Ethylene inhibitors enhance in vitro root formation on faba bean shoots regenerated on medium containing thidiazuron

The possible involvement of ethylene in in vitrorooting of faba bean (Vicia faba L.) shootsregenerated on medium containing thidiazuron wasinvestigated. The effects of the ethylene precursor1-aminocyclopropane-1-carboxylic acid (ACC) and threeethylene inhibitors, silver nitrate (AgNO₃),acetyl salicylic acid (ASA) and cobalt chloride(CoCl₂) on root formation were tested in vitrousing TDZ-induced shoots of faba bean accession 760.ACC inhibited root formation. In contrast, ethyleneinhibitors promoted root formation, AgNO₃ at theappropriate concentrations enhanced root emergence andincreased root number per shoot, root growth rate, androot length. Both CoCl₂ and ASA at theappropriate concentrations increased rootingefficiency. These promotive effects may result from areduction in ethylene concentration or inhibition ofethylene action. The results offer a new approach toimprove the rooting efficiency of TDZ-induced shootsof faba bean and possibly of other plant species.     

Stimulation of shoot regeneration from cotyledons of Helianthus annuus by the ethylene inhibitors,silver and cobalt

The effects of CoCl₂, AgNO₃ and ethylene released by exogenous 2-chloroethylphosphonic acid (Ethephon), were studied on shoot regeneration from cotyledons of Helianthus annuus cv. E8206R, a poorly regenerative cultivar. Inhibition of ethylene biosynthesis by CoCl₂, at concentrations of 20 K, provoked a substantial enhancement of shoot regeneration (30 %): the control was poorly regenerative. However, CoCl₂ had no effect when Ethephon was supplied. Inhibition of ethylene action by AgNO₃, at concentrations of 10–25 M, caused a significant increase in plant regeneration: 25 % instead of 1.2 % in the control. Furthermore, addition of Ethephon to AgNO₃-treated tissues failed to reduce the stimulation of shoot regeneration caused by AgNO₃. On the basis of these findings, it is suggested that ethylene inhibits the regeneration process from cotyledons of sunflower.Abbreviations NAA 1-naphthalene acetic acid - BAP 6-benzylamino-purine - GA₃ gibberellic acid - Ethephon 2-chloroethylphosphonic acid - MS Murashige and Skoog medium - AVG aminoethoxyvinylglycine     

Ethylene requirement during germination of Botrytis cinerea spores

Application of 2,5-norbornadiene, a competitive inhibitor of ethylene, effectively inhibited the germination of Botrytis cinerea Pers. ex Fr. spores. The transfer of spores from 2,5-norbornadiene to air relieved inhibition by norbornadiene, indicating that its effects are non-toxic and reversible. Ethephon (2-chloroethylophosphonic acid), which stimulates spore germination of B. cinerea , does not affect germination in the presence of norbornadiene. However, ethephon appeared to be effective in relieving inhibition, when norbornadiene was removed from the atmosphere surrounding spores. The addition of ethylene to an atmosphere enriched with norbornadiene, counteracted the inhibition of spore germination. The inhibition of spore germination by 2,5-norbornadiene and the reversal of this effect by ethephon or ethylene, indicate that the action of ethylene is indispensable for germination of B. cinerea spores.     

Micronutrient optimization results into highly improved in vitro plant regeneration in kodo (<Emphasis Type="Italic">Paspalum scrobiculatum</Emphasis> L.) and finger (<Emphasis Type="Italic">Eleusine coracana</Emphasis> (L.) Gaertn.) millets

Basal medium constituents and their concentration play an important role in growth and morphogenesis of plant tissues cultured in vitro. In this study effect of different inorganic nutrients (CoCl₂, MnSO₄, ZnSO₄, CuSO₄ and AgNO₃) on callus induction and plant regeneration in Paspalum scrobiculatum and Eleusine coracana was examined. A 5× and 3× increase in regeneration response at enhanced levels of CuSO₄ was noted for kodo and finger millets, respectively. Significant improvement in plant regeneration was also observed with the increase in levels of Co and Mn. Addition of AgNO₃ to the basal medium also had a stimulatory effect on callus induction and plant regeneration. Optimization of nutrient level in the basal medium has highly significant role in obtaining maximum regeneration response from explants and callus culture.     

Enhanced shoot regeneration from Brassica campestris by silver nitrate

Summary The morphogenetic response of Brassica campestris genotype R500 to inhibitors of ethylene biosynthesis and action was investigated. A medium containing 1.0 mg.l^–1 NAA, 2.0 mg.l^–1 BAP, and 30 or 60 M AgNO₃ significantly enhanced both the percentage shoot regeneration and the number of shoots per cotyledon expiant. Although callus proliferation occurred on hypocotyl segments, no shoots were formed in response to AgNO₃ with expiants older than five days. Cotyledons older than six days formed shoots only with AgNO₃. Cobalt chloride at 20 and 30 M increased cotyledon shoot regeneration but was inferior to AgNO₃. Hypocotyl segments were unresponsive. Salicylic acid at 25 and 50 M prevented both shoot regeneration and callusing without any obvious toxic effects. Removal of expiants from AgNO₃ after 12 days did not alter the percentage of shoot regeneration but increased the number of shoots per expiant. This response was dependent on the level of BAP. Percentage shoot regeneration and number of shoots per cotyledon explant were not affected by removal of CoCl₂. These results indicate that the poor regenerative capacity of this genotype may be related to ethylene biosynthesis or metabolism.Abbreviations NAA Naphthalene Acetic Acid - BAP 6-Benzylamino Purine - MS Murashige and Skoog Medium     

Evaluation of a closed system,diffusive and humidity-induced convective throughflow ventilation on the growth and physiology of cauliflower in vitro

The effects of ethylene inhibitors (silver nitrate – AgNO₃ and silver thiosulphate – Ag₂S₂O₃ as inhibitors of ethylene activity, cobalt chloride – CoCl₂ as inhibitor of ethylene biosynthesis) and ethylene stimulator (aminocyclopropane-1-carboxylic acid – ACC) were studied on the growth of cauliflower (Brassica oleracea L.) seedlings cultured in closed vessels (60 cm³). The addition of ethylene inhibitors have significant stimulatory effects on the growth and development of seedlings and the effects were greatest with 10 μM AgNO₃, the fresh weight of leaves was 2.6×, and the leaf area 2.8× those of the control (no additives). The effects of various methods of ventilation (humidity-induced convective through-flow ventilation, diffusive ventilation and sealed condition) on the growth and physiology of in vitrocauliflower seedlings were also investigated. The seedlings were cultured either in the presence or absence of AgNO₃ (inhibitors of ethylene activity) and ACC (a precursor). Ethylene and CO₂ levels in the head-space of the culture vessels were monitored. The humidity-induced through-flow ventilation system has shown to be effective for improving growth, leaf chlorophyll content and the rate of net photosynthesis and preventing symptoms of hyperhydricity, such as leaf epinasty, and franginess, reduction of leaf area etc. In contrast, the results also indicated that the sealing of culture vessels could have serious inhibitory effects on growth and development, induce hyperhydricity and reduce leaf chlorophyll content. In the light period, CO₂ depletion occurred in the head-space of the sealed vessels (ca. 40 μl l^-1), the CO₂ concentration increased with increasing efficiency of the ventilation. No ethylene accumulation was noticed in the head-space of the culture vessels when humidity-induced throughflow ventilation was applied; however, high ethylene accumulation occurred in sealed vessels. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of 2,5-norbornadiene on abscission and ethylene production in citrus leaf explants

Citrus ( Citrus sinensis L. Osbeck) leaf explants completely abscise within 48 h when exposed to saturating amounts of ethylene at 25°C. When 2,5-norbornadiene was added, 2000 μl 1⁻¹ reduced abscission of explants also exposed to 2 μl 1⁻¹ of ethylene to the level of the control, and 8000 μl 1⁻¹ reduced abscission in explants exposed to 10 μl 1⁻¹ of ethylene to the level of the control, but abscission was complete when 1 000 μl 1⁻¹ of ethylene was used in the presence of 8 000 μl 1⁻¹ of 2,5-norbornadiene. When explants were exposed to 2 μl 1⁻¹ of ethylene, 2000 μl 1⁻¹ of 2,5-norbornadiene prevented abscission if applied up to 10 h after exposure to ethylene. After 18 h, applied 2,5-norbornadiene had little effect on abscission at 48 h. A Lineweaver-Burk plot gave a 1/2 maximum value of 0.12 μl 1⁻¹ for ethylene on abscission, 2,5-Norbornadiene gave competitive kinetics with respect to ethylene with a K₁ value of approximately 120 μl 1⁻¹ of 2,5-norbornadiene. The presence of 2,5norbornadiene stimulated ethylene production, which progressively increased as the 2,5-norbornadiene concentration was increased from 250 to 8 000 μl 1⁻¹ 2,5-Norbornadiene also suppressed the induction of cellulase and polygalacturonase by ethylene. Together, 2,5-norbornadiene and 2,4-dichlorophenoxyacetic acid were more effective than either alone in reducing abscission. 2,5-Norbornadiene also was effective in preventing the reduction of indole-3-acetic acid transport induced by ethylene.     

Ethylene inhibitors enhance in vitro root formation from apple shoot cultures

Effects of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and three ethylene inhibitors, AgNO₃, aminoethoxyvinyglycine (AVG) and CoCl₂, on root formation were tested in vitro using shoot cultures of the apple (Malus×domestica Borkh.) cultivar Royal Gala. ACC inhibited root formation by delaying root emergence and increasing callus formation at the bases of shoots. In contrast, ethylene inhibitors promoted root formation. Both AgNO₃ and AVG at the appropriate concentrations increased the percentage of shoots producing roots and reduced callus formation at the base of these shoots. AgNO₃ stimulated root emergence and enhanced root growth, while AVG increased the number of roots per shoot. CoCl₂ slightly increased root number and rooting efficiency. These promotive effects may result from a reduction in ethylene concentration or inhibition of ethylene action. The results found in this study may be used to improve the rooting efficiency of other apple cultivars and rootstocks, and possibly of other plant species. Received: 2 March 1997 / Revision received: 1 July 1997 / Accepted: 18 July 1997     

Induction of secondary somatic embryogenesis in hybrid larch (Larix x leptoeuropaea) as related to ethylene

Induction of secondary somatic embryogenesis was studied with hybridlarch (Larix x leptoeuropaea)cotyledonary somatic embryos obtained after 3, 4, 5 and 6 weeks of culture on amaturation medium supplemented with abscisic acid. Almost all 3-week maturedcotyledonary somatic embryos can develop embryonal masses whereas only 78, 27and 12% of them are able to do so after 4, 5 and 6 weeks of maturation,respectively. During the first week of culture on the induction medium, somaticembryos with high embryogenic potential (i.e. 3-weekmatured) release little ethylene (less than 1.5 nL h^–1g^–1 FW), whereas those which have almost completelylosttheir ability to induce embryonal masses (i.e. 6-weekmatured) produce much more ethylene. Thereafter, ethylene production by bothtypes of embryos is very similar at around 5–6 nLh^–1 g^–1 FW. Enrichment of theatmosphere with ethylene, or addition of 2-chloroethylphosphonic acid(ethephon)or ACC in the induction medium strongly reduced the induction of secondarysomatic embryogenesis. Moreover, inhibitors of ethylene action(AgNO₃and 2,5-norbornadiene) improved the development of embryonal masses fromsomaticembryos, particularly from the 6-week maturated ones. The results obtainedclearly suggest that ethylene is involved in the regulation of somaticembryogenesis in hybrid larch. The possible relationship between somaticembryogenic potential and ethylene biosynthesis by the explants or sensitivityof the latter to ethylene is discussed.     

Implication of Ethylene in the Release of Secondary Dormancy in Amaranthus caudatus L. Seeds by Gibberellins or Cytokinin

Ethephon (Eth), gibberellin A₃, A_{4 + 7} (GA₃, GA_{4 + 7}), and 6-benzyladenine (BA) removed secondary dormancy of Amaranthus caudatus seeds. The GAs and BA potentiated the effect of ethephon or 1-aminocyclopropane-1-carboxylic acid (ACC), an ethylene biosynthesis precursor, in terms of the rate or final percent of germination. Aminoethoxyvinylglycine (AVG), an ACC synthase activity inhibitor, was observed to simultaneously inhibit the release from dormancy effected by GA₃ or BA as well as the ethylene production stimulated by these regulators. Breaking of secondary dormancy by GA₃, GA_{4 + 7} or BA was prevented by 2,5-norbornadiene (NBD), an inhibitor of ethylene binding. Ethylene completely or markedly reversed the inhibitory effect of NBD. We thus conclude that the removal of secondary dormancy in Amaranthus caudatus seeds by gibberellin or benzyladenine involves ethylene biosynthesis and action.     

Ethylene as an effector of wound-induced resistance to cellulase in oat leaves

Peeling the abaxial epidermis from oat leaves (Avena sativa var. Victory) induces the formation of wound ethylene and the development of resistance to cellulolytic digestion of mesophyll cell walls. Ethylene release begins between 1 and 2 hours after peeling in the light or dark. Aminoethoxyvinylglycine (AVG, 0.1 millimolar), CoCl₂ (1.0 millimolar), propyl gallate (PG, 1.0 millimolar) or aminooxyacetic acid (AOA, 1.0 millimolar) inhibits, whereas AgNO₃ stimulates wound ethylene formation. Incubation on inhibitors of ethylene biosynthesis (AVG, CoCl₂, PG, AOA) or action (AgNO₃, hypobaric pressure or the trapping of ethylene with HgClO₄) also prevents the development of wound-induced resistance to enzymic cell wall digestion. 1-Aminocyclopropane-1-carboxylic acid (ACC, 1.0 millimolar) reverses AVG (0.1 millimolar) inhibition of the development of resistance. Exogenous ethylene partially induces the development of resistance in unwounded oat leaves.

These results suggest that peeling of oat leaves induces ethylene biosynthesis, which in turn effects changes in the mesophyll cells resulting in the development of resistance to cellulolytic digestion.

     

Effect of silver nitrate on multiple shoot formation of Virginia-type peanut through shoot tip culture

Summary A protocol for micropropagation of Virginia-type peanut plants, an ancient crop of the New World, is reported. This study was conducted to explore the effect of silver nitrate (AgNO₃), alone or in combination with growth regulators, on multiple shoot formation from shoot tip culture. Incorporation of AgNO₃ into the medium, without growth regulators, induced regeneration of the explants (which did not develop at all in the AgNO₃-free medium), and stimulated the emergence of axillary shoots. When AgNO₃ was added in combination with cytokinins and α-naphthaleneacetic acid (NAA), maximum average shoot number per regenerating explant was recorded (6.3) in Murashige and Skoog (MS) medium containing 33 μM 6-benzyladenine, 5.3 μM NAA, and 23.54 μM AgNO₃. Moreover, AgNO₃ showed a positive and marked effect on both shoot elongation and the reduction of callus proliferation from the basal ends of shoot tips. Following a period of elongation, the shoots were rooted in hormone-free Ms medium, showing no residual effects due to the long-term culture in AgNO₃-containing media. Acclimatization was easily obtained after plantlets were transferred to pots under greenhouse conditions, with 90% survival.     

Interaction of karrikinolide and ethylene in controlling germination of dormant <Emphasis Type="Italic">Avena fatua</Emphasis> L. caryopses

Caryopses of Avena fatua L. are dormant after harvest and germinate poorly at 20 °C. Dormancy was released by after-ripening the dry caryopses in the dark at 25 °C for 3 months. Karrikinolide (butenolide, 3-methyl-2H-furo[2,3-c]pyran-2-one, KAR₁), in contrast to exogenous ethylene and the precursor of ethylene biosynthesis 1-aminocyclopropane-1-carboxylic acid (ACC), completely overcame dormancy. The effect of KAR₁ was not affected by aminoethoxyvinylglycine (AVG), α-aminoisobutyric acid (AIB) and CoCl₂, inhibitors of ACC synthase and oxidase, respectively. 2,5-Norbornadiene (NBD), a reversible inhibitor of ethylene binding to its receptor, counteracted the stimulatory effect of KAR₁. Ethylene, ethephon and ACC counteracted and AVG reinforced inhibition caused by norbornadiene. Inhibition due to norbornadiene, applied during the first 3 days of imbibition in the presence of KAR₁, disappeared after transfer to air or ethylene. The obtained results confirm that KAR₁ breaks dormancy and indicate that ethylene alone plays no role in releasing dormancy of Avena fatua caryopses. KAR₁ probably did not relieve dormancy via the stimulation of ethylene biosynthesis. Some level of endogenous ethylene is probably required for ethylene action, which might be required for releasing dormancy by KAR₁ or for subsequent germination of caryopses after removing dormancy.     

Headspace ethylene accumulation on Stizolobium hassjoo hairy root culture producing L-3,4-dihydroxyphenylalanine

The addition of 1-aminocyclopropane-1-carboxylic acid (ethylene precursor), or 2-chloroethylphosphonic acid (ethephon, an ethylene-releasing compound) decreased root dry weight and l-DOPA (l-3,4-dihydroxyphenylalanine) accumulation in hairy root cultures of Stizolobium hassjoo. The inhibition caused by ethephon-mediated ethylene release was alleviated by 0.5 mg CoCl₂ l^–1 as an inhibitor of ethylene biosynthesis. The action of ethylene was inhibited by 1.5 mg AgNO₃ l^–1. Ethylene thus lowers hairy root formation and l-DOPA production; CoCl₂ decreases ethylene formation leading to a considerably improved root dry weight and l-DOPA production.     

Involvement of Ethylene in Potato Microtuber Dormancy

Potato (Solanum tuberosum L.) single-node explants undergoing in vitro tuberization produced detectable amounts of ethylene throughout tuber development, and the resulting microtubers were completely dormant (endodormant) for at least 12 to 15 weeks. The rate of ethylene production by tuberizing explants was highest during the initial 2 weeks of in vitro culture and declined thereafter. Continuous exposure of developing microtubers to the noncompetitive ethylene antagonist AgNO₃ via the culture medium resulted in a dose-dependent increase in precocious sprouting. The effect of AgNO₃ on the premature loss of microtuber endodormancy was observed after 3 weeks of culture. Similarly, continuous exposure of developing microtubers to the competitive ethylene antagonist 2,5-norbornadiene (NBD) at concentrations of 2 mL/L (gas phase) or greater also resulted in a dose-dependent increase in premature sprouting. Exogenous ethylene reversed this response and inhibited the precocious sprouting of NBD-treated microtubers. NBD treatment was effective only when it was begun within 7 d of the start of in vitro explant culture. These results indicate that endogenous ethylene is essential for the full expression of potato microtuber endodormancy, and that its involvement may be restricted to the initial period of endodormancy development.     

Requirement of Ethylene for Growth of Callus and Somatic Embryogenesis in Medicago sativa L.

The role of ethylene in the growth of callus and somatic embryogenesisin Medicago sativa was examined. The application of 2,5-norbornadiene,a competitive inhibitor of ethylene action, during a 10 d inductionperiod to medium containing 2,4-D and kinetin inhibited thegrowth of callus but did not affect somatic embryogenesis, nordid it affect ethylene production during the induction stage.The exposure of tissue, incubated on differentiation medium,without hormones, to an atmosphere of 2,5-norbornadiene, inhibitedboth growth and embryo maturation and stimulated pigmentation.The inhibition of embryo maturation was observed even in thepresence of norbornadiene at a concentration which did not affectgrowth of tissue. It is suggested that the action of endogenous ethylene is necessaryfor the growth of the callus and embryo maturation. Key words: Medicago sativa, ethylene, callus growth, somatic embryogenesis     

1-Aminocyclopropane-1-Carboxylic Acid Enhanced Direct Somatic Embryogenesis from Oncidium Leaf Cultures

Influence of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and two ethylene inhibitors, silver nitrate (AgNO₃) and cobalt chloride (CoCl₂), on direct somatic embryogenesis were tested in vitro using leaf cultures of Oncidium cv. Gower Ramsey. Leaf cells of tips, adaxial sides and cut ends could directly form somatic embryos on a hormone-free 1/2-strength MS medium. The frequency of embryo-producing explants was 55, 52.5 and 30 %, respectively. The embryo numbers per embryo-producing explant was 20.3. ACC at lower concentrations (5 and 10 μM) significantly retarded direct embryo formation from cut ends. However, higher concentrations of ACC (20 and 50 μM) significantly promoted embryogenic response of leaf tips and adaxial sides. All concentrations of AgNO₃ and CoCl₂ significantly retarded direct embryo formation. The best response was found on 20 μM of ACC, and the frequency of embryo-producing explants were 90, 85 and 35 % on leaf tips, adaxial sides and cut ends, respectively. The embryo numbers per embryo-producing explant was 32.2. This revised version was published online in July 2006 with corrections to the Cover Date.     

Promotion ofin vitro shoot formation from excised roots of silktree (Albizzia julibrissin) by an oxime ether derivative and other ethylene inhibitors

Summary This report describes the regeneration response of excised seedling roots of silktree (Albizzia julibrissin) to added ethylene precursors/generators (1-amino-cyclopropane-1-carboxylic acid [ACC], 2-chloroethylphosphonic acid [CEPA]), biosynthesis inhibitors (aminoethoxyvinylglycine [AVG], an oxime ether derivative [OED={[(ispropylidene)-amino]oxy}-acetic acid-2-(methoxy)-2-oxoethyl ester], CoCl² [Co⁺⁺]), and an ethylene action inhibitor (AgNO₃ [Ag⁺]). When placed on B5 medium, about 50% of the control explants formed shoot buds within 15 days. Addition of ACC or CEPA (1–10 µM) to the culture medium decreased both the percentage of cultures forming shoots and the number of shoots formed per culture. In contrast, AVG and OED (1–10 µM) increased shoot formation to almost 100% and increased the number of shoots formed per culture. Likewise, both Co⁺⁺ and Ag⁺ (1–10 µM) increased shoot regeneration, but the number of shoots produced after 30 days was less than with AVG or OED. The inhibitors of ethylene biosynthesis were partially effective in counteracting the inhibitory effect of ACC on shoot formation. These results suggest that modulation of ethylene biosynthesis and/or action can strongly influence the formation of adventitious shoots from excised roots of silktree.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - CEPA 2-chloroethylphosphonic acid - OED oxime ether derivative     

Synergistic effect of ethylene inhibitors and putrescine on shoot regeneration from hypocotyl explants of Chinese radish (Raphanus sativus L. var. longipinnatus Bailey) in vitro

Summary The role of ethylene and putrescine on shoot regeneration from hypocotyl explants of Chinese radish (Raphanus sativus L. var. longipinnatus Bailey cv. Red Coat) was investigated. Explants were recalcitrant in culture, but exogenous application of ethylene inhibitor [20–30 M aminoethoxyvinylglycine (AVG) or AgNO₃] enhanced shoot regeneration of explants grown on medium supplemented with 2 mg/l N⁶-benzyladenine and 1 mg/l 1-naphthaleneacetic acid. The best regeneration occurred in the medium containing AgNO₃ in combination with AVG. Culture medium solidified with agarose in the presence of AgNO₃ but not AVG was also beneficial to shoot regeneration. Exogenous putrescine, 2-chloroethylphosphonic acid and 1-aminocyclopropane-1-carboxylate had no effect on shoot regeneration. However, regeneration was greatly promoted by 10–25 mM putrescine in combination with 30 M AgNO₃ or AVG. Explants with high regenerability grown in the presence of AgNO₃ or in combination with putrescine emanated high levels of ethylene throughout the 21-d culture period. By contrast, AVG or putrescine alone resulted in a decrease in ethylene production. For rooting of shoot cuttings, IAA and IBA at 1–5 mg/l were more effective than NAA.Abbreviations ACC 1-aminocyclopropane-1-carboxylate - AVG aminoethoxyvinylglycine - BA N⁶-benzyladenine - CEPA 2-chloroethylphosphonic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) medium - NAA 1-naphthaleneacetic acid - PAs polyamines - SAM S-adenosyl-L-methionine     

Ethylene as an endogenous inhibitor of root regeneration in tomato leaf discs cultured in vitro

We examined ethylene effects on root regeneration in tomato leaf discs cultured in vitro. Applied ethylene or Ethephon did not stimulate rooting in the leaf discs. In the presence of indoleacetic acid. 5 × 10^-6M, these substances significantly inhibited root formation. Ethylene production (nl C₂H₄· (24 h)^-1. flask^-1) was positively correlated with increased IAA concentrations at various times during the culture period and, as a consequence, with the rooting response after 168 h. However, separate testing of equimolar concentrations of seven different auxins and auxin-like compounds showed no positive correlation between the rate of ethylene production and subsequent rooting response. Aeration of gas-tight flasks containing leaf discs and absorption of ethylene evolved from the discs by mercuric perchlorate in gas-tight flasks or pre-treatment of leaf discs with AgNO₃ significantly enhanced IAA induced root regeneration. Thus, these studies indicate that ethylene is not a rooting hormone per se. Furthermore, ethylene (whether applied externally or synthesized by the tissue) does not appear to account for the ability of auxin to stimulate rooting.