Somatic Embryogenesis in Capparis decidua (Forsk) Edgew — A Multipurpose Agroforestry Plant

An efficient protocol for direct somatic embryogenesis in Capparis decidua has been developed. Mature zygotic embryos cultured in Murashige and Skoog (MS) liquid medium with 2,4-0 (0.1 mg l^?1) and BA (0.5 mg l^?1) produced somatic embryos directly without an intervening callus phase from the subepidermal cells. Treatment with ABA promoted maturation of somatic embryos and BA (1 mg l^?1)promoted germination. One zygotic embryo produced approximately 230 somatic embryos within 14 to 15 weeks. Embryos germinated in eight weeks and acclimatized plants were transferred to pots.     

In vitro regeneration in Trifolium. 1. Direct somatic embryogenesis in T. rubens (L.)

The origin and development of zygotic and somatic embryos of Trifolium rubens L. was studied with the aid of paraffin sections and light microscopy. Zygotic embryos were collected, fixed and prepared daily from one to ten days after cross-pollination. Somatic embryos were obtained by plating petiole sections on modified L2 medium with 0.015 mgl^-1 picloram and 0.1 mgl^-1 6-BAP. Cultured petioles were collected and fixed daily from one to 25 days after plating. Two regions in the vascular bundle sheath of cultured petioles gave rise to callus. The first region was adjacent to the phloem fibers and produced friable callus. The second region gave rise to compact callus that was connected to the fascicular cambium. Somatic embryos originated from single cells in the cortex directly without intervening callus formation and from single cells in the friable callus. In addition, embryos arose from meristematic regions in compact callus. Many early stages of embryogenesis (one, two and four-celled stages) were observed in the cortex and friable callus. Zygotic embryogenesis in Trifolium differs from other legumes in that the suspensor is short and has a broad attachment. This arrangement was observed in zygotic embryos of T. rubens and in many somatic embryos. However, a continuum of somatic embryogenesis was observed where some young embryos had a Trifolium suspensor-like arrangement while others were attached to a long narrow suspensor-like structure more characteristic of Medicago.     

Somatic embryogenesis versus zygotic embryogenesis in Ensete superbum

In vitro regenerated corm with a shoot incubated on MS medium with modified combination of vitamins supplemented with 2 mg l^–1 2,4-D, 1.5 mg l^–1 BA and 1000 mg l^–1 L-glutamine formed an embryogenic callus. On transfer to a hormone free medium the callus turned black and formed whitish spherical nodules on the peripheral region from which mature embryos grew out in about 40 days. Histological preparations at successive stages in development confirmed the origin of somatic embryos initiated from single cells of the callus. Detailed analysis of the ontogeny of the somatic embryogenesis and zygotic embryogenesis has been done in the present study. Comparison of the ontogenetic stages of the somatic embryogenesis to that of zygotic embryogenesis has shown that the early segmentation of the embryo, the organization of the embryonic apex, formation of cotyledon and epicotyl, the morphology and shape of the zygotic and somatic embryos of E. superbum at successive stages show remarkable similarities in spite of the different environments in which they have developed and differen-tiated.     

Single-cell origin and development of somatic embryos in Picea abies (L.) Karst. (Norway spruce) and P. glauca (Moench) Voss (white spruce)

The origin and development of somatic embryos in calli initiated from immature zygotic embryos of Picea abies (L.) Karst. (Norway spruce) and P. glauca (Moench) Voss (white spruce) was studied. Immature zygotic embryos cultured on callus induction medium produced two types of white calli that were phenotypically different from one another. The callus that proliferated from the hypocotyl region was white to translucent, glossy, mucilaginous and embryogenic. The callus mass which originated from the radicle end was reddish-white, nonmucilaginous and nonembryogenic. Whole mount preparations of the entire explant with two different types of calli showed the presence of embryogenic cells in the mucilaginous callus mass derived from the hypocotyl region of the zygotic embryo. The origin of somatic embryos in both Norway and white spruce could be traced to single cells of the hypocotyl callus.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine     

Somatic embryogenesis and plantlet formation from a rare and endangered tree species,Oplopanax elatus

We tested the possibility of plantlet formation via somatic embryogenesis with leaf segments and mature zygotic embryos from a rare and endangered tree species,Oplopanax elatus. To induce calli, expiants were cultured under darkness in a solid MS medium containing 3% sucrose, 1g L^-1 glutamine, and 0.3% gelrite. Treatment supplements included 2,4-D alone or in combination with thidiazuron. Generally, callus induction and growth were good from leaf expiants, whereas embryogenic calli could be induced only from zygotic embryos. These embryogenic calli were white or pale yellow and very friable. ABA and activated charcoal appeared to be important factors when inducing somatic embryos, with optimum levels being 0.1 mg L^-1 and 0.02%, respectively. Many somatic embryos showed abnormalities during their development on the germination medium, but 35% could be converted if placed on a medium containing gibberellic acid (GA₃). The germinating embryos sometimes formed secondary embryos at the lower portion of the hypocotyls. Normal or converted plantlets were acclimatized in an artificial soil mixture; their survival was about 60% after two months. This culturing system provides a feasible approach for regenerating plants, via somatic embryogenesis, from mature zygotic embryos.     

Changes of 6-benzylaminopurine and 2,4-dichlorophenoxyacetic acid concentrations in plant tissue culture media in the presence of activated charcoal

Somatic embryos and embryogenic callus were initiated from immature zygotic embryos of ginseng (Panax ginseng C.A. Meyer). These somatic embryos were multiplied by adventitious (secondary and tertiary) embryogenesis and their growth and development were dependent on growth hormones in the medium. Auxins, 2,4-d, NAA, and IAA at 1.0 mg l^-1 were effective in inducing secondary and tertiary somatic embryos, which proliferated directly from the apical or cotyledonary portions of the primary somatic embryos. Single somatic embryos or clusters or embryos developed from the explanted primary embryos. Cytokinin (Kn, BA) inhibited adventitious embryogenesis. Secondary somatic embryos developed to maturation and later regenerated into plantlets in two stage process; firstly elongation of the shoot axes on MS +1.0 mg l^-1 Kn, secondly formation of root on 1.0 mg l^-1 Kn+1.0 mg^-1 GA₃ medium.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - IAA in-doleacetic acid - Kn kinetin - BA benzylaminopurine - PSE primary somatic embryo - SSE secondary somatic embryo - TSE tertiary somatic embryo     

Somatic embryogenesis from callus cultures of <Emphasis Type="Italic">Terminalia chebula</Emphasis> Retz.: an important medicinal tree

Somatic embryogenesis was obtained from cotyledon and mature zygotic embryo callus cultures of Terminalia chebula Retz. Callus cultures of cotyledon and mature zygotic embryo were initiated on induction medium containing Murashige and Skoog (MS) nutrients with 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) either 0.01 or 0.1 mg/l Kinetin and 30 g/l sucrose. Induction of somatic embryogenesis, proliferation and development was obtained through different culture passages. Embryogenic cotyledon callus with globular somatic embryos was obtained on MS basal medium supplemented with 50 g/l sucrose. Globular somatic embryos were observed from mature zygotic embryo callus on induction medium. Different stages of somatic embryo development from cotyledon and mature zygotic embryo calluses were observed on MS basal medium supplemented with 50 g/l sucrose after 4 weeks of culture. Histological studies have revealed the developmental stages of somatic embryos. A maximum of 40.3±1.45 cotyledonary somatic embryos/callus was obtained from mature zygotic embryo compared to 7.70±0.37 cotyledonary somatic embryos/callus initiated from cotyledons. Germination of somatic embryos and conversion to plants were achieved. Highest frequency of germination (46.66±0.88) of somatic embryos was obtained on MS basal medium containing benzyladenine (0.5 mg/l) with 30 g/l sucrose.     

High frequency plant regeneration from zygotic-embryo-derived embryogenic cell suspension cultures of watershield (Brasenia schreberi)

An improved protocol for high frequency plant regeneration via somatic embryogenesis from zygotic embryo-derived cell suspension cultures of watershield (Brasenia schreberi) was developed. Zygotic embryos formed pale-yellow globular structures and white friable callus at a frequency of 80% when cultured on half-strength MS medium supplemented with 0.3 mg l⁻¹ 2,4-D. However, the frequency of formation of pale-yellow globular structures and white friable callus decreased slightly with increasing concentrations of 2,4-D up to 3 mg l⁻¹, where the frequency reached ~50% of the control. Cell suspension cultures from zygotic embryo-derived white friable callus were established using half-strength MS medium supplemented with 0.3 mg l⁻¹ 2,4-D. Upon plating of cell aggregates on half-strength MS basal medium, approximately 8.3% gave rise to somatic embryos and developed into plantlets. However, the frequency of plantlet development from cell aggregates was sharply increased (by up to 55%) when activated charcoal and zeatin were applied. Regenerated plantlets were successfully transplanted to potting soil and grown to normal plants in a growth chamber. The distinctive feature of this study is the establishment of a high frequency plant regeneration system via somatic embryogenesis from zygotic embryo-derived cell suspension cultures of watershield, which has not been previously reported. The protocol for plant regeneration of watershield through somatic embryogenesis could be useful for the mass propagation and transformation of selected elite lines.     

High-frequency plant regeneration via somatic embryogenesis and organogenesis and in vitro flowering of regenerated plantlets in Panax ginseng

 The morphogenesis ability of light yellowish globular callus derived from cotyledons of mature zygotic embryos of Panax ginseng was investigated. The optimal media for somatic embryogenesis and shoot organogenesis were MS medium containing 0.5 mg l^–1 2,4-dichlorophenoxyacetic acid, 0.1 mg l^–1 6-benzyladenine (BA), and 500 mg l^–1 lactoalbumin hydrolysate, and SH medium supplemented with 0.5 mg l^–1 α-naphthaleneacetic acid, 0.1 mg l^–1 BA, and 500 mg l^–1casein hydrolysate. The influences of glucose, mannose, fructose, and sorbose in the media on somatic embryogenesis and shoot organogenesis were revealed as differences in the numbers of somatic embryos and adventitious shoots per gram of morphogenic callus. The best regeneration of somatic embryos was obtained on medium containing glucose, with a mean of 8.7 somatic embryos per gram of callus. The best regeneration of shoots was observed on medium containing fructose, with an average of 12.2 adventitious shoots per gram of callus. Of the somatic embryos 95% were converted into regenerated plantlets, and 100% of adventitious shoots rooted to form regenerated plantlets. Regenerated plants were successfully established in soil. Flowering was observed in 5.7% of the regenerated plants derived from shoot organogenesis and in 1.4% of the regenerated plants derived from somatic embryogenesis. Received: 1 December 1998 / Revision received: 13 September 1999 / Accepted: 20 September 1999     

Histological comparison of single somatic embryos of maize from suspension culture with somatic embryos attached to callus cells

An embryogenic suspension culture of Zea mays, genotype 4C1, was obtained from friable callus that was cultured on solid medium and had been obtained from zygotic embryos. The suspension contained non-dividing elongated cells, clusters of dividing isodiametric cells, and globular, ovoid, and polar stages of somatic embryos. The single somatic embryos were blocked in shoot meristem formation: when transferred to regeneration medium they developed a root and, at the shoot side, a green cap with meristematic cells, but a scutellum and leaf primordia were not formed. In medium containing 2,4-dichlorophenoxy acetic acid, somatic embryos formed embryogenic callus aggregates, consisting of globular stage somatic embryos attached to each other via undifferentiated callus cells. These somatic embryos developed into mature embryos with the zygotic histological characteristics, such as scutellum and leaf primordia, in maturation medium, and then regenerated into plants in regeneration medium. By omitting the maturation phase, regeneration occurred via organogenesis. Polyembryos, i. e. embryos attached to each other without callus tissue in between, behaved as single somatic embryos. It is concluded that the attached callus tissue provides a factor that stimulates scutellum and leaf primordia formation.Abbreviations CMM callus maintenance medium - 2,4D 2,4-dichlorophenoxy acetic acid - PCV packed cell volume - MS Murashige and Skoog medium     

Plant regeneration through somatic embryogenesis from callus induced on immature embryos of Alstroemeria spp. L.

Summary The plant regeneration ability of callus obtained from zygotic embryos of the monocot Alstroemeria spp. was studied. The best explants for somatic embryogenesis were immature zygotic embryos in half-ovules when the endosperm was still soft and white. For 2 genotypes embryogenic callus was induced on callus induction medium with a success rate of 54%. The best callus induction period was 10 weeks. The morphology of embryogenic callus was nodular. Somatic embryos were formed after transfer of the callus to regeneration medium. These somatic embryos revealed later on the typical features of zygotic Alstroemeria embryos. The total duration of the plant regeneration protocol, from inoculation till rooted plantlets ready for transfer to the greenhouse, was 28 weeks.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) - NAA -naphthaleneacetic acid     

Plant Regeneration Through Somatic Embryogenesis in Taxus wallichiana

We describe an efficient process for regeneration of Taxus wallichiana (Zucc) plants 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 I^?1 6-benzyladenine (BA) and 1.0–2.0 mg I^?1 á-Napthaleneacetic acid (NAA) produced compact yellow (CY) callus after 4 weeks of culture. The 8-week-old CY call! (lines CY-A and CY-B) were initially slow growing but proliferated on transfer to WPM basal medium supplemented with 8.0 mg I^?1 2,4-D, 0.1–0.9 mg^?1 NAA and 0.3–1.0 mg^?1 BA after 4 weeks. Four morphologically distinct calli lines were obtained, of which only two call! lines, CY-B-FW and CY-B-FY were embryogenic. The 12-week-old callus line CY-B-FW developed globular somatic embryos on transfer to secondary medium after 8 weeks and matured in maturation medium after 4 weeks. Only 10% of the mature somatic embryos regenerated into complete plantlets after 4 weeks on conversion medium. Although the frequency of conversion was low, complete regenerated plantlets via somatic embryogenesis were obtained after 7–8 months of initiation of culture. Taxane analysis showed that the paclitaxel accumulation was higher in embryogenic callus than in non-embryogenic callus.     

Plant regeneration via somatic embryogenesis from in vitro tissue culture in two Tunisian Cucumis melo cultivars Maazoun and Beji

Hypocotyl, cotyledon and zygotic embryo explants from two Tunisian Cucumis melo L. cultivars Beji and Maazoun, cultured on the MS medium added with 2,4-D (0.25–1 mg l⁻¹) and BA (0.10–0.50 mg l⁻¹), produce calluses with somatic embryos after 3 weeks of culture. For Beji c.v. the highest percentage (62.50%) of embryogenesis was observed for cotyledons. The average embryo number per callus was 10.40. Embryogenesis induction for zygotic embryos reached 33.50% with 29 embryos per callus. The embryogenesis ability of hypocotyls did not exceed 12.50% (2.50 embryos per callus). Somatic embryogenesis for Maazoun c.v. explants was less efficient. Embryos formation was observed only for cotyledons (29%) and zygotic embryos (25%). Cotyledonary staged embryos, when transferred to hormone free MS medium, germinated. The maximum germination rates were 51.50 and 44.50%, respectively for Maazoun and Beji c.v. The highest percentage (36.50%) of survival plants was noted for Beji c.v. Regenerants were diploids (2n = 2x = 24) and morphologically similar to their parents issued from seeds.     

黄山栾树体细胞胚的发生和组织学观察

黄山栾树无菌苗的节间和叶柄离体培养后,其体细胞胚发生的结果表明：节间愈伤组织可诱导产生体细胞胚,而叶柄愈伤组织则生根：节间愈伤组织诱导培养基为MS＋3．0mg．L～2,4．D＋0．5～3．0mg．L-1NAA;节间胚性愈伤组织诱导培养基为MS＋2．0nag．L-2,4-D;胚性愈伤组织转移到无植物生长调节剂的MS培养基上可发育成正常植株。组织学观察表明,体细胞胚在胚性愈伤组织中有的发生于愈伤组织表层细胞,有的发生在愈伤组织内部。黄山栾树体细胞胚的形成经历球形胚、心形胚、鱼雷胚和子叶胚几个阶段,这与合子胚的发育途径相似。     

Herbicide resistant transgenic papaya plants produced by an efficient particle bombardment transformation method

A system for the production of transgenic papaya (Carica papaya L.) plants using zygotic embryos and embryogenic callus as target cells for particle bombardment is described. Phosphinothricin (bar ) and kanamycin (npt II) resistance genes were used as selectable markers, and the gus gene (uidA) as a reporter gene. Selection with 100 mg/l kanamycin and 4 mg/l phosphinothricin (PPT) yielded a total of over 90 resistant embryogenic colonies from three independent experiments using embryogenic callus as a target tissue. This represents an efficiency of 60 transgenic clones per gram of fresh weight callus bombarded. The efficiency of genetic transformation using zygotic embryos was lower, as only 8 independent resistant clones were recovered out of 645 bombarded zygotic embryos, giving a efficiency of 1.24%. Subsequent subculture of transgenic somatic embryos both from zygotic embryos and embryogenic callus led to the development of plants with apparently normal morphology. Histological, fluorimetric assay for GUS, NPT II assay and DNA analysis (Southern hybridization) showed that kanamycin /PPT resistant plants carried and expressed the transgenes.Abbreviations Gus -glucuronidase - NPTII neomycin phophotransferase II - bar phophinothricin acetyl transferase gene - Pat phosphinothricin acetyl transferase - PPT phosphinothricin - Km kanamycin - 2,4-D 2,4-dichlorophenoxyacetic acid - K kinetin - BAP benzylaminopurine - IBA indolbutyric acid     

Effects of type of explant and age,plant growth regulators and medium strength on somatic embryogenesis and plant regeneration in <Emphasis Type="Italic">Eucalyptus camaldulensis</Emphasis>

Plant regeneration was achieved through direct and indirect somatic embryogenesis in Eucalyptus camaldulensis. Callus was induced from mature zygotic embryos and from cotyledon explants collected from 10, 15, 25, and 30-day-old seedlings cultured on Murashige and Skoog (MS) basal medium supplemented with different concentrations of naphthaleneacetic acid (NAA). Maximum callus induction from mature zygotic embryos was obtained on MS basal medium containing 1 mg l⁻¹ NAA. The frequency of callus development varied based on the age of the cotyledon explants 10-day-old explants giving highest percentage on MS basal medium supplemented with 1 mg l⁻¹ NAA. Callus obtained from mature zygotic embryos gave highest frequency of somatic embryogenesis on MS basal medium containing 0.5 mg l⁻¹ benzyladenine (BA) and 0.1 mg l⁻¹ NAA. Separate age wise culture of the calli, obtained from cotyledons of different ages cultured separately, revealed high somatic embryogenic potential on callus from 10-day-old cotyledons. Direct somatic embryogenesis too was obtained from hypocotyl explants without an intervening callus phase on MS basal medium containing 0.5 mg l⁻¹ BA. The effects of abscisic acid (ABA), sucrose, and different strengths of MS medium on somatic embryo maturation and germination were also investigated. Number of mature somatic embryos increased with lower concentrations (0–1 mg l⁻¹) of ABA while no significant differences were observed at higher concentrations (2–5 mg l⁻¹) of ABA. Compared to basal medium containing lower concentrations of sucrose (1%), the MS medium supplemented with higher levels of sucrose (4%) showed significantly lower frequency of mature somatic embryos. Basal medium without any dilution gave the highest number of immature embryos. However, the number of mature embryos was high at higher medium dilutions.     

Somatic embryogenesis and plant regeneration in different organs ofEuterpe edulis mart. (Palmae): Control and structural features

Somatic embryogenesis and further plant regeneration were observed using zygotic embryos, young inflorescences and young leaves ofEuterpe edulis (Palmae) as explants. Both for the cultures of zygotic embryos and inflorescences, activated charcoal in the medium was essential for the establishment of viable cultures. Embryogenesis was induced by using a gelled basal medium with MS or Euwens salts supplemented by high 2, 4-D levels (50–100 mg L⁻¹). The embryogenic process was direct without a callus stage. For further development, cultures with globular or post-globular embryos were transferred to the basal medium with 2-iP (2.5 mg L⁻¹) and NAA (0.1 mg L⁻¹). To convert embryos to plantlets, cultures were transferred to a third medium in which sucrose and salts were reduced to the half-strenght of the basal medium, without growth regulators. In the case of liquid medium, with either 2, 4-D or NAA (10–20 mg L⁻¹). The developmental stage of each explant was critical for the induction of embryogenesis. The histological study of embryogenic cultures revealed that in the case of zygotic embryos, somatic embryos arise directly from the surface of the cotyledonar node, or from subepidermal tissues. In the inflorescences, a pro-embryogenic tissue is formed at the floral primordium region; in the leaves, the first morphogenic event is cell proliferation in the vascular parenchyma.     

High-frequency somatic embryogenesis from germinated zygotic embryos of <Emphasis Type="Italic">Schisandra chinensis</Emphasis> and evaluation of the effects of medium strength,sucrose, GA<Subscript>3</Subscript>, and BA on somatic embryo development

We developed a new protocol for highly efficient somatic embryogenesis and plantlet conversion of Schisandra chinensis. Friable embryogenic callus was induced from cotyledonary leaves and hypocotyls of germinated zygotic embryos on Murashige and Skoog (MS) agar medium containing 2,4-dichlorophenoxyacetic acid (2,4-D). Preculture of zygotic embryos on 2,4-D-containing medium increased embryogenic callus induction efficiency. The highest embryogenic callus induction frequency of 56.7% was obtained from shoot apical meristem-containing hypocotyl explants from 1-week-old germinated embryos on MS medium containing 4.0 mg l⁻¹ 2,4-D. Embryogenic callus proliferation, somatic embryo (SE) formation, and subsequent plantlet conversion occurred under optimal culture conditions. The effects of MS medium strength, sucrose, gibberellic acid (GA₃), and 6-benzyladenine (BA) on SE formation and plantlet conversion were evaluated. Low MS medium strength (1/4 to 1/2) was necessary for SE formation, and the optimal sucrose concentration was 2.0%. Supplementing medium with GA₃ negatively impacted SE formation and subsequent development. BA significantly increased the number of SEs and the plantlet conversion capacity. One-third-strength MS medium with 1.0% sucrose and 0.5 mg l⁻¹ BA produced the highest number of SEs (309 embryos from 9 mg embryogenic callus) and the highest frequency of plantlet conversion from germinated SEs (52.6%). When transplanted to soil, 90% of the regenerated plants developed into normal plants.     

Effects of growth regulator concentrations and explant size on shoot organogenesis from callus derived from zygotic embryos of sunflower (Helianthus annuus L.)

Effects of medium growth regulator composition and embryo size on shoot organogenesis of callus derived from globular- to torpedo-shaped zygotic embryos of five sunflower (Helianthus annuus L.) genotypes were examined. Forty growth regulator combinations composed of 0 to 5 mgl^-1 naphthaleneacetic acid (NAA) and 0 to 1 mgl^-1 6-benzylaminopurine (BA) were tested. The frequency of zygotic embryos forming shoot-regenerating callus was analysed according to categorical data modelling using a maximum-likelihood approach. Both NAA and BA must be present to induce the formation of morphogenic callus from zygotic embryos, but each growth regulator effect varied with the genotype. For four genotypes, NAA and BA effects were neither linear nor quadratic; whereas, they were linear for the fifth one. Most effective concentrations across genotypes were 0.1 mgl^-1 NAA and 0.5 mgl^-1 or 0.2 mgl^-1 BA. However, the optimal growth regulator combination depended on the genotype and an interaction between the two growth regulators. The frequency of shoot-regenerating callus also varied with the size of the embryo explant. For all five genotypes, 0.4 to 1.2 mm long heart-shaped zygotic embryos formed morphogenic callus more frequently than smaller less-developed ones.     

Regeneration of Acacia mangium through somatic embryogenesis

 Somatic embryogenesis and whole plant regeneration were achieved in callus cultures derived from immature zygotic embryos of Acacia mangium. Embryogenic callus was induced on MS medium containing combinations of TDZ (1–2 mg/l), IAA (0.25–2 mg/l) and a mixture of amino acids. Globular embryos developed on embryogenic callus cultured on the induction medium. Nearly 42% of embryogenic cultures with globular embryos produced torpedo- and cotyledonary-stage embryos by a two-step maturation phase. The first stage occurred on 1/2-strength MS basal medium containing 30 g/l sucrose and 5 mg/l GA₃ followed by the second stage on 1/2-strength MS basal medium containing 50 g/l sucrose. Of the cotyledonary-stage somatic embryos, 11% germinated into seedlings that could be successfully transferred to pots. Light- and scanning electron microscopy showed that the somatic embryos originated from single cells of the embryogenic callus. Further, a single cell layer could be detected beneath the developing somatic embryos that appeared to be a demarcation layer isolating the somatic proembryonic structure from the rest of the maternal callus. A suspensor-like structure connected the globular embryos to the demarcation layer. This is the first successful report of plant regeneration through somatic embryogenesis for this economically important tropical forest species. Received: 20 January 2000 / Revision received: 28 September 2000 / Accepted: 29 September