Somatic embryogenesis in Narcissus pseudonarcissus cvs. Golden Harvest and St. Keverne

Somatic embryos (SEs) have been produced from bulb and shoot culture leaf explants of Narcissus pseudonarcissus cvs. Golden Harvest and St. Keverne. Initial experiments with cv. Golden Harvest resulted in SEs from leaf lamina, leaf base, bulb scale and scape (flower stem) explants. Embryogenesis was induced on media with a range of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BAP) concentrations. There were significantly more SEs with media containing 5 μM 2,4-D and 0.5 μM or 5 μM BAP than any other growth regulator combination. Scape explants produced more early SEs than the other explant types, and when orientated with their basipetal surface away from the medium, they produced significantly more advanced SEs than those with this surface in contact with the medium. Leaf explants from shoot cultures of cv. Golden Harvest produced SEs on medium with BAP combined with 2,4-D or 1-naphthaleneacetic acid (NAA), but 4-amino-3, 5, 6-trichloro-2-pyridinecarboxylic acid (picloram) was ineffective. SEs converted to plantlets efficiently following a 4°C treatment in addition to 4.9 μM indole-3-butyric acid (IBA). These plantlets readily transferred to ex vitro conditions.     

Changes in protein profiles associated with somatic embryogenesis in peanut

The somatic embryogenesis potential of zygotic embryo axes of peanut (Arachis hypogaea L. cv. DRG-12) at different stages of development was evaluated by culturing on MS medium with 18.1 μM 2,4-dichlorophenoxyacetic acid (2,4-D). A 100 % frequency with 18.3 somatic embryos per explant was observed from 4 mm long immature zygotic embryo axes collected 31 – 40 d after pollination. Medium supplemented with 16.6 μM picloram resulted in slow development of somatic embryos whereas in the presence of 21.5 μM α-naphthaleneacetic acid (NAA), the explants underwent maturation with induction of roots after 30 d. The changes in protein profiles in zygotic embryo axes at different stages of development correlated with their potential to form somatic embryos. Immature zygotic embryo axes exhibited high frequency somatic embryogenesis in the stage preceding abundant accumulation of 22 and 65 kDa proteins. The content of 22 and 65 kDa proteins decreased immediately after culture on medium fortified with 18.1 μM 2,4-D and increased again after 12 d of culture coinciding with the development of somatic embryos on the explants. The content of 22 and 65 kDa proteins was low at 15 d of culture on medium supplemented with 16.6 μM picloram possibly due to slow development of the somatic embryos on the explant. On maturation medium containing 21.5 μM NAA, a marked increase in the content of 22 and 65 kDa proteins in 15 d-old cultures was observed.     

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.     

Morpho-histological study of direct somatic embryogenesis in endangered species Frittilaria meleagris

Direct somatic embryogenesis of Frittilaria meleagris L. was induced using leaf base explants excised from in vitro grown shoots. Somatic embryos occurred at the basal part of leaf explants 4 weeks after culture on a Murashige and Skoog (MS) medium supplemented with various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) or kinetin (KIN). The highest number of somatic embryos (SEs) were formed (9.74) from leaf explant on MS medium supplemented with 0.1 mg dm⁻³ 2,4-D after 4 weeks of culture initiation. An initial exposure to a low concentration of KIN in the medium also enhanced SEs induction. Our observations by light and scanning electron microscopy revealed that SEs originate directly from the epidermal and subepidermal layers of leaf explant. The developmental stages of somatic embryogenesis from the first unequal cell division through the meristematic clusters, multi-cellular globular somatic embryos to the fully formed cotyledonary embryos were determined. After 4 weeks on MS medium without plant growth regulators, SEs developed into bulblets.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration of the heavy metal tolerant and hyperaccumulator <Emphasis Type="Italic">Arabidopsis halleri</Emphasis> (Brassicaceae)

Plants were regenerated from root explants of Arabidopsis halleri (L.) O’Kane and Al-Shehbaz via a three-step procedure callus induction, induction of somatic embryos and shoot development. Callus was induced from root segments, leaflets and petiole segments after incubation for 2 weeks in Murashige and Skoog medium (MS) supplemented with 0.5 mg/l⁻¹ (2.26 μM) 2,4-D (2,4-dichlorophenoxyacetic acid) and 0.05 mg/l⁻¹ (0.23 μM) kinetin. Only calli developed from root segments continued to grow when transferred to a regeneration medium containing 2.0 mg/l⁻¹ (9.8 μM) 6-γ-γ-(dimethylallylamino)-purine (2ip) and 0.05 mg/l⁻¹ (2.68 μM) α-naphthalenacetic acid (NAA) and eventually 40 of them developed embryogenic structures. On the same medium 38 of these calli regenerated shoots. Rooting was achieved for 50 of the shoots subcultured in MS medium without hormones. The regeneration ability of callus derived from root cuttings, observed in this study, makes this technique useful for genetic transformation experiments and in vitro culture studies.     

In vitro morphogenesis in zygotic embryo cultures of neem (<Emphasis Type="Italic">Azadirachta indica</Emphasis> A. Juss.)

Immature zygotic embryo cultures of neem yielded highly regenerative cultures, with the response varying with the embryo stage at culture. Early dicotyledonous stage embryos were the most responsive followed by torpedo stage embryos. The embryo cultures differentiated three types of regenerants: somatic embryos (SEs), shoot buds and neomorphs. SEs exhibited morphological abnormalities such as pluricotyledony, fusion of cotyledons and absence of cotyledons. Although these SEs showed secondary embryogenesis, the occurrence of normal dicotyledonous embryos was extremely rare. On MS basal medium 3% of SEs developed a long tap root but a plumular shoot did not appear. However, it was possible to regenerate plantlets from immature zygotic embryo cultures of neem via neomorph formation and adventitious shoot bud formation. The transplantation survival of these plants was more than 80%.Abbreviations BAP 6-Benzylamino purine - CH Casein hydrolysate - 2,4-D 2,4-Dichlorophenoxyacetic acid - GA₃ Gibberellic acid - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - NAA -Naphthaleneacetic acid - SE Somatic embryoCommunicated by W. Harwood     

Somatic embryogenesis from leaf and zygotic embryo explants of Blighia sapida ‘Cheese’ ackee

Summary This study investigated factors affecting the production of somatic embryos in Blighia sapida (ackee). Explants obtained from fully expanded leaves or cotyledons of immature zygotic embryos excised from brown (BSCZE) or green seeds (GSCZE) were cultured on Murashige and Skoog medium supplemented with 9, 18 and 36&#x03BC;M 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4 or 22.1 &#x03BC;M benzylaminopurine (BAP) or 0.2&#x2013;19.9 &#x03BC;M thidiazuron (TDZ). Leaf explants grown on media supplemented with the different combinations of 2,4-D and BAP formed callus, but they were non-embryogenic, while explants were not responsive on TDZ-supplemented media. GSCZE explants grown in the presence of 2,4-D/BAP combinations of 9/4.4, 18/4.4 or 36/4.4 &#x03BC;M formed non-embryogenic callus profusely, but explants gave rise to organized globular protuberances (GPs) and non-embryogenic callus on media containing TDZ, with the best concentration at 0.4 &#x03BC;M. BSCZE explants grown on TDZ-supplemented media also formed callus, but no GPs were detected. When GPs were cultured on media containing TDZ and abscisic acid they (ABA), gave rise to the highest number of somatic embryos. The medium was also beneficial for the development of somatic embryos from the globular to cotyledonary stage.     

A three media transfer system for direct somatic embryogenesis from leaves ofSenecio x hybridus Hyl. (Asteraceae)

Bisected leaves were cultured on semi-solid Murashige & Skoog medium amended with 13.5 M 2,4-D and 4.5 M BA for 7–14 days, transferred to 0.5% activated charcoal medium (without growth regulators) for three days, then transferred to MS basal medium. Control explants remained on initiation medium for comparison to transferred explants. Twenty-eight days after initiation of cultures, explants exposed to 11–14 days on induction medium yielded the largest number and most developmentally advanced embryos. Mature, white somatic embryos from transferred explants were visible after 35 days. Conversely, somatic embryos on control explants were not morphologically mature until 2–4 months. Mature embryos from transferred explants germinated without a resting stage, whereas embryos from control explants appeared quiescent. Histological examination confirmed embryo anatomy, however embryos, regardless of treatment, had abnormal cotyledons and regenerated plants had multiple stems.Abbreviations AC activated charcoal - BA 6-benzylaminopurine - CrAF III chromium trioxide, acetic acid and formaldehyde - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige & Skoog medium - NAA 1-naphthalenacetic acid     

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.     

Effect of plant growth regulators on ovary culture of coconut (<Emphasis Type="Italic">Cocos nucifera</Emphasis> L.)

Coconut is a cross pollinating palm, propagated only by seeds. Tissue culture is the only vegetative propagation method available for coconut. Consistent callogenesis was obtained by culturing unfertilised ovaries at -4 stage in CRI 72 medium containing 100 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.1% activated charcoal. Callusing was improved by application of 9 μM thidiazuron (TDZ). Embryogenic calli were subcultured onto somatic embryogenesis induction medium containing 66 μM 2,4-D. Stunted growth was observed in the somatic embryos after subculture onto CRI 72 medium containing abscisic acid (ABA). Maturation of somatic embryos could be achieved in Y₃ medium without growth regulators. Conversion of somatic embryos was induced by adding gibberellic acid (GA₃) to conversion medium containing 5 μM 6-benzyladenine (BA) while 2-isopentyl adenine (2iP) increased the frequency of plant regeneration. A total of 83 plantlets was produced from 32 cultured ovaries.     

Plant regeneration of mulberry (<Emphasis Type="Italic">Morus indica</Emphasis>) from mesophyll-derived protoplasts

A protocol is presented for regenerating plants from protoplasts of tropical mulberry. Leaves from seedling node cultures maintained in vitro were used as donor tissue. Optimal cell wall digestion was achieved with a combination of cellulase (2%) and macerozyme (1%). The plant growth regulator (PGR) combination zeatin (2.3 μM) and 2,4-dichlorophenoxyacetic acid (2,4-D) (2.3 μM) resulted in the highest number (29%) of cell divisions. First cell divisions were observed at day 4 after plating. Only zeatin (2.3 μM) and 2-methoxy-3,6-dichlorobenzoic acid (dicamba) (13.5 μM) supplemented medium supported subsequent divisions in protoplast cultures. Microcolonies reached a cell number of approximately 50, after 40 to 42 days of culture. The cells of these colonies continued dividing, leading to formation of microcalli. Whole plants were obtained after culture of microcalli on Murashige and Skoog (MS) medium containing thidiazuron (TDZ) (4.5 μM) and indole-3-acetic acid (IAA) (17.1 μM). The regenerated shoots were rooted on MS medium supplemented with 4.9 μM indole butyric acid (IBA). With a low survival rate during acclimation, regenerated plants were established in the greenhouse.     

Somatic embryogenesis in cell cultures of Thapsia garganica

Cell cultures from different species of the genus Thapsia (Apiaceae) have been investigated. In one 4-yearold line of T. garganica L. spontaneous somatic embryogenesis up to the globular stage occurred in a suspension culture containing 1 mg l^–12,4-dichlorophenoxyacetic acid (2,4-D). Also callus cultures of this line, previously maintained on a medium containing 1 mg l^–1 2,4-D, when transferred to various media deprived of 2,4-D, produced somatic embryos that developed into plantlets. Cell culture, embryos and regenerated organs were analysed for their content of thapsigargins. The undifferentiated cell culture did not synthezise thapsigargins, but was found to produce a yet unidentified compound not present in planta. White embryos in the pre-cotyledonary stage did not synthezise thapsigargins either, but when the embryos developed to the cotyledonary stage and became green, the synthesis started. Regenerated roots and shoots also contained thapsigargins.Abbreviations BAP Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - EtOAc ethyl acetate - FDA fluorescein diacetate - IAA Indole-3-acetic acid - IBA indole-3-butyric acid - 2-iP 2-isopentenyladenine - NAA 1-Napthaleneacetic acid     

Anthocyanin production in callus cultures of Cleome rosea: Modulation by culture conditions and characterization of pigments by means of HPLC-DAD/ESIMS

Leaf and stem explants of Cleome rosea formed calluses when cultured on MS medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) or 4-amino-3,5,6-trichloropicolinic acid (PIC). The highest biomass accumulation was obtained in the callus cultures initiated from stem explants on medium supplemented with 0.90 μM 2,4-D. Reddish-pink regions were observed on callus surface after 6–7 months in culture and these pigments were identified as anthocyanins. Anthocyanins production was enhanced by reducing temperature and increasing light irradiation. Pigmented calluses transferred to MS1/2 with a 1:4 ratio NH₄⁺/NO₃⁻, 70 g L⁻¹ sucrose and supplementation with 0.90 μM 2,4-D maintained a high biomass accumulation and showed an increase of 150% on anthocyanin production as compared with the initial culture conditions. Qualitative analysis of calluses was performed by high performance liquid chromatography coupled to diode array detector and electrospray ionization mass spectrometry (HPLC-DAD/ESIMS). Eleven anthocyanins were characterized and the majority of them were identified as acylated cyanidins, although two peonidins were also detected. The major peak was composed by two anthocyanins, whose proposed identity were cyanidin 3-(p-coumaroyl) diglucoside-5-glucoside and cyanidin 3-(feruloyl) diglucoside-5-glucoside.     

High-frequency direct somatic embryogenesis from leaf tissues of <Emphasis Type="Italic">Drimiopsis kirkii</Emphasis> Baker (giant squill)

Whole plants were regenerated from excised leaves of Drimiopsis kirkii Baker (Lily of the Valley) through direct somatic embryogenesis. An initial exposure to a low level of 2,4-dichlorophenoxyacetic acid (2,4-D, 0.45 μM) in the medium was essential in inducing the direct formation of somatic embryos. A high concentration of 2,4-D (4.52 μM) in the proliferation medium reduced embryogenesis and enhanced callus formation. The presence of kinetin in the medium enhanced the somatic-embryogenesis-inducing effect of 2,4-D (0.45 μM). The maximum embryogenesis rate (4,026 somatic embryos per gram of leaf) was obtained in explants cultured for 30 d in medium supplemented with 2.33 μM kinetin and 0.45 μM 2,4-D (embryo induction medium). Kinetin (4.65 μM) also enhanced embryo germination (97.6%), but the presence of α-naphthalene acetic acid in the medium drastically reduced embryo germination. Following conversion, the regenerated plantlets were transferred to soil and showed normal morphological characteristics.     

Somatic embryogenesis and plant regeneration from leaflets of peanut,Arachis hypogaea

Somatic embryos were induced on peanut (Arachis hypogaea) leaflets from aseptically germinated embryo axes. Leaflet size influenced percent somatic embryogenesis; 5–8 mm long cut leaflets were superior to 2–3 mm long uncut leaflets. Maximum embryogenesis of 14.6% was obtained after a 15 d incubation on induction medium (modified MS with B5 vitamins, 30 g/l sucrose, 4 g/l Gel-Gro, 40 mg/l 2,4-D +0.2 mg/l kinetin) followed by transfer to a secondary medium with 5 mg/l 2,4-D+0.2 mg/l kinetin. Primary somatic embryos were fused along the axes with no distinct cotyledons, but secondary embryos had single axes with two cotyledons. Other treatments had lower percent embryogenesis, no secondary embryogenesis, and embryos with single axes with two cotyledons. Some somatic embryos converted into normal plants capable of greenhouse survival.Abbreviations MS Murashige and Skoog (1962) medium - B5 Gamborg et al. (1968) B5 medium - 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6benzylaminopurine - NAA 1-naphthaleneacetic acid     

Development of in vitro plant regeneration of Australian native waxflowers (Chamelaucium spp.) via somatic embryogenesis

Waxflowers (Chamelaucium spp.) are native to Australia and now are grown for the cut flower industry worldwide. As part of an effort to achieve somatic hybridization between the species to improve flower quality, somatic embryogenesis was achieved for Chamelaucium uncinatum and C. repens. Somatic embryos from young leaves of C. uncinatum and C. repens were induced in vitro on Murashige and Skoog (MS) agar medium containing 20 g/l sucrose and 2,4-dichlorophenoxyacetic acid (2,4-D). For C. uncinatum, up to 4% of explants developed somatic embryos at 20 μM 2,4-D and for C. repens, up to 3% developed somatic embryos at 5 μM 2,4-D. Somatic embryos of C. uncinatum were also induced from immature seeds—a maximum of 6% of seed explants producing somatic embryos on MS medium containing 0.05 μM 6-benzyladenine (BA) and 0.5 μM Naphthalene acetic acid (NAA). Somatic embryo cultures maintained on MS medium supplemented with 0.1 μM 2,4-D were induced to develop into plantlets after transfer to a hormone-free medium under light.     

Somatic embryogenesis from integument (perisperm) cultures of coffee

Somatic embryogenesis was induced in integument (perisperm) cultures of C x R hybrid cultivar of coffee, after a culture period of 15 months, using a sequence of 3 modifications of MS medium. Vigorously growing soft, white, watery crystalline calli were obtained on MS + TIBA (1 mg/l) + L-cysteine HCl (50 mg/l) + PVP (100 mg/l). After 45 d, the calli were subcultured to MS + IAA (0.5 mg/l) + 2,4-D (0.05 mg/l) + Kn (8.6 mg/l) and maintained for the next 9 months without any transfer. On this medium, the callus proliferation was initially vigorous which slowed down after 5–6 months, and then the calli turned light brown and somewhat compact. Later, when the calli were transferred to MS + thiamine HCl (10 mg/l) + pyridoxine HCl (3 mg/l) + nicotinic acid (2 mg/l) + 2,4-D (0.2 mg/l) + 2ip (2.5 mg/l) and cultured for 2 months, they turned darker, more compact and the proliferation almost stopped. These calli were subcultured onto fresh medium of the same composition. After another 2 months of culture cream-coloured, highly friable, embryogenic calli appeared, which in turn produced a few clearly identifiable SEs in another 1 month. Further proliferation and maturation of SEs was achieved by culturing the embryogenic calli on MS + ABA (1 mg/l) for 3 months. The SEs were germinated into 2 cm tall plantlets after 2–3 subcultures, each of 2 months duration on 1/2-MS + Kn (0.1 mg/l).Abbreviations MS Murashige and Skoog (1962) basal medium - ABA Abscisic acid - TIBA 2,3,5 -Triiodobenzoic acid - IAA Indole-3-acetic acid - 2,4-D 2,4-dichloro-phenoxyacetic acid - Kn Kinetin - 2ip N⁶-(2-isopentenyl) adenine - PVP Polyvinylpyrrolidone; - SEs Somatic embryos     

Plant regeneration from embryogenic suspension cultures of Musa acuminata cv. Mas (AA)

Embryogenic callus was established using immature male flower of Musa acuminata cv. Mas. After 5–6 months of culture, embryogenic callus was obtained at 21.75±11.9 from 750 immature male flower clusters with translucent somatic embryos proliferated from the whitish friable callus. It was observed that flower clusters ranging from 4 to 11 responded to form embryogenic callus and out of which 3–10 somatic embryos were formed per flower cluster. Embryogenic callus were obtained at a percentage of 10.00±0.3 on M1 medium initially supplemented with 18 M 2,4-dichlorophenoxyacetic acid (2,4-D) for 3 months and subsequently transferred to the same media with reduced 2,4-D (9 M) for the next 2–3 months. Embryos developed into translucent spheres and slightly torpedo shaped embryos in suspension cultures. Plantlets were obtained on medium M4 supplemented with 0.8M BA, at an average regeneration rate of 13.00±0.58.     

Observations on the combined effects of light,NAA and 2,4-D on somatic embryogenesis of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis>) hybrids

Somatic embryogenesis of cucumber was affected by auxin and light during the induction phase. In the light, 2,4-dichlorophenoxyacetic acid (2,4-D) alone induced little embryogenesis, while combined with naphthaleneacetic acid (NAA) it induced 1.25 somatic embryos (SEs) per callus. In the dark 2,4-D alone induced 5 times more SEs per callus.     

Regeneration of <Emphasis Type="Italic">Aeschynanthus radicans</Emphasis> via direct somatic embryogenesis and analysis of regenerants with flow cytometry

Plant regeneration through direct somatic embryogenesis in Aeschynanthus radicans ‘Mona Lisa’ was achieved in this study. Globular somatic embryos were formed directly from cut edges of leaf explants and cut ends or on the surface of stem explants 4 wk after culture on Murashige and Skoog (MS) medium supplemented with N-phenyl-N′-1, 2, 3-thiadiazol-5-ylurea (TDZ) with α-naphthalene acetic acid (NAA), TDZ with 2,4-dichlorophenoxyacetic acid (2,4-D), or 6-benzylaminopurine (BA) or kintin (KN) with 2,4-D. MS medium containing 9.08 μM TDZ and 2.68 μM 2,4-D resulted in 71% of stem explants producing somatic embryos. In contrast, 40% of leaf explants produced somatic embryos when induced in medium containing 6.81 μM TDZ and 2.68 μM 2,4-D. Somatic embryos matured, and some germinated into small plants on the initial induction medium. Up to 64% of stem explants cultured on medium supplemented with 9.08 μM TDZ + 2.68 μM 2,4-D, 36% of leaf explants cultured on medium containing 6.81 μM TDZ and 2.68 μM 2,4-D had somatic embryo germination before or after transferring onto MS medium containing 8.88 μM BA and 1.07 μM NAA. Shoots elongated better and roots developed well on MS medium without growth regulators. Approximately 30–50 plantlets were regenerated from each stem or leaf explant. The regenerated plants grew vigorously after transplanting to a soil-less substrate in a shaded greenhouse with more than a 98% survival rate. Three months after their establishment in the shaded greenhouse, 500 plants regenerated from stem explants were morphologically evaluated, from which five types of variants that had large, orbicular, elliptic, small, and lanceolate leaves were identified. Flow cytometry analysis of the variants along with the parent showed that they all had one identical peak, indicating that the variant lines, like the parent, were diploid. The mean nuclear DNA contents of the variant lines and their parent ranged from 4.90 to 4.99 pg 2C⁻¹, which were not significantly different statistically. The results suggest that the regenerated plants have a stable ploidy level, and the regeneration method established in this study can be used for rapid propagation of ploidy-stable Aeschynanthus radicans.