CALCULATIONS OF BIOELECTRIC POTENTIALS : IV. SOME EFFECTS OF CALCIUM ON POTENTIALS IN NITELLA

In Nitella the substitution of KCl for NaCl changes the P.D. in a negative direction. In some cases this change is lessened by adding solid CaCl₂ to the solution of KCl. This may be due to lessening the partition coefficient of KCl or to decreasing the solubility of an organic substance which sensitizes the cell to the action of KCl. Little or no correlation exists between this effect of calcium and its ordinary antagonistic action in producing a balanced solution which preserves the life of the cell indefinitely. CaCl₂ is negative to NaCl but positive to KCl. The effects of mixtures of KCl, NaCl, and CaCl₂ are discussed. The concentration effect of a mixture of KCl + CaCl₂ shows certain peculiarities due to action currents: these resemble those found with pure KCl. These studies and others on Nitella, Valonia, and Halicystis indicate that mobilities and partition coefficients are variable and can be brought under experimental control.     

Tailoring the biological response of zirconium implants using zirconia bioceramic coatings: A systematic review

BackgroundThe poor biological performance of zirconium implants in the human body resulting from their bio-inertness and vulnerability to corrosion and bacterial activity reflects the need for further studies on substitution or performing the surface modification. The suggestion of employing zirconia (ZrO₂) bioceramic coatings for surface modification seems beneficial.ObjectivesThis systematic review aims to identify and summarize existing documents reporting the biological responses for ZrO₂ coatings produced by the PEO process on zirconium implants.MethodsPubMed, Scopus, and Web of Science international databases were searched for the original and English-language studies published between 2000 and 2021. All publications reported at least one study about in-vitro (cellular and immersion studies), in-vivo (animal studies), and antibacterial topics for ZrO₂-PEO coated zirconium implants.ResultsThroughout the initial search, 496 publications were found, and 296 papers remained following the elimination of duplicates. Finally, after multiple screening and eligibility assessments, 25 publications were qualified and included in the review. Among them, 25 in-vitro (cellular and immersion in SBF and Hanks’ solutions studies), one in-vivo (animal studies), and eight antibacterial studies were found.ConclusionThe ZrO₂ coated samples demonstrate no cytotoxicity, high cell viability rate, and excellent biocompatibility. However, changing the solution composition and electrical parameters during the PEO procedures result in significant changes to in-vitro responses. As an instance, the ZrO₂ coating surface demonstrates greater biocompatibility after irradiated by UV, which makes the surface more suitable for cell growth. Due to weak apatite-forming ability, the zirconium sample shows low bioactivity in SBF. However, most cases (13 out of 16) show that the specific morphology and chemical composition of the ZrO₂ coating promote apatite-forming ability with good bioactivity in SBF. Nevertheless, few papers (three out of 16) showed that the ZrO₂ coatings immersed in SBF had no apatite precipitates and so no bioactivity. These cases limit the bioactivity enhancement to treatment by UV-light irradiation, hydrothermal and chemical treatment, thermal evaporation, and cathodic polarization post-treatment on ZrO₂ coatings. Both zirconium and ZrO₂ coated samples do not show apatite-forming ability in Hanks’ solution. The ZrO₂ coated implant with the bone together indicates a greater shear strength and rapid new bone formation ability during 12 weeks because of containing Ca-P compounds and porous structure. The UV post-treated ZrO₂ coating induces faster new bone formation and firmer connection of bond with bone than those of untreated ZrO₂ coatings. A stronger antibacterial activity of ZrO₂ coatings is confirmed in half of the selected papers (four out of eight studies) compared to the bare zirconium samples. The antibacterial protection of ZrO₂ coatings can be influenced by the PEO procedure variables, i.e., solution composition, electrical parameters, and treatment time. In three cases, the antibacterial activity of ZrO₂ coatings is enhanced by deposition of Zn, Ag, or Cu antibacterial layers through thermal evaporation post-treatment.     

THE COMPOSITION OF THE CELL SAP OF THE PLANT IN RELATION TO THE ABSORPTION OF IONS

1. Chemical examination of the cell sap of Nitella showed that the concentrations of all the principal inorganic elements, K, SO₄, Ca, Mg, PO₄, Cl, and Na, were very much higher than in the water in which the plants were growing. 2. Conductivity measurements and other considerations lead to the conclusion that all or nearly all of the inorganic elements present in the cell sap exist in ionic state. 3. The insoluble or combined elements found in the cell wall or protoplasm included Ca, Mg, S, Si, Fe, and Al. No potassium was present in insoluble form. Calcium was predominant. 4. The hydrogen ion concentration of healthy cells was found to be approximately constant, at pH 5.2. This value was not changed even when the outside solution varied from pH 5.0 to 9.0. 5. The penetration of NO₃ ion into the cell sap from dilute solutions was definitely influenced by the hydrogen ion concentration of the solution. Penetration was much more rapid from a slightly acid solution than from an alkaline one. It is possible that the NO₃ forms a combination with some constituent of the cell wall or of the protoplasm. 6. The exosmosis of chlorine from Nitella cells was found to be a delicate test for injury or altered permeability. 7. Dilute solutions of ammonium salts caused the reaction of the cell sap to increase its pH value. This change was accompanied by injury and exosmosis of chlorine. 8. Apparently the penetration of ions into the cell may take place from a solution of low concentration into a solution of higher concentration. 9. Various comparisons with higher plants are drawn, with reference to buffer systems, solubility of potassium, removal of nitrate from solution, etc.     

Silicon metabolism in diatoms. I. Evidence for the role of reduced sulfur compounds in silicon utilization

1. Cells of the fresh water diatom Navicula pelliculosa may be grown in a mineral medium containing a low concentration of silicon. When transferred to a fresh silicate solution and incubated under non-growing conditions such deficient cells rapidly take up silicon from the medium. 2. The utilization of silicon is an aerobic process. 3. When deficient cells are washed with distilled water or saline, their ability to utilize silicon is impaired whereas respiration is unaffected. 4. The ability of washed cells to take up silicon can be partially restored with sulfate or ascorbic acid, and is completely restored by Na₂S, Na₂S₂O₃, glutathione, l-cysteine, dl-methionine, or ascorbic acid plus sulfate. 5. The sulfhydryl reagent, CdCl₂, inhibits silicon utilization of unwashed cells at concentrations which do not affect respiration. This inhibition similarly is reversed by glutathione or cysteine. 6. However, sodium iodoacetate or sodium arsenite inhibits respiration and silicon utilization at the same concentrations. 7. The silicon taken up by deficient cells is deposited at the cell surface as a thickening of the existing silica frustules. 8. Sulfhydryl groups in the cell membrane may be involved in silicon uptake by diatoms.     

Radiorespirometer for continuous monitoring of chick embryo development.

A radiorespirometer is described that is capable of continuous monitoring of O₂ utilization, CO₂ and/or ¹⁴CO₂ production per minute, heart rate, and body activity of an embryonated egg while it develops for several days in a closed chamber with near normal pO₂ or other desired pO₂. Oxygen is supplied to the embryo by an electrolytic cell, and CO₂ is removed by a KOH solution flowing through a diffusion cell separated from the embryo chamber by a CO₂ permeable rubber membrane. The instrument permits selecting embryos for viability and developmental stage, according to their O₂ utilization per minute and respiratory quotient, without breaking the eggshell. Inoculation of the embryonated egg with ¹⁴C-labeled substrates, drugs, or toxins can occur without interfering with continuous recording of metabolic activity.     

THE PENETRATION OF BASIC DYE INTO NITELLA AND VALONIA IN THE PRESENCE OF CERTAIN ACIDS,BUFFER MIXTURES,AND SALTS

When living cells of Nitella are exposed to an acetate buffer solution until the pH value of the sap is decreased and subsequently placed in a solution of brilliant cresyl blue, the rate of penetration of dye into the vacuole is found to decrease in the majority of cases, and increase in other cases, as compared with the control cells which are transferred to the dye solution directly from tap water. This decrease in the rate is not due to the lowering of the pH value of the solution just outside the cell wall, as a result of diffusion of acetic acid from the cell when cells are removed from the buffer solution and placed in the dye solution, because the relative amount of decrease (as compared with the control) is the same whether the external solution is stirred or not. Such a decrease in the rate may be brought about without a change in the pH value of the sap if the cells are placed in the dye solution after exposure to a phosphate buffer solution in which the pH value of the sap remains normal. The rate of penetration of dye is then found to decrease. The extent of this decrease is the greater the lower the pH value of the solution. It is found that hydrochloric acid and boric acid have no effect while phosphoric acid has an inhibiting effect at pH 4.8 on stirring. Experiments with neutral salt solutions indicate that a direct effect on the cell (decreasing penetration) is due to monovalent base cations, while there is no such effect directly on the dye. It is assumed that the effect of the phosphate and acetate buffer solutions on the cell, decreasing the rate of penetration, is due (1) to the penetration of these acids into the protoplasm as undissociated molecules, which dissociate upon entrance and lower the pH value of the protoplasm or to their action on the surface of the protoplasm, (2) to the effect of base cations on the protoplasm (either at the surface or in the interior), and (3) possibly to the effect of certain anions. In this case the action of the buffer solution is not due to its hydrogen ions. In the case of living cells of Valonia under the same experimental conditions as Nitella it is found that the rate of penetration of dye decreases when the pH value of the sap increases in presence of NH₃, and also when the pH value of the sap is decreased in the presence of acetic acid. Such a decrease may be brought about even when the cells are previously exposed to sea water containing HCl, in which the pH value of the sap remains normal.     

Inactivation of Escherichia coli Cells in Aqueous Solution by Atmospheric-Pressure N2, He,Air, and O2 Microplasmas

Atmospheric-pressure N₂, He, air, and O₂ microplasma arrays have been used to inactivate Escherichia coli cells suspended in aqueous solution. Measurements show that the efficiency of inactivation of E. coli cells is strongly dependent on the feed gases used, the plasma treatment time, and the discharge power. Compared to atmospheric-pressure N₂ and He microplasma arrays, air and O₂ microplasma arrays may be utilized to more efficiently kill E. coli cells in aqueous solution. The efficiencies of inactivation of E. coli cells in water can be well described by using the chemical reaction rate model, where reactive oxygen species play a crucial role in the inactivation process. Analysis indicates that plasma-generated reactive species can react with E. coli cells in water by direct or indirect interactions.     

Cat Heart Muscle In Vitro : IV. Inhibition of transport in quiescent muscles

Cellular concentrations, [K]_i, [Na]_i, and [Cl]_i, and cell water contents were measured in vitro at 27°C in cat papillary muscles. Measurements were made with and without ouabain at varying concentrations of K and ouabain, at pH 5.2 and 9.0, in absence of O₂, and in NaCl-free solution. Large losses of cell K and increases of cell Na occurred in presence of ouabain, at 2–3°C, and in K-free medium. The dependence of inhibition of cation transport by ouabain on external K concentration, studied at constant initial [K]_i, was consistent with a competition between K and ouabain localized to the external face of the membrane. In NaCl-free sucrose solution [K]_i remained at its physiological value and was not affected by exposure to ouabain or low temperature, except when Ca was also omitted. Ouabain inhibition persisted at pH 9.0 and in Ca-poor media. Cells swelled and lost K at pH 5.2, and residual ouabain effect was small. At pH 9.0, or in absence of O₂, or in Ca-poor solutions cells became permeable to mannitol. The ion movements observed after inhibition of active transport are compatible either with a passive K distribution and a primary inhibition of Na extrusion or with inhibition of a coupled active transport of both K and Na.     

The Time Course of the Loss and Recovery of Contracture Ability in Frog Striated Muscle Following Exposure to Ca-Free Solutions

Using area under the contracture curve to quantitate contractures, the diffusion coefficient of calcium ions within the frog toe muscle during washout in a calcium-free solution and subsequent recovery after reintroduction of calcium to the bathing solution was calculated to be about 2 x 10^-6 cm²/sec. The diffusion coefficient measured during washout was found to be independent of temperature or initial calcium ion concentration. During recovery it was found to decrease if the temperature was lowered. This was likely due to the repolarization occurring after the depolarizing effect of the calcium-free solution. The relation between contracture area and [Ca]_o was found to be useful over a wider range than that between maximum tension and [Ca]_o. The normalized contracture areas were larger at lower calcium concentrations if the contractures were produced with cold potassium solutions or if NO₃ replaced Cl in the bathing solutions. Decreasing the potassium concentration of the contracture solution to 50 mM from 115 mM did not change the relation between [Ca]_o and the normalized area. If the K concentration of the bathing solution was increased, the areas were decreased at lower concentrations of Ca.     

The Reponse of Duck Erythrocytes to Norepinephrine and an Elevated Extracellular Potassium : Volume regulation in isotonic media

This paper presents evidence that duck erythrocytes regulate their size in isotonic media by utilizing a previously reported "volume-controlling mechanism." Two different experimental situations are examined. In the first, cells enlarge in a solution containing norepinephrine and an elevated [K]_o; and in the second, enlarged cells shrink to their original size if the norepinephrine and excess potassium are removed. As the erythrocytes enlarge, K, Cl, and H₂O accumulate. Shrinkage, in contrast, is accompanied by the controlled loss of K, Cl, and H₂O. These changes and the associated changes in membrane permeability resemble those reported previously when duck erythrocytes incubate in anisotonic media. There cells, after first shrinking or swelling, utilize a "volume-controlling mechanism" to reestablish their original size. The mechanism regulates cell size by adjusting the total number of osmotically active intracellular particles. The present studies indicate duck red cells use this mechanism to readjust their total monovalent cation content and thus their solute content in isotonic media as well. In addition, evidence is presented which indicates that the "volume-controlling mechanism" and ouabain-inhibitable cation pump differ functionally.     

Immobilisation and mechanical support of individual protoplasts

Mesophyl cell protoplasts of Vicia faba were suspended in a solution consisting of 10% sodium alginate and 0.4 M mannitol. The protoplasts could be immobilized by cross-linking the alginate in the presence of 100 mM CaCl₂. Changes in the osmolarity of the external medium led to reversible shrinkage and swelling of the entrapped protoplasts. It was demonstrated by using the pressure probe technique that a pressure gradient (cell turgor pressure) of several 100 mbar is built up when the immobilized cells were transferred to hypotonic solution. By complexing the Ca²⁺ in the alginate matrix with sodium citrate buffer the protoplasts could be released from the matrix. No morphological change or alteration of the membrane permeability of the immobilized protoplasts was observed after a storage period of up to 14 days at 4°C in the matrix.     

Induction of HLA expression in Daudi cells after cell fusion

The Daudi cell line, established from a Burkitt lymphoma, has recently been found to be HLA- andΒ ₂-microglobulin-negative, although it expresses B lymphocyte alloantigens. This report is concerned with the reexpression of HLA-A10, B38, and B17 on the Daudi cell, after cell fusion with another human cell line (Raji) or with mouse fibroblasts. In the latter fusion, the same HLA specificities are re-expressed, but not humanΒ ₂-microglobulin while mouseΒ ₂-microglobulin andH-2 could be detected. No such reexpression was observed when Daudi was fused with the F9 mouse teratocarcinoma, which lacks mouseΒ ₂-m andH-2. No HLA activity (alloantigenic and xenogenic activity) was detected in the membrane or cytoplasm of Daudi, using salt extraction and sonication. Therefore we postulate thatΒ ₂-microglobulin could be necessary for the expression and possible synthesis of the HLA antigen.     

Sorption of Heavy Metals to the Filamentous Bacterium Thiothrix Strain A1

A study was undertaken to determine the ability of the filamentous bacterium Thiothrix strain A1 to sorb heavy metals from solution. Cells of Thiothrix strain A1 were harvested, washed, and suspended in solutions of metals. After an equilibration period, biomass was separated from solution and the metal content in acid-digested cells and/or filtrates was determined by atomic absorption spectrophotometry. Sorption of nickel and zinc was very rapid; most of the sorbed metal was bound in less than 10 min. The sorption data for copper fit the Freundlich isotherm, and nickel and zinc data fit biphasic Freundlich isotherms. Sorption of both nickel and zinc was dependent on cell age. Cells harvested 24 h after inoculation sorbed approximately one-half of the amount of metal per gram cell protein than did cells harvested after 48, 72, or 96 h. Calcium and magnesium effectively competed with zinc for binding sites, whereas potassium had only a slight effect on the capacity of cells to sorb zinc. The primary mechanism of metal sorption apparently was ion exchange, because 66 to 75% of nickel or zinc could be desorbed by placing metal-laden cells in a solution of 5 mM CaCl₂. A competition experiment with nickel and zinc indicated that both metals occupied the same sorption sites. The strong chelating agents EDTA and NTA effectively prevented metal uptake, but lactate enhanced the uptake of nickel. Thiothrix strain A1 grown in nickel-containing medium had a relatively low uptake of nickel compared with uptake by resting cells suspended in a simple buffer solution.     

Origin of the short circuit decay profile and maintenance of the cation transport capacity of the larval lepidopteran midgut in vitro and in vivo

The larval midguts of Hyalophora cecropia and Manduca sexta contain two primary cell types: a columnar cell and a goblet cell. Employing scanning electron microscopy, goblet cells were found to contain within their cavities semi-viscous matrix plugs. Massive release or removal of goblet matrix plugs is noted following a variety of physiological insults to the tissues, including stretching, gaseous carbon dioxide anesthesia, gut evacuation, gut excision in the absence of cold anesthesia, and mounting the tissue in a chamber designed for the study of cation transport. A reduction in the capacity to actively transport cations in vitro or in vivo follows each of these treatments.When a current (short-circuit current = I_SC) is imposed across the isolated larval midgut that is equal but opposite in direction to the natural electromotive force generated by the tissue, a characteristic irreversible I_SC (and potential = P.D.) decay profile is obtained. This decay profile normally consists of three phases : a transient increase in I_SC, a rapid decay in I_SC and a slower but continuous decay in I_SC. The transient increase in I_SC is an artifact associated with anoxia. The duration of this transient increase in I_SC is related to elapsed time between mounting the midgut in a chamber designed for measuring I_SC and the time at which the hemolymph side of the tissue is bathed in oxygenated saline. The rapid decay is associated with massive release of matrix plugs, an increase in membrane K⁺ conductance and a reduced capacity to transport K⁺. The slower decay is associated with further loss of plugs coupled with cell death. Cell death is caused by inadequacies in the saline normally employed to bathe the midgut epithelium in vitro. These inadequacies promote tissue histolysis and disruption of the normal epithelial topology.     

Photosensitization of skin fibroblasts and HeLa cells by three chlorin derivatives: Role of chemical structure and delivery vehicle

The chemical nature of the sensitizer and its selective uptake by malignant cells are decisive to choose an appropriate biocompatible carrier, able to preserve the photosensitizing characteristics of the dye. In this paper we demonstrate the photodynamic properties of three chlorins, derived from chlorophyll a, and the usefulness of liposomal carriers to design pharmaceutical formulations. The chlorins have been quantitatively incorporated into stable liposomes obtained from a mixture of l-α-palmitoyloleoylphosphatidylcholine and l-α-dioleoylphosphatidylserine in a 13.5:1.5 molar ratio (POPC/OOPS-liposomes). The chlorin uptake by skin fibroblasts increases steadily, reaching in all cases a plateau level dependent on both the chlorin structure and the vehicle employed. The photophysical properties of the three chlorins in THF are nearly identical and fulfill the requirements for a PDT photosensitizer. Incorporation of chlorins into liposomes induces important changes in their photophysics, but does not impair their cellular uptake or their cell photosensitization ability. In fact we observe in the cells the same photophysical behavior as in THF solution. Specifically, we demonstrate, by recording the near-IR phosphorescence of ¹O₂, that the chlorins are able to photosensitize the production of ¹O₂ in the cell membrane. The cell-photosensitization efficiency depended on the chlorin and cell line nature, the carrier, and the length of pre-incubation and post-irradiation periods. The high photodynamic activity of chlorin-loaded liposomes and the possibility to design liposomal carriers to achieve a specific target site favors this approach to obtain an eventual pharmaceutical formulation.     

THE KINETICS OF OSMOTIC SWELLING IN LIVING CELLS

The rate of swelling of unfertilized sea urchin eggs in hypotonic sea water was investigated. Analysis of curves leads to the following conclusions. 1. The rate of swelling follows the equation, See PDF for Equation where V _eq., V ₀, and V_t stand for volume at equilibrium, at first instant, and at time t, respectively, the other symbols having their usual significance. This equation is found to hold over a wide range of temperatures and osmotic pressures. This relation is the one expected in a diffusion process. 2. The rate of swelling is found to have a high temperature coefficient (Q ₁₀ = 2 to 3, or µ = 13,000 to 19,000). This deviation from the usual effect of temperature on diffusion processes is thought to be associated with changes in cell permeability to water. The possible influence of changes in viscosity is discussed. 3. The lower the osmotic pressure of the solution, the longer it takes for swelling of the cell. Thus at 15° in 80 per cent sea water, the velocity constant has a value of 0.072, in 20 per cent sea water, of 0.006.     

Effects of spinal or general anesthesia on F₂-isoprostanes and isofurans during ischemia/reperfusion of the leg in patients undergoing knee replacement surgery

General and spinal anesthesia are used extensively in orthopedic surgery. However, these methods of anesthesia may result in different amounts of oxygen being delivered to the patient. Ischemia/reperfusion injury after release of the tourniquet initiates free radical-mediated oxidative stress. F₂-isoprostanes are reliable markers of in vivo lipid peroxidation. However, under conditions of high oxygen tension, isofurans are formed. We aimed to compare plasma isofurans and F₂-isoprostanes in spinal versus general anesthesia in patients undergoing knee-replacement surgery in a randomized, blinded study. Thirty-nine patients were randomized to spinal (SA; n = 19) or general anesthesia (GA; n = 20). Blood was collected before anesthesia, and a tourniquet was then applied to the limb during surgery. After release of the tourniquet, blood samples were collected at 30 min, 2 h, and 24 h for measurement of plasma F₂-isoprostanes and isofurans by gas chromatography–mass spectrometry. The two groups were comparable in age and body mass index. Plasma F₂-isoprostanes were significantly lower in the GA patients compared with the SA patients (p = 0.045). In contrast, the GA patients had significantly elevated plasma isofurans (p = 0.032). Increased isofurans during GA compared with SA are likely to reflect increased oxidative stress due to elevated oxygen concentrations during GA. Further studies are required to assess the implications of these findings on perioperative outcomes.     

Electrical potential differences in cells of barley roots and their relation to ion uptake

Single cell electropotentials of barley (Hordeum vulgare L., cv. `Compana') root cortex were measured at different external concentrations of KCl in the presence of Ca²⁺. The roots were low in salt from seedlings grown on 0.5 mm aerated CaSO₄ solution. Thus, the conditions were equivalent to those used to define the dual mechanisms found with radioactive tracer-labeled ion uptake. In 0.5 mm CaSO₄ alone, there is an increase with time of cell negativity from about -65 millivolts 15 minutes after cutting segments to about -185 millivolts in 6 to 8 hours. Two possible hypotheses, not mutually exclusive, are offered to explain this aging effect: that cutting exposes plasmodesmata which are leaky initially but which seal in time, and that some internal factors, e.g., hormones diffusing from the apex, have a regulatory effect on the cell potential, an influence which becomes dissipated in isolated segments and permits the development of a higher potential difference. In any case changes in selective ion transport must be involved. The cell potentials at KCl concentrations above 2.0 mm are more negative than would be expected for a passive diffusion potential. It is suggested that this discrepancy may be due to an electrogenic pump or to a higher K⁺ concentration in the cytoplasm than in the remainder of the cell, or perhaps to both. Whether there is a clear relationship between cell potential and mechanisms 1 and 2 of cation transport depends upon whether the cell potentials of freshly cut or of aged tissue represent the values relevant to intact roots.     

Calcium effect on the mycelial cell walls of Botrytis cinerea

The goal of this study was to determine whether calcium ion, (one of the electrolytes released after plant cell attack), may have a direct effect on fungal growth and chemistry of the fungal cell wall. B. cinerea was grown on Richard's solution containing different amounts of CaCl₂, and the cell walls were extracted from the mycelium after 7 days of growth. Mineral, neutral and aminosugar, protein and uronic acid contents were determined. At 1 g l⁻¹ CaCl₂, only the aminosugar content increased. At 2 g l⁻¹ CaCl₂, neutral sugar synthesis was reduced, whereas the uronic acid content increased. For higher CaCl₂ concentrations, the calcium ion content of the cell wall increased, resulting in reduced protein and neutral sugar contents. Meanwhile, the cell wall proportion of the mycelia increased on a dry weight basis due to an increase in uronic acid, Ca, P, Na and neutral sugar contents of the cell wall with increasing CaCl₂ in the media. The resulting thickening of the fungal cell wall caused by calcium ion may be an important factor in the host-pathogen relationship.     

The significance of the methods of stigmatal and placental pollinationin vitro inAntirrhinum majus L.; seed and callus formation on placentae

A study was made on certain problems connected with the application of methods of stigmatal and placental pollinationin vitro in the snapdragon. Germinable pollen without microbial contamination could be obtained only from the flowers which were left, after surface sterilization, on the stem till the dehiscence of anthers. The germination of pollen, and especially the growth of pollen tubes was better in a 0.3 M lactose solution with 10^?3 per cent H₃BO₃ than in usual sucrose media. The seedsin vitro could be obtained only after normal pollination and the successive artificial cultivation of the entire pistil (the method of stigmatal pollinationin vitro). The seeds did not differ either in their morphology or in their size from those developed under natural conditions, they were viable and without dormancy. Though the pollinated pistils were cultivated in the medium without growth substances, callus formation from uncovered ovules and placenta was observed in some cases. A dependence was revealed of the proliferation on pollination and on the cultivar employed. When using the method of placental pollinationin vitro we failed to obtain seeds. The main difficulty in the use of this method seems to consist in the fact that pollen tubes are not capable of growing from the surface of ovules and placenta into the micropyle.