Histological study of somatic embryogenesis induction on zygotic embryos of macaw palm (<Emphasis Type="Italic">Acrocomia aculeata</Emphasis> (Jacq.) Lodd. ex Martius)

The aim of this paper was to describe the histological events that led to somatic embryogenesis in macaw palm (Acrocomia aculeata (Jacq.) Lodd. ex Martius). Zygotic embryos were inoculated on Y₃ medium containing 9 μM 4-amino-3,5,6-trichloropicolonic acid (picloram). Somatic embryos regenerated from nodular callus on induction medium with activated charcoal under photoperiod or without activated charcoal under dark. Many proembryos originated from the fundamental meristem after 10–20 days of culture. When transferred to medium containing activated charcoal, under photoperiod, calli regenerated into somatic embryos of unicellular origin. These embryos had protoderm, plumule and procambial strands and some of them could germinate. After 30–40 days of culture, meristematic masses grew from procambial cells. The masses generated nodular callus, and after transfer to medium without activated charcoal, under dark, they generated somatic embryos of multicellular origin. Those embryos did not regenerate into plants.     

Somatic embryogenesis and plant regeneration from seeds of wild<Emphasis Type="Italic"> Dicentra spectabilis</Emphasis> (L.) LEM

Regeneration via somatic embryogenesis from callus was studied in Dicentra spectabilis. To obtain somatic embryogenic callus, we cultured D. spectabilis seeds on MS basal media supplemented with various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D). The highest percentage of embryogenic callus formation was observed on media containing 1.0 mg/l 2,4-D under dark conditions. Somatic embryogenesis was studied by transferring the callus onto MS basal medium containing different concentrations (0.0, 0.1, 0.5, 1.0, 2.0 mg/l) of KIN (kinetin) and/or BAP. Somatic embryogenesis on MS basal media with 1.0 mg/l of KIN was excellent under light conditions. Somatic embryos were rooted by transferring them to half-strength MS basal media containing 2 g/l Phytagel. About 64.2% of the somatic embryos converted to rooted plantlets, 4% showed secondary embryogenesis and 31.8% did not develop and died. Rooted plantlets showed a 46% survival rate when acclimatized ex vitro.Abbreviations BAP 6-Benzylaminopurine - 2.4-D 2,4-Dichlorophenoxyacetic acid - KIN Kinetin - SEM Scanning electron microscopyCommunicated by H. Lörz     

In vitro regeneration in Trifolium. 1. Direct somatic embryogenesis in T. rubens (L.)

The origin and development of zygotic and somatic embryos of Trifolium rubens L. was studied with the aid of paraffin sections and light microscopy. Zygotic embryos were collected, fixed and prepared daily from one to ten days after cross-pollination. Somatic embryos were obtained by plating petiole sections on modified L2 medium with 0.015 mgl^-1 picloram and 0.1 mgl^-1 6-BAP. Cultured petioles were collected and fixed daily from one to 25 days after plating. Two regions in the vascular bundle sheath of cultured petioles gave rise to callus. The first region was adjacent to the phloem fibers and produced friable callus. The second region gave rise to compact callus that was connected to the fascicular cambium. Somatic embryos originated from single cells in the cortex directly without intervening callus formation and from single cells in the friable callus. In addition, embryos arose from meristematic regions in compact callus. Many early stages of embryogenesis (one, two and four-celled stages) were observed in the cortex and friable callus. Zygotic embryogenesis in Trifolium differs from other legumes in that the suspensor is short and has a broad attachment. This arrangement was observed in zygotic embryos of T. rubens and in many somatic embryos. However, a continuum of somatic embryogenesis was observed where some young embryos had a Trifolium suspensor-like arrangement while others were attached to a long narrow suspensor-like structure more characteristic of Medicago.     

Direct somatic embryogenesis from shoot apical meristems of pea, and thidiazuron-induced high conversion rate of somatic embryos

Direct somatic embryogenesis from shoot apical meristems of pea is described. Somatic embryos were induced directly (without callus intervention) from meristematic tissues grown on a medium supplemented with 2.5 μM picloram. Within 4 to 5 weeks, fully morphologically developed somatic embryos were obtained. Somatic embryos originated from apical as well as from basal parts of meristem explants. The initiation and development of somatic embryos was asynchronous, basal somatic embryos developed more quickly than apical ones. Abundant secondary embryogenesis was observed after isolation of primary somatic embryos and culturing them on media for germination. Morphologically normal somatic embryos germinated on medium without growth regulators; the conversion rate was increased by application of 10 μM thidiazuron (TDZ). TDZ was also able to induce shoot bud regeneration on embryos without differentiated a shoot apex, allowing to germinate up to 78 % of all harvested somatic embryos with various morphology. The protocol was successfully tested in 47 out of 48 P. sativum and P. arvense cultivars as well as in two wild peas (P. elatius, P. jomardi). This revised version was published online in July 2006 with corrections to the Cover Date.     

Somatic embryogenesis and plant regeneration from immature embryo cultures of onion (Allium cepa L.)

Somatic embryos were obtained and plants regenerated from immature embryos of onion following culture on embryogenic induction media. Highest rates of somatic embrogenesis resulted from 0.5- to 1.5-mm immature embryos cultured on media containing 5 mg/l of picloram. Somatic embryos formed either directly on the surface of embryos or developed from compact cultures. The production of somatic embryos was significantly affected by the addition of auxin, embryo size and cultivar. The potential of somatic embryogenic cultures for plantlet regeneration has been maintained for over 1 year in some lines. Three types of immature-embryo-derived cultures were characterized by histology. Some cultures were morphologically similar to immature-embryo-derived embryogenic cultures of other monocotyledonous species. Cultures such as these have proven to be useful target tissues in transformation studies. Received: 16 December 1997 / Revision received: 23 February 1998 / Accepted: 13 March 1998     

Organogenesis and somatic embryogenesis from callus cultures in Muscari armeniacum Leichtl. ex Bak.

Summary Establishment of fast-growing, highly regenerable callus cultures was examined in Muscari armeniacum Leichtl. ex Bak. in order to develop an efficient genetic transformation system. High-frequency callus formation was obtained from leaf explants of cv. Blue Pearl on media containing 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthaleneacetic acid (NAA) or 4-amino-3,5,6-trichloropicolinic acid (picloram, PIC). Fast-growing, yellowish nodular callus lines and white friable callus lines containing a few somatic embryos were established on initiation medium supplemented with 4.5 μM 2,4-D and with 54 μM NAA, respectively. The yellowish nodular calluses vigorously produced shoot buds after transfer to media containing 0.44–44 μM 6-benzyladenine (BA), whereas the white friable calluses produced numerous somatic embryos upon transfer to plant growth regulator-free (PGR-F) medium. Histological observation of shoot buds and somatic embryos indicated that the former consisted of an apparent shoot meristem and several leaf primordia, and the latter had two distinct meristematic regions, corresponding to shoot and root meristems. Both shoot buds and somatic embryos developed into complete plantlets on PGR-F medium. Regenerated plants showed no observable morphological alterations. High proliferation and regeneration ability of these calluses, were maintained for over 2 yr.     

Somatic embryogenesis and direct as well as indirect organogenesis in Lilium pumilum DC. Fisch., an endangered ornamental and medicinal plant

Somatic embryogenesis and organogenesis in Lilium pumilum were successfully regulated by picloram, α-naphthaleneacetic acid (NAA), and 6-benzyladenine (BA). In organogenesis, the highest shoot regeneration frequency (92.5%) was obtained directly from bulb scales on Murashige and Skoog (MS) medium containing 2.0 mg L^?1 BA and 0.2 mg L^?1 NAA, while organogenic callus (OC) formed from leaves on MS medium supplemented with 1.0 mg L^?1 BA and 0.5 mg L^?1 NAA. Following subculture, 76.7% of OC regenerated shoots. In somatic embryogenesis, the combination of picloram and NAA increased the amount of embryogenic callus (EC) that formed with a maximum on 90.7% of all explants which formed 11 somatic embryos (SEs) per explant. Differences between EC and OC in cellular morphology and cell differentiation fate were easily observed. SEs initially formed via an exogenous or an endogenous origin. The appearance of a protoderm in heart-shaped SE and the bipolar shoot–root development in oval-shaped SE indicated true somatic embryogenesis. This protocol provides a new and detailed regulation and histological examination of regeneration pattern in L. pumilum.     

<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 and plant regeneration from immature embryos of hybrid sunflower (Helianthus annuus L.) on a high sucrose-containing medium

Somatic embryos of sunflower (Helianthus annuus) were obtained by placing immature zygotic embryos on a high sucrose (12%) containing medium. The somatic embryos were first observed 6 days after culture and a callus intermediate was not formed. Histological examination revealed the classical stages of embryo development. The somatic embryos proliferated directly from the surface of the zygotic embryos and germinated after placement on a low sucrose medium. Seeds have been obtained from regenerated plants.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA Indole-3-acetic acid - BAP 6-benzylamino purine. Salaries and research support were provided by State and Federal funds appropriated to OSU-OARDC. Journal Article No. 67–87     

In vitro plant regeneration and ethylmethanesulphonate (EMS) uptake in somatic embryos of date palm (Phoenix dactylifera L.)

The effect of the auxins dicamba (3,6-dichloro-2-methoxybenzoic acid) and picloram (4-amino-3,5,6-trichloropicolinic acid) on callus growth and embryogenesis in Phoenix dactylifera L. was investigated. Maximum callus fresh weight was obtained in nutrient medium enriched with 200 µm picloram. Somatic embryogenesis and subsequent plant regeneration was achieved following transfer of such calli to hormone-free medium. Germination of the somatic embryos was influenced by treatment with the chemical mutagen ethylmethanesulphonate (EMS). Uptake of the labelled mutagen ([¹⁴C]EMS) by the somatic embryos increased with increased incubation time. Presence of dimethyl sulphoxide (DMSO) as a carrier agent during mutagenic treatment was necessary for efficient mutagen uptake.     

Comparison of different liquid/solid culture systems in the production of somatic embryos from Narcissus L. ovary explants

Alternative procedures for the production of Narcissus L. somatic embryos were investigated. Somatic embryogenesis was initiated on ovary explants isolated from cv. Carlton bulbs, chilled for 12 weeks at 5°C. The explants were cultured on MS media with 3% sucrose and growth regulators: Picloram or 2,4-D (10 or 25 μM) and BA (1 or 5 μM) for 12 weeks in the culture systems: continuous cultivation on solid media, continuous cultivation in liquid media and sequential cultivation using cycles in liquid and solid media. Two types of somatic embryogenesis, indirect and direct, were observed. The developmental pathway depended on the period of exposure to liquid media. Somatic embryos were formed via embryogenic nodular callus on solid media. 2,4-D and BA stimulated the process. The 4-week and 8-week liquid medium treatments resulted in the development of somatic embryos directly from the ovary explant tissue. The highest number of somatic embryos was noted under the influence of 25 μM 2,4-D and 5 μM BA in explants cultivated for 8 weeks in liquid medium and then, for 4 weeks, on solid medium. The effects of inoculum density on biomass increase and the formation of somatic embryos in cultures obtained on a medium with 25 μM 2,4-D and 5 μM BA were also checked. The highest biomass increase was observed after subculturing in liquid medium containing 0.5 μM NAA and 5 μM BA when the density of inoculum was 0.5 g/25 ml of the medium. The highest number of somatic embryos was noted when the density of inoculum was 1.5 g/25 ml.     

Somatic embryogenesis from immature leaves of in vitro grown tea shoots

Immature leaves of in vitro grown shoots of tea were cultured on various levels of 2,4-D. Somatic embryos were induced directly on leaves or via embryogenic callus produced at the basal regions of the leaves. Induction of embryogenesis appeared to be correlated with the maturity of the leaf explants, with younger leaves responding better. The embryogenic response of leaf explants also was correlated with the period of culture in 2,4-D containing liquid medium. Embryogenic calli or repetitive somatic embryos maintained their regeneration capacity for more than 3 years. Histological observation revealed somatic embryos were formed on various regions of the leaf midrib. Somatic embryos germinated and developed into plantlets on agar medium containing BA and IBA.Abbreviations BA 6-benzylaminopurine - IBA indole-3-butyricacid - 2,4-D 2,4-dichloro phenoxyacetic acid     

The ability of Prunus avium× P. pseudocerasus `Colt' to form somatic embryos in vitro contrasts with the recalcitrance of P. avium

Somatic embryos were induced on roots excised from in vitro plants of Prunus avium× pseudocerasus `Colt'. On medium containing 6-benzylamino purine (BAP, 1.5 μM) and 2,4-dichlorophenoxyacetic acid (2,4-D, 15 μM), a mean of 25 (s.e. ± 2.0) somatic embryos were produced on intact root systems and 15 (s.e. ± 1.7) on roots systems cut into 10 mm pieces. Most somatic embryos were formed directly on intact roots and indirectly (from callus) on sectioned roots. A mean of 2.5 (s.e. ± 0.25) secondary embryos per primary embryo were formed directly on primary embryos after they were transferred to medium containing BAP (1.5 μM), indole-3-butyric acid (10 μM) and 2,4-D (5 μM). After transfer to a medium containing BAP (2 μM) and gibberellic acid (GA<sub>3</sub>, 3 μM), shoots developed in 75% (s.e. ± 7.3) of the embryos. Somatic embryos were not induced on explants of in vitro roots or shoots of P. avium, and were induced infrequently on zygotic embryos, although a wide range of media were tested. Possible reasons for the contrasting embryogenic ability of `Colt' and P. avium are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Somatic embryogenesis from immature embryos of redbud (Cercis canadensis)

Somatic embryos developed directly from 96 and 110 day post-anthesis Cercis canadensis L. (redbud) zygotic embryos from one of two trees sampled that were explanted onto modified Schenk and Hildebrandt medium amended with either 1, 2, 3 or 5 mg/1 2,4-D in combination with either 7.6 or 12. 6 mM ammonium ion. Although somatic embryogenesis was expressed on most media, the number of explants that produced somatic embryos and the mean number of embryos formed per explant were greatest on media that contained either 2 or 3 mg/1 2,4-D; 12.6 mM ammonium ion inhibited embryogenesis from 96 day post-anthesis explants. Zygotic embryos explanted 117 days after anthesis produced only callus and roots. Somatic embryos that were bottle-shaped or had distinct cotyledons organized roots on germination media, but only one embryo formed a shoot. No additional development occurred. Histological examination of somatic embryos showed that shoot apical meristems were poorly developed.Abbreviations 2,4-D 2, 4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - BAP 6-benzylaminopurine - FPA Formalin-propionic acid-ethanol (50%)     

Histology of somatic embryogenesis from floral tissues cocoa

Somatic embryogenesis fromTheobroma cacao L. flower buds, as previously reported on five Forastero hybrid genotypes, was tested on several other genotypes, belonging to the three cocoa-tree groups: Forastero, Trinitario and Criollo. The results gave evidence of genotypic efficiencies. Explants were cultivated under two successive conditions: callogenesis and expression media. The morphological and histological responses were different for embryogenic or non-embryogenic genotypes. For embryogenic genotypes, only staminodes and stamen filaments were able to produce somatic embryos: after a few days on the expression medium, groups of callus cells went through the meristematic and then embryonic stages, and finally formed somatic embryos. Many of them showed abnormalities. Simultaneously, some embryogenic cells were visible. These started to divide to form pro-embryos which however were unable to evolve into proper somatic embryos.     

Plant regeneration via somatic embryogenesis in Schlumbergera truncata

Somatic embryogenesis was induced from phylloclade explants of Schlumbergera truncata cv. Russian Dancer. Callus developed on phylloclade explants and sub-cultured over a period of 16 months on MS medium containing mainly cytokinins was superior for the induction of somatic embryos compared to callus grown for a shorter time in the establishment medium. Sub-culture of callus grown in SH-or MS-based liquid media supplemented with 7.0 μM kinetin and transferred onto solid MS-based medium with either 0.45 μM 2,4-D or without hormones resulted in the differentiation into somatic embryos. SH-based medium proved better than MS-based medium when used as the first medium for the induction of somatic embryogenesis. However, somatic embryogenesis, contrary to adventitious shoot formation, was reduced when 2,4-D was included in the MS-based medium used for final transfer compared to the medium without growth regulators, indicating that a critical hormonal balance was reached. Somatic embryos developed root and shoot poles when grown on G medium. On this medium approximately 70% germination was recorded in the embryos that were differentiated earlier from the callus that was grown for a longer time in the establishment medium. This callus was grown on either SH- or MS-based medium supplemented with 7.0 μM kinetin, and then transferred after 30 days (from SH medium) onto MS medium without hormones or after 40 days (from MS medium) onto MS medium with 0.45 μM 2,4-D. Furthermore, plants from somatic embryos were successfully potted in soil and showed further growth and formation of a second set of phylloclades (secondary phylloclades). Histological studies showed that somatic embryos had no detectable connection with the mother explants and that advanced stages of somatic embryos had a contained vascular system. In addition to the normal dicotyledonous embryos, anomalous embryos with multiple cotyledons and vase-like embryos were observed. Secondary embryos were also recorded in this study.     

Evidence of somatic embryogenesis from root tip explants of the rattan Calamus manan

Summary Somatic embryogenesis of Calamus manan, a single-stemmed rattan species, in tissue culture was scientifically demonstrated for the first time. Root tips of in vitro plantlets produced friable callus when the explants were cultivated for several mo. on a Murashige and Skoog induction medium containing 7.5 mg Picloram per l (31.1 μM). Histological analyses established the presence of proembryos within the callus which differentiated subsequently into somatic embryos using the same culture medium. Histological examination revealed that these somatic embryos completely lacked starch and protein reserves, which did not prevent them, however, from germinating, and showing bipolar development. These somatic embryos further developed into young plants, similarly to zygotic embryos.     

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.     

New approaches to improve the efficiency of somatic embryogenesis in oil palm (Elaeis guineensis Jacq.) from mature zygotic embryos

We developed an efficient and simple system for inducing somatic embryogenesis and regenerating plantlets from mature zygotic embryos of oil palm. Embryogenic calli were induced from mature zygotic embryos of oil palm on modified Murashige and Skoog medium with 2,4-dichlorophenoxyacetic acid or picloram, alone or in combination with activated charcoal. The greatest frequency of embryogenic callus induction (97.5%) was obtained by culturing mature zygotic embryos on callus induction medium with 450 μM picloram and 2.5 g?L^?1 activated charcoal. Embryogenic calli proliferated on a medium with a reduced concentration of picloram. Embryogenic calli were then subcultured on a medium supplemented with 12.3 μM 2-isopentenyladenine and 0.54 μM naphthaleneacetic acid, with subcultures at 4-wk intervals. Somatic embryos were regenerated on a medium with Murashige and Skoog macro- and micronutrients at half-strength concentrations supplemented with 20 g?L^?1 sucrose, 2.5 g?L^?1 activated charcoal, and 2.5 g?L^?1 Phytagel. Detailed histological analysis revealed that somatic embryogenesis followed an indirect pathway. Primary calli were observed after 4–6 wk of culture and progressed to embryogenic calli at 12 wk. Embryogenic cells exhibited dense protoplasm, a high nucleoplasmic ratio, and small starch grains. Proembryos, which seemed to have a multicellular origin, formed after 16–20 wk of culture and successive cell divisions. Differentiated somatic embryos had a haustorium, a plumule, and the first and second foliar sheaths. In differentiated embryos, the radicular protrusion was not apparent because it generally does not appear until after the first true leaves emerge.     

Somatic embryogenesis and plant regeneration in Acacia farnesiana and A. schaffneri

Summary Efficient plant regeneration systems via somatic embryogenesis have been developed for Acacia farnesiana and Acacia schaffneri [Leguminosae (Mimosoideae)]. The protocol used in this study consisted of placing immature, zygotic embryos of these species in Murashige and Skoog semi-solid basal medium supplemented with 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.65 μM kinetin to induce callus. Some parts of the callus were used for direct embryo differentiation and others for establishment of cell suspension cultures. In the first case, somatic embryos were produced on semi-solid differentiation media without growth regulators or with abscisic acid (ABA). The higher number of somatic embryos, 345 and 198 embryos per g callus in A. farnesiana and A. schaffneri, respectively was obtained in media without growth regulators, but adding ABA increased the percentage of embryos that reached more advanced differentiation stages. The production of somatic embryos was achieved starting from cell suspensions only when these suspensions were plated into the semi-solid differentiation medium. Somatic embryos germinated on medium containing 217 μM adenine sulfate with efficiencies of 69% in A. farnesiana and 47% in A. schaffneri. Some somatic embryos that developed into plantlets were acclimatized in the greenhouse, and they grew into normal plants.