Comparative assessment of the efficacy of bacterial and cyanobacterial phytohormones in plant tissue culture

Efficient callus and explant regeneration medium, using microbial extract (SPE purified) or supernatant has been formulated for Brassica oleracea L. var. capitata. Two cyanobacterial strains (Anabaena sp. Ck1 and Chroococcidiopsis sp. Ck4) and two bacterial strains, (Pseudomonas spp. Am3 and Am4) known to produce a number of cytokinins, tZ, cZ, ZR, DHZR and IAA were selected for the media formulation. Supernatant from strains with high cytokinin to IAA ratio, including Pseudomonas aeruginosa Am3 (2.08) and Chroococcidiopsis sp. Ck4 (0.8) efficiently induced compact calli which were turned green upon exposure to light. The strains producing lower cytokinins to IAA ratio (0.11–0.13) on the other hand induced friable callus which were unable to regenerate on the selected media combinations. Leaf, stem and root explants of Brassica oleracea L. regenerated on MS medium supplemented with phytohormones from microbial origin with efficiency comparable to standard cytokinins and IAA. Supplements from cyanobacterial origin proved to be the best for induction of adventitious roots and shoots on internodal and petiolar segments. Hypocotyl explants however, responded well on MS supplemented with bacterial metabolites. Induction of adventitious shoots on root explants was supported by phytohormones from both origin equally well. Callus induction on the seeds and regeneration of shoots on calli was also observed. Cyanobacteria based media were more efficient to induce calli capable of regeneration upon exposure to light. Internodal explants were highly amenable to regenerate shoot and roots simultaneously. Root explants were the less successful to regenerate shoots.     

ENDOGENOUS CYTOKININS IN THREE GENERA OF MICROALGAE FROM THE CHLOROPHYTA1

Nine axenic microalgal (Chlorophyta) strains from three genera (Protococcus, Chlorella, and Scenedesmus) were analyzed for endogenous cytokinins. Cytokinin‐like activity was detected using the excised cucumber cotyledon bioassay. Five strains showed no cytokinin‐like activity and four strains, low cytokinin‐like activity. Ethanolic extracts of the microalgae containing a mixture of deuterium‐labeled standards were purified using a combined DEAE‐Sephadex octadecysilica column and immunoaffinity column based on wide‐range specific mon‐oclonal antibodies and analyzed by HPLC linked to a micromass single quadrupole mass spectrometer with an electrospray interface and a photodiode array detector. There were similar trends in cytokinin profiles for the nine microalgal strains investigated, although concentrations did vary. Both isopentenyladenine and isopentenyladenosine were detected in all nine strains. cis‐Zeatin and cis‐zeatin riboside occurred at higher concentrations than the trans isomers, whereas trans‐zeatin‐O‐glucoside and trans‐zeatin riboside‐O‐glucoside were dominant over the cis isomers. Dihydrozeatin and its conjugates were not detected in any significant amounts. The aromatic benzyladenine always occurred at higher concentrations than benzyladenosine. The topolins were well represented with all three isomers (ortho, meta, and para) being detected, with ortho‐topolin and ortho‐topolin riboside occurring at higher concentrations than the other isomers. However, for the O‐glucosides, the meta isomers (meta‐topolin‐O‐glucoside and meta‐topolin riboside‐O‐glucoside) occurred at higher concentrations than the other isomers. No N‐glucosides were detected (isopentenyladenine‐9‐glucoside, zeatin‐9‐glucoside, dihydrozeatin‐9‐glucoside, benzyladenine‐9‐glucoside, ortho‐topolin‐9‐glucoside, and meta‐topolin‐9‐glucoside). Generally, zeatin and topolin conjugates were the dominant forms of isoprenoid and aromatic cytokinins, respectively. There was no distinct trend in the proportions of isoprenoid to aromatic cytokinins.     

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.     

Interactions of bacterial cytokinins and IAA in the rhizosphere may alter phytostimulatory efficiency of rhizobacteria

Phytohormones from rhizobacterial origin have been linked to their phytostimulation potential. However, while studying the efficacy of plant growth promoting bacteria, focus has always been on a single hormone. The role of plant hormones often overlay and they mutually modulate their effect. In current study focus was on the role of two hormones (cytokinins and indole acetic acid) in phytostimulation by rhizobacteria. Endogenous rhizosphere bacteria were isolated and screened for the presence of phytohormones. Bacterial strains from three different genera (Pseudomonas, Bacillus and Azospirillum) were screened positive for cytokinins and IAA. Phytohormones were simultaneously determined in SPE purified bacterial extract by ultra performance liquid chromatography (UPLC) coupled to a tandem mass spectrometer through electrospray interface. Cytokinins and IAA were determined in positive and negative mode, respectively with MRM scan. Zeatin, zeatin riboside and dihydrozeatin riboside were detected and quantified in the selected strains. Significant positive correlation between cytokinins and IAA in bacterial culture and plant endogenous hormones (r = 0.933 and r = 0.983; P = 0.01, respectively) was observed. However, strains with high IAA to cytokinins ratio could hardly enhance in-planta cytokinins, indicating antagonistic relation between the two hormones. Significant correlation of cytokinin with shoot length (r = 0.797; P = 0.01), fresh weight (r = 0.685; P = 0.01) and dry weight (r = 0.704; P = 0.01) was reported under axenic conditions. Bacterial IAA was correlated negatively to root length (r = 0.853; P = 0.01) and positively correlated to the number of roots (r = 0.964; P = 0.01). In natural conditions maximum increase in spike length (33%), number of tillers (71%) and weight of seeds (39%) was documented at final harvest in bacterially inoculated plants.     

Phytostimulation and biofertilization in wheat by cyanobacteria

Cyanobacteria are commonly used for the phytostimulation and biofertilization of agriculture crops due to their nitrogen-fixing ability. However, the contribution by their phytohormones has been neglected. This study focuses on the screening of rhizospheric and free-living cyanobacteria for in vitro phytohormones production and growth stimulation in wheat. Selected isolates were shown to release cytokinin and indole-3-acetic acid (IAA) by using UPLC coupled with MS/MS via an electrospray interface. The maximum cytokinin and IAA concentration was 22.7 pmol mg⁻¹ ch-a and 38 pmol mg⁻¹ ch-a, respectively, in the culture medium of Chroococcidiopsis sp. Ck4 and Anabaena sp. Ck1. The growth of wheat inoculated with cyanobacterial strains was stimulated under axenic as well as field conditions. Seed germination, shoot length, tillering, number of lateral roots, spike length, and grain weight were significantly enhanced in inoculated plants. The maximum increase in grain weight (43%) was demonstrated in wheat plants inoculated with Chroococcidiopsis sp. Ck4 under natural conditions. Positive linear correlation of cyanobacterial cytokinin with shoot length (r = 0.608; P = 0.01), spike length (r = 0.682; P = 0.01), and grain weight (r = 0.0.869; P = 0.01) was recorded. Similarly, cyanobacterial IAA was correlated with the root growth parameters shoot length (r = 0.588; P = 0.01), spike length (r = 0.0.689; P = 0.01), and weight of seeds (r = 0.480; P = 0.05). The endogenous phytohormones pool of the plant was enhanced significantly as a result of the plant–cyanobacteria association in the rhizosphere. It was concluded that cyanobacterial phytohormones are a major tool for improved growth and yield in wheat.     

Effect of phytohormones synthesized by rhizosphere bacteria on plants

New strains of rhizosphere microorganisms Azotobacter chroococcum Az d10, Bacillus megaterium Pl-04, and Bacillus mucilaginosus B-1574 were found to be able to synthesize cytokinins (CKs) and indolylacetic acid (IAA). Three forms of CKs—dihydrozeatin riboside, isopentenyl adenosine, and trans-zeatin riboside—were identified, whose ratio was different in the three bacterial cultures. Inoculation of cucumber (Cucumis sativus L.) plants increased the content of CKs and IAA in them by 35.6 and 21.3%, respectively, and also stimulated seed germination and increased the growth rate, the biomass of shoots, the number of lateral roots, and the root hair area, which ensured better plant nutrition. The IAA/CKs ratio shifted during bacterization towards CKs due to increase in the content of riboside forms, which apparently caused growth stimulation.     

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.     

HIGH‐THROUGHPUT SCREENING TECHNOLOGY FOR MONITORING PHYTOHORMONE PRODUCTION IN MICROALGAE1

New miniaturized techniques for multiplying microalgae and estimating their phytohormone production were developed; in these methods, the strains to be tested are cultivated in microtitre plates, and the phytohormones in suspensions of the cultures are measured by direct ELISAs. Specific and sensitive ELISAs for determining abscisic acid (ABA), indole‐3‐acetic acid (IAA), cis‐ and trans‐zeatin riboside, isopentenyladenosine (iPR), and other less common cytokinins were developed for this purpose. Polyclonal antibodies used in the ABA and IAA assays were raised against C1‐ and C1′‐ conjugates of the compounds with BSA, respectively, and thus were specific for the free acids and their respective C1‐derivatives. The use of cytokinin ribosides coupled via their sugar residues to BSA as haptens generally led to antibodies that bound free bases, 9‐glycosides and nucleotides, but with high specificity for the corresponding N⁶‐side chains. Using internal standards, dilution assays, and authentic [²H] and [³H] recovery markers, it was shown that the ELISAs could be used to estimate contents of the selected phytohormones in the cultures. The ELISAs provided reliable and very fast estimates of the selected phytohormones, at concentrations ranging from 0.01 to 10 pmol · mL^?1 in various microalgal strains. In addition, a recently developed HPLC selected ion monitoring mass spectrometry (HPLC‐SIM–MS) method was used to calibrate and validate the ELISA results and confirm the presence of the detected phytohormones in immunoaffinity‐purified extracts. Where independent validation of results is deemed necessary, the use of quantitative HPLC–MS is recommended for each new microalgal strain to be tested.     

Evaluation of a Bioassay for Cytokinins Using Soybean Hypocotyl Sections

The dose-response curves of several cytokinins were investigatedin a soybean hypocotyl bioassay. Zeatin riboside, zeatin-O-ß-D-glucoside,dihydrozeatin, and dihydrozeatin riboside produced linear responsesparallel to that for zeatin. The hypocotyl section assay wassuperior to the conventional soybean callus assay because theresponse (log₁₀ transformed data) was linear, exhibited lowvariability, and was more reproducible and more sensitive. Theassay was quicker to perform and required less cytokinin.     

High Performance Liquid Chromatographic Analysis of Cytokinins in Sorghum bicolor Leaves

High performance liquid chromatography with octadecylsilica (Bondapak C₁₈/Poracil B) column packing was used to purify and separate cytokinins in Sorghum leaf extracts. The column size was 56 × 0.21 cm i.d. By gradient elution, using acidified water with increasing amounts of methanol, the major peaks of cytokinin activity, as determined by the callus tissue bioassay. were effectively separated from large amounts of extraneous impurities. These cytokinins were further separated on a microoctadecylsilica column (μBondapak C₁₈, 30 × 0.4 cm i.d.) with a gradient of acidified water-acetonitrile. Zeatin and zeatin riboside gave distinct ultra violet absorption peaks which could be used for quantitative estimation. Biological activity corresponded to the elution of these peaks. These two cytokinins are the major cytokinins in Sorghum leaves.     

Physiology of Tuberization in Solanum tuberosum L: cis-Zeatin Riboside in the Potato Plant: Its Identification and Changes in Endogenous Levels as Influenced by Temperature and Photoperiod

Using high pressure liquid chromatography, the cucumber cotyledon bioassay, and mass spectrometry a cytokinin isolated from Solanum tuberosum L. cv. Katahdin plant tissues has been identified as cis-zeatin riboside. Zeatin riboside (ZR) levels in plants grown under inducing conditions (28 C day and 13 C night with a 10-hour photoperiod) were significantly higher than those in plants grown under noninducing conditions (30 C day and 28 C night with an 18-hour photoperiod). The highest level of ZR was noted in below-ground tissue after 4 days exposure to inducing conditions, with tuber initiation observed after 8 days. A companion study conducted to determine the effect of ZR on in vitro tuberization of noninduced rhizomes revealed that after 1 month in culture, controls exhibited 0% tuberization, while ZR treatments of 0.3 and 3.0 milligrams per liter showed 39 and 75% tuberization, respectively.     

Cytokinin production by fluorescent Pseudomonas in the presence of rice root exudates

Free-living bacteria may trigger the plant growth through production of phytohormones viz. gibberellins, auxins and cytokinins. A total 50 isolates of fluorescent Pseudomonas were screened for their ability to produce cytokinins such as isopentenyladenosine (IPA), dihydroxyzeatin riboside (DHZR) and zeatin riboside (ZR) as plant growth-promoting activity. Pseudomonas fluorescens AK1 and Pseudomonas aeruginosa AK2 were found higher phytohormones producing strains. Of the three cytokinins, IPA was the major cytokinin produced by both isolates in pure culture (5.5 and 2.9 pmol/ml, respectively) and with rice root exudates (5.9 and 3.4 pmol/ml, respectively). Production of ZR and DHZR for both organisms was found after 48 and 72 h. Amount of ZR and DHZR increased with time for both isolates in pure culture conditions. In presence of rice, production of ZR was increased 0.8 and 0.6 pmol/ml for P. fluorescence AK1 and P. aeruginosa AK2, respectively, in comparison with controls. There was no significant difference in the production of DHZR with rice exudates.     

The content of endogenous hormones and sugars in the process of early somatic embryogenesis in the tree fern <Emphasis Type="Italic">Cyathea delgadii</Emphasis> Sternb.

Somatic embryogenesis (SE) of Cyathea delgadii presents a model system for investigating the mechanisms associated with the acquisition of embryogenic competence by single epidermal cells of stipe explants cultured on plant growth regulator-free medium. The present work reveals relationship between endogenous hormone and sugar content in the process of early SE in C. delgadii. By comparing two types of initial explants, i.e. incapable (non-etiolated) and capable (etiolated) of SE, it was established that in etiolated explants, the glucose, fructose, sucrose, and abscisic acid (ABA) contents diminished, but indole-3-acetic acid (IAA) and cytokinins (CKs; i.e. cis/trans zeatin, cis/trans-zeatin riboside, kinetin, kinetin riboside, isopentenyladenosine) contents increased. The ratios between phytohormones revealed that a high concentration of ABA is the main factor inhibiting SE induction. Because of explant excision, a dramatic reduction in concentration of all phytohormones studied was observed, but hormonal balance and sugar content remained almost unchanged. During the 14-day-long culture, the ABA/CKs and ABA/IAA ratios remained constant, whereas the greatest differences were detected for the IAA/CKs and Z-type/iPA cytokinin ratios. Excluding day 6 of culture, cytokinins were found to be the predominant phytohormones over IAA. An almost 12-fold increase in soluble sucrose concentration at day 6 of culture might be the switch to the SE expression phase. Frequent cell divisions leading to somatic embryo formation are clearly associated with increase in trans-zeatin riboside content.     

The occurrence of free and bound cytokinins in clubroots and Plasmodiophora brassicae infected turnip tissue cultures

This paper deals with the quantitative determination of free and bound cytokinins in clubroot tissue and in Plasmodiophora brassicae Woron, infected Brassica campestris L. callus tissue. The fractions were separated in a butanol soluble fraction containing the free cytokinins such as zeatin and zeatin riboside and a water soluble fraction containing the bound cytokinins. The butanol fraction was extensively purified and analysed by high pressure liquid chromatography (HPLC). The butanol fraction contained cytokinins which cochromatographed with zeatin and zeatin riboside and not with dihydrozeatin. Zeatin and zeatin riboside were quantitatively determined by HPLC. Recovery of the cytokinins varied between 30–50%. Clubs contained 50–160 ng zeatin and 210–300 ng zeatin riboside per g dry weight. Callus tissue contained 133 ng zeatin and 169 ng zeatin riboside per g dry weight. Clubs, callus as well as healthy tissue contain large amounts of bound cytokinins. Upon treatment of the water soluble fraction first with alkaline phosphatase and then with β-glucosidase biologically active fractions were found which coeluted with zeatin and zeatin riboside on Sephadex LH₂₀ in 20% ethanol. Evidence is presented for a novel cytokinin in the water soluble fraction which yields free zeatin and glucose-6-phosphate after treatment with β-glucosidase.     

The effect of exogenous and endogenous phytohormones on the in vitro development of gametophyte and sporophyte in <Emphasis Type="Italic">Asplenium nidus</Emphasis> L.

The fern Asplenium nidus L. is in great demand as an ornamental plant. The aim of this work was to investigate the influence of phytohormones in promoting a gametophytic and sporophytic growth in homogenized sporophytes tissue. Exogenous application of 0.5 and 5 μM N ⁶-benzyladenine, 0.05 and 0.5 μM indole-3-acetic acid (IAA), and 0.3 and 3 μM gibberellic acid (GA₃) favoured sporophyte regeneration, whereas gametophyte regeneration took place when plant material was cultured in a hormone-free liquid MS medium. The endogenous contents of the auxin IAA, the cytokinins trans-zeatin, trans-zeatin riboside, dihydrozeatin, dihydrozeatin riboside, isopentenyladenine and isopentenyladenosine, and the gibberellins GA₁, GA₃, GA₄, GA₇, GA₉ and GA₂₀ in growing gametophytes and sporophytes were evaluated. Similar levels of the auxin and cytokinins and qualitative differences in the gibberellins were found between both generations.     

Fluctuation of endogenous cytokinins in leaves and roots of short-day and long-day tobacco associated with photoperiodic induction

As the dynamics of changes in phytohormones may be involved in photoperiodic regulation of the rates of growth and flowering, fluctuation of cytokinins was followed in long-day and short-day tobacco. Zeatin (Z) and zeatin riboside (ZR) were identified in leaves and roots using a GC-MSC system. In plants of the long-day tobaccoNicotiana silvestris increasing the number of long-day inductive for flowering (10, 20, 30, 40 LD) resulted in a rise in ZR activity. Half the plants reached a reproductive stage on the 40th day of induction. In short-day Mam moth tobacco plants, short-day floral induction (10, 20, 30, 40 SD) caused similar but less marked changes in ZR.     

Endogenous hormonal content and somatic embryogenic capacity of Corylus avellana L. cotyledons

Endogenous indole-3-acetic acid (IAA), abscisic acid (ABA) and cytokinins [zeatin (Z) zeatin riboside, dihydrozeatin, dihydrozeatin riboside, N⁶-isopentenyl adenine (iP) and N⁶-isopentenyladenine riboside] were evaluated in hazelnut (Corylus avellana L.) cotyledons of different developmental stage and genetic source for their somatic embryogenic capacity. There was an inverse correlation between the embryogenic potential of cotyledons and the degree of maturity of zygotic embryos, the first characteristic being associated with iP-type cytokinins and the second with Z-type cytokinins. Although the differences in total cytokinin, ABA and IAA contents between the cotyledons were small, the IAA/ABA and, mainly, the iP-type/Z-type cytokinin ratios were found to be two good indexes of the embryogenic competence of explants, suggesting that the endogenous hormonal balance is a very important factor defining the in vitro potential of hazelnut cotyledons. Received: 6 January 1997 / Revision received: 3 March 1997 / Accepted 1 April 1997     

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.     

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.