Seed germination,seedling inoculation and establishment ofAlnus spp. in containers in greenhouse trials

Methods for production of containerized seedlings ofAlnus species were developed which permit nitrogen-fixing nodules to form on the root systems prior to outplanting, in order to provide an early nitrogen input during seedling establishment. The methods are based on procedures for inoculating root systems with suspensions ofFrankia (Actinomycetales), applied either directly in the container cell as a soil drench at the time of seeding, or as a root dip for seedlings transplanted into the containers. Germination of dried, stored seed was enhanced by light and by presoaking for 16 h in water. Pretreatments to overcome seed dormancy or to eliminate fungal pathogens did not further enhance germination. Some loss of seedlings was recorded in the early stages of growth shortly after germination, which is a factor in calculating projected seedling yield. Nodulation and seedling growth were evaluated in terms of growth media characteristics. Seedlings performed well in peat-vermiculite, at soil pH between 5.5 and 8.0.     

APOLAR EMBRYOS OF FUCUS RESULTING FROM OSMOTIC AND CHEMICAL TREATMENT

Embryos of the brown alga Fucus vesiculosas L. were grown as populations in glass petri dishes in seawater at 15 C in continuous low-intensity unilateral fluorescent illumination for periods up to 2 weeks. A quantitative estimate of increase in nuclear number was made from acetocarmine squash preparations of samples taken at 12-or-24 hr intervals. Over the period of 2-6 days embryos showed a doubling time of about 12-18 hr. Under normal seawater culture conditions each embryo formed a single rhizoid. When grown in seawater supplemented with sugar concentrations above 0.4 m , Fucus embryos developed as multicellular spherical embryos lacking rhizoids. In 0.6 m sucrose-seawater, 97% of the embryos were apolar at 2 days; only 37% were apolar at 4 days, many having recovered from the sucrose inhibition. Some embryos remained apolar after growth in 0.6 m sucrose for 2 weeks. Nuclear counts showed that sucrose-seawater markedly inhibited the rate of cell division. Other sugars including D-glucose, D-fructose, D-galactose and the sugar alcohol D-mannitol were also effective. When apolar embryos grown in sucrose-seawater were returned to seawater, embryo growth resumed at the normal seawater rate, judged from nuclear counts. Such embryos formed multiple rhizoids, varying from two to eight rhizoids per embryo, which developed on the embryo quadrant or half away from the unilateral light. Each of the multiple rhizoids originated from a single small cell in the periphery of the multicellular spherica embryo. Thus the rhizoid-forming stimulus apparently had been subdivided among a number of the cells of the apolar embryos. The implications of this finding are discussed. Attempts to produce multiple rhizoids by treatment of embryos with indoleacetic acid or 2,4-dichlorophen-oxyacetic acid failed. However, embryos treated with 10⁻⁴ M or 5 × 10⁻⁵ m 2,3,5-triiodobenzoic acid formed 40 and 30% multiple rhizoids, respectively, suggesting that some chemical, perhaps hormonal, mechanism is involved in polarization and rhizoid initiation in Fucus embryogenesis.     

Nitric oxide synthesis in the CNS endothelium and macrophages differs in its sensitivity to inhibition by arginine analogues.

Inhibition of nitric oxide production by arginine analogues was examined in three cell systems; macrophages, CNS tissue and endothelial cells. Nitric oxide production was assessed indirectly using in vitro assays measuring nitrite production (macrophages), cGMP elevation (CNS) and acetylcholine-induced relaxation of aortic ring segments (endothelium). NG-monomethyl-L-arginine and NG-amino-L-arginine possessed similar inhibitory activity in all three assays, while NG-nitro-L-arginine displayed a striking selectivity for inhibition of brain and endothelial cell nitric oxide synthesis, with IC50 values of 0.05 microM in the CNS versus 200 microM in macrophages. These results suggest that distinct enzymes are responsible for nitric oxide synthesis in different cell types, and indicate that it may be possible to selectively modulate nitric oxide production in vivo.     

In vitro clonal multiplication of the actinorhizal plant Comptonia peregrina

Micropropagation of the actinorhizal plant Comptonia peregrina of the Myricaceae was achieved successfully by the induction of root buds in excised root culture with cytokinin (1.0 M benzyladenine). Excised root segments with initiated root buds were subcultured in Woody Plant Medium (WPM) lacking growth regulators, developing extensive callus which subsequently gave rise to multiple adventitious buds. Shoot elongation was facilitated by transfer of calluses to more aerated conditions. Root initiation was induced on shoots by brief treatment with auxin (<1 M indolebutyric acid) and transfer to WPM for plantlet development. Controlled light and aeration in liquid medium were critical conditions for successful micropropagation.     

Growth and anthocyanin accumulation rates of carrot suspension cultures grown with excess nutrients after semicontinuous culture with different limiting nutrients at several dilution rates, pHs, and temperatures

When carrot cell cultures, after growth in semicontinuous culture, were transferred to media containing excess nutrients, they grew at different rates. The growth rates were generally higher after semicontinuous culture at higher dilution rates. There appears to be a limit on dilution rate above which growth rate does not increase. These changes were also displayed by clones from the parental culture. The possibility that these changes in growth rate reflect a need for the cultures to adapt to their new conditions is discussed. The growth rates of the cultures is markedly increased at 25 degrees C compared with 22 degrees C with little further increase at 28 degrees C. Growth rate is altered by less than 20% when pH is changed from 4.5 to either 5.5 or 4.2. The rates of anthocyanin accumulation by the cultures were similar under all conditions tested except at 22 degrees C. They were larger than the rates of dry weight accumulation. In contrast, the amounts of anthocyanin accumulated in the clones and in the parental cultures grown at pH 5.5 instead of pH 4.5 were very different. The observations were interpreted as showing that the clones differ in the rate of metabolism but not in the rate of synthesis of anthocyanins and that at pH 5.5 the rate of metabolism of anthocyanins but not the rate of synthesis is higher than it is at pH 4.5. The use of semicontinuous cultures as a source of inoculum for batch cultures rather than as a source of biomass for extraction of chemicals is discussed.     

Hormonal Control of Cell Proliferation and Xylem Differentiation in Cultured Tissues of Glycine max var. Biloxi

The relationship between tracheary element differentiation, cell proliferation and growth hormones was examined in agar-grown soybean callus. The time course of cell division and tracheary element formation in tissues grown on a medium containing 5 x 10(-7)m kinetin and 10(-5)m NAA was determined by means of maceration technique. After a slight lag period, a logarithmic increase in cell number was observed through the twelfth day of the culture period. Cell numbers increased at a considerably slower rate after the twelfth day. The rate of tracheary element formation varied with the rate of cell proliferation. Tracheary elements increased logarithmically during the log phase of growth. As the rate of cell division decreased after the twelfth day of culture, the rate of tracheary element formation also decreased. In the presence of 10(-5)m NAA, cell number increased as the kinetin concentration was increased between 10(-9) and 10(-6)m. However, tracheary element formation was not initiated unless the kinetin concentration was 5 x 10(-8)m or above. When the Biloxi callus was subcultured repeatedly on media containing 10(-8)m kinetin, a tracheary element-free population of cells was obtained. This undifferentiated tissue produced tracheary elements upon transfer to a medium containing 5 x 10(-7)m kinetin. In the presence of 5 x 10(-7)m kinetin, NAA stimulated cell proliferation between 10(-7) and 10(-5)m, but no tracheary elements were formed without auxin, or with 10(-7)m NAA. Neither NAA nor kinetin at any concentration tested stimulated tracheary element formation in the absence of an effective level of the other hormone. However, 2,4-D at 10(-7) or 10(-6)m promoted both cell proliferation and tracheary element differentiation in the absence of an exogenous cytokinin.     

Amino Acid incorporation in developing fucus embryos

The incorporation of ¹⁴C-leucine and ¹⁴C-amino acid mixture into protein in unfertilized eggs and developing embryos of the brown alga Fucus vesiculosus L. was studied. Bacterial contamination was initially a problem, but it was found that the addition of 40 μg/ml chloramphenicol to the incubation medium would inhibit bacterial protein synthesis without affecting early development of the Fucus embryos. The kinetics of uptake and incorporation of ¹⁴C-leucine into the trichloroacetic acid-soluble and -insoluble fractions indicated that the exogenous precursor did not equilibrate with the main soluble leucine pool before incorporation into protein. Uptake and incorporation of leucine by embryos 90 to 175 minutes old were proportional to exogenous leucine concentration over the range 5 × 10⁻⁶ m to 5 × 10⁻³ m. Unfertilized eggs will incorporate ¹⁴C-leucine into protein. The rate of this incorporation increases dramatically in newly fertilized eggs with a maximum rate at 3.5 hours, a period of cell wall formation and increasing metabolic rates. Thereafter, the rate of incorporation declines until approximately 15 to 17 hours when it increases again concurrently with the onset of rhizoid initiation and cell division.