Role of purine metabolism in thidiazuron-induced somatic embryogenesis of geranium (Pelargonium×hortorum) hypocotyl cultures

Somatic embryogenesis in geranium ( Pelargonium × hortorum Bailey cv. Scarlet Orbit Improved) was achieved by culturing hypocotyl sections on Murashige and Skoog (1962) (MS) medium containing 10 μ M thidiazuron (TDZ) (induction medium) for 3 days and subsequently transferring the sections onto a basal medium lacking any plant growth regulators (expression medium). Addition of the purine analogue 2.6-diaminopurine (DAP) to the somatic embryo induction medium completely inhibited the embryogenic response as well as chlorophyll accumulation without affecting enlargement of the treated tissues. Addition of 20 μ M adenine sulphate to the expression medium, i.e during embryo growth and development phase, completely reversed the DAP-induced inhibition of the embryogenic response while addition during the induction phase caused only a 50% reversal of the inhibition. Analysis of endogenous levels of plant growth substances indicated that TDZ alone elevated the levels of auxins, cy-tokinins and abscisic acid while the presence of DAP during the induction phase caused a further increase in the levels of adenine and adenosine. These findings indicate a possible critical role for purines in embryogenesis from geranium hypocotyl tissues. However, the conversion of cytokinin bases to their corresponding nucleotide forms was not evident as the levels of isopentenyl adenine and zeatin increased during the second day of culture.     

Acetylsalicylic acid enhances and synchronizes thidiazuron-induced somatic embryogenesis in geranium (Pelargonium x hortorum Bailey) tissue cultures

Summary Thidiazuron (TDZ) effectively induced somatic embryogenesis in cultured hypocotyl explants of geranium (Pelargonium x hortorum Bailey) during only a 3-day period of induction. The presence of acetylsalicylic acid (ASA) during this period caused a two-fold increase in the number of somatic embryos and enhanced synchronization of embryo development compared to the TDZ treatment alone. Salicylic acid was ineffective in modulating similar embryogenic responses as ASA. The ASA-induced enhancement and synchronization of somatic embryogenesis could possibly be used as an experimental system to study the interplay of growth regulators in somatic embryogenesis.Abbreviations ASA acetylsalicylic acid - SA salicylic acid     

Role of endogenous purine metabolism in thidiazuron-induced somatic embryogenesis of peanut (Arachis hypogaea L.)

Somatic embryogenesis was induced at the hypocotyledonary notch region of intact peanut (Arachis hypogaea L.) seedings cultured on a medium containing 10 mol·L^–1 thidiazuron (TDZ). Inclusion of the purine analogs 2,6-diaminopurine (DAP), azaadenine or azaguanine to the thidiazuron amended medium inhibited the embryogenic response of the seedlings. DAP-mediated inhibition was not overcome by the addition of adenine sulphate. Inhibition of the embryogenic response by DAP provides evidence that the TDZ-induced accumulation of purine cytokinins is an essential component of TDZ-induced somatic embryogenesis process. Analyses of the endogenous level of purine metabolites indicated that supplementation of the media with TDZ resulted in an overall increase in the endogenous cytokinins while DAP inhibited the purine recycling resulting in decreased levels of endogenous adenine and zeatin.     

Morphoregulatory role of thidiazuron : substitution of auxin and cytokinin requirement for the induction of somatic embryogenesis in geranium hypocotyl cultures

Somatic embryogenesis was induced in hypocotyl explants of geranium (Pelargonium × hortorum) cultured on media supplemented with various concentrations of N-phenyl-N′-1,2,3-thiadiazol-5-ylurea (thidiazuron). In less than 2 weeks, somatic embryos were observed in treatments containing levels of thidiazuron (TDZ) ranging from 0.2 to 1.0 micromolar. The use of N⁶-benzylaminopurine in combination with indole-3-acetic acid also evoked embryogenesis, but the efficiency of somatic embryo production was significantly lower than that obtained with TDZ. Hypocotyl culture for only 2 days on TDZ-supplemented medium before transfer to a basal medium was sufficient for inducing somatic embryogenesis. This distinction between the induction and expression of embryogenesis may provide an experimental system for studying the developmental biology of somatic embryogenesis. Substitution of the auxin-cytokinin requirement for the induction of somatic embryogenesis by TDZ suggests the possibility of a novel mode of its action by modulation of endogenous growth regulators.     

Inhibitory effect of GA3 on the development of thidiazuron-induced somatic embryogenesis in geranium (Pelargonium xhortorum Bailey) hypocotyl cultures

Somatic embryogenesis in geranium (Pelargonium xhortorum Bailey cv Scarlet Orbit Improved) can be achieved by incubating hypocotyl explants on MS medium supplemented with thidiazuron (TDZ; 10 M for 3 days followed by subculture on medium devoid of any plant growth regulators. The presence of gibberellins (GAs) during both the induction and expression phases of embryogenesis was significantly detrimental to somatic embryo formation on the hypocotyl explants. The addition of the GA-synthesis inhibitors paclobutrazol, uniconazole or ancymidol during the period of growth and differentiation of somatic embryos increased the number of somatic embryos formed on each explant. However, paclobutrazol added during the period of induction had no significant influence on somatic embryo formation. Results suggest that both exogenously supplied as well as endogenous GAs play a role, albeit a negative one, on somatic embryogenesis of geranium.Abbreviations MS Murashige and Skoog (1962) medium - MSO basal medium devoid of any plant growth regulator - TDZ N-phenyl-N1,2,3-thidiazol-5-ylurea (thidiazuron)     

Smoke-saturated water promotes somatic embryogenesis in geranium

The effect of smoke saturated-water (SSW) on somatic embryogenesis was studied using geranium hypocotyl culture as a model system. Treatment of explants with 10% SSW or the inclusion of SSW with thidiazuron, a compound which induces somatic embryogenesis, enhanced the embryogenic potential of the geranium hypocotyl culture. Prolonged exposure to SSW was detrimental to embryogenesis. The SSW treatment also accelerated the rate of embryo development suggesting a growth regulatory role of SSW.     

Thidiazuron-induced somatic embryogenesis in intact seedlings of peanut (Arachis hypogaea): Endogenous growth regulator levels and significance of cotyledons

The regulatory role of thidiazuron (TDZ) and explant factors in imparting somatic embryogenic potential was assessed in relation to endogenous growth regulator levels in peanut ( Arachis hypogaea L. cv. Tango). TDZ induced somatic embryogenesis over a range of concentrations (0.5 to 10 μ M ). Culture of seedlings for just 2 days on TDZ-supplemented medium was sufficient to induce somatic embryogenesis. Seedling age and retention or removal of cotyledons during culture influenced morphogenic potential significantly. The younger the seedlings, the better was the embryogenic response to TDZ treatment. The embryogenic potential of seedlings was limited for explants with no cotyledons and they did not respond to increasing levels of TDZ. In contrast, retention of at least one or both the cotyledons resulted in increased response to TDZ. Endogenous levels of cytokinins, auxins and abscisic acid were influenced by TDZ treatment, while TDZ itself was detected only in the cotyledons. The cytokinin N⁶-(2-iso-pentenyl)adenine (2iP) fluctuated on a 10-day cycle in the cotyledons; TDZ treatment suppressed this change and caused an overall decrease in the pool of available 2iP in the tissue. It appeared that TDZ induced somatic embryogenesis in peanut by influencing endogenous levels of both auxin and cytokinins.     

Efficient plant regeneration through direct somatic embryogenesis from leaf explants of <Emphasis Type="Italic">Phalaenopsis</Emphasis> ‘Little Steve’

Summary Leaf segments of the orchid sp. Phalaenopsis ‘Little Steve’ were used as explants testing the effects of 2,4-dichlorophenoxyacetic acid (2,4-D; 0.45, 2.26, 4.52 μM), 6-furfurylaminopurine (kinetin; 2.32, 4.65, 13.95 μM), N⁶-benzyladenine (BA; 2.22, 4.44, 13.32 μM), and 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (TDZ; 2.27, 4.54, 13.62μM) on the induction of direct somatic embryogenesis. After 20–30 d of culture in darkness, clusters of somatic embryos formed from leaf surfaces and wounded regions of explants on half-strength Murashige and Skoog medium supplemented with BA and TDZ. However, kinetin had no response on direct embryo induction. In addition, 2,4-D highly retarded the frequency of embryogenesis that was induced by TDZ. Generally, adaxial surfaces near wounded regions had the highest embryogenic competency compared to other regions of explants. Histological sections revealed that somatic embryos mostly arose from epidermal cell layers of the explants. Secondary embryogenesis occurred at basal parts of embryos, and originated from outer cell layers. Following transfer of regenerated embryos onto growth regulator-free medium for 3.5–4 mo., plantlets with three to four leaves and several roots were obtained. This protocol provides a simple way to regenerate plants through direct somatic embryogenesis, and is suitable for further studies on embryo development and genetic transformation of Phalaenopsis.     

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

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

Somatic embryogenesis and plant regeneration from root sections of <Emphasis Type="Italic">Allium schoenoprasum</Emphasis> L.

A protocol has been developed for somatic embryogenesis and subsequent plant regeneration in Allium schoenoprasum L. Calli were induced from root sections isolated from axenic seedlings and cultivated on media containing either Murashige and Skoog’s (MS) or Dunstan and Short’s mineral solution supplemented with 5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) in combination with 6-benzylaminopurine (BA), 6-furfurylaminopurine (Kin) or thidiazuron (TDZ) at 1, 5 or 10 μM. The highest frequencies of callus induction were achieved on media with 5 μM 2,4-D in combination with 5 μM TDZ or 10 μM BA (78.9% and 78.4%, respectively). Calli were then transferred to 1 μM 2,4-D, where compact yellow callus turned to segmented yellowish callus with transparent globular somatic embryos at the surface. Calli that were previously grown on media with 5 μM 2,4-D in combination with 10 μM BA or 10 μM TDZ showed the highest frequencies of embryogenic callus formation (45% and 42%) as well as mean number of somatic embryos per regenerating callus. The choice of mineral solution formulation did not significantly affect callus induction or embryogenic callus formation. The embryos could complete development into whole plants on plant growth regulator (PGR)-free medium, but inclusion of Kin (0.5, 2.5 and 5 μM) in this phase improved somatic embryo development and multiplication. Subsequently transferred to 1/2 MS PGR-free medium, all embryos rooted and the survival rate of the plants in a greenhouse was 96%.     

Factors Affecting Somatic Embryogenesis Induction and Conversion in “Paradise Tree” (<Emphasis Type="Italic">Melia azedarach</Emphasis> L.)

Factors affecting somatic embryogenesis induction and conversion in paradise tree (Melia azedarach) were evaluated. Somatic embryogenesis was influenced by plant growth regulators, explant stage, carbohydrate source and concentration, gelling agents, light, and induction times. MS medium with 4.54 μM thidiazuron (TDZ) was optimal for the induction of embryogenic tissue. Zygotic embryos that were 1-1.5 mm long (torpedo and early cotyledonal stage) had a greater embryogenic response than smaller or larger embryos and better conversion of somatic embryos into plants. In general, embryos that formed in medium containing 1% or 5% carbohydrate were hyperhydrics or fused, respectively, whereas those that formed in medium with a carbohydrate concentration of 3% had better morphology. Raffinose at 3% yielded satisfactory somatic embryo induction with good morphology and the best values of conversion into plants. Induction and conversion of somatic embryos were superior on medium solidified with agar A-1296. The explants maintained under 160 μmol m⁻² s⁻¹ or 1 week in darkness and later 160 μmol m⁻² s⁻¹ produced a significantly higher embryogenic index. Only 4 days of treatment on induction medium, with either raffinose or sucrose at 3% as a carbohydrate source, were required to induce somatic embryogenesis, but longer exposure, until 18 days, increased the yield and improved the morphology of somatic embryos.     

Induction of high-frequency somatic embryogenesis in geranium (Pelargonium x hortorum Bailey cv Ringo Rose) cotyledonary cultures

Summary The cv Ringo Rose of hybrid seed geranium (Pelargonium x hortorum Bailey), previously shown to be recalcitrant in culture, produced somatic embryos when cotyledonary explants were cultured on regeneration medium containing thidiazuron (TDZ), forchlorfenuron (CPPU), or a combination of indole-3-acetic acid and N⁶ benzylaminopurine (IAA+BAP). Amendment of the basal medium with TDZ (0.5 M) was the most effective treatment. Addition of amino acids to the medium promoted the growth of somatic embryos. Retention of the proximal region of the cotyledon was crucial for regeneration, but the removal of the distal 1/3 to 1/2 cotyledon had no significant effect on somatic embryogenesis. Cotyledonary explants formed somatic embryos in higher frequency and much earlier than hypocotyl explants cultured on the same medium. The somatic embryos induced on cotyledonary explants were germinated on basal medium. More than 70% of the somatic embryos were converted into plants and transferred to soilAbbreviations BAP N⁶-benzylaminopurine - CPPU N-(2-chloro-4-pyridyl)-N'-phenylurea (forchlorfenuron) - IAA indole-3-acetic acid - TDZ N-phenyl-N'-1,2,3,-thiadiazol-5ylurea (thidiazuron)     

Somatic embryogenesis of <Emphasis Type="Italic">Merwilla plumbea</Emphasis> (Lindl.) Speta

A simple and efficient system was developed for rapid somatic embryogenesis from leaf explants of Merwilla plumbea, a traditional but threatened medicinal plant in South Africa. Friable embryogenic callus (FEC) was obtained from leaf explants on embryogenic callus induction medium containing agar-solidified Murashige and Skoog (MS) salts and vitamins, 8.3 μM picloram, 2.3 μM thidiazuron (TDZ) and 20 μM glutamine. FEC was subsequently incubated in embryogenic callus proliferation medium containing 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.1 μM picloram for 7 days before it was transferred to liquid somatic embryo medium (SEM_L) containing MS medium supplemented with 0.4 μM picloram and 0.9 μM TDZ. In SEM_L supplemented with 150 mg L⁻¹ haemoglobin, 5.4–35.6 somatic embryos per settled cell volume of 500 mg FEC were obtained. These embryos were at globular to cotyledonary developmental stages. Embryo maturation, germination and plant formation rate was 94.4% following transfer of SEs to half-strength MS medium supplemented with 1.4 μM gibberellic acid. Plantlets transferred into soil acclimatized in the misthouse and established successfully in the greenhouse (100%). This is the first report on induction of Merwilla plumbea somatic embryogenesis. The protocol developed offers controlled vegetative propagation by alleviating extinction threats, ensures germplasm conservation and provides a system for physiological, biochemical, molecular and cellular studies of embryo development.     

Endogenous hormone levels in explants and in embryogenic and non-embryogenic cultures of carrot

Carrot ( Daucus carota L. F1 hybrid Starca) excised hypocotyls were cultured on Murashige and Skoog medium with and without 2,4-dichlorophenoxy acetic acid (2,4-D) to determine the effect of this plant growth regulator on their further development and their endogenous hormone levels. Culture in the absence of 2,4-D stimulated root development at one end of the hypocotyl segments and increased the endogenous levels of free indole-3-acetic acid (IAA), zeatin/zeatin riboside and N ⁶(Δ²-isopentenyl) adenine/ N ⁶(Δ²-isopentenyl) adenosine, as determined by radio-immunoassay. On the other hand, the presence of 2,4-D in the culture medium promoted callus induction and proliferation, together with abscisic acid (ABA) accumulation, in the hypocotyl segments during the first weeks of culture. When the callus segments generated in the hypocotyl sections cultured in the presence of 2,4-D were cultivated further, the development of two callus types was observed, one composed of preglobular and globular embryos and the other translucent, watery and lacking any sign of organisation. The embryos of the first type germinated when callus segments were transferred to regeneration conditions, while no change was observed when the second type was induced to regenerate. Higher levels of free IAA and ABA were obtained in the embryogenic calli when compared to the non-embryogenic, while no differences were observed among callus types in the other hormones evaluated. The possible role of the different plant hormones during induction of somatic embryogenesis is discussed.     

Somatic embryogenesis and plant regeneration from floral explants of cacao (Theobroma cacao L.) using thidiazuron

Summary A procedure for the regeneration of cacao (Theobroma cacao) plants from staminode explants via somatic embryogenesis was developed. Rapidly growing calli were induced by culturing staminode explants on a DKW salts-based primary callus growth (PCG) medium supplemented with 20 g glucose per L, 9 μM 2,4-D, and thidiazuron (TDZ) at various concentrations. Calli were subcultured onto a WPM salts-based secondary callus growth medium supplemented with 20 g glucose per L, 9 μM 2,4-D, and 1.4 nM kinetin. Somatic embryos were formed from embryogenic calli following transfer to a hormone-free DKW salts-based embryo development medium containing sucrose. The concentration of TDZ used in PCG medium significantly affected the rate of callus growth, the frequency of embryogenesis, and the number of somatic embryos produced from each responsive explant. A TDZ concentration of 22.7 nM was found to be the optimal concentration for effective induction of somatic embryos from various cacao genotypes. Using this procedure, we recovered somatic embryos from all 19 tested cacao genotypes, representing three major genetic group types. However, among these genotypes, a wide range of variation was observed in both the frequency of embryogenesis, which ranged from 1 to 100%, and the average number of somatic embryos produced from each responsive explant, which ranged from 2 to 46. Two types of somatic embryos were identified on the basis of their visual appearance and growth behavior. A large number of cacao plants have been regenerated from somatic embryos and established in soil in a greenhouse. Plants showed morphological and growth characteristics similar to those of seed-derived plants. The described procedure may allow for the practical use of somatic embryogenesis for clonal propagation of elite cacao clones and other applications that require the production of a large number of plants from limited source materials.     

A novel system for rapid high frequency somatic embryogenesis in Medicago sativa

A novel, genotype dependent system for rapid high frequency somatic embryogenesis in Medicago sativa L. was developed in which the first embryos are visible as early as 15 days after the explant (hypocotyl, petiole, leaf) is put into culture. The simplest method involves culture of the explants on a single Murashige and Skoog (MS) medium supplemented with 2 g l−¹ casein hydrolysate, 9 μ M 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.2 μ M kinetin. An efficient two-step, two-medium system was developed to allow separation of the induction and differentiation phases. The explants are cultured on MS with 22.6 μ M 2,4-D and 4.7 μ M kinetin (induction medium) for 10 days and then on basal MS for 20 days. Embryo yields and embryo conversion to plantlets were strongly dependent on the 2,4-D and kinetin concentrations in the induction medium. Both petiole and leaf explants were highly embryogenic and very little callus proliferation occurred when this method was used. Selected clones from three ssp. falcata -based M. sativa cultivars showed a response very similar to the highly regenerable falcata clone F1.1, but it was not possible to produce large numbers of somatic embryos in tissue cultures of cv. Regen S, which is used in most M. sativa tissue culture research, with this procedure. These results suggest that there are two distinct developmental pathways for somatic embryogenesis in M. sativa , with Regen S cultures requiring extensive dedifferentiation during a prolonged callus phase, while the genotypes described in this report have no such requirement.     

Carbon source dependent somatic embryogenesis and plant regeneration in cotton, Gossypium hirsutum L. cv. SVPR2 through suspension cultures

Highly reproducible and simple protocol for cotton somatic embryogenesis is described here by using different concentrations of maltose, glucose, sucrose and fructose. Maltose (30 g/l) is the best carbon source for embryogenic callus induction and glucose (30 g/l) was suitable for induction, maturation of embryoids and plant regeneration. Creamy white embryogenic calli of hypocotyl explants were formed on medium containing MS basal salts, myo-inositol (100 mg/l), thiamine HCI (0.3 mg/l), picloram (0.3 mg/l), Kin (0.1 mg/l) and maltose (30 g/l). During embryo induction and maturation, accelerated growth was observed in liquid medium containing NH3NO4 (1 g/l), picloram (2.0 mg/l), 2 ip (0.2 mg/l), Kin (0.1 mg/l) and glucose (30 g/l). Before embryoid induction, large clumps of embryogenic tissue were formed. These tissues only produced viable embryoids. Completely matured somatic embryos were germinated successfully on the medium fortified with MS salts, myo-inositol (50 mg/l), thiamine HCl (0.2 mg/l), GA3 (0.2 mg/l), BA (1.0 mg/l) and glucose (30 g/l). Compared with earlier reports, 65% of somatic embryo germination was observed. The abnormal embryo formation was highly reduced by using glucose (30 g/l) compared to other carbon sources. The regenerated plantlets were fertile but smaller in height than the seed derived control plants.     

Efficient plant regeneration via somatic embryogenesis and organogenesis from the explants of <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

High-frequency plant regeneration of C. roseus cv. ‘little bright eye’ via somatic embryogenesis and organogenesis from five out of six explants was standardized. Two factors were found to be important for regeneration: (1) the type of explants, and (2) the combination and concentrations of plant growth regulators. The highest regeneration percentage through somatic embryogenesis was obtained from mature zygotic embryo in MS medium supplemented with 7.5 μM of thidiazuron (TDZ). The mature embryo also regenerated efficiently via organogenesis in MS medium supplemented with either 2.5 μM TDZ or 5.3 μM α-naphthalene acetic acid (NAA) and 2.2 μM 6-benzylaminopurine (BA). Hypocotyl and cotyledon did not induce somatic embryogenesis and organogenesis in TDZ-containing medium but gave a maximum percentage of shoots in MS medium supplemented with 5.3 μM NAA and 2.2 μM BA. Stem nodes and meristem tips showed better regeneration via organogenesis in the medium supplemented with NAA and BA and in lower concentrations of TDZ.     

Influence of boric acid on somatic embryogenesis of a cytosterile line of indica rice

The effect of boric acid on somatic embryo induction in rice was investigated using mature seeds of a widely used cytoplasmic male sterile line of indica rice IR-58025. Boric acid was added at a concentration of 100.00, 161.29, 241.93, 322.58 and 403.22 μM to the embryo induction medium containing basal salts, with 9.84 μM 2,4-dichlorophenoxyacetic acid. Boric acid was found to exert different effects on scutellum, coleoptile and root tissues. Increased growth of embryogenic masses with increasing boric acid concentrations was observed on coleoptile tissues of mature seed. Embryogenic response was observed at 161.29 μM boric acid on coleoptile and 241.93 μM boric acid on root tissue. Scutellum tissue showed embryonic response in 161.29 and 241.93 μM boric acid. The results indicate that the boric acid used in standard Murashige and Skoog medium is not sufficient to support induction of somatic embryogenesis in IR-58025 A. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cytokinins induce direc somatic embryogenesis of <Emphasis Type="Italic">Dendrobium</Emphasis> chiengmai pink and subsequent plant regeneration

Summary Four auxins (2,4-dichlorophenoxyacetic acid [2,4-D], indole-3-acetic acid [IAA], indole-3-butyric acid [IBA], and naphthaleneacetic acid [NAA]), and five cytokinins (N ⁶-[2-isopentenyl]-adenine [2iP], N ⁶-benzyladenine [BA], 6-furfurylaminopurine [kinetin], 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea [TDZ], and 6-[4-hydroxy-3-methylbut-2-enylamino]purine [zeatin]) were examined for their effects on direct embryo induction from leaf explants of Dendrobium cv. Chiengmai Pink cultured on 1/2 Murashige and Skoog (MS) medium. Whether in light or darkness, explants easily became necrotic and no embryos were obtained on growth regulator-free or auxin-containing media after 60 d of culture. By contrast, five cytokinins tested induced direct embryo formation from leaf explants, and explants cultured in light had a higher embryogenic response compared with those cultured in darkness. The best condition for direct embryo induction was at 18.16 μM TDZ cultured in light for 60 d, where 33% of explants formed a mean number of 33.6 embryos per explant. During subculture on growth regulator-free 1/2 MS medium, embryos gradually developed into plantlets. Secondary embryogenesis was occasionally found on sheath leaves of embryos. Regenerated plantlets were successfully transplanted and grown in a greenhouse environment.