Transformation efficiencies and progeny analysis after varying different parameters of direct gene transfer of Nicotiana tabacum protoplasts

Nicotiana tabacum protoplasts were transformed by polyethylene glycol (PEG)-mediated uptake and electroporation, with circular and linear DNA, and with or without X-ray irradiation. We investigated the influence on the transient expression by these parameters as well as on the frequencies for stable transformation. Plants were regenerated and selfed, and the progenies of the transformed plants were analysed and used to compare the pattern of gene integration by these different variations in transformation methods. The results from the transient expression as judged by glucuronidase (GUS) activity, showed electroporation to give higher and more reproducible results than PEG-mediated uptake. Using linear instead of circular DNA increased the rate of stable transformation about 3 times. Including a mild X-ray treatment gave an increase in the same range. When the inheritance of the transferred trait was investigated, it was found that protoplasts transformed with linear DNA resulted in the highest number of plants with single-copy insertions. Protoplasts transformed with circular DNA showed the highest incidence of losing the trait, while plants in which the transformation included an X-ray treatment, had the highest frequency of multicopy insertion events.     

Ionic processes underlying the decrease in osmotic potential of Chara L-cell fragments in dilute electrolyte solutions

Movements of ions are considered to be governed by the electroneutrality rule. Therefore, a cation moving across the cell membrane into the cell either passively or actively should move together with its counterion, an anion, in equal amounts of charge or in exchange for another cation inside the cell. This means that the net influx of the cation in question should be affected by the permeability of its counterion and/or another cation inside the cell. To examine osmotic and ionic regulation in Chara cells, cell fragments of Chara having a lower osmotic pressure than normal (L-cell fragments) were prepared. The L-cell fragments were individually put into various dilute electrolyte solutions and their osmotic potentials were measured with a turgor balance. Concentrations of K⁺, Na⁺, Ca²⁺, Mg²⁺, Cl^?, NO^?₃. and SO^2?₄. in the external electrolyte solutions in which L-cells had been incubated were also analysed by ion chromatography. The results showed that in 0.5 mM KCL + 0.1 mM CaCl₂ solution, Chara L-cell fragments absorbed K⁺ and Cl^? to maintain electroneutrality and then regained their osmotic potential very rapidly. When the anion was Cl, the cation absorbed at the highest rate was K⁺ On the other hand, when the cation was K, the anion absorbed at the highest rate was Cl, Other ions Ca²⁺, SO^2?₄ and NO^?₃ showed much less permeability than K⁺ and Cl ^?for the Chara plasma membrane. The conclusion from these findings was that due to the constraint of electroneutral transport, the uptake rate of a salt into L-cells is limited by the permeability of the least permeable ion.     

Effect of ammonium on the regulation of nitrate and nitrite transport systems in roots of intact barley (Hordeum vulgare L.) seedlings

The effect of NH ₄ ⁺ on the regulation of NO ₃ ^– and NO ₂ ^– transport systems in roots of intact barley (Hordeum vulgareL.) seedlings grown in NO ₃ ^– or NO ₂ ^– was studied. Ammonium partially inhibited induction of both transport systems. The inhibition was less severe in NO ₂ ^– -fed than in NO ₃ ^– -fed seedlings, presumably due to lower uptake of NH ₄ ⁺ in the presence of NO ₂ ^– . In seedlings pretreated with NH ₄ ⁺ subsequent induction was inhibited only when NH ₄ ⁺ was also present during induction, even though pretreated roots accumulated high levels of NH ₄ ⁺ . This indicates that inhibition may be regulated by NH ₄ ⁺ concentration in the cytoplasm rather than its total accumulation in roots. L-Methionine sulfoximine did not relieve the inhibition by NH ₄ ⁺ , suggesting that inhibition is caused by NH ₄ ⁺ itself rather than by its assimilation product(s). Ammonium inhibited subsequent expression of NO ₃ ^– transport activity similarly in roots grown in 0.1, 1.0, or 10 mM NO ₃ ^– for 24 h (steady-state phase) or 4 d (decline phase), indicating that it has a direct, rather than general feedback effect. Induction of the NO ₃ ^– transport system was about twice as sensitive to NH ₄ ⁺ as compared to the NO ₂ ^– transport system. This may relate to higher turnover rates of membraneassociated NO ₃ ^– -transport proteins.Abbreviations Mes 2(N-morpholino)ethanesulfonic acid - MSO L-methionine sulfoximine     

Long term lily scale bulblet storage: effects of temperature and storage in polyethylene bags

Collections of lily genotypes are usually maintained by yearly planting, harvesting and storage of the bulbs. To facilitate this maintenance, a storage method has been developed for a collection of lily genotypes, including Asiatic hybrids, Oriental hybrids, Lilium longiflorum and L. henryi. Scale bulblets were stored either dry, sealed air-tight in polyethylene bags, or in moist vermiculite in open polyethylene bags for a period of 2 yr. The decrease in mass, sprouting proportion and ion leakage or sprouting proportion alone were determined for treatments carried out at -2°C, °C and 17°C. Sealing scale bulblets in polyethylene bags at -2°C resulted in the smallest decrease in mass, the least ion leakage and the highest sprouting proportion after 2 yr of storage.     

Effects of salinity on uptake and distribution of Na+, Cl- and K+ in two wheat cultivars

Plants of two wheat (Triticum aestivum L.) cultivars differing in salt tolerance were grown in sand with nutrient solutions. 35-d-old plants were subjected to 5 levels of salinity created by adding NaCl, CaCl₂ and Na₂SO₄. Growth reduction caused by salinity was accompanied by increased Na⁺ and Cl^- concentrations, Na⁺/K⁺ ratio, and decreased concentration of K⁺. The salt tolerant cv. Kharchia 65 showed better ionic regulation. Salinity up to 15.7 dS m^-1 induced increased uptake of Na⁺ and Cl^- but higher levels of salinity were not accompanied by further increase in uptake of these ions. Observed increases in Na⁺ and Cl^- concentrations at higher salinities seemed to be the consequence of reduction in growth. Uptake of K⁺ was decreased; more in salt sensitive cultivar. This was also accompanied by differences in its distribution.     

Ecological optima and tolerances of Thelypteris limbosperma,Athyrium distentifolium,and Matteuccia struthiopteris along environmental gradients in Western Norway

The distribution and abundance of Thelypteris limbosperma, Athyrium distentifolium, and Matteuccia struthiopteris are modelled statistically in relation to 14 environmental variables along the major climatic, topographic, and edaphic gradients in western Norway. The data are from 624 stands from which measurements or estimates of mean January and mean July temperatures, humidity, altitude, aspect, and slope are available. From 182 of these stands eight soil variables have also been measured. The species responses are quantified by two numerical methods: Gaussian logit regression and weighted averaging (WA) regression. The estimated WA optima suggest that A. distentifolium has an ecological preference for low July and January temperatures, high altitudes, and soils of low-medium pH and base content. The species shows statistically significant Gaussian responses with summer temperature, humidity (= Martonnes humidity index), altitude, slope, aspect, pH, cation exchange capacity, and base saturation with optima of 8.7 °C, 188.9, 1220 m, 28°, 29°, 4.8, 13.77 mEq 100 g dry soil^-1, and 13.4%, respectively. These suggest that the occurrence and relative abundance of A. distentifolium are well predicted by summer temperature, topography, and soil pH and base status. T. limbosperma has WA optima that suggest that it favours moderately high winter and summer temperatures, high humidity, medium altitude, and soils of low pH and base content. It has significant Gaussian responses to summer temperature (optimum =12.6 °C), winter temperature (-1.8 °C), humidity (179.2), altitude (459.5 m), slope (22.5°), and Na (0.7 mg 100 g dry soil^-1). These suggest that climatic factors, altitude, and slope are significant predictors for its occurrence and abundance. M. struthiopteris has high WA optima for summer temperature, pH, Ca, Mg, K, Na, cation exchange capacity (CEC), and base saturation, and a low optima for humidity and winter temperature. Of these, summer temperature (16.0 °C), Ca (63.1 mg 100 g dry soil^-1), Mg (41.0 mg 100 g dry soil^-1), K (23.6 mg 100 g dry soil^-1), Na (5.0 mg 100 g dry soil^-1), CEC (60.7 mEq 100 g dry soil^-1), and base saturation (56.3%) have significant Gaussian logit responses, as do aspect (150.2°) and loss-on-ignition (9.4%). These results suggest that the occurrence and relative abundance of M. struthiopteris are well predicted by high soil base cations, a generally southern aspect, low organic content in the soil, and high July temperatures.     

Advantages of the scanning ion microscopy for mapping halogen corticoids in normal and transformed cells in culture

Summary— The intra-cellular distribution of eight halogen glucocorticoids was investigated by ion microscopy in two cellular varieties of cultured non-cancer cells (fibroblast 3T3) and cancer cells (human breast tumor cells MCF-7). Two types of ion microscopy helped to determine this distribution, a direct imaging ion microscope (SMI 300) with low spatial resolution, and a scanning ion microscope (IMS4F), featuring high resolution, serving to obtain maps representing the intra-cellular distribution of the fluorine elements and drugs present in these monolayer cultured cells. The fluorine images representative of the drugs containing fluorine showed that these drugs are essentially concentrated in the cell nuclei. In these nuclei, the distribution of these drugs is different from that of heterochromatin and of the nucleolus.     

Role of sodium ions for sulfate transport and energy metabolism in Desulfovibrio salexigens

The sodium ion gradient and the membrane potential were found to be the driving forces of sulfate accumulation in the marine sulfate reducer Desulfovibrio salexigens. The protonmotive force of –158 mV, determined by means of radiolabelled membrane-permeant probes, consisted of a membrane potential of –140 mV and a pH gradient (inside alkaline) of 0.3 at neutral pH_out. The sodium ion gradient, as measured with silicone oil centrifugation and atomic absorption spectroscopy, was eightfold ([Na⁺]_out/[Na⁺]_in) at an external Na⁺ concentration of 320 mM. The resulting sodium ionmotive force was –194 mV and enabled D. salexigens to accumulate sulfate 20000-fold at low external sulfate concentrations (<0.1 M). Under these conditions high sulfate accumulation occurred electrogenically in symport with three sodium ions (assuming equilibrium with the sodium ion-motive force). With increasing external sulfate concentrations sulfate accumulation decreased sharply, and a second, low-accumulating system symported sulfate electroneutrally with two sodium ions. The sodium-ion gradient was built up by electrogenic Na⁺/H⁺ antiport. This was demonstrated by (i) measuring proton translocation upon sodium ion pulses, (ii) studying uptake of sodium salts in the presence or absence of the electrical membrane potential, and (iii) the inhibitory effect of the Na⁺/H⁺ antiport inhibitor propylbenzilylcholin-mustard HCl (PrBCM). With resting cells ATP synthesis was found after proton pulses (changing the pH by three units), but neither after pulses of 500 mM sodium ions, nor in the presence of the uncoupler tetrachorosalicylanilide (TCS). It is concluded that the energy metabolism of the marine strain D. salexigens is based primarily on the protonmotive force and a protontranslocating ATPase.Abbreviations MOPS morpholinopropanesulfonic acid - TCS tetrachlorosalicylanilide - PrBCM propylbenzilylcholin-mustard HCl - Tris tris(hydroxymethyl)aminomethane - TPP⁺ bromide tetraphenylphosphonium bromide     

Predictions for oxygen supply control to enhance population stability of engineered production strains

The simultaneous growth and product formation in a microbial culture is an important feature of several laboratory, industrial, and environmental bioprocesses. Metabolic burden associated with product formation in these bioprocesses may lead to growth advantage of a nonproducing mutant leading to a loss of the producing population over time. A simple population dynamics model demonstrates the extreme sensitivity of population stability to the engineered productivity of a strain. Here we use flux balance analysis to estimate the effects of the metabolic burden associated with product secretion on optimal growth rates. Comparing the optimal growth rates of the producing and nonproducing strains under a given processing condition allows us to predict the population stability. In order to increase stability of an engineered strain, we determine processing conditions that simultaneously maximize the growth rate of the producing population while minimizing the growth rate of a nonproducing population. Using valine, tryptophan, and lysine production as specific examples, we demonstrate that although an appropriate choice of oxygenation may increase culture longevity more than twofold, total production as governed by economic criterion can be increased by several orders of magnitude. Choice of optimal nutrient and oxygen supply rates to enhance stability is important both for strain screening as well as for culture of engineered strains. Appropriate design of the culture environment can thus be used to enhance the productivity of bioprocesses that use engineered production strains. (c) 1994 John Wiley & Sons, Inc.     

Best Linear Unbiased Prediction (BLUP) for regional yield trials: a comparison to additive main effects and multiplicative interaction (AMMI) analysis

Multilocation trials are often used to analyse the adaptability of genotypes in different environments and to find for each environment the genotype that is best adapted; i.e. that is highest yielding in that environment. For this purpose, it is of interest to obtain a reliable estimate of the mean yield of a cultivar in a given environment. This article compares two different statistical estimation procedures for this task: the Additive Main Effects and Multiplicative Interaction (AMMI) analysis and Best Linear Unbiased Prediction (BLUP). A modification of a cross validation procedure commonly used with AMMI is suggested for trials that are laid out as a randomized complete block design. The use of these procedure is exemplified using five faba bean datasets from German registration trails. BLUP was found to outperform AMMI in four of five faba bean datasets.