Marine Brown Algae Requiring Vitamin B12

The brown algae Lithosiphon pusillus, Ectocarpus fasciculatus and Pylaiella litoralis were cultivated in bacteria-free cultures in artificial sea water, ASP 6F. The growth was tested with different additions of vitamins and other metabolites. Lithosiphon pusillus and Ectocarpus fasciculatus were found to require vitamin B₁₂ for optimal growth. The zoospores of Pylaiella litoralis, when cultured in medium Asp 6F with kinetin added, had an absolute requirement for vitamin B₁₂.     

The Demand for Iodine and Bromine of three Marine Brown Algae grown in Bacteria-free Cultures

Three marine brown algae have been cultivated with different additions of iodine and bromine in bacteria-free cultures. Ectocarpus jasciculatus appeared to have an absolute demand for iodine and was inhibited by a concentration of 64 μmol of KJ per 1. Lithosiphon pusillus had the best growth in the highest concentration tested (64 μmol/1) but there was always some growth in the series without iodine. Additions could be made either as inorganic iodine or as organically bound iodine. Additions of KJ to a culture medium consisting of vitamin-free Asp 6 F with B₁₂ (1 μg/1) and kinetin (20 μmol/1) remarkably increased the growth of the zoospores of Pylaiella litoralis. Lithosiphon pusilius proved to be indifferent to bromide additions in media containing KJ. In media lacking KJ addition of 1 μmol of KBr per 1 is stimulating but higher concentrations of KBr are inhibiting. The inhibiting effect is overcome by iodide addition.     

The Quantitative Yield in Purification of Cytokinins. Model-experiments with Kinetin, 6-Furfuryl-amino-purine

Model-experiments with kinetin, 6-furfuryl-amino-purine, to determine the quantitative yield in purification of cytokinin extracts have been performed. If kinetin in acidic water solution is partitioned three times with equal volumes of ethyl ether, about 50 per cent of the kinetin passes into the ether phases. In similar experiments with ethyl acetate more than 90 per cent of the kinetin goes over to the ethyl acetate phases. Accordingly, use of these two solvents in purification of cytokinin extracts leads to very large losses. Use of petroleum ether or n-hexane on the other hand leads to none or very small losses. Partitioning of alkaline kinetin solutions three times with equal volumes of 1-butanol results in an almost quantitatitve extraction of the kinetin from the water solution. About 7 per cent of the original amount of kinetin follows the acid auxin, when a saturated ether solution of kinetin is extracted according to a common method for acid auxins. Kinetin may thus interfere in later analyses for auxins. The dangers involved when cytokinins are “purified” according to normal practice are pointed out.     

Induced mutation to monocotyledony in periwinkle,Catharanthus roseus, and suppression of mutant phenotype by kinetin

A recessive EMS-induced mutation inherited in Mendelian fashion caused monocotyledonous embryo formation and seed germination on high salt medium inCatharanthus roseus. Availability during embryo development of exogenously supplied cytokinin kinetin suppressed the mutant phenotype. These observations suggest that, inC. roseus, (i) insufficiency in endogenous kinetin may lead to monocotyledonous embryo patterning and (ii) dicotyledonous embryo formation requires a critical amount of kinetin in certain cells of early embryos.     

Selective shift in the metabolic pathways of excised tomato cotyledons in response to mannitol induced water stress

Excised tomato cotyledons subjected to mannitol induced water stress had more total sugars than normal cotyledons during injury but less total sugars than normal cotyledons in the adaptive phase. During water stress injury, protein and UNA synthesis were reduced, activities of beta-fructofuranosidase and phosphofructokinase were reduced but activities of proteases, ribonuclease, hexokinase and glucose-6-phosphate dehydrogenase were increased. Additions of kinetin reversed the water stress effects on enzyme activities. It is concluded that during water stress injury, there was a fundamental reduction in cytokinin activities leading to the selective shift in the enzyme populations.     

Effect of growth regulators onVicia faba plants irrigated by sea water Leaf area,pigment content and photosynthetic activity

The antagonistic effects of some growth regulators [i.e. indol-3-yl-acetic acid (IAA), gibberellic acid (GA₃) or kinetin] on stress imposed by sea water on leaf area, pigment and photosynthetic activity in leaves of broad bean plants at different stages of development were investigated. Seed priming with GA₃ alleviated either partially or completely the effects induced by the two levels of sea water (10 and 25 %) used on leaf area at all experimental stages. However, IAA, GA₃ and kinetin inhibited leaf growth by themselves in almost all measurements. Seed pretreatment with kinetin alleviated the inhibition of pigment production in sea water-irrigated plants. Furthermore, GA₃ or kinetin nullified the deleterious effects imposed by irrigation with sea water particularly the high level (25 %) on photosynthetic¹⁴CO₂ fixation.     

Occurrence, biosynthesis and properties of kinetin (N6-furfuryladenine)

In this paper we review the data on the structure and properties of N⁶-furfuryladenine (kinetin, K) accumulated during the last forty years. In 1955, kinetin was isolated from DNA as an artifactual rearrangement product of the autoclaving process. Subsequently, its cytokinin activity has been established, demonstrating a wide variety of biological effects, including those on gene expression, inhibition of auxin action, stimulation of calcium flux, the cell cycle, and as an anti-stress and anti-ageing compound. Recently, our views on this very well known plant hormone have changed. There are new data, which show that it occurs in cellular DNA as the product of oxidative, secondary modification and a secondary reaction of DNA. Also new results on the biological function of kinetin have been reported. Various biological effects produced by this hormone in vitro and in vivo have made kinetin even more scientifically interesting and commercially attractive as an ingredient of many beauty cosmetics.     

SOMATIC EMBRYOGENESIS AND ORGANOGENESIS FROM CALLUS CULTURES OF SOLANUM CAROLINENSE

Culture of stem segments of Solanum carolinense L. on medium supplemented with 10 mg/1 2,4-dichlorophenoxyacetic acid and 1 mg/1 kinetin, induced callus formation. When subcultured on medium lacking 2,4-D but containing a cytokinin, the callus regenerated. The mode of regeneration depended on the type and concentration of cytokinin employed; high concentrations of benzyladenine and all concentrations of kinetin promoted organogenesis, while low concentrations of benzyladenine induced somatic embryogenesis in addition to organogenesis. With age and continued subculture on 2,4-D containing medium, callus progressively lost its ability to regenerate when the auxin was replaced by cytokinin. In conjunction with previous studies on regeneration from anther cultures of S. carolinense, it appears that in both cases, 2,4-D is required for callus initiation and proliferation but must be exchanged for a cytokinin before differentiation will occur. However, since it was not possible to induce embryogenesis in pollen-derived callus, developmental potential may be influenced by the ploidy level of responding cells in culture.     

The Inhibition by Cytokinin of Auxin-Promoted Elongation in Excised Soybean Hypocotyl

The experiments characterize the inhibition by kinetin of auxin-promoted elongation in excised hypocotyl sections of 3-day soybean seedlings (Glycine max cv. Hawkeye 63). It was found that concentrations of kinetin above 4.2 μM did not further inhibit auxin-promoted elongation. Kinetin is as potent an inhibitor of elongation as actinomycin D or cycloheximide. Tissue incubated for 3 or 5 h in the absence of auxin or cytokinin would, upon addition of auxin, exhibit a new growth rate similar to that of tissue grown in auxin for the entire incubation period. Similarly, tissue grown for 3 and 5 h in the presence of auxin would revert to the control rate of elongation upon addition of kinetin. A 10 to 30 min preincubation in kinetin yielded the tissue incapable, for the ensuing 6 h, of increasing its rate of elongation in response to auxin. Zeatin and isopentenyladenine were more potent than kinetin and benzyladenine in the inhibition of elongation. Levels of ethylene produced in the presence of auxin plus cytokinin indicated that it was not involved in this auxin-cytokinin interaction. Kinetin by itself did not promote elongation; nor did it enhance auxin-promoted elongation at low auxin concentrations.     

Das Verhalten von CO2-Assimilation,Nadpund PGS-Reduktion und ATP-Synthese Intakter Blattzellen in Abhängigkeit vom Wassergehalt

Summary The carrot-root tissue culture assay for cytokinin activity has been improved by changing the site of explant excision and eliminating certain vitamins from the basal medium. These modifications increased its sensitivity and enabled zeatin [6-(4-hydroxy-3-methylbut-trans-2-enyl)aminopurine] to be detected at concentrations less than 5×10^-5M. In the improved assay, zeatin was markedly more active than kinetin, 6-benzylaminopurine, 6-(o-methylbenzyl)aminopurine and 6-(3-methylbut-2-enyl)aminopurine.The activity of zeatin also exceeded that of kinetin in the etiolated bean-leaf disk expansion assay. Zeatin was markedly more effective than kinetin and 6-(3-methylbut-2-enyl)aminopurine in promoting frond expansion and increasing frond number of Spirodela oligorrhiza cultures grown under continuous illumination. Zeatin was also more active than kinetin and 6-(3-methylbut-2-enyl)aminopurine in increasing frond number of Spirodela cultures grown in darkness. In retarding the senescence of disks of leaves of several species, kinetin was considerably more effective than zeatin which was more active than 6-(3-methylbut-2-enyl)aminopurine. The allylic hydroxyl group in zeatin is therefore a structural feature associated with high cytokinin activity.The relative activities of cytokinins can be very different and even in reverse order in different bioassays. It is suggested that this is due to the mechanism of cytokinin action varying in the different biological systems used.Part IV: Shannon and Letham (1966).     

Abscisic Acid Inhibition of Kinetin Nucleotide Formation in Germinating Lettuce Seeds

Imbibing ‘Grand Rapids’ lettuce (Lactuca saliva L.) seeds take up ¹⁴C-kinetin, and metabolize this cytokinin to the 5′-nucleotide. The identity of the labeled nucleotide in seed extracts was verified by Sephadex LH-20 column chromatography, paper and thin layer chromatography, and high voltage paper electrophoresis. Incubations with kinetin in the presence of abscisic acid lead to an apparent specific inhibition of kinetin nucleotide formation. ABA has no effect on kinetin uptake, and does not inhibit kinetin nucleotide synthesis in vitro by a cell-free preparation from lettuce seeds. Additionally, ABA does not inhibit adenylate synthesis from exogenously supplied adenine. These results represent a specific cytokinin-ABA interaction, which might play a significant role in the hormonal regulation of lettuce seed germination.     

Kinetin inhibition of h-uracil and C-leucine incorporation by tobacco cells in suspension culture

The rate of ¹⁴C-leucine and ³H-uracil incorporation by tobacco cells (Nicotiana tabaccum var. Samsun N.N.) in suspension culture was simultaneously decreased by the addition of kinetin at concentrations above 2.5 × 10⁻⁵m. Ribosomal RNA was the first RNA species affected by kinetin. The purine derivatives, adenine and N⁶-methyl-aminopurine, which exhibit low cytokinin activity overcame the inhibitory effects of kinetin. However, purine derivatives without cytokinin activity, guanine, N^6,6-dimethyl-aminopurine, and 2-aminopurine, did not relieve kinetin inhibition.     

THE ROLE OF CYTOKININS IN THE “FASCIATION” DISEASE CAUSED BY CORYNEBACTERIUM FASCIANS

The disease caused by Corynebacterium fascians can be imitated by treatment with kinetin. Cultures of the bacteria were grown on a purine-free medium of known composition. They require thiamine. Two assays for cytokinin were developed; one depends on the release of lateral pea buds from apical dominance and is highly specific but of only moderate sensitivity. The other depends on the retention of chlorophyll in senescing oat leaves and is very sensitive (detecting 2 × 10^–4 μg of kinetin equivalents), though it is somewhat less specific and, therefore, requires care in usage. Both give results in 3–4 days and are thus far more rapid than tissue culture methods. With these assays C. fascians has been shown to produce a chloroform-soluble cytokinin active in both tests. The substance is stable to heat in acid or basic media, is soluble in non-polar solvents, and behaves as a base. It is precipitated from water solution by Ag ions and may, therefore, be a purine derivative. Pea tissue infected with C. fascians, but not uninfected tissue, yields a compound of similar solubilities and biological activity. Reasons are given for believing that the synthesis of cytokinins may be important, not only for C. fascians, but also for many other plant parasites.     

Short-Term Metabolism of Radioactive Kinetin during Lettuce Seed Germination

Exogenously applied 8-¹⁴C-kinetin is rapidly taken up by seeds of lettuce (Lactuca sativa L. cv. Grand Rapids). Radioactive metabolites were extracted and purified by solvent fractionation, column and paper chromatography. The primary metabolite was identified as the 9-riboside-5′-monophosphate. As germination proceeds, some kinetin is released from this bound storage form, giving a maximum level of free kinetin at 12 hours after imbibition. After this time the concentration of ribotide increases while the concentration of free base decreases. Other metabolites are the 9-riboside, AMP and IMP. It is suggested that a required amount of free base cytokinin is necessary by 12 hours after imbibition. This concentration of free cytokinin may act as a physiological trigger for later events during germination.     

Dimethyl sulfoxide: A solvent for cytokinins in theAmaranthus bioassay

Dimethyl sulfoxide present in the agar medium at concentration 0.2 % (v/v) and lower does not inhibit cytokinin-induced betacyanin synthesis in theAmaranthus caudatus seedlings. The activity of kinetin, N⁶-(Δ²-isopentenyl)adenine andtrans- zeatin is the same when these cytokinins are dissolved in either water or dimethyl sulfoxide and incorporated into the medium after autoclaving. A simple method is described which allows the cytokinin activity of slightly water-soluble and thermolabile compounds,e.g. aromatic urea and thiourea derivatives, to be determined in theAmaranthus bioassay.     

Effects of kinetin and related compounds on growth and sexual reproduction of Saprolegnia australis

Summary An isolate of Saprolegnia australis grew readily and produced abundant sex organs on a chemically defined medium. The optimal temperature for growth was higher (25°) than the optimal temperature for sexual reproduction (20°). Addition of the purines, adenine and hypoxanthine to the medium stimulated production of oogonia while addition of kinetin and 6-benzylaminopurine inhibited this process. When kinetin and adenine were added together, the two purines interacted in affecting oogonial production, and adenine overcame the inhibitory action of kinetin. It is suggested that purines may play some specific role in the synthesis of one or more of the hormones which probably initiate sexual reproduction in S. australis and that kinetin and 6-benzylaminopurine may inhibit hormone synthesis. Two other growth regulators of higher plants, indol-3-ylacetic acid and gibberellic acid, did not affect development of the fungus. No evidence was found to suggest that S. australis was producing any substance with cytokinin activity.     

Uptake, transport and metabolism of cytokinin in moss protonema

Uptake, transport and metabolism of cytokinin in the protonemaof Funaria hygrometrica were studied using labelled kinetin(6-furfurylamino [8-¹⁴C]-purine). All cells of the protonema,chloronema and caulonema, were able to take up kinetin, whichwas carried in the symplastic transport system from cell tocell. Radioactivity was especially accumulated in growing cellsof the protonema. Kinetin was metabolized immediately afteruptake. While only very little kinetin (less than 1%) remainedas free kinetin and one part was immobilized in chromatographicseparation [e.g. attached to proteins and incorporated intonucleic acids (17)], most of the remaining kinetin was metabolizedto adenine derivatives. Exogenously supplied adenosine changedthe metabolism of kinetin. In the caulonema, adenosine reducedthe turnover of kinetin to other adenine derivatives and enhancedthe content of labelling in the start fraction. Thus adenosinecan stimulate cytokinin-dependent bud formation in moss protonema. (Received November 24, 1977; )     

Toward the production of artemisinin through tissue culture: Determining nutrient-hormone combinations suitable for cell suspension cultures

Summary Artemisia annua L. is the source of a potent antimalarial, artemisinin. As part of a program to produce artemisinin through tissue culture, a series of 14 multifactorial experiments were conducted to determine suitable conditions for initiating and maintaining friable callus fromA. annua. In the first six experiments, three different nutrient formulations [Gamborg B5 (B5), Murashige and Skoog (MS), and Whetmore and Rier (WR)], each with 32 combinations of auxins and cytokinins [2,4-dichlorophenoxyacetic acid (2,4-D) with benzyladenine (BA), or 1-naphthaleneacetic acid (NAA) with 6-furfurylaminopurine (kinetin)], were tested. Both B5 and WR nutrients supported friable callus formation from leaf explants with some combinations of auxin and cytokinin. Inasmuch as friable callus seemed to be produced over a wider range of auxin and cytokinin concentrations in combination with B5, the remaining experiments were conducted solely with this nutrient formulation. In the remaining eight experiments, it was determined that friable callus formed when combinations of NAA with kinetin or 2,4-D and BA were used with B5 medium. Lighter colored, more friable callus formed in response to 2,4-D and BA than with NAA and kinetin. No single combination of concentrations of auxin and cytokinin seemed to be “ideal” for producing friable callus. Ranges of 2,4-D from 0.5 to 2.0 with BA between 0.025 and 0.1, or NAA between 0.5 and 2.0 with kinetin between 0.5 and 1.0 mg/liter, produced acceptable results.     

Promotion of stomatal opening in detached epidermis of Kalanchoe daigremontiana Hamet et Perr. by natural and synthetic cytokinins

The cytokinins kinetin and zeatin increased stomatal opening at 15°C in the dark by up to 50% in detached epidermis of the CAM plant Kalanchoe daigremontiana. Stomata opened maximally following incubation with 10 mmol m^-3 cytokinin. This study extends the range of species in which exogenous cytokinins promote stomatal opening.