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.     

Somatic embryogenesis of two new <Emphasis Type="Italic">Panax ginseng</Emphasis> cultivars,Yun-Poong and Chun-Poong

Somatic embryogenesis from single cells is important for normal plant regeneration of ginseng. Cotyledon explants from zygotic embryos of two new ginseng cultivars, Chun-Poong and Yun-Poong, produced somatic embryos on Murashige and Skoog (MS) basal medium and MS medium containing growth regulators. The highest frequency of single somatic embryo formation was obtained when cotyledon explants were excised from premature (cultured for 1 day) zygotic embryos (about 6 mm in length) of both cvs. Chun-Poong and Yun-Poong and then cultured on MS medium supplemented with 7% sucrose. The frequency of single somatic embryo formation was strongly enhanced when Chun-Poong cotyledons were subjected to plasmolysis with 0.1–0.5 M sucrose for 24 h and Yun-Poong cotyledons to plasmolysis with 1.0 M sucrose for 24 h and then cultured on MS medium with 2,4-D.     

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.     

Induction of somatic embryogenesis from female flower buds of elite <Emphasis Type="Italic">Schisandra chinensis</Emphasis>

Somatic embryogenesis (SE) was induced in female flower buds from mature Schisandra chinensis cultivar ‘Hongzhenzhu’. Somatic embryo structures were induced at a low frequency from unopened female flower buds and excised unopened on Murashige and Skoog (MS) agar medium containing 4.0 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D). Friable embryogenic calli were induced from somatic embryo structures after three to four subcultures on initiation medium. The frequencies of mature somatic embryo germination and plantlet conversion were low, but increased in the presence of gibberellic acid (GA₃). Some germinated somatic embryos could form friable embryogenic calli on medium without plant growth regulators (PGRs). The germination and conversion frequencies of somatic embryos from embryogenic calli induced using PGR-free medium were higher than for somatic embryos from embryogenic calli induced on medium containing 2,4-D. Most somatic embryos from 2,4-D-induced embryogenic calli had trumpet-shaped embryos, and most somatic embryos from PGR-free medium–induced embryogenic calli had two or three cotyledons. Histological observation indicated that two- and three-cotyledon embryos had defined shoot primordia, but most of the trumpet-shaped embryos yielded plantlets that lacked or had poorly developed meristem tissue. Cytological and random amplification of polymorphic DNA (RAPD) analyses indicated no evidence of genetic variation in the plantlets of somatic embryo origin.     

Primary and repetitive secondary somatic embryogenesis of <Emphasis Type="Italic">Lepidosperma drummondii</Emphasis> (Cyperaceae) and <Emphasis Type="Italic">Baloskion tetraphyllum</Emphasis> (Restionaceae) for land restoration and horticulture

Somatic embryogenesis was developed as a method of mass propagation for Lepidosperma drummondii (Cyperaceae), a difficult to propagate but important species for post-mining restoration in a region of high plant biodiversity, in the southwest of Western Australia. Cultures were initiated from excised zygotic embryos, shoot cultures to rhizomes. Only zygotic embryos of L. drummondii developed somatic embryos, with half strength Murashige and Skoog basal medium (BM) and 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) being the most effective combination. The first culture cycle yielded a mean of 30 somatic embryos per excised zygotic embryo forming an embryo cluster. After a further 6 wk in culture (on fresh BM with 1 μM 2,4-D), approximately 350 somatic embryos per starting embryo cluster were recorded. Following regular sub-culturing of primary somatic embryo clusters onto fresh media (every 4 wk), more than 74,000 secondary somatic embryos were estimated to have been produced after eight subculture periods. This translates to between 1,000 and 2,000 somatic embryos produced from an estimated 45 mg of starting tissue per culture plate or potentially 22,0000–44,000 somatic embryos per gram of tissue. This is a significant improvement over all previous methods used to propagate L. drummondii, in which typical in vitro shoot multiplication rates are as low as 1.43 per 8 wk. This also compared favourably with published data and concurrent experiments undertaken in this study (as an extra control measure) on somatic embryo production for a related species Baloskion tetraphyllum (using the same BM with 1 μM 2,4-D and coleoptile segments as explants). Various media combinations were investigated for efficacy in converting somatic embryos into plants with best results ranging from 86% to 100% conversion for B. tetraphyllum on BM without plant growth regulators. Development of L. drummondii somatic embryos into plants was not observed on BM without plant growth regulators. However, a best result of 39% conversion to plants was observed on BM with 1 μM thidiazuron. This is the first report of successful development of somatic embryogenesis and conversion of somatic embryos into plants using thidiazuron for the Australian cyperale L. drummondii.     

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

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

High Frequency of Plant Production via Somatic Embryogenesis from Callus or Cell Suspension Cultures inEleutherococcus senticosus

Hypocotyl segments ofEleutherococcus senticosuscultured on Murashigeand Skoog's (MS) medium with 4.5 µM2,4-D produced somaticembryos directly from the surface of explants without interveningcallus formation. When these somatic embryos were subculturedto the same MS medium with 4.5 µM2,4-D, friable embryogeniccalli were formed mainly from radicle tips of somatic embryos,but at a low frequency (5%). Selected embryogenic calli weremaintained on MS agar or liquid medium with 4.5 µM2,4-D.To induce somatic embryo development, embryogenic calli andcell clumps were transferred to MS medium lacking 2,4-D. Thefrequency of somatic embryo formation differed between culturetypes with 1570 embryos formed per Petri dish from callus cultureand 5514 embryos formed per flask from cell suspension cultures.Somatic embryos formed on agar medium had larger cotyledonsthan those of embryos formed in liquid medium. GA₃treatmentwas necessary to induce germination from somatic embryos. Therate of plant conversion was 97% in somatic embryos from callusculture and 76% in embryos from liquid culture. Regeneratedplantlets were successfully acclimatized in the glasshouse.Copyright1999 Annals of Botany Company Eleutherococcus senticosus, micro propagation, somatic embryogenesis.     

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.     

Plant regeneration through somatic embryogenesis of a medicinal plant, <Emphasis Type="Italic">Phellodendron amurense</Emphasis> Rupr.

Somatic embryogenesis and subsequent plant regeneration were established from hypocotyl and internode explants collected from in vitro-grown seedlings and in vitro-proliferated shoots, respectively. Somatic embryogenesis was significantly influenced by the types of auxin and cytokinin. Friable calluses with somatic embryos developed well in Murashige and Skoog basal (MS) medium supplemented with 0.8–8.8 μM 6-benzylaminopurine (BA) and 2.0–8.0 μM 2,4-dichlorophexoxyacetic acid (2,4-D) or α-naphthaleneacetic acid (NAA). The maximal frequency of embryogenic callus and somatic embryo formation were obtained when the MS medium was amended with 8.8 μM BA and 4.0 μM 2,4-D. The best embryo germination occurred in a hormone-free 1/2-MS medium. The highest percentage of shoot proliferation was observed in embryogenic calluses in MS medium containing 2.0 μM BA and 1.0 μM NAA. In vitro-grown shoots were rooted in MS medium with 0.5–2.0 μM indole-3-butyric acid. Regenerants were transferred to vermiculite and successfully established under an ex vitro environment in garden soil.     

Clonal plant production from self- and cross-pollinated seed families of <Emphasis Type="Italic">Pinus sylvestris</Emphasis> (L.) through somatic embryogenesis

Several factors affecting somatic embryogenesis (SE) in Pinus sylvestris from self- and cross-pollinated seed families were studied with the aim of producing large quantities of clonal plants. Somatic embryogenesis initiation from zygotic embryos was improved on a medium with lower than standard concentrations of 2,4-dichlorophenoxyacetic acid (2.2 vs. 9.5 μM) and 6-benzyladenine (2.2 vs. 4.5 μM). On this medium, initiation rates of four controlled crosses, including one self-cross, varied from 3% to 25%. Among the maturation factors tested, the concentration of abscisic acid (ABA 80, 120 μM) had no significant effect on the production of mature somatic embryos when the medium contained 0.1 M sucrose. When sucrose concentration was 0.2 M, however, 1.4 times more mature somatic embryos were produced on medium with 80 μM compared with 120 μM ABA. Under our best maturation conditions, mature somatic embryos accumulated amounts of storage proteins that were similar to the amounts in mature zygotic embryos. Activated charcoal exerted a beneficial effect on mature somatic embryo production of 24-week-old cultures; there was no evidence of such an effect in 8-week-old cultures. Thirty-seven embryogenic lines from a self-cross and an out-cross were chosen for clonal plant production. Highly embryogenic lines produced mature somatic embryos that were more likely to convert to plants than those from less embryogenic lines. After 4 months of growth in a shade house, plantlet survival rates exceeded 70% for 31 lines out of 35. This report describes an improved method for accelerated production of large quantities of Scots pine for clonal tests.     

Somatic embryogenesis and plant regeneration in Cedrela fissilis

Somatic embryos were obtained from immature zygotic embryos of Cedrela fissilis Well. (Meliaceae), after a culture period of 12 months, with regular subcultures every 6–8 weeks. Callus was developed on explants in 2 months on Murashige and Skoog (MS) medium containing 2,4 dichlorophenoxyacetic acid (2,4-D) or picloram (PIC). When the calli were transferred to fresh medium, embryogenic tissue appeared on MS + 45 μM 2,4-D, or 22.5 μM 2,4-D + 0.4 μM 6-benzyladenine (BA), or 20.7 μM PIC after 6 months. Sub-culture of embryogenic tissue in MS medium supplemented with 4.5 μM 2,4-D resulted in the differentiation into somatic embryos after further 4 months. Repeated secondary somatic embryogenesis was achieved by regular subculture on this medium. Maturation and conversion of somatic embryos into plantlets was achieved on MS medium without plant growth regulators and the conversion frequency was approximately 12.5 %. The plantlets were successfully acclimatized in pots with soil. Histological studies showed that somatic embryos had no detectable connection with the mother explants and that somatic embryos in advanced stages were bipolar with shoot and root apical meristems, they contained vascular system and showed typical characteristics of a somatic dicotyledonous embryo.     

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

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

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.     

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.     

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.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of the rare medicinal plant <Emphasis Type="Italic">Ceropegia candelabrum</Emphasis> L. through somatic embryogenesis

Summary Efficient in vitro propagation of Ceropegia candelabrum L. (Asclepidaceae) through somatic embryogenesis was established. Somatic embryogenesis depended on the type of plant growth regulators in the callus-inducing medium. Friable callus, developed from leaf and internode explants grown on Murashige and Skoog (MS) medium supplemented with 4.52μM2,4-dichlorophenoxyacetic acid (2,4-D), underwent somatic embryogenesis. Compared to solid media, suspension culture was superior and gave rise to a higher number of somatic embryos. Transfer of the friable callus developed on MS medium containing 4.52μM 2,4-D to suspension cultures of half- or quarter-strength MS medium with lower levels of 2,4-D (0.23 or 0.45 μM) induced the highest number of somatic embryos, which developed up to the torpedo stage. Somatic embryogenesis was asynchronous with the dominance of globular embryos. About 100 mg of callus induced more than 500 embryos. Upon transfer to quarter-strength MS agar medium without growth regulators, 50% of the somatic embryos underwent maturation and developed into plantlets. Plantlets acclimatized under field conditions with 90% survival.     

Improvement of somatic embryogenesis in zonal geranium

A number of media constituents including sucrose, ammonium nitrate and plant growth regulators were evaluated in an attempt to improve somatic embryo production in zonal geranium (Pelargonium ×hortorum) cv. Scarlet Orbit Improved. Somatic embryo production was characterized by the quantity and type of somatic embryo induced by the treatments. Sucrose at 4% supported the highest number of total somatic embryos while improving the proportion of the morphologically normal cotyledon-stage somatic embryos. Addition of ammonium nitrate also improved embryo production. With 1.89 mM ammonium nitrate, normal cotyledon-stage embryo development was increased by 53%; the proportion of normal cotyledon-stage embryos decreased and abnormal embryos with leaves or serrated margins in cotyledons (fringed-shoot type) increased with higher ammonium nitrate concentrations. The effect of plant growth regulators on somatic embryogenesis indicated that exogenous supply of indole-3-acetic acid (IAA) at a range of 0.25 to 4 µM failed to promote somatic embryogenesis. In contrast, benzyladenine (BA) up to 2.0 µM increased the total embryo number and the proportion of desirable cotyledon-stage embryos. There was no interaction between IAA and BA. Our research has demonstrated that improvement in both quantity and quality of somatic embryos can be achieved in zonal geranium.     

Improvement of somatic embryogenesis in zonal geranium

A number of media constituents including sucrose, ammonium nitrate and plant growth regulators were evaluated in an attempt to improve somatic embryo production in zonal geranium (Pelargonium ×hortorum) cv. Scarlet Orbit Improved. Somatic embryo production was characterized by the quantity and type of somatic embryo induced by the treatments. Sucrose at 4% supported the highest number of total somatic embryos while improving the proportion of the morphologically normal cotyledon-stage somatic embryos. Addition of ammonium nitrate also improved embryo production. With 1.89 mM ammonium nitrate, normal cotyledon-stage embryo development was increased by 53%; the proportion of normal cotyledon-stage embryos decreased and abnormal embryos with leaves or serrated margins in cotyledons (fringed-shoot type) increased with higher ammonium nitrate concentrations. The effect of plant growth regulators on somatic embryogenesis indicated that exogenous supply of indole-3-acetic acid (IAA) at a range of 0.25 to 4 μM failed to promote somatic embryogenesis. In contrast, benzyladenine (BA) up to 2.0 μM increased the total embryo number and the proportion of desirable cotyledon-stage embryos. There was no interaction between IAA and BA. Our research has demonstrated that improvement in both quantity and quality of somatic embryos can be achieved in zonal geranium.     

A rapid protocol for somatic embryogenesis from immature leaflets of groundnut (Arachis hypogaea L.)

Summary Plant regeneration via somatic embryogenesis was developed in two groundnut varieties. Somatic embryogenesis was induced from immature leaflets on MS medium with different concentrations of the auxins 2,4-dichlorophenoxyacetic acid (2,4-D) or naphthaleneacetic acid (NAA) in combination with 0.5 mg/l of the cytokinin BA. The highest frequency of somatic embryo formation occurred on MS medium fortified with 20 mg 2,4-D per l. Of the two auxins tested individually 2,4-D was more effective for induction of embryogenesis as well as production of embryos. Embryo development and maturation was achieved on MS medium supplemented with N⁶-benzyladenine (BA) (0.5–2.0 mg/l) and 2,4-D (0.5 mg/l). Plant conversion frequency from somatic embryos was highest in presence of 2.0 mg BA per l and 0.5 mg NAA per l. The frequency of embryogenesis and plant regeneration was higher in the VRI-2 cultivar than in the other cultivar tested. Regenerated plants were transferred to soil, grown to maturity, and produced viable seeds.     

The embryogenic competency and morphological changes during somatic embryogenesis in Iris pseudacorus

Embryogenic callus was obtained from bulb segments of Iris pseudacorus on Murashige and Skoog (MS) medium with 2,4-dichlorophenoxyacetic acid (2,4-D) alone or in combination with kinetin. When early globular somatic embryos were subcultured onto MS medium with 4.52 μM 2,4-D, high frequency of somatic embryogenesis was obtained. Deprivation of 2,4-D was required for maturation. Mature somatic embryos had an elongated scutellum with a notch on the base of scutellum. Separation of embryos from embryo clusters was necessary to enhance the frequency of germination. Germination was stimulated by separation of embryos from embryo clusters and transfer onto fresh half-strength MS medium with 3% sucrose. After acclimation in artificial soil in greenhouse for 2 months, 96.4% of plantlets survived.