Mineral Agar Research Articles

The Effect of Sucrose on the Differentiation of Excised Fern Leaf Tissue into Either Gametophytes of Sporophytes

Excised juvenile leaves of Microgramma vacciniifolia (Polypodiaceae) develop sporophytic regenerants when grown on mineral agar with sucrose. The ratio of sporophytes to gametophytes produced from the leaf tissue increases with higher percentages of sucrose such that at 4% sucrose, the induction of aposporous gametophytes is a rare occurrence. Experiments varying the osmotic potential with sorbitol and those holding the osmotic potential of the culture medium constant while varying the sucrose level indicate that the effect of sucrose on the differentiation of fern leaf tissue into either gametophyte or sporophyte is nutritional rather than osmotic. A significant effect of sucrose in altering the differentiation of fern leaf tissue is the increased rate of senescence promoted by high sucrose concentrations.

Test Tube Reproduction of Heterodera a Venae On Resistant and Susceptible Wheats

Tests for resistance to Australian Heterodera avenae confirmed a high resistance to cyst development in two introduced wheats, AUS. 10894 and AUS. 11577 (cv. Loros). A test tube method is described for the mono-axenic culture of wheat seedlings on mineral agar medium containing larvae hatched from individual cysts. In ten such cultures with susceptible Summit wheat, cyst counts per plant ranged from 27 to 44; on average over 50 % of the hatched larvae became sexually mature. In 100 similar cultures with the two resistant wheats, many developed no females and a maximum of nine white cysts was found, although large numbers of males developed. The results agreed with those from pot tests using natural soil infestation, but since test tube cultures facilitated total recovery of all eggs and nematodes from each cyst, the percentage of sexually mature males and females could also be determined. The percentages of males and females on susceptible roots were variable, but averaged 1.33 males to 1 female, whereas resistant wheats produced over 20 males to 1 female. No difference was found in the overall reaction of AUS. 10894 and AUS. 11577 to H. avenae sampled from seventeen locations. The method may be useful for various nematological investigations and for selection in backcross breeding programmes.

Soil streptomycetes and bacteria related to lysis of Blastomyces dermatitidis.

Twenty-eight microorganisms (22 streptomycetes and 6 bacteria) isolated from unsterilized soils baited with yeast cells of B. dermatitidis were purified on a mineral agar containing B. dermatitidis yeast cells as a carbon source. Twenty of these Streptomyces spp. and 2 species of the genus Bacillus when streaked individually on Sabouraud's Dextrose Agar, previously seeded with living B. dermatitidis yeast cells, inhibited mycelial growth. One species of Streptomyces was shown to be as inhibiting as Streptomyces nodosus Trejo (the Amphotericin-producing streptomycete). Six of the lytic Streptomyces spp. were individually shown to inhibit the growth of B. dermatitidis when they were introduced into sterile soil. Our limited experiments did not show that antibiotics caused inhibition of B. dermatitidis in media or in soil, but it was thought that the disappearance of B. dermatitidis from natural soils might be caused by inhibition of growth by antibiotics (or other metabolites), by enzymatic degradation of dead cells, and by autolysis from nutrient deprivation. Since living B. dermatitidis cells were not lysed directly by inhibitory microorganisms from natural soils, chemical soil factors essential for the destruction of walls of living fungal cells could have been destroyed in the sterilization of the soil.

Photomorphogenesis in Arabidopsis thaliana (L.) Heynh

Arabidopsis seeds were germinated on sterile mineral agar supplemented with 1% glucose and cultured under continuous light regimes. With 4-hour incandescent plus 20-hour monochromatic illumination in the region from 400 to 485 nanometers there was effective floral induction at an intensity of 100 microwatts per square centimeter. Exclusion of far red wave lengths from the 4-hour incandescent period sharply reduced the effectiveness of subsequent monochromatic blue light in promoting floral induction. Delayed floral induction occurred under continuous incandescent light lacking far red and was attributable to the blue wave lengths. Continuous 485 nanometer (100 microwatts per square centimeter) exposure without any white light treatment during the postgermination growth period was ineffective in floral induction and meristem development. Light at 730 nanometers under the same conditions was partially effective, whereas energy between 500 and 700 nanometers was completely ineffective. When continuous monochromatic light at a 3-fold higher energy level was administered, all photomorphogenic responses were accomplished with 485 nanometer light, including germination and 100% floral induction without any white light treatment at any time during the experiment. Almost equal quantum effectiveness was calculated when equivalent quantum flux densities in the region from 710 to 740 nanometers or at 485 nanometers were used. It is postulated that floral induction in Arabidopsis may be the result of a continuous excitation of a stable form of far red-absorbing phytochrome localized in or on a membrane, and that excitation can be either by direct absorption of energy by far red-absorbing phytochrome or by transfer from an accessory pigment.

UV sensitivity of germinating culture of Bacillus cereus.

The spores ofBacillus cereus can be germinated in reasonable synchrony in rich media. The survival of germinating cells after UV irradiation is strongly affected by repair. When plating on mineral agar a linear decrease of survival is obtained between 20–60 min of germination, whereas the survival on nutrient agar shows only minor fluctuations. The difference in survival on these two media which reached 1–2 orders of magnitude is due mostly to the shape of the shoulder on the survival curve. Survival on mineral agar may be increased by postincubation in complex media or by preincubation in mineral media. It was concluded that repression and derepression of biosynthetic pathways was responsible for the different efficiency of repair.

DEVELOPMENT OF VEGETATIVE AND FLORAL MERISTEMS OF ARABIDOPSIS THALIANA

Seeds were sown on mineral agar and grown under axenic constant conditions. Floral transition and flower primordia were observed at approximately 172 and 220 hr, respectively. The vegetative apex was flat in outline for 160 hr, after which time it was slightly dome shaped; cytohistological zonation was not readily apparent. The inflorescence apex (172 hr) was markedly convex and possessed 3–5 and 4–6 cell layers in the most axial and lateral portions of the apex, respectively. The nucleoli of cells of the T2 layer were larger than cells of the T1 and outer corpus during vegetative development; in contrast, they were equal in size at reproductive stages.

Changes in Behavior of Mosses Treated with Ultrasound

Short, growing filaments of moss protonema [Physcomitrium turbinatum (Mx.) Bridel] develop readily on mineral agar into normal plants; they produce branch filaments by a regular pattern of lateral divisions of the subapical cells and extend themselves linearly by divisions of the apical cell. Divisions of intercalary cells that produce two in-line daughters in the main axis are quite rare, elongation of the axis being essentially restricted to the apical cell. Brief, low-amplitude, topically applied sonic vibration of subapical, individual cells in the filament frequently results in the division of the treated cell or one of its near or distant intercalary neighbors so as to produce in-line daughters. Persistent differences in growth pattern as compared to normal plants have been observed in several cultures of moss plants derived from single, treated, apical cells; they are described.

LEAF-NODULE SYMBIOSIS. I. ENDOPHYTE OF PSYCHOTRIA BACTERIOPHILA.

Centifanto, Ysolina M. (University of Florida, Gainesville), and Warren S. Silver. Leafnodule symbiosis. I. Endophyte of Psychotria bacteriophila. J. Bacteriol. 88:776-781. 1964.-The leaf-nodule endophyte of Psychotria bacteriophila has been repeatedly isolated in pure culture from germinating seedlings and young leaves on a nitrogen-free mineral agar. Its morphological, serological, and cultural characteristics place it within the Klebsiella-Aerobacter group. The endophyte has been provisionally named Klebsiella rubiacearum. Pure cultures fix N(2) under anaerobic conditions with good efficiency (4.54 mug of N fixed/mg of glucose utilized in 3 days). Nitrogen fixation by pure cultures is intimately related to pyruvate and hydrogen metabolism.

Callus Tissues from the Mosses Polytrichum and Atrichum

Callus-like tissues, isolated from protonemal cultures of two species of mosses, grow vigorously and without marked differentiation on media containing sucrose, casamino acids, and coconut milk. On mineral agar and on media containing sorbitol the tissue from Polytrichum (found diploid) reverts to the growth pattern of apparently normal moss plants.

CYTOLOGICAL DIFFERENTIATION IN GAILLARDIA PULCHELLA

THE BLANKET FLOWER, a widespread composite of the southern United States, is morphologically and cytologically variable. No detailed studies of this species have been made, other than the taxonomic treatments by Rydberg (1915) and Biddulph (1944). Gaillardia pulchella has had a long and confused taxonomic history. It has been split into several distinct species by some workers (Rydberg, 1915; Nelson, 1926) and confused with the related Gaillardia aristata by others. METHODS.Eighteen collections of seed of Gaillardia pulchella (table 1) from different parts of the species range were grown in a standard habitat at the Indiana University experimental garden. Chromosome counts of these races were determined from microsporocyte and root-tip preparations. Morphological characters of these wild races and their artificially produced hybrids are given in fig. 1 and 2. Reciprocal hybrids between races were produced by bagging heads before the flowers opened, then transferring pollen to the fresh stigmas daily until all flowers had been pollinated. In the following discussion of hybrids, the formulae refer to both reciprocals. Mature embryos were cultured in order to facilitate germination. Embryos were limited in number, and the abnormal germination of some decreased their chances of survival under greenhouse conditions. No morphological differences were found in reciprocals, and the frequency of crossing in either direction was the same. In culturing embryos, Tottingham's modification of Knop's solution (Miller, 1938) was used in half the normal concentrotion with 1 per cent agar added. Achenes were sterilized in sodium hypoclorite (commercial Chlorox 1 part: distilled water 7 parts) for 1 hr., dissected, and embryos were planted vertically in 2.5 cc. of mineral agar in shell vials. Cotyledons began growth in two or three days, and seedlings were transplanted to 3-in. pots approximately four weeks later. When large enough, hybrids were transplanted to the garden. Hybrids which reached maturity grew much more vigorously and bloomed more profusely than either parent. They were morphologically intermediate in most respects, and 9 selected hybrids are evaluated in fig. 2. No character found in one parent clearly dominated the contrasting character in -the other parent. All hybrids were as tall or taller than

Frenching Symptoms Produced in Nicotiana Tabacum and Nicotiana Rustica with Optical Isomers of Isoleucine and Leucine and with Bacillus Cereus Toxin

It has been suggested by the writer that the stages leading to abnormal growth in frenching of field tobacco may be as follows: bacterial soil toxin ?? receptor -? isoleucine ?? frenching. The chemical composition of the bacterial soil toxin is unknown as yet. However, natural leucine as well as isoleucine was found to be capable of producing these growth abnormalities in Nicotiana rustica. Natural isoleucine can produce the symptoms of frenching in tobacco (1, 3) and in N. rustica (5). The natural isomer of leucine is also effective with N. rustica (5). The actions of other optical isomers of isoleucine and leucine are unknown. Frenching of tobacco in the laboratory has been produced with a common soil microorganism, Bacillus cereus Fr. and Fr. (2, 4). Though this bacillus is not known to invade the tissues of the tobacco plant, rhizosphere and rhizoplane counts increase by 65 and 200%, respectively, when frenching occurs in the field (6). Diffus?tes of B. cereus in aseptic culture appear to be capable of exercising a similar action (4). The absence of free amino acids in soil and the marked increase of free isoleucine in affected leaves led to the assumption that the bacterial toxin and isoleucine were probably not identical (7). No free amino acids could be detected in soil by the Van Slyke method for determining free amino acids which is sensitive to 0.01% or 100 p.p.m., the approximate concentration at which isoleucine would have to be present to be effective. Data in this paper cover the frenching responses of tobacco treated with optically active isomers of isoleucine, leucine, and two dipeptides of leucine. The effects of various impure preparations of bacterial toxin from B. cereus were also studied. Methods The tests upon seedlings of the action of organic compounds and of bacterial toxin were made necessarily in aseptic culture. The methods used have been published in previous papers (1, 2). Briefly, they consisted in growing tobacco seedlings in aseptic culture on mineral agar from sterilized seed. Either 200 ml. or 1000 ml. flasks were used. A light intensity of about 500 foot-candles was furnished by white fluorescent lamps. Usually, the experiments with N. tabacum were carried out in 200 ml. flasks at 25? C, and those with N. rustica in 1000 ml. flasks at room temperature. The 302

THE GROWTH CAPACITY OF THE SUNFLOWER HYPOCOTYL

agar to which indole-3-acetic acid (IAA) had been added, (3) on sucrose mineral agar after inoculation of the fragment with crown-gall bacteria. Five aspects of growth were measured: increase in length, breadth, fresh and dry weight, and production of adventitious roots. Sunflower seeds var. Russian Giant were sterilized by being immersed for two minutes in a 1% solution of sodium hypochlorite. They were then -washed in three changes of sterile distilled water and soaked in water for two hours. The embryos were then removed from their seed coats, transferred aseptically to tubes (25 mm. ? 200 mm.) each containing 10 ml. nutrient agar (2% sucrose + White's mineral solution+ 1% agar) and cultured at 25? C in continuous light from four 40 watt fluorescent lamps. When the hypocotyls attained a length of 100 mm. the embryos were drawn out of their tubes and the hypocotyls severed just above the root and immediately below the cotyledons. The hypocotyls were cut into segments five mm. long which were placed radicle end uppermost in agglutination tubes (10 mm. ? 75 mm.) containing three ml. of nutrient agar. They were incubated for 10 days in darkness at 25? C, then removed, photographed, and weighed. The photographs were made by projecting the image of the fragment through an enlarger onto printing paper. The magnification used was 1.7 diameters in most instances. The length and breadth of the fragment and the size of the tumors were estimated from these shadowgraphs. Agrobacterium tumefaciens, strain B.P., was used in these experiments, suspensions being prepared from 24 hour slopes. Indoleacetic acid was incorporated into the nutrient agar for the hypocotyl fragments after autoclaving. Growth of hypocotyl fragments on sucrose mineral agar Ten sunflower hypocotyls, each about 100 mm. long, were divided into 20 segments five mm. long; and every alternate fragment beginning with

Penicillin as a Plant Hormone

IN the course of investigations on the effects of some antibiotics on plant tissues in vitro, it was discovered that commercial penicillin sodium (Squibb) has a potent effect on the growth of excised fragments of sunflower stem tissue cultured on White's sucrose mineral agar. A concentration of 500 units per c.c. of this substance caused great proliferation of the cambial tissue without production of visible roots (Fig. a). 5 units per c.c. also caused proliferation of cambial tissue accompanied by an abundant production of roots (Fig.b). Stem tissue cultured in the absence of the antibiotic showed neither proliferation nor root production (Fig. c). As cambial proliferation and production of roots are both induced in sunflower stem tissue by indole acetic acid, fragments of stem tissue were also cultured in the presence of pure penicillins. The reactions of such stem fragments to different concentrations of these substances as well as to commercial penicillin and indole acetic acid are given in the accompanying table.