Transgenic turf-type tall fescue (Festuca amndinacea Schreb.) plants regenerated from protoplasts

To improve turfgrasses using genetic engineering, we have developed a transformation system in turf-type tall fescue, one of the most important turfgrass species. Embryogenic cell cultures were established after callus induction from embryos of mature seed. The agarose-bead method with nurse cells was used to culture protoplasts and plants were regenerated from protoplasts of tall fescue cultured cells. To develop transgenic tall fescue plants, the hygromycin resistance gene and the -glucuronidase gene were introduced into the tall fescue protoplasts by electroporation. A high concentration (200 mg/l) of hygromycin was required to select transformed cells because of the high level of endogenous resistance to the antibiotic in tall fescue. Most of the transformed cells exhibited GUS activity and several plants were regenerated from these cells. The presence of introduced genes was confirmed by Southern blot hybridization of PCR amplified DNA from transgenic plants.Abbreviations Adh alcohol dehydrogenase - BAP benzylaminopurine - bp base pair(s) - GUS -glucuronidase - Kb kilobase(s) - MS Murashige and Skoog's medium - PCR polymerase chain reaction     

Effect of microspore stage and media on anther culture of peanut (Arachis hypogaea L.)

This study was designed to study the effects of stage of microspore development and culture medium on androgenic response in peanut (Arachis hypogaea L.). Anthers of various developmental stages were cultured for 7 days, then fixed and observed cytologically. Three sets of media, involving different basal media, growth regulators, sucrose levels and glutamine concentrations, were tested. In all experiments, the stage of development of the microspores at the time of culture was highly significant. The early uninucleate microspores stage was identified as producing the highest anther response rating. The effect of media was nonsignificant in all experiments. However, the stepwise modification of the media through the course of the study resulted in an almost 8 x increase in anther response rating. Numerically, the best media tested was N₆ basal medium with 1 mg 1^-1 NAA, 0.1 mg 1^-1 BA, 5.5% sucrose, and 3.5 g 1^-1 glutamine. While no haploids were obtained, four-nucleate cells were observed, indicating the potential in peanuts for an androgenic reponse.     

Phloem unloading in soybean seed coats: dynamics and stability of efflux into attached ;empty ovules'

The time-course of sucrose efflux from attached seedcoats (having their embryos surgically removed) into aqueous traps placed in the `empty ovules' had three phases. The first phase lasted 10 minutes and probably was a period of apoplastic flushing. The second lasted 2 to 3 hours and is thought to be a phase of equilibration of seed coat symplast with the frequently refreshed liquid. The third phase of relatively steady efflux was postulated to reflect the continued import of sucrose from the plant, and hence to reflect the rate of sieve tube unloading. The average steady state efflux was equal under most conditions to the estimated rate of sucrose import. Efflux and import were unaffected by 150 millimolar osmoticum (mannitol or polyethylene glycol [molecular weight about 400]), by 0.5 millimolar CaCl<sub>2</sub>, or by pretreatments up to 20 minutes with p-chloromercuribenzenesulfonic acid (PCMBS); they were enhanced by 40 micromolar abscisic acid, 40 micromolar indoleacetic acid, 20 micromolar fusicoccin, and 1 millimolar dithiothreitol (DTT) and were inhibited by 100 micromolar KCN, by 0.03% H<sub>2</sub>O<sub>2</sub>, by 20 micromolar and 5 micromolar trifluoromethoxy (carbonyl cyamide) phenylhydrazone, by repeated 5 minutes per hour treatments with 5 millimolar PCMBS, and by 5 millimolar DTT. The `steady state' sucrose efflux was able to account for about half the rate of dry weight growth of the embryo, but stabilization of the system with <1 millimolar DTT taken together with other considerations is likely to give good correspondence between experimental unloading rates and in vivo growth rates.     

Inhalation anesthesia in adult cattle

An inhalation technique was used for anesthesia during ileal cannulation in five adult cows. Following sedation with intravenous acepromazine, anesthesia was induced intravenously with thiopental sodium in 5% glyceryl guaiacolate solution. Endotracheal intubation was performed and anesthesia maintained with halothane in oxygen via a circle system with a precision vaporizer. In all cases, induction was smooth and no difficulties were experienced during the maintenance of anesthesia. Total anesthesia time was 1.5 to 2.5 hours. Following completion of the surgical procedure, which was performed with the animal in left lateral recumbency, each cow was rolled to a sternal position and supported, if necessary. The endotracheal tube was left in place, with oxygen administration continued, until the animal was able to swallow. Recoveries were rapid and all animals were ambulatory within 30 minutes after completion of the surgery. The only post-operative complication due to anesthesia was transient mouth soreness in two cases, attributed to the use of a mouth speculum during intubation.     

Interspecies transduction of plasmids among Bacillus anthracis, B. cereus, and B. thuringiensis

Bacteriophage CP-51, a generalized transducing phage for Bacillus anthracis, B. cereus, and B. thuringiensis, mediates transduction of plasmid DNA. B. cereus GP7 harbors the 2.8-megadalton multicopy tetracycline resistance plasmid, pBC16. B. thuringiensis 4D11A carries pC194, the 1.8-megadalton multicopy chloramphenicol resistance plasmid. When phage CP-51 was propagated on these strains, it transferred the plasmid-encoded antibiotic resistances to the nonvirulent Weybridge (Sterne) strain of B. anthracis, to B. cereus 569, and to strains of several B. thuringiensis subspecies. The frequency of transfer was as high as 10(-5) transductants per PFU. Tetracycline-resistant and chloramphenicol-resistant transductants contained newly acquired plasmid DNA having the same molecular weight as that contained in the donor strain. Antibiotic-resistant transductants derived from any of the three species were effective donors of plasmids to recipients from all three species.     

Chromosomal mapping of Bacillus thuringiensis by transduction.

Three groups of linked markers were mapped in Bacillus thuringiensis 4042B by using two-, three-, and four-factor crosses mediated by the temperate bacteriophages TP-13 and TP-18. The order of markers was (trp-11, trp-2)-(leu-1, leu-2)-his-1-(lys-1, lys-2)-cys-1 in the first group; met-1-(argCl, argOl)-met-2-(pyr-1, pyrA2) in the second group; and met-3-pur-1-(nal-1, nal-2)-str-1-(pur-2, pur-4)-pur-3 in the third group. Electron microscopic measurements of head sizes suggested that the volume of the TP-13 phage head is seven times greater than that of the TP-18 phage head. The TP-18 genome was shown by DNA restriction analysis to have a molecular mass of 36 megadaltons. TP-13 was useful for scanning large segments of the B. thuringiensis chromosome, and TP-18 was effective for ordering markers too closely linked for simple resolution with TP-13.     

Nitrate reductase: an improved assay method for phytoplankton

A new assay for measuring the activity of nitrate reductasein phytoplankton, based upon the permeability of cells treatedwith toluene to substrates and products, is described. The methodis simple and, since the reaction is carried out directly ona glass fiber filter, can be easily performed in the field oron shipboard. In comparison with previous methods, this techniquegave higher absolute amounts of NO^–₂ formed per unit tuneand higher enzymatic activities per sample volume when testedwith axenic algal cultures and with natural phytoplankton populationsfrom Lake Kinneret, the River Jordan and the Eastern Mediterranean.