Somatic embryogenesis in mature zygotic embryos of <Emphasis Type="Italic">Picea likiangensis</Emphasis>

Somatic embryogenesis (SE) was successfully induced from mature zygotic embryos of seven families of Picea likiangensis (Franch.) Pritz after 20 weeks culture on initiation medium. Three basal media (one-half strength LM medium, one-half strength LP medium and improved LP medium) with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (6-BA) were tested but only one-half strength LM medium supplemented with 2,4-D and 6-BA was successful for the embryogenic cultures (EC) initiation. The initiation frequencies of EC varied greatly from different families when culturing on the same initiation medium. The highest frequency (41.3%) was induced from one of the families on one-half strength LM medium supplemented with 3 mg L⁻¹ 2,4-D and 1.5 mg L⁻¹ 6-BA and 16.83% on average for seven families. EC were subcultured and proliferated on the same medium as the initiation one every 10 days. 3 lines of EC induced from the same family were applied in maturation experiment. Cotyledonary somatic embryos were observed after EC were transferred to maturation media of one-half strength LM medium containing 20-80 mg L⁻¹ abscisic acid and 7.5% polyethylene glycol (PEG-4000). However, one-half strength LM medium supplemented with 40 mg L⁻¹ or 60 mg L⁻¹ ABA and 7.5% PEG gave the best maturation and the 3 lines showed different ability in maturation. Over 80% cotyledonary somatic embryos germinated normally on DCR medium containing 0.2% activated carbon. The success on SE induction of the species has provided an effective clonal propagation method for this important tree’s genetic improvement.     

Somatic embryogenesis from zygotic embryos of <Emphasis Type="Italic">Schisandra chinensis</Emphasis>

We describe the multi-step regeneration system of medicinal plant Schisandra chinensis (Turcz.) Baill. The seeds were pre-treated with 0.005 μM thidiazuron. Subsequently the zygotic embryos of the early heart stage were cultured on medium with 50 μM of 2,4-dichlorophenoxyacetic acid (2,4-D) and after three weeks the embryogenic calli were transferred to a medium with 10 μM of 2,4-D and 4 μM of 6-benzyladenine and were sub-cultured at the 4-week intervals. Abscisic acid (30 μM) and polyethyleneglycol (3 %) significantly influenced the synchronization of development of the somatic embryos (SEs) to the globular stage. The following culture on a medium without growth regulators resulted in full developed cotyledonary stage SEs. Indole-3-butyric acid (0.05 μ) contributed to their rapid conversion to plantlets.This experimental work was supported by project No. 521/02/1129 of the Grant Agency of the Czech Republic.     

Organogenesis from root explants of commercial populations of <Emphasis Type="Italic">Passiflora edulis</Emphasis> Sims and a wild passionfruit species, <Emphasis Type="Italic">P. cincinnata</Emphasis> Masters

Root explants of a wild passionfruit species (Passiflora cincinnata) and three P. edulis commercial populations (‘FB 100’, ‘FB 200’, and ‘FB 300’) were incubated on Murashige and Skoog (MS) medium supplemented with 4.44 μM 6-benzyladenine (BA) to induce shoot organogenesis. Shoots elongated in liquid medium with 2.89 μM gibberellic acid (GA₃) under agitation were rooted in coconut fiber and acclimatized followed by transfer to a greenhouse into pots containing mixture of coconut fiber and Plantmax^® (1:1). Explant samples were collected during organogenesis and submitted to light and scanning electron microscopy (SEM). Root explants of P. cincinnata responded earlier than those of P. edulis. However, on the third assessment, at 90 days, the genotype ‘FB 200’ showed shoot number significantly higher than ‘FB 100’ and ‘FB 300’, not differing from P. cincinnata. Organogenesis in P. cincinnata and P. edulis occurred via direct pathway, which was confirmed by anatomical studies and SEM. Flow cytometric analysis revealed no variation in DNA content of regenerated plantlets among all genotypes. Nuclear DNA (2C) values (pg) in regenerants of P. cincinnata (2.99 pg) and P. edulis (3.26–3.28 pg) were consistent with DNA amounts of seed-derived control plants.     

Somatic embryogenesis from immature zygotic embryos of annatto (<Emphasis Type="Italic">Bixa orellana</Emphasis> L.)

Summary In order to establish a protocol for somatic embryogenesis of annatto, Bixa orellana L., seeds (70 d after anthesis) from field-grown orchards had their coats dissected off, and immature zygotic embryos were excised aseptically from immature seeds collected from field-grown trees and used as explants. Embryos were cultured onto MS medium supplemented with or without different combinations of plant growth regulators and activated charcoal. Direct somatic embryogenesis was induced on explants incubated either in Murashige and Skoog (MS), 2,4-dichlorophenoxyacetic acid (2,4-D), and/or kinetin-supplemented media after 25 d of culture. The highest frequencies of embryogenesis and embryos per explant were obtained on medium containing 2.26 μM 2.4-D, 4.52μM kinetin, and 1.0 gl⁻¹ activated charcoal. The presence of charcoal was critical in increasing embryos per explant, to reduce the time to obtain somatic embryos, and mainly to prevent callus proliferation and subsequent indirect somatic embryogenesis. No embryogenic response was achieved when mature embryos were used. It was also observed that embryogenic response was significantly affected by genotype. Histological investigations revealed that primary direct somatic embryos differentiated exclusively from the protodermis or together with the outer ground meristem cell layers of the zygotic embryo axis, and from the protodermis of zygotic cotyledons. Diverse morphological differences, including malformed embryos, were observed among somatic embryos. In spite of the high frequencies of histodifferentiation of all embryo stages, a very low conversion frequency to normal plants from somatic embryos was observed.     

Somatic embryogenesis and plant regeneration from immature zygotic embryos of <Emphasis Type="Italic">Melia azedarach</Emphasis> (Meliaceae)

Summary A procedure for the regeneration of ‘paradise tree’ (Melia azedarach, Meliaceae) plants from immature zygotic embryos via somatic embryogenesis was developed. Somatic embryos were induced from explants cultured on Murashige and Skoog medium supplemented with 0.45, 4.54, or 13.62 μM thidiazuron. Histological examination revealed that somatic embryos were induced directly from the explants. Further development of somatic embryos was accomplished with Murashige and Skoog medium at quarter-strength with 3% sucrose. A large number of plants were regenerated from somatic embryos and successfully established in soil in a greenhouse. These plants are morphologically similar to those of seed-derived plants. This system may be beneficial for mass propagation as well as for genetic manipulation of the ‘paradise tree’.     

A novel regeneration system for a wild passion fruit species (<Emphasis Type="Italic">Passiflora cincinnata</Emphasis> Mast.) based on somatic embryogenesis from mature zygotic embryos

The objective of the present work was to induce somatic embryogenesis from zygotic embryos of Passiflora cincinnata Masters. Zygotic embryos formed calli on media with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.5 μM benzyladenine (BA) after 30 days of in vitro culture. A concentration of 18.1 μM 2,4-D resulted in the largest number of somatic embryos. Embryogenic calli were yellowish and friable, forming whitish proembryogenic masses. Morphologically, embryogenic cells were small and had large nuclei and dense cytoplasm, whereas non-embryogenic cells were elongated, with small nuclei and less dense cytoplasm. Calli cultured under white light on basal Murashige and Skoog’s medium with activated charcoal produced embryos in all developmental stages. There were differences among the treatments, with some leading to the production of calli with embryos and some only to callus formation. Some abnormalities were associated with somatic embryos, including fused axes, fused cotyledons and polycotyledonary embryos. Production of secondary somatic embryos occurred in the first cycle of primary embryo development. Secondary embryos differentiated from the surface of the protodermal layer of primary embryos with intense cell proliferation, successive mitotic divisions in the initial phase of embryoid development, and a vascular system formed with no connection to the parental tissue. This secondary embryogenic system of P. cincinnata is characterized by intense proliferation and maintenance of embryogenic competence after successive subcultures. This reproducible protocol opens new prospects for massive propagation and is an alternative to the current organogenesis-based transformation protocol.     

Somatic embryogenesis and plant regeneration from immature zygotic embryos of<Emphasis Type="Italic"> Cryptomeria japonica</Emphasis> D. Don

This report describes the successful plant regeneration via somatic embryogenesis from immature zygotic embryos of Cryptomeria japonica D. Don. For the induction of embryogenic tissue, we determined that the optimal medium contained N⁶-benzyladenine and 2,4-dichlorophenoxyacetic acid. Immature zygotic embryos that were collected at the end of June yielded embryogenic tissue at the highest frequency. Embryogenic tissues that had proliferated in liquid medium included small and loosely packed cells and elongating or elongated cells. We used ten cell lines to determine the optimal medium for the development of somatic embryos. Induced somatic embryos germinated with synchronous sprouting of cotyledons, hypocotyls and roots. Gibberellin A₃ in the germination medium had a positive effect on both the elongation of hypocotyls and the survival of seedlings. The frequencies of induction and germination of somatic embryos differed among the cell lines examined. Most of the seedlings grew normally. This system of somatic embryogenesis required 4–5 months for the regeneration of C. japonica plantlets from immature zygotic embryos.Abbreviations ABA Abscisic acid - BA N⁶-Benzyladenine - 2,4-D 2,4-Dichlorophenoxyacetic acid - GA₃ Gibberellin A₃Communicated by F. Sato     

Somatic embryogenesis in <Emphasis Type="Italic">Buchanania lanzan</Emphasis> spreng

Summary We report a protocol for somatic embryogenesis and plantlet regeneration of Buchanania lanzan Spreng (Family—Anacardiaceae), which is a tropical fruit tree widely distributed in the dry forests of India. Calluses were initiated from immature zygotic embryos cultured on Murashige and Skoog (MS) medium supplemented with various combinations of 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzyladenine (BA) and/or 1-naphthaleneacetic acid (NAA). The highest frequency (60%) of somatic embryo induction was obtained in cultures grown on MS medium fortified with 4.53 μM 2,4-D, 5.32 μM NAA and 4.48 μM BA. The medium supplemented with 15 μM abscisic acid (ABA) was most effective for maturation and germination of somatic embryos. This is the first report on somatic embryogenesis in B. lanzan, which may be helpful for in vitro propagation, ex situ conservation and genetic manipulation of this species.     

<Emphasis Type="Italic">In vitro</Emphasis> organogenesis of <Emphasis Type="Italic">Passiflora alata</Emphasis>

Passiflora alata in vitro organogenesis was studied based on explant type, culture medium composition, and incubation conditions. The results indicated that the morphogenic process occurred more efficiently when hypocotyl segment-derived explants were cultured in media supplemented with cytokinin and AgNO₃ incubated under a 16-h photoperiod. The shoot bud elongation and plant development were obtained by transferring the material to MSM culture medium supplemented with GA₃ and incubated in flasks with vented lids. Histological analyses of the process revealed that the difficulties in obtaining plants could be related to the development of protuberances and leaf primordia structures, which did not contain shoot apical meristem. Roots developed easily by transferring elongated shoots to 1/2 MSM culture medium. Plant acclimatization occurred successfully, and somaclonal variation was not visually detected. The efficiency of this organogenesis protocol will be evaluated for genetic transformation of this species to obtain transgenic plants expressing genes that can influence the resistance to Cowpea aphid borne mosaic virus.     

Somatic embryogenesis from rhizome explants of <Emphasis Type="Italic">Cymbopogon winterianus</Emphasis>

Plantlet regeneration through indirect somatic embryogenesis was attempted from rhizome derived callus of Cymbopogon winterianus Jowitt (cv. Jorlab2). Optimum callus was induced on Murashige and Skoog (MS) basal medium supplemented with 4 mg dm⁻³ 2,4-dichlorophenoxyacetic acid (2,4-D). Initially the callus was friable, shiny white and watery in nature. After subculturing on MS medium containing 2,4-D and kinetin (Kn), callus was transferred onto the MS medium supplemented with 2,4 -D, Kn and coconut water to induce somatic embryogenesis. Optimum somatic embryogenesis (78.33 %) was achieved on MS medium containing 3.0 mg dm⁻³ 2,4-D and 0.5 mg dm⁻³ Kn. High frequency (65 %) plantlet conversion from embryos was achieved in MS medium supplemented with 2 mg dm⁻³ N⁶-benzyladenine (BA), 0.5 mg dm⁻³ Kn, 0.2 mg dm⁻³ calcium pantothenate and 0.2 mg dm⁻³ biotin.     

Somatic embryogenesis from <Emphasis Type="Italic">Arabidopsis</Emphasis> shoot apical meristem mutants

Zygotic embryos of three Arabidopsis thaliana (L.) Heynh. mutants lacking an embryonic shoot apical meristem (SAM), shoot meristemless (stm), wuschel (wus) and zwille/pinhead (zll/pnh) were used as explants to establish embryogenic cell cultures. Somatic embryos of all three mutants showed the same mutant phenotypes as their zygotic equivalents. These results provide genetic evidence that the developmental program of somatic and zygotic embryos is indistinguishable. They also suggest that a functional SAM is not required for somatic embryogenic cell formation in Arabidopsis.     

Somatic embryogenesis in oak (<Emphasis Type="Italic">Quercus</Emphasis> spp.)

Summary The present review summarizes the factors involved in controlling the process of oak somatic embryogenesis as a method for vegetative plant propagation and includes also data on artificial seed production, cryopreservation and transformation. One major limitation, the inability to initiate embryogenic cultures from mature trees, has been recently overcome. Leaves from selected cork oak trees with an age of 50 yr and more have been used to initiale somatic embryogenesis (SE) with a frequency of up to 20%. These findings offer encouraging prospects for cloning proven superior plant material and to integrate this propagation system into tree improvement programs. Once the process of SE has been initiated, the multiplication cycle proceeds via secondary embryogenesis, which can be maintained indefinitely. Problems are reported by the formation of anomalous embryos. The mutability of somatic embryogenic cell lines of various oak species has been monitored by flow cytometry and molecular markers. No somaclonal variation was detected applying random amplified polymorphic DNA (RAPD) or amplified fragment length polymorphism (AFLP) markers, whereas DNA-content measurements via flow cytometry revealed tetraploidy in some cell lines after several years of continuous subculture. Maturation and low germination frequencies are the main bottlenecks for a broader use of this technique. Recently attention has been on embryo quality and parameters for conversion capacity such as high endogenous cytokinin level and low abscisic acid (ABA) level. Although oak is probably the species that is the most well-developed system for a broadleaved forest tree, data on growth performances of somatic embryo-derived plants are rare.     

Somatic embryogenesis and plant regeneration from immature zygotic embryo cultures of mountain ash (<Emphasis Type="Italic">Sorbus pohuashanensis</Emphasis>)

Plant regeneration through somatic embryogenesis was achieved using immature zygotic embryos (ZE) of Sorbus pohuashanensis as explants. Over 50% of immature ZEs from immature seed collected at 30 days after pollination produced direct somatic embryos (SEs) on Murashige and Skoog (MS) medium supplemented with 0–0.44 μM 6-benzyladenine (BA) in combination with 5.73 μM naphthaleneacetic acid (NAA) or with 0.91–2.26 μM 2,4-dichlorophenoxyacetic acid (2,4-D) alone. Fourteen to 23 SEs per explant were regenerated on MS medium supplemented with BA 0.44 μM in combination with NAA 5.73 μM. SE formation decreased when sucrose concentrations were higher than 40 g L⁻¹. Repetitive embryogenesis occurred following culture on solid MS medium containing 12 μM abscisic acid, 75 g L⁻¹ polyethylene glycol, and 20 g L⁻¹ sucrose at 25 ± 1°C under a 16-h photoperiod with a light intensity of 40 μmol m⁻² s⁻¹. Over 40% of the mature SEs germinated on solid MS medium under light condition described previously. Up to 40% of the regenerated plantlets were successfully acclimatized under greenhouse conditions. Plantlets derived from SEs grew vigorously with similar morphology as those germinated from ZEs. Histological studies of explants at various developmental stages of somatic embryogenesis revealed that SEs passed through globular, heart, torpedo, and mature stages. Similar to ZE suspensors, similar structures of SE degenerated in later stages of embryo development. ZE and SE are a effective means of regenerating tissue culture plantlets for S. pohuashanesis.     

Somatic embryogenesis of <Emphasis Type="Italic">Myrciaria aureana</Emphasis> (Brazilian grape tree)

The aim of this research was to establish a long-term somatic embryogenic cultures that could be used for cryopreservation. For the induction of somatic embryogenesis, different levels of 2,4-D as well as the combination of 2,4-D and indole-3-acetyl-l-aspartic acid (IASP) were tested on cotyledons of zygotic embryos. The somatic embryogenic cultures were established and maintained up to 2 years through frequent subculturing on a medium containing 2,4D + IASP. Light, activated charcoal, and polyethylene glycol (PEG) were tested for the regeneration and maturation of somatic embryos, and the mature embryos were germinated in JADS medium. The combination of light and PEG provided the highest number of mature embryos. The somatic embryos obtained were smaller than zygotic embryos and lacked starch. There was an interaction between 2,4-D and IASP on the induction and regeneration of somatic embryo in Myrciaria aureana. The combination of light and PEG increased the number of mature embryos; however, charcoal was detrimental to the process.     

Somatic embryogenesis and <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation in <Emphasis Type="Italic">Bixa orellana</Emphasis> L.

Establishment, maintenance, regeneration, and transformation of somatic embryos by both direct and indirect means (callus-mediated) was achieved for Bixa orellana, a tropical plant whose seeds produce commercially edible ‘annatto pigment,’ which mainly constitutes an apocarotenoid called bixin. Callus-mediated methodology was found to be efficient in producing a greater number of embryos in a short time. The maximum of 28 somatic embryos were produced in 16–18 weeks when immature zygotic embryonic stalks were inoculated onto Murashige and Skoog (MS) medium containing B5 vitamins supplemented with 0.44 μM benzyladenine (BA), 0.054 μM α-naphthaleneacetic acid (NAA), 2.89 μM gibberellic acid (GA₃), 0.02 μM triiodobenzoic acid (TIBA), and 0.011 μM triacontanol (TRIA). Callus initiation from hypocotyl explants was obtained on MS medium supplemented with 1.07–2.14 μM NAA and 10.2 μM BA. In 3 months, somatic embryos were produced when callus was inoculated onto MS medium supplemented with 4.44 μM BA, 40 μM AgNO₃, and 0.011 μM TRIA. Somatic embryos were efficiently regenerated on MS basal solid and liquid media supplemented with 0.44–4.4 μM BA, 0.54–2.69 μM NAA, 4.92 μM 2iP, 2.1 μM calcium d-pantothenate, 0.21 μM biotin, 227.7 μM cysteine HCl monohydrate, and 108.6 μM adenine sulfate. Agrobacterium tumefaciens GV 3101 harboring pCAMBIA 1305.2 binary vector-mediated stable transformation of somatic embryos exhibited a transformation frequency of 2.56%. As somatic embryogenesis in any perennial system is useful in terms of both commercial and scientific nature, this somatic embryo-based transformation protocol for the commercially important dye-yielding tropical plant B. orellana is useful for its improvement through genetic engineering.     

Somatic embryogenesis and regeneration of <Emphasis Type="Italic">Vigna radiata</Emphasis>

An efficient regeneration protocol via somatic embryogenesis was optimized for mung bean [Vigna radiata (L.) Wilczek; cv. Vamban 1]. Primary leaf explants were used for embryogenic callus induction in MMS medium (Murashige and Skoog salts with B5 vitamins) containing 2.0 mg dm⁻³ 2,4-dichlorophenoxyacetic acid (2,4-D), 150 mg dm⁻³ glutamine and 3 % sucrose. Fast growing, highly embryogenic cell suspensions were established from 21-d-old calli in MMS medium supplemented with 0.5 mg dm⁻³ 2,4-D and 50 mg dm⁻³ proline (Pro), and maximum recovery of globular (39.0 %), heart-shaped (26.3 %) and torpedo-stage (21.0 %) somatic embryos were observed in this medium. Mature cotyledonary-stage somatic embryos were cultured for 5 d in half strength B5 liquid medium containing 0.05 mg dm⁻³ 2,4-D, 20 mg dm⁻³ Pro, 5 μM abscisic acid, 1000 mg dm⁻³ KNO₃, 50 mg dm⁻³ polyethylene glycol (PEG 6000) and 30 g dm⁻³ D-mannitol. Mature somatic embryos were germinated after dessication for 3 d and complete development of plantlets accomplished in MMS medium containing 30 g dm⁻³ maltose, 0.5 mg dm⁻³ benzyladenine and 500 mg dm⁻³ KNO₃. Profuse lateral roots, and regeneration frequency (up to 60 %) were observed in half-strength MMS medium containing 0.5 mg dm⁻³ indolebutyric acid (IBA). The regenerated plants were grown to fruiting and were morphologically normal and fertile.     

Rapid regeneration of <Emphasis Type="Italic">Phaseolus angustissimus</Emphasis> and <Emphasis Type="Italic">P. vulgaris</Emphasis> from very young zygotic embryos

A rapid regeneration protocol for proembryos of Phaseolus angustissimus as young as 1 day after pollination (DAP) involving pod culture for 1 week followed by embryo culture for 2 weeks and embryo germination for 1 or 2 weeks is provided. Optimization of the media was conducted with pods collected 3 DAP. The best pod culture medium was composed of basal medium [(Phillips and Collins 1979) salts with (Geerts et al. 2001) vitamins], 1000 mg l⁻¹ glutamine, 1000 mg l⁻¹ casein hydrolysate, 3% sucrose and 0.5% agar. Embryo culture medium consisted of basal medium with 500 mg l⁻¹ glutamine, 250 mg l⁻¹ casein hydrolysate, 1.9 μM ABA, 3% sucrose and 0.5% bacto-agar. Embryos developed into plantlets on germination medium containing basal medium with 0.25 μM BA, 3% sucrose and 0.7% bacto-agar. Fertile, normal plants were recovered from direct embryogenesis and from micrografted embryo-derived shoots. Embryos obtained from pods collected 3 DAP regenerated plantlets at a rate of 29.3%, while embryos from pods collected 2 DAP and 1 DAP regenerated at rates of 20.2 and 4%, respectively. A second accession of P. angustissimusregenerated at a rate of 26.2%. Using this 5-week protocol for P. vulgaris resulted in a plantlet regeneration rate of 12.5%.     

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.     

Somatic embryogenesis,organogenesis and plant regeneration in taro (<Emphasis Type="Italic">Colocasia esculenta</Emphasis> var. <Emphasis Type="Italic">esculenta</Emphasis>)

Callus was initiated in three different “esculenta” taro cultivars by culturing corm slices in the dark on half-strength MS medium supplemented with 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) for 20 days followed by subculture of all corm slices to half-strength MS medium containing 1.0 mg/l thidiazuron (TDZ). Depending on the cultivar, 20–30% of corm slices produced compact, yellow, nodular callus on media containing TDZ. Histological studies revealed the presence of typical embryogenic cells which were small, isodiametric with dense cytoplasms. Somatic embryos formed when callus was transferred to hormone-free medium and ~72% of the embryos germinated into plantlets on this medium. Simultaneous formation of roots and shoots during germination, and the presence of shoot and root poles revealed by histology, confirmed that these structures were true somatic embryos. Plants derived from somatic embryos appeared phenotypically normal following 2 months growth in a glasshouse. This method is a significant advance on those previously reported for the esculenta cultivars of taro due to its efficiency and reproducibility.