Recurrent somatic embryogenesis and plant regeneration from immature zygotic embryos of cabbage (Brassica oleracea var. capitata) and cauliflower (Brassica oleracea var. botrytis)

A simple and rapid protocol was established for repetitive somatic embryogenesis and subsequent plant regeneration in two important Brassica oleracea varieties, cabbage and cauliflower. Direct regeneration of somatic embryos (SEs) was achieved from immature zygotic embryos cultured on B5 plant growth regulator (PGR)-free (B5-0) induction medium and on B5 medium supplemented with 1 mg l^?1 2,4-dichlorophenoxyacetic acid (2,4-D) (B5-D). Zygotic embryos of both cabbage and cauliflower at the cotyledonary (C) stage (1.8 mm long) incubated on B5-0 medium displayed the highest embryo-forming capacities (EFCs) of 11.84 and 11.95, respectively. Secondary somatic embryos (SSEs) appeared on the cabbage and cauliflower’s primary embryos at a high frequency (83.3 and 87.5 %, respectively), and this process continued in a repetitive way on PGR-free Murashige and Skoog (MS-0) medium. The embryogenic potential of the cultures with a gradual diminution was maintained for 10 months (ten cycles). A total of 20 % of the mature SSEs from cabbage and 55 % from cauliflower spontaneously regenerated plantlets on MS-0 medium. The addition of 1 mg l^?1 6-benzyladenine (BA) or 6-furfurylaminopurine (Kin) in the regeneration medium significantly improved somatic embryo conversion into plantlets by up to 56 % in cabbage and 79 % in cauliflower. Regenerated plants acclimated successfully to ex vitro conditions and displayed morphological and reproductive characteristics similar to seed-derived plants. Effective recurrent somatic embryogenesis may be an appropriate practical solution for clonal propagation and genetic modifications of cabbage and cauliflower.     

Somatic embryogenesis in Alnus glutinosa (L.) Gaertn

Induction of somatic embryos and plant regeneration was demonstrated for the first time in Alnus glutinosa. Somatic embryos were initiated from zygotic embryos collected 1–3 weeks post-anthesis (WPA), i.e., when they were at globular or early cotyledonary stage and were 0.5–1 mm in length. Induction frequency (16.6 %) and the mean number of somatic embryos (4.5 embryos/explant) were highest after culture of zygotic embryos, collected at 3 WPA, on Murashige and Skoog medium (MS) supplemented with 0.9-μM 2,4-dichlorophenoxyacetic acid and 2.22-μM benzyladenine (BA). No embryogenic induction was observed on medium with BA alone. Initial somatic embryos differentiated indirectly from callus tissue formed at the surface of the zygotic embryos. Embryogenic competence was maintained by secondary embryogenesis, which was affected by explant type, plant growth regulators and genotype. Secondary embryogenesis was induced by culture of small groups of whole somatic embryos or isolated cotyledon explants on medium consisting of MS medium (half-strength macronutrients) supplemented with 0.44-μM BA. Histological study of isolated cotyledon explants revealed that secondary embryos developed directly from differentiated embryogenic tissue on the surface of cotyledons. Somatic embryos at successive stages of development, including cotyledonary-stage embryos with shoot and root meristems, were evident. For plantlet conversion, somatic embryos were transferred to maturation medium supplemented with 3 % maltose, followed by 6 weeks of culture in Woody Plant Medium supplemented with 0.44-μM BA and 0.46-μM Zeatin (Z). This novel protocol appears promising for mass propagation, conservation and genetic transformation of black alder.     

Plant regeneration through direct somatic embryogenesis from immature zygotic embryos of the medicinal plant,Paris polyphylla Sm.

A protocol for induction of direct somatic embryogenesis and subsequent plant regeneration for the medicinally important and endangered plant Paris polyphylla Sm. has been developed for the first time. Immature zygotic embryos (IZEs) were cultured on different media namely Gamborg (B5), ½ B5, Murashige and Skoog (MS), ½ MS, Chu et al. (N6), ½ N6, Schenk and Hildebrandt (SH) and ½ SH. Highest frequency of somatic embryogenesis (32.6 %) and mean number of somatic embryos (SEs) per explant (28.7 ± 1.7) were obtained on ½ MS medium directly without an intermediate callus phase. The frequency of SE induction was significantly increased to 40.7 % when ½ MS medium was solidified with gelrite compared to agar (32.6 %). Secondary somatic embryos (SSEs) appeared on the primary SEs in a repetitive way on plant growth regulator-free ½ MS medium but with a gradual decrease in embryogenic potential during subsequent subcultures. Plasmolyzing pre-treatment of SSEs with 1.0 M mannitol for 12 h effectively maintains its embryogenic capacity. Primary embryos at the elongated dimpled and early cotyledonary stage displayed the highest embryo forming capacity of 26.94 and 27.87, respectively. High frequency of SE germination (94.0 %) occurred on ½ MS medium with 0.5 mg/l gibberellic acid. Highest percentage of seedling to plantlet conversion was observed in the medium supplemented with 0.05 mg/l 6-benzylaminopurine and 0.1 mg/l α-naphthalene acetic acid. Regenerated plants displayed morphological characteristics similar to that of the wild plants. Flow cytometry analysis showed ploidy stability of the regenerated plants.     

Plant regeneration of common ash (<Emphasis Type="Italic">Fraxinus excelsior</Emphasis> L.) by somatic embryogenesis

This is the first report on somatic embryogenesis in common ash (Fraxinus excelsior L.). Experiments on somatic embryogenesis induction were carried out on zygotic embryos at different phases of development and maturation. The embryo axes were isolated and cultured on media containing different plant growth regulators (PGRs). Embryogenic tissues were obtained from embryos collected at an incomplete maturation phase and cultured on a modified Murashige and Skoog medium containing 8.8 μM 2,4-dichlorophenoxyacetic acid and 4.4 μM benzyl-adenine (BA). Embryos isolated from seeds at an advanced stage of maturation showed only organogenetic phenomena. Embryogenic tissues were successfully subcultured and multiplied on medium containing a reduced concentration of PGRs. After their isolation, somatic embryos were induced to develop and mature by transfer to PGR-free medium and subsequent culture on medium containing 0.1 μM BA. Somatic embryos developed completely and also germinated spontaneously. Embryo germination and conversion were significantly improved when subjected to a period of storage at 4°C and transplant onto woody plant medium. Plantlets were successfully transferred to soil and acclimatized in a “misted” greenhouse.     

花楸合子胚诱导体细胞胚胎发生研究

分别以完整成熟胚、切去一个子叶的成熟胚和切下的子叶为外植体,以MS为基本诱导培养基、1/2MS为基本分化培养基,进行了花楸体细胞胚胎发生研究。结果表明:以完整合子胚作为外植体的体胚诱导率最高,为100%,最佳植物生长调节剂组合为5 mg.L-1NAA+2 mg.L-16-BA;NAA和6-BA浓度及二者的交互作用对愈伤组织和体胚诱导率的影响极显著;光照配合延长继代间隔时间有利于体胚发生。实体观察结果表明,花楸体胚发生方式有直接发生和间接发生两种;体胚发育经历了球形期、心形期、鱼雷形期和子叶期。组织学观察结果表明,体胚具有两极性,子叶期体胚结构完整。     

Plant regeneration via direct somatic embryogenesis in Panax japonicus

Panax japonicus is one of the important medicinal plants. Here, we established the protocol for plant regeneration of P. japonicus via direct somatic embryogenesis. Somatic embryos were directly obtained from the segments of zygotic embryos on MS medium with 4.4 μM 2,4-D. Thereafter, somatic embryos were produced by repetitive secondary somatic embryogenesis. The secondary somatic embryo formation was enhanced by plasmolyzing pretreatment (1.0 M mannitol for 10 h). Frequency of secondary somatic embryo formation from cotyledon segments was lowered by plasmolyzing pretreatment, but the number of somatic embryos per explants was greatly increased. Plasmolyzing pretreatment resulted in retardation of embryo growth and required subculture to fresh medium for further growth of embryos into cotyledonary stage. Without plasmolyzing pretreatment, cotyledonary embryos were obtained after 8 weeks of culture. All the cotyledonary somatic embryos germinated by 5 μM GA₃ treatment, but only 15.3% were germinated on hormone-free medium. After 2 months of culture on 1/2 strength WPM medium, plantlets produced flowers spontaneously. In the anthers of in vitro flowers, microsporogenesis occurred normally with low number of pollen grains.     

Somatic embryogenesis and plant regeneration in zygotic embryo explant cultures of rugosa rose

Rugosa rose (Rosa rugosa) is cultivated as a garden flower and an important genetic resource for the breeding of roses (R. hybrida). This study describes culture conditions for high frequency plant regeneration from zygotic embryo explants via somatic embryogenesis in rugosa rose. Mature zygotic embryo, cotyledon, and radicle explants formed embryogenic calluses at frequencies of 38, 6.7, and 8.8% when cultured on half-strength Murashige and Skoog medium (½MS) supplemented with 2.26, 9.05, and 9.05 μM 2,4-dichlorophenoxyacetic acid, respectively. Embryogenic calluses produced numerous somatic embryos, which then developed into plantlets on ½MS without growth regulators. Regenerated plantlets were grown to whole plants in a growth chamber.     

Recurrent somatic embryogenesis and plant regeneration from seedlings of <Emphasis Type="Italic">Hepatica nobilis</Emphasis> Schreb.

A method for secondary somatic embryogenesis was developed on embryos derived from embryogenic callus formed on Hepatica nobilis seedlings. Somatic embryogenesis (SE) was induced on seedlings (on the hypocotyl and epicotyl parts) grown on the Murashige and Skoog (1962) medium (MS) supplemented with 1 µM naphthaleneacetic acid (NAA), and/or 0.1 µM 6-benzyladenine (BA) and on medium without plant growth regulators (PGR). The best response of embryogenic callus formation was observed on the medium containing 1 µM NAA alone or with 0.1 µM BA. Individual somatic embryos, formed on embryogenic callus on the medium without PGR (MS0), at heart, torpedo and cotyledonary stage, were transferred to the media where secondary somatic embryo formation and development into plantlets occurred. Although the most efficient repetitive cycles of secondary SE were recorded for all stages of somatic embryos (heart, torpedo, cotyledonary) on the MS0 medium (77.8–87.4 %), secondary somatic embryos were also obtained on all media supplemented with cytokinins. The best rate of somatic embryos germination was achieved on MS media with 0.2 µM NAA and 2 µM BA, and 0.1 µM NAA and 1 µM BA (48.8–52.0 %) when more mature embryos (cotyledonary stage) were used. Plantlets grown from somatic embryos were successfully acclimatized to greenhouse conditions.     

Somatic embryogenesis and plant regeneration in zygotic embryos of Trifolium nigrescens (Viv.)

This study developed a plant regeneration protocol for Trifolium nigrescens (Viv.) via somatic embryogenesis (SE). Immature zygotic embryos at torpedo (TsE) and cotyledonary (CsE) stage were cultured on media with different auxins and cytokinins at different concentrations. The cultural requirements for SE differed between the explants used: the addition of 6-furfurylaminopurine (kinetin) or N⁶-[2-isopentenyl]-adenine (2iP) along with 2,4-dihydrophenoxyacetic acid (2,4-D) or 1-naphthaleneacetic acid (NAA) was needed to elicit the embryogenic response of CsE, but an exogenous cytokinin totally inhibited 2,4-D-induced SE from TsE. When applied alone, neither the cytokinin nor NAA induced SE in TsE or CsE. In all effective cultures the first somatic embryos appeared directly from the upper part of the hypocotyl (TsE and CsE) and from the margin of cotyledons (TsE) on day 7. Embryogenic callus occurred on CsE after 10 days. At comparable concentrations 2,4-D was a more potent SE inducer than NAA, but most of the embryoids induced on media with 2,4-D displayed morphological abnormalities, whereas those produced in the presence of NAA generally resembled zygotic embryos. Plant regeneration was achieved after transfer of somatic embryos or embryo-derived first shoots to medium without plant growth regulators (PGRs). The frequency of plant recovery was about 30% for embryoids obtained on media containing 2,4-D, and for material from media with NAA the recovery rates were 44–68% (somatic embryos) and 72–100% (embryoid-derived shoots). Regenerants appeared identical to each other and to wild plants; they produced flowers and had the chromosome complement typical for the species, 2n = 16, in root tip cells.     

Histology of Organogenic and Embryogenic Responses in Cotyledons of Somatic Embryos of Quercus Suber L

In cork oak (Quercus suber L.), recurrent embryogenesis is produced in vitro through autoembryony without exogenous plant growth regulators (PGRs); secondary embryos appear on the embryo axis but seldom on cotyledons. Focusing mainly on the histological origin of neoformations, we investigated the influence of the embryo axis and exogenous PGRs on the embryogenic potential of somatic embryo cotyledons. Isolated cotyledons of somatic embryos became necrotic when cultured on PGR-free medium but gave secondary embryos when cultured on media containing benzyladenine and naphthaleneacetic acid. Cotyledons of cork oak somatic embryos are competent to give embryogenic responses. Isolated cotyledons without a petiole showed a lower percentage of embryogenic response than did those with a petiole. In petioles, somatic embryos arose from inner parenchyma tissues following a multicellular budding pattern. Joined to the embryo axis, cotyledons did not show morphogenic responses when cultured on PGR-free medium but revealed budlike and phylloid formations when cultured on medium with PGRs. The different morphogenic behavior displayed by somatic cotyledons indicates an influence of the embryo axis and indicates a relationship between organogenic and embryogenic regeneration pathways.     

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 immature zygotic embryo cultures of Houttuynia cordata Thunb via somatic embryogenesis

This study reports high-frequency plant regeneration from immature zygotic embryo cultures of Houttuynia cordata Thunb via somatic embryogenesis. Numerous green globular structures were directly formed on the surfaces of cotyledons and radicles from 2-week-old immature zygotic embryos at a frequency of 42.1 % when cultured on Murashige and Skoog (MS) medium supplemented with 2 mg l^?1 of α-naphthaleneacetic acid (NAA) and 1 mg l^?1 of 6-benzyladenine (BA). In comparison, white globular structures and pale-yellow calluses were formed simultaneously at a frequency of 28.3 % when cultured on MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D). The pale-yellow calluses were transferred to MS liquid medium supplemented with 2,4-D to establish embryogenic cell suspension cultures consisting of round, isodiametric cells that formed cell aggregates. Upon plating of these cell aggregates on half-strength MS medium without growth regulators under light conditions, cell aggregates gave rise to numerous globular embryos at a frequency of 56 %. Of the globular embryos, 15 % were successfully converted into cotyledonary embryos when cultured on half-strength MS medium under light conditions. The plant regeneration system of H. cordata established in this study will be useful for the selection, genetic transformation, and mass proliferation of elite clones with medicinal potential.     

Direct somatic embryogenesis and plantlet regeneration in oil palm

Direct embryogenesis without an intervening callus phase from cotyledonary nodes of germinated immature zygotic embryos of hybrids viz. DG1 and DG21 of oil palm (Elaeis guineensis) is reported here. Direct embryogenesis was achieved when the cotyledonary nodes of germinated immature zygotic embryos were cultured in dark for 8 weeks on Eeuwens media (Y3) supplemented with 40 μM 2,4-Dichlorophenoxyacetic acid (2,4-D), 40 μM α-Naphthaleneacetic acid (NAA), 10 μM 2,4,5-Trichorophenoxyacetic acid (2,4,5-T), 10 μM Thiadiazuron (TDZ), 10 μM 6-Benzyladenine (BA). The globular embryos with clear suspensor region appeared directly on the explants and multiplied. On subculture to fresh media, the other stages such as torpedo and heart shaped embryos were seen. On transfer to light in Y3 media containing BA (2 μM) and ABA (1 μM) they matured into complete plantlets. In 2% of the cultures secondary embryogenesis also was seen. Along with several other advantages of direct somatic embryogenesis this protocol opens up the prospect of genetic transformation in this important commercial crop.     

Changes in competence for somatic embryogenesis in Norway spruce zygotic embryo segments

Embryogenic capacity of Norway spruce zygotic embryo sections was drastically altered by a preinduction transfer to hormone-free medium for 7 or 14 days. An increase in competence for somatic embryogenesis was found with the cotyledons, while the hypocotyl sections completely lost their competence. These changes in competence were not dependent on physical contact between plant sections, and could not be correlated to the developmental stage of each section. The increase of competence in cotyledonary material was due not only to an increase of genotypes initiating somatic embryos, but also to an increase in embryogenicity of cotyledons.     

Enhanced production of recombinant human gastric lipase in turnip hairy roots

Somatic embryogenesis is a reliable and important tool, and the relevant genes controlling this process act as vital roles through the whole development of somatic embryos. However, regeneration via somatic embryogenesis in Chinese chestnut has been impeded and its molecular mechanism is not known. Therefore, firstly we described a protocol for somatic embryo initiation, development, maturation and germination. Embryogenic calli were obtained in embryo initiation medium containing 1.8 μM 2,4-D and 1.1 μM 6-BA, and then were transferred to embryo development medium without any hormones for at least 4 weeks, until cotyledonary embryos appeared. Next, the somatic embryos were transferred to embryo maturation medium containing Gamborg’s B-5 Basal Salt Mixture with 0.5 μM NAA and 0.5 μM 6-BA for 3 weeks. Finally, these mature embryos were germinated in embryo germination medium consisting of WPM with 0.5 μM NAA and 0.5 μM 6-BA, resulting in shoot regeneration with a 2.1% conversion rate. Additionally, eight embryogenesis-related genes were identified, and the expression profiles of these genes during embryogenesis were analyzed via quantitative real-time RT-PCR (qRT-PCR). The CmSERK, CmLEC1, CmWUS and CmAGL15 genes exhibited high expression in the initial embryo stages, which inferred that these genes played key roles during the initiation of embryogenesis. Studies on embryogenesis-related genes will provide an insight for further elucidating molecular mechanism during somatic embryogenesis of Chinese chestnut. Furthermore, the successful establishment of a somatic embryo regeneration system for Chinese chestnut will lay a significant foundation for a stable genetic transformation system and genetic improvement.     

Somatic embryogenesis and plantlet regeneration from mature zygotic embryos of Manchurian ash (Fraxinus mandshurica Rupr.)

Using mature cotyledonary explants of Fraxinus mandshurica, an efficient plant regeneration system was developed via somatic embryogenesis. More than 67 % of mature cotyledons of zygotic embryos yielded 23–159 somatic embryos (SEs) per explant when incubated on medium consisting of half-strength Murashige and Skoog (MS) salts and vitamins (MS1/2) supplemented with 8.88 μM 6-benzyladenine (BA), 26.84 μM naphthaleneacetic acid (NAA), 75 g L^?1 sucrose, and 400 mg L^?1 casein hydrolysate (CH). Approximately, 82 % of induced SEs were observed on browning cotyledonary explants. Histological studies of cotyledon explants at various stages of somatic embryogenesis revealed that the SEs originated from single epidermal cells and developed to the globular, heart, torpedo, and cotyledonary stage embryos. Secondary somatic embryos (SSEs) formed on the surface of radicle tips of the SEs. Addition of low concentrations of NAA and 200–400 mg L^?1 CH to MS1/2 medium increased SSE induction. Cotyledonary SSEs were cultured on MS1/2 medium with 10 mM abscisic acid in the presence of light to promote maturation, and >92 % of mature SSEs were able to germinate with normal shoots. After 8 weeks in culture in the presence of light on medium with one-third of the MS macroelements as well as 0.06 μM NAA, >94 % of the germinated SSEs converted into plantlets. Plantlets acclimatized successfully to ex vitro conditions and developed normal phenotypes under field conditions.     

Handling culture medium composition for optimizing plant cell suspension culture in shake flasks

Douglas-fir is a conifer species of major economic importance worldwide, including Western Europe and New Zealand. Herein we describe some characterization and significant refinement of somatic embryogenesis in Douglas-fir, with focus on maturation. The most typical structures observed in the embryonal masses were large polyembryogenic centres (up to 800–1500 µm) with a broad meristem, creating a compact cell “package” with suspensor cells. Singulated somatic embryos composed of both a embryonal head (300–400 µm) and long, tightly arranged suspensor were also frequent. Embryo development was enhanced following embryonal mass dispersion on filter paper discs at low density (50–100 mg fresh mass). Moreover, increasing gellan gum concentration in maturation medium (up to 10 g L^?1) improved both the quantity and quality of cotyledonary somatic embryos (SEs), which were subsequently able to germinate and develop into plantlets at high frequency. Embryogenic yield was highly variable among the seven embryogenic lines tested (27–1544 SE g^?1 fresh mass). Interestingly secondary somatic embryogenesis could be induced from cotyledonary SEs of both low- and highly-productive lines with some useful practical outcomes: secondary lines from low-performance lines (30–478 SE g^?1 fresh mass) displayed significantly higher embryogenic yield (148–1343 SE g^?1 fresh mass). In our best conditions, the total protein content in cotyledonary SEs increased significantly with maturation duration (up to 150 µg mg^?1 fresh mass after 7 weeks) but remained below that of mature zygotic embryos (300 µg mg^?1). The protein pattern was similar in both somatic and zygotic embryos, with major storage proteins identified as 7S-vicilin- and legumin-like proteins.     

Large-scale somatic embryogenesis and regeneration of <Emphasis Type="Italic">Panax notoginseng</Emphasis>

An efficient system for inducing somatic embryogenesis in Panax notoginseng was established using shaker flasks and bioreactor cultures; furthermore, regenerated plantlets were successfully transferred to ex vitro soil conditions. Embryogenic callus was induced from segments of adventitious roots incubated on Murashige and Skoog (MS) medium containing 1.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) after 5 weeks of culturing. The highest frequency (100%) of somatic embryogenesis, with a mean of 32.7 somatic embryos per callus, was obtained on embryogenic callus incubated on a medium containing 0.5 mg/L 2,4-D. To scale-up somatic embryo formation, 10 g (~1.65 × 10⁴) of early globular-stage somatic embryos were incubated in a 3 L airlift bioreactor containing 1.5 L 1/2 MS medium without plant growth regulators (PGRs) for a period of 4 weeks; these globular-stage somatic embryos then developed into cotyledonary embryos. When maintained on PGR-free medium, the cotyledonary embryos developed roots but did not develop shoots. However, when they were treated with gibberellic acid (GA₃), they continued to germinate and transformed into plantlets after 2 weeks of culture. Plantlets with well-developed shoots and roots were transferred to an autoclaved vermiculite and perlite mixture, acclimatized for a period of 3 months and successfully transferred to forest mountain soil. Following overwintering, these plants produced new growth.     

Somatic embryogenesis in <Emphasis Type="Italic">Pinus nigra</Emphasis>: embryogenic tissue initiation,maturation and regeneration ability of established cell lines

The effect of plant growth regulators (PGR), 6-benzyladenine (BA), 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthaleneacetic acid (NAA) and sugars (sucrose, maltose, glucose, fructose) on the initiation of somatic embryogenesis of Pinus nigra Arn. was investigated. Megagametophytes containing immature zygotic embryos have been used as explants. The experiments were done in the years 2000 and 2001. Higher initiation frequencies were obtained in 2001 when the zygotic embryos showed uniformity, being in the precotyledonary stage of development. Embryogenic tissue initiation occurred on all the media tested, including PGR-free medium. Relatively high initiation frequencies were obtained on media containing either NAA (9.09 %) or 2,4-D (7.14 %) alone. Somatic embryos were present as bipolar structures and showed differences in morphological features among cell lines. Plantlet regeneration occurred in cell lines containing bipolar somatic embryos composed of compact meristematic embryo “head” and suspensor organized into bundles.We highly appreciate the financial support from VEGA, Slovak Grant Agency, project No. 2/2089/22.