Role of polyamines on de novo shoot morphogenesis from cotyledons of Brassica campestris ssp. pekinensis (Lour) Olsson in vitro

Summary The promotive effect of ethylene inhibitors (Els), i.e. AgNO₃ and aminoethoxyvinylglycine (AVG) on de novo shoot regeneration from cultured cotyledonary explants of Brassica campestris ssp. pekinensis cv. Shantung in relation to polyamines (PAs) was investigated. The endogenous levels of free putrescine and spermidine in the explant decreased sharply after 1–3 days of culture, whereas endogenous spermine increased, irrespective of the absence or presence of Els. AgNO₃ at 30 M did not affect endogenous PAs during two weeks of culture. In contrast, explants grown on medium containing 5 M AVG produced higher levels of free putrescine and spermine which increased rapidly after three days and reached a peak at 10 days. An exogenous application of 5 mM putrescine also resulted in a similar surge of endogenous free spermine of the explant. More strikingly, shoot regeneration from explants grown in the presence of 1–20 mM putrescine, 0.1–2.5 mM spermidine, or 0.1–1 mM spermine was enhanced after three weeks of culture. However, exogenous PAs generally did not affect ethylene production, and endogenous levels of 1-aminocyclopropane-1-carboxylate (ACC) synthase activity and ACC of the explant. This study shows the PA requirement for shoot regeneration from cotyledons of B. campestris ssp. pekinensis in vitro, and also indicates that the promotive effect of PAs on regeneration may not be due to an inhibition of ethylene biosynthesis.Abbreviations PAs polyamines - AVG aminoethoxyvinylglycine - SAM S-adenosylmethionine - ACC 1-aminocyclopropane-1-carboxylate - Els ethylene inhibitors     

Role of Ethylene on de Novo Shoot Regeneration from Cotyledonary Explants of Brassica campestris ssp. pekinensis (Lour) Olsson in Vitro

The promotive effect of AgNO₃ and aminoethoxyvinylglycine (AVG) on in vitro shoot regeneration from cotyledons of Brassica campestris ssp. pekinensis in relation to endogenous 1-amino-cyclopropane-1-carboxylic acid (ACC) synthase, ACC, and ethylene production was investigated. AgNO₃ enhanced ACC synthase activity and ACC accumulation, which reached a maximum after 3 to 7 days of culture. ACC accumulation was concomitant with increased emanation of ethylene which peaked after 14 days. In contrast, AVG was inhibitory to endogenous ACC synthase activity and reduced ACC and ethylene production. The promotive effect of AVG on shoot regeneration was reversed by 2-chloroethylphosphonic acid at 50 micromolar or higher concentrations, whereas explants grown on AgNO₃ medium were less affected by 2-chloroethylphosphonic acid. The distinctive effect of AgNO₃ and AVG on endogenous ACC synthase, ACC, and ethylene production and its possible mechanisms are discussed.     

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     

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     

Ethylene and CO2 affect direct shoot regeneration from the petiolar ends of Paulownia kawakamii leaves cultured in vitro

The effects of ethylene and CO₂ on shoot regeneration in excised leaf cultures of Paulownia kawakamii were examined. When both the gases were prevented from accumulating in the headspace of cultures using mercuric perchlorate and potassium hydroxide traps, shoot regeneration frequency improved and callus production was reduced compared to the control and cultures with only one of the gases trapped. Incorporation of either aminoethoxyvinylglycine (AVG) or 1-amino-cyclopropane-1-carboxylic acid (ACC) in the culture medium caused significant reduction in shoot regeneration. There was profuse callus production in the presence of high amounts of ACC, which was accompanied by over sixfold increase in the rate of ethylene production. However, in the presence of AVG callus production was delayed and shoot regeneration decreased, suggesting that low levels of ethylene might be needed for de novo shoot bud induction in Paulownia cultures.Abbreviations IAA Indole-3-acetic acid - MP mercuric perchlorate - AVG aminoethoxyvinylglycine - ACC 1-aminocyclopropane-1-carboxylic acid     

Silver nitrate and aminoethoxyvinylglycine promote in vitro adventitious shoot regeneration of pomegranate (Punica granatum L.)

A protocol is presented for direct adventitous shoot organogenesis and complete plant regeneration from seedling-derived explants of pomegranate (Punica granatum L.), a tropical fruit tree. Murashige and Skoog (1962) (MS) medium enriched with 8.9 mumol/L benzyladenine (BA), 5.4 mumol/L naphthaleneacetic acid (NAA) and 10% coconut water (CW) induced adventitious shoot bud differentiation in axenic seedling-derived cotyledons as well as hypocotyl segments. The cotyledons were more responsive than the hypocotyls. Addition of ethylene inhibitors such as AgNO3 (10-40 mumol/L) and aminoethoxyvinylglycine (AVG) (5-15 mumol/L) to the medium markedly enhanced regeneration frequency as well as number of shoots obtained per explant. The promotive effect of AVG and AgNO3 on shoot organogenesis was observed only in cotyledon explants. The regeneration medium containing AgNO3 (20 mumol/L) or AVG (10 mumol/L) induced adventitious shoot buds from 57% or 53% of the cotyledon explants respectively. These shoot buds developed into shoots upon transfer to a regeneration medium without AgNO3 and AVG. The promotive effect of AVG on shoot regeneration was reversed by exogenous application of 20 mumol/L 2-chloroethylphosphonic acid (CEPA), an ethylene releasing compound. On the other hand, shoot regeneration stimulated by AgNO3 was relatively less affected by CEPA. Regenerated shoots were rooted in half-strength MS medium (1/2 MS) containing 0.54 mumol/L NAA. The well rooted plantlets were acclimatized and eventually established in soil.     

Effect of AgNO3 and aminoethoxyvinylglycine on in vitro shoot and root organogenesis from seedling explants of recalcitrant Brassica genotypes

The presence of 1–10 M aminoethoxyvinylglycine (AVG) or 5–30 M AgNO₃ markedly enhanced shoot regeneration from cotyledon and hypocotyl cultures of eight recalcitrant Brassica campestris and B. juncea genotypes tested. Expiants of B. campestris ssp. chinensis and ssp. parachinensis grown with a high AVG concentration (20 M), regenerated poorly. All cytokinins tested were equally effective in promoting shoot formation, except that kinetin was inhibitory to shoot regeneration from hypocotyls of B. campestris ssp. pekinensis (cv. Wong Bok). Both AgNO₃ and AVG had no effect on percent rooting and number of roots per rooted cutting of Wong Bok, White Sun and Leaf Heading, but AgNO₃ was inhibitory to rooting of India Mustard. However, root elongation of all cuttings was markedly inhibited by AVG at concentrations of 5 and 10 M.Abbreviations AVG aminoethoxyvinylglycine - BA benzyladenine - IBA indole-3-butyric acid - 2ip 6-{ie195-01}-{ie195-02}-dimethylallylamino purine - MS Murashige and Skoog - NAA naphthaleneacetic acid     

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     

Endogenous ethylene requirement for adventitious root induction and growth in tomato cotyledons and lavandin microcuttings in vitro

The role of ethylene in the formation of adventitious roots in vitro was studied in tomato (Lycopersicon esculentum Mill. cv. UC 105) cotyledons and lavandin (Lavandula officinalis Chaix × Lavandula latifolia microshoots. Both systems were able to form roots on hormone-free medium evolving low amounts of ethylene. The addition of 20–50 M indole-3-acetic acid (IAA) inhibited root formation in tomato cotyledons while increasing ethylene production. Naphthaleneacetic acid (NAA, 3 M) stimulated root number in lavandin explants and induced a transient rise in ethylene evolution. Enhanced ethylene levels via the endogenous precursors 1-aminocyclopropane-1-carboxylic acid (ACC, 25–50 M) drastically impaired root regeneration and growth in tomato. In lavandin, 10 M ACC stimulated ethylene production and significantly inhibited the rooting percentage and root growth. Conversely, ACC enhanced the root number in the presence of NAA only. Severe inhibition of rooting was also caused by ethylene reduction via biosynthetic inhibitors, aminoethoxyvinylglycine (AVG, 5–10 M) in tomato, and salicylic acid (SA, 100 M) in lavandin. A strict requirement of endogenous ethylene for adventitious root induction and growth is thus suggested.Abbreviations LS Linsmaier and Skoog medium - BA N⁶-benzyladenine - NAA 1-naphthaleneacetic acid - IAA Indole-3-acetic acid - AVG Aminoethoxyvinylglycine - SA Salicylic acid - ACC 1-aminocyclopropane-1-carboxylic acid     

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     

Rhizome induction and plantlet regeneration of Cymbidium goeringii from flower bud cultures in vitro

Apical flower buds of Cymbidium goeringli Reichenbach fil. (ca 2 mm long) exeised from infloreseences (ca 5 cm long) were explanted on modified Murashige & Skoog medium (=MS medium) supplemented with N⁶-benzyladenine (BA) and -naphthaleneacetic acid (NAA). Within 107 days of culture, swelling growth, chlorophyll synthesis, and subsequent rhizome differentiation were observed. MS medium containing 0.1 mg l^-1 BA and 10 mg l^-1 NAA was found to be optimal for initiating rhizome development and subsequent plantlet regeneration.Explants cultured on MS medium supplemented with 1 mg l^-1 NAA alone formed a mass of rhizome branches. Multiple shoots of rhizome branches were induced from apical segments when rhizomes were transferred to MS medium containing 0.1 mg l^-1 BA and 10 mg l^-1 NAA.Abbreviations NAA -naphthaleneacetic acid - BA N⁶-benzyladenine     

The effect of ethylene inhibitors （AgNO3, AVG）, an ethyleneliberating compound （CEPA） and aeration on the formation of protocorm-like bodies of hybrid Cymbidium （Orchidaceae）

Protocorm-like bodies （PLBs） or thin cell layers （TCLs） derived from PLBs of hybrid Cymbidium Twilight Moon ＇Day Light＇ can induce new or neo-PLBs on Teixeira Cymbidium （TC） medium, which contains 0.1 mg/L a- naphthaleneacetic acid, 0.1 mg/L kinetin, 2 g/L tryptone and 20 g/L sucrose, and is solidified with 8 g/L Bacto agar. This study aimed to assess the response ofneo-PLB formation to an ethylene-liberating compound （2-chloroethylphosphonic acid （CEPA））, to two ethylene inhibitors （silver nitrate （AgNO3） and aminoethoxyvinylglycine （AVG））, and to aeration （made possible by using Milliseal~ or autoclaved filter paper）. AgNO3 at 1 or 2 mg/L in TC medium significantly increased the fresh weight of PLBs while 1 mg/L of AgNO3 also showed a significant increase in the number ofneo-PLB from both half-PLBs and from tTCLs. In contrast, AVG and CEPA inhibited neo-PLB formation. Neo-PLB formation from half-PLB or TCL explants in the presence of aeration resulted in significantly lower neo-PLB weight. The use of AgNO3 and aeration are alternative means to mass produce neo-PLBs for micropropagation purposes.     

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.     

Ethylene production during anther culture of Brussels sprouts (Brassica oleracea var gemmifera) and its relationship with factors that affect embryo production

The ethylene inhibitor silver nitrate (AgNO₃) is known to overcome the poor response of the Brussels sprouts cultivar Hal to anther culture. Ethylene production by Hal anthers after 6 h of culture at 35°C was on average 10- and 20-fold greater than from anthers of the highly responsive cultivars Gower and GA1 x RDF2. The initial 24 h period at 35°C necessary for embryogenesis in anther culture of Brussels sprouts generally reduced ethylene production by the anthers after 6, 24, 48 and 72 h of culture, although the effect was not seen in 2 out of 3 Hal experiments until 24 h, and after 6 h was only found with 1 of 3 GA1 x RDF2 experiments. Embryo production was inhibited by the inclusion of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC) or the ethylene-releasing compound, ethephon in the media. Silver nitrate (AgNO₃) and the ethylene biosynthesis inhibitor aminoethoxyvinylglycine (AVG) promoted embryogenesis but did not substitute for the high temperature treatment. The relevance of ethylene production during anther culture to the effects of genotype and high temperature on anther culture embryogenesis is discussed.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine     

Stimulation of in vitro shoot proliferation from nodal explants of cassava by thidiazuron, benzyladenine and gibberellic acid

Multiple shoots were produced from nodal explants of cassava (Manihot esculenta Crantz) by a two-step procedure: a 6- to 8-day exposure to 0.11–0.22 µM thidiazuron (TDZ) in liquid Murashige and Skoog (MS) medium followed by culture on agar-solidified MS medium supplemented with 2.2 µM 6-benzyladenine (BA) and 1.6 M gibberellic acid (GA₃). TDZ caused the nodal explants to expand and this expansion (growth) continued during culture with BA and GA₃. From this expanded explant, clusters of buds and fasciated stems developed continuously and these gave rise to shoots. The shoot proliferation process was open-ended, yielding an average of 31.5 shoots per nodal explant after 10 weeks of culture with genotype CG 1–56. A positive response was also obtained from seven other genotypes evaluated with this protocol.Abbreviations BA 6-benzyladenine - BM basal medium - DPU 1,3-diphenylurea - GA₃ gibberellie acid - 2iP isopentenyladenine - MSM multiple shoot medium - NAA 1-naphthaleneacetic acid - PGR plant growth regulator - TDZ thidiazuron - Z zeatin     

Enhancement of shoot regeneration from cotyledon explants of Brassica rapa ssp oleifera through pretreatment with auxin and cytokinin and use of ethylene inhibitors

The presence of benzyladenine or naphthaleneacetic acid in seed germination medium markedly enhanced subsequent shoot regeneration from the base of the excised cotyledon explants of Brassica rapa cv. Horizon. Cotyledon explants from younger seedlings (3 or 4-day old) produced more shoots than those from older seedlings. Addition of the ethylene inhibitor aminoethoxyvinylglycine (1.0 M) to the regeneration medium improved shoot regeneration three fold.Abbreviations AVG aminoethoxyvinylglycine - BA benzyladenine - MGBG methylglyoxal-bisguanylhydrazone - MSBN ms (murashige & skoog 1962) medium supplemented with 4.4 m BA & 5.4 m NAA, 2% sucrose - NAA naphthaleneacetic acid     

De novo shoot morphogenesis and plant growth of mustard (Brassica juncea) in vitro in relation to ethylene

Role of ethylene in de novo shoot morphogenesis from explants and plant growth of mustard ( Brassica juncea cv. India Mustard) in vitro was investigated, by culturing explants or plants in the presence of the ethylene inhibitors aminoethoxyvinylglycine (AVG) and AgNO₃. The presence of 20 μ M AgNO₃ or 5 μ M AVG in culture medium containing 5 μ M naphthaleneacetic acid and 10 μ M benzyladenine were equally effective in promoting shoot regeneration from leaf disc and petiole explants. However, AgNO₃ greatly enhanced ethylene production which reached a maximum after 14 days, whereas ethylene levels in the presence of AVG remained low during 3 weeks of culture. The promotive effect of AVG on shoot regeneration was overcome by exogenous application of 25 μ M 2-chloroethylphosphonic acid (CEPA), but AgNO_3-induced regeneration was less affected by CEPA. For whole plant culture, AVG did not affect plant growth, although it decreased ethylene production by 80% and both endogenous levels of 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC by 70–80%. In contrast, AgNO₃ stimulated all 3 parameters of ethylene synthesis. Both AgNO₃ and CEPA were inhibitory to plant growth, with more severe inhibition occuring in AgNO₃. Leaf discs derived from plants grown with AVG or AgNO₃ were highly regenerative on shoot regeneration medium without ethylene inhibitor, but the presence of AgNO₃ in the medium was inhibitory to regeneration of those derived from plants grown with AgNO₃.     

In vitro propagation of loblolly pine via direct somatic organogenesis from mature cotyledons and hypocotyls

A high-efficiency two-step culture procedure for direct somaticorganogenesis in loblolly pine (Pinus taeda L.) resulting inthe formation of multiple shoot structures induced on cotyledons andhypocotyls of mature zygotic embryos is described. Mature zygoticembryos of eight genotypes of loblolly pine were used as explants toinduce direct somatic organogenesis with this two-step culture method,involving the induction and the differentiation of direct adventitiousshoots. After mature zygotic embryos of eight genotypes of loblolly pinewere cultured on induction medium containing 2,4-dichlorophenoxyaceticacid (2,4-D) or -naphthaleneacetic acid (NAA), 6-benzyladenine(BA), and kinetin for 2–3 weeks, embryos were transferred todifferentiation medium. Adventitious shoot regeneration via directsomatic organogenesis with the frequency of 8.7–27.8% wasobtained from mature zygotic embryo cultures of the genotypes tested.The highest mean number of 32.6 adventitious shoots per mature zygoticembryo was produced from genotype La. The tissue culture protocol of invitro shoot regeneration via direct somatic embryogenesis was optimizedafter examining the periods of the induction culture, chillingtreatment, glutamine concentration, and basic medium levels. Rooting wasachieved on TE medium supplemented with 0.5 mg/l indole-3-butyric acid(IBA), 0.5 mg/l gibberellic acid (GA₃), and 1 mg/l6-benzyladenine (BA), and regenerated plantlets were established insoil. These results suggested that adventitious shoot regeneration viadirect somatic organogenesis could be useful for clonal micropropagationof some genotypes of loblolly pine and for establishing a transformationsystem of this coniferous species.     

Ethylene involvement in vegetative bud formation in tobacco thin layers

Summary The role of ethylene in vegetative bud regeneration was studied in cultured tobacco (Nicotiana tabacum L. cvSamsun) thinlayer expiants. The experimental approach consisted in supplementing the bud-inducing medium with an inhibitor of ethylene biosynthesis, aminoethoxyvinylglycine (AVG), an ethylene antagonist, silver thiosulphate (STS), or an ethylene-releasing compound, 2-chloroethylphosphonic acid (CEPA), at various concentrations. The organogenic response was assessed both macroscopically (percentage of bud-forming expiants, final number of buds per expiant) and cytohistologically (number, characteristics, and localisation of meristemoids and bud primordia). The time course of ethylene production during culture was also evaluated. At the end of culture (day 27) all the expiants treated with these compounds had a lower number of buds compared to controls. STS was detrimental to meristemoid initiation at all the concentrations tested. In contrast, 0.5 M AVG, which strongly inhibited ethylene production, provoked a large increase in the formation of meristemoids early in culture and the appearance of anomalous (twin) buds. CEPA reduced meristemoid formation but, at the lower concentrations (1 and 10 M) speeded up bud emergence. On the whole it mainly favoured disorganised growth and xylogenesis. The results of this work highlight the contrasting effects of ethylene in relation to the two critical stages of the organogenic process, i.e., meristemoid formation and bud primordium development.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - STS silver thiosulphate - CEPA 2-chloroethylphosphonic acid - IAA indole-3-acetic acid - BA benzyladenine - HF hormone-free     

An efficient in vitro method for mass propagation of a woody ornamentalIxora coccinea L.

Various factors that affect culture establishment, shoot growth, proliferation and rooting ofIxora coccinea L., a woody shrub, were studied. Stem cuttings (decapitated shoot, three nodes) were the most suitable explants for multiple-shoot proliferation, and when cultured on a woody plant medium (WPM) containing 2.5 M BA produced axillary shoots which branched repeatedly, yielding an average of 27 shoots per explant after 6 weeks in culture. Kinetin, 2-iP, zeatin and thidiazuron all induced multiple-shoot formation, but were less effective than BA. While the presence of IAA in the multiplication medium was detrimental to shoot proliferation, shoot growth was not affected by IAA. The production of large amounts of basal callus and vitrification of shoots were the major problems to be avoided in proliferating shoot cultures. Addition of TIBA to the multiplication medium markedly reduced basal callusing, while sealing the culture vessels with a fluorocarbon polymer (tetrafluoroethyleneperfluoroalkyl vinyl ether) film (Neoflon PFA film) almost completely eliminated vitrification. A reduction in the number of vitrified shoots was also achieved with AVG treatment. Following this protocol of using BA-supplemented WPM and Neoflon film, it would be possible to produce more than 100,000 plants from a single stem cutting in 1 year.Abbreviations AVG Aminoethoxyvinylglycine - BA N⁶-benzyladenine - BM basal medium - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2-tiP N⁶-(2-isopentenyl)adenine - KIN kinetin - MS Murashige and Skoog medium - NAA -naphthaleneacetic acid - SRM shoot regeneration medium - TDZ thidiazuron - TIBA 2,3,5-triiodobenzoic acid - WPM woody plant medium - ZEA zeatin