Somatic embryogenesis in cell suspension cultures of pigeonpea (Cajanus cajan)

Suspension cultures of calli derived from seedling leaf explants of Cajanus cajan L. var. Vamban-1 produced somatic embryos. The highest embryogenic frequency was induced on semisolid MS (Murashige and Skoog, 1962) medium supplemented with 6.78 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The maximum frequency of somatic embryogenesis was observed when this callus was transferred to MS liquid medium supplemented with 4.52 μM 2,4-D. Further studies on ontogeny of somatic embryos showed that the cells destined to become somatic embryos divided into spherical proembryos. Subsequent divisions in the proembryo led to globular, heart and torpedo-shaped somatic embryos. The germination of somatic embryos occurred on auxin-free MS basal medium. Effects of various auxins, cytokinins and carbohydrates on induction and frequency of somatic embryogenesis were studied. A medium supplemented with 4.52 μM of 2,4-D and 87.64 mM sucrose was effective in inducing a higher frequency of somatic embryos, whereas cytokinin had no effect and led to recallusing of embryos. About 5–6% of embryos converted into plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of auxins,cytokinins, carbohydrates and amino acids on somatic embryogenesis induction from shoot apices of pea

Studies were conducted to test the effects of various auxins, cytokinins, carbohydrates and amino acids on somatic embryogenesis from shoot apices of pea (Pisum sativum L.) cultured on a sole medium. Picloram (4.5 M) and 4-chlorophenoxyacetic acid (45 M) were the most effective auxins. Addition of cytokinins (benzyladenine, zeatin, kinetin) to auxin-containing medium reduced embryo production. Amino acids (glutamine, alanine, proline) did not improve somatic embryogenesis. Carbohydrate seemed to be a critical factor. Embryogenic efficiency and embryo development were promoted by high carbohydrate concentration. The best results were obtained with fructose (252–504 mM); the number of somatic embryos per cultured explant was 3- to 4-fold higher compared to the control (84 mM sucrose). From these results, an optimized induction medium is proposed.Abbreviations BA benzyladenine - 4-CPA 4-chlorophenoxyacetic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - MS Murashige & Skoog - EE embryogenic explants - G globular somatic embryos     

Effect of growth regulators on microspore embryogenesis in coconut anthers

The effect of growth regulators on induction of androgenesis in coconut was investigated using seven different growth regulators at various concentrations and combinations. Three auxins (1-naphthalene acetic acid—NAA, indoleacetic acid—IAA, picloram) and three cytokinins (2-isopentyl adenine-2-iP, kinetin, zeatin) were tested either alone or in combination with 2,4-dichlorophenoxyacetic acid (2,4-D), using modified Eeuwens Y3 liquid medium as the basal medium. Among the tested auxins, 100 μM NAA in combination with 100 μM 2,4-D enhanced the production of calli/embryos (123) whereas IAA and picloram showed negative and detrimental effects, respectively, for androgenesis induction over 100 μM 2,4-D alone. Kinetin and 2-iP enhanced the production of calli/embryos when 100 μM 2,4-D was present in the culture medium. Both cytokinins at 10 μM yielded the highest frequencies of embryos (113 and 93, respectively) whereas zeatin (1 or 2.5 μM) had no impact on microspore embryogenesis. When calli/embryos (produced from different treatments in different experiments) were sub-cultured in somatic embryo induction medium (Y₃ medium containing 66 μM 2,4-D), followed by maturation medium (Y₃ medium without growth regulators) and germination medium (Y₃ medium containing 5 μM-6-benzyladenine—BA and 0.35 μM gibberellic acid—GA₃), plantlets were regenerated at low frequencies (in most treatments ranging from 0% to 7%).     

Somatic embryogenesis in Agave tequilana Weber cultivar azul

Somatic embryogenesis was achieved from leaves of Agave tequilana Weber cultivar azul utilizing MS medium supplemented with L2 vitamins and the addition of cytokinins: 6-benzylaminopurine (BA), 1-phenyl-3(1,2,3-thiadiazol-5-yl)urea (TDZ), 6-(γ-γ-dimethylamino)purine (2ip) and 6-furfurylaminopurine (KIN), combined with the auxin 2,4-dichlorophenoxyacetic acid (2,4-D). Differences among the six genotypes studied with regard to their embryogenic response in culture were found. Embryos produced by genotype S3 under a hormone regime of high cytokinin (44.4 to 66.6 μM BA) compared to auxin (4.5 μM 2,4-D) contained chlorophyll, whereas those produced when auxin was high compared to cytokinin (9.0 and 13.6 μM 2,4-D and 1.3 and 4.0 μM BA, respectively) were whitish and morphologically similar to their zygotic counterparts. Somatic embryos matured and germinated after transferring the embryogenic calli to maturation and germination medium without growth regulators and enriched with organic nitrogen. Microscopic observations demonstrated a unicellular origin for production of indirect somatic embryos.     

Somatic embryogenesis in cell suspension cultures of Acacia sinuata (Lour.) Merr.

Summary Suspension cultures initiated from calluses derived from seedling leaf explants of Acacia sinuata (Lour.) Merr. produced somatic embryos. Embryogenic callus was induced on semisolid MS (Murashige and Skoog, 1962) medium supplemented with 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.22 μM 6-benzylaminopurine. A high frequency of somatic embryos was induced in MS liquid medium supplemented with 4.52 μM 2,4-D and 10% coconut water. Further studies on ontogeny of somatic embryos showed that the cells destined to become somatic embryos divided into spherical proembryos. Subsequent development led to the formation of globular, heart, torpedo-shaped and cotyledonary-stage embryos. The conversion of somatic embryos occurred on auxin-free MS medium. Effects of various auxins, cytokinins, carbohydrates and amino acids in enhancing productin, of somatic embryos were studied. MS medium supplemented with 87.64 mM sucrose and 342.46 μM glutamine promoted higher somatic embryo production whereas cytokinin had no effect and led to recallusing of embryos. About 8–10% of embryos converted into plants.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration via somatic embryogenesis through cell suspension cultures of horsegram [<Emphasis Type="Italic">Macrotyloma uniflorum</Emphasis> (Lam.) verdc.]

Summary In vitro regeneration of plants via somatic embryogenesis through cell suspension culture was achieved in horsegram. Embryogenic calluses were induced on leaf segments on solid Murashige and Skoog (MS) medium with 9.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Differentiation of somatic embryos occurred when the embryogenic calluses were transferred to liquid MS medium containing 2,4-D. Maximum frequency (33.2%) of somatic embryos was observed on MS medium supplemented with 7.9 μM 2,4-D. Cotyledonary-torpedo-shaped embryos were transferred to liquid MS medium without growth regulators for maturation and germination. About 5% of the embryos germinated into plants, which grew further on solid MS medium. The plants were hardened and established in soil. Effects of various auxins, cytokinins, carbohydrates, amino acids, and other additives on induction and germination of somatic embryos were also studied. A medium supplemented with 7.9 μM 2,4-D, 3.0% sucrose, 40 mg l⁻¹ L-glutamine, and 1.0 μM abscisic acid was effective to achieve a high frequency of somatic embryo induction, maturation, and further development.     

Somatic embryogenesis in tulip (<Emphasis Type="Italic">Tulipa gesneriana</Emphasis> L.) flower stem cultures

In the present study, the procedures for induction of somatic embryogenesis (SE) in an in vitro culture of the tulip have been developed. SE was initiated on flower stem explants isolated from “Apeldoorn” bulbs during their low-temperature treatment. Bulbs had not been chilled or had been chilled for 12 or 24 weeks at 5°C. The explants were cultured with exogenous auxins 2,4-dichlorophenoxyacetic acid (2,4-D), 4-amino-3,5,6-trichloropicolinic acid (Picloram), α-naphthaleneacetic acid (NAA) at 1–100 μM and cytokinins: benzyladenine (BA) and zeatin (ZEA) at 0.5–50 μM. Increase in auxin concentrations caused an intensive enlargement of the explant parenchyma, which changed into homogenous colorless callus. On the same media, vein bundles developed into yellowish, nodular callus. Picloram was more efficient in inducing the formation of embryogenic nodular callus than 2,4-D, whereas the latter stimulated formation of colorless callus. The base of the lower part of the flower stem isolated from bulbs chilled for 12 weeks proved to be the best explant for callus formation. The highest number of somatic embryos was produced on medium with 25 μM Picloram and 0.5 μM BA. Development of adventitious roots was noticed in the presence of 2,4-D. Globular embryos developed into torpedo stage embryos under the influence of BA (5 μM) and NAA (0.5 μM). Morphological and anatomical data describing development of callus and somatic embryos are presented.     

Plant regeneration in vitro from embryogenic cultures of spring- and winter-type barley (Hordeum vulgare L.) varieties

Summary Immature embryos of 41 lines of barley were screened in vitro for callus induction and somatic embryogenesis on different media to establish totipotent cultures. The use of modified MS and CC media, both supplemented with 1 g/l casein hydrolysate, and the substitution of agarose for agar resulted in the highest frequencies of somatic embryo induction. Embryogenic callus was induced and plants regenerated from 23 of the lines tested. The auxins 2,4-D, dicamba, picloram and 2,4,5-T were suitable for embryogenic callus induction. High frequencies of somatic embryo germination occurred on CC medium supplemented with 1 mg/l IAA and 0.05 mg/l zeatin. A strong genotypic effect on the capacity and frequency of embryogenic callus formation was found. Cultivar Golden Promise always gave the best results. Experiments with field grown material in 3 consecutive years showed that environmental factors also strongly influenced the induction of somatic embryogenesis and plant regeneration.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - dicamba 3,6-dichloro-o-anisic acid - picloram 4-amino-3,6,6-trichloropicolinic acid - NAA naphtaleneacetic acid - IAA indole-3-acetic acid - ABA abscisic acid - BAP 6-benzyl amino purine - 2iP 6-(3-methyl-2 butenyl 1-amino)purine - GA₃ gibberellic acid     

Direct somatic embryogenesis and plant regeneration from immature explants of chickpea

A protocol for plant regeneration via somatic embryogenesis was developed in two chickpea (Cicer arietinum L.) cultivars ICCV-10 and Annigeri. Somatic embryos were induced from immature cotyledons on Murashige and Skoog’s (MS) medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), α-naphthaleneacetic acid (NAA) and picloram alone or in combination with 0.5 — 2.0 mg dm⁻³ N⁶-benzylaminopurine (BA) or kinetin (KIN). NAA was better for somatic embryo induction compared to other auxins. The well formed, cotyledonary shaped embryos germinated into plantlets with 36.6 % frequency on MS medium supplemented with 2.0 mg dm⁻³ BA + 0.5 mg dm⁻³ abscisic acid (ABA). The frequency of embryogenesis and plantlet regeneration was higher in cv. ICCV-10 as compared to cv. Annigeri. Regenerated plants were transferred to soil (40 % survival) and grown to maturity. Histological studies of explants at various developmental stages of somatic embryogenesis reveled that somatic embryos developed directly from the cotyledon cells and they were single cell origin.     

<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.     

Somatic embryogenesis and plant regeneration inCoronilla varia L. (crownvetch) long-term tissue cultures

Spontaneous recovery of regeneration abilities was observed in a long-term (about two-year-old) crownvetch (Coronilla varia L.) tissue culture permanently grown on MS medium containing 1 mg 1^-1 IAA. Somatic embryos and later complete plants differentiated from initially regenerating roots. The formation and development of embryos was accompanied by a 10- to 20-fold increase in the content of cardioactive glycosides hyrcanoside and deglucohyrcanoside in the culture biomass. The effect of auxins (IAA, NAA, 2,4-D) and cytokinins (6-BAP) on calogenesis and somatic embryogenesis was examined; further development of somatic embryos was enhanced by light. Primary explants which were newly isolated from sterile R₁ plantlets showed differential, organ-specific ability of somatic embryogenesis which was highest in root cuttings. Regenerated plants were transferred to field culture; two-year-old cultures of regenerated plants showed in most cases phenotypic deviations from the original material, especially dwarfism.     

Repetitive somatic embryogenesis and plant regeneration in Zizyphus jujuba Mill

Summary A procedure for regenerating Zizyphus jujuba Mill. (Chinese date) plants through repetitive somatic embryogenesis (RSE) was developed. Primary somatic embryos were produced from cotyledon-derived cultures of germinated plants in vitro. The highest induction frequency of primary somatic embryogenesis (PSE) was obtained with a combination of 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 0.49 μM indole-3-butyric acid (IBA), and 0.44 μM benzyladenine (BA) (17.4%). These primary somatic embryos were multiplied by RSE on media with different plant growth regulator (PGR) combinations. The highest RSE frequency (51.3%), was obtained with 0.58 μM gibberellic acid (GA₃). However, the highest number (4.4 per primary somatic embryo) of repetitive somatic embryos was obtained with 0.98 μM 6-r-r-dimethylallylaminopurine (2-iP). For germination of somatic embryos, different PGRs, cold and desiccation treatment were tested. Desiccation of somatic embryos at 25±1°C for 2 wk was the best treatment for germination with epicotyl elongation and root development. Of over 256 plants regenerated, 237 (92.6%) survived.     

Somatic embryogenesis and plant regeneration from leaf tissue of jojoba

A protocol was developed for the induction, maturation and germination of somatic embryos from leaf tissue of jojoba [Simmondsia chinensis (Link) Schneider]. Explants were placed on their adaxial sides in Petri dishes and maintained in darkness on half-strength Murashige and Skoog basal medium (MS/2). Combinations of 2,4-dichlorophenoxyacetic acid (1.35–4.52 μM) with 6-benzylaminopurine (1.33–4.43μM) and 2 synthetic cytokinins, N-(2-chloro-4pyridyl)-N′-phenylurea (1.21–4.03μM) or (E)-6-[3-(trifluoromethyl)-but-2-enylamino] purine (1.11–3.71μM) resulted in formation of embryogenic cultures and somatic embryos. After two 30-day subcultures, embryogenic cultures were transferred onto MS/2 medium supplemented with different auxins and cytokinins. Somatic embryo maturation, germination and plantlet formation were achieved using 1-naphthaleneacetic acid (3.75μM) or indole-3-butyric acid (3.44μM) in combination with BA (0.44 or 1.33μM) or F3iP (0.37 or 1.11μM). Histology confirmed each stage of development. This revised version was published online in June 2006 with corrections to the Cover Date.     

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 effect of auxins, time exposure to auxin and genotypes on somatic embryogenesis from mature embryos of wheat

The effects of different factors on the embryogenesis and plant regeneration from mature embryos of Russian spring and winter genotypes were studied. Embryogenic callus induction was achieved on MS medium supplemented with different concentrations of 2,4-D (2,4-dichlorophenoxyacetic acid), 2,4,5-T (2,4,5-trichlorophenoxyacetic acid) or Dicamba (3,6-dichloro-o-anisic acid). Although all auxins were able to induce callus from explants with high frequency (98–100%), Dicamba was more effective for the induction of embryogenic callus (21.8–38.3%). Maximum embryogenic callus formation and high number of regenerated plants were observed at 12 mg l⁻¹ of Dicamba. The time exposure to Dicamba (7, 14, 21 and 28 days) had a significant effect on efficiency of somatic embryogenesis. When contact of explants with callus induction medium was increased from 7 to 21 days the rate of somatic embryogenesis and number of regenerated plants per embryogenic callus gradually increased from 13.0 to 38.4% and 3.6 to 8.0%, respectively. Supplement of additional auxins (indoleacetic acid (IAA), indolebutyric acid (IBA), and naphthaleneacetic acid (NAA)) to callus induction medium with Dicamba had a positive effect on the rate of embryogenic callus formation, while the average number of regenerated shoots was not affected. The best rate of somatic embryogenesis was observed at the addition of 0.5 mg l⁻¹ IAA with Dicamba (61.0%). The optimum combination of Dicamba and IAA increased the efficiency of somatic embryogenesis and plant regeneration from seven spring and winter wheat genotypes, thought overall morphogenic capacity was still genotype dependent.     

2,4,5-Trichlorophenoxyacetic acid promotes somatic embryogenesis in the rose cultivar ‘Livin’ Easy’ (<Emphasis Type="Italic">Rosa</Emphasis> sp.)

Somatic embryogenesis (SE) offers vast potential for the clonal propagation of high-value roses. However, some recalcitrant cultivars unresponsive to commonly employed SE-inducing agents and low induction rates currently hinder the commercialization of SE technology in rose. Rose SE technology requires improvement before it can be implemented as a production system on a commercial scale. In the present work, we assessed 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), a synthetic auxin not previously tested in rose, for its effectiveness to induce SE in the rose cultivar ‘Livin’ Easy’ (Rosa sp.). We ran a parallel comparison to the commonly used 2,4-dichlorophenoxyacetic acid (2,4-D). We tested each auxin with two different basal media: Murashige and Skoog (MS) basal medium and woody plant medium (WPM). MS medium resulted in somatic embryo production, whereas WPM did not. 2,4,5-T induced SE over a greater concentration range than 2,4-D's and resulted in significantly greater embryo yields. 2,4,5-T at a concentration of 10 or 25 μM was better for embrygenic tissue initiation than 2,4,5-T at 5 μM. Further embryo development occurred when the tissue was transferred to plant growth regulator (PGR) free medium or media with 40% the original auxin concentration. However, the PGR-free medium resulted in a high percentage of abnormal embryos (32.31%) compared to the media containing auxins. Upon transfer to germination medium, somatic embryos successfully converted into plantlets at rates ranging from 33.3 to 95.2%, depending on treatment. Survival rates 3 months ex vitro averaged 14.0 and 55.6% for 2,4-D- and 2,4,5-T-derived plantlets, respectively. Recurrent SE was observed in 60.2% of the plantlets growing on germination medium. This study is the first report of SE in the commercially valuable rose cultivar ‘Livin’ Easy’ (Rosa sp.) and a suitable methodology was developed for SE of this rose cultivar.     

Induction of somatic embryos in cotyledonary tissue of soybean,Glycine max L. Merr

A method for the induction of somatic embryos in soybean tissue cultures is described. Cotyledons from immature embryos were utilized as explant source. Supplementing the culture medium with auxins (2,4-D, MCPA, 2,4,5-T, NAA, IAA, IBA) caused formation of meristematic tissue on cotyledon explants. The extent of meristematic tissue formed depended on the kind and concentration of auxin in the culture medium. With 2,4-D and MCPA, embryoids originated from meristematic tissue. Embryoid formation rates were influenced by the developmental stage of the embryos serving as explant source and auxin concentration. Addition of cytokinins to the medium containing 2,4-D or supplementing it with high sugar concentrations inhibited the formation of meristematic tissue and of embryoids on cotyledon explants.Abbreviations BA 6-benzyladenin - 2,4-D 2,4-dichlorphenoxyacetic acid - IAA indoleacetic acid - IBA indolebutyric acid - MCPA 2-methyl-4-chlorophenoxyacetic acid - NAA -naphthaleneacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - L2 Phillips and Collins (1979) medium Present and correspondence address: Akademie der Landwirtschaftswissenschaften der DDR, Institut für Pflanzenernährung, DDR-6909, Jena     

High-frequency somatic embryogenesis of koala fern (<Emphasis Type="Italic">Baloskion tetraphyllum,restionaceae</Emphasis>)

Summary Baloskion tetraphyllum is a member of the Restionaceae and is an important species for the rehabilitation of disused mine sites and wetland areas, and is also highly prized as a cut flower. Its use for restoration of disturbed land is, however, severely limited, due to very poor propagation success by conventional methods. A study was conducted to evaluate the potential of somatic embryogenesis for the large-scale propagation of this species. A variety of auxins (at different concentrations) were investigated for their efficacy in stimulating somatic embryogenesis. Somatic embryos were successfully induced from excised coleoptiles of B. tetraphyllum on half-strength Murashige and Skoog (1/2MS) medium supplemented with 1 μM 2,4-dichlorophenoxyacetic (2,4-D). To scale up the production, proliferation of secondary somatic embryos was achieved using primary somatic embryos as the tissue source, on 1/2MS+1 μM 2,4-D resulting in a 30-fold increase in somatic embryo, numbers. Almost all the somatic embryos developed into plants and were established ex vitro. The other auxins investigated, including p-chlorophenoxyacetic acid, indole-3-acetic acid, α-naphthaleneacetic acid, and picloram, were not as effective as 2,4-D. The age, of the explant material significantly influenced somatic embryogenesis with white, young coleoptiles (5–7 d) producing 50% more somatic embryos than green, more mature (8–14 d) coleoptiles. The protocol developed for B. tetraphyllum has the potential to be commercially viable, with an estimated 22 000 somatic embryos produced from 1 g of plant material. This research may also have a positive impact on the propagation of other important Restionaceae species.     

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.     

Somatic embryogenesis and plant regeneration in a woody species: the European Spindle Tree (Euonymus europaeus L.)

Somatic embryogenesis and subsequent plant regeneration of Euonymus europaeus L (European Spindle Tree) were obtained from square pieces of mature zygotic embryos with an intervening callus phase. Callus and somatic embryos were induced using a Murashige and Skoog's semi-solid basal medium supplemented with several combinations of auxins and cytokinins. The greatest number of somatic embryos was obtained with a continuous exposure to 22.8 M indoleacetic acid and 0.046 M kinetin. The frequency of somatic embryogenesis from zygotic embryos depends on the cold conservation time of seeds. The embryos frequently germinated on the same medium. Further development of somatic embryos into plantlets was achieved on a medium devoid of growth regulators.Abbreviations MS Murashige and Skoog's medium - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - BAP 6-benzylaminopurine - RH relative humidity