Inhibition ofEnt-kaurene oxidation by cytokinins

Cytokinins, which have some structural similarities to ancymidol, a plant growth retardant, were tested for their effects on the cell-free oxidation ofent-kaurene. Results indicate that several cytokinins inhibit this reaction in microsomal extracts of liquid endosperm from immature wild cucumber seeds. N⁶-cyclohexanemethyladenine was the most active (inhibiting 50% of the controlent-kaurene oxidation at 2×10^?6 M). N⁶-isoamyladenine, N⁶-benzyladenine, N⁶-(Δ²-isopentenyl)adenine and dihydrozeatin were active at successively higher concentrations. Zeatin, kinetin, adenine, N⁶-benzyladenosine, and N⁶-(isopentenyl)adenosine were inactive in this system. The basis for the inhibition ofent-kaurene oxidation by cytokinins may be similar to that of ancymidol: interaction with cytochrome P-450. A binding spectrum similar to that of ancymidol with cytochrome P-450 from wild cucumber endosperm microsomes was obtained with four active cytokinins. The cytokinin binding properties of this protein are currently under investigation. No metabolism of N⁶-benzyladenine could be detected under conditions in which the cytokinin inhibited the oxidation ofent-kaurene toent-kaurenol.     

Cytokinin inhibition of Arabidopsis root growth: An examination of genotype, cytokinin activity, and N6-benzyladenine metabolism

The effects of cytokinins on the in vitro growth of the roots of Arabidopsis thaliana seedlings were examined. Root growth was inhibited in a manner dependent upon the type of cytokinin compound, the cytokinin concentration, the Arabidopsis genotype, and the duration of exposure to cytokinin. For the cytokinins N ⁶-benzyladenine (BA), isopentenyl adenine (iP), or dihydrozeatin (DHZ), the concentration required for 50% root growth inhibition differed for each cytokinin and in each of three Arabidopsis genotypes tested. iP was the most active cytokinin in inhibiting the root growth of the Ler-0 genotype, whereas iP and BA had equal activity when tested with the Col-2 and Columbia genotypes. DHZ had the lowest activity of the three cytokinins tested in all three genotypes. A brief 1-day exposure of seeds to a root-inhibiting concentration of BA increased root growth compared with seedlings grown without BA; exposure to BA for 3–6 days inhibited root growth. BA metabolism was evaluated after 6 h and 1, 3, and 6 days in Columbia seedlings. BA, N ⁶-benzyladenosine (BAR), and N ⁶-benzyladenosine-5-monophosphate (BAMP) decreased with time, whereas N ⁶-benzyladenine-7--d-glucopyranoside (BA-7-G) and N ⁶-benzyladenine-9--d-glucopyranoside (BA-9-G) accumulated in the growing seedlings. Seven aromatic cytokinins were compared at 5 m for their effect on Col-3 root growth. BA, BAR, N ⁶-(m-hydroxybenzylamino)adenine, and N ⁶-(o-hydroxybenzylamino)adenine were highly effective in inhibiting root growth, whereas N ⁶-(p-hydroxybenzylamino)adenine produced only a slight decrease in root growth. BA-7-G and BA-9-G did not affect root growth.Abbreviations BA N ⁶-benzyladenine - iP isopentenyl-adenine - DHZ dihydrozeatin - BAR N ⁶-benzyladenosine - BAMP N ⁶-benzyladenosine 5-monophosphate - BA-7-G N ⁶-benzyladenine-7--d-glucopyranoside - BA-9-G N ⁶-benzyladenine-9--d-glucopyranoside - m-OH BA N ⁶-(m-hydroxybenzylamino)adenine - o-OH BA N ⁶-(o-hydroxybenzylamino)adenine - p-OH BA N ⁶-(p-hyrdoxybenzylamino)adenine - HPLC high performance liquid chromatography - gFW grams fresh weight     

The effect of growth retardants on phytochrome-induced lettuce seed germination

Experiments were carried out to explore the involvement of the plant hormone gibberellin (GA) in the light-induced germination of lettuce seeds. Three growth retardants known to be inhibitors of GA biosynthesis were tested for their effect on red-light-induced germination. Chlormequat chloride (CCC) and AMO-1618 had no effect, but ancymidol was strongly inhibitory. Moreover, the inhibition caused by ancymidol was completely overcome by GA₃. CCC and AMO-1618 inhibit the formation ofent-kaurene, while ancymidol blocks the oxidation ofent-kaurene toent-kaurenoic acid. Ancymidol also was found to inhibit GA-induced dark germination of lettuce seeds, and this inhibition was partially reversed by higher levels of GA. Therefore, the results suggest two possibilities for the relationship between phytochrome and GA in this system: first, the rate-limiting step in the germination of light-sensitive lettuce seeds, that which is regulated by phytochrome, is the oxidation ofent-kaurene toent-kaurenoic acid. Alternatively, red-light treatment may result in the release of active GAlike substances which, in turn, induce germination. In either case the results presented here support the view that phytochrome exerts its effect on lettuce seed germination by means of GA rather than via an independent pathway.     

Presence of endogenous ent-kaurene in a microsomal preparation from Cucurbita maxima L. Endosperm and implications for kinetic studies of ent-kaurene oxidase

Microsomal and soluble cell-free extracts prepared from liquid endosperm of Cucurbita maxima L. were found to contain high concentrations of endogenous ent-kaurene and ent-kaurenol by gas chromatography-mass spectrometry-chemical ionization with deuterated internal standards. Increases in the levels of ent-kaurenol, ent-kaurenoic acid, and ent-7-hydroxykaurenoic acid are correlated with a decline in the amount of endogenous ent-kaurene following a 10 min incubation of microsomes with NADPH and FAD. The rate of oxidation of radiolabeled ent-kaurene by the microsomal fraction was determined, and the need to account for endogenous substrate is shown. Endogenous ent-kaurene present in soluble extracts had the effect of diluting the [¹⁴C]ent-kaurene synthesized from [¹⁴C]mevalonic acid, resulting in reduced specific radioactivity of the product. The dilution of [¹⁴C]ent-kaurene was more pronounced in extracts with higher endogenous ent-kaurene levels or when the reactions were run in the presence of O₂ and NADPH. Evidence is presented that suggests differential metabolism of endogenous ent-kaurene and radiolabeled ent-kaurene in both microsomal and soluble extracts.Abbreviations Kaurene ent-kaur-16-ene - MVA mevalonic acid - kaurenol ent-kaur-16-en-19-ol - kaurenoic acid ent-kaur-16-en-19-oic acid - EtOAc ethyl acetate - MeOH methanol - GC-MS-CI gas chromatography-mass spectrometry-chemical ionization - 13-OH KA ent-13-hydroxykaur-16-en-19-oic acid - 7-OH kaurenoic acid ent-7-hydroxykaur-16-en-19-oic acid - kaurenal ent-kaur-16-en-19-al - Me(x) methyl ester of x - TMS(x) trimethylsilyl ether or ester of x - GA(x) gibberellin A(x)     

Photoreduction of Sulfite by Spinach Leaf Preparations in the Presence of Grana System

The β-anomer of N⁶-benzyladenosine has been long known as an artificial cytokinin.¹⁾ Recently, however, this compound was isolated from a cytokinin-autotrophic cell culture of anise, Pimpinella anisum L.²⁾ Thus, this compound now constitutes the third member of the naturally-occurring cytokinins possessing an N⁶-benzyladenine structure, together with N⁶-(O-hydroxybenzyl)adenosine isolated from leaves of Poplus robusta³⁾ and N⁶-(o-hy-droxybenzyl)-2-methylthio-9-β-d-glucofuranosyl-adenine isolated from fruits of Zantedeschia aethio-pica.⁴⁾ No information has been available concerning whether or not the a-anomer of N⁶-benzyladenosine possesses cytokinin activity.

We report here the synthesis and cytokinin activity of N⁶-benzyl-α-adenosine (N⁶-benzyl-9-α-d-ribofuranosyl adenine).     

Enzyme immunoassays of N 6-benzyladenine and N 6-(meta-hydroxybenzyl)adenine cytokinins

Enzyme-linked immunosorbent assays (ELISAs) were developed for determination of N ⁶-benzyladenosine, N ⁶-(meta-hydroxybenzyl)adenosine, and structurally related cytokinins. The use of the ELISAs allowed detection over the range of 0.05–70 pmol for N ⁶-benzyladenine and 0.01–20 pmol for the N ⁶-(meta-hydroxybenzyl)adenine cytokinins. Polyclonal antibodies used in the assays were specific for N ⁶-benzyladenine and N ⁶-(meta-hydroxybenzyl)adenine and their corresponding N ⁹-substituted derivatives. By the use of internal standardization, dilution assays, authentic [2-³H]cytokinin recovery markers, and immunohistograms, the ELISAs have been shown to be applicable for the estimation of N ⁶-benzyladenine and N ⁶-(meta-hydroxybenzyl)adenine-type cytokinins in plant tissues. For the analysis of cytokinins in the tissues of young poplar leaves and Solarium teratoma shoot culture, the extracts were fractionated by high performance liquid chromatography (HPLC) and the fractions analyzed by ELISAs. Immunohistogram ELISA analysis of fractions from different HPLC systems indicated major peaks of immunoreactivity co-chromatographing with the labeled and unlabeled standards of N ⁶-benzyladenine, N ⁶-meta-hydroxybenzyl)adenine, and their N ⁹-glycosides in these tissues.Abbreviations ELISA enzyme-linked immunosorbent assay - FW fresh weight - (mOH)[9R]BAP N ⁶-(meta-hydroxybenzyl)adenosine - HPLC high performance liquid chromatography - TBS Tris-buffered saline - TEAA triethylammonium acetate - [9R]BAP N ⁶-benzyladenosine     

Inhibition of dopamine-β-hydroxylase by cytokinins

The activity of cell-free preparations of dopamine--hydroxylase from a mammalian source was inhibited by a number of N⁶-substituted adenine derivatives that are hormonally active as cytokinins in plant systems. The synthetic cytokinin N⁶-cyclohexylmethyladenine exhibited inhibitory activity equivalent to that of 1-phenyl-3-(2-thiazolyl)-2-thiourea (PTTU), a compound known to be a potent inhibitor of dopamine--hydroxylase activity. PTTU itself was found to exhibit cytokinin activity in the tobacco callus bioassay and to inhibit the activity of the plant enzyme, cytokinin oxidase. The possible significance of these observations is discussed in relation to known effects of cytokinins on phenethylamine metabolism.     

Inhibition of gibberellin biosynthesis by paclobutrazol in cell-free homogenates ofCucurbita maxima endosperm andMalus pumila embryos

The plant growth retardant paclobutrazol, (PP333) (2RS, 3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)pentan-3-ol, inhibits specifically the three steps in the oxidation of the gibberellin-precursorent-kaurene toent-kaurenoic acid in a cell-free system fromCucurbita maxima endosperm. The KI₅₀ for this inhibition is 2×10^–8 M. The KI₅₀ values for the separated2S, 3S, and2R, 3R enantiomers of paclobutrazol in this system are 2×10^–8 M and 7×10^–7 M, respectively. A cell-free preparation from immatureMalus pumila embryos convertsent-kaurene to gibberellin A₉, whereas no conversion occurs in a similar preparation fromMalus endosperm. The conversion ofent-kaurene by the embryo preparation is inhibited by paclobutrazol with KI₅₀ values for the2S,3S and2R,3R enantiomers of 2×10^–8 M and 6×10^–8 M, respectively.     

The effect of auxin on cytokinin levels and metabolism in transgenic tobacco tissue expressing an ipt gene

The ipt gene from the T-DNA of Agrobacterium tumefaciens was transferred to tobacco (Nicotiana tabacum L.) in order to study the control which auxin appears to exert over levels of cytokinin generated by expression of this gene. The transgenic tissues contained elevated levels of cytokinins, exhibited cytokinin and auxin autonomy and grew as shooty calli on hormone-free media. Addition of 1-naphthylacetic acid to this culture medium reduced the total level of cytokinins by 84% while 6-benzylaminopurine elevated the cytokinin level when added to media containing auxin. The cytokinins in the transgenic tissue were labelled with ³H and auxin was found to promote conversion of zeatin-type cytokinins to ³H-labelled adenine derivatives. When the very rapid metabolism of exogenous [³H]zeatin riboside was suppressed by a phenylurea derivative, a noncompetitive inhibitor of cytokinin oxidase, auxin promoted metabolism to adenine-type compounds. Since these results indicated that auxin promoted cytokinin oxidase activity in the transformed tissue, this enzyme was purified from the tobacco tissue cultures. Auxin did not increase the level of the enzyme per unit tissue protein, but did enhance the activity of the enzyme in vitro and promoted the activity of both glycosylated and non-glycosylated forms. This enhancement could contribute to the decrease in cytokinin level induced by auxin. Studies of cytokinin biosynthesis in the transgenic tissues indicated that trans-hydroxylation of isopentenyladenine-type cytokinins to yield zeatin-type cytokinins occurred principally at the nucleotide level.Abbreviations Ade adenine - Ados adenosine - BA 6-benzylaminopurine - C control - Con A concanavallin A - CP cellulose phosphate - IPT isopentenyl transferase - NAA 1-naphthylacetic acid - NP normal phase - NPPU N-(3-nitrophenyl)-N-phenylurea - RIA radioimmunoassay - RP reversed phase We wish to thank Dr. J. Zwar for supplying phenylurea derivitives.     

Cyclin-dependent kinase inhibitor,roscovitine, in combination with exogenous cytokinin,N6-benzyladenine,causes increase of cis-cytokinins in immobilized tobacco cells

Dynamics of the response of tobacco cells (line BY-2) to exogenous cytokinin, N ⁶-benzyladenine, and cyclin-dependent kinase inhibitor, roscovitine, was followed using alginate-immobilized cells packed into a column. N ⁶-Benzyladenine (1.25 M) increased the synthesis of the physiologically-active endogenous cytokinin, isopentenyladenosine, in the effluent up to 0.1 nM. Simultaneously, conversion of the excess of endogenous cytokinins to biologically inactive derivatives of cis-zeatin occurred, up to 0.8 nM. Roscovitine (50 M) further increased cis-cytokinins, up to 2.2 nM.     

Kinetin-induced stimulation of electrogenic pumping in soybean suspension cultures is unrelated to signal transduction

Primary modes of action of cytokinins have been thought to involve stimulation of the electrogenic H⁺ pump and-or opening of plasmamembrane Ca²⁺ channels. In order to test these hypotheses, rapid changes in membrane transport in response to cytokinin application were studied in heterotrophic suspension-cultured callus of soybean (Glycine max (L.) Merr.) using electrophysiological techniques. Kinetin (N⁶-furfurylaminopurine; 2 M) elicited membrane hyperpolarization of 13±1 mV. This effect occurred even at membrane poteintials more negative than the most negative ionic equilibrium potential, and therefore might have resulted either from stimulation of the electrogenic pump, or from closure of ionic channels. The former mechanism of action appears most likely because (i) kinetin-induced membrane hyperpolarization is not accompanied by a significant change in plasma-membrane resistivity and (ii) hyperpolarization is abolished by cyanide, which inhibits electrogenic pump activity by depletion of cellular ATP.Electrogenic pumping is also activated by two other cytokinins: N⁶-(benzyl)adenine and trans-zeatin. However, it is unlikely that the hormonal effect on electrogenesis is directly related to transduction of the cytokinin signal, for the following reasons: (i) hormonally inactive, but chemically related compounds (cis-zeatin, adenine) also elicited membrane hyperpolarization; (ii) hormonally active, N⁹-substituted cytokinins failed to stimulate electrogenesis; (iii) the chemically unrelated cytokinin N,N-diphenylurea also failed to stimulate electrogenesis.The results imply that the kinetin effect on electrogenic pumping is related to adenine, or its metabolism, and not hormonal action. Adenine was absorbed by soybean cells, but not in sufficient quantities to have a significant effect on adeninenucleotide pools. It appears likely that the control of electrogenesis requires either the presence of a purine free base (i.e. no substituents at the N⁹ position) or phosphoribosylation of the free base. No evidence was found for cytokinin-induced Ca²⁺-channel opening, though it is argued that such an event might be physiologically relevant, yet undetectable with the methods employed. It is essential that future studies on cytokinin signal transduction — especially as they relate to membrane transport — take into account the possibility that metabolic effects unrelated to hormone action are dominant.Abbreviations and symbols bzl⁶Ade N⁶ (benzyl)adenine - SRB Soybean Recording Buffer - V_m membrane potential     

Regulation of cytokinin oxidase activity in tobacco callus expressing the T-DNA ipt gene

There are indications that the cytokinin content in transgenic tissues expressing the cytokinin biosynthetic ipt gene is under metabolic control, which prevents the accumulation of cytokinins to lethal levels. The objective of this study was to investigate the relationships between the content of endogenous cytokinins and the activity of cytokinin oxidase (which is believed to be a copper-containing amine oxidase, EC 1.4.3.6.) in ipt transgenic tobacco callus. In addition, the effect of exogenously applied N-benzyladenine (BA) on this relationship was examined. Endogenous cytokinin concentrations were measured in callus of Nicotiana tabacum L. cv. Petit Havana SRI transformed with the ipt of Agrobacterium tumefaciens under the control of a light-inducible promoter and in non-transformed tissue using LC-tandem mass spectrometry. The activity of cytokinin oxidase was estimated by measuring the conversion of [2,8-³H]N⁶-(Δ²-isopentenyl)adenine to [³H]adenine by enzyme preparations in vitro. The 14-day-old ipt-transformed callus contained a 25-fold higher amount of cytokinins as compared to the non-transformed tissue. Mainly zeatin- and dihydrozeatin-types of cytokinins (free bases, ribosides, nucleotides and O-glucosides) accumulated in the ipt transgenic tissue. The cytokinin pool of both ipt-transformed and non-transformed tissues consisted predominantly of cytokinins that are either resistant to cytokinin oxidase attack (nucleotides and O-glucosides of cytokinins and cytokinins bearing N⁶-saturated side chain) or have a low affinity for the enzyme (zeatin and its riboside). The former represented 71.6 and 74.8% and the latter 27.7 and 24.4% of the pool of endogenous cytokinins in ipt-transformed and non-transformed tissues, respectively. Enzyme preparations from ipt-transformed tissue exhibited 1.5-fold higher cytokinin oxidase activity compared with that observed in control tissues. Application of exogenous BA affected the total levels of cytokinins of the two tissue lines in different ways. The cytokinin content increased by 1.7- and 1.5-fold in ipt-transformed tissues 6 and 12 h after BA application, respectively, while it declined in the non-transformed control by 1.6- to 2.0-fold between 3 and 12 h after BA application. The increase in cytokinin content in the ipt callus is due to an increase of zeatin- and dihydrozeatin-type cytokinins (nucleotides, ribosides and free bases) leading to an enhanced accumulation of O-glucosides after 12 h. Following BA treatment, the cytokinin oxidase activity increased up to 1.8-fold in ipt-transformed and 1.6-fold in non-transformed tissues. The levels of isopentenyl-type cytokinins were near the detection limit; however, the enhancement of cytokinin oxidase activity after BA treatment in both tissue lines was correlated with the content of preferred substrate of the enzyme, N⁶-(Δ²-isopentenyl)adenosine.     

ent-Kaurene biosynthesis in a cell-free system from wheat (Triticum aestivum L.) seedlings and the localisation of ent-kaurene synthetase in plastids of three species

A cell-free system capable of converting [¹⁴C]geranylgeranyl diphosphate to ent-[¹⁴C]kaurene and to an unidentified acid-hydrolysable compound was obtained from the basal portions of 5-d-old shoots of wheat seedlings (Triticum aestivum L.). By means of marker enzyme activities, the synthesis of ent-kaurene and the unknown compound could be quantitatively assigned to a plastid fraction obtained by Percoll-gradient centrifugation of the homogenate. The enzyme activities were located within the plastids, probably in the stroma, because they withstood trypsin treatment of the intact plastids, and the plastids had to be broken to release the activity, which was then obtained in soluble form. Plastid membranes had no activity. Plastid stroma preparations obtained from pea (Pisum sativum L.) shoot tips and pumpkin (Cucurbita maxima L.) endosperm also yielded ent-kaurene synthetase activity, but did not form the unknown compound. The exact nature of the active plastids was not ascertained, but the use of methods for proplastid isolation was essential for full activity, and the active tissues are all known to contain high proportions of proplastids, developing chloroplasts or leucoplasts. We therefore believe that ent-kaurene synthesis may be limited to these categories. Mature chloroplasts from the wheat leaves did not contain ent-kaurene synthetase activity and did not yield the unknown component. Incorporation of [¹⁴C]geranylgeranyl diphosphate into ent-[¹⁴C]kaurene and the unknown component was assayed by high-performance liquid chromatography with on-line radiocounting. ent-[¹⁴C]Kaurene was identified by Kovats retention index and full mass spectra obtained by combined gas chromatography-mass spectrometry. The unknown component was first believed to be copalyl diphosphate, because it yielded a compound on acid hydrolysis, which migrated like copalol on high-performance liquid chromatography and gave a mass spectrum very similar to that of authentic copalol. However, differences in the mass spectrum and in retention time on capillary gas chromatography excluded identity with copalol. Furthermore, the unhydrolysed compound was not converted to ent-kaurene by a cell-free system from C. maxima endosperm as copalyl diphosphate would have been.Abbreviations ADH alcohol dehydrogenase - AMO 1618 2isopropyl-4-(trimethylammoniumchloride)-5-methylphenyl piperi-dine-1-carboxylate - BSA bovine serum albumin - DTT dithioth-reitol - GA_n gibberellin A_n - GAPDH NADP⁺-glyceraldehyde 3-phosphate dehydrogenase - GC-MS combined gas chromatography-mass spectrometry - GGPP all trans-isomer of geranyl-geranyl diphosphate - KS ent-kaurene synthetase - MDH malate dehydrogenase - MAA mevalonate activating activity - SOR shikimate oxidoreductase We thank Mrs. Gudrun Bodtke and Mrs. Dorothee Dasbach for able technical assistance, Prof. L.N. Mander (Australian National University, Canberra, Australia) for ent-[²H₂]kaurene and Dr. Yuji Kamiya (RIKEN, Saitama, Japan) for geranylgeraniol and copalol. The work was supported by the Deutsche Forschungsgemeinschaft.     

Cytokinin-induced hypocotyl elongation in light-grown<Emphasis Type="Italic"> Arabidopsis</Emphasis> plants with inhibited ethylene action or indole-3-acetic acid transport

Cytokinins inhibit hypocotyl elongation in darkness but have no obvious effect on hypocotyl length in the light. However, we found that cytokinins do promote hypocotyl elongation in the light when ethylene action is blocked. A 50% increase in Arabidopsis thaliana (L.) Heynh. hypocotyl length was observed in response to N⁶-benzyladenine (BA) treatment in the presence of Ag⁺. The level of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid was strongly increased, indicating that ethylene biosynthesis was up-regulated by treatment with cytokinin. Furthermore, the effects of cytokinins on hypocotyl elongation were also tested using a series of mutants in the cascade of the ethylene-signal pathway. In the ethylene-insensitive mutants etr1-3 and ein2-1, cytokinin treatment resulted in hypocotyl lengths comparable to those of wild-type seedlings treated with both Ag⁺ and BA. A similar phenotypical response to cytokinin was observed when auxin transport was blocked by -naphthylphthalamic acid (NPA). Applied cytokinin largely restored cell elongation in the basal and middle parts of the hypocotyls of NPA-treated seedlings and at the same time abolished the NPA-induced decrease in indole-3-acetic acid levels. Our data support the hypothesis that, in the light, cytokinins interact with the ethylene-signalling pathway and conditionally up-regulate ethylene and auxin synthesis.     

Inhibition of growth by ancymidol and tetcyclacis in the gibberellin-deficientdwarf-5 mutant ofZea mays L. and its prevention by exogenous gibberellin

Leaf sheath length and shoot dry matter of the gibberellin-deficientdwarf-5 mutant ofZea mays L. were further reduced by micromolar concentrations of two putative gibberellin biosynthesis inhibitors, ancymidol [-cyclopropyl--(p-methoxyphenyl)-5-pyrimidine methyl alcohol] and tetcyclacis [5-(4-chlorophenyl)-3,4,5,9,10-pentaazatetracyclo-5,4,1,0^2,6,0^8,11-dodeca-3,9-diene]. Growth retardant action was prevented by the subsequent application of gibberellin (GA₄₊₇). Plants treated with both gibberellin and growth retardants were identical in all outward respects to those treated with gibberellin alone. Although thedwarf-5 mutant is blocked in the synthesis ofent-kaurene and does not contain detectable quantities of gibberellin, the above results are consistent with the interpretation that biologically active levels of endogenous gibberellin are present in the dwarf which can be decreased by biosynthesis inhibitors.Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the United States Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.     

Inhibition of gibberellin biosynthesis by paclobutrazol in cell-free homogenates ofCucurbita maxima endosperm andMalus pumila embryos

The plant growth retardant paclobutrazol, (PP333) (2RS, 3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)pentan-3-ol, inhibits specifically the three steps in the oxidation of the gibberellin-precursorent-kaurene toent-kaurenoic acid in a cell-free system fromCucurbita maxima endosperm. The KI₅₀ for this inhibition is 2×10^?8 M. The KI₅₀ values for the separated2S, 3S, and2R, 3R enantiomers of paclobutrazol in this system are 2×10^?8 M and 7×10^?7 M, respectively. A cell-free preparation from immatureMalus pumila embryos convertsent-kaurene to gibberellin A₉, whereas no conversion occurs in a similar preparation fromMalus endosperm. The conversion ofent-kaurene by the embryo preparation is inhibited by paclobutrazol with KI₅₀ values for the2S,3S and2R,3R enantiomers of 2×10^?8 M and 6×10^?8 M, respectively.     

Micropropagation ofUraria picta, a medicinal plant, through axillary bud culture and callus regeneration

Summary Micropropagation ofUraria picta, a leguminous herb, was achieved through axillary bud culture and nodal callus culture. Bud break was best when nodes were cultured on Murashige and Skoog (1962) (MS) medium supplemented with 2.6 μM α-naphthalene acetic acid and 4.4 μM N⁶-benzyladenine. Optimum shoot multiplication was observed in adenine sulphate at 2.47 μM concentration. Competent callus was initiated around the nodal ring of the explant on the basal medium supplemented with cytokinins and auxin (α-naphthalene acetic acid and N⁶-benzyladenine), which regenerated into new profusely growing shoots on transferring to 0.13 μM N⁶-benzyladenine. Shoots elongated to 5 node length with 1.11 μM N⁶-benzyladenine were rooted on half-strength MS basal medium. The rooted plants were successfully established with 80% survival. About 400 such plants were transferred to the field.     

The effect of an elevated cytokinin level using the ipt gene and N 6-benzyladenine on single node and intact potato plant tuberization in vitro

Two models of potato (Solanum tuberosum L.) tuberization in vitro (intact plants and single nodes) were used to study the role of cytokinins in this process. We applied hormone in two different ways. The exogenous addition of 10 mg · L^-1 N ⁶-benzyladenine (BA) into the tuberization medium resulted in advanced tuber formation in intact plants, and microtubers appeared 10–20 days earlier than in the experiments in which no cytokinin was supplied. Transformation with the Agrobacterium tumefaciens ipt gene provided potato clones with endogenously elevated cytokinin levels (3–20 times higher zeatin riboside content in different clones). The onset of tuberization in intact ipt-transformed plants with low transgene expression was advanced in comparison with control material, and exogenously applied BA further promoted the tuberization process. On the contrary, tuberization was strongly inhibited in ipt-transformed nodes, and an external increase of the cytokinin level caused complete inhibition of expiant growth. In untransformed (control) nodes cytokinin application resulted in primary and secondary tuber formation, which depended on the BA concentration in cultivation media.Abbreviations BA N ⁶-benzyladenine - PCR polymerase chain reaction - HPLC high performance liquid chromatography - ELISA enzyme-linked immunosorbent assay - NAA -naphthylacetic acid     

Growth of and quassin accumulation by cultures of Quassia amara

Callus and suspension cultures were established from Quassia amara, a member of the Simaroubaceae. Analysis of the tissue culture showed that quassin was present in both callus and suspension cultures. The effect of variation in auxin and cytokinins on both callus growth and the presence of quassin was examined. The suspension culture was grown in a 7 liter bioreactor when good yields of quassin were achieved.Abbreviations IAA indole-3-acetic acid - IBA indolebutyric acid - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - 6BA 6-benzyladenine - IpA N⁶ (-isopentenyl) adenine - IpAR N⁶ ( isopentenyl) adenine riboside - td doubling time