Defined Nutrient Medium Research Articles

Clonal propagation of Eucalyptus by tissue culture

Multiple adventitious buds were induced on cotyledons, shoot tips and nodal stem segments of Eucalyptus species cultured on a defined nutrient medium supplemented with the cytokinin zeatin and the auxin indole-3-butyric acid (IBA). The adventitious buds could be recycled on cytokinin medium to produce more buds, thus providing the possibility of producing large clones from selected genotypes. The adventitious shoots were rooted in auxin medium and some of the resulting propagules were outplanted in the field. These techniques have the potential for use in the genetic improvement of Eucalyptus.

Establishment of Proliferating Callus from Roots, Cotyledons and Hypocotyls of Carob (Ceratonia siliqua L) Seedlings

Roots, cotyledons and hypocotyls of Ceratonia siliqua, excised from sterile seedlings produced callus on agar with a defined nutrient medium. Callus production takes place in 2-month old cultures. Percentage callus formation in cotyledons and hypocotyls was greater than in roots. In the cotyledons, callus induction was stimulated in the veins with differentiation of both shoots and roots. Callus growth in the second explant was greatly increased when coconut milk was added to the medium where organogenesis eventually occurred.

Citrus Tissue Culture

An in vitro bioassay for chemicals that affect Citrus abscission was used to identify three inhibitors of stylar abscission in lemon pistil explants incubated on defined nutrient media. The three inhibitors (picloram, 4-chlorophenoxyacetic acid, and 3,5,6-trichloropyridine-2-oxyacetic acid) are all auxins, and the most potent of them (i.e. picloram) was found to be at least 10 times more active in the bioassay than 2,4-dichlorophenoxyacetic acid. Picloram (2 micromolar) also was shown to be effective in inhibiting stylar abscission in pistil explants from other Citrus cultivars such as mandarin, Valencia, and Washington navel oranges and grapefruit. To study the physiology of auxins active as abscission inhibitors versus inactive auxins in lemon pistils, the transport and metabolism of [1-(14)C]-2,4-dichlorophenoxyacetic acid was compared with that of [2-(14)C]indole-3-acetic acid, which is without effect in the bioassay over the range from 0.1-100 micromolar. Insignificant quantities of labeled indole-3-acetic acid and/or labeled derivatives were found to reach the presumptive zone of stylar abscission under the test conditions. Labeled 2,4-dichlorophenoxyacetic acid and/or labeled derivatives also were transported slowly through pistils, but some radioactivity could be detected in the stylar abscission zone as early as 24 hours after the start of incubation. Extensive conversion of [2-(14)C]indole-3-acetic acid to labeled compounds tentatively considered to be glycoside and cellulosic glucan derivatives was found with the use of solvent extraction methodology. A significantly smaller percentage of the radioactivity in pistils incubated on [1-(14)C]-2,4-dichlorophenoxyacetic acid was found in fractions corresponding to these derivatives. Both transport and metabolism appear to be important factors affecting the activity of auxins as abscission inhibitors in the bioassay.

Vesicle formation and acetylene reduction activity in Frankia sp. CPI1 cultured in defined nutrient media

It has been reported that certain species of free-living Rhizobium were capable of producing nitrogenase1–5, thereby demonstrating conclusively that the nif genes reside in the bacterial component of the dinitrogen-fixing symbiosis between Rhizobium and leguminous host plants. In the comparable dinitrogen-fixing symbiosis between the actinomycete Frankia and the actinorhizal woody dicotyledons that serve as nodulated host plants, the endophytic filamentous bacteria have only recently been brought into culture6–8. We report here evidence that free-living Frankia sp. grown in appropriate nutrient conditions show nitrogenase activity (acetylene reduction) associated with a distinctive morphological structure formed in effective nodules and also developed in culture.

Influences of Environmental and Physiological Conditions on Floral Bud Formation of Torenia Stem Segments Cultured in vitro

Internodal segments of Torenia fournieri Lind. differentiated floral buds when they were grown on a defined nutrient medium lacking NH 4 NO 3 . Two environmental factors, temperature and light, strongly affected floral bud formation. A temperature of 25 ± 2 °C, 8 hr short day photoperiod, and a light intensity of 5000 lux gave the best flowering response. Physiological factors such as the size of explants, the age of mother plants and the stem portions from which explants were taken, also influenced floral bud formation. The highest percentage of floral bud formation was obtained with 4 mm long explants prepared from the 2nd stem internodes of 12 week old plants, of which 91% of cultures produced floral buds.

In vitro culture of western red cedar (Thuja plicata). II. Induction of male strobili from vegetative shoot tips

Vegetative shoot tips of mature western red cedar (Thuja plicata Donn) were cultured on defined nutrient media. In the presence of high concentrations of various gibberellins, microsporangiate strobili were induced from the vegetative tips during an 8-week period under continuous illumination. No strobili were induced by GA3 under short-day (SD; 8-h photoperiod) conditions or continuous darkness. The ontogenetic stages of strobilus induction and development in culture were similar to those reported for intact plants. Prolonged culture produced externally mature male strobili. However, histological examination of the sporogenous tissues revealed either abortion of the tissues or an apparent arrest in early meiosis. Wide concentration ranges of either IAA or NAA in the presence or absence of 1 × 10−4 M GA3 did not qualitatively modify the early development of male strobili or induce female strobili, although 1 × 10−4M IAA completely inhibited induction of the male strobili by GA3. Benzylaminopurine or kinetin suppressed the induction of male strobili by GA3, while Ethrel or abscisic acid at the concentrations tested had no apparent qualitative effects on microsporangiate strobili formation.

In vitro Culture of Western Redcedar (Thuja plicata Donn). I. Plantlet Formation

Juvenile and mature tissues of Western Redcedar (Thuja plicata Donn) were induced to form multiple adventitious buds when cultured in continuous light at 25 C on defined nutrient media containing 1 x 10-6M to 1 x 10-5M BAP in the presence or absence of 1 x 10-7M NAA. Excision and transfer of adventitious buds onto a wide range of defined and undefined media allowed shoot elongation and leaf formation to occur. Subsequent transfer of the shoots onto media containing 5 x 10-6M to 5 x 10-5M IBA induced adventitious root formation within 4-6 wk. The resulting plantlets exhibited normal shoot morphology and were successfully transferred from tissue culture flasks to greenhouse conditions.

Interaction of Boron with Components of Nucleic Acid Metabolism in Cotton Ovules Cultured in Vitro

Cotton (Gossypium hirsutum L.) ovules grown in a defined nutrient medium undergo normal morphogenesis, including fiber production. In identical medium lacking boron, ovules callus and accumulate brown substances. Boron deficiency-like symptoms were induced by 6-azauracil and 6-azauridine in ovules growing in boron-sufficient media. Other nucleoside base analogs either reduced or had no effect on over-all growth, but did not cause typical boron-deficient callus growth of cotton ovules. Orotic acid and uracil countered the effects of 6-azauracil. Actinomycin D, fluorodeoxyuridine, and ethidium bromide reduced not only fiber production on ovules growing in boron-sufficient media but also callusing of ovules in boron-deficient media.Similarities between symptoms of boron deficiency and 6-azauracil injury, and the ability of uracil to suppress both, suggest that boron deficiency symptoms are related to reduced activity in the pyrimidine biosynthetic pathway. Growth inhibition by most nucleoside base analogs tested, actinomycin D, fluorodeoxyuridine, and ethidium bromide, as compared to callusing brought on by boron deficiency and 6-azauracil, indicates that boron deficiency symptoms are not related to a reduction in nucleic acid biosynthesis. Based on this information, a discussion of the possibility that boron deficiency causes reduced synthesis of UDP-glucose is presented.

PROMOTION OF ROOT INITIATION BY GIBBERELLIC ACID IN LEAF DISCS OF TOMATO (LYCOPERSICON ESCULENTUM) CULTURED IN VITRO

SUMMARYGibberellic acid (GA3) stimulated the rooting of tomato leaf discs cultured on a defined nutrient medium in continuous darkness. In the presence of 2 × 10−4 M tryptamine, GA3 significantly enhanced root initiation over the concentration range of 1 × 10−8 M to 1 × 10−4 M. GA3 could be replaced by GA4/7 but not by kinetin. Indole‐3‐lactic acid was a highly active indole derivative in the induction of rooting in the tomato leaf discs. This effect was significantly enhanced by GA3. Possible reasons for this observation are discussed.

Stoffwechselprodukte von Mikroorganismen

At low iron(III)-concentrations (less than 10(-5) M) the fungus Aspergillus viridi-nutans Ducker & Thrower excretes desferri-ferricrocin as the main sideramine into the culture medium. While this compound accounts for 95% of the sideramines produced, small amounts of additional sideramines may also be detected. In a search for an inexpensive nutrient medium for optimum production of desferri-ferricrocin, experiments using shake flasks with good aeration were undertaken initially. The best medium conditions were then employed in a fermentor system. In a 20-1 fermentor with "intensor" system, it was shown that at certain growth rates there was an inverse correlation between rate of growth and rate of sideramine production. A defined nutrient medium of glucose plus acetate as carbon sources, and urea or ammonium acetate as nitrogen sources was used. Two different feeding regimens were used in response to changes of pH or to changes of partial pressure of oxygen in the submerged culture: acetic acid/urea or acetic acid/ammonium acetate additions regulated these conditions. The rate of sideramine production under such feeding achieved a maximum of 20 mg 1(-1) h-1 over a period of several days.

Defined nutrient medium for the in vitro maintenance ofXenopus laevis oocytes

A procedure is described for the isolation and culture of large numbers of follicle cell-free Xenopus laevis oocytes in all stages of development. The isolation procedure involves the incubation of pieces of ovary in a calcium-free solution OR2 containing 0.2% collagenase. A defined nutrient medium for the maintenace of the oocytes in vitro is presented. It is shown that this medium, referred to as DNOM, can maintain certain morpological and functional characteristics of oocytes for periods up to 3 weeks.

Wachstum und N‐Stoffwechsel sowie Anderungen des Isoenzymspektrums der Glutamatdehydrogenase (GDH) in batchvermehrten, licht‐ und dunkelkultivierten Zellsuspensionen von Pisum sativum

Abstract Growth, N‐metabolism and isoenzyme pattern of glutamate dehydrogenase in batch‐cultures of Pisum sativum cells under light and dark conditions.Cell suspension cultures of Pisum sativum L. derived from root and shoot sections of seedlings have been prepared and cultured in defined nutrient medium. Both the cells and the media were analysed daily for the N‐fractions and carbohydrates during the growth period.The data obtained indicate specific correlations between growth and nitrogen and carbohydrate metabolism. At the beginning of the growth cycle ammonia as compared to nitrate was favoured in uptake. An increased uptake of nitrate occurred at the end of the linear growth phase when carbohydrate in the media was depleted. The uptake of sucrose was rapid during the whole growth cycle, only in the range of the linear growth phase the uptake stagnated for 3 or 4 days. During increased biosynthesis of nitrogenous compounds at the beginning of the growth cycle up to seven isoenzymes of the glutamate dehydrogenase could be separated by polyacrylamide gel electrophoresis. The isoenzyme pattern changed during the stationary growth phase, especially when the carbohydrate content in the medium decreased. There is some evidence that the isoenzyme pattern is influenced by carbohydrate metabolism.

Growth and metabolism of cells and tissue of jack pine (Pinus banksiana). 3. Growth of cells in liquid suspension cultures in light and darkness

Friable, jack pine (Pinus banksiana Lamb.) callus on agar plates with a defined nutrient medium was transferred to a liquid medium of the same composition to establish cell suspension cultures. Dry weights and patterns of growth for daughter cells were estimated in continuous light or darkness, with or without conditioned media and arginine supplement. In all treatments, growth was near-exponential. Greatest final size was obtained with basal medium under continuous light.The distribution of clump sizes at the later stages of growth fits a stochastic model reflecting two types of daughter cell behavior described by the probability of one type remaining with the clump as opposed to sloughing off into the medium. In light, cells contained many more chloroplasts, thicker cell walls, and more compacted clumps than in darkness: yet cellular clumping patterns both in light and darkness were largely similar. Nearly 20% of the clumps showed polarity and symmetry and contained actively streaming suspensor-like cells. The remaining clumps were spherical and produced unorganized growth patterns. The progress of growth was eventually dominated by the tendency of daughter cells to remain attached to clumps compared with the initial tendency for clumps to fragment and cells to be released into the medium.

Growth and metabolism of cells and tissues of jack pine (Pinus banksiana). 1. The establishment and some characteristics of a proliferated callus from jack pine seedlings

No more than 2% of the embryos from dry seeds of jack pine (Pinus banksiana Lamb.) produced callus on agar with a defined nutrient medium. When contact of the embryo with the nutritive female gametophyte was maintained during imbibition, callus production by excised seedlings on the medium increased to over 80%. Callus production was optimal with 5-day-old seedlings. Percentage callus formation was greater when the initial contents of the explant in dry weight, glutamate N, amide N, total nucleotides, RNA, adenylate energy charge, and oxygen uptake were also greater. Small initial increases in levels of these components over those found in dry embryos and resulting from imbibition substantially improved callus production. Subsequent increases, concomitant with germination and seedling development, gave only slightly more callus. By contrast, inverse or poor relations were observed with DNA, total soluble protein, and aminoacyl tRNA synthetase activity. In the initial explants the most conspicuous cytological events, leading to optimal callus production, were the onset of the first wave of mitosis and prominence of nucleoli. Seed reserves were nearly consumed and ribosome-like particles, which were associated with chromatin, appeared as new cytoplasm was being synthesized during the onset of proliferation of cells on the agar medium.

In vitro induction of embryoids in tissue cultures of Nigella damascena

Callus derived from the pedicels and floral buds of Nigella damascena L. when broken down to single cells and small clumps produced large numbers of embryoids. These embryoids passed through stages comparable to zygotic embryogeny in a defined nutrient medium.

IN VITRO BUD FORMATION IN STEM SEGMENTS OF AILANTHUS ALTiISSIMA

SUMMARYExcised internodal segments of Ailanthus altissima were grown aseptically on a defined nutrient medium which lacked added growth regulators. Vascular cambium, phloem or cortex, depending upon which of these tissues is exposed, undergoes tangential cell divisions and produces new tissue on top of the explant. Buds appear in surface layers of the newly formed tissue. Seasonal controls of this process are evident from the greater amount of regeneration in samples collected in spring (March‐May).

Bud formation and embryo differentiation in in vitro cultures of Petunia

Summary Excised stem and leaf explants of Petunia inflata and two varieties of Petunia hybrida, „rose du ciel“ and „cascade“ were grown on a defined nutrient medium. The morphogenetic responses of the expiants varied with the growth substances added to the medium. There was either the direct regeneration of shoot buds or the development of a friable callus which differentiated into embryos. Plantlets were produced from the embryos.

Variations in the Metabolism of the Daughter Sporocysts of Microphallus pygmaeus in a Chemically Defined Medium

A comparison is made of the variations in the mean size (length), reduced weight, oxygen uptake, metabolic rate and the number of contained fully formed metacercariae undergoing autolysis, in mature daughter sporocysts of Microphallus pygmaeus, in sea water, artificial sea water and in a chemically defined nutrient medium (modified medium 199). The work indicates that the sporocysts begin to degenerate almost immediately in the non-nutrient media but have a higher metabolic activity and remain healthy for up to 36 days in the nutrient medium.

Plant Virus Multiplication in Callus Tissues with Special Reference to the Effect of Fluorescent Light

Tobacco stem tissues were isolated from plants systemically infected with tobacco mosaic virus (TMV) and grown on a chemically defined nutrient medium containing various growth substances. The effect of fluorescent light on TMV multiplication in callus tissues was investigated. The growth rate of TMV infecting callus tissues was higher in light conditions than in dark conditions. It is suggested that the stimulation in growth of TMV-infected callus tissues in light conditions is caused by the CO2 fixation.

Factors influencing premature induction of UDP-glucuronyltransferase activity in cultured chick embryo liver cells.

Very young (5-day-old) chick embryo livers during organ culture on rafts over a chemically defined nutrient medium precociously develop, from zero, adult levels of UDP-glucuronyltransferase activity. Induction of enzyme does not require DNA synthesis or mitosis, or exposure to hormones, serum, or tissue extracts. Thus, this functional differentiation of embryo liver is apparently not dependent on cell proliferation or on extrinsic substances other than simple nutrients.UDP-glucuronyltransferase activity develops rapidly also in embryo liver cells cultured as monolayers or aggregates; specific activity of enzyme on a protein basis increases at the same rate in cells undergoing widely different rates of cell division, and net protein synthesis. These findings suggest that precocious development of UDP-glucuronyltransferase activity in chick embryo liver is due, not to incidents of culture, but to removal from the embryonic environment. Supporting this, chick embryo liver grafted to the chorioallantoic membrane does not develop UDP-glucuronyltransferase activity until subsequently transferred to an in vitro cell culture system.