Knop's Medium Research Articles

Establishment and growth characterization of suspension culture of cells from the moss, Sphagnum imbrication

A green‐pigmented callus of the moss, Sphagnum imbricatum Hornsch. ex. Russ., was induced and a chlorophyllous cell suspension culture was established using a modified Murashige and Skoog's medium without plant hormones. Cell growth in the light in the presence of glucose started after a short lag and was exponential for 12 days. The chlorophyll level was about 15 μg (mg cell dry weight)−1 and photosynthetic activity ca 20 to 50 μmol O2 (mg chlorophyll)−1h−1. Cell growth in the light was negligible in the absence of glucose under ordinary air, but photoautotrophic growth was possible under elevated CO2 concentrations. In the dark, the moss cells grew heterotrophically and continued to synthesize chlorophyll, although at a much reduced rate. The suspension‐cultured cells redifferentiated protonemata and shoots when transferred to solid Knop's medium. In contrast to the callus cells, which could not assimilate nitrate, redifferentiated plantlets could use nitrate as the sole nitrogen source.

Callus formation and induction of a cell suspension culture from Acacia senegal

Cell proliferation has been induced from the cambial zone of a branch of Acacia senegal, kept on the basal Knop and Ball medium in the presence of auxin. Transferred on the more complete nutrient medium of Schenk and Hildebrandt, the colonies gave rise to several cell lines. One of the friable lines, consisting of aggregates of parenchymatous cells, gave a cell suspension culture in an agitated liquid medium which is maintained as a strain of illimited growth. The heterogeneous suspension did not undergo noticeable changes after eight transfers. Aggregates collected on a 1000-μm nylon seive were able to grow on a solid medium and gave back a friable callus similar to the initial colonies.

The Hormonal Regulation of Protonema Development in Mosses III. Auxin-Resistant Mutants of the Moss Funaria hygrometrica Hedw.

Summary Mutants altered in their response to auxins were isolated from the moss Funaria hygrometrica by screening the mutagenized spores for growth on high concentrations of naphthaleneacetic acid. These mutants differ in the extent of their inability to form caulonema on Knop's medium (minimal medium without auxin), a behaviour also exhibited by wild type protonema grown in dim light. Of the four auxin-resistant mutants initially chosen, NAR-2 is the most distinct from the wild type by remaining totally in chloronema state on minimal medium. It exhibits caulonema differentiation only when subjected to exogenous auxins or the precursors of IAA. The sensitivity to IAA is not changed in comparison to the wild type. The auxin deficiency of the mutant NAR-2 seems neither to be due to a block of the auxin biosynthetic pathway nor to a block in its uptake/transport mechanism. However a 3 times higher activity of IAA oxidase has been found in the mutant as compared to the wild type. Auxin treatment stimulates the auxin oxidase activity in soluble and particulate fractions. In contrast, peroxidase activity is reduced in the mutant. The results support the hypothesis that the morphogenetic step of caulonema formation is regulated by the internal auxin level.

Relations entre hypolignification et état vitreux chez Salix babylonica en culture in vitro. Rôle de la nutrition ammoniacale

The relationship between nitrogen-fed willow cuttings cultured in vitro and the appearance of vitreous plants has been studied. By using two basal media (Murashige and Skoog medium and Knop medium half diluted) an in vitro culture scheme has been developed which allows the control of the vitrification process by varying the nitrogen supply in the culture media. The results obtained agree that the vitrification of the cloned plants is directly associated with the ammonium content of the culture medium. In vitrified plants, lignin content of the shoot is reduced by 50%. Correlatively, histological controls at different levels of the shoot of the plantlets show that the reduction of the xylem tissue can reach 70%.

Promotion of Fern Rhizoid Elongation by Metal Ions and the Function of the Spore Coat as an Ion Reservoir

The perine, or outer coat, of spores of the fern Onoclea sensibilis L. may be chemically removed by a brief treatment with dilute NaClO. Treated spores germinate normally on glass-redistilled H(2)O, but elongation of the rhizoid which is differentiated during germination is severely limited. Rhizoid elongation in perine-free spores, however, is normal when the spores are germinated on Knop's mineral medium or on single-salt solutions of Ca(2+), Mn(2+), or Mg(2+). In intact spores which retain their perine, rhizoid elongation is normal on distilled H(2)O, and the perine serves as a source of ions which are available to the spores and can sustain rhizoid elongation, even when the external medium is deficient. Electron micrographs show that there are structural differences in the rhizoid wall between perine-free spores germinated on distilled H(2)O or on nutrient solutions, and also a difference in the number of vesicles in the apical cytoplasm. Localization of Mg(2+) and Ca(2+) in the elongating rhizoid can be visualized with chlorotetracycline fluorescence. No concentration of these ions can be detected by this technique in the small rhizoid initial cell before cell elongation begins.

Developmental Morphology of Platycerium coronarium (Koenig) Desv

The growth habit and certain developmental aspects of Platycerium coronarium (stag's horn fern) are described. Starting from the primordial stage, the nest and pendulous leaves develop to maturity in 90 and 80 days respectively. The fertile lobe, which is part of the pendulous leaf, reaches its maximum size in 40 days. The morphogenesis of the nest leaf is more variable, and it matures and deteriorates earlier than the pendulous leaf. The acrostichoid sorus formation is completed in 3 weeks from inception and spore dispersal takes place when the fertile lobe is about 100 days old. The area of the fertile lobe and number of spores produced were determined. On Knop's agar medium the gametophytes develop in 2 months and 85 per cent of them are unisexual (both male and female) and 15 per cent bisexual. Less than 1 per cent of the gametophytes give rise to sporophytes. The juvenile leaves are simple, displaying open dichotomous venation; the first nest and pendulous leaves are produced 24 months after the date of spore germination.

Development of moss plants from isolated and regenerated protoplasts

Proroplasts were enzymatically isolated from protonema of Funaria hygrometrica L. SIBTH., cultured from the spores on Knop's solid medium. The protoplasts were incubated in hanging drops in liquid Knop's medium with 0.3 M mannitol and 0.5% glucose. Isolation of protoplasts was obtained successfully, through the action of Macerozyme®, cellulase and Rohament P® mixed with mannitol. Cultured protoplasts began to regenerate walls within 24 h, followed by the enlargement and divisions within 48 h. These protoplasts started producing active and long strands of protonema within 5 days, while well-differentiated leafy gametophytes were developed just in 20–23 days.

Behavior of downy mildew fungus of Japanese radish on modified Knop's medium.

Behavior of downy mildew fungus of Japanese radish on modified Knop's medium.

Prothallus Morphology in Some Tectarioid Ferns

Among the tectarioid ferns, studies of the prothalli have been made in Arcypteris and Pleocnemia (Atkinson, 1970), Quercifilix (Nayar, 1960), and Tectaria (Kachroo, 1956; Mahabale & Venkateswaran, 1959; Stokey, 1960; Nayar & Kaur, 1964; Srivastava, 1968). In other tectarioid fern genera no such studies have been made, although Stokey (1960, pp. 82-84) reported the hair types occurring in some genera, including Ctenitis, Heterogonium, and Pteridrys. The present study concerns ten species of tectarioid ferns: Ctenitis ampla (Humb. & Bonpl. ex Willd.) Ching,2 C. currori (Mett. ex Kuhn) Tardieu, C. eriocaulis (Fee) Alston, C. recedens (J. Smith ex Moore) Copel., Pteridrys australis Ching ex C. Chr. & Ching, Tectaria devexa (Kunze) Copel., T. fuscipes (Wall. ex Bedd.) C. Chr., T. heracleifolia (Willd.) Underw., T. leuzeana (Gaud.) Copel., and T. variolosa (Wall. ex Hook.) C. Chr. Spores of all the species except T. fuscipes and T. leuzeana were obtained by Prof. B. K. Nayar, of Calicut University, during his visit to England in 1972 from plants grown at the Royal Botanic Gardens, Kew. Spores of T. devexa, T. fuscipes, and T. variolosa were taken from plants grown at the National Botanic Gardens, Lucknow. Observations were made on prothalli raised on sterilized Knop's agar medium maintained at 23-25?C under 600 ft-c light intensity for 8 hours daily. OBSERVATIONS

Redetermination of cultured root tips to leafy shoots in Selaginella willdenovii

Analysis of root growth in Selaginella willdenovii has indicated that a new root is initiated every 6.8 days (S.E. ± 0.42). The distribution of root growth on intact plants was examined and it was found that the zone of most rapid growth occurred about 3.6 mm from the tip. Our previous studies have shown that auxin is transported acropetally in root segments and intact roots. Thus the net movement of auxin in roots is toward the growing tip. When root tips were grown in sterile culture on Knop's medium, about 20% of them developed leaf primordia at or along the flanks of the root tip and continued to grow as ots. When 1 mm root tips were grown on Knop's medium supplemented with 0.5 μmM naphthaleneacetic acid (NAA) only roots developed and shoots were never observed. Histological preparations have been made of root tips which have become re-determined as shoots and the appearance is that of a shoot primordium with a distinctive apical dome and leaf primordia developing from the peripheral zone.

In vitro Post‐germination Growth and Development of Embryos of Alectra (Scrophulariaceae)

Abstract In vivo, seeds of the obligate root parasite Alectra vogelii Benth. (Scrophulariaceae) germinate only after being soaked in water for a period of time (pretreatment) followed by stimulation by certain factors exuded from a host root. Germinated seedlings do not develop beyond radicle emergence, and finally die, unless their radicles make contact with and penetrate into a host root conductive system. In vitro, germinated embryos obtained by exposing sterilized and pretreated seeds to root exudate of Vigna unguiculata were aseptically cultured on Knop's, White's and Murashige and Skoog's media. The embryos grew into seedlings with shoots and roots on a medium containing mineral salts and sucrose, but not on mineral salts alone. Seedling performance was generally not improved when the mineral salts‐sucrose media were supplemented with vitamins. Shoot extension growth was better on Murashige and Skoog's mineral salts‐sucrose medium than on Knop's or White's medium. However, seedling development was greatly boosted when cultivated on White's minerals salts‐sucrose medium supplemented with coconut milk. Seedlings turned green on transfer to light but did not flower. The successful culture of these embryos and seedlings on a simple, chemically defined medium of mineral salts and sugar suggests that these nutrient components are the minimal external requirements for stimulation and support of normal seedling growth. These may be obtained in vivo by the parasite's tapping of the host root conductive system.

In vitro studies on the induction of sporogenous tissue on leaves of cinnamon fern. III. The role of reduced nitrogen

Excised set III leaf primordia of cinnamon fern cultured on either Knop's or Knudson's medium containing ammonium sulfate produced fertile leaves provided that the media included the appropriate concentration of sugar and the cultures were exposed to the optimal environmental factors. The highest percentage of fertility (about 80%) was obtained on leaves cultured on media containing 6% sucrose and the usual concentration (250 mg/liter) of ammonium sulfate, and placed in continuous darkness at 26 °C. However, leaf primordia cultured under the same conditions without ammonium sulfate or this substance included at concentrations 3 × or higher than the usual amount produced sterile leaves only.From other experimental evidence, a possible mechanism by which sugar supply and ammonium ions influence development is postulated and discussed for the induction of sporangia on cultured cinnamon fern leaves. However, further research is necessary to uncover the biochemical role of reduced nitrogen and carbohydrate in leaf fertility.

Förderung von protonemawachstum und knospenbildung bei dem laubmoos Funaria hygrometrica durch pilze

Summary Protonema of the moss Funaria hygrometrica was grown on a Knop medium plus agar together with one of the fungi: Aspergillus niger, Alternaria solani, Alternaria spec. and Penicillium spec. Growth was increased by the fungi. Early addition of the fungus hastened bud formation. The same was true when the mycelium was added later. The only exception made Penicillium spec. which inhibited growth when added 8 days after moss germination. One gets the same growth promotion after having completely removed the fungus from the agar where it was grown during 13 days before transfering the moss spores to this agar. The effect of factor H (bud forming and growth promoting factor extracted from moss protonemata [ Klein 1967]) either added directly to the substrate or indirectly by preculturing moss on the substrate is not changed by the fungus. The fungal effect is therefore due to a substance produced by the fungus. The effect is not caused by degradation of regulators produced by the moss.

Morphology of the Spores and Prothallus of Christiopteris tricuspis

Christiopteris Copel. is a small genus of epiphytic, polypodiaceous ferns that has morphologically better-known sporophytes than many other genera of leptosporangiate ferns. Three species are recognized, one of Sikkim, Annam, and Malaya, another of the Philippines, and the third of New Caledonia. Bower (1928, pp. 213-218) was much impressed by the diplodesmic vascular system of the reduced fertile lamina of C. tricuspis (Hook.) Christ, and he made a somewhat detailed study of the sporophyte, concluding that it represents a primitive form in the polypodioid phylum, along with Cheiropleuria and Dipteris. Holttum (1954, p. 211) and Copeland (1947, p. 179) regard the genus as probably related to Crypsinus. Fertile material of this rather rare genus is not easily obtained, and little is known regarding the spores and prothalli. Tn October, 1964, Prof. R. E. Holttuml sent me a bit of a fertile frond of C. tricuspis that was cultivated at Kew (originally collected in Malaya). Spores from this sample were promptly cultured on Knop's Agar medium (Nayar, 1962a) maintained at a temperature of 24 ? 2?C under a light intensity of 600 ft-c. No prothalli grew to the point of archegonium formation, although they were in culture for nearly a year. Further samples of spores were obtained in 1965, but again the prothalli could not be maintained long enough to produce archegonia. Spores (stored in plastic bags in a refrigerator) lost their viability in about two months. Techniques followed in the study of the spores are the same as reported earlier (Nayar, 1964; Nayar and Devi, 1964a). For the study of fresh spores, samples mounted in acetocarmine were used in addition to those mounted in glycerine jelly.

STUDIES ON STRIGA SENEGALENSIS III. IN VITRO CULTURE OF SEEDLINGS. ESTABLISHMENT OF CULTURES

Striga senegalensis seeds were cultured on Knop's and modified Murashige‐Skoog's medium. An aqueous root exudate/extract of the host (Sorghum vulgare) was found essential for the germination of seeds in vitro. The seedlings grew better on a medium containing sucrose than on mineral salts alone. Only limited growth was achieved on Knop's medium. Better growth was supported by Murashige‐Skoog's medium. By transfer to larger culture vessels seedlings were kept in the latter for several months. The seedlings developed chlorophyllous shoots in light and flowered after 4–4½ months. The successful culture of these seedlings in a simple medium of only mineral salts and sugar indicates that the parasitic seedling depends on the host for only water, mineral salts and sugar and not for more elaborate substances.

AN INDOLEACETIC ACID 'OXIDASE' SYSTEM IN THE MYCELIUM OF MELAMPSORA LINI (PERS.) LEV.

Flax rust mycelium was grown on cotyledons of flax in a modified Knop's medium. The mycelium produced and apparently secreted a typical peroxidase-mediated indoleacetic acid 'oxidase'. Decarboxylation of radioactive indoleacetate (—C14OOH) was stimulated by Mn++ (10−4 and 10−3M), dichlorophenol (10−4M), and resorcinol (10−4M). Catechol (10−4M) and pyrogallol (10−4M) inhibited the reaction. Moreover, pyrogallol competitively inhibited the reaction in the presence of resorcinol. All four phenols increased oxygen uptake by the mycelium, catechol and pyrogallol being the most effective. Cu++ (10−3M) inhibited the reaction by 60%; cyanide (10−3M) and diethydithiocarbamate (2 × 10−4M) were almost completely inhibitory. The results are discussed briefly in relation to host–parasite interactions and the control of indoleacetic acid oxidase activity in vivo.

A Contribution Toward Lichen Synthesis

The synthesis of lichens in the laboratory, beginning with the isolated algal and fungal symbionts, has long been the dream of lichenologists. Despite numerous attempts (a good history is given by Werner 1927) and a few reported successes (Bonnier 1889, Thomas 1939), the problem still remains unsolved and is as enigmatic today as it was some 90 years ago when the true nature of lichens was discovered. The experiments described in this paper have resulted in some interesting observations regarding the initial stages of lichen synthesis. Several of the results obtained deviate quite markedly from those published by earlier workers, notably those of Gaston Bonnier, who has claimed to have achieved many true lichen syntheses. Bonnier described synthetic culture in which 5 days after germination the clasping hyphae had laid hold of the algae and symbiotic growth had begun, a small lichen thallus being formed some 50 days later, with subsequent apothecial formation. In the light of my experiments this is indeed a remarkable feat. Bonnier's experiments contain numerous inconsistencies and unexplained points (Thomas 1939, p. 187); these, coupled with the fact that no one has been able to duplicate his experiments, make one hesitant in accepting his results. The lichen used for our experiments was Lecanora dispersa (Pers.) Rohl., an urban, saxicolous form growing quite conveniently on the concrete ledge of the basement window of the Farlow Herbarium. The procedure, carried out under sterile conditions and using sterile apparatus and cultures, may be outlined as follows: A non-nutrient agar (Noble's Agar) containing Knop's mineralsolution medium (pH 7.3) was employed. Immediately after autoclaving, this medium was pipetted into a van Tieghem cell and a carefully washed cover-slip was then placed over this filled cell and lifted off again

Production of aerial mycelium and uredospores by Melampsora lini (Pers.) Lev. on flax leaves in tissue culture.

Dense, felt-like growth of aerial mycelium of Melampsora lini (Pers.) Lév. was obtained when surface-sterilized, rust-infected cotyledons of flax were put on a modified Knop medium, containing fresh, ripe coconut milk, sucrose, and Difco Bacto-Agar. The mycelium remained fully dependent on the living host tissue, but could easily be collected free from host material in quantities sufficient for respiration and small scale chemical studies. Slight modification of the method allowed production of appreciable quantities of uredospores free from contaminating microorganisms.

Some Physical Properties of the Nuclear Membrane

During the last 50 years several investigators have described processes which bring into evidence such physical properties of nuclear membranes as resistance to deformation, extensibility, elasticity, etc. (Albrecht, Mottier, Kite, Chambers, Nemec). Our observations made on resting nuclei of the embryonic region of the onion root tip, centrifuged at 30,000 g., (35,000 r.p.m., radius: 22 mm.) are a contribution to the stock of facts already accumulated by these authors. The root tips, cut off at a length of 18 mm. were fastened by their cut end in a brass tube 15 mm. long and slightly wider than the roots; their free ends were in the centrifugal direction. The tubes were fixed firmly to the rotor of a Sharpies electric super-centrifuge. The rotor contained some Knop's plant medium in order to keep the roots steadily immersed. The centrifugation lasted from 10 minutes to 2 hours. The roots were then fixed in Flemming's fluid; and stained either with Heidenhain's iron haematoxylin or Flemming's triple stain. After a short centrifugation, the nuclear content is concentrated at the bottom of the nuclei and a clearer space appears at the top. Then the whole nuclei are thrown to the bottom of the cells. (Fig. 1 A.) So far these results are the same as those obtained by previous investigators working with a much lower centrifugal force. After a longer centrifugation the nucleus flattens against the floor of the cell, taking a more or less hemispheric shape, eventually forming an angle of less than 90° between the horizontal diameter of the hemisphere and the vertical portion of the nuclear membrane (Fig. 1B). The diametric line is perfectly straight and shows no evidence of any folding or shrinking. At the top of these hemispheric nuclei one can almost always observe an elevated portion of the membrane which encloses a transparent region and reminds one of a blister.