An improved disk bioassay for determining activities of plant growth regulators

An improved filter paper disk bioassay for determining activities of plant growth regulators was developed and evaluated. Indole-3-acetic acid (IAA), (±)-abscisic acid (ABA), and 6-furfurylamino-purine (kinetin) were dissolved in 95% ethanol at fixed dilutions. Specific concentrations of each growth regulator were then evenly dropped onto individual 6-cm paper disks and the solvent evaporated. The activities of the above three water-insoluble plant growth regulators in both solution and the disk assay were compared using the excised cucumber (Cucumis sativus L.) cotyledon root formation bioassay (IAA), wheat (Triticum aestivum L.) coleoptile straight growth bioassay (ABA), and cucumber cotyledon expansion bioassay (kinetin), and similar results were obtained at each concentration. The possible principle of this method has been studied using the cucumber cotyledon expansion bioassay. The results suggested that the disks, as carriers, had highly dispersed kinetin molecules on them and greatly accelerated the dissolution and diffusion of kinetin from disks to water.     

ffects of Some Inhibitors on Potassium-and IAA-Induced Adventitious Root Formation of Excised Cucumber Cotyledon

The effects of some inhibitors on potassium- and IAA-induced rooting were studied adopting the root-formation bioassay in the excised cucumber ( Cucumis sativus L. ) cotyledon. 5-fluomuracil at 7 Ï 10-4 – 7 Ï10-1 mmol/L and cycloheximide at 3.5 Ï 10-4 – 1.05 Ï10-2 mmol/L significantly inhibited potassium- and IAA-induced adventitious root formation of the excised cucumber cotyledons, respectively. Na3VO4 at 0.1 – 1.0 mmol/L obviously inhibited potassium and IAA-induced adventitious rooting of the excised cucumber cotyledons, and similar inhibitory effect was found with 2,3,5-triiodobenzoic acid (TIBA) at 2 Ï 10-4 – 2 Ï 10-l mmol/L.There is a close relationship between potassium and IAA-induced adventitious rooting and the promotive effect of potassium on rooting is possibly brought about via affecting the endogenous level of IAA.     

Comparative Study on the Bioassay between N6, O2'-Dibutyryl Cyclic Adenosine 3,5-Monophosphate and Cytokinin

The physiological activity of diBcAMP by various classical bioassay methods of cytokinin was studied. It exhibits the cytokinin-like activity in many ways of biological response, such as: the unfolding of etiolated barley leaf, the expansion of radish cotyledon, the expansion and greening of cucumber cotyledon and the inhibition of the elongation of mung-bean hypocotyl, etc. The activity of 1 mM diBcAMP was approximately the same as the activity of 0.044 mM benzyladenine in the most of the tests. However, the activity of 1 mM diBcAMP was found to be higher than the optimal concentration (0.044 mM) of benzyledenine in the radish cotyledon under the expansion test. There was no toxicity of diBcAMP in all of the bioassay methods used, even with higher concentration.     

IAA及KCN在黄瓜子叶离体叶绿体氧化还原作用过程中的相互作用

ＩＡＡ对于不同时期的叶绿体ＤＣＰＩＰ光还原均有促进效应。而对叶绿体的放氧效率的促进则随子叶叶岭的增长而下降至零。ＣＮ＾－（１０＾－３ｍｏｌ／Ｌ）存在时不同程度地抑制叶绿体的ＤＣＰＩＰ光还原和氧化放氧。而ＩＡＡ（１０＾－５,１０＾－４ｍｏｌ／Ｌ）则能消除ＣＮ＾－的抑制作用而仅表现ＬＡＡ原有的促进作用。同时,ＩＡＡ可以释放照光和不照光条件下ＫＣＮ对离体叶绿体分解外源Ｈ２Ｏ２的抑制。叶绿体光系统的光还原     

Rapid Determination of Cytokinins and Auxin in Cyanobacteria

Five cyanobacterial strains, Anabaena sp. Ck1, Oscillatoria sp. Ck2, Phormidium sp. Ck3, Chroococcidiopsis sp. Ck4, and Synechosystis sp. Ck5 were selected for their positive cytokinins-like activity using cucumber cotyledon bioassay and GUS assay in Arabidopsis ARR5::GUS. Classical cucumber cotyledon bioassay was modified for direct screening of cyanobacteria avoiding need for extraction and purification. Cytokinins from cyanobacteria were absorbed onto filter paper which was then assayed for cytokinins-like activity. A rapid chromatographic method was developed for the simultaneous determination of cytokinins and indole-3-acetic acid (IAA). Cyanobacterial biomass (50–100 mg) and cell-free culture filtrate were extracted in Bieleski buffer and purified by solid-phase extraction. The extract was used to determine phytohormones by ultra performance liquid chromatography and electrospray ionization-tandem mass spectrometry in positive and negative modes, respectively, with multiple reactions monitoring. Stable isotope-labeled cytokinins and IAA standards were added in the samples to follow recovery of the compounds and method validation. Five cytokinins determined in the selected strains were Zeatin (cis and trans isomers), Zeatin riboside, Dihydrozeatin riboside, and zeatin-o-glucoside. The strains were shown to accumulate as well as release the phytohormones.     

Stimulation of cytokinins and chlorophyll synthesis in cucumber cotyledons by triadimefon

Triadimefon [1-(4-chlorophenoxy)-3,3-dimethyl-1-(1,2,4-triazol-1-yl)-2-butanone] changes the morphology and partitioning of dry matter in cucumber ( Cucumis sativus L. cv. National Pickling) seedlings. The dry weights, potassium and cytokinin levels in the cotyledons and roots of the treated seedlings were higher, whereas the hypocotyl weights were lower than the controls. When etiolated intact seedlings or cotyledons excised from triadimefon-pretreated dark-grown seedlings were exposed to light, chlorophyll synthesis in the pretreated cotyledons was stimulated. Triadimefon does not have cytokinin-like activity in the cucumber cotyledon greening bioassay, but appears to induce the plants to produce more cytokinims, probably by stimulating root growth. Hence it is proposed that the stimulation of chlorophyll production by triadimefon in cucumber cotyledons is mediated by maintaining high levels of potassium and cytokinins in the cotyledons.     

The Roles of the Cotyledons and Auxin in the Adventitious Root Formation of Hypocotyl Cuttings of Light-Grown Cucumber Seedlings

Adventitious root formation in encumber hypocotyl cuttings was studied. Root formation was quantitatively related to the amount of the cotyledons attached to the hypocotyl. Complete removal of the cotyledons diminished root formation entirely. Hut the removal of the apical bud had no effect. Treatment of the hypocotyl with triiodobenzoic acid resulted in the inhibition of root formation. On t he other hand, IAA promoted root formation. Promoting concentration of IAA was 1 mg/1 for the cuttings with intact cotyledons and 10 mg/1 for those with l/8th of the cotyledons. The first two or three days of treatment was most effective. The presence of auxin (IAA-like. substance) in cucumber seedlings was demonstrated by paper chromatography and the pea straight test. It is concluded that the cotyledon is necessary for root formation in cucumber hypocotyl cuttings and that auxin is at least one of the factors supplied from the cotyledons.     

A simple bioassay for abscisic acid using cucumber hypocotyls

A simple bioassay based on the inhibition by abscisic acid (ABA) of cucumber (Cucumis sativus L., cv. National Pickling) hypocotyl elongation was developed. Sections of 3-day-old dark-grown cucumber hypocotyl taken from 0–5 mm immediately below the cotyledon were used for the assay. A dark incubation period of 20 h was followed by an exposure to light for 24 h. Under these conditions, the inhibition of hypocotyl elongation is proportional to the abscisic acid applied. The minimum detectable level of abscisic acid was 10^–9 M, and the range of linear response to abscisic acid was between 10^–7 and 10^–3 M. This assay is 10 times more sensitive than the cucumber cotyledon greening bioassay for abscisic acid.     

An improved bioassay for cytokinins using cucumber cotyledons

The cucumber cotyledon greening bioassay is frequently used for detecting cytokinins. Beneficial modifications of the original technique included using 5-day-old cucumber (Cucumus sativus L., cv. National Pickling) cotyledons treated with combinations of 40 millimolar KCl and various concentrations of cytokinins. A dark incubation period of 20 hours was followed by an exposure to light for 3.5 hours. Under these conditions, extremely low (0.0001 milligram per liter) concentrations of N⁶-benzyladenine, zeatin, kinetin, or zeatin riboside can be detected. Of the four cytokinins tested, kinetin appeared to be the least active. With these improvements, the assay is 10 times more sensitive than is the previously described cucumber cotyledon greening bioassay for cytokinins.     

表油菜素内酯对黄瓜幼苗下胚轴过氧化物酶和IAA氧化酶活性的影响

黄瓜幼苗用0.1～1 ppm表油菜素内酯(epiBR)处理1～3d后,下胚轴中过氧化物酶活性明显低于对照;随着处理浓度的增加和处理时间的延长,与对照之间的差别愈趋增大。当浓度高于1ppm时,过氧化物酶的活性不再继续降低。表油菜素内醋对过氧化物酶活性的这种抑制作用需经约1O h的滞后期。IAA氧化酶的活性变化与过氧化物酶相似,epiBR处理时间愈长酶活性增加愈趋缓慢。 经IAA处理的下胚轴,过氧化物酶和IAA氧化酶的活性变化与对照无明显差异。这提示油菜素内酯与IAA促进生长可能是通过不同的作用方式。     

茶梅体细胞胚胎发生和植株的再生

在山茶属植物中,我们曾报道过金花茶和云南山茶组织培养形成胚状体和再生植株。茶梅(Camellia sasanqua)也是山茶属的一种重要的观赏植物,其植株矮小,花色鲜艳且具香味,除供观花外,还可做成不同造型的盆景供观赏,在园艺上也可用作茶花嫁接的砧木。茶梅组织培养通过体细胞胚状体产生植株,至今尚未见报道。现把部份实验结果报道如下。     

The etiolated cucumber hypocotyl weight-growth test: A sensitive,easy and ultrafast bioassay for IAA

A sensitive, easy and fast bioassay is described for the detection of IAA. The bioassay consists of measuring the weight increase of hypocotyl sections from etiolated cucumber seedlings incubated in a simple medium containing 2 mM KCl, 0.1 mM CaCl and 10 mg/l chloramphenicol (pH 7). The sensitivity of the test is comparable to that of the Avena first internode test, exhibiting a significant response at an IAA concentration as low as 1 ng/ml. The bioassay requires only 3 h, and is easy to perform. The slope of the log-linear concentration-response curve is moderately steep giving good accuracy. The assay is insensitive to the pH of the media between 4 to 7 and can be performed in the presence or absence of buffer. The growth of the etiolated hypocotyl sections is insensitive to GA and to kinetin except at high concentrations.     

An improvement of cucumber cotyledon greening bioassay for cytokinins

The cucumber cotyledon greening bioassay for cytokinins was modified by using 95% acetone-ethanol instead of 80% acetone as extraction solvent. The cotyledons were extracted directly with a 2:1 (v/v) acetone-ethanol solution in dark for 24 hours, omitting the homogenization and centrifugation operations of the previous bioassay. The modified bioassay is more convenient and especially useful in screening cytokinin-active substance from a large number of samples.     

The Effects of Cytokinins on Unfolding of Cotyledons and Opening of Hypocotylar Hooks of Cucumber

Benzyladenine (BA), zeatin and kinetin caused the unfolding of cucumber (Cucumis sativus L. Var. Jing-ye No. 4) cotyledons. But the other plant hormones e.g. gibberellic acid (GA3), indoleacetic acid(IAA), ethylene (ethrel), abscisic acid (ABA) and other related compound e.g. KCl, adenine were not so. This response was caused by even lower concentration (0.01 ppm). Therefore, it could be used as a bioassay for cytokinins. KCl affaeted synergistically on cytokinin-induced unfolding. Basally applied BA (10 ppm) stimulated the opening of the intact hypocotylar hook. The light affacted synergistically on BA-induced opening. However, basally applied BA prevented the opening of decapitated hooks, especially in the light. The cotyledons were found to play a part of effects in maintaining the hook.     

与芒果子叶切段不定根形成有关的活性物质初探

在芒果子叶维管渗出物中检测到多种物质,其中生物碱和甾体两种物质为维管渗出物所特有.芒果子叶的甲醇提取液可提高黄瓜子叶不定根形成的百分率、生根数和根鲜重, 促进小麦胚芽鞘的伸长.     

Promotive effect of chrysanthemic acids on adventitious root formation in some plants

Adventitious root formation in excised cucumber (Cucumis sativus L.) cotyledons was significantly promoted by (±)-cis-chrysanthemic acid at 0.006–1.8 mM. The effect of (±)-cis-chrysanthemic acid on indole-3-acetic acid (IAA)-induced rooting was additive. Rooting in excised cucumber cotyledons was significantly promoted by several isomers of chrysanthemic acid and sodium (±)-cis-chrysanthemate at 0.18 mM. Rooting in mung bean (Phaseolus radiatus L.) hypocotyls was also stimulated by the sodium salt at 0.06–0.6 mM. Rooting of kidney bean (Phaseolus vulgaris L.) hypocotyls was also clearly enhanced by sodium (±)-cis-chrysanthemate at 0.18–6 mM.     

INDUCTION OF APOGAMY IN MEGAGAMETOPHYTES OF ZAMIA INTEGRIFOLIA

Ovules of Zamia integrifolia Ait. were dissected and megagametophytes and embryos excised and cultured. Induction of apogamous, haploid roots and leaves by auxin and kinetin, in combination with glutamine, asparagine and alanine, was observed. The organs resembled their normal diploid counterparts. 2,4-D at 1 ppm was more effective than 1 ppm IAA, but less effective than 5 ppm IAA. The highest percentage of apogamy was 59% after 5 months of culture. In several cultures embryoids were formed both by diploid excised embryos and by haploid megagametophtyes.     

The Effect of Potassium on Cotyledon Expansion Induced by Cytokinins

Potassium has been found to enhance greatly the expansion response of cucumber cotyledons to cytokinins. A reduction of the response to kinetin is obtained with increasing age of the cotyledons. The lesser response is associated with lower levels of potassium remaining in the cotyledon. A high level of KCI in the incubation medium offsets the lower potassium content of the tissue and enables a much larger response to the cytokinins. At 40 mM KCI the response to kinetin is 4.2 times greater than in the absence of KCI. Calcium increases the effect of potassium on the response to kinetin. When incubated in 40 mM KCI and 10 mM CaCI₂ with 10 mg/I 6-benzylamino-purine, the final weight of the cotyledons is 6.8 times the initial weight after just 4 days. This KCI-CaCI₂ combination is also found to promote chlorophyll synthesis in the usual cucumber cotyledon bioassay.     

Growth response of excised ovaries of reseda odorata in sterile culture

In the basal growth medium only the ovaries excised 15 days after pollination reached maturity. In a medium containing IAA (5ppm) and kinetin (0.5ppm) ovaries excised even 10 days after polliration produced viable seeds. Young ovaries formed in the pedicel region a callus, which later differentiated into roots; older ovaries (excised 15 days after pollination) produced callus tissue and roots from the ovary wall.