Stimulation of shoot regeneration in Triticum aestivum and Nicotiana plumbaginifolia Viv. tissue cultures using the ethylene inhibitor AgNO3

Silver nitrate effectively promoted shoot regeneration in wheat (Triticum aestivum L.) callus cultures derived from immature embryos. This effect could be observed in both weakly and strongly regenerating cultivars, and in using material from both field and greenhouse grown plants. The role of silver ions as an inhibitor of ethylene action was supported by a reversal of the inhibitory effects of 2,4-D and ethylene on morphogenesis in wheat callus cultures.Enhancement of shoot regeneration by silver nitrate was also observed in callus cultures of non-regenerating or weakly regenerating mutants of Nicotiana plumbaginifolia Viv. derived from cell cultures.Abbreviations BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - Ethrel 2-chlorethylphosphonic acid - NAA 1-naphthaleneacetic acid - NR^– nitrate reductase deficient     

Effect of l-aminocyclopropane-l-carboxylic acid,silver nitrate,and norbornadiene on plant regeneration from maize callus cultures

The effect of the ethylene antagonists norbornadiene and silver nitrate and the ethylene precursor l-aminocyclopropane-l-carboxylic acid (ACC) on Zea mays plant regeneration was studied. A 12-fold increase in plant regeneration, as measured by number of plants obtained per gram fresh weight from callus cultures of maize inbreds Pa91 and H99, was obtained by 250 M norbornadiene and 100 M silver nitrate treatments. An increase in amout of nonregenerable tissue and a 68% decrease in plant regeneration were associated with callus treated with 1 mM ACC. Ethylene emanation from 1 mM ACC treated callus reached a maximum of 170 nl g^–1 h^–1 after 3 days compared to 7 nl g^–1 h^–1 for the control. The free proline content was up to 80% lower in 1 mM ACC treated callus grown for 30 days on medium with or without 12 mM proline, respectively, as compared to each control. These studies indicate that ethylene action inhibitors such as norbornadiene and silver nitrate can be used to increase plant regeneration efficiency from maize callus cultures.Abbreviations ACC l-aminocyclopropane-l-carboxylic acid - gfw gram fresh weight     

Ethylene precursors and antagonists increase embryogenesis of Hordeum vulgare L. anther culture

The role of ethylene in microspore embryogenesis and regeneration was analyzed by studying the effects of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and the ethylene antagonists silver nitrate and silver thiosulphate on the androgenic response of in vitro cultured anthers of seven genotypes of barley. Incorporation of either ACC or silver salts in the culture medium lead to a significant increase in callus induction for five of the seven genotypes tested. The treatment that increased callus induction depended upon genotype. Only anthers cultured on 1 mg l^–1 silver thiosulphate gave rise to fertile plants in all seven genotypes tested.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole acetic acid - PAA phenyl acetic acid - STS silver thiosulphate - ACC 1-aminocyclopropane-1-carboxylic acid     

Influence of different ethylene inhibitors on somatic embryogenesis and secondary embryogenesis from <Emphasis Type="Italic">Coffea canephora</Emphasis> P ex Fr.

The effect of cobalt chloride, salicylic acid, and silver nitrate for embryogenesis was studied in in vitro cultures of Coffea canephora. Murashige and Skoog (in Physiol. Plant. 15:473–497, 1962) medium containing 20 and 40 μM either of cobalt chloride, silver nitrate, or salicylic acid supplemented with 1.1 μM N ⁶ benzyladenine and 2.85 μM indole-3-acetic acid was used for the study. At 20 and 40 μM silver nitrate treatment, 35–48% explants responded for embryogenesis, and 38 ± 7 and 153 ± 27 embryos were produced from each callus mass, respectively, whereas only 5% control explants responded on medium devoid of silver nitrate, cobalt chloride, or salicylic acid. Secondary embryogenesis was observed in 70–90% of the explants, and around 100–150 embryos were produced from each explant cultured on a medium containing silver nitrate, and only a 3% response was noticed in control embryo explants. Yellow friable embryogenic calluses were obtained from the cut edges of most of the tissues grown in a medium supplemented with cobalt chloride. The results clearly demonstrated that, among the tested ethylene inhibitors, silver nitrate is very effective in reprogramming the cellular machinery toward embryogenesis.     

Polyamines influence morphogenesis and caffeine biosynthesis in in vitro cultures of <Emphasis Type="Italic">Coffea canephora</Emphasis> P. ex Fr.

The influence of polyamines, polyamine inhibitors and ethylene inhibitors were tested in Coffea canephora for in vitro morphogenetic response and caffeine biosynthesis. Coffea canephora produced non-embryogenic and embryogenic calli. Somatic embryos were produced only from the embryogenic callus. Endogenous polyamine pools were estimated in these tissues. Somatic embryos were subjected to secondary embryogenesis under the influence of putrescine, silver nitrate and specific inhibitors of polyamine biosynthesis. Estimation of endogenous total polyamines revealed that embryogenic callus contained 11-fold more spermine and 3.3-fold higher spermidine when compared to non-embryogenic callus. Incorporation of polyamines resulted in 58% explant response for embryogenesis when compared to control with 42% response. Incorporation of silver nitrate resulted in 65% response for embryogenesis. Incorporation of polyamine biosynthetic pathway inhibitors DFMO and DFMA resulted in 83% reduction in embryogenic response with concomitant increase in caffeine levels by two-fold as compared to control. These results have clearly demonstrated that polyamines play a crucial role in embryogenesis and caffeine biosynthesis.     

Involvement of ethylene on growth and plant regeneration in callus cultures of Heliconia psittacorum L.f.

The level of ethylene accumulated in morphogenic callus cultures of Heliconia psittacorum L.f. was only one quarter that of non-morphogenic cultures. The rate of ethylene production in the morphogenic callus cultures during early stages of differentiation of protocorm-like bodies leading to plantlet regeneration was 10-fold higher than that during callus proliferation. In cultures sealed with gastight serum caps, fresh weight gain was reduced 2-to 3-fold compared to those that were closed with Kaputs. Treatment with 1-aminocyclopropane-1-carboxylic acid ( 100 M) caused complete inhibition of plant regeneration from the morphogenic callus on subsequent culture under inductive conditions. Silver nitrate and aminoethoxyvinylglycine also reduced plant regeneration. These results indicate that while high levels of ethylene were inhibitory, a low level of endogenous ethylene production may be necessary during the plant regeneration phase in callus cultures of Heliconia.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - AC activated charcoal - ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - BM basal medium - CH casein hydrolysate - DM development medium - MM maintenance medium - PLB protocorm-like body     

Ethylene influences green plant regeneration from barley callus

The plant hormone ethylene is involved in numerous plant processes including in vitro growth and regeneration. Manipulating ethylene in vitro may be useful for increasing plant regeneration from cultured cells. As part of ongoing efforts to improve plant regeneration from barley (Hordeum vulgare L.), we investigated ethylene emanation using our improved system and investigated methods of manipulating ethylene to increase regeneration. In vitro assays of regeneration from six cultivars, involving 10 weeks of callus initiation and proliferation followed by 8 weeks of plant regeneration, showed a correlation between regeneration and ethylene production: ethylene production was highest from ‘Golden Promise’, the best regenerator, and lowest from ‘Morex’ and ‘DH-20’, the poorest regenerators. Increasing ethylene production by addition of 1-aminocyclopropane 1-carboxylic acid (ACC) during weeks 8–10 increased regeneration from Morex. In contrast, adding ACC to Golden Promise cultures during any of the tissue culture steps reduced regeneration, suggesting that Golden Promise may produce more ethylene than needed for maximum regeneration rates. Blocking ethylene action with silver nitrate during weeks 5–10 almost doubled the regeneration from Morex and increased the Golden Promise regeneration 1.5-fold. Silver nitrate treatment of Golden Promise cultures during weeks 8–14 more than doubled the green plant regeneration. These results indicate that differential ethylene production is related to regeneration in the improved barley tissue culture system. Specific manipulations of ethylene were identified that can be used to increase the green plant regeneration from barley cultivars. The timing of ethylene action appears to be critical for maximum regeneration.     

Direct and indirect somatic embryogenesis on cotyledon explants of <Emphasis Type="Italic">Quassia amara</Emphasis> L., an antileukaemic drug plant

Summary In vitro propagation of Quassia amara L. (Simaroubaceae) was attempted using mature and juvenile explants. Attempts to establish in vitro culture using leaf and internode explants from a plant more than 15yr old were unsuccessful due to severe phenolic exudation. Plant regeneration through direct and indirect somatic embryogenesis was established from cotyledon explants. Murashige and Skoog (MS) medium with 8.9 μM N⁶-benzyladenine (BA) and 11.7 μM silver nitrate induced the highest number (mean of 32.4 embryos per cotyledon) of somatic embryos. Direct somatic embryogenesis as well as callus formation was observed on medium with BA (8.9–13.3 μM). Semi-mature pale green cotyledons were superior for the induction of somatic embryos. Embryos developed from the adaxial side as well as from the point of excision of the embryonic axis. More embryos were developed on the proximal end compared to mid and distal regions of the cotyledons. Subculture of callus (developed along with the somatic embryos on medium with BA alone) onto medium containing 8.9 μM BA and 11.7 μM silver nitrate produced a mean of 17.1 somatic embryos. Primary somatic embryos cultured on MS medium with 8.9 μM BA and 11.7μM silver nitrate produced a mean of 9.4 secondary somatic embryos. Most of the embryos developed up to early cotyledonary stage. Reduced concentration of BA (2.2 or 4.4 μM) improved maturation and conversion of embryos to plantlets. Ninety percent of the embryos converted to plantlets. The optimized protocol facilitated recovery of 30 plantlets per cotyledon explant within 80d. Plantlets transferred to small cups were subsequently transferred to field conditions with a survival rate of 90%.     

Synthesis of Silver Nanoparticles Using Callus Extract of Carica papaya — A First Report

This is the first report of synthesis of silver nanoparticles by using callus extract of Carica papaya. MS medium supplemented with the growth hormones, 2.0 mg l^?1 IBA and 0.5 mg l^?1 BAP was found to be more suitable for the induction of callus and multiple shoots in papaya. The extract of callus obtained by grinding showed ability of synthesis of silver nanoparticles when treated with silver nitrate (1 mM). The formation of brown colour in the reaction mixture indicates the synthesis of silver nanoparticles. The further detection and characterization of these synthesized silver nanoparticles was carried by spectrophotometry. FTIR spectrum analysis showed peaks between 1000–2000 cm^?1 which confirmed the presence of proteins and other ligands required for the synthesis and stabilization of silver nanoparticles. SEM micrograph confirmed the synthesis of spherical silver nanoparticles in the size range of 60–80 nm.     

Probing the role of endogenous ethylene in the degreening of citrus fruit with ethylene antagonists

The ethylene antagonists, 2,5-norbornadiene (NBD) and silver nitrate, were used to probe the involvement of endogenous ethylene in the natural degreening of citrus fruit. Mature-green, detached Shamouti orange (Citrus sinensis L. Osbeck) fruit were treated with NBD vapor or dipped in solutions of silver nitrate. More than 80% of the chlorophyll was lost from control fruit after 8 days. NBD (0.11 mmole/liter) inhibited the loss of chlorophyll by 60%. NBD also antagonized the degreening induced by exogenous ethylene by 50%. Silver nitrate (0.1 mM) inhibited the loss of chlorophyll by 55%. Ethylene evolution of mature, green detached fruit was <2 nl.fruit^-1.h^-1 (ca. 13.5 nl.Kg^-1FW.h^-1) and did not change significantly for 7 days after harvest. NBD concentrations up to 0.22 mmole/liter did not enhance ethylene evolution. Not with-standing the extremely low amounts of ethylene evolved, the inhibition of degreening by NBD and silver nitrate suggests that endogenous ethylene is involved in the control of this process in mature citrus fruit.     

Growth of embryogenic sweet orange callus on media varying in the ratio of nitrate to ammonium nitrogen

Embryogenic callus from Citrus sinensis (L.) Osbeck cv. Hamlin was cultured for 28 days on 20 media arranged in a 5×2×2 factorial varying in the ratio of nitrate to ammonium nitrogen, total inorganic nitrogen, and benzyladenine. Fresh weight increase of callus and final medium pH were significantly affected by total inorganic nitrogen and the ratio of nitrate to ammonium. The nitrate to ammonium ratio accounted for 55% of the variation in the fresh weight increase of the callus and 93% of the variation in the final medium pH. Varying the NO₃ ^-:NH₄ ^- ratio provided adequate pH control.Abbreviation BA benzyladenine     

Ethylene and nitric oxide are involved in maintaining ion homeostasis in <Emphasis Type="Italic">Arabidopsis</Emphasis> callus under salt stress

In the present study, the role of ethylene in nitric oxide (NO)-mediated protection by modulating ion homeostasis in Arabidopsis callus under salt stress was investigated. Results showed that the ethylene-insensitive mutant etr1-3 was more sensitive to salt stress than the wild type (WT). Under 100 mM NaCl, etr1-3 callus displayed a greater electrolyte leakage and Na⁺/K⁺ ratio but a lower plasma membrane (PM) H⁺-ATPase activity compared to WT callus. Application of exogenous 1-aminocyclopropane-1-carboxylic acid (ACC, an ethylene precursor) or sodium nitroprusside (SNP, a NO donor) alleviated NaCl-induced injury by maintaining a lower Na⁺/K⁺ ratio and an increased PM H⁺-ATPase activity in WT callus but not in etr1-3 callus. The SNP actions in NaCl stress were attenuated by a specific NO scavenger or an ethylene biosynthesis inhibitor in WT callus. Under 100 mM NaCl, the NO accumulation and ethylene emission appeared at early time, and NO production greatly stimulated ethylene emission in WT callus. In addition, ethylene induced the expression of PM H⁺-ATPase genes under salt stress. The recovery experiment showed that NaCl-induced injury was reversible, as signaled by the similar recovery of Na⁺/K⁺ ratio and PM H⁺-ATPase activity in WT callus. Taken together, the results indicate that ethylene and NO cooperate in stimulating PM H⁺-ATPase activity to modulate ion homeostasis for salt tolerance, and ethylene may be a part of the downstream signal molecular in NO action.     

Enhanced plant regeneration in pearl millet (Pennisetum americanum) by ethylene inhibitors and cefotaxime

Cefotaxime, a cephalosporin antibiotic, and different ethylene inhibitors, such as silver nitrate, cobalt chloride, nickel chloride and O-acetyl salicylic acid, significantly delayed the loss of regeneration potential in embryogenic cultures of Pennisetum americanum. In the presence of these chemicals, ethylene content in the atmosphere of the culture vessel was less than that of the control. Cefotaxime, silver nitrate and O-acetyl salicyclic acid did not have any effect on callus growth based on fresh weight, while growth based on dry weight was enhanced by O-acetyl salicyclic acid.Abbreviations ASA O-acetyl salicylic acid - BA benzyladenine - CW coconut water - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid - MS Murashige and Skoog     

Polyamines in plants infected by citrus exocortis viroid or treated with silver ions and ethephon

The levels of polyamines in leaves of Gynura aurantiaca DC and tomato, Lycopersicon esculentum Mill. cv Rutgers, infected with citrus exocortis viroid (CEVd) or treated with silver nitrate or ethephon (2-chloroethylphosphonic acid) were measured by HPLC in relation to development of symptoms. Previously it had been demonstrated that treatment of G. aurantiaca plants with silver nitrate or ethephon closely mimicked the effects of viroid infection in the plants. In the studies reported here, a marked decrease in putrescine level was observed in plants infected by CEVd or treated with silver ions or ethephon. There was no significant change in either spermidine or spermine levels. Treatment of G. aurantiaca plants with specific inhibitors of ethylene biosynthesis (aminoethoxyvinylglycine, Co²⁺) or ethylene action (norbornadiene) prevented the decrease of putrescine associated with silver nitrate treatment and had no effect on spermidine or spermine levels. The development of viroid-like symptoms, the production of associated pathogenesis-related proteins, and the rise in protease activity induced by silver nitrate, were all suppressed by exogenous application of putrescine. The decreased level of putrescine as an ethylene-mediated step in the transduction of the viroid and silver or ethephon signaling is discussed.     

An increase in ethylene sensitivity is associated with jasmonate-promoted senescence of detached rice leaves

The role of ethylene in jasmonate-promoted senescence of detached rice leaves was investigated. Ethylene production in methyl jasmonate-treated leaf segments of rice was lower than in the control leaves. Treatment of leaf segments with silver nitrate or/and silver thiosulfate, inhibitors of ethylene action, inhibited methyl jasmonate-, jasmonic acid-, linolenic acid-, and abscisic acid-promoted senescence of detached leaves. We suggest that an increase in ethylene sensitivity, but not ethylene level, is the initial event triggering the enhanced senescence by jasmonates of detached rice leaves.Abbreviations JA jasmonic acid - MJ methyl jasmonate - STS silver thiosulfate - ABA abscisic acid     

Improvement of embryogenic callus induction and shoot regeneration of buffalograss by silver nitrate

Addition of the ethylene antagonist, silver nitrate (AgNO₃), into callus induction medium significantly enhanced embryogenic callus production (both induction frequency and callus growth) of field-collected male immature inflorescence cultures of buffalograss NE84-45-3 and 'Texoka'. No stimulatory effect of AgNO₃ was observed on embryogenic callus induction for female immature inflorescence culture of a female genotype `609' and `Texoka'. Calli initiated on AgNO₃-containing media had more shoot-regenerating calli than those initiated on AgNO₃-free media, when they were transferred to the regeneration media. Benzyladenine at 2.2 μM gave the best response for regeneration, regardless of the callus source. Although average number of shoots regenerated per callus was lower for calli initiated on AgNO₃-containing media, total number of shoots regenerated was higher. The stimulatory effect, however, was environment and genotype dependent. While the addition of AgNO₃ significantly stimulated embryogenic callus induction of NE84-45-3 immature inflorescences collected in Fall 1995 and May 1997, it only slightly increased the embryogenic callus induction frequencies in May 1996 when rainy conditions occurred. For male inflorescences of `Texoka' collected in early May, AgNO₃ significantly enhanced embryogenic callus production consistently over the two-year period (1996, 1997). Published as Journal Series No. 1351, Agricultural Research Division, University of Nebraska. This revised version was published online in June 2006 with corrections to the Cover Date.     

Metabolic Discrimination of Catharanthus roseus Calli According to Their Relative Locations Using 1H-NMR and Principal Component Analysis

Creating a plant-cell suspension culture involves first transferring the callus into liquid media, but there are no objective criteria for selecting the location of the callus to be transferred. In this study, inner and outer cells of Catharanthus roseus with various elicitors in solid-state cultures were differentiated by ¹H NMR (nuclear magnetic resonance) spectrometry and principal component analysis (PCA). It was found that the samples of various elicitors and relative locations could be separated in PCA-derived score plots. Especially, there was a clear separation between nontreated samples and those cotreated with silver nitrate and methyl jasmonate. Loading-plot analysis was therefore applied to data obtained from nontreated samples and those cotreated with silver nitrate and methyl jasmonate to determine the separation of major metabolites on score plots. The levels of valine, lactic acid, threonine, alanine, arginine, acetic acid, malic acid, succinic acid, citric acid, asparagine, choline, lactose, fumaric acid, phenylalanine, tryptophan, and formic acid were higher in the inner callus than in the outer callus, whereas 2-oxoglutaric acid, oxalacetic acid, sucrose, and glucose dominated in the outer callus. The results obtained in this study suggest that inner and outer calli can be differentiated by ¹H-NMR-based metabolomic analysis.     

Effects of ethylene on somatic embryogenesis and galanthamine content in <Emphasis Type="Italic">Leucojum aestivum</Emphasis> L. cultures

The influence of ethylene on in vitro morphogenesis of Leucojum aestivum and galanthamine accumulation was studied. Calli were cultivated on Murashige and Skoog (MS) medium supplemented with 25 μM 4-amino-3,5,6-trichloropicolinic acid (picloram) and 0.5 μM benzyladenine (BA). During incubation under these conditions, callus cultures produced ethylene (9.5 nL/g fresh weight: F.W.) whereas no ethylene was found in somatic embryos cultivated on medium supplemented with 0.5 μM α-naphthalene acetic acid (NAA) and 5 μM zeatin. Application of the precursor of ethylene 1-aminocyclopropane-1-carboxylic acid (ACC) increased ethylene production in both cultures, and decreased callus growth by a factor of 1.2, whereas callus growth was enhanced by a factor of 1.1 in the presence of an inhibitor of ethylene silver nitrate (AgNO₃) or by a factor of 1.2 with an absorbent potassium permanganate (KMnO₄). ACC enhanced the induction of somatic embryos and the development of globular embryos. Removal of ethylene by KMnO₄ during somatic embryogenesis led to the development of plants with greater length. Silver thiosulphate (STS) induced galanthamine production in callus cultures (0.1% dry weight), whereas ACC induced galanthamine production in somatic embryo cultures (2% dry weight).     

Effects of silver nitrate on the tissue culture of immature wheat embryos

The immature embryos of four common wheat (Triticum aestivum L.) genotypes with desirable agronomic traits were evaluated for their tissue culture response to ethylene antagonist, silver nitrate, added to callus-inductive and subculture media at six concentrations. The addition of AgNO₃ significantly improved embryogenic callus frequency and callus growth, but reduced the necroses and almost did not affect callus induction frequencies. Strong genotypic effects on callus induction, embryogenic callus formation, and necrosis frequency were observed. It was also found that there were significant interactions between genotype and AgNO₃ concentrations affecting embryogenic callus frequency and callus growth index. In general, 10 mg/l concentration may be considered most favorable for embryogenesis and prevention of necroses; at the same time, it did not reduce callus induction and promoted callus growth. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 4, pp. 592–596. The text was submitted by the authors in English.     

Elicitation of anthocyanin production in callus cultures of <Emphasis Type="Italic">Daucus carota</Emphasis> and the involvement of methyl jasmonate and salicylic acid

The effect of methyl jasmonate (MeJA) and salicylic acid (SA) on the anthocyanin accumulation, endogenous titres of polyamines and ethylene production in callus cultures of Daucus carota were studied. The interaction of these signaling molecules with elicitors from Aspergillus niger was investigated and the involvement of MeJA was elucidated through the use of the jasmonic acid (JA) biosynthetic inhibitor ibuprofen. MeJA and SA were both found to stimulate the anthocyanin production in the callus cultures. The highest levels of anthocyanin was observed in the cultures treated with 200 μM SA 0.36 % and 0.01 μM MeJA 0.37 %. The MeJA and SA treatments were also found to result in higher activity of Ca²⁺ ATPase suggesting that the enhancement of anthocyanin by SA and MeJA could be mediated through the involvement of the calcium channel. The treatment of the callus cultures with SA was found to result in marginally higher titres of endogenous polyamines (PAs) whereas MeJA resulted in lower levels of PAs as compared to the control. The SA treatment was found to result in lower ethylene production and the treatment with MeJA stimulated the ethylene production. These results suggest that the stimulation of anthocyanin production by MeJA and SA in callus cultures of D. carota is not related to the ethylene production.