Auxin and nutritional stress coupled somatic embryogenesis in <Emphasis Type="Italic">Oldenlandia umbellata</Emphasis> L.

Somatic embryos were induced from internodal segment derived callus of Oldenlandia umbellata L., in MS medium supplemented with different concentrations of 2,4-Dichlorophenoxy acetic acid (2,4-D). Initially calli were developed from internodes of microshoots inoculated in 2.5 µM NAA supplemented medium. Then calli were transferred to 2,4-D added medium for somatic embryogenesis. Nutritional stress coupled with higher concentration of 2,4-D triggered somatic embryogenesis. Nutritional stress was induced by culturing callus in a fixed amount of medium for a period up to 20 weeks without any external supply of nutrients. Addition of 2.5 µM 2,4-D gave 100% embryogenesis within 16 weeks of incubation. Callus mass bearing somatic embryos were transferred to germination medium facilitated production of in vitro plantlets. MS medium supplemented with 2.5 µM benzyl adenine and 0.5 µM α-naphthalene acetic acid produced 15.33 plants per culture within 4 weeks of culture. Somatic embryo germinated plants were then hardened and transferred to green house.     

Efficacious somatic embryogenesis and fertile plant recovery from shoot apex explants of onion (Allium cepa. L.)

An efficient somatic embryogenesis and regeneration system was developed for the first time in onion using shoot apex explants. These explants were used to initiate callus in Murashige and Skoog (MS) medium supplemented with 4.0 mg l^?1 2,4-dichlorophenoxyacetic acid. The induction frequency of primary callus in this medium was 85.3%. The primary calli were then transferred onto medium supplemented with 2.0 mg l^?1 2,4-dichlorophenoxyacetic acid. Following two biweekly subcultures, embryogenic callus formed. Inclusion of a low concentration of 6-benzylaminopurine in the subculture medium promoted the formation of embryogenic callus. The addition of 2.0 mg l^?1 glycine, 690 mg l^?1 proline, and 1.0 g l^?1 casein hydrolysate also increased the frequency of callus induction and embryogenic callus formation. The highest frequency of embryogenic callus (86.9%) and greatest number of somatic embryos (26.3 per callus) were obtained by the further addition of 8.0 mg l^?1 silver nitrate. Somatic embryos formed plantlets on regeneration medium supplemented with 1.5 mg l^?1 6-benzylaminopurine; addition of 2.0 mg l^?1 glycine to the regeneration medium promoted a high frequency of regeneration (78.1%) and plantlet formation (28.7 plants per callus). The regenerated plantlets were transferred to half-strength MS medium supplemented with 1.5 mg l^?1 indole-3-butyric acid for root development; the maximum frequency of root formation was 87.7% and the average number of roots was 7.6 per shoot. The regenerated plantlets were successfully grown to maturity after hardening in the soil. This is the first report of somatic embryogenesis and regeneration from shoot apex explants of onion.     

Somatic embryogenesis and plant regeneration from cultured mature caryopses of bahiagrass (Paspalum notatum Flugge)

Callus cultures were initiated from mature excised caryopses of bahiagrass (Paspalum notatum Flugge) on Murashige & Skoog medium supplemented with 20 gl^–1 sucrose and 2 mg l^–1 2,4-D. Excised mature caryopses readily germinated and callus developed at the base of coleoptiles. There was considerable variation in the amount of non-embryogenic callus among the cultures. Most of the explants produced non-embryogenic translucent callus consisting of thin-walled cells and unorganized tissue. Some of these calli gave rise only to roots. Other explants formed embryogenic calli which were distinguished morphologically as white, globular and friable. Somatic embryos developed and germinated precociously when embryogenic calli were transferred to a 2,4-D-free medium. Somatic embryogenesis was confirmed by histological sections and scanning electron microscopy. Of the 300 cultures, 35 were embryogenic but only 10 produced plants that were successfully grown to maturity.     

High frequency plant regeneration via somatic embryogenesis in cell suspension cultures of coriander (Coriandrum sativum L.)

Hypocotyl segments and zygotic embryos of coriander formed embryogenic calli at frequencies of up to 75% when cultured on MS medium supplemented with 1 mgl^–1 2,4-D. Calli were transferred to MS liquid medium with 1 mgl^–1 2,4-D to initiate cell suspension cultures. Embryogenic cells became finely dispersible in the medium as the subculture proceeded. Cultures were transferred to a nitrogen compound enriched liquid MS medium containing 2% sucrose and 0.1 mgl^–1 2,4-D, and cultured two weeks before plating on MS basal medium. Approximately 75% of cell aggregates (1 to two mm in diameter) underwent development into globular to cotyledonary somatic embryos after two weeks of plating. Most of the embryos were subsequently regenerated into plantlets. Regenerants were successfully transplanted to potting soil and grown to maturity in a phytotron.Abbreviations MS Murashige and Skoog - MS1D MS medium + 1 mgl^–1 2,4-D     

Plant regeneration through somatic embryogenesis from mature leaf explants of Eryngium foetidum,a condiment

Eryngium foetidum L. is an important plant cultivated as a leafy vegetable and for its essential oil, which are of high economic value in international trade market. Plants were regenerated through somatic embryogenesis from mature leaf explants of field grown plants. Leaf explants produced dark brown, compact callus on Linsmaier and Skoog (LS) medium with the combination of 1.0 mg l^-1 2,4-dichlorophenoxy acetic acid (2,4-D) and 1.0 mg l^-1 benzylaminopurine (BAP). Somatic embryos were induced from embryo-forming callus cultures on Murashige and Skoog (MS) medium supplemented with 0.1 mg l^-1 2,4-D, 2.0 mg l^-1 BAP and 1.0 mg l^-1 gibberellic acid (GA₃). Subsequently, conversion of these somatic embryos into plantlets occurred on MS medium supplemented with 1.0 mg l^-1 GA₃ and/or 0.1 mg l^-1 BAP. The regenerated shoots were rooted and elongated on MS medium supplemented with 0.1 mg l^-1 IAA and 1.0 mg l^-1 GA₃. These plantlets were hardened and transferred to the soil. This revised version was published online in June 2006 with corrections to the Cover Date.     

Thidiazuron required for efficient somatic embryogenesis from suspension-cultured cells ofPimpinella brachycarpa

To maintain embryogenic cell lines ofPimpinella brachycarpa, we suspension-cultured friable and rapidly growing yellowish calli in an MS liquid medium containing 0.2 ~ 2,4-D and 0.5pM BAP. Efficient somatic embryogenesis was achieved when selected cells were then transferred to an MS medium (0.2% gelrite) that contained 0.2gM 2,4-D, 0.5 uM BAP, and 10.0 laM TDZ (thidiazuron). These cells were cultured at 27°C under continuous illumination (21.5 I~E m^-2 s^-l). Embryogenic calli expanded about four-fold, and developed into pale yellow calli. Somatic embryogenesis was initiated only from glossy and nodular-type calli. After two more weeks of culture, globular embryos appeared on the surface of calli grown in the MS medium that contained 10.0 /aM TDZ only, or in combination with 0.5 gM NAA. Experimenting with 2,4-D, an auxin, to promote embryogenic calli resulted in excessive browning and death. We overcame this problem by growing glossy embryogenic and nodular calli on media that contained 10.0 gM TDZ. Calli that were not treated with TDZ turned dark brown and were not viable. Up to 74% of the calli showed somatic embryos when the medium was supplemented with 10.0 uM TDZ and 0.5 uM NAA. Embryos from these TDZ-induced, somatic embryogenic calli grew efficiently, forming multiple shoots and developing into normal plants. Therefore, efficient differentiation of suspension-cultured cell clusters into embryogenic calli, along with treatment of subsequent somatic embryos by TDZ, suggests that TDZ probably helps in establishing the optimum cytokinin-auxin ratio required for induction and expression of somatic embryogenesis.     

Formation of plantlets through somatic embryogeny in the Himalayan blue poppy,Meconopsis simplicifolia (Papaveraceae)

Summary Exuberant and subculturable calli could be induced from only hypocotyl and leaf segments of ca 4-month-old seedlings of Meconopsis simplicifolia cultured on Murashige & Skoog's medium supplemented with 10^–6M kinetin + 10^–5M -naphthalene acetic acid. Suspension cultures were initiated from the calli in a similar medium but with 10^–5M 2,4-dichlorophenoxy acetic acid in place of -naphthalene acetic acid. In ca 80% of the suspension cultures somatic embryos differentiated freely (80–85%) as well as on the surface of small clumps of tissue (15–20%). Somatic embryos that developed beyond heart-shaped stage were transferred to agar-solidified Murashige & Skoog's medium free of growth substances. When maintained in 10 h light and 14 h dark the somatic embryos developed into plantlets bearing cauline leaves. From seed sowing to raising normal plantlets via callus required 28 weeks; on average 80 plantlets were obtained from one explant in three passages.Abbreviations 2,4-D 2,4-dichlorophenoxy acetic acid - FAA formalin-acetic acid-alcohol - GA₃ gibberellic acid - Kn kinetin - MS Murashige & Skoog's medium (Murashige and Skoog 1962) - NAA -naphthalene acetic acid     

Somatic embryogenesis in Abutilon indicum (L.) Sweet and assessment of genetic homogeneity using SCoT markers

This study describes an efficient plant regeneration protocol for Abutilon indicum via somatic embryogenesis from 2,4-dichlorophenoxyacetic acid (2,4-D)-induced leaf-derived callus on MS medium, fortified with 13.32 μM 6-benzyladenine (BA), 2.68 μM α-naphthalene acetic acid (NAA), 200 mgl^?1-activated charcoal, and 11.54 μM ascorbic acid. This combination produced the highest (15.5 ± 0.7) number of somatic embryos after four weeks of culture. Further, the embryogenic calli were transferred to MS medium supplemented with 13.32 μM BA, 1.44 μM gibberellic acid (GA₃), and 3% (w/v) sucrose and showed highest rate of germination (76.3 ± 7.0%). The germinated somatic embryos showed maximum plantlet conversion (62.6 ± 1.90%) on ½ MS medium supplemented with 4.92 μM indole-3-butyric acid and 6.0% sucrose (w/v). The highest frequency of secondary somatic embryogenesis (34.4 ± 0.82) was observed on ½ MS medium, supplemented with 133 μM FeSO₄·7H₂O, 74 μM ethylene diamine tetraacetic acid disodium dihydrate (disodium EDTA), and 15% polyethylene glycol-4000 (PEG-4000) after three weeks of subculture. Scanning electron microscopy observations also substantiated the development of primary and secondary somatic embryos from embryogenic calli. Start codon targeted polymorphism (SCoT) marker analysis of 214 somatic embryo-derived plantlets amplified 167 numbers of bands ranging from 230 to 2125 bp. The homogeneous banding pattern confirmed the genetic uniformity of this sample of somatic embryo-derived plantlets as compared with the donor plant.     

Somatic embryogenesis in cell suspension cultures of olive <Emphasis Type="Italic">Olea europaea</Emphasis> (L.) ‘Chetoui’

Somatic embryogenesis of olive Olea europaea (L.) ‘Chetoui’ was studied using cell suspension cultures initiated from mature leaf-derived calli. Calli were developed on half-strength MS medium supplemented with 10 μM NAA and 2.25 μM 2i-P in the dark. Different combinations of three plant growth regulators (2,4-D, NAA and zeatin) were tested to determine cell proliferation and somatic embryogenesis induction and differentiation. Embryogenic suspension cultures were established in olive-modified medium for embryogenesis (OMe) containing 2.5 μM 2,4-D and 2.5 μM zeatin. Pre-globular and globular embryos were induced from mature olive tissue in liquid medium. In addition, the nitrogen form as inorganic (reduced; (NH₄)₂SO₄ or oxidized; KNO₃) and organic (CH) was used separately or in combination to improve the cell growth and proliferation. The most effective growth rate and cell proliferation were obtained with the medium containing inorganic and organic nitrogen forms.     

Plant Regeneration Through Somatic Embryogenesis in Leaf Derived Callus of Plumbago Rosea

Regeneration of Plumbago rosea L., a rare medicinal plant, via somatic embryogenesis in callus cultures derived from leaf explants was described. Optimum callus formation was achieved on semi-solid Murashige and Skoog (MS) medium supplemented with 0.25 mg dm^–3 kinetin and 2.0 mg dm^–3 1-naphthaleneacetic acid (NAA). Somatic embryogenesis was achieved upon transferring the 4-week-old callus to a medium containing 1.0 mg dm^–3 kinetic (Kn), 0.5 mg dm^–3 gibberellic acid (GA₃) and 0.1 mg dm^–3 NAA. Embryo maturation and germination was achieved on the half-strength MS basal salts supplemented with 0.01 – 0.25 mg dm^–3 Kn and 2 % (m/v) saccharose. An average of 50 – 60 plantlets were obtained from 150 mg of embryogenic callus within 4 week of subculture. Out of the 50 plantlets about 28 survived in the greenhouse.     

Micropropagation ofPanax notoginseng by somatic embryogenesis and RAPD analysis of regenerated plantlets

Somatic embryogenesis was induced in callus tissues derived from young flower buds ofPanax notoginseng via callus within 18 weeks of culture. The mature somatic embryos were germinated on half-strength Murashige and Skoog's (MS) medium supplemented with gibberellic acid A₃(GA) and 6-benzyladenine (BA). The most suitable medium for optimal root formation proved to be MS medium supplemented with 1-naphthaleneacetic acid (NAA). Total DNA was extracted from the leaves of the regenerated plantlets ofP. notoginseng. Analysis of random-amplified polymorphic DNA (RAPD) using 21 arbitrary oligonucleotide 10-mers, showed the genetic homogeneity ofP. notoginseng. The amplification products were monomorphic for all of the plantlets ofP. notoginseng regenerated by embryogenesis, suggesting that somatic embryogenesis can be used for clonal micropropagation of this plant.     

Somatic embryogenesis from mature zygotic embryos of Distylium chinense (Fr.) Diels and assessment of genetic fidelity of regenerated plants by SRAP markers

The objective was to establish an efficient regeneration protocol for Distylium chinense based on somatic embryogenesis and evaluate the genetic stability of plants regenerated in vitro. To induce callus mature zygotic embryos were cultured on Murashige and Skoog’s (MS) medium that was supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and N⁶-benzyladenine (BA). After 20 days, the highest rate of callus formation (88.9 %) occurred on MS medium supplemented with 0.5 mg l^?1 2,4-D and 0.1 mg l^?1 BA. It was observed that light-yellow, compact, dry, nodular embryogenic calli had formed. These calli were then subcultured on fresh MS medium supplemented with 0.1 mg l^?1 BA and 0.5 mg l^?1 α-naphthaleneacetic acid (NAA) for proliferation for an additional 30 days. To induce somatic embryos and plant regeneration, the embryogenic callus was transferred to fresh MS medium that was supplemented with different concentrations of BA and NAA. After 30 days, 0.5 mg l^?1 BA in combination with 0.5 mg l^?1 NAA produced the best result in terms of somatic embryogenesis (%), shoot differentiation (%), number of shoots per callus and shoot length. Next, the plantlets were transferred to the field for 5 weeks and a 95 % survival rate was observed. The sequence-related amplified polymorphism markers confirmed genetic stability of plants regenerated in vitro. To our knowledge, this is the first report that describes a plant regeneration protocol for D. chinense via somatic embryogenesis to be used for germplasm conservation and commercial cultivation.     

籼稻幼穗细胞悬浮培养物的胚胎发生和植株再生

籼稻幼穗诱导的愈伤组织,培养在MS基本成分并附加1—7 mg/l 2,4-D和5％椰子汁的液体培养基中,得到胚性小细胞团。这些细胞的特征是:形状小而圆,细胞壁薄,细胞内为稠密的胞质所充满,没有或具小的液泡,具淀粉粒。把它们转移到含1 mg/l BA的琼脂培养基中,在扫描电镜下观察到球形、心形、梨形和成熟胚,两周后萌发成具根、芽的完整植株。10—14天龄的悬浮培养物具有较高的再生植株能力。     

Plant regeneration through somatic embryogenesis from callus cultures of <Emphasis Type="Italic">Dioscorea zingiberensis</Emphasis>

A new method was established for somatic embryogenesis and plant regeneration from callus cultures of Dioscorea zingiberensis C.H. Wright. Primary callus was induced by culturing stems, leaves and petioles on Murashige and Skoog (MS) medium supplemented with 0.5–2.0 mg l^–1 N⁶-benzyladenine (BA) and 0–2.0 mg l^–1 -naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D) for 1 month. The highest frequency (87%) of callus formation was achieved from stem explants treated with 0.5 mg l^–1 BA and 2.0 mg l^–1 2,4-D. Somatic embryos were obtained by subculturing embryogenic calli derived from stem explants on MS medium supplemented with 2.0–4.0 mg l^–1 BA and 0–0.4 mg l^–1 NAA or 2,4-D for 3 weeks. The optimum combination of 4.0 mg l^–1 BA and 0.2 mg l^–1 NAA promoted embryo formation on one-third of the calli. After a further month of subculture on the same medium, mature embryos were transferred to MS medium supplemented with 0–4.0 mg l^–1 BA, NAA or indole-3-butyric acid (IBA) for further development of plantlets and tuber formation. Plant growth regulators had a negative effect on the development of mature embryos.     

Recovery of avocado (<Emphasis Type="Italic">Persea americana</Emphasis> Mill.) plants transformed with the antifungal plant defensin gene PDF1.2

Embryogenic avocado cultures derived from ‘Hass’ protoplasts were genetically transformed with the plant defensin gene (pdf1.2) driven by the CaMV 35S promoter in pGPTV with uidA as a reporter gene and bar, the gene for resistance to phosphinothricin, the active ingredient of the herbicide Finale® (Basta) (Bayer Environmental Science, Research Triangle Park, Durham, NC ). Transformation was mediated by Agrobacterium tumefaciens strain EHA105. Transformed cultures were selected in the presence of 3.0 mg l^?1 phosphinothricin in liquid maintenance medium for 3–4 mo. Liquid maintenance medium consisted of modified MS medium containing (per liter) 12 mg NH₄NO₃ and 30.3 mg KNO₃ and supplemented with 0.1 mg l^?1 thiamine HCl, 100 mg l^?1 myo-inositol, 30 g l^?1 sucrose, 3.0 mg l^?1 phosphinothricin, and 0.41 μM picloram. Somatic embryo development from transformed cultures was initiated on MS medium supplemented with 45 g l^?1 sucrose, 4 mg l^?1 thiamine HCl, 100 mg l^?1 myo-inositol, 10% (v/v) filter-sterilized coconut water, 3.0 mg l^?1 phosphinothricin, and 6.0 g l^?1 gellan gum. Limited plant recovery occurred from somatic embryos on semi-solid MS medium supplemented with 3.0 mg l^?1 phosphinothricin, 4.44 μM 6-benzylaminopurine (BA), and 2.89 μM GA₃; transformed shoots were micrografted on in vitro-grown seedling rootstocks. Approximately 1 yr after acclimatization in the greenhouse, transformed shoots were air-layered to recover transformed roots. Genetic transformation of embryogenic cultures, somatic embryos, and regenerated plants was confirmed by polymerase chain reaction (PCR), Southern blot hybridization, the XGLUC reaction for uidA, and application of the herbicide Finale® to regenerated plants.     

Plant Regeneration Through Somatic Embryogenesis in Taxus wallichiana

We describe an efficient process for regeneration of Taxus wallichiana (Zucc) plants from callus cultures derived from zygotic embryos. Zygotic embryos cultured on half strength Lloyd and McCown’s basal medium supplemented with SH vitamin (1/2 WPMSH), 0.5 mg I^?1 6-benzyladenine (BA) and 1.0–2.0 mg I^?1 á-Napthaleneacetic acid (NAA) produced compact yellow (CY) callus after 4 weeks of culture. The 8-week-old CY call! (lines CY-A and CY-B) were initially slow growing but proliferated on transfer to WPM basal medium supplemented with 8.0 mg I^?1 2,4-D, 0.1–0.9 mg^?1 NAA and 0.3–1.0 mg^?1 BA after 4 weeks. Four morphologically distinct calli lines were obtained, of which only two call! lines, CY-B-FW and CY-B-FY were embryogenic. The 12-week-old callus line CY-B-FW developed globular somatic embryos on transfer to secondary medium after 8 weeks and matured in maturation medium after 4 weeks. Only 10% of the mature somatic embryos regenerated into complete plantlets after 4 weeks on conversion medium. Although the frequency of conversion was low, complete regenerated plantlets via somatic embryogenesis were obtained after 7–8 months of initiation of culture. Taxane analysis showed that the paclitaxel accumulation was higher in embryogenic callus than in non-embryogenic callus.     

Somatic embryogenesis and organogenesis in callus cultures of a tree legume — Albizia richardiana King

Hypocotyl explants of 1 and 10 mm lengths were excised from 12-day-old in vitro-grown seedlings of Albizia richardiana. The larger pieces, after 40 days of culture, developed shoots along with green calli on B₅ + BAP (10^–7–10^–5M), while the smaller segments produced only green calli on B₅+BAP (10^–7–10^–4M) medium. Some of the green calli turned morphogenic and started producing somatic embryos with the 2nd sub-culture and shoots from 7th sub-culture onwards. Calli retained the morphogenic potential even after repeated sub-culturing for over two years. The number of embryos in an embryogenic culture varied from 2 to 20 per callus mass of 5–6.5 cm³. Sucrose at the 2% level in MS medium was optimal for embryogenesis while 4% was optimal for shoot bud differentiation. Higher levels of sucrose (6–10%) caused browning of green calli and also inhibited differentiation into embryos and shoot buds. By selective sub-culturing of 0.1 cm³ pieces of embryogenic calli on MS+10^–5M BAP, 46% of the cultures produced somatic embryos. The latter germinated into plantlets on Knop's medium.Abbreviations BAP 6-benzylaminopurine - B₅ Gamborg et al., 1968 medium - IAA Indole-3-acetic acid - MS Murashige and Skoog's (1962) medium     

The effect of explant material on somatic embryogenesis of Cyclamen persicum Mill

Somatic embryos of Cyclamen persicum Mill. could be produced through a callus phase from juvenile explant material including anthers, ovaries and zygotic embryos. The auxin 2,4-D (1.0–1.5 mg l^-1) and coconut milk (10% v/v) in MS medium were important factors for the induction of somatic embryogenesis. Somatic embryos germinated into plantlets in MS medium without growth regulators. The plants grew well in the greenhouse and flowered normally. The plants were phenotypically identical to the mother plants with a few exceptions.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthylacetic acid - IAA 3-indoleacetic acid - BA 6-benzyladenine - ABA abscisic acid - CM coconut milk     

Somatic embryogenesis from leaf protoplasts of Rauvolfia vomitoria shoot cultures

Rauvolfia vomitoria mesophyll protoplasts have been isolated from axenic shoot cultures and cultured (10⁵-10⁶ protoplasts per ml) in Murashige and Tucker liquid medium containing growth regulators. Within 6–8 weeks, a mixed population of calli and proembryos were obtained and transferred on solid media. Calli produced shoots; however, rooting did not occur. Somatic embryos achieved different patterns of development. In particular, whole plantlets have been obtained either directly through germination of primary embryos or via embryogenic calli.Abbreviations B5 Gamborg et al. (1968) medium - BA N⁶ (benzyl) adenine - 2,4-D 2,4 dichlorophenoxyacetic acid - MT Murashige and Tucker (1969) medium - NAA naphthalene acetic acid - Z zeatin - K kinetin     

Plant regeneration via somatic embryogenesis from solid and suspension cultures of Vitis vinifera L. cv. ‘Manicure Finger’

Vitis vinifera L. cv. ‘Manicure Finger’ is one of the major table grape varieties in China. To provide a strong foundation for genetic transformation with potential for crop improvement, we undertook plant regeneration via somatic embryogenesis. Anthers and gynoecia were harvested from immature flowers and used as explants to induce embryogenic calli. Explants cultured in MS1 medium (based on Murashige and Skoog basal salts), supplemented with 4.5-μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4-μM 6-benzylaminopurine (6-BA) showed the highest rates of embryogenic callus induction (3.7%?±?1.3% for anthers and 4.8%?±?2.5% for gynoecia). After several months, somatic embryos were produced from embryogenic calli cultured in plant growth regulator-free MS2 medium (with reduced sucrose). Somatic embryos (SE) at the cotyledonary stage were isolated and cultured on three different media (MS2, MS3, or B) for conversion into plantlets, the efficiency of which ranged from 63.9%?±?4.8% to 83.9%?±?8.4%. After 1 mo of in vitro culture, 80% of plants with at least six leaves were successfully transplanted into soil. SE was repeatedly induced from previously induced somatic embryos for up to 1.5 yr. Using embryogenic calli as starting material, suspension cultures containing embryogenic cell aggregates were also established in liquid MS medium supplemented with 4.5-μM 2,4-D. The embryogenic cell aggregates continued to proliferate without differentiating for successive subculture cycles. After transfer to 2,4-D-free liquid medium for 4 wk, an average of 63.7%?±?9.0% mature SEs were produced per 20 mL of liquid medium. More than 40% of somatic embryos at cotyledonary stage, derived from the suspension cultures, successfully germinated into plants using solid medium.