Effects of activated charcoal in anther cultures

Embryogenesis in anther cultures of Anemone Canadensis L., Anemone hupehensis Lemoine and Nicotiana tabacum L. was shown to be inhibited by abscisic acid added to the medium. However, this inhibition was reduced in the presence of activated charcoal (AC).
The presence of AC in the culture medium strongly promoted embryogenesis in anther cultures of Anemone Canadensis compared with other media combinations. Treatment of the agar medium with AC, which was removed before inoculation of the anthers, also stimulated embryogenesis, but treatment of the water constituent did not.
The number of embryos produced in anther cultures of Anemone Canadensis and Nicotiana tabacum was shown to be positively related to the length of lime of incubation on medium supplemented with AC. In the case of Anemone Canadensis the stimulating effect of AC was most pronounced when the first visible embryos had emerged.
The presence of anther-derived embryos from Anemone Canadensis in anther cultures of Anemone Canadensis and Nicotiana tabacum was shown to inhibit embryogenesis. It was also demonstrated that embryos from anther cultures of Anemone canadensis, Papaver setigerum DC and Clematis viticella L. produced phenolic substances, and that the concentration of these substances was higher in culture medium lacking AC. Treatment of such medium with AC could reduce the concentration of phenolic substances by more than 80%.     

Improvement of anther culture technique: Activated charcoal bound in agar medium in combination with liquid medium and elevated CO2 concentration

Anthers of different species of the genera Anemone, Clematis, Papaver and Nicotiana were cultured by floating on a liquid medium which overlay an agarified charcoal medium . This technique proved to be superior to conventional methods i.e. culture on either solid or liquid media. Cold treatment of Anemone anthers for 7 days after inoculation on the double layer medium gave about the same frequency of embryos per anther as corresponding cultures cold treated before inoculation. An elevation of the CO₂ concentration to 2% stimulated embryogenesis in anther cultures of Anemone canadensis, Anemone vitifolia, Papaver setigerum and Papaver radicatum . Cold treatment of cultures of Anemone canadensis inhibited embryogenesis if the ensuing culture was performed in 2% CO_2. On the other hand, cold treatment was stimulating, with an optimum of about 20 days, if the cultures were maintained in normal air. Chemical analysis of untreated anthers of Anemone canadensis showed the presence of abscisic acid (2.2 × 10⁻⁶ g/g anthers). Cold treatment reduced the concentration of abscisic acid to 0.6 × 10⁻⁶ g/g anthers. By use of assays with Lemna gibba as test organism, activated charcoal was shown to adsorb abscisic acid that was added to the medium. Medium treated with charcoal before inoculation of anthers of Anemone canadensis provided to inhibit embryo production.     

In vitro culture of the seagrass Halophila decipiens

The seagrass Halophila decipiens Ostenfeld was grown axenically in a culture medium consisting of 20% artificial seawater, f/4 nutrients (except that glutamic acid was the nitrogen source), and 1% sucrose (w:v). The culture medium was adjusted to pH 5.0. A root–rhizome layer was created by solidifying a portion of the medium with 0.9% agar (w:v) and 1% activated charcoal (w:v). The rhizome layer also contained the following vitamins: 0.5 mg l⁻¹ nicotinic acid, 0.5 mg l⁻¹ pyridoxine, 0.5 mg l⁻¹ biotin, 0.5 mg l⁻¹ cyanocobalamin and 0.1 mg l⁻¹ of thiamine HCl. The liquid overlay (without vitamins or charcoal) was poured onto the agar-solidified root–rhizome layer. Growth of H. decipiens was not improved by the addition of the auxins indoleacetic acid (IAA), indolebutyric acid (IBA) or naphthaleneacetic acid (NAA) at either of the tested concentrations (10 and 50 μM). At a concentration of 10 μM, the cytokinins 6-(γ,γ-dimethylallylamino) purine (2iP) and benzylaminopurine (BA) stimulated shoot and branch production compared to controls with no cytokinins. Among the tested nitrogen sources, growth was best on 1.7 mM glutamic acid. Cultures grown on 1.7 mM NH₄Cl showed the same growth rates as those grown on glutamic acid, but the leaves were smaller and curled, suggesting incipient ammonium toxicity. Use of nitrate or urea led to mortality of the cultures. Long term axenic culture of H. decipiens appears to require the added vitamins. Hence, H. decipiens is the first seagrass known to need exogenous vitamins. Cultures of H. decipiens died when grown suspended in liquid cultures or in a biphasic medium system without activated charcoal in the root–rhizome layer. The use of more highly charged κ-carrageenan could not replace the use of activated charcoal and agar in the root–rhizome layer.     

Factors influencing somatic embryogenesis induction in <Emphasis Type="Italic">Eucalyptus globulus</Emphasis> Labill.: basal medium and anti-browning agents

The low induction rates of somatic embryogenesis (SE) in Eucalyptus globulus hamper scaling up the process for commercialization. We analyzed the effectiveness of several media (MS, 1/2MS, B5, WPM, DKW and JADS) during SE induction and expression. MS and B5 were the best media for SE induction and embling regeneration. In general, MS was the best medium for expression, independently of the medium previously used during induction. Several anti-browning compounds (ascorbic acid, charcoal, DTE, DTT, PVP, PVPP and silver nitrate) were added to the expression medium (MS), but all decreased SE potential and only DTE, charcoal and silver nitrate reduced explant browning. When added only during the induction period, anti-browning agents reduced accumulation of phenolics but also severely reduced SE potential. Continuous exposure completely inhibited the SE response. The negative impact of anti-browning agents on SE potential raises a question about the role of production/accumulation of phenolics in the SE process.     

The role of ethylene and reducing agents on anther culture response of tetraploid potato (Solanum tuberosum L.)

Summary The role of ethylene in embryogenesis of cultured potato anthers was studied indirectly by testing various substances known to affect ethylene formation. The reducing agents ascorbic acid and L-cysteine prevented browning of anther cultures and significantly stimulated embryogenesis. Embryogenesis was also promoted by the use of the ethylene inhibitors AgNO₃ and n-propyl-gallate and by the polyamines spermidine and putrescine. The use of the ethylene releasing compound ethrel significantly inhibited embryogenesis.Abbreviations MS Murashige & Skoog - PVP polyvinylpyrrolidone - MW molecular weight - ACC 1-aminocyclopropane-1-carboxylic acid - ethrel 2-chloroethylphosphonic acid (ethephon)     

Enhancement of somatic embryogenesis in camphor tree (Cinnamomum camphora L.): osmotic stress and other factors affecting somatic embryo formation on hormone-free medium

The aim of this study was to improve the direct somatic embryogenesis and initiate embryogenic callus formation in camphor tree (Cinnamomum camphora L.) on hormone-free medium. The influence of osmotic stress pretreatment of immature zygotic embryos (0.5 and 1.0 M solution of sucrose for 12, 24, 48, 72, 96, 120, and 144 h at 4 or 25°C) before cultured on hormone-free medium, on embryogenesis efficiency was assessed. The embryogenesis frequency was improved from 16.29 to 93.27%, while the average number of somatic embryos per explant increased from 3 to 12.57. Activated charcoal (AC), medium renewal, basal medium, light conditions and sucrose concentration in culture medium were also evaluated for their effect on somatic embryogenesis. AC addition and 10-day medium renewal did not increase embryogenesis efficiency significantly, and Murashige and Skoog (MS) medium proved to be more beneficial for somatic embryo formation than others. No differences were found between embryogenesis frequencies when cultured in darkness or under light, but culturing under light yielded more embryos. After the sucrose solution pretreatment, high level concentration of sucrose in induction medium was not needed for somatic embryogenesis, for it had a negative effect on somatic embryo formation when the concentration of sucrose was higher than 50 g l⁻¹. The derived embryogenic lines were maintained via repetitive embryogenesis on hormone-free medium. Low ratio formation of embryogenic callus was observed on the surface of somatic embryos both on induction and proliferation medium. Plantlets derived from somatic embryos grew vigorously with normal appearance similar to germinated zygotic embryos.     

The role of activated charcoal in plant tissue culture

Activated charcoal has a very fine network of pores with large inner surface area on which many substances can be adsorbed. Activated charcoal is often used in tissue culture to improve cell growth and development. It plays a critical role in micropropagation, orchid seed germination, somatic embryogenesis, anther culture, synthetic seed production, protoplast culture, rooting, stem elongation, bulb formation etc. The promotary effects of AC on morphogenesis may be mainly due to its irreversible adsorption of inhibitory compounds in the culture medium and substancially decreasing the toxic metabolites, phenolic exudation and brown exudate accumulation. In addition to this activated charcoal is involved in a number of stimulatory and inhibitory activities including the release of substances naturally present in AC which promote growth, alteration and darkening of culture media, and adsorption of vitamins, metal ions and plant growth regulators, including abscisic acid and gaseous ethylene. The effect of AC on growth regulator uptake is still unclear but some workers believe that AC may gradually release certain adsorbed products, such as nutrients and growth regulators which become available to plants. This review focuses on the various roles of activated charcoal in plant tissue culture and the recent developments in this area.     

Vitamin and Amino Acid Requirements of Pediococcus soyae and Pediococcus acidilactici Kitahara’s Strain

Nutritional requirements of three strains of Ped. soyae, one strain of Ped. acidilactici (Kitahara’s strain) were determined from a complete synthetic medium. Upon inspection by the single omission method, Ped. soyae required glycine-betaine, uracil, riboflavine, pyridoxine or pyridoxal, pantothenic acid, nicotinic acid, leucovorin, biotin, glutamic acid, arginine, histidine, isoleucine, leucine, phenyl alanine, valine, tryptophane, methionine, cystine and serine with exception of the P-factor. In addition to the above mentioned nutrients, a representative strain of the organism required xanthine, folic acid, thiamine, aspartic acid and threonine for the minimum synthetic medium. Ped. acidilactici Kitahara’s strain required riboflavine, pyridoxine, pantothenic acid, nicotinic acid, biotin and all components of the basal medium consisting of amino acids except methionine, but it did not require leucovorin, glycine-betaine and organic bases. Nutritional requirements of Ped. pentosaceus, Kitahara’s strain was proved to be quite identical with Ped. cerevisiae Pederson (= Ped. pentosaceus Mees).     

The effects of water-soluble vitamins on the expansion of rabbit blastocysts in vitro

The vitamin requirements for culture of rabbit morulae to expanded blastocysts were examined. Early morulae were cultured for 5 days either in a control complete medium containing all the 11 water-soluble vitamins of F10 culture medium (biotin, pantothenate, choline, inositol, niacinamide, pyridoxine, riboflavin, thiamine, folic acid, B12, and lipoic acid) or in media with each vitamin omitted individually. Blastocyst diameters were measured at the end of culture. The omission of inositol, pyridoxine, riboflavin, and niacinamide resulted in large statistically significant decreases in blastocyst expansion. The omission of B12 resulted in a significant increase in blastocyst expansion indicating that the level present in F10 is toxic to rabbit blastocysts.     

Effect of exogenous cytokinins on growth and somatic embryogenesis in anise cells (Pimpinella anisum L.)

A cell culture of anise was grown in the presence or absence of 2,4-dichlorophenoxyacetic acid (2,4-D). Application of isopentenyladenine or isopentenyladenosine (4·10^-8 to 4·10^-7 M) to the proembryonic culture (+2,4-D) yielded an increase of the cell density, in contrast to a proembryonic culture grown without exogenous application of cytokinins. Embryogenesis was induced by transferring the cells to a hormone-free medium. Embryo development was promoted by isopentenyladenine and isopentenyladenosine (5·10^-8 to 5·10^-7 M), higher concentrations (5·10^-6 M) inhibited embryogenesis. The effect of cytokinins on embryogenesis was only promotive until the third day of culture, i.e. coincident with cell growth rather than differentiation.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - i6Ade isopentenyladenine - i6Ado isopentenyladenosine     

Microspore embryogenesis in anther cultures of two Indian cultivars of Brassica juncea (L.) Czern.

Direct microspore-derived embryo formation in anther cultures of two cultivars of Brassica juncea was obtained. Preliminary culture of anthers at 35°C for 1–5 days prior to maintenance at 25°C stimulated embryogenesis. Embryogenesis was also stimulated by an initial culture at 5°C for 3 days. Analysis of squashed anthers revealed that approximately 10% of the microspores began dividing, but less than 1% developed into macroscopic embryos. All embryos transferred to embryo culture medium survived, but only 30% of these developed directly into normal plantlets. The androgenic plants were haploid (2n=18).     

Levels of water-soluble vitamins in methanogenic and non-methanogenic bacteria

The levels of seven water-soluble vitamins in Methanobacterium thermoautotrophicum, Methanococcus voltae, Escherichia coli, Bacillus subtilis, Pseudomonas fluorescens, and Bacteroides thetaiotaomicron were compared by using a vitamin-requiring Leuconostoc strain. Both methanogens contained levels of folic acid and pantothenic acid which were approximately two orders of magnitude lower than levels in the nonmethanogens. Methanobacterium thermoautotrophicum contained levels of thiamine, biotin, nicotinic acid, and pyridoxine which were approximately one order of magnitude lower than levels in the nonmethanogens. The thiamine level in Methanococcus voltae was approximately one order of magnitude lower than levels in the nonmethanogens. Only the levels of riboflavin (and nicotinic acid and pyridoxine in Methanococcus voltae) were approximately equal in the methanogens and nonmethanogens. Folic acid may have been present in extracts of methanogens merely as a precursor, by-product, or hydrolysis product of methanopterin.     

In vitro regeneration of Camellia sinensis (L.) O. Kuntze by somatic embryogenesis

Within 3 weeks of culture, excised cotyledon expiants of Camellia sinensis (L.) O. Kuntze produced somatic embryos without intermediate callus when cultured in Murashige and Skoog's basal medium with 30 g^–1 sucrose. In medium without plant growth regulators, up to 60% of the cultures developed somatic embryos. Embryogenic competence was reduced by increasing concentrations of plant growth regulators tested (i.e. kinetin, 6-benzylaminopurine, and indole butyric acid). The somatic embryos developed, grew to maturity without being subcultured within 6–8 weeks. Secondary embryogenesis was not observed. Germination of isolated mature somatic embryos was low in medium without plant growth regulators. Up to 53% and 60% germination occurred when medium impregnated with kinetin at 1.8 mgl^–1 or 1.0 mgl^–1 6-benzylaminopurine were used respectively. Callus was also routinely produced when cotyledons were cultured in MS basal medium with auxins (2,4-dichlorophenoxyacetic acid and indole acetic acid). Callus induction was however, also achieved in plant growth regulator free medium. Indirect somatic embryogenesis was not induced in the present study.Abbreviations K kinetin - BAP 6-benzylaminopurine - IBA indole butyric acid - IAA indole acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA Naphthalene acetic acid - Fe-EDTA Ethylenediaminetetra-acetic acid (Ferric monosodium salt)     

Effects of Activated Charcoal on Growth and Morphogenesis in Cell Cultures

The effects of activated charcoal on growth and morphogenesis in plate cultures of different plant cells have been studied. It was shown that medium containing charcoal induced embryogenesis in cultures of Daucus carota in which embryo formation could not be brought about by omitting auxin from the medium. Charcoal-medium also induced abundant root formation in older cultures of Allium cepa, which normally did not produce roots. The growth of cultures of Glycine max and Haplopappus gracilis was totally inhibited by charcoal. It is thought that activated charcoal removes substances from the medium, one of which might be auxin.     

Somatic embryogenesis, plant regeneration, and cryopreservation for Torreya taxifolia, a highly endangered coniferous species

Torreya taxifolia Arn., an ancient evergreen tree, is on the brink of extinction from attack by a fungal disease, recently reported to be caused by a novel isolate of Fusarium. We report the development of a somatic embryogenesis tissue culture system that can be used for cryogenic storage of T. taxifolia cultures and subsequent plant regeneration. Initiation of embryogenic tissue from immature zygotic embryos occurred on a conifer tissue culture medium containing 0.25?% activated charcoal, 43.8?mM maltose, 0.5?mM 2,4-dichlorophenoxacetic acid, 0.2?mM 6-benzylaminopurine, 0.2?mM kinetin, 0.1???M brassinolide, 3.8???M abscisic acid, 20.5???M biotin, 1.13???M folic acid, 1.28?mM 2(n-morpholino)ethanesulfonic acid and 0.69?mM pyruvic acid. Embryo induction ranged from 60 to 100?% across six seed sources. Somatic embryo development occurred on a medium containing 43.8?mM maltose, 1?% activated charcoal, 37.8???M abscisic acid, 20.5???M biotin, 0.1???M brassinolide, 0.205?mM folic acid, 1.28?mM 2(n-morpholino)ethanesulfonic acid and 0.69?mM pyruvic acid. Germination of somatic embryos ranged from 64 to 82?%. Embryogenic tissue cultures from 30 genotypes representing seed from six mother trees were cryopreserved, and culture recovery was demonstrated after freezing. In contrast to many other coniferous tree seeds, the measured water potential (?MPa) of T. taxifolia megagametophyte tissue rose greatly during seed after-ripening. Duplication of this rise in vitro allowed development of somatic embryos to the cotyledonary stage.     

Inhibitory conditioning for carrot somatic embryogenesis in high-cell-density cultures

Carrot somatic embryogenesis was strongly inhibited in high-cell-density cultures. This inhibition was not caused by depletion of nutrients or physical damage but by factor(s) released into the culture medium from cells during culture. A conditioned medium prepared by eliminating cells after high-cell-density culture inhibited somatic embryogenesis. The degree of inhibition increased with the amount of conditioned medium. A dialysis experiment revealed that the molecular weight of the inhibiting factor(s) was below 3,500. We also found that the conditioned medium contained a high-molecular-weight factor(s), which stimulated somatic embryogenesis. Received: 13 March 1998 / Revision received: 19 May 1998 / Accepted: 1 June 1998     

Microspore embryogenesis of high sn-2 erucic acid Brassica oleracea germplasm

Brassica oleracea accessions possess traits that would be useful in commercial Brassica crops. These traits can be studied more effectively through the production of doubled haploid plants. Nineteen B. oleracea accessions from several subspecies possessing significant sn-2 erucic acid were screened for suitability for microspore culture using techniques well established for Brassica. Fifteen of the 19 accessions produced embryos. Genotypic differences were observed with embryogenesis ranging from 0 to 3000 embryos/100 buds. Embryogenesis was improved for two of four accessions by initiating cultures in NLN medium with 17% sucrose, then reducing sucrose to 10% after 48 h. An increase in embryogenesis for the same two accessions was observed when microspores were cultured at a density of 100 000/ml rather than 50 000 microspores/ml. A culture temperature of 32 °C for 48 h was beneficial for three of the four accessions when compared to a longer incubation period (72 h) or a higher temperature (35 °C). One accession line, Bo-1, was found to produce microspore-derived embryos which contained triacylglycerols with significant proportions of erucic acid at the sn-2 position. This revised version was published online in June 2006 with corrections to the Cover Date.     

A Revised Medium for Growth of Pea Mesophyll Protoplasts

The nutrient requirements of mesophyll protoplasts from Pisum sativum L. cv. Timo have been investigated and a synthetic and completely defined medium has been designed. A high calcium concentration (12 mM) stimulated both protoplast survival and cell division. The content of iron and zinc was also critical. Additions of nicotinic acid, pyridoxine and thiamine were necessary. The protoplast growth was enhanced when some amino acids were included in the medium. An absolute requirement for auxin and cytokinin was shown. In the revised medium about 90% of the isolated protoplasts survived and formed a cell wall. The first divisions were observed after 5 days and after 1 week 10–20% of the cells had divided at least once.     

Stimulation of somatic embryogenesis in carrot by ethylene: Effects of modulators of ethylene biosynthesis and action

Endogenous levels of ethylene appeared to he suhoptimal for somatic embryogenesis in a suspension culture of carrot. Low concentrations of 1-aminocyclopropane-1-carboxylic acid (ACC). 2-chloroethylphosphonic acid (ethephon) and elhylene stimulated embryogenesis whereas higher concentrations were inhibitory. The stimulation by ACC was through its conversion to ethylene. whereas the inhibition by ACC was not. Low concentrations of AgNO₃. an inhibitor of ethylene action, inhibited embryo-genesis but stimulated ethylene production. Aminoethoxyvinylglycine (AVG) and aminooxyacetic acid (AOA). commonly used inhibitors of ACC synthase. inhibited both embryogenesis and ethylene production. However, the inhibition of embryogenesis was not related to the inhibition ote ethylene production. Very low concentrations of AVG stimulated embryo production in a way unrelated to its effect on ethylene production. Salicylic acid and CoCl₂. inhibitors of ACC oxidase in other systems, inhibited embryogenesis but. again, in way(s) unrelated to their inhibition of ethylene production. In fact, low concentrations of salicylic acid stimulated rather than inhibited ethylene production. The results show that in suspension-cultured cells, caution is warranted in the interpretation of results obtained with agents presumed to inhibit ethylene biosynthesis. The stimulation of somatic embryogenesis by ethylene unequivocally shows that the inhibition of embryo development by 2.4-dichlorophenoxyacetic acid (2.4-D) and other auxins cannot be through their stimulatory effect on ethylene production.     

Initiation and development of microspore embryogenesis and plant regeneration of Brassica nigra

Brassica nigra is generally regarded as a recalcitrant species for microspore culture among Brassica crops. Conditions for reliable induction of microspore embryogenesis of B. nigra were studied in this context. Flower bud length and microspore developmental stage were correlated with further embryogenesis. The optimal bud size range was 2.0–2.5 mm for the highest proportion of totipotent, late uninucleate microspore and the highest frequency of microspore embryogenesis. Treatment of a short heat shock by incubating the microspore culture at 32°C for 24 h was suitable for the microspore survival, sustained cell divisions, and further induced embryogenesis. Subsequently, the use of NLN medium with the addition of 13% sucrose and 0.1% activated charcoal (AC) provided the optimal conditions for the development of microspore-derived embryos (MDEs). The early cotyledonary (EC) stage embryos cultured on MS medium fortified with 4.6 μM zeatin (ZT) and 0.12 μM indole-3-acetic acid (IAA) resulted in the most efficient rates of plantlet regeneration. The ploidy levels of regenerated plants of B. nigra were determined by flow cytometry, revealing that 50.6% were diploid. The results enable the advancement of breeding programs and genetic studies in B. nigra.