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.     

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.     

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.     

An auxin-auxotrophic mutant of Nicotiana plumbaginifolia

Summary Auxin (indole-3-acetic acid) is considered to be an important signalling molecule in the regulation of plant growth and development but neither auxin synthesis nor its mode of action is clearly understood. To identify genes involved in these processes, mutations were sought that altered the auxin requirement of plant tissues for growth. For the first time mutant plants were obtained that carry a recessive mutation at a single nuclear locus (auxl) which results in an absolute requirement for exogenous auxin for normal growth. In the absence of auxin treatment, mutant plants undergo premature senescence and die.Abbreviations BAP 6-benzylaminopurine - BUdR 5-bromodeoxyuridine - 2,4-D 2,4-dichlorophenoxyacetic acid - FUdR 5-fluorodeoxyuridine - IAA-EE indole-3-acetic acid ethyl ester - IMS indole-3-methanesulfonic acid     

Indole-3-acetic acid metabolism in hormone-autotrophic, embryogenic callus of Inmil® cherry rootstock (Prunus incisa×serrula'GM 9') and in hormone-dependent, nonembryogenic calli of Prunus incisa×serrula and Prunus domestica

Callus derived from the roots of Inmil® cherry rootstock ( Prunus incisa × serrula ) proliferates in a hormone-free solid medium. When transferred to a hormone-free liquid medium, such callus forms somatic embryos. On the other hand, leaf-derived callus of P. incisa × serrula and leaf- and root-derived calli of P. domestica require exogenous auxin for sustained growth and never form embryos. Levels of free and esterified indole-3-acetic acid (IAA) were similar in both types of calli grown on a solid medium, whereas the amide-conjugated IAA was higher in the root-derived embryogenic one. Transfer to a liquid medium did not affect the level of both free and conjugated IAA in the nonembryogenic callus, but in the embryogenic callus it decreased the level of amide-conjugated IAA. Isotopic dilution of 13 C-IAA taken up from a medium was faster in the embryoegenic than in the nonembryogenic calli, which shows that the rate of IAA metabolism was higher in embryogenic callus. Besides IAA, indole-3-ethanol and indole-3-acetyl-N-aspartate were detected in nonembryogenic calli and in in vitro-grown shoots of P. domestica , whereas in embryogenic callus and in in vitro - grown shoots of P. incisa × serrula indole-3-acetamide was detected.     

Auxin structure and activity on tomato morphogenesis in vitro and pea stem elongation

In order to understand better the relationship between auxin structure and activity on morphogenesis and cell elongation, six different auxins were tested on the regeneration of tomato (Lycopersicon esculentum Miller var. Alice) from cotyledons and on pea (Pisum sativum L. var. Alaska) stem elongation. The auxins were: indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), 1, 2-benzisoxazole-3-acetic acid (BOA), 1,2-benzisothiazole-3-acetic acid (BIA), 1-naphthalenacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D). All these compounds obey the minimum requirement rules for auxin activity and all were effective on cell elongation. At the dose of 10 M and in the absence of cytokinin, they all, except 2,4-D, induced roots, while in the presence of cytokinin they induced shoots, roots, hairy root-like filaments (HRLF) or callus depending on their concentration. The morphogenetic pattern did not change by varying cytokinin concentration. We conclude that auxin structure plays a minor role in morphogenesis or cell elongation, because it is only responsible for variations in the level of auxin activity.     

The nature of spontaneous changes in growth rate in isolated coleoptile segments

About 4 hours after they are cut from the seedling, corn (Zea mays L.) coleoptile segments mounted vertically show a strong increase in growth rate. This increase occurs in water or various buffers near pH 7 and is not accompanied by the accumulation of a growth promoter in the medium. The increase in growth rate is prevented by 1 mmp-fluorophenylalanine and is strongly inhibited by 0.1 mmp-chlorophenoxyisobutyric acid.The increased growth rate is accompanied by a 95% increase in the ability of tissue extracts to catalyze the conversion of (14)C-tryptophan to (14)C-indole-3-acetic acid and by a nearly 3-fold increase in indole-3-acetic acid oxidase activity. The increase in growth rate is also observed in segments from coleoptiles grown aseptically.The spontaneous increase in growth rate is completely but reversibly inhibited by 1 mum indole-3-acetic acid. Cytokinins have little effect on the spontaneous growth response, whereas gibberellic acid is observed to extend the latent period and reduce the magnitude of the response. It is tentatively concluded that the increase in endogenous growth rate may result from increased auxin production upon derepression of the auxin biosynthesis pathway after isolating the tissue from the normal supply of auxin from the tip.     

Effect of different plant growth regulators on micro-tuber induction and plant regeneration of Pinellia ternate (Thunb) Briet

An efficient micropropagation system for Pinellia ternate (Thunb) Briet, a traditional Chinese medicinal plant, has been developed. Petiole and lamina of P. ternate were used as explants and cultured on Murashige and Skoog (MS) medium containing different concentrations of different plant growth regulators. The results indicated that low concentration of 2,4-dicholorophenoxy acetic acid (2,4-D), indole-3-acetic acid (IAA) and α-naphthalene acetic acid (NAA) were suitable for micro-tuber induction, but callus induction rate increased with increasing concentrations of growth regulators. Tubers induction rates of petiole and leaf were (81.8 %–100 %) and (89.4 %–96.0 %) respectively, when 0.2 mg l⁻¹ 2, 4-dicholorophenoxy acetic acid, indole-3-acetic acid or α-naphthalene acetic acid were present in the medium. Tubers induction rates of petiole and leaf cultured on MS medium supplemented with 0.2–0.5 mg l⁻¹ 6-benzyl amino purine (6-BAP) were (94.1 %–100 %) and (96.0 %–100 %) respectively. When the concentration of 2,4-dicholorophenoxy acetic acid, α-naphthalene acetic acid and 6-benzyl amino purine was increased to 2.0 mg l⁻¹, callus induction rates of petiole and leaf were 100 % and 98.2 %, 91.0 % and 36.0 %, 62.3 % and 70.0 %, respectively. Different concentration of kinetin (KT) and zeatin (ZT) had no significant effect on micro-tuber induction of petiole. Most petioles showed polarity during the cultivation of explants, when supplemented with different concentrations of auxin or cytokinin in the MS medium.     

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.     

Callus culture and in vitro biosynthesis of cardiac glycosides from Calotropis gigantea (L.) Ait

Calotropis gigantea (L.) Ait., belonging to the family Asclepiadaceae, is a source of many cardiac glycosides (CGs) and their steroidal moieties (genins). These CGs have been reported to have anti-proliferative activity on tumor cell lines and are potential targets for cancer chemotherapy. However, the abundance of CGs in wild plants is particularly restricted and it is difficult to isolate the desired compound in required quantities. This study is the first attempt to standardize the induction and proliferation of callus from various explants of C. gigantea specifically for the production of CGs. Callus growth was accompanied by CG measurement using high-performance liquid chromatography-tandem mass spectrometry. Murashige and Skoog (MS) and modified Murashige and Skoog (MMS) media were optimized with various combinations and concentrations of auxin and cytokinin for induction and growth of calli from a range of explant sources. While leaves and stem explants resulted in greatest callus induction, MMS medium was found to be optimal. However, no CG was produced from callus grown on this medium. In contrast, the induction and proliferation of callus on MS medium were optimum at primary stages, but growth slowed during the third subculture. Therefore, calli were transferred to MMS medium to promote callus proliferation and production of CGs. As a result, three CGs and two genins were biosynthesized. Furthermore, the callus induction data in MS medium indicated that among different auxins, 2,4-dichlorophenoxyacetic acid was the best for callus induction compared to 1-naphthylacetic acid and indole-3-acetic acid. The data also revealed that the cytokinin/auxin ratio was critical rather than their independent presence for the induction of callus. Thus, the in vitro biosynthesis of targeted CGs may offer an alternative pathway for new source of anti-proliferative agents in required quantities.     

Micropropagation of Mandevilla moricandiana (A.DC.) Woodson

A protocol was developed for micropropagation of Mandevilla moricandiana (A.DC.) Woodson, a native plant from Brazil. Shoots, obtained from in vitro plantlets were used as source of nodal segments for shoot production from axillary buds. The nodal segments were grown on Murashige and Skoog medium supplemented with different concentrations of 6-benzyladenine and/or indole-3-acetic acid to induce axillary bud elongation. After a 2-mo culture period, the medium supplemented with 1.0 mg?L^?1 6-benzyladenine gave the largest number of nodal segments per explant. The nodal segments obtained from plants developed under these conditions were grown on medium supplemented with different concentrations indole-3-acetic acid, ??-naphthaleneacetic acid, and indole-3-butyric acid. The use of the medium supplemented with indole-3-acetic acid and indole-3-buryric induced shoot elongation and shoot development, formation of basal callus, and/or indirect organogenesis of roots. Following transfer of shoots to soil, the plants with only basal callus showed 10% survival and developed roots from callus, while in vitro-rooted plants had a maximum 40% survival rate ex vitro. Regardless of the auxin added to the rooting medium, the acclimatization period allowed the plants rooted in vitro to develop their shoots fully. The protocol developed here is suitable for the production of shoots and rooted plantlets of M. moricandiana.     

Relationship of indole-3-acetic acid and tryptophan concentrations in normal and 5-methyltryptophan-resistant cell lines of wild carrots

A 5-methyltryptophan(5-MT)-resistant cell line of wild carrot (Daucus carota L.), W001, that exhibited auxin-independent callus growth, was found to accumulate indole-3-acetic acid (IAA) and tryptophan (trp). Anthranilate-synthetase activity in W001 cell extract was less sensitive to feedback inhibition by trp than in the original 5-MT-sensitive cell lines. It is hypothesized that the resistant enzyme allowed more trp synthesis and accumulation which, in turn, affected the IAA concentration in the cell. Since carrot cultures cannot regenerate in the presence of exogenous auxin, the elevated IAA concentration in W001 may be responsible for its drastically reduced capacity to regenerate. The relationship between trp and IAA levels was further investigated by examining the effect of 2,4-dichlorophenoxy acetic acid (2,4-D) on the endogenous concentration of trp and IAA. In general, the IAA level was reduced but the trp concentration was elevated when 2,4-D was present in the culture medium.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - 5-MT 5-methyltryptophan - 5-MT^r 5-MT-resistant - 5-MT^s 5-MT-sensitive - trp tryptophan     

Rapid multiplication of Sapium sebiferum Roxb. by tissue culture

Multiple shoots formation and elongation was induced from stem explants of Sapium seedlings on media containing cytokinins. Leaf explants produced callus on a medium containing cytokinins, auxin, casein hydrolysate and coconut milk, which could be induced to form multiple shoots on transfer to a medium lacking casein hydrolysate, coconut milk and auxin. Rooting of isolated shoots by treatment with an auxin mixture (indole-3-acetic acid, indole-3-butyric acid and indole-3-propionic acid) and transfer of the plantlets to field have also been successful.     

The apparent loss of tissue culture competence during leaf differentiation in yams (Dioscorea bulbifera L.)

Explants taken from the leaves of yams (Dioscorea bulbifera L.) at different stages of development were cultured in vitro on a checkerboard using various combinations and/or concentrations of auxin (2,4-d) and cytokinin (6-BAP). An addition of cytokinin to the culture media was not essential for callus induction from explants derived from young leaves in the very early stages of expansion. When the leaves expanded further they required cytokinin and the requirement increased considerably during expansion. Explants taken from fully expanded leaves were no longer able to proliferate, even when extremely high concentrations of cytokinins were applied. Callus grown from highly immature leaves was able to continue proliferating in the absence of cytokinin when subcultured. Callus, that initially required cytokinin in the medium, proliferated in the absence of exogenous cytokinin when subcultured.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - 6-BAP 6-benzylamino purine - 1-NAA 1-naphthalenacetic acid     

Auxin, Gibberellin, Cytokinin and Abscisic Acid Production in Some Bacteria

Summary In this study, auxin (indole-3-acetic acid), gibberellin, cytokinin (zeatin) and abscisic acid production were investigated in the culture medium of the bacteria Proteus mirabilis, P. vulgaris, Klebsiella pneumoniae, Bacillus megaterium, B. cereus, Escherichia coli. To determine the levels of these plant growth regulators, high performance liquid chromatography (HPLC) technique was used. Our findings show that the bacteria used in this study synthesized the plant growth regulators, auxin, gibberellin, cytokinin and abscisic acid.     

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; )     

Reversal of cytokinin action by riboflavin during stage II micropropagation: The role of 3-methyleneoxindole

Summary Nicotiana tabacum explants were grown on stage II micropropagation medium containing 6-[γ,γ dimethylallyl amino]-purine (2iP), a cytokinin, and indole-3-acetic acid (IAA), an auxin, under a photon fluence rate of ≈60 μmol m⁻² s⁻¹. In the presence of riboflavin the explants showed growth patterns suggesting that IAA had assumed a dominant regulatory role, even though the medium contained a high 2iP:IAA molar ratio. It was determined that this effect was produced by 3-methyleneoxindole, a product of the riboflavin-catalyzed photooxidation of IAA. Methyleneoxindole was recovered from the riboflavin-treated agar medium and found to be much more active than IAA in supporting stage II and stage III micropropagation of tobacco explants. Reversal of cytokinin action by riboflavin did not result from the inhibition of any cytokinin-specified growth function because, in the presence of riboflavin, normal stage II growth was obtained if naphthaleneacetic acid was used as the auxin.     

Anthocyanin accumulation and changes in activities of phenylalanine ammonia-lyase and chalcone synthase in roselle (Hibiscus sabdariffa L.) callus cultures

Time-course changes in anthocyanin accumulation, phenylalanine ammonia-lyase activity and chalcone synthase activity were examined in roselle callus tissues incubated under different culture conditions. Phenylalanine ammonia-lyase activity was not affected by either the kind of auxin supplemented to the medium or light regime. In contrast, chalcone synthase activity was markedly suppressed when the callus was cultured with a medium containing indole-3-acetic acid instead of 2,4-dichlorophenoxyacetic acid (2,4-D) or in the dark. The results imply that in roselle callus cultures chalcone synthase plays a more important role in anthocyanin biosynthesis regulated by 2,4-D and light irradiation than phenylalanine ammonialyase.Abbreviations LS Linsmaier and Skoog - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - PAL phenylalanine ammonia-lyase - CHS chalcone synthase     

Role of Auxin in Induction of Polarity in Fucus vesiculosus Zygotes

We studied the effects of auxin (indole-3-acetic acid) on formation of the primary polarity axis in zygotes of the brown algae Fucus vesiculosusL. Within the first 2.5 h after fertilization, the zygotes release this phytohormone in the ambient medium. The treatment of developing zygotes with the inhibitor of indole-3-acetic acid transport from the cell 2,3,5-triiodobenzoic acid at 5 mg/l arrests the auxin secretion and leads to its accumulation in the cells. This causes a significant delay in zygote polarization. The treatment of zygotes with the exogenous indole-3-acetic acid at 1 mg/l stimulates cell polarization and formation of a rhizoid protuberance. When auxin was added to the medium with triiodobenzoic acid, the inhibitory effect of the latter was eliminated. It has been proposed that the content of indole-3-acetic acid in the ambient medium is a key factor in the induction of polarity of the F. vesiculosus zygotes.     

The influence of genomes on autonomous growth of pith cultures of Nicotiana glauca-Langsdorffii hybrids

Summary A differential influence of the two parental genomes on cell proliferation and morphogenesis in pith tissue explants can be observed among the various tumorous hybrid combinations between Nicotiana glauca Grah. and N. langsdorffii Weinm.: the F₁ hybrid (GL), its amphiploid (GGLL), and two different triploids (GGL and GLL). This influence was evident when the explants were cultured in the presence of exogenous auxin (indole-3-acetic acid, 2.5 M), supplied either continuously or for a brief period of time. Compared with the F₁ and the amphiploid, the higher proportion of N. glauca genomes in GGL cells resulted in greater growth, the higher proportion of N. langsdorffii genomes in GLL cells in lesser growth. In addition, shoots are produced on the GGL callus, while only roots are formed on calli of the other types in the same medium. When, in addition to auxin, a cytokinin [6-(3-methyl-2-butenyl-amino)purine] was added to the culture medium, the differential growth of the different tissue types was less pronounced; at 1.0 M of the cytokinin, all tissues grew at about the same rate and remained undifferentiated, regardless of their genomic composition.