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Auxins and auxin inhibitors from tissue extracts of normal Nicotiana plants, Nicotiana glavca, N. langsdorffii and their hybrid (which spontaneously produces tumors) were separated by ascending paper chromatography with n-butanol-distilled water. An Avena curvature test was used for demonstrating growth-promoting and growth-inhibiting substances. IAA could be found in extracts of the parents and the hybrid (RF 0.75). Hybrid tissue yielded the highest amount (37.10), N. glauca tissue less (30.80), and N. langsdorffii tissue the least amount (8.50) of IAA. A second growth promoter (RF 0.35) could be separated from the tissue extracts of the parents and the hybrid, but it showed only low activity in the Avena test. Three inhibitors were present in extracts from N. langsdorffii and the hybrid at RF 0.25, 0.45, and 0.85, whereas N. glauca showed only two of them (RF 0.25 and 0.85). The inhibitor with an RF of 0.45 seemed to be identical with the acidic, benzene-insoluble "inhibitor i" of Bennet-Clark and Kefford (1953). The inhibitor (neutral, benzene-soluble) at RF 0.85 could be found in some tissue extracts of the parents and the hybrid, but showed only little activity in the curvature tests. From neutral and from acidic plant extracts within a pH range of 4.4 to 5.8 a third inhibitor with an RF of 0.25 could be separated. It seems that the high concentration of natural IAA in the hybrid is regulated by a variety of inhibitors with different specificities in the growth-regulating process. Nicotiana langsdorffii tissue has much less auxin but the same variety of inhibitors as the hybrid, whereas N. glauca tissue contains less auxin than the hybrid and only two of the three inhibitors found in N. langsdorffli and hybrid

IN CERTAIN. sections of Louisiana after the backwater from the Atchafalaya and Ouchita Rivers receded this past spring (1937), an unusual deposit of thick, cobwebby, or, in many cases, paper-like material was seen hanging from weeds and other vegetation in the areas which had been flooded. The amount of this deposit was variable in different locations. In one section just north of Jonesville, Louisiana, the deposit was so great as to attract unusual attention. In this section the paper-like deposit not only clung to vegetation, but covered the ground and fences, making certain fields appear as though they were blanketed with snow (fig. 1-3). The inhabitants in the vicinity where the accumulation was abundant were so attracted by the phenomenon that a sample of the material was sent in on April 17, 1937, to the Botany Department, Louisiana State IUniversity, for identification. Microscopic examination of the white paper-like tissue revealed that it was composed of unbranched filaments interwoven very similarly to the fibers of lens paper (fig. 4). These fila'ments appeared to be of algal origin, but since no cell contents (protoplasts) were present, definite determination could not be made. In order to make a positive identification, the writer made a trip to Jonesville. In several places along the roadside en route the material was noted-first, where the backwater from the Atchafalaya River had receded sou-th of Melville and again along Little River east of Pollock. Just north of Jonesville, where the -greatest display was noted, a study revealed what probably had happened. The particular field in question had been planted in cotton the previous year, and the old stalks, which were three to four feet in height,' were still standing during the inundation. At the peak of the rise the 'water probably was four or five feet in depth over the field and contained a luxuriant growth of free-floating filamentous algae. There was evidence of a flow of water over this area, and apparently as the water p'assed through the branches of cotton stalks, weeds, and wire fences, the algal filaments were

SMITH (1937) uses analysis of variance in the study of stoima number in Phaseolus vulgaris. It is hoped that some criticism of the statistics in this paper will help to clarify their uses and interpretation and to indicate some of the commoner errors in statistical work. In Smith's table 1 an analysis of variance for each of six regions of the leaf is presented. There seems to be no reason for analyzing the regions separately, and the analysis could have been presented as in table 1 of this paper. (For interpretation of table 1 see Snedecor (1937), especially section 10.12.) Other points concerning Smith's table are (1) the eighth analysis does not add; (2) one fifth of the total sum of squares for the seventh analysis should be the sum of squares between pieces in the eighth analysis, but these figures are 75.7 and 74.6, respectively, a poor agreement; (3) adding the sum of squares within pieces for the six regions gives 168.8 and should coincide with the sum of squares within pieces in the eighth analysis, but the figure given is 179.0; (4) from the means of regions given in the text, the sum of squares between regiolns is 56.6, but 56.3 appears in the seventh analysis; (5) though the mean squares are given to two decimals, the second decimal is incorrect in many of them, as may be expected from the preponderance of ciphers in the second decimal; and (6) the last 5 per cent F should be approximately 1.52.

RECENTLY MORELAND (1937) described an instance of the formation of natural paper by the matting and drying of algal filaments, mostly Tribonema, in fields inundated by the overflowing of the Atchafalaya and Ouchita Rivers in Louisiana. This spring two similar cases of the formation of natural paper from algal remains were discovered in temporary marshes, and in both instances the paper was made up predominantly of the alga Tribonema.2 On May 7, at the intersection of Crystal Street and Harrison Avenue in Harrison, New York, I came upon a swamp in which Tribonemna was grow

It is still an open question whether or not wood placed in contact with alkaline soils has a longer or a shorter life than wood placed in contact with ordinary soils. Since there are enormous areas throughout the West in which the soil is more or less alkaline, and since relatively large amounts of wood in the form of railroad ties, fence posts, telegraph posts, wood pipe, flumes, etc., are continually in contact with such soil, the question is one of considerable importance. The action of alkaline soils upon wood may be either direct or indirect. By a direct effect is meant the chemical action of the salts composing the soil "alkali " upon the wood. The only salt found in alkaline soils that need be considered in this connection is sodium carbonate or sodium bicarbonate. Sodium chlorid and sodium sulphate, the other important component salts of soil "alkali," have no important chemical effect upon wood substance or structure. The action of any strong base upon wood is well known, but whether or not the concentration of sodium carbonate or bicarbonate ever becomes great enough in an alkaline soil to injure chemically wood in contact with it is more or less a matter of conjecture, although there seems to be a priori some evidence in favor of the affirmative side of this question. It is obvious that, if wood that is saturated with the soil solution of any alkaline soil containing sodium carbonate or sodium bicarbonate in considerable amounts should gradually dry out, the concentration of the salt would gradually increase with the evaporation of the solvent until the solution became quite concentrated. It is conceivable that a constant repetition of this process may have an ultimate effect on the wood. It is not, however, the purpose of this paper to consider the direct effect of alkaline soils upon wood placed in contact with them. The present paper considers only the indirect effect of the salts making the "alkali" of alkaline soils upon wood. By an indirect effect is meant

Freshly excised Pelargonium pith tissue lacks peroxidase activity toward guaiacol or benzidine, but it develops such activity within 24-36 hr in aseptic culture. All the activity is manifested as a single enzyme moving toward the cathode during electrophoresis on starch gel at pH 9.0. This development of peroxidase activity is at first (up to ca. 50 hr in culture) inhibited and later (ca. 100-150 hr in culture) promoted by IAA. This dual effect of IAA resembles that previously reported for specific isoperoxidases in tobacco pith cells. Kinetin alone also inhibits peroxidase formation, but in the presence of IAA those concentrations which enhance growth enhance peroxidase formation as well. RECENTLY it has been shown that the various organs of peas (Ockerse, Siegel, and Galston, 1966; Siegel and Galston, 1967) and tobacco (Galston, Lavee, and Siegel, in press) contain distinctive patterns of electrophoretically separable isoperoxidases, and that auxin can both enhance and inhibit the formation of specific members of the isozyme complex. The results with cultured tobacco pith were particularly instructive, since these initially homogeneous cells can be caused to differentiate into lignified cells, roots and shoots (Skoog and Miller, 1957), and the development of particular isoperoxidases is frequently correlated with the new growth and differentiation patterns. Since similar morphogenetic changes were recently elicited in cultured Pelargonium pith (Chen and Galston, 1967), experiments were undertaken to determine the pattern of peroxidase activity in this tissue, and its changes under various culture conditions. The results are detailed below. M\ETHODs-Plant material-Shoots of greenhouse-grown Pelargonium 'Nittany Lion' were excised at the base and the pith removed aseptically from the middle 10 cm of stem by means of a sterile no. 1 cork borer (diam 2.5 mm). The excised cylinders were sliced transversely every 2-3 mm before implantation on the medium. In vitro cultures-A slightly modified semisolid White's medium (21-MW of Lavee, 1960) was used throughout. The cultures were initiated by explants weighing ca. 25 mg and were maintained for 6 weeks at 25 C in ca. 100 ft-c white fluorescent light in tubes containing 10 ml of medium to which were added 2 mg/liter of an I Received for publication 24 November 1967. 2 Permanent address: The Volcani Institute of Agricultural Research, P.O. Box 15, Rehovot, Israel. appropriate auxin, such as indole-3-acetic acid (IAA) or a-naphthaleneacetic acid (NAA) and, where appropriate, 0.2 mg/liter kinetin. Peroxidase activity-Sap was expressed from ca. 20 mg of tissue by squeezing the tissue directly onto a porcelain plate with a spatula. Three 5-mm2 pieces of 3 MM Whatman filter paper were then used to absorb the expressed sap. Each square, containing 6,u liter sap, was separately analyzed. Total peroxidase was determined by eluting the enzyme from one such filter paper square in 1 ml 0.1 M pH 5.8 phosphate buffer at room temperature for 2 hr, then adding 3 ml of 5 mm guaiacol and H202 (Galston et al., in press) in phosphate buffer. Optical density readings at 470 nm, taken at 15to 60-sec intervals for 3-10 -min in a Bausch and Lomb Spectronic-20 colorimeter, indicated the course of the reaction. Results are expressed as A OD/min. For electrophoretic separation and detection of isoperoxidases, one filter paper square was introduced into the middle of an 18 X 14 X 0.6cm starch-gel plate, prepared as previously described (Siegel and Galston, 1967), and containing NaOHborate buffer at pH 9.0. The electrophoretic separation proceeded for 70 min in a gradient of 1OV/cm. To visualize the separated isoperoxidases, the gels were sliced transversely and each half developed by flooding with guaiacol-H202 reagent. The resulting brown spots were photographed for the permanent records, and the intensity of the reaction estimated visually directly from the stained gel. Three replicate cultures were sampled for each value; all experiments were repeated at least three times and all yielded similar results. Data of representative experiments were chosen for presentation. RESULTS-Pith segments removed at 6-cm

The relation between the aerobic and the anaerobic production of carbon dioxid has been the subject of much discussion and is still far from settled. It would seem that the problem can be attacked by comparing the aerobic and the anaerobic behavior of the same organism under a variety of conditions which affect respiration. If the aerobic and the anaerobic methods of production of carbon dioxid are essentially alike, they should vary in similar fashion when subjected to such influences. But if they are fundamentally different, this fact should become clear when a sufficient variety of conditions has been studied. No quantitative studies of this kind have yet been made, so far as the writer is aware, with the exception of a recent paper by Lyon.' The present paper describes a quantitative investigation of the effect of certain anesthetics upon the production of carbon dioxid by wheat. No attempt has been made to investigate the subject in all its bearings, since the purpose was merely to make certain exploratory experiments in order to discover whether any striking differences exist in the behavior toward reagents under aierobic and anaerobic conditions. The method employed is that described by Osterhout.2 In the anaerobic experiments the oxygen was driven out of the apparatus as far as possible by means of a current of nitrogen (from a cylinder of compressed nitrogen). Before entering the apparatus it passed in succession through a series of towers containing (NH4)2C03, conqentrated NH40H plus copper gauze, and dilute H2S04. The current of nitrogen was allowed to flow through the apparatus for half an hour before starting the experiment. A bottle of alkaline pyrogallol was placed in the apparatus; through this the circulating gas constantly bubbled. If it became so dark as to indicate leakage of oxygen from outside, the experiment was rejected. Not infrequently there was some darkening during the course of an experiment, indicating the presence of traces of oxygen, which, however, were so promptly removed by the pyrogallol that they did not appear to interfere with the results.3

Despite the potential colntribution suggested from chromatographic studies of many groups, taxonomic conclusions based on paper chromatography alone may carry some inherent errors. In many instances chemical differences between taxa cannot be characterized by two-dimensional chromatography of crude extracts. Pigment diversity may go unnoticed in such a study, and conclusions based on simple chromatographic results may show too much similarity between the taxa being compared. The resolution of chromatographic separations of the often very complex mixtures encountered in crude plant extracts can be greatly improved by a preliminary column separation, and in this study a method of pigment analysis employing both preliminary column and subsequent paper chromatography is used. While it is usually impractical to identify all of the components of a flavonoid complex, each member should be characterized to the degree that it can be distinguished from all others. Here, the characterization of a component includes its rate of movement in the column, relative position and color under ultraviolet light on the chromatogram, the absorption spectrum in ethanol and the change in the absorption spectrum when a reagent is added to the ethanol which has a particular effect on the spectral properties of that flavonoid. With very few exceptions no one criterion of characterization is definitive for any component. More than 20 flavonoid pigments have been observed in the two tetraploid Gossypium species, G. barbadense and G. hirsutum. Only 10 of the constituents were common to the two species. The results of a previous study based on two-way paper chromatography of crude extracts makes the two species appear much more similar in flavonoid composition than is indicated by the more detailed column-paper chromatography presented here. The degree of difference between G. hirsutum and G. barbadense is surprisingly large in light of the generally accepted theory of their common origin. The value of chromatographic-chemotaxonomic studies is greatly increased if the chemical systems involved are characterized.

Very little has been published on the shore vegetation of lakes in Colorado. Brief references were made to lakes of the Pike's Peak region a number of years ago by Clements (I, 2). A somewhat extended account by the writer and W. W. Robbins (9) described the associations at Redrock Lake, Boulder County, Colorado, in the subalpine zone. Later, a short paper (5) pointed out certain features of shore vegetation in the montane zone. Recently Dr. Robbins has given a most careful and illuminating description (ii) of a number of lakes in the montane zone near Tolland, Colorado. The present writer, dealing with sedges of northern Colorado, has named and characterized (7) certain of the plant associations of lake shores at different altitudes. In a paper by Dodds (3) on the plankton crustacea of Colorado lakes there are some references to vegetation and a very good account of physiography and climate. The following pages give the results of a study of subalpine lakes, the study carried on chiefly from the University of Colorado Mountain Laboratory (8) at Tolland, Colorado, during the last ten years. A later paper will take up alpine lakes. PHYSIOGRAPHY

The seeds were sterilized by immersion in a 0.25-percent solution of Uspulun for one hour, and were placed in germinators between layers of moist filter paper April 24 and were planted in sterilized quartz sand (No. 3) in 7-inch bulb pots April 26 (i). There were four cultures of each of three types of wheat. Two of each of the three sets of cultures were given the nutrient solution containing nitrates, and two of each set the solution lacking nitrates. The cultures were placed in the bell jar apparatus described in the preceding paper (2) and were sealed air tight with DeKhotinsky cement. One of each of the pairs of cultures was given approximately 0.4 percent carbon dioxid in the incoming air and the other was placed in a chamber receiving air lacking carbon dioxid. A reduced pressure equal to 0.004 atmospheres was maintained for all of the cultures. Fresh nutrient solutions were given every third day and the moisture content of the sand was maintained during intervening times by the addition of freshly distilled water. The cultures were removed from the apparatus May 6 and were immediately placed in darkened moist chambers until the seedlings could be weighed and measured. The temperature and light conditions were both highly favorable for rapid growth. The days were all clear except two which were only partly cloudy.

STAFFORD, HELEN A. (Reed Coll., Portland, Oregon.) Distribution of tartaric acid in the leaves of certain angiosperms. Amer. Jour. Bot. 46(5): 347-352. 1959.-An optically active isomer of tartaric acid has been definitely identified and quantitatively analyzed in the leaves of 9 species of angiosperms, and trace amounts have been tentatively identified in about 22 others out of 49 species examined. In species where identification was positive, the quantity of tartrate varied from 5 to 200 gmoles/g. fresh wt. of leaf tissue; identification was based on paper chromatography and on the metavanadate colorimetric test. The 9 species include Vitis vinifera, V. labruscana, V. californica, Parthenocissus tricuspidata, P. quinquefolia, Pelargonium hortorum, Bauhinia malabarica, Phaseolus vulgaris, and Coleus blumei. Optical rotation data of the isolated acid indicate that the tartrate in Pelargonium and Parthenocissus quinquefolia is the (+) -isomer similar to that reported for Vitis vinifera, but the opposite to that reported for Bauhinia reticulata. The relationship of tartrate to other organic acids is discussed. WHILE MALIC ACID is generally the dominant organic acid that accumulates in the cells of higher plants, occasionally tartaric, citric, or oxalic acid is found in as great or even greater amounts (Pierce and Appelman, 1943). Although tartaric acid has been reported to be present in numerous species (Buchs, 1957), the large majority of older identifications are questionable, and actual quantitative data have been lacking except in a few isolated instances (Franzen and Helwert, 1923; Thimann and Bonner, 1950; Vickery and Palmer, 1954). Furthermore, since the work of Pasteur (1848) on the isomers of tartrate, the optical activity of the tartrate isolated from plants rarely has been checked. One exception is the confirmation of the presence of the (+) -isomer in wine made from the fruits of Vitis vinifera, and the first report of large quantities of the opposite or (-)-isomer in both fruits and leaves of Bauhinia reticulata (Rabate and Gourevitch, 1938). So far, the mesoisomer never has been identified in any plant tissue, although fungi appear to possess enzymes capable of converting epoxysuccinate to mesotartrate (Martin and Foster, 1955). Since some of the isomers of tartrate are active in cell-free enzyme systems isolated from higher plants (Stafford, 1957), the present investigation to restudy the problem of the distribution of tartrate was undertaken, based on more rigid criteria to establish its presence. Leaves, rather than fruits, were analyzed because they are more suitable for research on cell-free enzymes in plants with low pH values, and because there is some indication that the major site of the origin of tartrate is in the leaf (Amerine, 1956). Identification of tartaric acid was considered positive only if all 3 of the following criteria were 1 Received for publication October 27, 1958. This investigation was supported by a grant from the National Science Foundation (NSF-G4651). The author is indebted to Miss Maryanna Pearson for her assistance throughout this work. met: (1) the identification by paper chromatography of an acid spot at the expected Rf value; (2) the detection of an orange spot at the same Rf value after spraying with a solution of sodium metavanadate; (3) the isolation of sufficient amounts for a quantitative colorimetric analysis based on the metavanadate method. This test is relatively specific for either the (+) or (-)-isomer but will not distinguish between them and will not detect mesotartrate. MATERIALS AND METHODS. Leaves were obtained either from greenhouse or native sources. In a few cases, as indicated, air-dried specimens were used and the values for dry wt. were converted to fresh wt. by arbitrarily assuming that the dry wt. was 15% of the fresh wt. Fresh samples were either extracted immediately upon picking or were first frozen for storage. Rootstocks of grape plants were supplied by Dr. C. J. Alley of the Department of Viticulture at the University of California, Davis, California, while seeds of Bauhinia malabarica were made available by Dr. R. B. Ledin of the University of Florida Sub-tropical Experiment Station. About 3 g. fresh wt. of leaves were used as the starting material for extraction. The leaves were ground in a small Waring blendor in hot 50% ethyl alcohol, and Dowex 50 (H+) was added to decationize the sample. After centrifugation, the supernatant and washings of the residue were evaporated to dryness under reduced pressure at 50?C. Only rarely can the crude extract be used directly in the quantitative analysis based on the metavanadate test; generally a partial purification is necessary. The residue was taken up in about 7 ml. of water and suitable aliquots (0.4-3.0 ml.) were applied as a 30-cm.-long streak to filter paper and were chromatographed in an ether-formic acid-water mixture (5:2:1 v/v). A small strip 0.5 cm. wide was cut from the center of the paper and sprayed with a plh indicator (0.1%o bromcresol green) to detect the position of the acids. Afterwards, a second

It has long been known that the rate of transpiration from the leaves of different species of plants is not the same, even if the plants are growing under identical environmental conditions. Obviously such variations in the rate of transpirational water loss must be due to conditions and factors which are internal in the leaf. The relative resistance of the leaves of a species to water-vapor loss may be an important factor in determining the water relations of that plant, i.e., whether it is a mesophyte, a hydrophyte, or a xerophyte. It has been assumed that a plant the leaves of which were relatively resistant to water-vapor loss was better fitted to withstand conditions of diminished water supply than one with leaves not so resistant. It is doubtful whether the ability of a plant to conserve water in its leaves under conditions adverse to the maintenance of a favorable water balance may be accepted as a universal criterion of relative xerophytism, but this is probably a factor. A simple, rapid, and reasonably accurate method for measuring the rate of water-vapor loss from leaves, especially if it be adapted to field use, is a necessity in many studies of physiological ecology. The cobalt chlorid paper method first devised by Stahl (7), and considerably improved by later workers, has been developed upon a quantitative principle which appears to give satisfactory results in field measurements of the rate of water-vapor loss from leaves. The general principle of this method is well known. Absorbent paper, such as filter paper, is soaked in a dilute solution of cobalt chlorid and dried. When dry this paper is blue in color, but upon exposure to moist air the cobalt chlorid rapidly takes up water vapor and becomes hydrated. This hydration is accompanied by a change in the color of the paper from blue to a pale pink. If a small piece of this cobalt chlorid paper is applied to a leaf surface, and protected from atmospheric moisture by glass or some other transparent substance, the time required for the paper to change from blue to pink is a measure of the rate at which that leaf surface is losing water vapor to the paper. Livingston and his associates (3, 4) introduced a number of improvements in this method of determining the rate of water-vapor loss from leaves. For the details of their methods the original papers should be consulted. A standard evaporating surface was used by these workers as a basis for comparative measurements. A free water surface was first used as a standard

were tested in various controlled atmospheres of He, N2, and CO2. Atmospheres of 16.7 %, 33.3 %, and 88.9 % of each of these gases (thus reducing 02 concentrations to 17.5 %, 14.0 % and 2.3 % respectively) were introduced into 1000-ml stoppered vacuum flasks, each containing 100 seeds placed on substrates of either sterilized soil or filter paper. The flasks were placed in growth chambers with an 8-hr light period at 20 C and a 16-hr dark period at 5 C. The germination rate was accelerated directly with increased concentrations of He and N2 but was retarded by increased percentages of CO,. Control flasks eventually reached nearly the same percentages of germination as those of He and N2 but at a later point in time. THE ANNUAL GRASS Heleochloa alopecuroides (Pill and Mitterp) Host is characteristic of the dry phase of periodically inundated areas. This grass is important as waterfowl food but has little other economic value. Perhaps for that reason little is known of its ecology. Distribution records are incomplete in the United States for this introduced species, which is native to the Mediterranean region (Lorch, 1962). The goal of this study was to determine what role 02 and CO2 concentrations play in Heleochloa germination. An attempt was made to reduce 02 concentrations on some seeds and to increase CO2 concentrations on other seeds. Both He and N, were used to reduce

A B S T R A C T The quantitative composition of major sugars in the nectars of 35 plant species was compared with that of 12 fruit juices. The use of specific quantitative assays for glucose, fructose, and sucrose made it possible to analyze large samples without quantitative chromatographic separations. Sucrose ranged from 0-99%. In most nectars, glucose and fructose were present in approximately equal amounts. No evidence was found for the presence of maltose, raffinose, and other oligosaccharides. LITTLE INFORMATION is available on the sugar composition of nectars. This may be due to the small size of the samples usually available and the lack of specific microanalytical techniques. Wykes (1952) used paper chromatography to separate the main sugars from nectars. Fructose and glucose were eluted from the paper and quantitated by reduction of copper. Maltose was claimed to be present in a few samples. The nonreducing sucrose was not determined. Furgala, Gochnauer, and Holdaway (1958) used a similar method to separate the sugars of some northern clover nectars but assayed the eluates with a phenol sulfuric acid reagent, which reacts also with sucrose. Their results suggest that maltose comprises 3-10 % of the total sugars present. Percival (1961) separated a large number of nectars by paper chromatography and estimated the preponderance of glucose, fructose, and sucrose after spraying with anisidine. Small amounts of material with RF values corresponding with maltose, melibiose, or raffinose were found in several species, but these oligosaccharides were not further

THE PRESENT communication is a sequel to a previous paper (Steward et al., 1954) which described an investigation, by quantitative paper chromatography, of the nitrogen compounds which, when free, comprise the soluble nitrogen fraction and, when combined, the protein nitrogen of certain shoot apices. The earlier investigation revealed general effects of two kinds. First, the angiosperm shoot apices which were investigated differed greatly from the fern apex studied with respect to the range of soluble amino-nitrogen compounds that they contained. Second, there were evident signs that differences in composition in these apices occurred pari passu with development. The data here reported extend our knowledge with respect to Adianlum pedatum. The shoot apex of Adiantum as earlier reported was somewhat remarkable in contrast to that of Lupinus in that the alcohol soluble nitrogen consisted of relatively few identifiable amino acids and amides present in quantity (aspartic, glutamic, /aminobutyric acids, valine, leucine and glutamine) and a substance, not identified, which was present in far greater quantity than any of these. The reexamination of the apex of Adiantum had the following objectives: (i) To investigate the apex at a time when it was most actively growing. (ii) To determine the composition of various regions of the apex which were sampled according to their state of maturity. (iii) To shed further light on the chemical nature of the hitherto unidentified nitrogen compound that occurs in the Adiantum apex.

IN A PREVIOUS paper (Phillips, 1947), the author showed that barley seedlings and older barley plants produce considerably more CO2 than alcohol under anaerobic conditions. The "extra" CO2 is in part due to a release of CO2 bound in bicarbonates, but the origin of much of it is unexplained. In the present study, the rate of carbohydrate loss during an anaerobic period was measured and compared with the alcohol and CO2 production in an attempt to determine the origin of the extra CO2. MATERIALS AND METHODS. Roots of barley plants 2-4 weeks old were used as experimental material. The roots were not sterile, but experiments lasted only 4 hr., and bacterial contamination did not appear to be serious. For a nutrient solution, Lake Mendota water was buffered to pH 5.0 with KH2PO4. This water is high in most mineral nutrients and supported good growth for 4 weeks. The plants were grown in pyrex crystallizing dishes 17 cm. in diameter and 9 cm. in depth, with mosquito netting fastened across the top on which the seeds were placed. The dishes were covered with a bell j ar until the roots reached the nutrient solution, then uncovered and the solution aerated vigorously through capillary tubes. After about 3 days the plants were transferred from a darkroom to the greenhouse, and aeration continued. The mat of roots weighed about 30 g. at the end of 2 weeks. Experimental procedure.-The roots were clipped just below the netting, washed well, dried gently on filter paper, weighed into 10-20 g. samples, and placed with 200 ml. of distilled water in 500 ml. wash bottles with standard taper joints. Wash bottle heads were inserted, and a stream of pure N2 was passed through the flask at a rapid rate. The N2 was freed of 02 and CO2 by the method of Peters and Van Slyke (1932). The gas from one flask was passed through an absorber containing alkali for CO2 determinations. At the end of 4 hr., the roots were removed and dried in a vacuum oven at 80 ?C. for sugar analyses, or phospho-tungstic acid was added as a killing agent and the contents of the flask distilled for alcohol analysis. Control sets of roots were killed in a similar manner at the beginning of each experiment. For experiments in the Warburg respirometer, the roots were cut into lengths of about 1 in., mixed well, dried on filter paper, and weighed into samples of proper size. Analyses.-For determination of CO2, the gas from one flask of roots was passed through a sintered glass aerator into 100 ml. of 0.05 N NaOH. The carbonate formed was measured by titration to 1Received for publication August 16, 1948. Most of the work reported in this paper was performed in the laboratories of the Department of Botany, Universitv nf Wisnsin. the phenolphthalein and methyl orange end points with 0.05 N HCI. A blank determination was performed in which N2 was bubbled through a flask containing only water, and this value was suttracted in every case. Bound CO2 was released from the tissue by treatment with 10 per cent trichloracetic acid, collected in alkali, and titrated in the same manner. Carbon dioxide production was also measured in a standard Warburg respirometer. The flasks, containing about 0.5 g. of root segments in 2 ml. of distilled water, were flushed with purified N2, closed, and placed in a water bath at 25?C. Carbon dioxide production was calculated from readings on the manometer in the usual manner. Alcohol was determined by the method of Harger (1935) on a solution obtained by distilling 150 ml. from the original flask, adding alkali, and redistilling until 100 ml. were collected. This method includes acetaldehyde in the results. Reducing sugars and sucrose were determined by the method of Hassid (1937). These are combined as "total sugars" and expressed in mM. of glucose. The material was analyzed for starch by the method of Hassid et al. (1940) but the starch content of the roots was below the limits of this method, and insignificant in comparison with the sugars.

It is hardly possible to give in a short paper a convincing account of a theory which must be based on an extensive array of facts, only a part of which (i) have thus far been presented. The problem has lately been much discussed from the ontogenetic point of view (see the exhaustive bibliography in Foster, 7). The morphological and phylogenetic standpoints, however, have been very much neglected. The ontogenetic investigations partly support my theory but more often contradict it. This, however, is of no consequence, as a morphological problem must be solved primarily by comparative morphological investigations as well as by the evolutionary history of plant organs. Even Gliick (9, p. 637-638), a prominent member of Goebel's school, correctly pointed out that ontogenetic facts do not always agree with the true morphological character of the organ. Thus he mentions that the stamens of some plants (Euphorbia, Casuarina, Najas, Typha, Cyclanthera) though they clearly belong ontogenetically to the axis, are undoubtedly metamorphosed phyllomes. Many other cases (see Velenovsky, i i) of a similar nature have been emphasized, and Schr6dinger (io, p. I85-I86) points out that in not less than five genera of the Cunoniaceae the stipules arise " nachweislich echt freiachsenbuirtig, d.h. aus selbstandigen Primordien, fur deren Vorw6lbung im Innern des Sprossgipfels vollkommen selbstandige Zellvermehrungsherde differenziert werden." Hardly any botanist, however, would assume that they do not belong to the respective leaves but that they are organa sui generis. It is hardly necessary to repeat in detail my theory of the phylogenetic differentiation of the phyllome according to which the most primitive form is a one-membered, broadly inserted leaf which in further evolution differentiated into a lower member (the sheath) as the predominantly mechanical organ and the blade as an organ designed for assimilation. In further evolution the auricles of the sheath, which are of rather common occurrence, lengthen while the sheathing part shortens (stipulae adnatae, called by Gluck more appropriately stipulae vaginales); finally, after total disappearance of [The Journal for March (I8: 147-235) was issued March 23, I93I.]

A B S T R A C T Wood samples from Populus deltoides Marsh. were used to investigate the relationship of gelatinous fibers to the size and number of other wood elements. An increase in the amount of gelatinous fibers was r elated to a decrease in diameter and an increase in wall thickness of non-gelatinous fibers. The relative sizes of rays, vessels, and fibers were inter-related, but all of these wood elements decreased in size with an increase in the amount of gelatinous fibers. Apparently environmental conditions controlling the differentiation of gelatinous fibers also influence the development of associated wood elements. Thus, the adverse physical pioperties of reaction wood must be attributed to all of these structural differences in the wood and not merely to the presence of large numbers of gelatinous fibers. THE EASTERN cottonwood (Populus deltoides 1\'Iarsh.) is among the most important of our world poplars for the manufacture of paper pulp, box venieer, shipping containers, and furniture parts. Its light color permits clear printing. Its low weight, resistanice to splitting, and toughness add to its value for making shipping containers. But not all cottonwood meets minimum standards for industrial use. Large numbers of gelatinous fibers are related to fuzzy surfaces, difficult machining, and faulty printing on lumber and veneer. Gelatiilous fibers are also associated with excessive longitudinal warping, some collapse in lumber and the buckling of veneer (Wahlgren, 1957). These fibers apparenitly occur in all cottonwoods but are most abundanit on the upper side of leaning and crooked trees (Kaeiser and Pillow, 1955; Berlyn, 1961). Gelatinous fibers in eastern cottonwood have been studied by a niumber of workers (Jayme, 1951; Kaeiser, 1955; Wahlgren, 1957; Berlyn, 1961), but the authors know of no studies of the relationship of gelatinous fibers to the size and number of other wood elemenits. Using small wood samples from 3 selected trees, the authors inivestigated correlations between the amoulit of gelatinous fibers aiid the size and number of other wood elements. Significant correlations were established between the amount of gelatinous fibers and the size of the wood elements, the iiunber of wood elemenits, and the wall thickness of the non-gelatinous fibers. The results also indicate that environmental conditions controlling the formation of gelatinous fibers also influence the development of the other associated wood elements.

Approximately 1,700 plants representing five species of Tragopogon (Compositae) and their F1 and F2 hybrids were analyzed by two-dimensional descending paper chromatography. Each species, or population within a species, was chromatographically distinct. Often, however, the differences were more quantitative than qualitative. The chromatographic data generally supported the species relationships which had been determined from previous morphologic, hybridization, and fertility studies. Inheritance of the flavonoid compounds was usually additive in the Fl's. Segregation and recombination of the genes controlling the synthesis of these compounds sometimes approximated 3: 1 or 9:7 ratios in the F2's. Occasionally parental compounds were missing from some of the hybrids. "Hybrid" compounds which had not been found in either parent were absent from the F1 but did occur in several F2 populations. Two linkage groups were present. The first contains genes controlling the synthesis of three compounds and the second, four compounds.

UNDERSTANDING the metabolism of the developing maize endosperm is a problem of practical and theoretical importance. Carbohydrate metabolism in the maize endosperm has been investigated extensively by means of various biochemical and genetic techniques (Culpepper and Magoon, 1924, 1927; Bernstein, 1943; Sprague et aL, 1943; Andrew et al., 1944; Cameron, 1947; Kramer and Whistler, 1949), but nitrogen metabolism of the endosperm has remained relatively unexplored. Zeleny (1935) and Watson (1949) have shown that endosperm proteins (largely zein and glutelin) are synthesized during the entire kernel maturation period, but that most of the glutelin (alkali-soluble) proteins are synthesized during the first half, and most of the zein complex of proteins (alcohol-soluble) during the last half of the endosperm maturation period. Biochemical genetics studies (Showalter and Carr, 1922; Frey et al., 1949; Frey, 1951) and analyses of various varieties and genotypes of maize endosperm (Hansen et al., 1945) show that there is a direct correlation between the content of zein and of total protein in the endosperm; this mav indicate that alcohol-soluble proteins are the major storage proteins of the endosperm. Interrelations of tryptophan, niacin and indoleacetic acid in the developing maize endosperm have been investigated by Stehsel and Wildman (1950). Their studies are perhaps the first attempt to explore the nitrogen metabolism of the developing maize endosperm on the level of individual enzymesubstrate-product reactions. It is only on this level that one can investigate adequately the various metabolic processes of the developing endosperm. Thus, a working knowledge of the kinds and amounts of nitrogenous components in the endosperm throughout its development is fundamental to understanding endosperm protein synthesis. This study is a preliminary investigation of the free amino acids and amides of the developing endosperm of four varieties of corn. Partition paper chromatography, an analytical technique proven to be useful for rapid surveys of kinds and approximate amounts of amino acids in other plant tissues (Dent et al., 1947) has been adapted for use in this investigation. MATERIALS AND METHODS.-Four morphologically dissimilar varieties of maize: Longfellow Flint

OBSERVATIONS ON the kinds of fungi found on cotton fibers and cotton cloth are numerous. In many instances, the investigators have been primarily concerned with fungi of the more superficial types growing on the sizing and other non-cellulosic carbon compounds of the fabric. The reports, however, of the fungi responsible for the decomposition of fabric such as awnings and tentage under actual conditions of outdoor exposure when not in contact with the soil, are much more limited (Broughton-Alcock, 1919; Thaysen and Bunker, 1927) and have not been quantitatively evaluated. Techniques employed for isolating organisms supposed to be primarily responsible for the actual decomposition of cellulose in cotton fabric have frequently erred on two major counts. Those methods employing nutrient agars with sugars as a source of carbon favor the rapid growth of any fungus or bacterium suited to that substratum, whether or not it be a cellulose decomposer. The method using filter paper and mineral salts agar while isolating some cellulose decomposers fails to detect many others because it does not take into account the fact that cotton fibers in gray duck supply vitamins B1, B6, and biotin (Robbins and Ma, 1942), as well as other possible nutritional constituents for fungi having special requirements (Robbins and Schmitt, 1945). Gray duck in contrast to bleached duck retains the protoplasmic contents left in the lumina of the natural fibers, as well as fragments of seedcoats, leaves, and the like. Bleaching and scouring largely remove these remnants. The natural cotton fiber, on a moisture-free basis, contains the following percentages of elements expressed as means of several readings (Beeson, 1941): 0.59 per cent K, 0.13 per cent Ca, 0.09 per cent Mg, 0.054 per cent P, 0.05 per cent S. Raw cotton on a moisture-free basis is reported (Marsh and Wood, 1942) to contain 0.2 per cent N, and bleached cotton none. Of the data available on the minor elements, iron and manganese are both present on a moisture-free basis in raw cotton as 190 parts per million. It is presumed, however, that other minor elements are present.

THE HERBICIDAL activity of plant growth substances has been investigated extensively in recent years, but the metabolic basis of herbicidal action remains obscure. The results reported here concern the influence of growth substances on certain respiratory enzymes from bean plants. Parallel studies also have been made in our laboratory on a monocotyledonous plant (Miller and Burris, 1951) to establish whether or not differences exist in their responses that may help to explain the differences in sensitivity to herbicides of diand monocotyledonous plants. An effect upon respiratory enzymes following herbicidal treatment has been suggested by a number of workers. One of the secondary effects of plant growth substances has been described as a depletion of the carbohydrate reserve, which may cause the death of the plant. This response would seem to indicate some change in the respiratory enzymes concerned with carbohydrate metabolism in the plant. Mitchell et al. (1949) have reviewed briefly the literature concerned with the relationship of growth substances and respiratory metabolism of plants. METHODS AND MATERIALS.-Sensation No. 2 yellow wax bean seeds were planted in 10-in. clay pots in pit sand, about twelve seeds per pot. They were supplied at weekly intervals with Hoagland and Arnon's (1938) nutrient solution No. 1 and were watered as necessary with tap water. Threeto 4-week-old plants were used in these experiments. Enzyme preparations having a relatively low rate of respiration without added substrate were made from bean leaves in the following manner: about 20 g. fresh weight of trifoliate leaves from which the petioles had been removed were ground with white silica sand in a glass mortar, and the sap was expressed through a double layer of cheesecloth. The pH of the expressed crude sap was usually 6.2-6.5. The pulp then was reground with 10 ml. of 0.1 M phosphate buffer, pH 8.0, and the liquid again expressed. The sap was adjusted to pH 8.0 with 1 N NaOH and centrifuged for 15 min. at about 560 X G; a better separation of chloroplasts and other cellular debris was obtained by centrifuging the sap at pH 8.0 rather than at pH 6.2-6.5. For certain experiments the centrifuged sediment was worked up with about 10 ml. of 0.1 M phosphate buffer, pif 8.0. These preparations

IN INVESTIGATING the role of zinc, manganese, and copper in plant nutrition, the use of suitable experimental methods for growing plants assumes particular importance. The amounts of these socalled microelements required by plants are so small that they are frequently supplied in considerable part or in toto by the incidental impurities included in the culture medium. A successful demonstration of the indispensability, as well as any study of the quantitative aspects of the importance of microelements in the nutrition of higher plants, depends directly on the removal of contaminants from the culture medium. The purpose of this investigation was to develop an experimental technique which, with the use of ordinary laboratory and greenhouse facilities, would permit the undertaking, on a fairly large scale, of experiments to study the physiological importance of zinc, manganese, and copper in the nutrition of higher plants. The object was attained by using the water culture method and, by proper selection of materials and purification procedures, reducing the contaminations derived from water, chemicals, and containers. The use of very sensitive chemical tests to determine the amounts of contaminants present in the culture medium after purification made it possible to prepare nutrient solutions of equal degrees of purity. It was possible, using this technique, to obtain consistent and reproducible responses in plants from adding minute amounts of metals to the nutrient medium (for example, 1 part of zinc in 200,000,000 parts of culture solution, which amounted to 1 y [0.001 mg.] of zinc to a plant). GENERAL CONSIDERATIONS.-The metal contaminants found in nutrient solutions are derived from three sources: containers, water, and chemicals. Previous investigators (Steinberg, 1919, 1935; Hopkins, 1933), studying the effect of microelements on lower plants, have described successful methods for nutrient solution purification in which metal impurities were removed by precipitation and adsorption at high temperatures and slightly alkaline reactions in the presence of calcium and phosphate ions. They have applied these purification procedures to either the complete nutrient solution (Steinberg, 1919, 1935) or to one divided into two portions (Hopkins, 1933), removing in one or two operations such impurities as were derived from the several nutrient compounds, water, and containers.2

Attention has recently been drawn to the necessity of a physiologically balanced salt solution for water cultures. This necessity arises from the fact that in order to know the effect on organisms of certain single salts or mixtures of salts in solution, one must use a control or check solution, called by True,1 a normal physiological solution. This solution or medium should cause no disturbance of the regular functions of the seedling. Such a culture would afford an ideal standard for comparison and one much to be desired. The usual method, however, has been to use distilled water. That such cultures for biological studies are unsuitable was shown some time ago. In the first place, 'distilled water' is an indefinite term. As soon as the seedling roots are introduced the conductivity of the water rises rapidly, showing that salts are leached from the roots and the water remains no longer pure. Secondly, the extraction of salts, it is affirmed, suggests the means by which pure samples of distilled water exert their harmful effects. Some authors affirm a starvation theory; others, like Loeb,2 the loss of certain necessary ion-proteid compounds; and still others, as True,3 that certain necessary constituents, partly inorganic, are dissociated from their proper attachments in the complicated chemical and physical mechanism of the living cell. Thirdly, the harmfulness of distilled water is attributed by others to the presence in it of toxic substance derived from the apparatus or because of the method of preparation. * Received for publication April I0. 1 True, R. H. Distilled Water in the Laboratory. Science n. ser. 39: 296.

This paper is concerned with the relationship between cell wall orientation in dividing tissues and stress. An examination of the possible orientations of a cell-plate in a cell under axial stress reveals that only one orientation passing through the center of the cell can be completely free from shear stress, thus providing a favored plane for the positioning of an hypothetical, shear-sensitive, cell-plate precursor. From this principle of shear-free partitioning, and the three basic rules of stress behavior along free boundaries, it is possible to predict certain features of cell wall orientation along free edges and epidermal surfaces and in some simple apices. The possible significance of the widespread phenomenon of axillary induction of bud formation is discussed in terms of the generation of stresses at the base of the axillant leaf, and the general efficacy of mechanical stress as a spontaneously arising morphogenetic trigger is considered. IN PREVIOUS PAPERS a correlation was established between cell wall patterns and principal stress trajectories, as studied in the maturing ovule of Gossypium hirsutum L. This was made possible by the accurate description of principal stress trajectories in a suitable plastic model by means of photoelastic stress analysis. The role of the integuments in the ontogeny of the ovule was discussed in terms of providing for the growth of the embryo-sac in a space free from the external compressive stresses generated by the growth of

FUNGAL TAXONOMISTS have known for 100 years that certain ascomycete and basidiomycete fruit bodies contain cells which stain blue-black or purple with iodine. This is the amyloid reaction. In previous papers (McCracken and Dodd, 1971; Dodd and McCracken, 1972) we have described the molecular structure of the amyloid material contained naturally in several basidiomycete genera. As detailed in these papers, amyloidity is a character which can be found in both ascomycetes and basidiomycetes. There are many genera which have amyloid material restricted to specialized portions of their fruit bodies, e.g., basidiospore walls and the apical rings of asci. There are a few basidiomycete fruit bodies, such as those of Hericium and Lentinellus, which contain prominent amyloid tissue. In our laboratory we are currently investigating various aspects of starch production in fungi. The present report describes the results of experiments to determine whether or not amyloidity could be induced in nonamyloid hyphae.

A B S T R A C T This paper, the first of a series of three, raises the general question of the relationship between stress, organogenesis, and cell wall orientation in a growing structure. It outlines a short descriptive chronology of events in the prepollination development of the ovule in cotton and proceeds to a brief experimental comparison of nucellar development in tissue culture with and without integuments. The significance of the integuments as mechanical effectors is discussed in terms of stresses which they induce in the growing ovule. THE EARLY EMBRYOLOGY of the cotton ovule represents a classical botanical problem of differentiation. The origin and maintenance of the female gametophyte within the diploid tissues of the nucellus involves an unambiguous act of singlecell differentiation: that of the single archesporial cell within the genetically homogeneous cell mass of the immature nucellus. The consequences of this event are readily visible in the mature embryo sac, which is the female gametophyte. Current understanding of differentiation is founded upon morphological, physiological, or genetic definitions of the differentiating system. The purpose of this paper then, is to outline a picture of the cotton ovule as a mechanical system. Since the work of Gore (1932) and Ramchan

HANLIN, RICHARD T. (Georgia Experiment Station, Experiment.) Studies in the genus Necwria. II. Morphology of N. gliocladioides. Amer. Jour. Bot. 48(10): 900-908. Illus. 1961.-Swollen tips of vegetative hyphae develop into multicellular archicarps from which multinucleate ascogonia forrn. From basal cells of each archicarp arise hyphae which grow up into a prosenchymatous, true perithecial wall; around this wall is formed a thin pseudoparenchymatous stroma of compacted hyphae. The ascogonia give rise to ascogenous cells from which croziers and asci form directly. At the same time, an apical meristem forms cells that grow downward into the centrum. These are pseudoparaphyses. Asci grow up among the pseudoparaphyses, which deliquesce as the ascocarp matures. The ascus tip contains a thick ring with a pore and lateral thickening of the ascus wall. Ascospores are forcibly ejected. The chromosome number is 4. This species conforms to the Nectria Developmental Type of Luttrell. THE TAXONOMIC position of those Pyrenomycetes having brightly colored, fleshy ascocarps has been variouslv interpreted by different workers. Some (Lindau, 1897; Gaumann, 1926; Bessey, 1950; Miller, 1949; Luttrell, 1951) have placed them in a separate order, the Hypocreales, while others (Miller, 1928; Clements and Shear, 1931; Gaumann, 1952; Von Arx and Muller, 1954; Munk, 1957), not considering color and texture sufficient basis for separation, included them in the Sphaeriales in one or more families. In recent years, developmental data have become increasingly important in taxonomic arrangements. In 1951, Luttrell proposed a system based on types of development of the ascocarp. He recognized 8 different developmental patterns, 1 of which, the Nectria Developmental Type, served as the basis for the order Hypocreales. The extent of this developmental type among hypocreaceous fungi can be determined only by detailed studies of the different species. The few studies which have been carried out are mostly incomplete and, therefore, of little value in determining the position of these fungi in a taxonomic system based on developmental morphology. The genus Nectria is a good example of this. Of the 4 species studied previously (N. ribis Tode ex Rab. [Vincens, 1917]; N. galligena Bres. 1 Received for publication May 10, 1961. Paper No. 1121, Department of Botany, University of Michigan; and Paper No. 397, Journal Series, Georgia Experiment Station. This research was supported in part by National Science Foundation Grant G-10727. Portion of a dissertation submitted to the faculty of The University of Michigan in partial fulfillment of the requirements for the degree of Doctor of Philosophy. I am grateful to Dr. L. E. Wehmeyer for his guidance during this study and for reviewing the manuscript. [Cayley, 1921]; N. ipomoeae Halst. [Cook, 1923]; N. flava Bon. [Gilles, 1947]), only the work on N. flava approaches the detail necessary for modern taxonomic work. The present study was conducted to determine the position of N. gliocladioides Smalley & Hansen on the basis of its development. MATERIALS AND METHODS-Nectria gliocladioides was originally isolated from decaying bulbs of Lilium auratum Lindl. (Smalley and Hansen, 1957). The material used in this study was derived from a culture obtained from Dr. E. B. Smalley. Cultures were grown on complete mineral-medium agar plates containing 2% glucose under continuous fluorescent ("Daylight") light at 25 C. Suitable material was cut up into blocks approximately 5 mm2, then killed and fixed in Allen-Bouin Solution, Type II (Sass, 1951). Dehydration was carried out bv means of the tertiary butyl alcohol (TBA) method (Sass, 1951). Embedding was done in Fisher Tissuemat (mp 54-56 C). Sections were cut at a thickness of from 8 to 10 u, the ribbons were floated on a 3% solution of formaldehyde in distilled water, and affixed to the slides with Haupt's Adhesive (Johansen, 1940). A number of stains were tried but the majority gave poor results. The method finally selected was a modification of that used by Robinow (1957). Sections were stained with the Feulgen technique (Johansen, 1940), then counterstained in 0.5% acetocarmine (45% acetic acid). Optimum time for both hydrolysis and counterstaining was 3 min. A longer time intensified the stain too much, obscuring detail. Studies of asci were made with fresh material. Squash mounts were made in 1 % acetoorcein (60% acetic acid) or 1% acetocarmine Fig. 1-13. All magnifications X 1700 unless otherwise stated.-Fig. 1. Typical Gliocladium conidiophore.-Fig. 2. Typical Verticillium conidiophore. X 850.-Fig. 3-4. Conidiophores intermediate in structure. X 850.-Fig. 5, left to right. Stages in conidial development, from branch to mature conidium. X 3000.-Fig. 6. Two mature conidia.-Fig. 7. Two conidia germinating after 6 hr in distilled water.-Fig. 8. Three mature ascospores, 2 with germ tubes after 6 hr.Fig. 9. Germinating conidium after 10 hr.-Fig. 10-12. Germinating ascospores after 14 hr.-Fig. 13. Tip of mature ascus showing wall thickening and apical ring with pore. X 3000.

INFORMATION ON the biochemical properties of plant mitochondria and microsomes has recently been reviewed by Goddard and Stafford (1954) and by Hackett (1955). Little work has been reported on the identification of these properties with specific types of particles defined by various physical and chemical criteria, or on the relationship of the isolated particles to intracellular structures. During an investigation of oxidative phosphorylation in particulate fractions isolated from peanut cotyledons, the authors found it of value to explore additional properties of the fractions. Accordingly, the particles isolated by differential centrifugation have been characterized by their appearance in the phase contrast and electron microscopes, response to janus green B, succinoxidase and palmitic-_-C'4 oxidase activities, and 2-hr. hydrolyzable ribonucleic acid content. The present paper includes the results of this study. In this paper, mitochondria are considered to be the microscopically visible particles with Krebscycle activity, and microsomes to be submicroscopic particles with high ribonucleic acid content but without Krebs-cycle activity. MATERIALS AND METHODS.-Isolation of the fractions.-Cotyledons were obtained from five-day-old etiolated peanut seedlings (Arachis hypogaea var. 'Jumbo') grown in steam-sterilized Vermiculite at 30?C. About 40 cotyledons were ground in a cold mortar with acid-washed quartz sand in 20 ml. of a cold solution containing 0.5 M sucrose and 0.01 M phosphate buffer at pH 7.4. Homogenization and the other steps in the isolation of each particulate fraction were performed at approximately 0?C. The cold, crude homogenate was filtered through a double layer of cheese cloth into a chilled plastic centrifuge tube and centrifuged at 500 g for 5 min. in an International refrigerated centrifuge to remove unbroken cells, cell walls, nuclei, etc. In much of the work, the supernatant from this preliminary centrifugation was decanted and separated with the high-speed attachment of the centrifuge into three successive fractions consisting predominantly of starch, mitochondria, and microsomes, respectively, by the procedure shown in the accompanying flow sheet (fig. 1). The starch fraction was isolated by accelerating the centrifuge as

As is well known, the curvature in the Avena test (Went, 1928) is dependent on concentration of the applied auxin only up to a certain limiting angle. The value of this angle, the so-called "maximum angle," was shown by Schneider and Went (1938) to depend on the IDP (the Interval between the first Decapitation and the Putting on of the auxinagar block). When the IDP is short the maximum angle may be of the order of 450 but, as the IDP approaches two hours, this maximum angle passes through a minimum value of about 10-15 ?. Coleoptiles such as would give this 150 maximum angle are the basic experimental objects of this paper. The auxin content of coleoptiles used in maximum angle curvatures is probably dependent on the IDP. Thus for short IDP the content is high as a result of auxin production by the tip before the first decapitation, for IDP about three hours long the content is moderately high as a result of auxin production by the "regenerated physiological tip," and at intermediate intervals the auxin content passes t-hrough a minimum value corresponding to the lack of auxin production before regeneration of the physiological tip. The variation of the size of the maximum angle with IDP parallels this probable auxin content of the coleoptiles so closely as to lead to the suggestion that the size of the maximum angle is directlv related to the auxin content of the coleoptile at the beginning of the period of curvature. The investigations reported here test this suggestion that the maximum angle curvature varies with the auxin content of the coleoptile. The content of the coleoptiles is varied by artificially supplying auxin before the maximum angle auxin-agar blocks are applied. Schneider and Went (1938) have performed comparable experiments in which the auxin content was artificially increased to verv high levels. The experiments reported here deal only with concentrations comparable to those normally in the coleoptiles. The coleoptiles most used were those whose auxin content was lowered by removing the normal source of auxin, i.e., by decapitation. The auxin content was then brought up to anv desired level by artificially supplying auxin before performing the curvature tests, i.e., by auxin pre-treatments. Many other auxin pre-treatments have been reported in the literature. For the rooting of cuttings, Cooper (1938) and Went (1939) performed pretreatments leading them to the conclusion that auxin 1 Received for publication March 22, 1940. The author wishes to express his appreciation to Professor K. V. Thimann for stimulating advice and criticism in carrying out this investigation. is instrumental in the translocation of some second factor essential for root initiation. For the pea test, Went (1939a) developed a pre-treatment such that the sensitivity of the pea test was increased. For straight growth of the Avena coleoptile, Went (1 939b) performed pre-treatments from which he concludes that one of the activities of auxin is to cause the mobilization of a second factor (food factor) which is necessary for growth (by cell elongation). The results of the present paper are consistent with Went's conclusion of a mobilization of a "food factor" by auxin, although other explanations are not excluded. The pre-treatments reported here are unlike those of earlier investigations in three different ways. The assay of pre-treatment effects depends primarily on maximal growth responses. The pre-treatments cause no increase of sensitivitv to threshold concentrations. Physiological concentrations of highly active auxins are the most efficacious for the pre-treatments; that is, the pre-treatment is effected with just the kind and quantities of auxins that greatly accelerate cell elongation. VARIABILITY.-For each of the types of measurements quoted herein there has been some vari-ation in magnitude of response even for duplicate sets of experiments. Perhaps these could be best considered after the experimental evidence, but they are mentioned here in order that the reader may consider them along with the experiments. Thus for curvatures, the maximum angle response shown in figure 5 is ten degrees lower than that of figures 1 and 2. For straight growth, the response shown in figure 6 is considerably less than that shown in figures 3 and 4. These variations are comparable to those commonly encountered in the Avena test (see, e.g., Went and Thimann, 1937). A most striking variation occurs in the comparison of the effectiveness of different auxins for pre-treatment. For example, in experiments that were the duplicates of the one in figure 7, the positions of the curves for 3-indole-propionic and phenvl-acetic acids, were sometimes reversed. But perhaps this is not too surprising for similar variations of sensitivity of coleoptiles to these auxins are reported by Thimann and Schneider (1939). Curiously, the response of plain agar pre-treated controls was also variable (fig. 1, 5, 7). Efforts to find variation in the agar itself produced only negative results. It will be noticed that in the experiment of figure 1, the agar controls gave the same response as the plain controls, i.e., the agar pre-treatment was without effect, but in the experiments of figures 5 and 7 the agar pre-treatment had a marked effect. To a limited extent, this agar pre-treatment effect

WHEN BOILED grass leaves are used as bait for the saprophytic chytrids in soil and water collections, Pythium is often found growing on the grass leaves. This method of collecting has proved effective in obtaining a number of species of Pythium and parasites of Pythium, which frequently are species of Pseudolpidium or Olpidiopsis. Pseudolpidium gracile Butler, the resting bodies of which lack companion cells, has been recollected and studied. In addition, two new species of Olpidiopsis, the resting bodies of which possess companion cells, have been found parasitizing Pythium. In a number of collections, however, the grass leaf bait yielded a species of Aphanomyces which was parasitized by what appeared to be Pseudolpidium Aphanomycis, in that all the resting bodies were without companion cells. These four species of Pseudolpidium and Olpidiopsis will be discussed in this paper. Recently taxonomic studies of the genus Olpidiopsis have been published by Shanor (1939, 1940) and McLarty (1939, 1941). Shanor (1939) suggests that the genus Pseudolpidium should be combine'd with Olpidiopsis for "none of the species originally placed in the genus when it was established can remain unquestionably as Pseudolpidium species. The continuance of the genus seems to rest on onlv two species, P. Pythii and P. gracile, both described by Butler. Even these two species need further critical study." McLarty (1941) merges the genus Pseudolpidium with the genus Olpidiopsis and amends Olpidiopsis "to include species with smooth and spiny zoosporangia which may form either sexual or asexual spores." Of Pseudolpidium Pythii and P. gracile McLarty states, "Very little is known of these species, although each has been observed since Butler's time. The resting spores of these species, as they are illustrated in the literature, appear very similar in appearance to spiny zoosporangia. Until they are re-collected and critically examined, these species may be transferred to Olpidiopsis as strictly asexual species." No opinion is expressed here as to the validity of the genus Pseudolpidium. The combining of Pseudolpidium and Olpidiopsis by McLarty (1941) on the basis of his having found both sexual and asexual spores in monospore cultures of his species, Olpidiopsis Achlyae, is justifiable. Nevertheless, the studies presented in this paper will emphasize the fact that in certain forms, formerly considered as species of Pseudolpidium and now to be placed in Olpidiopsis, according to McLarty, the lack of a companion cell on the resting spore is a constant and consistent characteristic of the species. In this connection two species, Pseudolpidium gracile Butler and P. Aphanomycis (Cornu) Fischer are now to be discussed. I Received for.publication May 1P, 1942. This work was supported by a grant from the Penrose Fund of the American Philosophical Society. Pseudolpidium gracile Butler.-A soil collection made by Dr. H. R. Totten at Key Largo, Florida, on December 27, 1940, yielded Pythium rostratum (fig. 15-20), parasitized by a species of Pseudolpidium (fig. 7 and 8). Because of the length of the spines and the yellowish color of the resting bodies, the parasite was identified as Pseudolpidium gracile Butler, though Butler (1907) lists Pythium rostratum as the host of Pseudolpidium Pythii. Pythium intermedium is given as the host of both P. Pythii and P. gracile, so it is possible that Pythium rostratum is likewise susceptible to both species of Pseudolpidium. Pseudolpidium gracile has been kept under observation in culture on its host since the date of its collection. Resting bodies are constantly produced in large numbers, but not a single resting body with a companion cell has been seen. The resting body is yellowish in color and covered with slender spines which attain a maximal length of 4 u (fig. 3). In shape the resting body varies from spherical to oval and measures 12 to 27 u in diameter, exclusive of the spines. Butler states that the wall of the resting body of his species is rather thin and in his figures of the resting body the wall does appear to be very thin. It is' for this reason that Shanor and McLarty have suggested that what Butler figures as resting bodies may be only spiny-walled zoosporangia. It is possible that Butler drew the resting bodies before the wall had attained its mature thickness for the contents of the resting body as illustrated by Butler appear to be undifferentiated. A mature resting body from the cultures of our P. gracile contains a single central oil globule surrounded by a peripheral layer of vacuolated protoplasm (fig. 3). Butler does not give any measurements of the thickness of the exospore and endospore walls of the resting bodies of P. gracile but measurements made on the resting bodies in our cultures show that the endospore may attain a thickness of 0.7 to 1.2 u while the exospore varies from 1.7 to 2.5 u in thickness. The development of the zoospotangia and the discharge of the spores do not differ from the descriptions of Butler. A period of vacuolization, during which one or two exit papillae appear on the sporangium, is terminated by the sudden disappearance of the vacuoles. Very soon after the disappearance stage the spores are cut out and discharged. The zoospores are biflagellate. The walls of some of the zoosporangia in older cultures are often covered with very short, fine spines (fig. 9). Pseudolpidium Aphanomycis (Cornu) Fischer.A culture of Aphanomyces cladogamous Dreschler was obtained from soil collected by Philip Couch near Petahatche, Mississippi. The A. cladogamous was parasitized by a species of Olpidiopsis which formed resting bodies that lacked companion cells (fig. 4 and 24). The identification of this species constitutes a difficult problem for two species of

THE PROCESSES of growth and differentiation in multicellular plants involve changes not only in individual cells but in intercellular relationships. Young meristematic cells, all much alike in size and character, soon become differentiated into a wide variety of elements. Some remain small, others increase in volume even to several thousandfold. Some become elongated, others flattened, and others modified in various ways. How the changes which take place in one cell are related to those in neighboring cells is evidently an important problem, since it involves the more general question as to the developmental relations of cell and organism. As to how the intercellular readjustments made necessary by differentiation are brought about there is no general agreement. Several possibilities suggest themselves. (1) Cells might slide along the surfaces of their neighbors and thus alter their positions. This commonly occurs in animal development, where cells may migrate for some distance. Ever since the work of Krabbe (1886) and other early students of the problem, a number of botanists have presented evidence that such "sliding growth" occurs in plant tissues, though this has been seriously questioned by others. (2) Some cells might cease division and begin expansion while their neighbors were still dividing, and thus become relatively large and make contacts with an increased number of cells. This method is certainly operative in many cases, though by no means in all. (3) Adjustments might be brought about by differential growth within cells, one portion of a cell exactly keeping pace with a rapidly growing neighbor cell and another portion with a more slowly growing cell, so that different parts of the same cell would thus grow at different rates. Such unequal or localized growth certainly takes place in root hairs, tyloses, and other tissues where one portion of the cell is free, but is more difficult to demonstrate in cells which are surrounded by others. (4) For a brief period when cells are very young and their walls mav still be subject to surface forces, readjustments in length and relative position of the walls may take place which do not involve either sliding or differential growth in the strict sense of these terms. It is often difficult to determine by a study of sections of killed material alone (the method heretofore chiefly employed) which of these possibilities of intercellular readjustment are actually operative. To obtain convincing evidence, it is obviously necessary to make successive observations of a group of cells in their living condition as they grow and differentiate. The purpose of the present paper is to bring 1 Received for publication June 7, 1939. This investigation was carried on by the aid of a grant from the Special Fund for Research of Columbia University. forward such evidence from a study of the developing surface layer of the root in a number of species. METHODS.-One of the authors (Sinnott, 1939) has recently described a technique by which it is possible to study living plant meristems directly. Seeds of certain small-seeded grasses are grown on damp lens paper in moist chambers. The terminal root meristems are sufficiently translucent so that the cells of the outermost layer and the one directly beneath it can be observed under high magnifications. The members of a group of young meristematic cells, and of all their descendents, may be identified in this way from their earliest condition near the extreme tip of the root, through the period of cell division and cell enlargement until their final size is established and differentiation is complete, a process occupying from 24 to 48 hours. By successive camera lucida drawings made at intervals, a complete recor.d may thus be obtained of every cell in an entire cell lineage, both as to its own changes and as to its relations with neighboring cells. Roots of Phleum pratense, Poa trivialis, Sporobolus cryptandrus, and Chloris gayana were the ones chiefly studied. Aside from the fact that it can be observed in the living condition, such material has certain other advantages. The cells are rectangular and in regular longitudinal rows. Cell divisions in the surface layer (the tissue chiefly studied) are all anticlinal and with rare exceptions are at right angles to the long axis of the root. Cell enlargement is entirely in the longitudinal direction. A considerable degree of differentiation occurs, for the cells which are to develop root hairs are in many respects unlike those which do not do so. In Phleum and Poa this differentiation is more pronounced and occurs earlier than in Sporobolus and Chloris (Sinnott and Bloch, 1939). The exact points of origin of new cell walls, the increase in size of individual cells, and any alterations in the relationships between cells, can thus be studied under the simplest of conditions and can be expressed in quantitative terms. RESULTS.-The basic results can be set forth most directly by presenting a series of drawings of the same group of cells at successive intervals from early development to maturity. This has been done for a group of surface cells in two roots of Phleum pratense in figures 1, A-J, and 2, A-D. In the first drawing in each root every cell is given a number, and since a region was always chosen in which one or more distinctively shaped cells were present, the original cells or their descendants can always be identified in later drawings. After division has ceased, all the cells elongate greatly, but they show no change in their transverse dimensions. It will be observed that numerous cell divisions occur and that following the last division the apical daughter cell ordinarily develops a root hair but that the basal one

DE TERRA, NOEL, and E. L. TATUM. (Rockefeller Inst., New York, N. Y.) A relationship between cell wall structure and colonial growth in Neurospora crassa. Amer.Jour. Bot. 50(7): 669-677. Illus. 1963.-Cell walls were isolated from morphological mutants of Neurospora crassa and from their corresponding wild-type strains. Acid hydrolysates prepared from these cell walls were then studied, qualitatively and quantitatively, with respect to their reducing sugar content. Paper chromatography revealed the presence of glucose and glucosamine in the cell walls of all strains studied. Quantitative analysis has shown that a group of 4 colonial mutants which strongly resemble each other in morphology all have significantly less glucose and more glucosamnine per unit weight of cell wall than do their corresponding wild-type strains. These data strongly suggest that a particular type of morphological aberration in Neurospora is associated with similar changes in cell wall composition. ABNORMAL morphological forms of Neurospora have been described by many investigators (Barratt and Garnjobst, 1949; Tatum, 13arratt, and Cutter, 1949; Srb, 1957). The "colonial" growth pattern is perhaps the most widely known of these aberrant forms. While normal growth is sparse and spreads loosely over an agar plate (Fig. 1), colonuial morphology is characterized by the formation of dense, restricted colonies (Fig. 2). Colonial growth may be induced in wild-type strains of Neurospora by the addition of L-sorbose or surface-active agents to the culture medium (Tatum et al., 1949). Microscopically, the hyphae of sorbose-grown coloniies are characterized by a great increase in the number of branches anid cross-walls per unit length of hypha and by the induction of branches at the hyphal tip, a pheniomenon rarely seen in normally growing young cultures (Fig. 3, 4). In liquid cultures inoculated with conidia, normal hyphae grow into a loosely branched network of filaments, while sorbosegrown hyphae form discrete, spherical colonies, with each sphere representing a single center of growth. Many colonial mutants of Neurospora have been isolated (13arratt and Garnjobst, 1949; Murray and Srb, 1962) and shown by genetic techniques to result from mutations at single loci. Some of the colonial mutants are almost identical in morphology to sorbose-grown cultures, while others differ in one or more important respects. The rigid cell wall of Neurospora must obviously play an important part in determiniing the morI Received for puiblication November 24, 1962. Supported in part by grant C-3610 from the National Cancer Institute, U.S. Public Health Service. 2 Present address: Department of Biophysics and Nuclear Medicine, Laboratory of Nuclear Medicine and Radiation Biology, University of California Medical Center, 900 Veteran Avenue, Los Angeles 24, California. 3Part of this work was performed during the tenure of a U.S. Public Health Service post-doctoral fellowship of the National Cancer Institute. phology of this organism, for spherical, osmotically sensitive protoplasts may be obtained from Neurospora hyphac by treatment with snail digestive juice (13achmainn and 13onner, 1959), ati enizyme preparation rich in carbohydrases (IfHoldein anid Tracy, 1950) which has been shown to digest at least one of the cell wall polysaccharides of Neurospora (de Terra and Tatum, 1961). In a previous paper (de Terra and Tatum, 1961), an attempt was made to investigate the part played by the cell wall in determining the morphology of Neurospora. L-Sorbose, an agent which induces colonial growth in Neurospora, was found to brirg about significant changes in the composition of the cell wall. Acid hydrolysates of cell walls isolated from sorbose-grown (colonial) hyphae were found to contain 184% as much glucosamine and 68% as much glucose per unit weight of cell wall as hydrolysates obtained from the cell walls of normally growing hyphae. In addition, snail digestive juice, an agent known to alter the structure of the cell wall by digesting at least one of its major components (glucati), was found to iniduce colonial growth when used in concentrations somewhat lower than those required for production of protoplasts. These firdii)gs suggested that at least some types of colonial growth are caused by structural changes in the cell wall. If this is indeed the case, one might expect some genetically determi!ied morphological forms of Neurospora, such as the colonial mutants, to show changes in cell wall composition similar to those induced by L-sorhose. The puirpose of this paper is to describe and discuss the results obtained from qualitative anid quantitative analysis of the sugars present in acid hydrolysates of cell walls prepared from 8 different morphological mutan)ts of Neurospora crassa. MATERIALS AND METHODS-Young, healthy hyphae of the desired strains of Neurospora crassa were grown in liquid shake culture in 0.2% sucrose miniiral rredium (Ryan, Beadle, and

Dormancy in plants is common in three organs, seeds, spores, and buds. In this paper I shall limit myself entirely to the discussion of dormancy in seeds for three reasons: far more critical and analytical work has been done on dormancy in seeds than upon the other two organs; obscure correlation effects do not play as prominent a part as in buds; and I am best acquainted with this phase of the subject. The title of this paper might imply that I am aligning myself with the mechanists as against the vitalists; such is not the case. The vitalists can justly claim that in growth we have as yet explained so little by known chemical and physical laws and have so much till to explain on this basis or on the basis of newly discovered laws that the title "Mechanism of Growth" would imply a very meager discussion of our knowledge of this process. To date we have to discuss growth mainly in physiological rather than mechanistic terms. The situation is rather different with the dormancy of seeds. Here so far as the work is analytical, it indicates that dormancy is brought about by factors inhibitory to general processes preceding or accompanying growth. In plants of the temperate zone, the seeds generally have a rest period. Howard' finds that more than 75 percent of the species, wild and cultivated, growing around Columbia, Missouri, have a distinct period of dormancy. The rest period is more general and much more persistent among wild than cultivated forms. Selection resulting from methods of cultivation have largely disposed of this character in domesticated forms, as is well illustrated by the two closely related species, Avena fatua and A. sativa.2 The former is delayed in its germination until the spring following ripening, con

It is well known that the corolla of many different flowers lives for only a short time, sometimes for only a few hours, though the fruit forms normally if fertilization has taken place. The cause of this ephemery of floral structures is unknown. In earlier papers I have pointed out that living matter is composed of very unstable chemical compounds which may be decomposed not only by chemical agents but also by heat, strong light, and even purely mechanical effects. These compounds are so unstable as to be compared with explosive substances.' It is known that explosives may decompose spontaneously, without harmful effect, if they are kept for a long time. We may therefore expect that the principal compounds of living matter may also decompose spontaneously. Such decomposition must lead to death, however, if the principal compounds are not restored by synthetic processes. Indeed, no plant or animal can be kept alive forever if its growth and metabolism has stopped and the renewal of living matter has ceased. Lower organisms, such as bacteria, lichens, infusoria, and rotatoria, and seeds, remain alive in a dry state or at extremely low temperatures for a long time, but they cannot be kept thus indefinitely. If the destructive processes in protoplasm proceed more rapidly than the synthetic ones every organism dies at length regardless of whether it is in a state of anabiosis or not. From this standpoint we may consider the death of the corolla of ephemeral flowers, but in this case the decomposition of the principal compounds of living matter evidently proceeds especially rapidly, and these compounds therefore seem to be particularly unstable. In the present paper experiments are described which have been designed to test the correctness of this supposition.

THE SECOND paper of this series (Beadle, 1945b) presents some details on the production and isolation of a number of mutants of Neurospora. In most cases investigated, the new strains differ from the wild type parent by a single gene and this gene is normally concerned with a step in the sequence of reactions leading to the production of an essential growth substance. It is considered that the gene controls the production of a specific enzyme that in turn catalyzes a specific reaction. As a rule, the mutations obtained in Neurospora appear to involve a complete inactivation or an actual physical deficiency in one gene, but recent studies on a group of temperature-sensitive mutants suggest that intermediate modifications of genes also exist. Oiily one mutant has been found that requires the vitamin riboflavin (isolation number 51602), and this requirement is a function of the temperature at which the mold is allowed to grow. That is, it grows and synthesizes riboflavin at a rate approaching that of a wild type strain at temperatures below 250C. while an external source of the vitamin is necessary above 28?C. The present paper is concerned with the inheritance of this mutant type and its growth and its temperature characteristics as well as some consideration of gene action in riboflavin synthesis in Neurospora. GENETIC ANALYSIS.-Mutant 51602 was crossed to wild type and the eight spores from each of twenty asci were isolated in order. In all twenty cases the temperature-sensitive, riboflavin-requiring mutant was recovered from four spores and wild type from the other four. Crosses to four mutants known to differ from wild type by a single gene gave similar results, and here too the mutant was recovered intact in its temperature-sensitivitv and nutritional requirements from four of the spores in each of 80 asci. Therefore, it may be concluded that mutant 51602 differs from wild type by a single gene. Experiments on linkage will be reported in a general paper on this subject. GROWTH.-Experiments on growth were carried out using the same general technique that has been extensively applied in this laboratory (Horowitz and Beadle, 1943; Beadle, 1944). Twenty ml. of medium contained in 125 ml. Erlenmeyer flasks were inoculated with a dilute conidial suspension and the mold allowed to grow at designated tem1Received for publication September 10, 1945. This work was supported by funds supplied by the Nutrition Foundation and the Rockefeller Foundation. The compounds lumichrome, lumiflavin, 3,4-dimethylphenyl-d, l'-ribamine and 2-amino-4, 5 dimethyphenyl-d, 1'-ribamine were obtainedthrough the courtesy of Drs. Jackson W. Foster and Max Tishler of Merck and Company. Riboflavin-adenine dinucleotide was generously furnished, from a very small supply, by Dr. F. M. Strong of the University of Wisconsin. peratures and periods of time. The mycelial pads were then removed, pressed on filter paper, placed in order on cardboard or filter paper, dried for three hours at 850C., cooled and finally weighed. The medium, containing inorganic salts, sucrose, biotin and water, has been described a number of times (Horowitz and Beadle, 1943; Beadle, 1945b). The appropriate supplement required to proiduce growth of the mutant was added in 2 ml. or less of water. At 310C. mutant 51602 does not grow without addition of riboflavin to the minimal medium that supports growth for wild type Neurospora. With increasing quantities of added riboflavin an Sshaped growth curve is obtained. The range of concentration varies with the temperature but extends from approximately 0.1 to 2.5 micrograms of riboflavin. Growth curves for various temperatures can be determined from the data represented in figure 1.

IN REGIONS of low rainfall, such as in the western grasslands of the United States, plants useful for soil conservation must not only be adapted to binding soil, but they must also be physiologically suited to withstand periods of drought without excessive injury. It is desirable that they should be economical in their use of the limited supply of moisture. In this paper the transpiration ratios of three western grasses commonly used for soil-conservation purposes are considered. Aaropfron Smithii Rydb., Bromus marginatus Nees., and Agropyron ciliare (Trin.) Franch. were the grasses used in this study. The first two of these are native to the western part of the United States and have been shown to be good soil-conserving plants in at least certain restricted areas. They produce good seed crops with a high percentage of fertility, and their forage values are satisfactory (Sampson, 1924). Agropyron ciliare is an Asiatic species which has been introduced into the United States and has been grown at several experiment stations. All three species are perennials. The term "transpiration ratio" is used in this paper to indicate the ratio between the amount of water transpired by the plant during its growth and the weight of dry matter produced. This term is synonvmous with the "water requirement" of Briggs and Shantz (1913b). Briggs lnd Shantz and co-workers (Briggs and Shantz, 1914, 1917; Shantz and Piemeisel, 1927; Dillman, 1931) made extensive studies of the transpiration ratios of plants adapted to the dry regions of the United States. Several grasses that occur commonly in the Great Plains were included in their investigations. Their work was done at Akron, Colorado, Newell, South Dakota, and Mandan, North Dakota. Agropyron Smi.thii was found to be one of the most extravagant plants, requiring more water to produce a gram of dry weight than alfalfa and considerably more than the grain crops. Agropyron cristatum and Bromus inermis were somewhat more efficient. The short grasses (Bouteloua gracilis and Buchloe dactyloides) were very economical in their use of water. Sorghum vulgare var. sudanense had a low transpiration ratio. Several other investigators have found this grass to be particularly economical with respect to water utilization (Miller, 1916; Tnlaikov, 1926; Ballard, 1933). 1 Received for publication October 23, 1939. Paper No. 697 from the Department of Botany and Botanical Gardens, University of Michigan. The author desires to take this opportunity to express his gratitude to Professor F. G. Gustafson for his advice and interest in these investigations, and to Dr. C. A. Connaughton and Dr. R. E. McArdle,,Director and former Director, respectively, of the Rocky Mountain Forest and Range Experiment Station, for their suggestions and for supplying the Fort Collins loam used in this investigation. Sampson and Weyl (1918) and SamDson (1919) are the only other investigators who have reported the transpiration ratio for any of thewesterngrasses. They obtained the value 1,339 for Bromus marginatus on eroded soil, and 1,110 on soil that had not been eroded. Sampson also reports the transpiration ratio for this grass as 803, 516, and 756 in oak-brush, aspen-fir, and spruce-fir associations, respectively. In each of these investigations the transpiration ratios of the grasses studied were found, with a few exceptions, to be nmaterially higher than those for other types of plants characteristic of the western grasslands. However, Sampson (1919) pointed out that the root growth of grasses forms a much higher percentage of the total plant growth than does the root growth of crop plants, such as wheat and peas. Consequently, the common practice of using only the dry weight of tops produced as a basis for the transpiration ratios of such plants gives misleading results, unless forage production alone is being considered. METIIODS.-Experiments of 1937.-In these experiments plants were grown in sealed impermeable pots which were weighed frequently to determine the quantity of transpiration. At the time of each weighing the transpired water was replaced, and at the end of the growing season the total loss of water was computed. The transpiration ratio was obtained by dividing the total transpiration by the dry weight produced. The potometers used were made of hleavily galvanized iron (fig. 1). They measured 35 cm. in diameter and 50 cm. in height and had a capacity of about 35 kg. of oven-dry soil. The soil used was a fertile sandy loam which had been prepared for greenhouse purposes. The surface of the soil in each can was covered with a disk of heavy cardboard which had been thoroughly impregnated and coated with paraffin. The melting point of this paraffin was 70?C. A preliminary experiment demonstrated that this type of cover, when sealed to the can with Plasteline, did not allow measurable quantities of water to evaporate from the soil. A disk of painted cardboard placed over the cover prevented the melting of the paraffin on excessively warm days. Water was introduced into the pots through a large-diameter glass tube which was situated near one edge of the pot. One end of this tube passed through the paraffined cover, and the other end terminated in the soil with a U-shaped extension, the two arms of which followed the contour of the pot for several inches. The ends of the U-tube were enclosed in galvanized-iron wire gauze wrapped in absorbent paper which served as a wick to disperse the water rapidly. The U-tubes with the wicks were buried in a one-half inch layer of sand which furtiher aided in dispersing the water rapidly and evenly

THESE INVESTIGATIONS were started in order to study quantitatively the auxin production of coleoptiles. Special attention was given to the extreme tip (1 mm.) which is the location where the auxin is produced in the intact coleoptile. In this study both the diffusion and the extraction technique were employed. These techniques, as will be shown below, are not interchangeable. Whereas the extraction technique will only produce data on the content of active auxin of the tissues at the time of extraction ,2 the diffusion technique as described here will yield data on the amount of potential auxin, generally only a fraction of which is active auxin at a given moment. The diffusion technique also will produce data on the rate of auxin production. Although a large amount of indirect evidence pointed to a production of auxin by the tip of coleoptiles, Thimann (1934, p. 29) is the only one to mention (without detail) an experiment which seems conclusive. That caution in this respect is necessary has been shown lately by investigations of root tips (Thimann 1934, van Overbeek 1939). Here it was shown that excised root tips of germinating oats and peas do not actually produce auxin when placed on agar blocks, although many investigators concluded from indirect evidence that they did. In the course of the investigations it appeared that by combining the diffusion and the extraction technique, manv other interesting conclusions could be drawn; for instance in regard to the amount of inactive auxin precursor. In addition to the study of auxin production in coleoptile tips the production of auxin in decapitated seedlings was also studied. 1. GENERAL METHOD.-Zea and Avena seedlings were used, which were grown in a physiological darkroom at 24?C. and 85 per cent humidity. The darkrooms were occasionallv illuminated with orange light (Went and Thimann, 1937). The Zea seedlings were mostly used five days after they were planted in moist sand. The Avena seedlings were three days old, at which stage thev are also used for Avena tests. Normal or F1 plants of Zea strains segregating nana, lazy and brachytic were used. The auxin production of these various strains was discussed in an earlier paper (van Overbeek, 1938a). 1 Received for publication April 29, 1940. Part of work was carried out with the assistance of the Work Projects Administration, Official Project No. 65-1-07-98, Work Project No. 11534. 2 This may not be generalized because it is not impossible that in certain seeds, leaves, etc., inactive auxin precursor is changed into active auxin during the process of ether extraction. This, however, cannot be the case to a large extent in coleoptile tips, as the results given in this paper will indicate. The Avena seedlings were of the imported Swedish Victory oats and cultivated in the standard way in glass holders with their roots in water unless otherwise indicated. All agar used was 1.5 per cent "Difco." The blocks were poured from a sterile stock at the beginning of each experiment. The volume of the blocks was 140 mm.3 for. Zea and 70 mm.3 for Avena. Each of these blocks was cut into twelve equal parts before being tested in the Avena test. Auxin extractions were made with peroxide-free ether exclusively; the tips were neither ground nor acidified. In general a similar technique was followed as outlined in previous papers (van Overbeek, 1938b, 1938c; van Overbeek and Bonner, 1938), but instead of extracting by standing, a small Soxhlet apparatus was used. In this manner all extractable auxin is removed from coleoptile tips.3 All auxin determinations were carried out with the standard Avena test, using three hours between decapitations, and photographs were taken ninety minutes after the blocks were put on. The minimum number of Avena test plants used for each single determination was twelve, but in many instances thirty-six or forty-eight plants were used. If results of the diffusion and extraction in one experiment were to be compared, all tests were run on the same dav. The variation is expressed as the standard error of the mean.4 The standard error was not calculated in such cases where there was no question as to the significance of a particular difference. For the Avena test as carried out in these laboratories, this error is on the average 5 per cent, but not less than 10 of curvature. EXPERIMENTS WITH ZEA.-2. Diffusion. The diffusion method has often been used to determine auxin production. However, the experiment was usually not continued until the coleoptile tips stopped giving off auxin. This was done in the experiments described in this paper by hourly transferring ten to twelve coleoptile tips of Zea to fresh agar blocks. The experiments were done in Petri dishes lined with moist filter paper in order to prevent drying out. The agar blocks containing auxin were saved in the refrigerator and tested the following day. The results are plotted in figure 1. The abscissa indicates the hourly transfers and the ordinate the concentration of auxin present in the agar 3Soxhlet extraction of auxin from 48 corn tips 5 mm. long, yielded after 24 hours (taken up in 0.3 cc. agar) 14.10; after a second period of 24 hours (taken up in 0.3 cc. agar) 0.00. ?

A new species of Spongiochloris, S. typica, was isolated from a Connecticut soil. It can be distinguished from other species by the presence of many pyrenoids; by the cell size of light-grown cells (up to 80), usually with thin walls; and by the vacuolate older cells. Zoospores present favorable material for observation of withdrawal of flagella upon quiescence. The organism was reintroduced into sterile soil, the soil was then air-dried, and the viability of the organism in soil was measured during 1 year. Each month, a 0.5-g sample of soil was heated at 100 C for 1 hr prior to incubation in basal medium; a control sample was incubated, along with the treated soil, in continuous illumination. By means of 10-fold dilutions of such cultures, it was determined that more than 10,000 cells per 0.5 g of soil survived heat pretreatment and more than 100,000 cells per 0.5 g were viable in the desiccated soil after 1 year. STARR (1955) described the genus Spongiochloris, distinguishing it from other unicellular, zoospore-producing chlorococcacean algae on the basis of plastid type, presence of pyrenoids, and type of zoospore produced. Two species were described at that time, S. spongiosa and S. excentrica. MXIore recently Deason and Bold (1960) and Chantanachat and Bold (1962) characterized 3 additional species. All can be distinguished easily by means of both morphological and physiological attributes (Chantanachat and Bold, 1962). Survival of algal cells in dry soil, resistance to desiccation, and the morphology of cells in dry soils have been studied frequently (Bristol 1920, 1927; Fritsch, 1922; Fritsch and Haines, 1923). Trainor (1962) demonstrated that some algae in dry soils were extremely resistant to high temperatures. A number of organisms survived heat pretreatment at 100 C for 1 hr, and at least 1 organism in each of 2 different soils was viable after 1 hr at 140 C. The present study was undertaken in order to investigate the possibility of reintroducing an organism into sterile soil, to explore further its resistance to both airand oven-drying, as well as initiate a study of the role of this alga in soil. The organism in question is new to science and will be described. It was initially chosen because it can grow well over a wide temperature range and is also capable of growth in both light and darkness. MATERIALS AND METHODS-The alga described in this paper was isolated from a soil sample collected from a cornfield in Storrs, Connecticut, in October, 1958. In the initial isolation procedure, 0.5 g of soil was placed in a sterile 8-oz soft-glass bottle which contained 50 ml of basal medium, IN I Received for publication May 2, 1963. This investigation was supported by N S F grant G16106. (Trainor, 1958). This bottle was incubated under continuous illumination at 26 i 1 C. Although presence of algae was noted within a week, the organism was not isolated until the 6th week. Three other organisms, not discussed in this paper, also were isolated. The organism was established in axenic culture by using the standard techniques of repeated streaking and isolation of colonies from agar p ates. All experiments, other than initial incubation, were conducted at 24 -4 1 C. When continuous illumination was used, the intensity of fluorescent illumination was 400 ft-c. Slower growth of the organism for maintenance of stock cultures, as well as for observation under a more "natural" light cycle, was achieved under 12-hr diurnal illumination at an intensity of 200 ft-c. The organism was grown routinely in IN medium (Trainor, 1958). For comparison with other known species, soil extract (Pringsheim, 1949) and standard bacteriological nutrient broth were used. All ages of cells, from recently released zoospores to cells from 6-month-old cultures, were observed microscopically. These cultures were grown under both continuous and diurnal illumination. The following procedure was used for the introduction of the alga into soil. Twenty grams of soil, from which the alga had been isolated initially, were placed in a deep Petri dish and drysterilized at 160 C for 2 hr, and then allowed to cool. Fifty ml of a young, actively growing culture of Spongiochloris typica, with approximately 2 million cells/ml, were added aseptically. Thorough mixing was achieved by swirling the dish. The cover was then propped up slightly to facilitate drying. The dish was incubated under continuous illumination and was visibly dry within 1]0 days.

BERTSCH, WALTER F., and WILLIAM S. HILLMAN. (Yale U., New Haven, Conn.) The photoinhibition of growth in etiolated stem segments. I. Growth caused by sugars in Pisum. Amer. Jour. Bot. 48(6): 504-511. Illus. 1961.-Etiolated pea stem segments were used to study the red, far-red reversible photoinhibition of stem growth. The growth of immature, apical tissue was insensitive to light after excision from the seedling, unless a growth-promoting sugar (sucrose, glucose, fructose) was included in the medium. Red light specifically inhibited the growth caused by the sugars. Growth promotions of the same magnitude caused by indoleacetic acid or gibberellic acid were not reduced by irradiation of the tissue. The effects of various other sugars were tested. Arabinose and galactose, which do not promote growth, did not cause photosensitive growth in the isolated tissue. Mannose and xylose promoted growth at low concentrations, and this small promotion was inhibited by irradiation. Photoinhibition occurred even at inhibitory concentrations of mannose and xylose. The distinctness of light-sensitive growth is discussed, and the photoinhibition of a growth process which is limited by availability of sugars is suggested. THE PHOTOCONTROL of etiolation in higher plants is exerted at least partly through the red, far-red reversible photoreaction (Downs, 1955; Withrow, Klein and Elstad, 1957; Fujii, 1957; Thomson, 1959). Experimental systems involving excised tissues should be valuable for studying the processes that link the photoreaction itself to the final effects on growth. Most such studies have been conducted with tissues in which red light promotes growth (e.g., Liverman, Johnson and Starr, 1955; Klein, 1959); the results below, however, were obtained with etiolated pea stem segments in which red light inhibits growth under suitable conditions (Hillman, 1959). They indicate that the photosensitivity of the growth of such tissue depends on the composition of the medium used and that it is not the same if different substances are used to promote the same amount of growth. The photosensitivity of growth caused by sugars is the subject of the present paper; subsequent papers (Bertsch, in preparation) will deal in detail with the effects of metal ions and of indoleacetic and gibberellic acids in the same system. MATERIALS AND METHODS-Plant growth conditions-Seeds of Pisum sativum L., variety 'Alaska,' were obtained from Asgrow Inc., of New Haven, 1Received for publication January 10, 1961. 2Represents a portion of a dissertation presented by W.F.B. to the Faculty of the Graduate School of Yale University in partial fulfillment of the requirements for the degree of Doctor of Philosophy. W.F.B. was supported by pre-doctoral fellowship CF-9318 of the National Cancer Institute, U. S. Public Health Service; by a Yale University fellowship; and by funds provided to Yale University by the American Cancer Society Institutional Grant. W.S.H. was supported by National Science Foundation grant G-4433 to A. W. Galston. The authors are indebted to Professor Galston for his advice and criticism. 3Present address: Laboratoire du Phytotron, Gif-surYvette (Seine-et-Oise), France. Connecticut. They were soaked for 3-4 hr in tap water, sown in washed vermiculite (Mica-Gro Type B, supplied by Platt Seed Co., Branford, Connecticut), and grown in plastic flats in darkness at 2628 C. The developing seedlings were protected from the darkroom safe-lights by black cloth, and exposed only when the cloth was lifted to inspect or remove the plants. All manipulations were performed under dim-green safe-lights consisting of green fluorescent tubes wrapped with 4 layers of dark-green and 6 layers of amber cellophane. Selection of seedlings and preparation of tissueSeedlings were allowed to develop until a majority of the third internodes were about 30 mm long (7-8 days). Pea seedlings of the variety 'Alaska' obtained from the Asgrow Company developed a recurved hook when grown in darkness at temperatures between 26 and 28 C. In order to keep the material as homogeneous as possible, sections were harvested only from seedlings which had an apical curvature of 220?-280?, and third internodes from 15 mm to 65 mm long. Figure 1 is a picture comparing "acceptable" and "unacceptable" seedlings which were grown in the same flat. Seedlings were harvested by cutting above the vermiculite with a razor blade. A 5-mm segment was cut just below the apical hook of those seedlings which met the above requirements, and placed in buffer (0.02 M P04; KH2PO4-Na2HPO4, pH 6.0) in a shaded Petri dish. The device used to cut the segments was a lucite block with removable razor blades placed 5 mm apart. In certain experiments, tissues other than the apical 5 mm were used, and in these instances the particular tissue will be described. After enough segments had been cut, they were randomized by mixing in buffer. Appropriate ir

LIKE THE other vegetative organs of the peanu (Arachis hypogaea L.), the stem has been describe( only in a cursory manner by earlier authors an( largely in terms of its external features. Its histo logical features have not been adequately describe( and in particular there is no account of the anatom ical changes which occur in the stem during matur ation of the plant, nor any morphological evalu ation of the tissues of the stem in relation to the other vegetative organs of the plant. This papei presents a study of the form and structure of thstem of Arachis hyp,ogaea and, together with twc preceding papers (Yarbrough, 1949, 1957), com pletes an account of seedling development in ithi species. The materials used and methods followed here were identical with those described in the latte paper (1957). FORM OF THE STEM.-The stem of Arachis hac been described by Waldron (1919) and Reed (1924) as presenting no features unusual amono herbaceous dicotyledons. It is green and pubesceni with simple hairs having two basal cells. Very young internodes (i.e., those within the last 4-> expanded leaves) of the main axis show some angu larity in transverse section (fig. 4) while youn& internodes of lateral branches are, in contrast usually flattened elliptical in form. In both cate gories lower internodes, where the pith begins ta break down, may be angular in appearance. Stil' older internodes in which the vascular cambium has formed abundant secondary tissue are usually circular in cross-section (fig. 5). The orientatior of stems of Arachis has been described (Gregory et al., 1951) and is different in erect and prostrate varieties. The main axis, however, in all varietieJ is erect. ANATOMY OF THE STEM.-The epidermis of the stem is composed of small cells (fig. 2, 3) with a layer of cutin somewhat thicker than that found or most herbs. Stomates and crystal-bearing cells, de scribed earlier for the leaf (Yarbrough, 1957), are also found in this tissue. The hypodermis consist, of collenchyma, largely of the simple, "angular' type. Adjacent to the stomates the collenchyma i9 only a single layer in thickness while in interstomatal areas it may consist of 3-4 cell layers. The cortex consists of two main types of tissue, (1) the specializ,ed chlorenchyma which is concentrated beneath and lateral to the stomates but may be con. tinuous in branching lines of cells between these points, and (2) the inner, larger, thin-walled paren.

WALKER, S. (Liverpool U., England.) Cytogenetic studies in the Dryopteris spinulosa complex. I. Amer. Jour. Bot. 48(7): 607-614. Illus. 1961.-A comparison of the D. spinulosa complex, as represented in North America, is made with that in Europe. Although in North America, other Dryopteris species are known to hybridize with members of the complex, the report has been limited to species which compare directly with those in Europe. Three tetraploid species and 2 diploids, the latter representing at least 1 ancestral genome in the complex, are discussed together with the interspecific hybrids. Synthetic hybrids have been produced, involving taxa from both sides of the Atlantic. Evidence from these hybrids suggests that D. intermedia, of North America, and D. maderensis, of Madeira, are conspecific. CYTOGENETICAL investigation of the genus Dryopteris has revealed a reticulate pattern of evolution. The results of studies in the Dryopteris spinulosa complex in Europe were published in an earlier paper (Walker, 1955). Similar work on this complex, as represented in North America, has led to a much wider investigation involving many other associated Dryopteris species and hybrids (briefly reported, Walker, 1959). It has become impossible to present all the data obtained in a single paper and, to facilitate a direct comparison with the earlier work, the present report covers only those representative species and hybrids found in North America which are comparable with the members of the complex in Europe. The relationships between the various taxa on both sides of the Atlantic have been investigated by the synthesis of a number of hybrids between them. Details of the wider investigation into the genus Dryopteris in North America will be published separately. The D. spinulosa complex in Europe includes 3 allotetraploid species, D. cristata (L.) Gray, D. spinulosa (O. F. Muell.) Watt and D. dilatata (Hoffm.) Gray, which require 4 ancestral diploids to account for their origin. One of these ancestral diploids, the genome of which is common to both D. spinilosa and D. dilatata, is now represented by 2 distinct species (previously regarded as diploid forms of "D. dilatata"); one, D. assimilis S. Walker,2 is found in Britain and on the continent of Europe, the other, D. maderensis Alston,3 on the island of Madeira. These species are very different in their pinnation and spore size, but cytogenetic evidence indicates they are descendants from the same ancestral diploid (Walker, 1955). From crosses between D. assimilis and D. maderensis, 2 1 Received for publication January 24, 1961. 2 Dryopteris assimilis, sp. nov., S. Walker. Based on Lastrea dilatata var. alpina. Moore, Handbook Brit. Ferns, 3rd ed., 126 (1857). Dryopteris dilatata var. alpina Moore, Brit. Ferns, I, 225 (1859). 3 Dryopteris maderensis Alston, published erroneously as D. maderensis (Milde) Alston (Alston, 1956), but Milde used 'forma maderensis' as a descriptive phase, not as a taxonomic forma (Milde, 1867). plants were produced showing normal meiotic pairing but approx. 40% abortive spores which suggests gene imbalance (Walker, unpublished). More hybrids are necessary to establish this. Wild hybrids occur within the complex both at the tetraploid and triploid levels. Chromosome counts for some members of the complex in North America have been reported previously (Britton, 1953; Manton and Walker, 1953), and detailed spore studies have been carried out (Crane, 1953). More material has been sampled by me and additional counts made. This complex has caused confusion to taxonomists; hence R. C. Benedict, with a long-standing interest (1924) in the spinulose dryopterids, referred to them (1954) as "one of the most disputed of our native fern groups." Separation of the various taxa into distinct species or varieties still remains a problem to most authors; this is particularly exemplified in the new Britton and Brown Flora (Gleason, 1952) and Gray's Manual of Botany, 8th ed. (Fernald, 1950). The presentation of the latter was criticized by Wherry (1952). Nomenclature for the various taxa is at present under consideration. The use of the species epithet, C"austriaca,"' in place of "dilatata,." as in the Flora of the British Isles (Clapham, Tutin and Warburg, 1952), and in the new Britton and Brown Flora (Gleason, 1952) has already been criticized (Walker, 1955; cf. Morton, 1958). The name. D. spinulosa (0. F. Muell.) Watt, is also invalid but is retained in this paper since, so far, no satisfactory replacement has been published. Neither D. lanceolata-cristata (Hoffm.) Alston (Alston, 1957) nor D. carthusiana (Villars) H. P. Fuchs (Fuchs, 1958) appear to be satisfactory substitutes. The following is a list of the taxa discussed in this paper: D. cristata (L.) Gray; D. spinulosa (0. F. Muell.) Watt; D. campyloptera (Kunze) Clarkson; (syn. D. spinulosa var. americana [Fisch.] Fernald),; D. intermedia (Muhl.) Gray (syn. D. spinulosa var. intermedia [Muhl.] Underw.); "D. dilatata" (Hoffm.) Gray; D. X uliginosa (A.