Plantlet regeneration via somatic embryogenesis from subcultured callus of mature embryos ofPicea abies (Norway spruce)

Summary Embryogenic callus was initiated from radicles of mature embryos removed from imbibed seeds (24 h). Embryogenic and other nonembryogenic types of callus proliferated on a modified half-strength Murashige-Skoog medium (MS) basal medium (BM) supplemented withmyo-inositol, casein hydrolysate (CH), L-glutamine (gln) and growth regulators kinetin (KN), N⁶-benzyladenine (BAP) each (20×10⁻⁶ M), 2,4-dichlorophenoxyacetic acid (2,4-D) (50×10⁻⁶ M) Embryogenic callus bearing suspensor-like cells in a mucilaginous gel matrix was isolated and maintained by subculture every 10 to 12 days on BM with KN, BAP each (2×10⁻⁶ M) and 2,4-D (5×10⁻⁶ M). Somatic embryos developed spontaneously from the callus on this medium at 23±1° C. Closer examination revealed that numerous polyembryonic clusters, comprised of elongated cells (suspensors) and small dense cells with large nuclei (somatic embryos), occurred in the viscous gel. When this enriched embryonal-suspensor mass was subcultured to low 2,4-D (1×10⁻⁶ M), globular embryos developed by 40 to 60 days. Upon transfer to a liquid medium without growth regulators, the embryos elongated and developed cotyledons and shoots with needles. Plantlet development was completed by 30 days in a basal medium without CH, gln and growth regulators. The total culture time was 150 days. Approximately 40±10 embryos were formed from 500 mg of initial callus. Somatic embryogenesis became aberrant if embryos remained attached to the callus mass and were not subcultured within 10 to 12 days according to the described protocol. Somatic embryos were encapsulated in an alginate gel and stored at 4° C for nearly two months without visible adverse effects on viability. Editor's Statement This paper presents advances in the in vitro regeneration of a commercially useful plant species from stored seeds. In addition, data is presented on short-term storage of the plantlets, and long-term proliferation of the embryonal mass in vitro.     

<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.     

Direct somatic embryogenesis from cotyledon and cotyledonary node explants in bishop’s weed <Emphasis Type="Italic">Trachyspermum ammi</Emphasis> (L.) sprague

A three-stage procedure for embryogenesis in Trachyspermum ammi was developed from cotyledon and cotyledonary node explants cultured in Murashige and Skoog (MS) liquid medium supplemented with 0.2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D). Globular somatic embryos without intervening callus phase developed in 4 wk. The development of embryos to heart and torpedo stages required second-stage subculture of the explants (along with developing embryos) in liquid medium with lower concentrations of 2,4-D. Further development of embryos required a third-stage subculture in hormone-free liquid medium supplemented with 100 mg l⁻¹ casein hydrolysate. Regeneration of complete plantlets occurred after the fully developed somatic embryos were transferred to solidified half-strength MS medium supplemented with 1 mg l⁻¹ gibberellic acid.     

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.     

Secondary somatic embryogenesis and plant regeneration in myrtle (Myrtus communis L.)

Immature seeds, as well as hypocotyls and cotyledons excised from seedlings of Myrtus communis L., were cultured on media containing half-strength Murashige and Skoog macronutrients (MS/2) with combinations of auxins and cytokinins, in order to study their morphogenetic competence. Somatic embryogenesis was obtained from cotyledons, hypocotyls and 2-month-old immature seeds with 0.1 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). The percentage of explants showing this primary somatic embryogenesis ranged from 4% for hypocotyls to 12% for 2-month-old immature seeds. In the latter, somatic embryogenesis was also obtained in media containing 2,4-D plus a cytokinin, and with only a cytokinin. Somatic embryos obtained from hypocotyls, cotyledons or immature seeds were able to develop on MS/2 medium without plant growth regulators. Subculture of primary somatic embryos obtained from immature seeds on MS/2 medium without plant growth regulators gave rise to clusters with secondary somatic embryos and embryogenic calli. These clusters were subcultured every 8 weeks, and they were the source of highly embryogenic cultures. An average of 10% of the secondary somatic embryos developed into plantlets in each subculture. Therefore, the same culture on MS/2 medium without growth regulators yielded both plantlets and de novo secondary embryos. Received: 6 April 1998 / Revision received: 10 July 1998 / Accepted: 21 July 1998     

Repetitive somatic embryogenesis from peanut cultures in liquid medium

Summary A regeneration system based on repetitive somatic embryogenesis was developed for peanut (Arachis hypogaea L.). Embryogenic suspension cultures were initiated using individual somatic embryos induced from immature cotyledons cultured on a modified Murashige and Skoog medium containing 40 mg/l 2,4-D for 30 days. After transfer to a modified MS liquid medium, the somatic embryos produced masses of secondary and tertiary embryos which continued to proliferate following manual separation and subculture of the embryogenic clumps. The cultures exhibited exponential growth, and have been maintained for over one year without apparent loss of embryogenic potential. Further embryo development, germination, and conversion were achieved by placing embryo clumps onto hormone-free, solid medium. The inclusion of a desiccation period during embryo development enhanced conversion four-fold. Plants have been established in soil and appear to be phenotypically normal.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - BA 6-benzylaminopurine - MSO Modified Murashige and Skoog basal medium - EM embryogenic masses     

Influence of culture conditions on embryo formation and maturation in auxin-induced embryogenic cultures of Digitalis obscura

Somatic embryogenesis was induced in hypocotyls of Digitalis obscura using indoleacetic acid or 2,4-dichlorophenoxyacetic acid with different culture and subculture conditions. Indoleacetic acid-induced embryogenic cultures were used to investigate the effects of amino acids, polyamines and growth regulators on embryo differentiation and maturation. Supplementation of the media with amino acids, polyamines or abscisic acid did not influence or had an adverse effect on embryogenic response. Gibberellic acid at 1.4 M in either culture (30 days) or subculture medium was effective in promoting both differentiation and normal embryo development. The efficiency of somatic embryogenesis was greatly enhanced when isolated indoleacetic acid-induced proembryogenic masses were subcultured in liquid medium with reduced auxin content.Abbreviations ABA abscisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - GA gibberellic acid - IAA indoleacetic acid - Ptr putrescine - Spd spermidine - Spn spermine     

Enhancing the productivity of hybrid yellow-poplar and hybrid sweetgum embryogenic cultures

Summary High-frequency embryogenesis systems were established for hybrid yellow-poplar (Liriodendron tulipifera×L. chinense) and hybrid sweetgum (Liquidambar styraciflua×L. formosana) by modifying a medium originally developed for embryogenic yellow-poplar cultures. Embryogenic cultures of both hybrids, consisting of proembryogenic masses (PEMs), were initiated from immature hybrid seeds on an induction-maintenance medium (IMM) supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D), benzyladenine (BA), and casein hydrolyzate (CH). For hybrid yellow-poplar, as many as 2100 germinable somatic embryos per 4000 cells or cell clumps were produced when PEMs were grown in liquid IMM lacking CH, at a pH that varied with genotype (3.5 or 5.6), followed by size fractionation and plating on semisolid embryo development medium (DM; IMM lacking 2,4-D and BA) without CH, but supplemented with 4.0 mgl⁻¹ (15 μM) abscisic acid. For hybrid sweetgum, up to 1650 germinable somatic embryos per 4000 cells or cell clumps were produced when PEMs were grown in liquid IMM without CH, but with 550 mgl⁻¹ l-glutamine, 510 mg l⁻¹ asparagine, and 170 mg l⁻¹ arginine at pH 5.6. Somatic embryos developed from cell clumps on DM without any plant growth regulators or other supplements. Hundreds of somatic embryos of both hybrids were germinated on DM without CH, transferred to potting mix, and hardened off in a humidifying chamber for transfer to the greenhouse.     

Rapid multiplication of Polyscias filicifolia by secondary somatic embryogenesis

Efficient plant regeneration through somatic embryogenesis was achieved in Polyscias filicifolia. Embryogenic calluses were induced on Murashige and Skoog (MS) basal medium supplemented with 0.5 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg l⁻¹ benzylaminopurine (BAP; type I callus) and on MS medium with 2.0 mg l⁻¹ 2,4-D and 0.01 mg l⁻¹ kinetin (type II callus) from leaf explants of a 2-yr-old plant. Primary somatic embryos (PSEs) developed after four passages of suspension culture established from embryogenic callus when cultured in liquid half-strength MS medium (1/2 MS) without growth regulators. PSEs in the cotyledonary stage were multiplied by adventitious embryogenesis. Single secondary somatic embryos (SSEs) or their clusters developed at the base of PSE hypocotyls and regenerated into plantlets in a one-step process on plant growth regulator-free 1/2 MS medium. Low sucrose concentration of 15 g l⁻¹ promoted development of normal SSEs. All SSEs regenerated into single, well-rooted plantlets on a Nitsch and Nitsch medium supplemented with 0.5 mg l⁻¹ kinetin, 0.1 mg l⁻¹ indole-3-butyric acid, and 10 mg l⁻¹ adenine sulfate. Subsequent two subculture cycles on the same medium were necessary to obtain plantlets sufficiency developed to allow successful transfer to the soil. Rooted plantlets were established in a peat mixture with 90% survival, with the plants showing normal morphological characteristics.     

Control of direct and indirect somatic embryogenesis by exogenous growth regulators in immature zygotic embryo cultures of rose

Immature zygotic embryos of rose (Rosa hybrida L.; cv. Sumpath) did not form somatic embryos or embryogenic calluses when cultured on half-strength Murashige and Skoog's medium supplemented with various con-centrations of 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole growth regulator. However, the zygotic embryos produced somatic embryos without an intervening callus phase at a frequency of 27.3% on medium with 4.44 M 6-benzyladenine (BA) alone. Immature zygotic embryos formed embryogenic calluses at a frequency of 25% on medium with a combination of 1.36 M 2,4-D and 4.44 M BA. Upon transfer to medium without growth regulators, embryogenic calluses produced numerous somatic embryos that subsequently developed into plantlets. Somatic embryos were induced directly from immature zygotic embryos, or indirectly via an intervening callus phase, by manipulating the exogenous growth regulators. Plantlets were successfully transplanted to potting soil and grown to maturity in a greenhouse.     

Plant regeneration from protoplasts of mango (Mangifera indica L.) through somatic embryogenesis

 This report describes a protocol for the regeneration of plants from protoplasts isolated from proembryogenic masses (PEMs) in a suspension culture derived from the nucellar callus of mango (Mangifera indica L. cv 'Amrapali'). The maximum yield (24.6±1.1×10⁶), with 81.04±4.1% viable protoplasts per gram PEMs, was obtained with an enzyme mixture containing 1.2% cellulase, 1.0% hemicellulase and 0.6% pectinase. An optimum density of 5×10⁴ cultured protoplasts per milliliter culture medium was required for the highest frequency (88.89±5.40%) of division. Dividing protoplasts developed into microcalli that proliferated on medium supplemented with growth regulators (auxins or kinetin alone, or auxins with kinetin) and produced somatic embryos after transfer to a growth regulator-free medium. The protocallus on 2,4-D-containing medium produced the maximum number (102.50±6.93) of somatic embryos. Maturation of somatic embryos depended upon the presence, and the nature and combination of growth regulators in the medium during proliferation of the callus. The mature somatic embryos germinated and developed into plants that were transferred to soil. Received: 1 April 1999 / Revision received: 27 July 1999 / Accepted: 23 August 1999     

Somatic embryogenesis in cell cultures of birch (Betula pendula Roth.)

Somatic embryogenesis was induced in cell cultures of birch (Betula pendula Roth.) derived from juvenile tissue of seed embryos and from mature leaf tissue. Embryos were formed in liquid and on solidified medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-furfurylaminopurine (kinetin). Sometimes somatic embryos formed only after transfer to medium devoid of growth regulators. The embryos germinated on hormone-free medium and were potted in soil and grown in the greenhouse.Finnish Forest Research InstituteUniversity of Helsinki, School of Pharmacy;     

Plant regeneration from leaf protoplasts of <Emphasis Type="Italic">Solanum virginianum</Emphasis> L. (<Emphasis Type="Italic">Solanaceae</Emphasis>)

Leaves of Solanum virginianum plants were used for protoplast isolation. To support cell wall formation and cell division, protoplasts were cultured in thin alginate layers floated in liquid medium. When protoplasts were plated at a density of 1.0 × 10⁶/ml in Kao and Michyaluk (KMp8) medium supplemented with 0.5 mg/l zeatin, 1.0 mg/l 2,4-dichlorophenoxyacetic acid, and 1.0 mg/l α-naphthaleneacetic acid, 42.3% of the dividing cells developed microcalli in 3–4 weeks. Shoot formation via organogenesis of protoplast-derived calli was achieved for 28% of calli transferred to solidified KMp8 medium supplemented with 2.0 g/l zeatin and 0.1 mg/l 3-indol acetic acid in about 2 weeks. Further shoot development was observed in Murashige and Skoog (MS) medium without growth regulators and roots were induced after transfer to MS medium containing 1.0 mg/l 3-indol butyric acid. Regenerated plants have normal morphology.     

Improvement of somatic embryogenesis in highland-papaya cell suspensions

Axillary buds (2 mm) from 3-year-old Carica pubescens Lenné et Koch (highland papaya) fruit-bearing plants grown in the greenhouse were cultivated in NN-medium supplemented with different growth regulators naphthaleneacetic acid and indoleacetic acid in combination with Zeatin, benzyladenine, Kinetin and thidiazuron. Several responses were observed within 2–3 months; namely, sprouting of the preformed axillary buds, bud branching into multiple shoots, callus formation at the basal end of the explant and somatic embryogenesis in the preformed callus. Somatic embryogenesis was frequent in most of the tested growth regulator combinations, with the exception of thidiazuron which showed no effect. A much higher yield of somatic embryos could be obtained in suspensions. Somatic embryogenesis was enhanced by the occurence of adventive embryogenesis on single embryos as globular embryo clusters. This was observed in cell suspensions initially grown in a WPM-medium with 2,4-dichlorophenoxyacetic acid, or in combination with benzyladenine or zeatin, for 6 days, then maintained in a growth regulator-free medium under continuous agitation (50 RPM) on an orbital shaker for 3 months. Single cells grown in the absence of 2,4-dichlorophenoxyacetic acid did not initiate embryogenesis and de-differentiated into callus. Plantlets were recovered after transfer of mature embryos from cell suspensions into Magenta flasks. In a second subculture, adventitious embryogenesis occurred spontaneously in clusters at the globular embryo stage under the same growth conditions, yielding a high number of embryos. The culture conditions described above allowed initiation of a large number of somatic embryos directly from cell suspensions through adventive somatic embryogenesis and indirectly from callus on axillary buds.Abbreviations 2,4-d dichlorophenoxyacetic acid - CH casein enzymatic hydrolysate - BA benzyladenine - FAA formalin:acetic acid:alcohol - Glu l-glutamine - IAA indoleacetic acid - NAA naphthaleneacetic acid - NN Nitsch and Nitsch-medium (1969) - TDZ thidiazuron - SD standard deviation     

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     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Ophiorrhiza prostrata</Emphasis> through somatic embryogenesis

In vitro propagation of an anticancerous drug synthesizing plant, Ophiorrhiza prostrata D. Don, was established through indirect somatic embryogenesis. Friable embryogenic calluses were initiated from O. prostrata leaf and internode explants on Murashige and Skoog (MS) media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) either alone or in combination with N⁶-benzyladenine (BA) or kinetin (KIN). Somatic embryos were developed after subculture of the friable calluses onto half strength MS media containing 0.45 or 2.26 μM 2,4-D alone or in combination with BA or KIN. Medium supplemented with 2.26 μM 2,4-D and 2.22 μM BA was optimal, supporting the production of a mean of 5.8 globular embryos. Subculture of globular embryo-bearing calluses on half strength MS medium without growth regulators produced the highest embryo frequency, and the majority of them developing to early torpedo stage. Somatic embryos underwent maturation and converted to plantlets at high frequency (90 %) on half strength MS medium supplemented with 0.44 μM BA. Somatic embryo-derived plantlets with well-developed roots were established in field conditions with a 90 % survival rate.     

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.     

Organogenesis and somatic embryogenesis in Codiaeum variegatum (L.) Blume cv. “Corazón de Oro”

Summary Adventive organogenesis and somatic embryogenesis were induced from leaf explants taken from in vitro or in vivo plants of Codiaeum variegatum cv. “Corazón de Oro.” Shoot multiplication occurred with N⁶-benzyladenine (BA) alone, where the simultaneous production of adventitious buds and somatic embryos occurred at the fourth subculture, and on leaves not in contact with the medium. A medium with BA and 2,4 dichlorophenoxy acetic acid (2,4-D) produced the largest organogenic response, for both in vivo- and in vitro-produced explants. Somatic embryogenesis was only induced when such explants were transferred to a medium lacking 2,4-D. Thus, a medium with BA only produced the largest percentage of explants with shoots and embryos. Replacing BA with thidiazuron induced up to 100% bud regeneration on in vitro-produced explants by 60 d, but was slower for in vitro-grown explants. Both types of embryos exhibited growth arrest that was partially overcome by transfer to hormone-free basal medium with activated charcoal. Rooted plants from all explants were successfully obtained on a medium with indole-butyric acid (IBA).     

Somatic embryogenesis in Ranunculus asiaticus L. hybr. thalamus cultivated in vitro

Calli derived from in vitro cultivated thalamus of Ranunculus asiaticus L. were initiated and maintained for 75 days on Murashige & Skoog's medium containing five concentrations of 2,4-d (0.1, 0.2, 0.4, 0.8, 1.6 mg l^-1). Embryoid differentiation occurred on calli initiated on 1.6 mg l^-1 2,4-d 75 days after subculture onto hormone-free medium. Calli which were initiated and maintained for 75 days on lower 2,4-d concentrations, then transferred to medium without hormones for 75 days, showed the first embryoids one month after further subculture on medium containing 0.05 mg l^-1 2,4-d. All the somatic embryos developed into plants, and 96% survived transplantation to in vivo growth conditions.     

In vitro regeneration of Camellia sinensis (L.) O. Kuntze by somatic embryogenesis

Within 3 weeks of culture, excised cotyledon expiants of Camellia sinensis (L.) O. Kuntze produced somatic embryos without intermediate callus when cultured in Murashige and Skoog's basal medium with 30 g^–1 sucrose. In medium without plant growth regulators, up to 60% of the cultures developed somatic embryos. Embryogenic competence was reduced by increasing concentrations of plant growth regulators tested (i.e. kinetin, 6-benzylaminopurine, and indole butyric acid). The somatic embryos developed, grew to maturity without being subcultured within 6–8 weeks. Secondary embryogenesis was not observed. Germination of isolated mature somatic embryos was low in medium without plant growth regulators. Up to 53% and 60% germination occurred when medium impregnated with kinetin at 1.8 mgl^–1 or 1.0 mgl^–1 6-benzylaminopurine were used respectively. Callus was also routinely produced when cotyledons were cultured in MS basal medium with auxins (2,4-dichlorophenoxyacetic acid and indole acetic acid). Callus induction was however, also achieved in plant growth regulator free medium. Indirect somatic embryogenesis was not induced in the present study.Abbreviations K kinetin - BAP 6-benzylaminopurine - IBA indole butyric acid - IAA indole acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA Naphthalene acetic acid - Fe-EDTA Ethylenediaminetetra-acetic acid (Ferric monosodium salt)