Immersion Frequency Research Articles

PROPAGATION OF PLANTS IN BIOREACTORS: PROSPECTS AND LIMITATIONS

Bioreactors provide a rapid and efficient plant micropropagation system but as yet are not fully exploited commercially for agricultural crops and forest trees. The culture system was applied to herbaceous and woody plant species utilizing liquid media to overcome intensive manual handling. Large-scale liquid cultures were used for propagation through organogenesis or somatic embryogenesis pathways depending on the species. Bioreactors have several advantages over agar cultures with a better control of the culture conditions. Direct contact of the plant tissue with the medium, optimal nutrient and growth regulators supply, continuous aeration and circulation, filtration of the medium for controlling exudates and contamination and the production of clusters of buds, meristems or protocorms for automated dispensing. The major issue imposed by liquid media in bioreactors is the phenomenon of hyperhydricity, morphogenic shoot and leaf malformation, due to the continuous immersion of the tissue in the medium. The malformations are manifested in glossy hyperhydrous leaves with distorted anatomy. The very nature of liquid tissue culture is an imposing stress to which the plants respond to the environmental signals in developmental aberration. The submerged tissue was found to exhibit oxidative stress symptoms, with elevated levels of reactive oxygen species that were associated with a change in anti-oxidant enzyme activity. These changes greatly affected the anatomy and physiology of the plants and their survival after transplanting. Two major solutions for malformation control were proposed: the use of growth retardants to control rapid proliferation and temporary immersion bioreactors. Growth retardants reduced water uptake during cell proliferation, decreased vacuolation and rapid growth, shortened stems and inhibited leaf expansion, inducing the formation of clusters. In tuber, bulb and corm producing plants growth retardants were found to enhance storage organs formation. Temporary immersion bioreactors were used to provide an improved aeration system to reduce hyperhydricity, by shortening plant duration in the liquid phase. The frequency of immersion and the medium composition improved plant development. Future prospects of micropropagation in bioreactors for optimal plant production will depend on both basic and applied studies. The former contributing to our understanding of plant responses to microenvironment signals to overcome the limitations of liquid cultures and the latter to provide specific culture manipulation to control the morphogenesis of plants in vitro improving survival ex vitro.

Improved shoot regeneration from nodules of Charybdis numidica in a temporary immersion system

SummaryA procedure for improved shoot regeneration from meristematic nodules of Charybdis numidica in a temporary immersion culture system was developed by optimizing immersion frequencies, volume of the nutrient medium, and alternating application of growth regulators. Modified liquid MS medium with 3% sucrose, 20 μM BAP and 5 μM NAA (shoot induction medium) was used to induce microshoot formation on 15 g of nodules in 1000 ml bottles. Volumes of medium (250 or 500 ml) and immersion frequency (5 min every 12 or 24 h) did not significantly influence shoot regeneration rates. Shoot induction medium additionally supplemented with 5 μM paclobutrazol in most cases led to less shoots but this effect was not significant, either. Microshoots formed under these conditions were severely hyperhydrated. Nearly complete elimination of hyperhydricity and enhanced formation of properly elongated shoots were achieved by running a shoot induction step with induction medium containing paclobutrazol followed by an elongation step with a medium supplemented only with 5 μM gibberellic acid GA3. This two-step procedure yielded about 900 healthy shoots per bottle after a two-month cultivation period. Root induction was performed ex vitro during acclimatization and the plantlets could be established in the greenhouse with good success.

High-yielding repetitive somatic embryogenesis and plant recovery in a selected tea clone, 'TRI-2025', by temporary immersion.

Methods for improving the efficiency of repetitive somatic embryogenesis and plant recovery from somatic embryos of clonal tea, TRI 2025 [Camellia sinensis (L.) O. Kuntze] were investigated by optimising the immersion frequencies of the explants using a modified temporary immersion system (TIS). The relative efficiencies of three conventional methods for multiplying embryos were compared with the temporary immersion method. The highest rate of multiplication of secondary embryos (24-fold) was achieved using the TIS. By controlling the immersion cycles, we achieved more consistent, synchronised multiplication and embryo development with a high level of plant recovery. A one-step computer-programmed immersion protocol based on a single, simple medium with no growth regulators was developed, enabling multiplication, maturation, germination and plant recovery within 17 weeks. Plantlets recovered through this method were hardy, with 2- to 5-cm-long shoots containing a minimum of 2-4 lush green leaves and a well-formed taproot. Callus formation, hyperhydricity and other developmental abnormalities were not observed at any stage in the process. Plantlets produced using this method were successfully acclimatised to glasshouse conditions. This protocol avoids culture transfers, and thus minimises the risk of contamination and reduces labour costs. This technique could have significant commercial implications in tea propagation as it has the potential for large-scale production with considerably reduced production costs.

In vitro Quantitative Microhardness Assessment of Enamel with Early Salivary Pellicles after Exposure to an Eroding Cola Drink

To assess the erosive effect of a cola drink on enamel incorporating early salivary pellicles, 72 groups of human enamel slabs were immersed in fresh cola, with groups differing in that slabs were (1) incubated in whole or in clarified saliva, (2) under three regimes of frequency intake (1, 5 and 10 times/day); (3) immersed with or without agitation, and (4) the pellicle was incubated for 20 min, 6 or 24 h. Quantitative assessments were done over an 8–day interval using surface microhardness testing. Results for primary enamel showed a sharp decrease from baseline (344.2±32.4 Vickers Units; mean ±SD) to day 1 (268.9±36.8), and reached 155.2±68.6 on day 8. Results for permanent enamel were 350.8±42.2, 315.9±39.2, and 149.8±85.2. Microhardness was affected by agitation, level of intake and type of saliva, but not by the pellicle incubation interval nor the type of enamel. The joint effect of static baths and higher immersion frequency was the most important factor in decreasing hardness.

Atmospheric deposition to forests along an elevational gradient at Whiteface Mountain, NY, U.S.A.

Abstract Atmospheric deposition rates of pollutants are known to be greater at high elevations than at low elavations in the same region, but the pattern of the deposition rate increase with elevation has not been established. This study was conducted to estimate nutrient and pollutant transfer rates from the atmosphere to forests along an elevational gradient at a site in the Adirondack Mountains of NY, U.S.A. Widely used models of cloud droplet, dry aerosol and gas phase SO 2 and HNO 3 deposition processes were modified for use in this application. An extensive data set describing the elevational variation in forest canopy composition and structure and microclimatic variables was assembled from measurements made by us during seven years of research activities at Whiteface Mountain, NY. Model estimates of total atmospheric deposition of S and N increased by factors of 4 and 5, respectively, over the elevational range 600–1275 m. Steep gradients in wind speed and cloud immersion frequency contributed to a nearly exponential increase in ion deposition by cloud water interception, which was responsible for most of the increase in total ion deposition rates with elevation. An additional factor contributing to increased deposition rates at high elevations was an increasing percentage of total leaf area attributable to coniferous vegetation, which is more effective at scavenging aerosol particles and cloud droplets than broad-leaved vegetation. Dry deposition contributed 13% and cloud water 61% of the 29.5 kg N ha −1 yr −1 estimated total N deposition at 1275 m elevation as opposed to 22 and 5% of the 7 kg N ha −1 yr −1 total N deposition estimated for surrounding low-elevation (600 m) forests. The dry deposition component accounted for 5% and the cloud water component 64% of the estimated 31 kg S ha −1 yr −1 total deposition at 1275 m elevation, in comparison with a 21% dry and 5% cloud contribution to the 8.4 kg S ha −1 yr −1 estimated deposition to low-elevation forests.

ブリ類結節症に対する浸漬法ワクチンの処理回数と経口法併用による有効性の比較

Efficacy of various direct immersion methods was investigated for the development of a practical vaccine against pseudotuberculosis in cultured yellowtail. Formalin-killed Pasteurella piscicida was used to immunize fish, one, two, three times by immersion and a combination of single immersion and oral vaccination methods. An artificial infection at three doses of P. piscicida to determine efficacy of various administered methods was carried out three weeks after the first vaccination. The relative percent survival at ten days after artificial infectionwere:a) 8.0×104 cfu/ml:0% in one immersion, 7% in two immersion, 13% in three immersions 17% in immersion+oral methods; b) 8.0×103 cfu/ml: 22% in one immersion, 39% in two im-mersions, 51% in three immersions and 65% in immersion+oral methods and c) 8.0×102 cfu/mt: 30% in immersion+oral methods. Agglutination titers against P. piscicida in the serum and skin mucus were determined in vaccinated fish prior to artificial infection and were observed to be enhanced with increasing frequency of immersion. In addition, agglutination titers against P. piscicida were observed in the intestinal mucus of fish vaccinated by three times immersion and a combination of immsersion and oral methods. Efficacy of direct immsersion methods increased with increasing administration times and combined with oral vaccination methods.

Preliminary results on factors causing zonation in Enteromorpha using a tide simulating apparatus

Cultures of an Enteromorpha sp. grown on glass plates have been subjected to artificial tidal action by mechanically lowering and raising the plates into a tank of sea water on a twelve-hour cycle or fractions thereof. Preliminary results show that at levels on the plates with the same percentage exposure to air, greater frequency of immersion gives zones with higher upper limits. Field data are given to show that in Ghana, Enteromorpha produces motile swarmers most frequently two days either before or after spring tides. Resistance to desiccation is linearly related to the salinity of the sea water in which the plants are grown.