Comparative Effects of Abscisic Acid and Methyl Jasmonate in Greening Cucumber Cotyledons and Its Application to a Bioassay for Abscisic Acid

In the cucumber cotyledon greening system, abscisic acid (ABA)is more potent inhibitor of growth and chlorophyll productionand/or destruction than methyl jasmonate (MJ). The inhibitoryeffect of ABA is apparent within 5 h of exposure to light whereasMJ is ineffective at all concentrations tested (10^–6 to10^–3 M). With longer exposure of 24 h to light and inthe presence of 40 mM KC1, the inhibition of growth and chlorophyllproduction by ABA is more pronounced whereas MJ does not inhibitgrowth and inhibits chlorophyll levels only at the higher concentrations.Both benzyladenine and KC1 stimulate chlorophyll productionand increase the fresh weights of the cucumber cotyledons andeither one of these compounds reverse the inhibitory effectsof ABA. Inhibition of chlorophyll production by ABA is valuableas a simple and rapid bioassay for abscisic acid. Under similarconditions cytokinins increase chlorophyll production and hencethe cucumber cotyledon greening system is ideal for detectingboth ABA which inhibits and cytokinins which stimulate chlorophyllproduction. (Received December 6, 1982; Accepted June 9, 1983)     

In vitro plantlet regeneration from cotyledons of the tree-legume Leucaena leucocephala

Two plant regeneration methods applicable to Leucaenaleucocephala were developed. In the first method, involvingorganogenesis via callus formation, cotyledon, hypocotyl and root segments wereinitiated on MS medium containing different concentrations ofN⁶-benzyladenine (BA), 2,4-dichlorophenoxyacetic acid (2,4-D), andnaphthaleneacetic acid (NAA). Both compact (type I) and friable (type II) calliwere obtained from the cotyledon and hypocotyl explants treated with differentconcentrations of the growth regulators. Shoots were generated only from thefriable calli formed from the cotyledon explants. The calli formed from thehypocotyl explants did not generate shoots and the root explants died withoutforming callus. Cotyledon explants from 3–4 day old seedlings showedmaximum callus induction compared to those from older seedlings. In a secondmethod involving direct organogenesis, excised cotyledons were cultured on 1/2MS medium containing 10–35 mg l^–1N⁶-benzyladenine (BA) for 7–14 days. Transfer of thecotyledonsto regeneration medium containing low BA resulted in callus formation andsubsequent shoot regeneration from the base of the excised cotyledon explants,with up to 100% frequency. Regenerated shoots rooted best on a basal mediumcontaining no growth regulators.     

Introduction of Resistance to Herbicide Basta® in Savoy Cabbage

Resistance to herbicide Basta® was introduced into pure inbred lines of Savoy cabbage (Brassica oleracea L. var. sabauda) by cocultivation of cotyledon and hypocotyl explants with Agrobacterium tumefaciens strains AGL1/pDM805 and LBA4404/pGKB5 (LB5-1). Shoot regeneration occurred on Murashige and Skoog medium supplemented with 1 mg dm^–3 6-benzyladenine and 0.5 mg dm^–3 indole-3-butyric acid at 42.3 % and 71.4 % of hypocotyl explants treated with AGL1/pDM805 and LB5-1, respectively. Putative transformants that survived selection on 10 mg dm^–3 phosphinothricin (L-PPT) supplemented medium were confirmed by GUS assay and PCR analysis. The transformation rate was 58 % with AGL1/ pDM805 and 25 % with LB5-1. Rooted plantlets were acclimated and then again screened for Basta®-resistance by spraying with 15 – 60 mg dm^–3 L-PPT. Surviving plants were selfed and Basta®-resistance was demonstrated in T₁ progeny.     

Organic nitrogen stimulates caulogenesis from hypocotyl callus of Phyllanthus fraternus

Factors involved in promoting caulogenesis from hypocotyl explants of Phyllanthus fraternus were studied. Hypocotyl explants were cultured on B₅ medium supplemented with 2,4-D or NAA in the presence and absence of BAP (at concentrations 0, 10^–7, 10^–6 and 10^–5M). Adventitious shoots differentiated from callus developed from the cut ends of 12.5% of the hypocotyl segments cultured on medium supplemented with 10^–6M BAP in combination with 10^–6M 2,4-D or 10^–6M NAA. Profuse rooting occurred from the hypocotyl explants on medium supplemented with 10^–6M BAP + 10^–6M NAA. Incorporation of casein hydrolysate in B₅ medium along with 10^–6M BAP + 10^–7M 2,4-D enhanced the frequency of cultures with adventitious shoots upto 68.0%. Glutamine, glutamic acid or proline could partially substitute for the effect of casein hydrolysate. Amongst the hypocotyls from 3–14 d old seedlings, the best caulogenesis was obtained with hypocotyls from 7 d old seedlings both in presence or absence of casein hydrolysate. Best rooting of shoots was achieved on half-strength B₅ medium supplemented with 10^–6M IBA. After hardening, plantlets were successfully transferred to the soil.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2, 4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - NAA 1-naphthaleneacetic acid - CH casein hydrolysate - Arg L-arginine - Glu L-glutamic acid - Gln L-glutamine - Leu L-leucine - Lys L-lysine - Pro L-proline     

Synthesis and Biological Activity of N-Acyl O-Indolylalkyl Ethanolamines

The plant-growth regulators, indole-3-carboxylic acids, were introduced into N-acyl ethanolamines, and a series of N-acyl O-indolylalkyl ethanolamines were prepared. Their biological activities to regulate rape hypocotyl elongation, cucumber cotyledon expansion and common wheat coleoptile growth were tested. The results indicate that the title compounds inhibited rape hypocotyl elongation, especially the indole-3-propionic acid derivatives, whose bioactivity was better than that of indole-3-acetic acid.     

Abscisic acid: a role in shoot enhancement from loblolly pine (Pinus taeda L.) cotyledon explants

Enhancement of shoot regeneration from loblolly pine (Pinus taeda L.) cotyledon explants was studied by addition of abscisic acid (ABA) to Gresshoff-Doy (GD) shoot induction medium containing benzylaminopurine (BA) and naphthaleneacetic acid (NAA). Addition of ABA (10^–7 M) doubled the morphogenic area of cotyledons and increased the fresh weight of cotyledon explants by 40 to 45% after 4 weeks. A 4-week exposure to ABA resulted in a larger morphogenic area per cotyledon than 3, 2, or 1 week(s) respectively. The enhancement by ABA was related to the explant seed source and was not increased by prolonged exposure. Compared to controls, shoot number was enhanced by 31% and 56% with 2 and 4 weeks of ABA (10^–7 M) exposure, respectively. Abscisic acid has a role in enhancing shoot morphogenesis in loblolly pine.Abbreviations ABA Abscisic acid - BA 6-Benzylaminopurine - GD Gresshoff and Doy (1972) nutrient medium - NAA -Naphthaleneacetic acid.     

Regulation of cabbage (Brassica oleracea capitata) hypocotyl elongation, and infection by Peronospora parasitica, with B-indolylacetonitrile and chloramphenicol

Untreated excised segments of the hypocotyls of dark grown cabbage seedlings are always systemically infected when inoculated with the conidia of the obligate parasite Peronospora parasitica (Downy mildew). Cabbage hypocotyl elongation is promoted by 10^–4 M indolylacetonitrile (IAN) and this elongation is inhibited by 100 g mL^–1 chloramphenicol (CAM). The fungus remains localized in 5–8 day old hypocotyl segments exposed to CAM, but this inhibition is reversed by IAN. Indol-3-acetic acid (IAA) has the same effect as IAN. Both gibberellic acid and kinetin inhibit systemic infection. Conidial spore germination is not reduced by the CAM concentration used in these experiments. The success of the pathogen in the host is not correlated with host elongation, but is probably related to a common metabolic site in either host or pathogen affected by both CAM and IAN.Abbreviations IAN indolylacetonitrile - CAM chloramphenicol - IAA indol-3-acetic acid - G gibberellic acid - K kinetin     

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.     

Inhibition of abscisic acid biosynthesis inCercospora rosicola by triarimol

The fungicide triarimol was tested for its effect on abscisic acid (ABA) accumulation in growing culturesof Cercospora rosicola. ABA accumulation was reduced by approximately 50% with 10^–8 M triarimol. Growth ofC. rosicola, as measured by dry weight accumulation, was inhibited by triarimol concentrations at or greater than 10^–7 M. These results are compared with those obtained with clomazone, ancymidol, and paclobutrazol, which inhibit ABA accumulation by 50% at concentrations of 5 × 10^–5, 5 × 10^–6, and 5 × 10^–7 M, respectively. Triarimol, therefore, is among the most potent inhibitors of ABA biosynthesis reported to date. Feeding studies with [¹⁴C]mevalonic acid confirmed the inhibition of ABA biosynthesis by 5 × 10^–8 M triarimol. These results support previous suggestions that one or more of the steps in the ABA biosynthetic pathway from mevalonic acid is catalyzed by cytochrome P-450. Feeding studies with 1-deoxy-[²H]-ABA in resuspended cultures ofC. rosicola show that the conversion of this substrate is not inhibited by triarimol.     

Rapid in vitro differentiation of Sesbania bispinosa plants — a leguminous shrub

Multiple shoots differentiated from hypocotyl explants of Sesbania bispinosa (Jacq.) W.F. Wight, a leguminous woody shrub, when cultured on Gamborg's basal medium alone or in combination with 6-benzyl aminopurine (10^–7–10^–4 M). For cotyledonary explants 6-benzyl aminopurine (10^–6–10^–4 M) was necessary. The shoots rooted when cultured on Gamborg's basal medium containing indole-3-butyric acid (10^-5 M). Plantlets thus formed were transferred to soil where they have flowered and also set fruits.     

Water deficit-induced changes in abscisic Acid, growth, polysomes, and translatable RNA in soybean hypocotyls

Soybean seedlings (Glycine max L.) were germinated and dark-grown in water-saturated vermiculite (water potential = −0.01 megapascal) for 48 hours, then transferred either to water-saturated vermiculite or to low water potential vermiculite (water potential = −0.30 megapascal). A decrease in growth rate was detectable within 0.8 hour post-transfer to low water potential vermiculite. A fourfold increase in the abscisic acid content of the elongating region was observed within 0.5 hour. At 24 hours post-transfer, hypocotyl elongation was severely arrested and abscisic acid reached its highest measured level: 3.7 nanograms per milligram dry weight (74-fold increase). A comparison of the polyA⁺ RNA populations isolated at 24 hours post-transfer from the elongating region of water-saturated and low water potential vermiculite-grown seedlings was made by two-dimensional (isoelectric focusing-sodium dodecyl sulfate) polyacrylamide gel analysis of in vitro translation products. It revealed both increases and decreases in the relative amounts of a number of translation products. Rewatering seedlings grown in low water potential vermiculite at 24 hours post-transfer led to a total recovery in growth rate within 0.5 hour, while abscisic acid in the elongating hypocotyl region required 1 to 2 hours to return to uninduced levels. Application of 1.0 millimolar (±) abscisic acid to well-watered seedlings resulted in a 48% reduction in hypocotyl growth rate during the first 2 hours after treatment. Plants treated with abscisic acid for 24 hours had a lower polysome content than control plants. However, hypocotyl growth inhibition in abscisic acid-treated seedlings preceded the decline in polysome content.     

Occurrence and bioassay responses of G

A growth regulator (G) occurs at high concentrations in adult leaves of E. grandis Maiden. Low concentrations of G are present in juvenile leaves of this species and also in adult leaves of some other Myrtaceae. Low concentrations of G (5×10^–6 and 10^–5 M) promote rooting in mung-bean cuttings and elongation in Avena coleoptile sections; high concentrations (5×10^–4 M) inhibit. These and other bioassay results indicate that G may have auxin-like activity.     

Effects of gibberellic acid and zeatin on the growth response of cucumber cotyledons

In contrast to cytokinin, gibberellic acid has no effect on the growth of the isolated cucumber cotyledon in darkness. Like cytokinins in light, gibberellic acid causes increases in fresh weight and area of the cotyledon at concentrations from 10^–7 to 10^–3 M. Radiant energies in the blue, red, and far-red regions of the spectrum all induce the growth responses to gibberellic acid. The effect of the far red is greater than that of the red, which is greater than that of the blue. Gibberellic acid is ineffective in the promotion of chlorophyll development, whereas cytokinins are very effective. Although zeatin and gibberellic acid both cause an increase in fresh weight and area of the cotyledons in light, they appear to have entirely separate actions in the growth responses.     

Effects of hygromycin on cotton cultures and its application in <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated cotton transformation

We have evaluated the effects of the antibiotic hygromycin B on cotton (Gossypium hirsutum L.) callus induction, callus proliferation, and seed germination. Nontransgenic cotyledon and hypocotyl showed obvious variance in tolerance to hygromycin. Cotyledons were more sensitive to hygromycin than hypocotyls. Hygromycin at 7.5 and 20 mg l⁻¹ completely inhibited callus initiation from cotyledon and hypocotyl explants, respectively. Nontransformed calli did not grow on media supplemented with 10 mg l⁻¹ hygromycin and were killed at 15 mg l⁻¹. In seed germination assay, the presence of 20 mg l⁻¹ hygromycin significantly suppressed shoot and root elongation of seedlings. This hygromycin concentration was applied to select regenerated transgenic plantlets and their progenies. Based on these results, we developed an efficient hygromycin selection protocol for Agrobacterium-mediated cotton transformation and regeneration.     

Influence of silver nitrate (ethylene inhibitor) on cucumber in vitro shoot regeneration

The effect of addition of silver nitrate (AgNO₃) on organogenesis of proximal and distal cotyledon and hypocotyl explants of five cucumber (Cucumis sativus L.) cultivars was investigated. Distal cotyledon and hypocotyl were unresponsive while only poor shoot regeneration was observed in proximal cotyledon and hypocotyl explants of all cucumber cultivars. The addition of different concentrations of AgNO₃ (10, 30 and 50 μM) to the medium, however, induced shoot regeneration in distal cotyledon except Suyo Long cultivar and effectively increased shoot regeneration response as well as the number of shoots per explant in proximal cotyledon and hypocotyl of all cucumber cultivars. This revised version was published online in June 2006 with corrections to the Cover Date.     

Inducible effects of abscisic acid on sporophyte discs from Laminaria japonica Areschoug (Laminariales, Phaeophyceae)

The effects of exogenous abscisic acid (ABA;10^–7–10^–5 M), a knownplantgrowth regulator, on reproduction and growth were investigated by culturingdiscs from sporophytes of Laminaria japonica Areschoug.ABAplays a role in triggering sorus formation, and it was found that sorusformation of discs was fastest in 10^–5 M ABA. Theapplication of 10^–5 M ABA to culturing discs alsosuppressed the expansion of surface area. ABA contents in sorus and vegetativeparts of the sporophyte were determined by bioassay. The mean ABA content insorus parts obtained from sporophytes was 0.222 ± 0.053g equivalent-ABA g wet weight^–1, which wasabout five times higher than the content found in vegetative parts (0.048± 0.009 g equivalent-ABA g wetweight^–1). Taken together these results suggest that sorusdevelopment requires an elevated level of ABA and is associated with decreasingvegetative growth and that the ABA level of the sporophyte may play a crucialrole in reproduction.     

Effects of fusicoccin and abscisic acid on glucose uptake into isolated beetroot protoplasts

Uptake of glucose, 3-O-methylglucose and sucrose into beetroot protoplasts is considerably stimulated by 10^–6M fusicoccin. This effect is decreased in the presence of 10mM Na⁺ or K⁺, 2 mM Mg²⁺ or Ca²⁺. Whereas fusicoccin causes no change in the pH-optimum of the sugar uptake (pH 5.0), the apparent K_m of this uptake which obeys a biphasic kinetics is decreased by the action of fusicoccin. In the protoplast suspension, fusicoccin induces an acidification which is suppressed by uncoupling agents. Correspondingly, uncouplers as well as vanadate and diethylstilbestrol markedly inhibit the effect of fusicoccin on sugar uptake. The present data support the view that glucose uptake into beetroot protoplasts depend on the proton-pumping activity of the plasmalemma-ATPase. cis–Abscisic acid diminishes significantly the fusicoccin-enhanced glucose uptake. By using a radioimmunoassay, the internal abscisic acid content of the protoplast was estimated to be in the range of 10^–6 M. Protoplasts isolated from bundle tissue contain twice as much abscisic acid as those derived from storage parenchyma. Because protoplasts from the bundle tissue were shown to take up sugars much faster than those from the storage cells, the observed effect of abscisic acid might reflect an involvement of this hormone in the regulation of carbohydrate partitioning in the beet.Abbreviations ABA cis–abscisic acid - bundle protoplast protoplasts isolated from the conducting tissue of beetroots - DES diethylstilbestrol - FC fusicoccin - 3-OMG 3-O-methylglucopyranose - PCMBS p–chloromercuribenzenesulfonic acid - storage protoplasts protoplasts isolated from storage parenchyma     

The Effect of Photosynthetic Enhancement on Photorespiration in Sinapis alba

High external concentrations of potassium were found to promote light-induced growth of cucumber cotyledons similarly to the effects previously observed for the growth induced by cytokinins. At 40 mM KCl, the response to white light was 3.6 times greater than in the absence of KCl. The promotive effect of calcium on the growth induced by cytokinin was not observed for light. In 40 mM KCl and 10 mM CaCl₂, the responses to light and cytokinin were similar and additive. Both near-red and far-red light induced growth at low intensities. The response to white light at low intensities was sharply increased with higher intensity up to 24 μE m^-2 s^-1 and only slightly increased above that level. Abscisic acid was found to inhibit strongly the responses to light and cytokinin. The inhibition was greater in the presence of KCl than in its absence and thus abscisic acid appeared to inhibit primarily by its interference with potassium uptake. Kinetic analysis found the light response promoted by potassium to be inhibited competitively by abscisic acid, with the light response having a K_m of 34 mM KCl and a V_max of 115 mg/cotyledon × 4 days. The inhibition of the cytokinin response by abscisic acid was noncompetitive in relation to potassium, having a K_m of 1 mM KCl and a V_max of 50 mg/cotyledon × 4 days. It is suggested that cytokinin, light and abscisic acid have primary properties affecting membrane permeability and that their interaction with potassium is an explanation of many similarities between light and cytokinin responses and their inhibition by abscisic acid.     

In vitro high frequency plant regeneration of a tree legume Tamarindus indica (L.)

Optimal culture conditions for high frequency plant regeneration from excised cotyledons of Tamarindus indica were established. Maximum shoot bud differentiation (100%) occurred when the adaxial surface of the entire cotyledon (excised from 12-d old seedlings) was in contact with MS medium containing 5×10^–6M BAP. On MS alone only roots were formed. Shoot or root formation was confined to nodal tissue at the top of the notch present on the adaxial surface at the proximal end of the cotyledon. Thirty-four to 95 shoots were regenerated in a 4 month period from individual cotyledons. Shoots were rooted on MS with 5.7×10^–6M IAA. IAA (5.7×10^–7M) alone induced complete plant formation. Regenerated plants were established in the soil with 70% success.Abbreviations BAP 6-benzylaminopurine - KIN kine-tin - 2-iP 6-Y-Y-dimethylallyl aminopurine - AdS adenine sulphate - IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA 1-naphthalene acetic acid - MS Murashige and Skoog (1962) medium     

Adventitious Shoot Regeneration and Micropropagation in Calendula officinalis L.

Hypocotyl, cotyledon and cotyledonary node explants of Calendula officinalis L were cultured on Murashige and Skoog (MS) media supplemented with various concentrations of thidiazuron (TDZ), kinetin (KIN), -naphthaleneacetic acid (NAA) and indole-3-butyric acid (IBA) to induce adventitious shoot regeneration and micropropagation. The highest frequency of adventitious shoot regeneration was achieved from hypocotyl and cotyledon explants on MS media supplemented with 0.75 mg dm^–3 TDZ and either 0.25 or 0.50 mg dm^–3 IBA. Efficient in vitro clonal propagation was also induced from cotyledonary nodes on a range of media supplemented with 0.75 mg dm^–3 TDZ and 0.05 mg dm^–3 NAA or 2 mg dm^–3 KIN and 1 mg dm^–3 NAA. Regenerated shoots were excised and rooted in MS medium supplemented with 1 mg dm^–3 NAA. The rooted plantlets were finally transferred to pots.