<Emphasis Type="Italic">In vitro</Emphasis> regeneration through somatic embryogenesis and organogenesis using cotyledons of <Emphasis Type="Italic">Cassia angustifolia</Emphasis> Vahl

In vitro regeneration through somatic embryogenesis as well as organogenesis using cotyledon of a woody medicinal legume, Cassia angustifolia is reported. The cotyledons dissected from semi-mature seeds, if inoculated on Murashige and Skoog’s medium (MS) supplemented with auxin alone or in combination with cytokinin, produced direct and indirect somatic embryos. A maximum of 14.36 ± 2.26 somatic embryos per 20 mg of explants including callus were produced in 70% cultures on MS medium with 2.5 μM benzyladenine (BA) + 10 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Although the percentage of embryogenic cultures was higher (83.33%) at 10 μM 2,4-D + 1 μM BA, the average number of somatic embryos was much less (7.6 ± 0.85) at this level, whereas at 2.5 μM BA and 5 μM 2,4-D, there was a simultaneous formation of both somatic embryos and shoots. The somatic embryos, although started germinating on the same medium, developed into full plantlets only if transferred to MS basal with 2% sucrose. Cytokinins alone did not induce somatic embryogenesis, but formed multiple shoots. Five micromolar BA proved optimum for recurrently inducing shoots in the competent callus with a maximum average of 12.04 ± 2.10 shoots and shoot length of 2.26 ± 0.03 cm. Nearly 91.6% shoots (2–2.5 cm in size) organized an average of 5.12 ± 0.58 roots on half strength MS + 10 μM indole-3-butyric acid. All the plantlets have been transferred successfully to soil. Types of auxin and its interaction with cytokinin significantly influenced somatic embryogenesis.     

Shoot development in Quercus suber L. somatic embryos

Summary N⁶-Benzyladenine (BA; 0.04–4μM) application to germinated Quercus suber somatic embryos considerably increased caulinar apex elongation frequency and maintained active growth in the plantlets, although it did not have a significant effect on the percentage of shoots with normal morphology. The addition of 0.5 μM indoleacetic acid together with the cytokinin did not have any effect. The use of a low concentration (0.04 μM) of BA allowed the appropriate radicle elongation in all germinating somatic embryos, but higher concentrations arrested this elongation.     

Effect of maturation duration on desiccation tolerance in hybrid larch (Larix×leptoeuropaea dengler) somatic embryos

Summary Cotyledonary somatic embryos ofLarix &#x00D7; leptoeuropaea that developed after various maturation times on media containing abscisic acid showed different frequencies of conversion into plants. Drying of these somatic embryos under high relative humidity (RH) before germination improved plantlet recovery and eliminated differences in the performance of somatic embryos matured for different times. However, dehydration of somatic embryos under 98% RH to a water content below that of zygotic embryos excised from mature seeds (0.97 and 1.36 g H₂O/g dry weight, respectively) showed a strong positive correlation between longer maturation time and desiccation tolerance. Drying somatic embryos at 4&#x00B0; C under 59% RH for 1 wk resulted in desiccation to a water content of 0.30 g H₂O/g dry weight, which was the closest to the hydration state of zygotic embryos in dried, stored seeds (0.20 g H₂O/g dry weight). Under this condition, only somatic embryos matured for 5 wk germinated and produced plantlets at a relatively high frequency (73 and 41%, respectively).     

Abscisic acid and ancymidol promote conversion of somatic embryos to plantlets and secondary embryogenesis inAsparagus officinalis L.

Summary The effects of abscisic acid (ABA) (0, 0.09 μM, 0.19 μM, 0.28 μM, and 0.38 μM) or ancymidol (0, 0.98 μM, 1.95 μM, 2.93 μM, 3.90 μM) in embryo germination medium on the conversion of primary embryos to plantlets and secondary embryogenesis were evaluated for asparagus. ABA and ancymidol each significantly enhanced both responses. ABA was more effective than ancymidol in promoting the conversion of primary embryos to plantlets, while the converse was true for the production of secondary embryos. The most effective treatments for embryo conversion were 0.19 and 0.28 μM ABA; 75–77% bipolar and 55–57% globular embryos converted to plantlets. For secondary embryogenesis, the most effective treatments were 1.95 and 2.93 μM ancymidol; 99–101 and 84–86 somatic embryos were produced from 10 globular and 10 bipolar embryos, respectively. Bipolar embryos generally converted to plantlets better than globular embryos, but more secondary embryos were produced from globular embryos than from bipolar embryos in all treatments. ABA and ancymidol also affected the morphology of the plantlets produced. The plantlets from the embryos incubated on the medium with ancymidol had strong and thick shoots and roots, while those on the medium with ABA had long, thin shoots and short thin roots.     

Plant regeneration from embryogenic cultures initiated from mature loblolly pine zygotic embryos

Summary Mature zygotic embryos of eight (open-pollinated) families of loblolly pine (Pinus taeda L.) were cultured on eight different basal salt formulations, each supplemented with 36.2 μM 2,4-dichlorophenoxyacetic acid, 17.8 μM 6-benzyladenine, 18.6 μM kinetin, 500 mg l⁻¹ casein hydrolysate, and 500 mg l⁻¹ l-glutamine for 9 wk; embryogenic tissue was formed on cotyledons, hypocotyls, and radieles of mature zygotic embryos. Callus was subcultured on the callus proliferation medium, the same as the induction medium but with one-fifth concentration of auxin and cytokinin for 9 wk. On this medium a white to translucent, glossy, mucilaginous embryogenic callus containing embryogenic suspensor masses (ESMs) was obtained. The highest frequency of explants forming embryogenic tissue, 17%, occurred on a modified Murashige and Skoog salts basal medium containing the concentration of KNO₃, Ca(NO₃)₂·4H₂O, NH₄NO₃, KCl, ZnSO₄·7H₂O, and MnSO₄·H₂O, 720, 1900, 400, 250, 25.8, and 25.35 mg l⁻¹, respectively. Embryogenic suspension cultures were established by culturing embryogenic callus in liquid callus proliferation medium. Liquid cultures containing ESMs were transferred to medium containing abscisic acid, polyethylene glycols, and activated charcoal for stimulating the production of cotyledonary somatic embryos. Mature somatic embryos germinated for 4–12 wk on medium containing indole-butyric acid, gibberellic acid, 6-benzyladenine, activated charcoal, and reduced sucrose concentration (15 g l⁻¹). Two hundred and ninety-one regenerated plantlets were transferred to a perlite:peatmoss:vermiculite (1∶1∶1) mixture, then the plants were transplanted to soil in the earth, and 73 plantlets survived in the field.     

Germination and plantlet regeneration from encapsulated somatic embryos of mango (Mangifera indica L.)

 Cotyledonary-stage somatic embryos (3–5 mm in length) originating from nucellar explants of Mangifera indica L. cv. Amrapali were encapsulated individually in 2% alginate gel. The encapsulated somatic embryos (ESEs) germinated successfully on 0.6% agar-gelled medium containing B5 macrosalts (half strength), Murashige and Skoog microsalts (full strength), 3% sucrose and 2.9 μM gibberellic acid. The percentage of germination of ESEs was higher than that of naked somatic embryos of the same size on the same medium. The germinability of ESEs was increased (73.61±7.08%) when the medium was supplemented with full-strength B5 macrosalts. Of the germinating ESEs, 45.83±3.40% developed into plantlets. Abscisic acid at 0.004 and 0.02 μM had no significant influence on germination and plantlet development, but caused a 3-week delay in germination. Well-developed plantlets regenerated from ESEs have been successfully established in soil. Received: 9 February 1998 / Accepted: 22 March 1999     

Improved germination of somatic embryos and plant recovery of European chestnut

For the mass production of chestnut trees with selected, hybrid, or genetically engineered genotypes, one potentially desirable propagation strategy is based on somatic embryogenesis. Although methods exist for the initiation of embryogenic cultures of Castanea sativa from immature zygotic embryos or leaf explants, the embryos produced have had low rates of conversion into plantlets. This study explored the possible benefits for somatic embryos that have already undergone maturation and cold treatments, of (a) partial slow or fast desiccation, and (b) of the addition of plant growth regulators or glutamine to the germination medium. Germination response was evaluated in terms of both conversions to plantlets and through embryos developing only shoots (shoot germination) that could be rooted following the micropropagation protocols developed for chestnut. Two or 3 wk slow desiccation in sealed empty Petri dishes resulted in a slight reduction in water content that nevertheless increased total potential plant recovery, shoot length, and the number of leaves per plantlet. However, best results were achieved by 2 h fast drying in a laminar flow hood, which reduced embryo moisture content to 57–58% and enhanced the potential plant recovery and quality of regenerated plantlets. Plant yield was also promoted by addition of 0.44 μM benzyladenine and 200–438 mg/l of glutamine to the germination medium, and plantlet quality (as evidenced by root, shoot, and leaf growth) by the further addition of 0.49 μM indole-3-butyric acid.     

Plant regeneration from alginate-encapsulated shoot tips of Phylianthus amarus schum and thonn, a medicinally important plant species

Summary A method was developed for plant regeneration from alginate-encapsulated shoot tips of Phyllanthus amarus. Shoot tips excised from in vitro proliferated shoots were encapsulated in calcium alginate beads. The best gel complexation was achieved using 3% sodium alginate and 75 mM CaCl₂·2H₂O. Maximum percentage response for conversion of encapsulated shoot tips into plantlets was 90% after 5 wk of culture on Murashige and Skoog (MS) medium without plant growth regulator. The regrowth ability of encapsulated shoot tips was affected by the concentration of sodium alginate, storage duration, and the presence or absence of MS nutrients in calcium alginate beads. Plantlets with well-developed shoot and roots were transferred to pots containing an autoclaved mixture of soilrite and peat moss (1∶1). The conversion of encapsulated shoot tips into plantlets also occurred when calcium alginate beads were directly sown in autoclaved soilrite moistened with 1/4-MS salts. Encapsulation of vegetative propagules in calcium alginate beads can be used as an alternative to synthetic seeds derived from somatic embryos.     

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.     

Somatic embryo initiation and germination in diploid cotton (<Emphasis Type="Italic">Gossypium arboreum</Emphasis> L.)

Summary The diploid cotton species can constitute a valuable gene pool for the more agronomically desirable cultivated tetraploid cultivars and offer better opportunities to study gene structure and function through gene knockouts. In order to exploit these advantages, a regeneration system is required to achieve these transformation-based goals. Carbohydrate source and concentration were evaluated to improve somatic embryo (SE) production and desiccation treatments to improve the conversion efficiency of SEs to plants in a diploid Gossypium arboreum accession, A₂-9 (PI-529712). Improved SE numbers and their subsequent conversion into plantlets was achieved with a Murashige and Skoog (MS)/sucrose-based medium M₂ [0.04M sucrose, 0.3 μM α-naphthaleneacetic acid (NAA)] On this medium, 219 embryos per g initiated, and close to 11% of these embryos germinated into plantlets. Neither a 5-d desiccation treatment of embryogenic callus previously cultured in liquid medium nor filter paper insertion improved the numbers of SEs induced or their conversion to plantlets. A 3-d desiccation period resulted in improved plant regeneration. When immature G. arboreum SEs induced on M₁ (0.2M glucose, 2.6 μM NAA, and 0.2 μM kinetin) medium underwent a 3-d desiccation treatment, 49% of these immature SEs were converted to plantlets after a 4-wk period on M₂ medium. These improved results will help to pave the way for future genetic transformation and associated gene structure and function studies utilizing G. arboreum. These results, in particular the 3-d desiccation treatment, can also be incorporated into regeneration protocols to improve the regeneration efficiency of other Gossypium species.     

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.     

Somatic embryogenesis and plant regeneration in <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

Embryogenic callus in Catharanthus roseus was initiated from hypocotyl on Murashige and Skoog’s (MS) medium supplemented with 1.0–2.0 mg dm⁻³ of 2,4-dichlorophenoxyacetic acid (2,4-D) or chlorophenoxyacetic acid (CPA). Calli from other sources were non-embryogenic. Numerous somatic embryos were induced from primary callus on MS medium suplemented with naphthalene acetic acid (NAA) within two weeks of culture. Embryo proliferation was much faster on medium supplemented with 6-benzylaminopurine (BAP). After transfer to medium with gibberellic acid (GA₃, 1.0 mg dm^{− 3}) mature green embryos were developed and germinated well into plantlets on MS liquid medium supplemented with 0.5 mg dm⁻³ BAP. Later, embryos with cotyledonary leaves were subjected to different auxins treatments for the development of roots. Before transfer ex vitro, plantlets were cultivated on half strength MS medium containing 3 % sucrose and 0.5 mg dm⁻³ BAP for additional 2 weeks. Additionally, the effect of liquid medium has been evaluated at different morphogenetic stages.     

Influence of freezable/non-freezable water and sucrose on the viability of <Emphasis Type="Italic">Theobroma cacao</Emphasis> somatic embryos following desiccation and freezing

Encapsulated cocoa (Theobroma cacao L.) somatic embryos subjected to 0.08–1.25 M sucrose treatments were analyzed for embryo soluble sugar content, non-freezable water content, moisture level after desiccation and viability after desiccation and freezing. Results indicated that the higher the sucrose concentration in the treatment medium, the greater was the extent of sucrose accumulation in the embryos. Sucrose treatment greatly assisted embryo post-desiccation recovery since only 40% of the control embryos survived desiccation, whereas a survival rate of 60–95% was recorded for embryos exposed to 0.5–1.25 M sucrose. The non-freezable water content of the embryos was estimated at between 0.26 and 0.61 g H₂O g⁻¹dw depending on the sucrose treatment, and no obvious relationship could be found between the endogenous sucrose level and the amount of non-freezable water in the embryos. Cocoa somatic embryos could withstand the loss of a fraction of their non-freezable water without losing viability following desiccation. Nevertheless, the complete removal of potentially freezable water was not sufficient for most embryos to survive freezing.     

Maturation and germination of walnut somatic embryos

Walnut somatic embryos were multiplied by repetitive embryogenesis on a solid basal DKW medium at 25°C in the dark. When the embryos were isolated at early cotyledonary stage (1–2 mm long) from the primary embryos and cultured on the medium for 3 weeks, they developed into mature embryos showing white, enlarged cotyledons and shoot and root apex. After transfer to light on solid germination medium, however, few mature embryos (0–5%) germinated. Germination percentage increased to about 10% when the mature embryos were pretreated by a storage at 4°C in the dark for 2 months, or by desiccation at 25°C in the dark for 3 or 5 days under an air-humidity conditioned by saturated salt solutions (Mg(NO₃)₂.6H₂O, or ZnSO₄.7H₂O). Similar results were obtained by the addition of gibberellic acid (GA₃) to the germination medium. When mature embryos were desiccated and then placed on medical cotton compresses in liquid germination medium, 45% of the embryos germinated into complete plantlets. These plantlets continued their growth after transplanting to a mixture of peat and vermiculite in pots.Abbreviations GA₃ gibberellic acid - DKW medium Driver & Kuniyuki Walnut medium     

Somatic embryogenesis and plantlet regeneration in American ginseng (Panax quinquefolium L.)

Summary Somatic embryogenesis in American ginseng (Panax quinquefolium L.) was investigated from three explant sources (root, leaf and epicotyl) with Murashige and Skoog (MS) medium containing different growth regulators. Mature roots and leaves obtained from 3- to 5-yr-old field-grown plants, and seedling leaves and epicotyls from plantlets grownin vitro, were evaluated. From root and epicotyl explants, callus development was optimal with 3,6-dichloro-o-anisic acid (dicamba) (9.0 μM) and kinetin (KN) (5.0 μM) as the growth regulators. When these calluses were transferred after 3 mo. to dicamba alone (9.0 μM), somatic embryo formation was observed at an average frequency of 15.6% in root explants after an additional 3 mo., and 2% in epicotyl explants after an additional 6 mo. No plantlets were recovered because the embryos germinated to form shoots with no roots. From leaf explants, callus growth was optimal with α-naphthaleneacetic acid (NAA) at 10.0 μM and 2,4-dichlorophenoxyacetic acid (2,4-D) at 9.0 μM. Somatic embryos developed on this medium, with the highest frequency (40%) obtained after 3 mo. from seedling-leaf explants. Calluses on mature leaves formed somatic embryos after 7 mo. with NAA/2,4-D at an average frequency of 30%. Transfer of these somatic embryos to 6-benzyladenine/gibberellic acid (4.4/2.9 μM) promoted shoot development but no roots were observed. Up to 100% of germination was observed within 6 wk on half-strength MS salts containing activated charcoal (1%) and on NAA/2,4-D (5.0/4.5 μM) with charcoal (1%). On the latter medium, somatic embryos enlarged and frequently gave rise to new somatic embryos after a brief callusing phase. The embryos germinated through a two-stage process, involving the elongation of the root followed by the formation of a shoot. The highest recovery of ginseng plantlets from germinated embryos was 61.0%. Following transfer to potting medium and maintenance under conditions of high humidity and low light intensity, the plantlets elongated and developed new leaves. A high percentage (50%) of these plants have been acclimatized to soil.     

Improvement of somatic embryogenesis and plant recovery in cassava

Methods for improving the efficiency of plant recovery from somatic embryos of cassava (Manihot esculenta Crantz) were investigated by optimizing the maturation regime and incorporating a desiccation stage prior to inducing germination. Somatic embryos were induced from young leaf lobes of in vitro grown shoots of cassava on Murashige and Skoog medium with 2,4-dichlorophenoxy acetic acid. After 15 to 20 days of culture on induction medium, the somatic embryos were transferred to a hormone free medium supplemented with activated charcoal. In another 18 days mature somatic embryos became distinctly bipolar and easily separable as individual units and were cultured on half MS medium for further development. Subsequent desiccation of bipolar somatic embryos resulted in 92% germination and 83% complete plant regeneration. The plants were characterized by synchronized development of shoot and root axes. Of the non-desiccated somatic embryos, only 10% germinated and 2% regenerated plants. Starting from leaf lobes, transplantable plantlets were derived from primary somatic embryos within 70 to 80 days.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - BA Benzyl aminopurine - GA Giberellic acid - MS Murashige and Skoog - NAA Naphthalene acetic acid     

Rapid propagation of Eleutherococcus senticosus via direct somatic embryogenesis from explants of seedlings

Explants from three different parts (cotyledon, hypocotyl or root) of one week-old seedlings of Eleutherococcus senticosus were cultured on Murashige and Skoog (MS) medium with 1.0 mg l^-1 2,4-D. Somatic embryos were formed directly from the surfaces of explants. The frequency of direct somatic embryo formation was the highest in the hypocotyl segments (75%) as compared to cotyledon (56%) or root segments (12%). When hypocotyl explants from 3 different stages of seedlings (zero, one or three week-old) were cultured on MS medium with 1.0 mg l^-1 2,4-D, the frequency of somatic embryo formation rapidly declined as the zygotic embryos germinated. However most somatic embryos (93%) from explants of zygotic embryos developed as fused state (multiple embryo), whereas somatic embryos (over 89%) from more developed seedlings developed into single state (single embryo). Single embryos germinated and regenerated into plantlets with both shoots and roots, while multiple embryos only regenerated into only multiple shoots. Plantlets that regenerated from single embryos of E. senticosus were acclimatized in a greenhouse. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

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.     

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.