Effects of N-1-naphthylphthalamic acid on the growth and bud formation of tobacco callus grown in vitro

The effect of N-1 -naphthylphthalamic acid (NPA), indole-3-aceticacid (IAA) and kinetin on callus growth and bud formation wasstudied mainly by a tobacco callus culture method. Callus producedfrom Nicotiana tabacum var. Wisconsin 38 was used as the testplant material. Callus growth on nutrient agar containing 2mg/liter of IAA was promoted by NPA added at a concentrationof 0.5 mg/liter with 0.4 mg/liter of kinetin or by NPA addedat 5 mg/liter in the absence of kinetin. At a high concentrationof 50 mg/liter, however, NPA inhibited growth on the mediumcontaining 2 mg/liter IAA and no kinetin. Kinetin reduced thisNPA inhibition. In the presence of 0.4 mg/liter kinetin and2 mg/liter IAA, when the concentration of NPA was 50 mg/liter,buds were initiated after calluses were grown on the test mediumfor 7 weeks in dim light, but no buds formed when NPA was omittedfrom the above medium. The control of callus growth and bud initiation is based onthe active ratio of auxin (IAA) to cytokinin (kinetin) in themedium and NPA added to the medium can promote or inhibit callusgrowth and induce bud formation. Therefore, it is proposed thatNPA can itself reduce auxin activity or enhance cytokinin activityand hence change the active ratio of the two regulators. NPAmay enhance the activity of cytokinin (here supplied as kinetin)but cannot substitute for it. ¹Present address: Department of Biology, Wisconsin State University,Oshkosh, Wisconsin 54901, U. S. A. (Received March 10, 1969; )     

Regulation of Tracheary Element Differentiation by Exogenous L-Methionine in Callus of Soya Bean Cultivars

The relationship between the induction of tracheary elementdifferentiation and exogenous L-methionine was examined in agar-growncultures of soya bean callus initiated from Glycine max L. ‘Wayne’and ‘Clark 63’. Although Wayne is a normal cultivarsoya bean, seedlings of Clark 63 exhibit abnormal growth at25 °C due to exessive ethylene biosynthesis at this temperature.Wayne callus showed increased xylogenesis in the presence ofexogenous L-methionine (3.7 µg 1^–1) in comparisonto IAA–KN controls at both 20 and 25 °C. Clark 63callus produced greater numbers of tracheary elements in responseto exogenous L-methionine only at 25 °C. The induction ofxylem differentiation was independent of the maintenance temperatureof the stock cultures of both cultivars. Xylogenesis initiatedbyan IAA–KN medium was inhibited by the addition of AgNO₃(20 mg 1^–1) to the extent of 76.5 per cent in cv. Wayneand 6 per cent in cv. Clark 63. The inhibitory effect was partiallyreversed by the addition of L-methionine (3.7 µg 1^–1)to the IAA–KN–AgNO₂ medium. These data support thehypothesis that xylogenesis in vitro involves auxin, cytokininand ethylene. differentiation, xylogenesis, L-methionine, ethylene, Glycine max L., soya bean, callus culture, auxin, kinetin     

Role of Cytokinin in Vessel Regeneration in Wounded Coleus Internodes

Cytokinin was found to be a controlling or limiting factor invessel regeneration around a wound in internodes of Coleus blumeiBenth. in which the endogenous cytokinin level was minimized.The cytokinin was applied in aqueous solution to the base ofexcised, mature internodes that had an active vascular cambium.Each internode also received IAA in lanolin at its apical end.Under low (0.1 %, w/w) or high (10%, w/w) auxin concentrations,the control internodes (without exogenous cytokinin) exhibitedsmall amounts of vessel regeneration. At appropriate concentrationszeatin, kinetin and 6-benzylamino-purine (BAP) induced a significantincrease in vessel regeneration around the wound. The threecytokinins also induced novel patterns of supplementary regenerationfurther from the wound surface. Kinetin and BAP showed the strongestpromoting effect at 5 and 10 µg ml^–1, while zeatinwas most effective at 20 µg ml^–1. At a low (0.1%) auxin level zeatin was the most effective cytokinin, whereaskinetin was the most effective one at high (1 %) auxin. An inhibitoryeffect on vessel regeneration was observed at the highest kinetinconcentration tested (50 µg ml^–1). The regenerationof vessels induced by cytokinin was very polar. Many more regeneratedvessel members differentiated below the wound than above it,and the regeneration process proceeded acropetally from thebase of the internode to its upper parts. Our results implya basipetal polar increase in cambium responsiveness along thestem axis from internode 5 to 7. The possible significance ofsuch a basipetal increase in cambium sensitivity in wood formationin trees is discussed. Auxin, Coleus blumei, cytokinin, vascular differentiation, vessel regeneration, wound xylem     

Expression of a KDEL-tagged dengue virus protein in cell suspension cultures of <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> and <Emphasis Type="Italic">Morinda citrifolia</Emphasis>

Hypericum perforatum L. (St. John’s wort) produces a number of phytochemicals having medicinal, anti-microbial, anti-viral and anti-oxidative properties. Plant extracts are generally used for treatment of mild to medium cases of depression. Plant regeneration can be achieved in this species by in vitro culture of a variety of explants. However, there are no reports of regeneration from petal explants. In this report plant regeneration from petal explants of St. John’s wort was evaluated. Petals of various ages were cultured on agarized Murashige and Skoog 1962 (MS) medium supplemented with auxin and cytokinin (kinetin), maintained in the dark and callus and shoot regeneration determined after 28 days. At an auxin to cytokinin ratio of 10:1, callus and shoot formation were induced by all levels of indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA), while 2,4-dichlorophenoxyacetic acid (2,4-D) induced only callus formation. The optimum level of auxin for shoot regeneration was 1.0 and 0.1 mg/l kinetin, where the regeneration frequency was 100 percent for all three auxins. The highest number of shoots per explant (57.4 and 53.4) was obtained with IAA and IBA, respectively. In the absence of auxin, kinetin levels of 0.1 and 0.25 mg/l induce callus and shoot formation at low frequency but not at lower levels. Callus and shoot formation did not occur in the absence of growth regulators. Petal-derived shoots were successfully rooted on half-strength MS medium without a requirement for exogenous auxin and flowering plants were established under greenhouse conditions. From these results it can be concluded that auxin type is a critical factor for plant regeneration from petal explants of Hypericum perforatum and there is no absolute requirement for high levels of cytokinin.     

Boron Nutrition of Cultured Tobacco BY-2 Cells: I. Requirement for and Intracellular Localization of Boron and Selection of Cells that Tolerate Low Levels of Boron

Cultured cells of tobacco (Nicotiana tabacum L. cv. Bright Yellow2) grown under the standard culture conditions (1 mg boron liter^–1medium as boric acid) contained boron at a concentration of2.26 mg boron kg^–1 oven-dried cells and the protoplastcontained 1.26% of the boron in the cells. The cells requiredboron for growth and the half-maximum growth rate was obtainedwith 0.056 mg of boron liter^–1 medium. Subculturing thecells in media with lower concentrations of boron allowed selectionof cells that can grow even in the presence of 1 µg boronliter^–1 medium. Cell walls of the selected cells seemedto be thicker than those of the control cells and Golgi bodieswere accompanied by more secretory vesicles than those in thecontrol cells. (Received May 25, 1992; Accepted September 10, 1992)     

Cytokinins in tobacco crown gall tumors

Bacteria-free tobacco ($\text{Nicotiana tabacum}$ cv. Wisconsin #38) crown gall strains incited by $\text{Agrobacterium tumefaciens}$ C58, 27, B6, CGIC, and AT4 have been analyzed for cytokinin content with the tobacco callus bioassay. All tumor strains contained high total levels of cytokinins ranging from 4–810 kinetin equivalents per kg fresh weight compared to 0.5 kinetin equivalents per kg for normal callus growing on medium with 0.1 μM N6-benzyladenine. Fractionation on a column of Sephadex LH-20 separated cytokinin activity from B6 tumors into a number of components among which ribosyl-$\text{trans}$-zeatin has been purified and characterized based on uv spectrum, biological activity and mass spectrum.     

The effect of auxin starvation on the growth of auxin-dependent tobacco cell culture: dynamics of auxin-binding activity and endogenous free IAA content

The growth of a cell strain derived from the stem pith of tobacco(Nicotiana tabacum L., cv. Virginia Bright Italia) was investigatedin subcultures grown at various levels of synthetic auxins.Both partial and complete auxin starvation resulted in a decreaseof the frequency of cell division. For these treatments theendogenous free indole-3-acetic acid content increased substantiallyat the commencement of the exponential growth phase. The possibilitythat the receptivity of the cells to auxin changed during thegrowth cycle was examined by measuring the activity of a membrane-boundauxin-binding site. In subcultures grown in a medium with anoptimal auxin concentration the maximum auxin-binding activitywas restricted to the end of the exponential growth phase. Inthe cells cultivated in partially or completely auxin deprivedmedia the auxin-binding activity increased to varying extents.These results probably reflect mechanisms controlling both theintracellular content of free auxin and the sensitivity of thecells to exogenous auxin supply (including auxin binding) withrespect to the cell division and/or growth Key words: Nicotiana tabacum L., plant cell culture, IAA, auxin-binding site, cell division     

Functional Correlation between Endogenous Phytohormone Levels and Hormone Autotrophy of Transformed and Habituated Soybean Cell Lines

We analysed the time course of the endogenous free IAA and cytokininlevels in hormone requiring and hormone autotrophic (both transformedand untransformed) Glycine max. L. Merr. cv. Mandarin tissuecultures. The auxin habituated line showed an enhanced endogenous IAAlevel, whereas the IAA as well as the cytokinin concentrationsin the cytokinin habituated line differed not significantlyfrom the non-habituated hormone requiring soybean callus. It were only the auxin habituated cells that could be inducedto fully habituated cells, from which a pale and a green typewas isolated. The phytohormone autotrophic growth of the paletype was sustained by enhanced IAA levels, whereas the greentype was characterised by elevated cytokinin concentrations. These results on the phytohormone content of partially and fullyhabituated soybean calli were compared with soybean crown galllines and discussed in view of the positive effect of exogenouslyapplied cytokinins on the endogenous IAA levels. ³Recipient of an Instituut voor Wetenschappelijk Onderzoek inNijverheid en Landbouw (I.W.O.N.L.) grant. ⁴Senior Research Associate Nationaal Fonds voor wetenschappelijkOnderzoek (N.F.W.O.). (Received March 25, 1988; Accepted July 7, 1988)     

Hormonal control of tobacco crown gall tumor morphology

The endogenous levels of auxin and cytokinin in teratoma and unorganized tobacco (Nicotiana tabacum L. var Wisconsin #38) crown gall tumor tissues were determined. Teratoma tissues contain levels of auxin and cytokinin favorable for shoot formation, whereas unorganized tumors contain levels of auxin that suppress shoot formation. This conclusion is based upon the observation that when levels of auxin and cytokinin similar to those found in a teratoma were added to the growth medium of nontumorous tobacco tissue, shoot formation resulted; when levels similar to those found in unorganized tumors were added, the normal tissue grew as unorganized callus.     

Hormonal Control of Xylogenesis in Pith Parenchyma Explants of Lactuca

The time course of xylem differentiation was determined in explantsof lettuce pith parenchyma (Lactuca sativa L. cv. Romana) culturedon Murashige and Skoog (1962) medium using different concentrationsof auxin (IAA) and one cytokinin (zeatin or kinetin). Increasinglevels of auxin from I mg 1^–1 to 15 mg 1^–1 in thepresence of a constant level of a cytokinin (zeatin or kinetin)yielded up to 10 mg 1^–1 IAA, an increase in the numberof tracheary element formations. Cytokinin concentrations aboveand below o.1 mg 1^–1 interacting with an optimal xylogenicamount of auxin inhibited xylogenesis. The IAA (10 mg 1^–1)-zeatin(0.1 mg ^1–1) treatment produced the greatest number oftracheids, while kinetin compared to zeatin did not producesuch an effect. The different effectiveness of zeatin and kinetinin inducing tracheary element formations was not due to a differentcapacity of the two cytokinins to stimulate cell division butit seems likely that zeatin, because of interaction with IAA,is more active than kinetin in the determination of the dividingcells in a specific type of cytodifferentiation. The IAA (10mg 1^–1)-zeatin (0.1 mg 1^–1) treatment produced about6.9 per cent tracheids with respect to cell division while IAA(10 mg 1^–1)-kinetin (0.1 mg 1^–1) produced 4.2 percent. These results are discussed with reference to the problemsof hormonal control of xylem differentiation.     

Sterol composition of normal and habituated sugarbeet callus (Beta vulgaris L.Altissima)

Summary A detailed qualitative and quantitative analysis of the sterol content of normal (auxin and cytokinin requiring) and habituated (auxin and cytokinin independent) sugarbeet callus (Beta vulgaris L.altissima) was made using mass spectrometry with gas chromatography. The total sterol content of the two lines did not differ significantly. Δ⁷-Sterols were the most important class of sterols in the two sugarbeet callus lines, as in allChenopodiaceae. Elevated levels of Δ⁸-sterols were found in the habituated callus. These sterols are considered to be badly integrated in the membrane of eucaryotic cells. A partial blocking of Δ⁸-Δ⁷-isomerase is hypothesized in the habituated cell line.     

Two effects of cytokinin on the auxin requirement of tobacco callus cultures

Cytokinin affects the requirement for auxin of a strain of tobacco callus (Nicotiana tabacum) which is cytokinin-autotrophic when grown on Murashige and Skoog medium with 11.4 mum of indole-3-acetic acid but requires cytokinin 6-(3-methyl-2-butenylamino)purine (i(6) Ade) when grown on the same medium with <3 mum indole-3-acetic acid. As the exogenous concentration of cytokinin (i(6) Ade) is increased, the concentration of indole-3-acetic acid required for growth is decreased. A second effect of cytokinin, observed sporadically in cultures with 2.5 mum or 5 mum i(6) Ade, is the transformation of some of the callus pieces to auxin-autotrophic growth. Strains, both callus-forming and bud-forming tissues, that arise in this manner are not permanently altered in their auxin requirement because subcultures on medium without cytokinin still require exogenous auxin.     

Hormonal induction and antihormonal inhibition of tracheary element differentiation in Zinnia cell cultures

Mechanically isolated mesophyll cells of Zinnia elegans L. cv Envy differentiate to tracheary elements when cultured in inductive medium containing sufficient auxin and cytokinin. Tracheary element differentiation was induced by the three auxins (alpha-naphthaleneacetic acid, indole-3-acetic acid, and 2,4-dichlorophenoxyacetic acid) and four cytokinins (6-benzyladenine, kinetin, 2-isopentenyladenine and zeatin) tested. Tracheary element formation is inhibited or delayed if the inductive medium is supplemented with an anticytokinin, antiauxin, or inhibitor of auxin transport.     

The Effect of Mutations in the T-DNA Encoded Auxin Pathway on the Endogenous Phytohormone Content in Cloned Nicotiana tabacum Crown Gall Tissues

We analyzed the endogenous auxin and cytokinin levels of clonedNicotiana tabacum SR 1-lines induced either by the wild-typeAgrobacterium tumefaciens C58 strain or by mutants affectedin the T-DNA-encoded IAA biosynthesis pathway. The wild-typeSR1-C58 line contained up to 20 times more IAA than a nontransformedSRI-callus line. The mutant lines affected in gene 1 (iaaM^–)or gene 2 (iaaH^–) contained intermediate levels of IAA. Analysis of the endogenous levels of indole-3-acetamide (IAM)in the nontransformed SR 1 callus line, the wild-type SR1-C58and the two mutant lines confirmed the T-DNA-induced IAA biosynthesispathway in the transformed tumor cells. Supplementing auxinto the mutant lines resulted in complete suppression of theshoot-forming ability, but no changes in the endogenous IAAlevels. There was no marked difference in the cytokinin level betweenthe nontransformed callus line and the wild type tumor line.The two mutant lines, however, showed a 20- to 30-fold highercytokinin level which was not affected by the addition of NAA.The T-DNA encoded hormone biosynthetic pathways are discussedin relation to pathways of the host plant. (Received July 29, 1986; Accepted February 14, 1987)     

Cytokinin and Ethylene Affect Auxin Transport-Dependent Rhizogenesis in Hypocotyls of Common Ice Plant (<Emphasis Type="Italic">Mesembryanthemum crystallinum</Emphasis> L<Emphasis Type="Italic">.</Emphasis>)

Hypocotyl explants of Mesembryanthemum crystallinum regenerated roots when cultured vertically with either the apical end (AE) or basal end (BE) in media containing indole-3-acetic acid (IAA). IAA alone induced roots regularly from the basal end of the explants, either from the cut surface immersed in the medium or from the opposite side. The inhibitors of auxin efflux carriers, α-naphthylphthalamic acid (NPA) and 2,3,5-triiodobenzoic acid (TIBA), inhibited rhizogenesis only from AE-cultured explants, indicating the role of polar auxin transport in root regeneration in this system. Cytokinin (zeatin, kinetin, BAP) added to auxin-containing medium reduced rhizogenesis from the explants maintained with BE and AE and additionally changed the IAA-induced pattern of rooting in AE-cultured explants by favoring rooting from the apical end and middle part of the hypocotyl with its concomitant reduction from the basal end. The addition of kinetin did not influence the content of IAA in the explants maintained with AE, suggesting that the cytokinin effect on root patterning was not dependent on auxin biosynthesis. Kinetin, however, strongly enhanced ethylene production. The importance of ethylene in regulating PAT-dependent rhizogenesis was tested by using an ethylene antagonist AgNO₃, an inhibitor of ethylene synthesis aminoethoxyvinylglycine (AVG), and a precursor of ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC). AgNO₃ applied together with IAA or with IAA and kinetin strongly reduced the production of ethylene, inhibited rhizogenesis, and induced nonregenerative callus from BE, suggesting the need for ethylene signaling to elicit the rhizogenic action of auxin. A reduction of rhizogenesis and decrease of ethylene biosynthesis was also caused by AVG. In addition, AVG at 10 μM reversed the effect of cytokinin on root patterning, resulting in roots emerging only from BE on the medium with IAA and kinetin. Conversely, ACC at 200 μM markedly enhanced the production of ethylene and partly mimicked the effect of cytokinin when applied with IAA alone, thus confirming that in cultured hypocotyls of ice plant, cytokinin affects IAA-induced rhizogenesis through an ethylene-dependent pathway.     

Tiba inhibition of <Emphasis Type="Italic">in vitro</Emphasis> organogenesis in excised tobacco leaf explants

Summary Triiodobenzoic acid (TIBA), an anti-auxin, was found to inhibit both shoot and root formation in cultured excised leaf explants of tobacco (Nicotiana tabacum L.). The shoot formation (SF) medium used required only exogenous cytokinin (N⁶-benzyladenine) and the root formation (RF) medium required both auxin (indole-3-butyric acid) and cytokinin (kinetin). By transferring the explants from SF or RF media to SF or RF media with TIBA (4.0×10⁻⁵ M), respectively or vice versa, at different times in culture, it was found that TIBA inhibition was at the time of meristemoid formation and after determination of organogenesis. This indicates that TIBA interfered with endogenous auxin involvement in organized cell division.     

Comparison of zeatin indoleacetate with zeatin and indoleacetic Acid in the tobacco bioassay

Zeatin indole-3-acetate, 6-[4-(indole-3-acetoxy)-3-methyl-trans-2-butenylamino]purine, is at least as effective as zeatin on a molar basis in satisfying the cytokinin requirement for growth and bud formation in tobacco bioassays. It is less effective than indole-3-acetic acid and is needed as a variable function of the cytokinin concentration for satisfying the optimal requirement of an auxin. Comparisons of the types of growth and yield of tissue obtained with serial concentration of the ester and with equimolar mixtures of its free base and acid indicate that the relative requirement for auxin changes with the concentration of cytokinin and is related to the types of callus growth and differentiation which occur. The results also suggest that the ester serves as a source of auxin only after modification, presumably by hydrolysis to indoleacetic acid.     

In vitro Hormonal Regulation of Laticifer Differentiation in Calotropis procera

Cultures of Calotropis procera were maintained on MS mediumsupplemented with 4·6 µM FAP and 3 µM NAA.Laticifer initials were observed in 2-week-old cultures whichwere converted into matured, branched, non-articulated laticifersin 4 weeks. It was observed that laticifer differentiation increasedwith the age of cultures, from 2·78% (in the second passage)to 15·11% (in the 12th passage). It has been establishedthat laticifer differentiation in vitro is a cytokinin-dependentprocess and among the cytokinins, FAP was more effective thanBA and 2-iP. But the type of auxin and its concentration alsoplay an important role in modifying the effect of cytokinin.Among the different auxins used IAA was more effective for laticiferdifferentiation than IBA and NAA, while 2,4D was inhibitory.Maximum laticifer differentiation (17·01% was observedon MS medium supplemented with 4·6 µM FAP and 1µM IAA.Copyright 1995, 1999 Academic Press Calotropis procera, callus culture, laticifer, differentiation, hormonal regulation     

Plant regeneration from petal explants of <Emphasis Type="Italic">Hypericum perforatum</Emphasis> L

Hypericum perforatum L. (St. John’s wort) produces a number of phytochemicals having medicinal, anti-microbial, anti-viral and anti-oxidative properties. Plant extracts are generally used for treatment of mild to medium cases of depression. Plant regeneration can be achieved in this species by in vitro culture of a variety of explants. However, there are no reports of regeneration from petal explants. In this report plant regeneration from petal explants of St. John’s wort was evaluated. Petals of various ages were cultured on agarized Murashige and Skoog 1962 (MS) medium supplemented with auxin and cytokinin (kinetin), maintained in the dark and callus and shoot regeneration determined after 28 days. At an auxin to cytokinin ratio of 10:1, callus and shoot formation were induced by all levels of indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA), while 2,4-dichlorophenoxyacetic acid (2,4-D) induced only callus formation. The optimum level of auxin for shoot regeneration was 1.0 and 0.1 mg/l kinetin, where the regeneration frequency was 100 percent for all three auxins. The highest number of shoots per explant (57.4 and 53.4) was obtained with IAA and IBA, respectively. In the absence of auxin, kinetin levels of 0.1 and 0.25 mg/l induce callus and shoot formation at low frequency but not at lower levels. Callus and shoot formation did not occur in the absence of growth regulators. Petal-derived shoots were successfully rooted on half-strength MS medium without a requirement for exogenous auxin and flowering plants were established under greenhouse conditions. From these results it can be concluded that auxin type is a critical factor for plant regeneration from petal explants of Hypericum perforatum and there is no absolute requirement for high levels of cytokinin.     

Effects of cytokinin on adventitious root formation in callus cultures ofVigna unguiculata (L.) walp

Summary Yellowish compact callus, induced from cowpea hypocotyls on Murashige and Skoog(MS) medium (1962) containing 0.2 mg/l(0.93 μM) kinetin and 0.4 mg/l (1.81 μM) 2,4-dichlorophenoxyacetic acid (2,4-D), was subcultured on MS medium containing cytokinin alone, auxin alone, or auxins plus cytokinins in order to determine the effect of cytokinins on root organogenesis in callus cultures. The callus actively proliferated on the same medium but did not show any organogenic activity macroscopically as well as microscopically. On medium with N⁶-benzyladenine (BA) and 1-naphthaleneacetic acid (NAA), the yellowish compact callus first changed to pale green compact callus and then many green spots appeared on its surface under light culture. But the yellowsih compact callus remained yellowish and white spots appeared on its surface in dark culture. These spots gradually became white nodular structures. Adventitious root formation from the nodular structures occurred not only on the same medium, but also on medium with either auxin or cytokinin but not both. Yellowish compact callus on medium with auxin alone was transformed to yellowish friable callus, which did not develop adventitious roots. The yellowish friable callus could gain rhizogenic activity only after morphological modification to pale green compact callus on medium with auxin plus cytokinin. The modified callus did not form adventitious roots on medium with auxins but only with cytokinins. Therefore, it is suggested that cytokinins have stimulating effects on root formation from callus that previously did not show rhizogenic activity on medium with auxins alone. In addition, the rhizogenic potential of cowpea callus was discriminated from that of leaf explants, which formed adventitious roots directly on medium with auxin alone.