Efficient plant regeneration from leaves of rapeseed (Brassica napus L.): the influence of AgNO3 and genotype

Factors influencing reliable shoot regeneration from leaf explants of rapeseed (Brassica napus L.) were examined. Addition of AgNO₃ to callus induction medium was significantly effective for shoot regeneration in all three genotypes initially tested. When 48 genotypes subsequently were surveyed, a large variation of shoot regenerability was observed, ranging from 100 to 0% in frequency of bud formation and from 7.5 to 0 in the number of buds per explant. A significant correlation (r=0.84) was observed between the frequency of bud formation and the number of buds per explant. The shoot regenerability from leaf explants was not related to that from cotyledonary explants (r=0.28). Histological observations showed that an organized structure developed from calluses produced at vascular bundle tissues after 7 days of culture on callus induction medium, and they developed shoot apical meristems one week after transfer onto shoot induction medium. Regenerated plantlets were obtained 2 months after the initiation of culture and they normally flowered and set seeds. No alterations of morphology or DNA contents were observed in regenerated plants and their S1 progenies.     

Organogenesis and plant regeneration in Taxus wallichiana (Zucc.)

We describe an efficient process for regeneration of Taxus wallichiana plants via shoot organogenesis from callus cultures derived from zygotic embryos. Zygotic embryos cultured on half strength Lloyd and McCown's basal medium supplemented with SH vitamin ((1/2) WPMSH), 0.5 mg l(-1) 6-benzyladenine (BA) in combination with 1.0-2.0 mg l(-1) 2,4-dichlorophenoxyacetic acid (2,4-D) or alpha-Napthaleneacetic acid (NAA) produced two morphologically distinct types of calli-compact, green callus (CG) and compact, yellow (CY) callus after 4 weeks of culture. Optimum frequency (63%) of adventitious shoot bud induction was achieved in CG callus (3.0+/-0.67 shoot buds per gram of CG callus) when cultured on (1/2) WPMSH basal medium supplemented with 2.5 mg l(-1) BA after 4 weeks. The inclusion of 1% activated charcoal (AC) to (1/2) WPMSH basal medium (shoot elongation medium) led to maximum shoot elongation (2.15 cms). Microshoots rooted in high frequency (40%) in MS basal medium in which the concentration of nitrates was reduced to one-fifth the normal concentration after 4 months of culture.     

Correlation of Endogenous Gibberellic Acid with Initiation of Mango Shoot Growth

Stems of mango (Mangifera indica L.) rest in a nongrowing, dormant state for much of the year. Ephemeral flushes of vegetative or reproductive shoot growth are periodically evoked in apical or lateral buds of these resting stems. The initiation of shoot growth is postulated to be primarily regulated by a critical ratio of root-produced cytokinins, which accumulate in buds and by leaf-produced auxin, which decreases in synthesis and transport over time. Exogenously applied gibberellic acid (GA3) delays initiation of bud break but does not determine whether the resulting flush of growth is vegetative or reproductive. We tested the hypothesis that endogenous GA3, which influences release of these resting buds, may decrease in stem tips or leaves with increasing age of mango stems. GA3 and several other GAs in stem tip buds and leaves were identified and quantified in stems of different ages. The major endogenous GAs found in apical buds and leaves of vegetative mango stems were early 13-hydroxylation pathway gibberellins: GA1, epi-GA1, GA3, GA19, GA20, and GA29, as identified by gas chromatography-mass spectrometry (GC-MS). A novel but unidentified GA-like compound was also present. The most abundant GAs in apical stem buds were GA3 and GA19. Contrary to the hypothesis, the concentration of GA3 increased within buds with increasing age of the stems. The concentrations of other GAs in buds were variable. The concentration of GA3 did not change significantly with age in leaves, whereas that of most of the other GAs declined. GA1 levels were greatest in leaves of elongating shoots. These results are consistent with the concept that rapid shoot growth is associated with synthesis of GAs leading to GA1. The role of GA3 in delaying bud break in mango is not known, but it is proposed that it may enhance or maintain the synthesis or activity of endogenous auxin. It, thereby, maintains a high auxin/cytokinin ratio similar to responses to GA3 that maintain apical dominance in other plant species.     

Correlation of Endogenous Gibberellic Acid with Initiation of Mango Shoot Growth

Stems of mango (Mangifera indica L.) rest in a nongrowing, dormant state for much of the year. Ephemeral flushes of vegetative or reproductive shoot growth are periodically evoked in apical or lateral buds of these resting stems. The initiation of shoot growth is postulated to be primarily regulated by a critical ratio of root-produced cytokinins, which accumulate in buds and by leaf-produced auxin, which decreases in synthesis and transport over time. Exogenously applied gibberellic acid (GA3) delays initiation of bud break but does not determine whether the resulting flush of growth is vegetative or reproductive. We tested the hypothesis that endogenous GA3, which influences release of these resting buds, may decrease in stem tips or leaves with increasing age of mango stems. GA3 and several other GAs in stem tip buds and leaves were identified and quantified in stems of different ages. The major endogenous GAs found in apical buds and leaves of vegetative mango stems were early 13-hydroxylation pathway gibberellins: GA1, epi-GA1, GA3, GA19, GA20, and GA29, as identified by gas chromatography-mass spectrometry (GC-MS). A novel but unidentified GA-like compound was also present. The most abundant GAs in apical stem buds were GA3 and GA19. Contrary to the hypothesis, the concentration of GA3 increased within buds with increasing age of the stems. The concentrations of other GAs in buds were variable. The concentration of GA3 did not change significantly with age in leaves, whereas that of most of the other GAs declined. GA1 levels were greatest in leaves of elongating shoots. These results are consistent with the concept that rapid shoot growth is associated with synthesis of GAs leading to GA1. The role of GA3 in delaying bud break in mango is not known, but it is proposed that it may enhance or maintain the synthesis or activity of endogenous auxin. It, thereby, maintains a high auxin/cytokinin ratio similar to responses to GA3 that maintain apical dominance in other plant species.     

Changes in Cytokinins and Gibberellin-Like Substances in Pinus radiata Buds during Lateral Shoot Initiation and the Characterization of Ribosyl Zeatin and a Novel Ribosyl Zeatin Glycoside

Based on detection and quantitation by bioassay, endogenous gibberellin-like substances (GAs) and cytokinins (CKs) in Pinus radiata D. Don buds during sequential shoot initiation shift from less polar to more polar forms (GAs) and from conjugated to free forms (CKs). As the terminal bud moves from the production of “short shoots” (needle fascicles) to “long shoots” (lateral branches or female conebuds), a more polar GA appears while a glucoside-conjugate of zeatin riboside is reduced, and zeatin riboside levels increase markedly.     

罗汉果花芽分化过程中形态及其激素水平变化特征

采用石蜡切片和酶联免疫法(ELISA)对罗汉果雄性、雌性、两性花芽分化过程的形态和激素水平变化进行观测,为罗汉果开花调控和品种选育提供科学依据。结果表明:(1)罗汉果雄性、雌性、两性花的花芽分化过程均可分为花芽未分化期、花芽分化初期、花序分化期、萼片原基分化期、花瓣原基分化期、雄蕊原基分化期和雌蕊原基分化期7个阶段。雄蕊原基分化期前,3种花芽分化过程无明显差异,各时期形态特征均依次为:茎端呈圆锥状(花芽未分化期)→茎端经半球形变成扁平状(花芽分化初期)→距茎端5~7节位处分化出穗状花序(花序分化期)→小花原基周围形成5个萼片原基(萼片原基分化期)→萼片原基内侧形成5个花瓣原基(花瓣原基分化期)。雄蕊和雌蕊原基分化期,3种花芽分化过程存在明显差异,雄蕊原基内侧出现雌蕊原基后,雄花芽雄蕊原基继续发育成雄蕊,雌蕊原基停滞生长,退为一个小突起;雌花芽雌蕊原基继续发育成雌蕊,雄蕊原基生长缓慢,退化为小花丝;两性花芽雌蕊和雄蕊原基均继续发育,形成外观正常的雌蕊和雄蕊。(2)内源激素脱落酸(ABA)、赤霉素(GAs)和玉米素核苷(ZR)含量在3种花芽分化过程中变化规律相似,即ABA含量在花芽生理分化期降低,花芽形态分化期升高,而GAs和ZR含量则基本保持不变;吲哚乙酸(IAA)含量在3种花芽分化过程中变化存在明显差异,雌花芽IAA含量在花芽生理分化期升高,花芽形态分化期逐渐降低,而雄性和两性花芽的IAA含量则基本保持不变。ABA/GAs、ABA/IAA、ZR/IAA和ZR/GAs激素含量比值在3种花芽分化过程中变化规律相似,ABA/GAs在花芽生理分化期降低,花芽形态分化期升高,而BA/IAA、ZR/IAA和ZR/GAs则基本保持不变。研究认为,罗汉果花芽分化过程经历一个"两性期",高ABA含量和ABA/GAs比值有利于罗汉果花芽分化,IAA可能对罗汉果花性分化具有重要作用。     

Involvement of endogenous gibberellins in potato tuber dormancy and early sprout growth: a critical assessment

The role of endogenous gibberellins (GAs) in the regulation of potato (Solanum tuberosum) tuber dormancy was examined by determining: 1. changes in endogenous GA levels during natural dormancy progression, 2. the effects of GA biosynthesis inhibitors on tuber dormancy duration and 3. the dormancy status and tuber GA levels in a dwarf mutant of potato. The tubers (cv. Russet Burbank) used in these studies were still completely dormant after 98 days of storage. Between 98 and 134 days of storage, dormancy began to end and tubers exhibited limited (< 2 mm) sprout growth. Tuber dormancy weakened with further storage and tubers exhibited greater rates of sprout growth after 187 days of storage. Tubers stored for 212 days or longer were completely non-dormant and exhibited vigorous sprout growth. Immediately after harvest, the endogenous contents of GA19, GA20, and GA1 were relatively high (0.48-0.62 ng g fresh weight(-1)). The content of these GAs declined between 33 and 93 days of storage. Internal levels of GA19, GA20, and GA, rose slightly between 93 and 135 days of storage reaching levels comparable to those found in highly dormant tubers immediately after harvest. Levels of GA19, GA20, and GA1 continued to increase as sprout growth became more vigorous. Neither GA4 nor GA8 was detected in any tuber sample regardless of dormancy status. Dormant tubers exhibited a time-dependent increase in apparent GA sensitivity. Freshly harvested tubers were completely insensitive to exogenous GAs. As postharvest storage continued, exogenous GAs promoted premature dormancy release with GA1 and GA20 eliciting the greatest response. Injection of up to 5 microg tuber(-1) of kaurene, GA12, GA19 or GA8 had no effect on dormancy release. Sprout growth from non-dormant tubers was also promoted by exogenous GA in the following sequence of activity: GA1 = GA20 > GA19. Kaurene, GA12, and GA8 were inactive. Continuous exposure of developing tubers to inhibitors of GA biosynthesis (AMO-1618, ancymidol, or tetcyclasis) did not extend tuber dormancy but rather hastened dormancy release. Comparison of tuber dormancy and GA1 content in tubers of a wild-type and dwarf mutant of S. tuberosum ssp. andigena revealed a near-identical pattern of dormancy progression in spite of the absence of detectable levels of GA1 in tubers of the dwarf sibling at any time during dormancy progression. Collectively, these results do not support a role for endogenous GA in potato tuber dormancy release but are consistent with a role for GAs in the regulation of subsequent sprout growth.     

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     

Enhancement of lateral bud growth in Coleus blumei BENTH. by (2-chloroethyl) trimethylammonium chloride (CCC)

The relationship of GA to apical dominance in Coleus was examinedby substituting 1 % IAA, in lanolin, for the shoot apex of CCC-treated,control and GA-treated plants containing, theoretically, hyponormal,normal and hypernormal GA levels, respectively. The greatestinhibition of lateral bud growth was obtained in the treatmentcombining 1 % IAA and 100 ppm GA, suggesting that GA may beimportant in the apical dominance of Coleus. CCC inhibited main axis growth, reduced the level of endogenousGA and caused a marked release of lateral buds from apical dominance. The significant stimulation of lateral bud growth by CCC couldnot be ascribed to reduced endogenous GA since it was not reversedby exogenous GA, or by GA plus IAA, whereas 100 ppm GA overcamethe inhibition of main axis growth by CCC. It was also shownthat the CCC stimulation was not a result of compensatory growth,that is, enhanced lateral bud growth resulting from reducedapical bud growth. The CCC effect on lateral buds was interpretedas involving a system independent of auxin and GA or else apossible immobilization of auxin in addition to inhibition ofGA biosynthesis. (Received December 5, 1967; )     

Expression of two gibberellin-regulated cDNAs during early flower development in tomato (Solanum lycopersicon). Effect of grafting and paclobutrazol

To study the role of translocation of gibberellin (GA) intermediates or bioactive GAs from other plant parts to buds during early flower development in tomato ( Solanum lycopersicon ), the effect of grafting and paclobutrazol (PAC) treatment on the expression of tgas100 and tgas118 , two GA-regulated mRNAs, was analysed. Both mRNAs accumulated in a dose-dependent fashion. Application of 0.5 ng GA₃ per bud to developmentally arrested flower buds of a GA-deficient mutant of tomato ( gib-1 ) induced tgas100 expression, while the tgas118 abundance increased. For obtaining normal flower development through anthesis in the mutant, a single GA₃ treatment was required of at least 5 ng GA₃ per bud. In wild-type flower buds, PAC decreased the abundance of tgas100 and tgas118 mRNAs either when PAC was sprayed on whole plants or directly applied to buds. When only the wild-type buds were treated with PAC, the expression profiles characteristic for untreated buds were not restored by translocation of endogenous GAs. Grafting of gib-1 scions onto wild-type donor plants did not result in normal flower development or expression profiles like in wild-type buds. We conclude that the role of GA transport in early flower development of tomato is negligible and that the GAs required for development have to be synthesized in the flower bud itself.     

细枝木麻黄离体培养过程中愈伤组织和再生芽的细胞组织学观察

为探讨细枝木麻黄(Casuarina cunninghamianaMiq.)愈伤组织分化过程的细胞组织学,对离体培养条件下的愈伤组织进行扫描电子显微镜和石蜡切片观察,分析愈伤组织的细胞分裂、分化以及芽再生的发生过程。结果表明,新鲜外植体培养于愈伤组织诱导培养基上,伤口处的薄壁细胞开始脱分化,培养1周后形成明显的愈伤组织;继续培养2周后,胚性愈伤组织形成,且表层细胞启动分化形成芽原基;培养4周,可肉眼观察到胚性芽原基,数量增多并逐渐分化形成不定芽;培养至第6周,生成不定芽,并大量增殖和分化。因此,细枝木麻黄是通过愈伤组织分化形成胚状体的途径进行植株再生的,为建立细枝木麻黄组织培养高效再生体系提供了理论依据。     

The role of plant growth regulators on direct and indirect plant regeneration from various organs of <Emphasis Type="Italic">Leucaena leucocephala</Emphasis>

Prolific differentiation of shoot buds of Leucaena leucocephala was induced from the different plant parts viz. cotyledon, hypocotyl and leaf. Adventitious shoot bud formation was recorded with prudent application of N⁶-2- (isopentenyl) adenosine and 15% (v/v) coconut water. Coconut water alone was unable to produce any beneficial effect with regard to the shoot bud proliferation but the response was augmented with the increase in concentration of N⁶-2- (isopentenyl) adenosine. However supra-optimal level of N⁶-2-(isopentenyl) adenosine was inhibitory. Best response was recorded from the cotyledon explant at 2 mg dm⁻³ N⁶-2-(isopentenyl) adenosine compared to the other two explants. Comparative assessment was undertaken following the same experimental protocol in liquid shake culture. The regenerated shoot buds were subcultured in plant growth regulator-free medium where leafy shoot emergence was recorded. Optimum regeneration of roots was observed in these shoots in presence of 1 mg dm⁻³ α-naphthalene acetic acid. Plantlets were finally hardened following standard procedures before transplantation to the field. In another experimental set up, the de-embryonated cotyledons regenerated shoot buds via callus formation. The regenerated shoots and plantlets obtained through callus mediated organogenesis could be used for rapid multiplication and also for the genetic improvement of individual clones of Leucaena leucocephala.     

Gibberellins regulate seed germination in tomato by endosperm weakening: a study with gibberellin-deficient mutants

The germination of seeds of tomato [Lycopersicon esculentum (L.) Mill.] cv. Moneymaker has been compared with that of seeds of the gibberellin-deficient dwarf-mutant line ga-1, induced in the same genetic background. Germination of tomato seeds was absolutely dependent on the presence of either endogenous or exogenous gibberellins (GAs). Gibberellin A₄₊₇ was 1000-fold more active than commercial gibberellic acid in inducing germination of the ga-1 seeds. Red light, a preincubation at 2°C, and ethylene did not stimulate germination of ga-1 seeds in the absence of GA₄₊₇; however, fusicoccin did stimulate germination independently. Removal of the endosperm and testa layers opposite the radicle tip caused germination of ga-1 seeds in water. The seedlings and plants that develop from the detipped ga-1 seeds exhibited the extreme dwarfy phenotype that is normal to this genotype. Measurements of the mechanical resistance of the surrounding layers showed that the major action of GAs was directed to the weakening of the endosperm cells around the radicle tip. In wild-type seeds this weakening occurred in water before radicle protrusion. In ga-1 seeds a similar event was dependent on GA₄₊₇, while fusicoccin also had some activity. Simultaneous incubation of de-embryonated endosperms and isolated axes showed that wild-type embryos contain and endosperm-weakening factor that is absent in ga-1 axes and is probably a GA. Thus, an endogenous GA facilitates germination in tomato seeds by weakening the mechanical restraint of the endosperm cells to permit radicle protrusion.Abbreviations GA(s) gibberellin(s) - GA₃ gibberellic acid     

八角莲组织培养的器官发生途径研究

为探究小檗科植物八角莲组织培养的器官发生方式,该研究以八角莲离体叶片、叶柄在MS培养基上诱导产生的愈伤组织、不定芽、不定根为对象,用连续石蜡切片技术分析八角莲组织培养的器官发生途径。结果表明:八角莲愈伤组织形成的解剖学特征是靠近表皮的薄壁细胞经激素刺激恢复分裂能力,继续培养形成拟分生组织。拟分生组织可形成许多分化中心。通过对八角莲组织培养产生的不定芽细胞组织学观察发现芽原基起源于愈伤组织外侧的几层薄壁细胞,芽原基背离愈伤组织中央生长形成不定芽,故八角莲脱分化形成的芽起源方式为外起源。而八角莲的根原基起源于组织深处髓部薄壁细胞和部分维管形成层细胞,进而形成类似球形或楔形并朝韧皮部突起的根原基轮廓,根原基继续发育会突破表皮生成不定根,起源方式为内起源。八角莲离体再生途径为器官发生型,在组培苗生长过程中先诱导形成不定芽,再诱导形成不定根,在愈伤组织上形成维管组织将不定芽和不定根连接成完整植株。     

Effect of thidiazuron,light fluence rates and kanamycin on in vitro shoot organogenesis from excised Rubus cotyledons and leaves

The relationship between genotype, tissue age and endogenous cytokinin levels on adventitious bud formation on Lachenalia leaf tissue were investigated. The genotypes studied, showed a variation in bud formation. The hybrid explants responded differently to factorial combinations of BA and NAA. The growth regulators could not substitute for the regeneration potential of the genotype. Tissue age had a pronounced effect on regeneration potential. Young tissue formed the largest number of buds. An interaction between tissue age and genotype was detected. Cytokinin levels in young leaf tissue were higher than in older tissue. In young tissue no relationship was observed between the cytokinin level and the number of buds formed. However, in older tissue it appears as if a relatively low endogenous cytokinin level enhanced bud formation.Abbreviations BA benzyladenine - NAA naphthalene-1-acetic acid - Z zeatin - ZR ribosylzeatin     

Elevated endogenous isopentenyl adenine content is correlated with an extremely shooty rice phenotype

The shooty rice (Oryza sativa L.) mutant produced on average 192 buds from a single shoot within one month. Numerous shoots carrying the same phenotype were easily regenerated from a small piece of any growing bud after dedifferentiation and callus induction. These traits were inherited by subcultures of all the generations without loss of the vigorous multiplying capability. The shooty phenotype was correlated with an elevated endogenous isopentenyl adenine (2iP) content. Addition of 2iP to the growth media promoted differentiation of wild-type rice callus to large extent, but had little effect on plant regeneration. This mutant may hold a key toward understanding of monocotyledonous (cereal) plant regeneration.     

Further experiments on spermidine-mediated floral-bud formation in thin-layer explants of Wisconsin 38 tobacco

We studied the effects of various polyamines on bud regeneration in thin-layer tissue explants of vegetative and floweringNicotiana tabacum L. cv. Wisconsin 38, in which application of exogenous spermidine (Spd) to vegetative cultures causes the initiation and development of some flower buds (Kaur-Sawhney et al. 1988 Planta173, 282). We now show that this effect is dependent on the time and duration of application, Spd being required from the start of the cultures for about three weeks. Neither putrescine nor spermine is effective in the concentration range tested. Spermidine cannot replace kinetin (N⁶-furfurylaminopurine) in cultures at the time of floral bud formation, but once the buds are initiated in the presence of kinetin, addition of Spd to the medium greatly increases the number of floral buds that develop into normal flowers. Addition of Spd to similar cultures derived from young, non-flowering plants did not cause the appearance of floral buds but rather induced a profusion of vegetative buds. These results indicate a morphogenetic role of Spd in bud differentiation. Dedicated to Professor Hans Mohr on the occasion of his 60th birthday     

Rapid plant regeneration and analysis of genetic fidelity of in vitro derived plants of Chlorophytum arundinaceum Baker—an endangered medicinal herb

An efficient in vitro multiplication system via multiple shoot bud induction and regeneration has been developed in Chlorophytum arundinaceum using shoot crown explants. Optimum regeneration frequency (87%) and desirable organogenetic response in the form of de novo organized multiple shoot buds without an intervening callus phase was obtained on Murashige and Skoog's (MS) minimal organics medium containing 3% sucrose (w/v) supplemented with 4×10⁻⁶ M Kn and 2×10⁻⁶ MIBA. Axenic secondary explants with multiple shoot buds on subculturing elicited best response with 1×10⁻⁵ M Kinetin (Kn) and 5×10⁻⁶ M indole-3-butyric acid (IBA) giving rise to an average of 18.74 shoots per culture with mean shoot length of 7.6 cm ± 1.73. Varying molar ratios of either Kn/IBA or Kn/NAA revealed statistically significant differences in the regeneration frequencies among the phytohormone treatments. It was observed that the shoot bud differentiation and regeneration was influenced by the molar ratios of cytokinins/auxin rather than their relative concentrations. Healthy regenerated shoots were rooted in half strength MS basal medium containing 3% sucrose (w/v) supplemented with 5×10⁻⁶ M IBA. Following simple hardening procedures, rooted plantlets, were transferred to soil-sand (1:1; v/v) with more than 90% success. Genetic fidelity was assessed using random amplified polymorphic DNA (RAPD), karyotype analysis and meiotic behaviour of in vitro and in vivo plants. Five arbitrary decamers displayed same banding profile within all the micropropagated plants and in vivo explant donor. The cytological and molecular analysis complemented and compared well and showed no genomic alterations in the plants regenerated through shoot bud differentiation. High multiplication frequency, molecular, cytological and phenotypic stability ensures the efficacy of the protocol developed for the production and conservation of this important endangered medicinal herb.     

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.