The effect of the type of closure on the gas composition of the headspace and the growth of GF 677 peach × almond rootstock cell suspension cultures

Summary The influence of culture flask closures, i.e., cotton plugs and rubber and aluminum-foil caps, on headspace gas composition and growth of leaf petiole callus-derived GF 677 cell suspensions was comparatively tested. Oxygen concentration always remained comparable to that of the lab atmosphere and CO₂ and C₂H₄ levels were slightly higher when flasks were closed with cotton plugs. By contrast, the gas environment markedly changed within 72 to 96 h in culture inside rubber and aluminum-capped flasks. Under rubber caps, CO₂ increased up to 18%, with a net production of about 0.8 mmol CO₂, oxygen decreased to 6% within 72 h, and ethylene accumulated up to 9 μl liter⁻¹ after 96 h. When aluminum foil closures were used, C₂H₄ and CO₂ increased over the first 72 h, reaching concentrations of about 6 μl liter⁻¹ and 7% (0.3 mmol produced), respectively, and decreasing after 192 h to 0.1 μl⁻¹ and 2%, respectively. The gaseous environment markedly affected cell growth. The exponential growth period of suspensions cultured in flasks closed with cotton plugs and aluminum foil caps started after about 72 h and the stationary phase after 240 h, the cell dry weight reaching its maximum at about threefold the initial weight. Large cell aggregates were found in flasks closed with cotton plugs, slight aggregation with aluminum closures, and no aggregates with rubber caps. However, hardly any growth, progressive browning of the suspensions, and the death of most cells occurred in rubber-capped flasks. A type of closure allowing low gas exchange rates, like aluminum caps, and frequent subcultures thus seems most conducive to rapid growing, more homogeneous GF 677 cell suspension cultures, and the prevention of aggregates, if undesired.     

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.     

Does ethylene play a role in thidiazuron-regulated somatic embryogenesis of geranium (Pelargonium × hortorum bailey) hypocotyl cultures?

Summary The accumulation of ethylene in headspace of hypocotyl cultures of geranium (Pelargonium × hortorum Bailey) and its possible role in thidiazuron-mediated somatic embryogenesis was investigated. The action of ethylene as determined by various ethylene synthesis and action inhibitors was varied. Silver nitrate (AgNo₃), aminoethoxyvinylglycine (AVG), and silver thiosulphate (STS) had no significant influence on the embryogenic response, while 1-methylcyclopropene (1-MCP) applied during the initial 3 d of induction or the expression phase, significantly increased the number of somatic embryos formed. Thidiazuron-treated tissues accumulated large quantities of ethylene within 6 h of culture, but the levels decreased after 12 h and reached very low levels after 3 d in culture. In the presence of acetylsalicylic acid (ASA), the levels of ethylene decreased by 20 to 50% during the first 48 h of culture. Analysis of endogenous auxin, cytokinins, and abscisic acid (ABA) indicated possible interactions of ethylene with other phytohormones during the induction of somatic embryos on geranium hypocotyl explants. Thidiazuron (10 μM) increased, while ASA decreased the levels of endogenous auxin, cytokinins, and abscisic acid during this period of induction.     

Micropropagation of American chestnut: Increasing rooting rate and preventing shoot-tip necrosis

Summary A three-step medium sequence was developed for rooting microcuttings of American chestnut [Castanea dentata (Marsh.) Borkh.]. First, individual shoots or clumps of shoots were cultivated on shoot-elongation medium for 4–8 wk until shoots were 2–3 cm long. The medium consisted of modified Woody Plant Medium, 500 mg/l polyvinylpyrrolidone (MW 40,000), and 0.89 μM benzyladenine. Microcuttings were then excised, vertically split at the base to approximately 2 mm through the pith, dipped in 5 or 10 mM indolebutyric acid for 1 min, and cultivated on half-strength Murashige and Skoog basal medium plus 0.2 g/l charcoal for 2 wk. During that time, roots were induced and became visible. Finally, the microcuttings were transferred back to shoot-elongation medium and cultivated for 3 wk, allowing growth of both roots and shoots. Using this protocol with 3 genotypes derived from one mature tree and two 1-yr-old seedlings, 57 to 73% rooting was obtained with less than 23% shoot-tip necrosis.     

An improved procedure for the propagation in vitro of grapevine (t Vitis vinifera cv. Pinot noir) using axillary-bud microcuttings

A method has been developed for rapid propagation in vitro of Vitis vinifera cv. 'Pinot noir' from axillary-bud microcuttings harvested from plantlets initially cultured in vitro. The requirement of plant growth regulators for the different stages of the micropropagation was examined. BA at 8.9 μM added to MS basal medium was found to be optimal for culture establishment, while for subcultures, the concentration of BA was reduced to 4.4 μM to prevent hyperhydricity. Among the two auxins (NAA and IBA) tested for rooting, IBA at 2.5 μM was found to induce a good rooting- system in 100% of the shoots. The advantages of this method, using microcuttings from established plants in vitro, is discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Role of protein kinase C in growth stimulation of primary mouse colonic epithelial cells

Summary The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (160 nM) and the secondary bile acid, deoxycholic acid (50 μM) stimulated DNA synthesis in quiescent primary epithelial cells from the normal mouse colon as measured by autoradiographic analysis of [³H]thymidine incorporation. The purpose of the present study was to investigate the involvement of protein kinase C in the proliferative response of the normal colonic cells. The protein kinase C inhibitor, bisindolylmaleimide GF 109203X, efficiently blocked the proliferative response of the cells to the phorbol ester and caused a dose-dependent decrease in the response to deoxycholic acid. While the phorbol ester-induced proliferation was unaffected by another inhibitor, H-7, the response of the cells to deoxycholic acid was blocked. Pretreatment of the cells with the phorbol ester (160 nM) for 24 h blocked the proliferative response to deoxycholic acid. Measurement of the intracellular distribution of protein kinase C activity showed a time-dependent and significant translocation of the enzyme activity from the soluble to the particulate cell fractions after exposure to 12-O-tetradecanoylphorbol-13-acetate. While exposure to the bile acid indicated a similar time-dependent translocation of the enzyme activity, the effect was not significant. The phorbol ester induced a time-dependent accumulation of c-fos mRNA and protein as measured by solution hybridization and immunocytochemistry, respectively. No effect of deoxycholic acid on c-fos expression could be observed in the present study. The data support a role for protein kinase C in the growth stimulating effect of physiological concentrations of deoxycholic acid on normal colonic epithelial cells. However, differences in the mechanisms underlying phorbol ester- and bile acid-induced proliferation are indicated.     

Effects of exogenous methyl jasmonate in elicited anthocyanin-producing cell cultures of ohelo (Vaccinium phalae)

Summary Elicitation of anthocyanin-producing cells of ohelo (Vaccinium pahalae) by both biotic (purified β-glucan and chitosan) and abiotic [sodium ferric ethylenediamine di-(o-hydroxyphenylacetate) FeEDDHA, and CuSO₄] elicitors resulted in significant enhancement of anthocyanin accumulation. Anthocyanin production increased up to 1.8 and 1.5-fold over the control in the presence of abiotic elicitors (90 μM FeEDDHA and 20 μM CuSO₄, respectively), and increased 1.9 and 1.6-fold in the presence of biotic elicitors (10 mg L⁻¹ β-glucan and 100 mg L⁻¹ chitosan). Maximum anthocyanin production with the two most effective elicitors was achieved when cultures were treated on Day 3 (β-glucan) or Day 0 (FeEDDHA) after the initiation of fresh cell cultures. A concentration-dependent response was exhibited by cultures treated with exogenous methyl jasmonate (MJ). The addition of 0.5 μM MJ alone provoked a 2–3-fold increase in anthocyanin production over that of the control; however, no additive effect on anthocyanin production was observed in any treatments which combined MJ and β-glucan or FeEDDHA. Conditioning of the cells with a preculture in either MJ, β-glucan, or FeEDDHA similarly did not enhance anthocyanin production. Inoculation of cultures elicited by MJ or β-glucan with ibuprofen, a reported inhibitor of jasmonate biosynthesis, dramatically stimulated, rather than inhibited, anthocyanin production, resulting in levels of accumulation beyond any of the tested elicitor combinations. Hypotheses for the observed influence of ibuprofen in this system are discussed.     

Ethylene inhibitors promote in vitro regeneration of cowpea (Vigna unguiculata L.)

Summary Ethylene is a plant growth regulator that is known to influence in vitro morphogenesis. This study investigated the effects of three ethylene inhibitors, silver nitrate (AgNO₃), 2,5-norbornadiene, and cobalt chloride (CoCl₂), on the regeneration of cowpea from cotyledon explants. Significant increases in the percentage of regeneration occurred as a result of adding either 50 μM AgNO₃ or 100 μM 2,5-norbornadiene. The number of shoots produced per explant was enhanced by adding 25 μM CoCl₂ or 100 μM norbornadiene. Maximum shoot elongation was obtained with 25 μM of either CoCl₂ or norbornadiene. The effect of the duration of exposure to AgNO₃ was also determined. The greatest percent regeneration was obtained with the addition of 60 μM AgNO₃ either to both the initiation and regeneration stages, or to only the regeneration stage. The promotive effects on organogenesis in response to ethylene inhibitors suggests an important role for ethylene in the process of in vitro morphogenesis of cowpea and may contribute to its normally low regeneration frequency.     

Adventitious root induction inCorylus avellana L. cotyledon slices

Summary An experimental rooting system was developed to study in vitro adventitious root formation in hazelnut cotyledons. Experiments involveda) assay of several culture media,b) use of different developmental status of the seeds (germinated and ungerminated),c) cotyledons subjected to various light regimes, andd) different size of cotyledons slices. It was observed that higher rooting was induced in cotyledonary portions of 5- or 7-mm thickness (250 and 350 mm³, respectively) cultured on half-strength basal medium supplemented with 50 μM indole-3-butyric acid and 5 μM kinetin. Rooting was affected by light and the developmental state of seeds. Preinitiation, initiation, and root manifestation stages were defined according to specific culture periods and in relation with morphologic and histologic changes. The first histologic changes (cell division and root primordia induction) were observed after 12 days in culture. At 30 days of culture in rhizogenic medium root primodia were fully differentiated with well-developed vascular tissues.     

Cadmium elevates level of protein, amino acids and alters activity of proteolytic enzymes in germinating rice seeds

When seeds of two rice cvs. Ratna and Jaya were germinated under increasing levels of cadmium nitrate (0, 100 and 500 μM) in the medium, a marked decrease in germination percentage was observed with Cd treatments, as compared to controls. There was more absorbed Cd in embryo axes than in endosperms. More uptake resulted with increasing Cd levels in the growth medium in embryo axes. In both rice cultivars, during a germination period of 0 – 120 h, an increased level of protein as well as free amino acids was noted in Cd treatments. Protease activity in general decreased in both embryo axes as well as endosperms due to Cd treatment. In vitro studies showed an enhancement in protease activity in Cd treatments at low Cd levels (50–100 μM), whereas concentrations above this caused inhibition in enzyme activity. Under 500 μM Cd treatments in vivo there was about 30 to 50 percent decline in leucine aminopeptidase (LAP) activity in endosperms, however, carboxypeptidase activity showed a marked increase in endosperms beyond 24 h under Cd treatments. In embryo axes of germinating seeds there was always a decline in peptidase activities, under the influence of cadmium. The leucine amino peptidase and protease activity were always greater in embryo axes in cv. Ratna than cv. Jaya. However, the carboxypeptidase activity was higher in Jaya when compared to Ratna in endosperms under Cd treatments. The results suggest possible suppression of protease and peptidase activities due to Cd treatments in germinating rice seeds leading to altered levels of protein and amino acids.     

Short-term in vitro storage of t Miscanthus x ogiformis Honda 'Giganteus' as affected by medium composition,temperature, and photon flux density

Miscanthus x ogiformis Honda 'Giganteus' shoot cultures were stored in vitro on proliferation or rooting medium for up to 27 weeks at temperatures of 8, 12, 16, or 20 °C and photosynthetic photon flux densities of 5, 10, or 20 μmol m⁻² s⁻¹. Plants survived storage much better on rooting medium than on proliferation medium. Plants stored on rooting medium for 1 week survived well when survival was assessed immediately after storage or after 14 days of acclimatization, but had the lowest survival 28 days after transplantation. With increasing storage period on rooting medium increasing survival was found 28 days after transplantation. This was probably a result of the development of rhizomes and/or roots during storage. Best survival was observed at 20 μmol m⁻² s⁻¹ and a temperature of 8-16 °C. Increasing the temperature to 25 °C during the last week of storage improved survival considerably. Root formation was slow at 8 °C, but after 27 weeks of storage the rooting percentage was the same at all storage temperatures. An increasing number of shoots per plant 28 days after transplantation was found with increasing PPFD during storage.Miscanthus shoot cultures can be stored in vitro for at least 27 weeks with limited losses when stored on rooting medium at 20 μmol m⁻² s⁻¹, a temperature of 16 °C, and given a 1-week end-of-storage treatment of 25 °C. This revised version was published online in June 2006 with corrections to the Cover Date.     

Expression of an antisense 1-aminocyclopropane-1-carboxylate oxidase gene stimulates shoot regeneration in Cucumis melo

The role of ethylene in shoot regeneration was investigated using transgenic Cucumis melo plants expressing an antisense 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene. ACC oxidase catalyses the last step of ethylene biosynthesis. Leaf and cotyledon explants from the transgenic plants exhibited low ACC oxidase activity and ethylene production, whereas the regeneration capacity of the tissues was greatly enhanced (3.5- and 2.8-fold, respectively) compared to untransformed control tissues. Addition of ethylene released by 50 or 100 μm 2-chloroethylphosphonic acid dramatically reduced the shoot regeneration rate of the transgenic tissues. The results clearly demonstrate that ethylene plays an important role in C. melo morphogenesis in vitro. Received: 23 April 1997 / Revision received: 9 June 1997 / Accepted: 2 July 1997     

Structural Changes and Aluminium Distribution in Maize Root Tissues

Growth and structural responses of primary roots of Zea mays L. to aluminium chloride were studied. The treatment of seedlings with 50 μM AlCl₃ resulted in high accumulation of Al, partial inhibition of root growth, occurrence of surface lesions in peripheral tissues, root thickening caused by expansion of inner cortical cells, reduced root cap length, extensive vacuolation, cell distortion, and increased synthesis of callose within 24 h. This revised version was published online in July 2006 with corrections to the Cover Date.     

In vitro regeneration and propagation of chickpea (Cicer arietinum L.) from meristem tips and cotyledonary nodes

Summary Two representative cultivars ofCicer arietinum, the desi-type cv.Annigeri and the kabuli-type cv.ICCV6, were regenerated in vitro and clonally propagated from cotyledonary nodes and meristem tips. The explants were dissected from 1-wk-old seedlings aseptically germinated on WH medium. In both cultivars, all nodes cultured on B5 medium supplemented with 4.4μM 6-benzylaminopurine developed up to seven shoots per node within 3 wk. Meristem tips were much better suited for multiple shoot formation. Cultured on DKW-C-a medium supplemented with 4.4μM 6-benzylaminopurine and 0.05μM indole-3-butyric acid, 96% of the meristem tips produced up to 10 shoots per explant. A new method in improving clonal propagation was subdividing the meristem tips. Doing so, multiple shoot formation was considerably enhanced: up to 90 shoots per original explant could be obtained with cv.Annigeri, and up to 50 with cv.ICCV6. Indole-3-butyric acid proved to be the best rooting factor. From several media tested, the best root induction and development was achieved on WH medium supplemented with 2.5μ M indole-3-butyric acid: 72% rooting with cv.Annigeri and 68% rooting with cv.ICCV6. With both cultivars there were no differences in rooting capacity between shoots of nodal origin and those derived from meristem tips. The plantlets obtained were transferred into soil and kept under greenhouse conditions. The survival frequency was 28% with cv.Annigeri and 23% with cv.ICCV6. R₀ plants remained smaller than seed-grown controls and produced only a few fertile seeds. There was no difference between R₁ plants and controls in growth, development, and seed set.     

Effect of ethylene and culture environment on development of papaya nodal cultures

Papaya (Carica papaya L.) nodal cultures modified the atmosphere of the headspace of the vessel used for culture maintenance by producing ethylene. Under culture maintenance nodal cultures grew poorly and leaves senesced. Incubating nodal cultures under a range of ethylene concentrations suggested that this poor performance was caused in part, by the production of ethylene and its accumulation in the headspace of the vessel. To further evaluate the role of ethylene accumulation in growth suppression, aminoethoxyvinylglycine (AVG), 1-aminocyclopropane-1-carboxylic acid (ACC) and silver thiosulphate (STS), were added to the nutrient medium and ethylene measurements performed during culture growth. The ethylene-suppressant, AVG, (1.2 μM) and the ethylene-antagonist, STS, (0.3 mM) significantly improved nodal culture growth (283 and 289% respectively), leaf area production (350 and 211% respectively) and reduced leaf senescence, while the ethylene-precursor, ACC, (1.5 mM) significantly decreased culture growth (71%), leaf area production (88%) and promoted leaf senescence. Furthermore, nodal culture growth was significantly better at 20 °C than 30 °C since ethylene production and accumulation were less in these conditions. Better control or management of ethylene accumulation produces healthier nodal cultures for micro-propagation and may be a way of improving productivity of other papaya shoot culture systems. This revised version was published online in June 2006 with corrections to the Cover Date.     

Carbendazim as an alternative plant growth regulator in tissue culture systems

Summary Carbendazim is the fungitoxic ingredient of different fungicides. In our experiments it was used as a supplement to stage II (multiplication) media for the micropropagation ofCordyline terminalis andPrunus avium. The product can be autoclaved without any loss of activity and there is no degradation of the product over a normal culture period of 32 days. WithC. terminalis andP. avium no phytotoxic effect was revealed up to 160μg/ml. ForC. terminalis shoot height was reduced and the number of shoots smaller than 15 mm increased significantly. Presented in the Session-in-Depth Novel Plant Growth Regulators at the 1992 World Congress on Cell and Tissue Culture, Washington, DC, June 20–25, 1992.     

Transformation of carnation with genes related to ethylene production and perception: towards generation of potted carnations with a longer display time

Potted carnation (Dianthus caryophyllus L. cv. Lillipot) plants were transformed with cDNAs for carnation 1-aminocyclopropane-1-carboxylate (ACC) synthase (DC-ACS1, s/aACS transgenes) or ACC oxidase (DC-ACO1, s/aACO transgenes) in sense or antisense orientation or mutated carnation ethylene receptor cDNA (DC-ERS2′) by Agrobacterium-mediated gene transfer. The presence of acetosyringone at 100 μM in media for shoot culture prior to leaf explant preparation and preculture of Agrobacterium in addition to the conventional method of addition to media for infection and coculture, and the use of water instead of nutrient media for infection and coculture increased the transformation efficiency to 4.0% compared to the 0.1% obtained by the conventional method. PCR analysis as well as Southern blot analysis confirmed the integration of the ethylene-related transgenes. Leaflet segments of cultured shoots of some lines transformed with s/aACO transgenes had less activity to convert ACC to ethylene than that of the non-transformed control plant, indicating that the integrated s/aACO transgenes reduced the expression of endogenous ACC oxidase gene (DC-ACO1) in the cultured shoots.     

Herbicidal Activity of Toxin from Fusarium avenaceum GD-2 against Wild Oats (Avena fatua L.)

The herbicidal activity potential of toxin from Fusarium avenaceum GD-2 was evaluated against wild oats (Avena fatua L.) in this study. The toxin was assayed in vitro to evaluate its inhibition against seed germination of A. fatua. The toxin of F. avenaceum GD-2 was shown to have an inhibitory effect of around 77.54% at 5 mg/mL against germination of A. fatua seeds. The inhibitory effect shown by the toxins against radicle had higher activity than plumule under the same concentration. The toxin of F. avenaceum GD-2 significantly diminished the plant length the part on the ground with various treatments when treated with the toxin under greenhouse conditions. However, there were no significantly different reductions in plant length and the weed fresh weight with different treatments. In detached leaf injection bioassay, the toxic metabolite was characterized after the culture filtrates crude extraction with petroleum ether, chloroform, ethyl acetate and n-butanol. The residues left after solvent evaporation were evaluated separately for their toxicity against the target weed. Residue (5 mg/mL) obtained from n butanol fraction showed the highest toxic activity when compared with others. Moreover, a host range experiments on the sensitivity of 10 plant species revealed that barnyard grasses and goosefeet were more sensitive to the toxin of the culture filtrate. Three herbicidal active compounds were isolated and purified from cultural filtrate with the same UV absorption peak. The recent results showed potential for the development of the toxins produced by F. avenaceum GD-2 as a bio herbicidal source to control and eliminate A. fatua weed.     

Accumulation characteristics of biomass and nitrogen and critical nitrogen concentration dilution model of cotton reproductive organ

Several nitrogen (N) field experiments were carried out in Nanjing and Anyang, China, to study the dynamic characteristics of biomass accumulation and N uptake, and to define the dilution curve for critical N concentration in cotton reproductive organ over the growth period. The results show that the total biomass and N accumulation were affected significantly by the rate of N application, exhibiting a sigmoid curve over time. The beginning time of fast N accumulation was 1–5 d earlier than that of biomass accumulation. The cotton lint yield was correlated with N concentration in the reproductive organ and fluctuated with varying N concentration, indicating the existence of luxurious N consumption in the cotton reproductive organ. The N concentration increased with increasing N application rates, and decreased gradually during the growth period. The relationship between biomass and N concentration can be described with a power equation. The patterns of the N concentration dilution model were consistent at both experimental sites, but the model parameter values of a differed. The results presented in this paper indicate that a critical N concentration dilution curve for cotton reproductive organ is independent of ecological region and can be described with a power equation.     

Impacts of small hydropower plants on macroinvertebrate communities

Several nitrogen (N) field experiments were carried out in Nanjing and Anyang, China, to study the dynamic characteristics of biomass accumulation and N uptake, and to define the dilution curve for critical N concentration in cotton reproductive organ over the growth period. The results show that the total biomass and N accumulation were affected significantly by the rate of N application, exhibiting a sigmoid curve over time. The beginning time of fast N accumulation was 1–5 d earlier than that of biomass accumulation. The cotton lint yield was correlated with N concentration in the reproductive organ and fluctuated with varying N concentration, indicating the existence of luxurious N consumption in the cotton reproductive organ. The N concentration increased with increasing N application rates, and decreased gradually during the growth period. The relationship between biomass and N concentration can be described with a power equation. The patterns of the N concentration dilution model were consistent at both experimental sites, but the model parameter values of a differed. The results presented in this paper indicate that a critical N concentration dilution curve for cotton reproductive organ is independent of ecological region and can be described with a power equation.