Secondary Somatic Embryogenesis in Abies numidica

The induction of secondary somatic embryogenesis in Abies numidica De Lann. was achieved. Precotyledonary, cotyledonary, and desiccated cotyledonary embryos were used as explants. Cotyledonary embryos before desiccation were the most suitable. The most beneficial was induction medium Schenk and Hildebrandt (SH) with 1 mg dm⁻³ thidiazuron and 1000 mg dm⁻³ myo-inositol. Initiation frequency was from 1 to 34 %. Maturation of somatic embryos was achieved on modified Murashige and Skoog medium supplemented with 40 g dm⁻³ maltose, 100 g dm⁻³ polyethylene glycol-4000 and 10 mg dm⁻³ abscisic acid. Mature somatic embryos after three weeks of desiccation germinated on SH medium with 10 g dm⁻³ charcoal and 10 g dm⁻³ sucrose. This revised version was published online in July 2006 with corrections to the Cover Date.     

Somatic embryogenesis and plant regeneration from immature zygotic embryos of Hinoki cypress (Chamaecyparis obtusa Sieb. et Zucc.)

We established a plant regeneration system for Hinoki cypress (Chamaecyparis obtusa) via somatic embryogenesis. Embryogenic tissues were successfully induced on three kinds of Smith media from megagametophyte explants containing pre-cotyledonary embryos of C. obtusa plus-trees. Factors affecting somatic embryo maturation were examined. The concentration of polyethylene glycol 4000 in the medium was a critical factor for embryo maturation and its effective concentration was 150 g/l. The addition of 30 g/l maltose to the medium had a positive effect on embryo maturation, but sucrose was ineffective. The mature somatic embryos germinated at a germination frequency of approximately 60%, and the presence of activated charcoal was effective in stimulating plantlet growth. The plantlets acclimatized successfully in a greenhouse. To our knowledge, this is first report describing details of a plant regeneration method for C. obtusa via somatic embryogenesis.Abbreviations ABA Abscisic acid - PEG Polyethylene glycol 4000 - SM1 Smith Standard Embryonic Tissue Capture Medium - SM2 Smith Standard Embryogenesis Medium - SM3 Smith Embryo Develop Medium     

Somatic embryo proliferation, maturation and germination in Catharanthus roseus

In the present study an efficient somatic embryogenesis method has been developed in Catharanthus roseus. Friable embryogenic callus was induced from hypocotyl of in vitro germinated seeds on Murashige and Skoog basal nutrient media supplemented with various auxins particularly 2,4-D (1.0 mg l⁻¹). However, only NAA (1.0 mg l⁻¹) produced somatic embryos in cultures. Embryo proliferation was even high on the same medium added with BAP. Cotyledonary somatic embryo germinated and converted into plantlets in BAP (0.5 mg l⁻¹) added medium following a treatment with gibberellic acid (1.0 mg l⁻¹) for maturation. Carbon sources and concentrations had a marked influence on maturation process. Plantlet conversion was better achieved when embryos were matured on 3% fructose or 3–6% maltose. The result discussed in this paper indicates that somatic embryos were produced in numbers and converted plantlets can be used as raw material, genetic modification to embryo precursor cell may improve alkaloid yield further.     

Repetitive somatic embryogenesis and plant regeneration in Garcinia indica choiss

Summary Immature seeds of Garcinia indica Choiss, were exeised from immature fruits and cultured on Lloyd and McCown (1980), woody plant medium (WPM) with different combinations of auxins and cytokinins. Somatic embryos were obtained on the media supplemented with 6-benzy laminopurine (BA; 2.2–22.1 μM) alone or in combination with α-naphthalene acetic acid (NAA; 2.6 μM) with 80% frequency within a period of 2–3 wk. Subculture of embryos on medium containing BA (16.0 μM) supplemented with indole-3-acetic acid (IAA: 2.8–5.7 μM) and/or kinetin (4.6 μM) gave rise to clusters of secondary somatic embryos along with maturation of primary embryos. In subsequent subculture on hormone-free half-strength WPM, the embryo clusters germinated with an increase in the number of secondary somatic embryos. About 70% of somatic embryos germinated into complete plantlets, which were successfully established under greenhouse conditions.     

Anatomical study of zygotic and somatic embryos of Tilia cordata

A comparative anatomical study was carried out on zygotic and somatic embryos of Tilia cordata Mill. to evaluate the effect of growth conditions on their development. Zygotic embryos (heart-shaped, torpedo, cotyledonary), collected during two autumn periods, were examined to investigate the effect of growing season on embryo development. In comparison, the influence of growth conditions on the development of somatic embryos in vitro was also studied. Treatment with abscisic acid (ABA) and polyethylene glycol-4000 induced the development of somatic cotyledonary embryos similar to zygotic embryos with respect to morphology and anatomy, as illustrated by the differentiation of the apical meristems and of the procambium. The pattern of accumulation of starch and protein was also similar in these embryos. Somatic cotyledonary embryos that developed spontaneously without ABA showed defective accumulation of storage material and a general failure to form the shoot apical meristem, leading to very low germination rates. Vacuolar phenolic deposits were observed along the procambium of both zygotic and somatic embryos regardless of the maturation stage. Tracheid formation was observed only in somatic embryos formed without ABA in the medium and in precociously germinated somatic embryos. Phenolic vacuolar inclusions were frequently observed in epidermal cells of these embryos. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of Abscisic Acid, Osmoticum, and Desiccation on Synthesis of Storage Proteins during the Development of White Spruce Somatic Embryos

The effect of abscisic acid (ABA), non-permeating osmoticumand desiccation treatment on storage protein synthesis duringmaturation of somatic embryos of Picea glauca (Moench) Voss.was examined. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis(SDS-PAGE) and Western blot analysis demonstrated that someof the major crystalloid and matrix polypeptides were absentfrom somatic embryos maturing on medium containing ABA and lowosmoticum. However, treatment with polyethylene glycol-4000(PEG) in combination with ABA resulted in the synthesis of aspectrum of storage polypeptides resembling that of mature zygoticembryos. These storage proteins accumulated throughout an 8-weekculture period, resulting in a threefold higher protein contentthan somatic embryos maturing for the same time in the absenceof PEG. The structure and distribution of protein bodies incells of these osmotically treated somatic embryos was similarto that in cells of mature zygotic embryos. Treatment with 5·0-7·5%PEG prevented catabolism of the accumulated storage polypeptidesduring desiccation. The optimal culture conditions for somaticembryo maturation and storage protein deposition was 16 µMABA and 7·5% PEG for 8 weeks followed by desiccation.Analysis of mRNAs by in vitro translation and immunoprecipitationof translated products showed that the crystalloid protein mRNAprofiles of zygotic and those of somatic embryos maturing on16 µM ABA in the absence of PEG were similar. The differencesobserved in the pattern of accumulated polypeptides in thesesomatic embryos and those of mature zygotic embryos, therefore,indicates that storage-protein synthesis in response to osmoticumis in part regulated at the translational level. During regenerationof somatic embryos to plantlets the storage polypeptides wererapidly utilized in a manner similar to that in zygotic seedlings.Copyright1993, 1999 Academic Press Desiccation, osmotic stress, storage proteins, Picea, embryogenesis—somatic, mRNA (crystalloid protein)     

Comparison of induction frequency, maturation capacity and germination of Abies numidica during secondary somatic embryogenesis

Efficiency of the method for improving repetitive somatic embryogenesis and plant recovery of Algerian fir (Abies numidica De Lann.) was investigated by evaluating of induction frequency, maturation capacity and germination. Individual zygotic embryos differed only slightly in induction frequencies (6.8 %) from somatic embryos of the first (5.7 %) and second cycle (5.5–9.0 %). The yield of mature embryos differed significantly among the cell lines of the same cycle and among cell lines of the different cycles. Percentage of abnormalities was lowest in the first cycle of somatic embryos, whereas the second and the third cycles of somatic embryos were branded by a higher frequency of abnormalities. The differences in germination of well developed somatic embryos depended on cell lines rather than on cycle of somatic embryos.     

Somatic embryo productions by liquid shake culture of embryogenic calluses in <Emphasis Type="Italic">Vigna mungo</Emphasis> (L.) Hepper

The regeneration of plants via somatic embryogenesis liquid shake culture of embryogenic calluses was achieved in Vigna mungo (L.) Hepper (blackgram). The production of embryogenic callus was induced by seeding primary leaf explants of V. mungo onto Murashige and Skoog (MS) (Physiol Plant 15:473–497, 1962) medium supplemented (optimally) with 1.5 mg/l 2,4-dichloro-phenoxyacetic acid. The embryogenic callus was then transferred to liquid MS medium supplemented (optimally) with 0.25 mg/l 2,4-dichloro-phenoxyacetic acid. Globular, heart-shaped, and torpedo-shaped embryos developed in liquid culture. The optimal carbohydrate source for production of somatic embryos was 3% sucrose (compared to glucose, fructose, and maltose). l-Glutamine (20 mg/l) stimulated the production of all somatic embryo stages significantly. Torpedo-shaped embryos were transferred to MS (Physiol Plant 15:473–497, 1962) liquid medium containing 0.5 mg/l abscisic acid to induce the maturation of cotyledonary-stage embryos. Cotyledonary-stage embryos were transferred to 1/2-MS semi-solid basal medium for embryo conversion. Approximately 1–1.5% of the embryos developed into plants.     

Manipulation of conditions for the culture of somatic embryos of white spruce for improved triacylglycerol biosynthesis and desiccation tolerance

In order to enhance post-germinative vigour, somatic embryos of Picea glauca (Moench) Voss. were matured under in-vitro conditions that stimulated triacylglycerol (TAG) biosynthesis. In P. glauca seeds over 90% of the TAG was stored within the megagametophyte, and isolated zygotic embryos contained twice the amount of TAG of somatic embryos cultured for four weeks on basal medium containing 16 M abscisic acid (ABA). Polyethylene glycol-4000 (PEG) as a non-permeating osmoticum with ABA promoted TAG biosynthesis by somatic embryos and sustained maturation throughout an eight-week culture period. Treatments that promoted TAG biosynthesis also prevented precocious germination and promoted desiccation tolerance. Thus, the optimal culture conditions for maturation, desiccation survival, and plantlet regeneration were 16–24 M ABA and 7.5% PEG for eight weeks, followed by desiccation. Under these conditions the levels of TAG per somatic embryo were raised ninefold to about five times the zygotic-embryo level, and the TAG fatty-acid composition became similar to that of zygotic embryos. A study of sectioned material, using light and transmission electron microscopy, showed that the structure and distribution of lipid bodies within these somatic embryos and the degree of embryo development were similar to mature zygotic embryos. Up to 81% of the desiccated somatic embryos regenerated to plantlets during which time the TAG was utilised in a manner similar to zygotic seedlings.Abbreviations ABA abscisic acid - PEG polyethylene glycol - TAG triacylglycerol - TL total lipid - TEM transmission electron microscopy Plant Research Centre contribution No. 1383We are grateful to Dawn Moore and Ken Stanley for technical assistance, and thank Pat Rennie for the electron microscopy. We acknowledge financial support through an NSERC/Forestry Canada/Weyerhaeuser Canada Ltd (Prince Albert, Sask.) research partnership programme.     

Induction of embryogenic tissue and maturation of somatic embryos in Pinus brutia TEN

Embryogenic tissues (ET) of Turkish red pine (Pinus brutia TEN) were initiated from immature precotyledonary zygotic embryos sampled from 15 different plus trees. Seven collections were made weekly from June 10 to July 22, 2003. DCR basal medium supplemented with 13.6 μM 2,4–dichlorophenoxyacetic acid (2,4-D) and 2.2 μM benzylaminopurine (BAP) was used for initiation and maintenance of ET. Overall initiation frequency of ET in the study was 11.6%, initiation rates ranging between 4.7% and 24.1% per tree. Out of 12,940 explants tested, 3.4% were converted into established cell lines (ECL) following five subcultures. Of the maturation treatments tested, 80 μM ABA, sucrose (3%) and maltose (3% and 6%), and 3.75% PEG combined with 1% gellan gum were the most suitable combination for somatic embryo maturation.     

Polyethylene glycol and maltose enhance somatic embryo maturation in loblolly pine (Pinus taeda L.)

Summary A culture medium that can efficiently produce mature somatic embryos was developed for loblolly pine (Pinus taeda L.). The medium contained maltose as a carbohydrate source and polyethylene glycol as an osmoticum. This medium formulation significantly enhanced embryo maturation efficiency compared to a medium with only maltose, or with sucrose combined with polyethylene glycol. Maltose at 4% and polyethylene glycol at 6% resulted in the highest embryo maturation efficiency; an average of around 100 cotyledonary embryos were produced from 1 g of embryogenic tissue. These results suggested that previous ineffective embryo maturation in loblolly pine may be due to the lack of the proper combination of osmoticum and carbohydrate source. This embryo maturation method also improved morphology of cotyledonary embryos of loblolly pine.     

Optimization of horse chestnut (<Emphasis Type="Italic">Aesculus hippocastanum</Emphasis> L.) somatic embryo conversion

Factors affecting conversion of horse chestnut (A. hippocastanum L.) somatic embryos into plantlets were evaluated. Anther filament derived embryogenic tissue developed bipolar structures with two cotyledons and a well-developed shoot and root apical meristem upon auxin omittance from the culturing medium. The impact of carbohydrate type (glucose, fructose, sucrose and maltose) and concentration (3 and 6%) on somatic embryo maturation and conversion were evaluated. Although conversion frequencies were high for all treatments, overall quality of regenerated plantlets was poor. Increasing the carbohydrate concentration in the maturation medium did not increase conversion of somatic embryos or quality of regenerated plantlets in terms of shoot height. On the contrary, addition of PEG (polyethylene glycol) in maturation media had a beneficial effect on shoot quality of regenerated plantlets. Sucrose was a superior carbon source when PEG was included in the maturation medium, in terms of conversion rate (65.7%) as well as of shoot quality of plantlets (43.8% of plantlets had shoots >2 cm). Clonal fidelity of the different development stages of somatic embryogenesis and of converted plantlets was assessed by flow cytometry and no major ploidy changes were found.     

Direct and indirect somatic embryogenesis on cotyledon explants of <Emphasis Type="Italic">Quassia amara</Emphasis> L., an antileukaemic drug plant

Summary In vitro propagation of Quassia amara L. (Simaroubaceae) was attempted using mature and juvenile explants. Attempts to establish in vitro culture using leaf and internode explants from a plant more than 15yr old were unsuccessful due to severe phenolic exudation. Plant regeneration through direct and indirect somatic embryogenesis was established from cotyledon explants. Murashige and Skoog (MS) medium with 8.9 μM N⁶-benzyladenine (BA) and 11.7 μM silver nitrate induced the highest number (mean of 32.4 embryos per cotyledon) of somatic embryos. Direct somatic embryogenesis as well as callus formation was observed on medium with BA (8.9–13.3 μM). Semi-mature pale green cotyledons were superior for the induction of somatic embryos. Embryos developed from the adaxial side as well as from the point of excision of the embryonic axis. More embryos were developed on the proximal end compared to mid and distal regions of the cotyledons. Subculture of callus (developed along with the somatic embryos on medium with BA alone) onto medium containing 8.9 μM BA and 11.7 μM silver nitrate produced a mean of 17.1 somatic embryos. Primary somatic embryos cultured on MS medium with 8.9 μM BA and 11.7μM silver nitrate produced a mean of 9.4 secondary somatic embryos. Most of the embryos developed up to early cotyledonary stage. Reduced concentration of BA (2.2 or 4.4 μM) improved maturation and conversion of embryos to plantlets. Ninety percent of the embryos converted to plantlets. The optimized protocol facilitated recovery of 30 plantlets per cotyledon explant within 80d. Plantlets transferred to small cups were subsequently transferred to field conditions with a survival rate of 90%.     

Regeneration of plant by somatic embryogenesis in <Emphasis Type="Italic">Pinus rigida</Emphasis>×<Emphasis Type="Italic">P. taeda</Emphasis>

Zygotic embryos at different developmental stages were tested for their potential in the initiation of embryogenic suspensor mass (ESM) lines using immature seeds of Pinus rigida × P. taeda. The highest frequency (1.1%) of ESM was obtained with explants from cones collected on July 1. All excised embryos of the July 1 collection were at the early proembryo stage. Two different culture media were compared. Forty-eight ESM lines were initiated on Pinus taeda basal medium (P6) (0.97%) with 13.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4 μM benzyladenine (BA). However, only four ESM were obtained on a modified Murashige and Skoog medium (MSG; 0.55%). Most of the ESM arose from the seeds that were at the stages ranging from late cleavage polyembryony to the early staged proembryo. Out of 52 lines (0.46%) that were produced from 11,388 explants, only two viable lines (0.018%) (PRT11 and PRT28) survived. As for somatic embryo maturation, the highest number (224/g⁻¹ FW) of matured cotyledonary somatic embryos (line PRT 28) was obtained on a medium containing 100 μM abscisic acid (ABA), 0.2 M maltose, and 1.2% gellan gum. For germination of the somatic embryos, the cotyledonary somatic embryos derived from maturation medium were transferred on half-strength Litvay medium (LM) plus 0.4% gellan gum. The germination rates were high (71.4–96.3%) regardless of the concentrations of either ABA or gellan gum in the maturation medium. Approximately 500 somatic plants were recovered from the germination medium and transferred to the green house; finally most of them were transplanted successfully to the experimental field.     

Peroxidase activity of desiccation-tolerant loblolly pine somatic embryos

Summary Desiccation tolerance can be induced by culturing somatic embryos of loblolly pine (Pinus taeda L.) on medium supplemented with 50 μM abscisic acid (ABA) and/or 8.5% polyethylene glycol (PEG₆₀₀₀). Scanning electron microscopy of desiccated somatic embryos showed that the size and external morphology of the desiccation-tolerant somatic embryos recovered to the pre-desiccation state within 24–36 h, whereas the non-desiccation-tolerant somatic embryos did not recover and remained shriveled, after rehydration. Peroxidase activity of desiccated somatic embryos increased sharply after 1 d of desiccation treatment at 87% relative humidity (RH), and desiccation-tolerant somatic embryos had higher peroxidase activity compared to sensitive somatic embryos. Higher peroxidase activity of desiccation-tolerant somatic embryos may have allowed them to catalyze the reduction of H₂O₂ produced by drought stress, and protected them from oxidative damage.     

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.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Echinacea purpurea</Emphasis> L.: Enhancement of somatic embryogenesis by indolebutyric acid and dark pre-incubation

Summary The embryogenic potential of different Echinacea purpurea tissues, viz. leaf, cotyledon, and root, was investigated. Maximum embryo-induction was achieved from leaf dises cultured on Murashige and Skoog medium supplemented with benzylaminopurine (5.0 μM) and indolebutyric acid (2.5 μM) where 95% of the explants responded, yielding an average of 83 embryos per explant within 4 wk of culture. Incubation of cultures in the dark for an initial period of 14 d significantly increased the frequency of somatic embryogenesis (6–8-fold in leaf explants). Exposure of the abaxial surface of leaves to the medium significantly increased the number of embryos. Transfer of somatic embryos to a medium devoid of growth regulators resulted in 80% germination within 7 d. Over 73% of the somatic embryos developed roots within 28 d of culture on a medium containing naphthaleneacetic acid (10 μM) with a maximum root number of 9.8 per plantlet. Transplanting ex vitro and acclimatization for a period of 7 d were sufficient to promote establishment of plants in the greenhouse, and more than 90% of the regenerated plants survived.     

Somatic embryogenesis and plant regeneration in date palm <Emphasis Type="Italic">Phœnix dactylifera</Emphasis> L., cv. Boufeggous is significantly improved by fine chopping and partial desiccation of embryogenic callus

Plant regeneration through somatic embryogenesis from young leaf explants (5–10 mm long) adjacent to the apex of 5–6 year old offshoots of Tunisian date palm (Phœnix dactylifera L.), cultivar Boufeggous was successfully achieved. Factors affecting embryogenic callus initiation, including plant growth regulators and explant size, were investigated. The highest induction frequencies of embryogenic calli occurred after 6–7 months on MS medium supplemented with 10 mg l⁻¹ 2,4-D and 0.3 mg l⁻¹ activated charcoal. The subculture of these calli onto maintenance medium resulted in the formation of proembryos. Fine chopping and partial desiccation (6 and 12 h) of embryogenic calli with proembryos prior to transfer to MS medium supplemented with 1 mg l⁻¹ ABA stimulated the rapid maturation of somatic embryos. Maturated somatic embryo yield per 0.5 g FW of embryogenic callus was 51 embryos with an average maturation time of 55 days. This was increased to 422 with finely chopped callus, and 124 and 306 embryos following 6 and 12 h desiccation treatments, respectively. The average time to maturation for these 3 treatments was 35, 43 and 38 days, respectively. Subsequent substitution of ABA in MS medium with 1 mg l⁻¹ NAA resulted in the germination and conversion of 81% of the somatic embryos into plantlets with normal roots and shoots. The growth of regenerated somatic plants was also monitored in the field.     

Somatic embryogenesis and plant regeneration in yakutanegoyou, <Emphasis Type="Italic">Pinus armandii</Emphasis> Franch. var. <Emphasis Type="Italic">amamiana</Emphasis> (Koidz.) Hatusima,an endemic and endangered species in Japan

Induction of somatic embryogenesis in Pinus armandii var. amamiana, an endemic and endangered species in Japan, was initiated from megagametophytes containing immature zygotic embryos on both media with and without plant growth regulators. Across nine open-pollinated families initiation frequency ranged from 0 to 20%, with an average of 1.5%. Embryogenic cultures were maintained and proliferated on a medium supplemented with 2,4-dichlorophenoxyacetic acid (3 μM) and 6-benzylaminopurine (1 μM). Maturation of somatic embryos occurred on medium containing maltose (50 g l⁻¹), activated charcoal (2 g l⁻¹), abscisic acid (100 μM), and polyethylene glycol (100 g l⁻¹). The frequencies of germination and plant conversion of somatic embryos differed among the embryogenic lines from 16 to 51% and from 12 to 40%, respectively. Growth of regenerated somatic plants has been monitored in the field.     

Induction of somatic embryos from cultures of <Emphasis Type="Italic">Agave vera-cruz</Emphasis> Mill.

Somatic embryogenesis from cultures of shoot apices, cotyledon and young leaves of in vitro shoots of Agave vera-cruz Mill. was studied. Embryogenic callus was obtained when explants were cultured on Murashige and Skoog’s (MS) medium (1962) supplemented with L₂ vitamins, 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-d) or 5.37 μM ∝-naphthalene acetic acid (NAA). Somatic embryos differentiated from this embryogenic callus upon subculture to maturation/conversion medium containing cytokinin either alone or with auxin and l-glutamine. The best combination of growth regulators for development of somatic embryos was found to be 5.37 μM naphthalene acetic acid plus 0.91 μM zeatin and 40 g/l sucrose. The conversion frequency of somatic embryos to plantlets varied from 46–50%. Rooted plantlets were transferred directly to pots containing a soil, sand, and manure mixture without any hardening phase with 96–98% survival of the plantlets. Based on the histological observations, the potential origin of the somatic embryo is discussed.