2. Total estrogen concentrations of the three groups 1 d post partum did not differ significantly. It is generally recognized that estrogens play an important role in the maturation process of the placentomes. Our investigation demonstrates that not only is the magnitude of the prepartum rise in estrogens of great influence of the maturation process but the duration of this rise is likewise important. These two factors are vital for a normal expulsion of the fetal membranes.     

Changes in quality of stallion spermatozoa during cryopreservation: Plasma membrane integrity and motion characteristics

Better procedures for freezing and thawing equine sperm are needed since variable fertility is obtained when cryopreserved sperm are used. To evaluate current methods of freezing equine sperm, we examined spermatozoal quality by means of two new techniques. These measured the integrity of plasma-acrosomal membranes by immunofluorescent analyses of binding of an antibody specific to the acrosome and evaluated eight parameters of spermatozoal motion using a fully automated computerized system. Five ejaculates from each of eight stallions were processed for freezing in egg yolk-lactose extender with 4% glycerol. Spermatozoal quality was assessed at four different points: at less than 15 min after collecting and before processing (Step 1); after centrifugation and just before freezing (Step 2); immediately after thawing less than 3 h after freezing (Step 3); and immediately after thawing 10 to 20 d after freezing (Step 4). Acrosome-specific monoclonal antibody detected differences (P <0.05) among steps and ejaculates within stallions. All parameters of spermatozoal motion, including the percentage of motile sperm, percentage of progressively motile sperm, curvilinear velocity, straight line velocity, linearity, amplitude of lateral head displacement, and radius of the average path for circularly swimming sperm, differed (P <0.05) among steps, and most of these parameters differed among ejaculates within a stallion and among stallions. For Steps 2 and 3, 62 and 37% of the sperm were motile, and 56 and 23% of all motile sperm had a curvilinear velocity of >100 mum/sec. Most damage to sperm occurred as a result of freezing-thawing, whereas centrifugation of sperm caused only minor damage.     

A spectrophotometric procedure for the determination of objective measurements of equine spermatozoan motility

A spectrophotometric procedure was developed and evaluated for the objective measurement of equine spermatozoan motility. A 100 mul sample of a sperm suspension, prepared by the removal of seminal plasma, was layered under a column of optically clear medium in a specially designed spectrophotometric cuvette maintained at 37 degrees C. Changes in light transmittance above the interface of the sperm suspension and medium were recorded on chart paper. As sperm cells swam into the medium, a decrease in light transmittance was recorded as a deflection on the chart paper. Chart recordings were analyzed for the height (cm) and time (min) to the peak deflection. To standardize the procedure, a fixed number of cells (1x10(9)) were used to prepare suspensions of 300x10(6) cells/ml. Coefficients of variation for mean values obtained under these conditions after the evaluation of five ejaculates from a given stallion were estimated at between 10 and 12%. Correlations between swim-up measurements and computer-assisted semen analysis demonstrated that the percentage of motile cells and mean velocity (mum/sec) of motile cells influenced swim-up measurements. Described here is a simple and inexpensive procedure to determine objective measurements of spermatozoan motility that may have application in semen evaluation and fertility testing in the stallion.     

Resonance Raman spectra of chlorophylls dissolved in liquid crystal matrices. III. Orientational distribution function of chlorophyll b in an MBBA + EBBA liquid crystalline matrix

Polarized resonance Raman spectra of chlorophyll (Chl) b oriented in a mixture of p-methoxybenzylideno-p'-butylaniline (MBBA) and p-ethoxybenzylideno-p'-butylaniline (EBBA) have been measured. The spectra have been analyzed and the second- and fourth-rank order parameters and the orientational distribution function of Chl b are presented.     

BCG: a modifier of immune responses to parasites

The use of BCG (Bacille Calmette-Guerin) as an adjuvant is well-established for vaccination against leprosy and tuberculosis. Dominique Frommel and Phillippe Lagrange discuss the effects of BCG in the control of parasite infections, particularly leishmaniasis, and the possibility of the development of anti-parasite recombinant BCG vaccines.     

Recycle use of immobilized glucoamylase by tangential flow filtration unit

Various properties of glucoamylase immobilized onto corn stover supporting material and separation of immobilized enzyme by tangential flow filtration unit were studied. Optimum pH and temperature of immobilized enzyme were 3.5 and 60 degrees C, respectively. Enzyme stability was studied in a packed-bed column. The starch conversion rate was attained at 81% for 15 days; after that, the hydrolysis rate gradually decreased. Size of supporting material proved to be an important factor, with higher activity and good loading yield resulting from smaller supporting material. Glucoamylase immobilized onto supporting material less than 44 mum was used for hydrolysis of 10% soluble starch at pH 3.5 and 40 degrees C for 3 h. Then immobilized glucoamylase was separated from the product by means of a tangential flow filtration unit using a 0.2-mum pore size Nylon 66 membrane filter. This operation was continued until 180 ml filtrate was obtained from a 260-mL starting volume. Then, the next batch was started by adding 180 mL starch substrate into the reactor. The batchwise experiments were repeated 20 times. The average filtration rate of each batch was determined and found to sharply decline during the first four batches. Thereafter, it gradually decreased from batch to batch. The cause of decreasing filtration rate appeared to be due to retrogradation of starch. The percentage of starch hydrolysis within 20 batches was in the range 89-96%. The filtration rate becomes higher if the hydrolyzation time is extended to 14 h. Resistance to filtration was also investigated. Almost all of the total resistance is related to insoluble materials, with the significant part of this from the resistance due to insoluble materials deposited on a surface of membrane and boundary layer resistance. Using a microscopic method, no microorganisms were found in the filtrate.