The initiation of somatic embryos and adventitious roots from developing zygotic embryo explants of Cercis canadensis L. cultured in vitro

Zygotic embryo explants of Cercis canadensis L. cultured in vitro responded to 1 and 5 M 2,4-dichlorophenoxyacetic acid (2,4-D) or 50 M -naphthaleneacetic acid (NAA) by initiating somatic embryos and adventitious roots. Somatic embryos and adventitous roots were formed from developing zygotic embryos, while fully developed embryos collected from mature seed initiated only adventitious roots. Following 2,4-D application, the number of somatic embryos decreased while adventitious roots increased with increasing developmental age of the explant. The greatest number of somatic embryos were initiated with a 5 to 20 day exposure to 5 M 2,4-D from zygotic embryos collected between 75 and 82 days post-anthesis in 1987. Somatic embryos formed directly from epidermal and subepidermal cells, while adventitious roots developed from interior cortex cells. Normal somatic embryos were recovered after a 20 day exposure to 5 M 2,4-D and acclimated to greenhouse conditions.     

Somatic embryogenesis and plant regeneration from zygotic embryo culture in blue spruce (Picea pungens Engelman.)

Summary Somatic embryogenesis and plantlet formation have been achieved from cultured mature zygotic embryos of blue spruce (Picea pungens Engelman.). The effect of three basal media LP, LM, and BLG, all used at half-strength, was tested at the induction phase. LM medium induced somatic embryogenesis to a higher extent than LP whereas BLG did not produce any embryonal-suspensor mass representing stage 1 somatic embryos. The embryonal-suspensor mass was induced on a wide range of auxin/cytokinin ratios. However, media containing either 2 M NAA and 10 M BA, or 10 M NAA and 5 M BA produced somatic embryos that gave the highest frequency of plantlets. The level of ABA required in the maturation medium for somatic embryos to mature properly varied with the auxin/cytokinin levels in the induction medium on which the somatic embryos were derived. Inclusion of AgNO₃ (10 – 100 M) in the induction medium reduced somatic embryogenesis and embryo conversion.Abbreviations NAA naphthalene-acetic acid - BA N⁶-benzylaminopurine - ABA abscisic acid     

Norway spruce somatic embryogenesis: high-frequency initiation from light-cultured mature embryos

Somatic embryos and rooted plantlets have been regenerated from light-initiated embryogenic callus derived from mature embryos of Picea abies. Under a 16 h photoperiod, mature zygotic embryos were cultured on a modified half-strength Murashige & Skoog medium without NH₄NO₃ and supplemented with 5 mM glutamine, 4.5 M N6-benzyladenine and 10.7 M naphthaleneacetic acid or 10 M 2,4-dichlorophenoxyacetic acid. White translucent embryogenic callus, proliferating from the callusing hypocotyl region after 3 weeks incubation, was isolated from the green non-embryogenic tissue and subcultured for over 12 months. Upon transfer of the embryogenic callus through a specific sequence of media, somatic embryos proceeded to mature, elongating and forming rings of cotyledonary leaves similar to those of zygotic embryos. Transferred to medium without growth regulators, the somatic embryos germinated and produced plantlets with green cotyledons, elongated hypocotyls and primary roots.     

High efficiency adventive embryogenesis on somatic embryos of anther,filament and immature proembryo origin in horse-chestnut (Aesculus hippocastanum L.) tissue culture

Adventive embryogenesis was successfully induced in cultures of zygotic and somatic embryos on MS medium supplemented with BA and NAA. A procedure has been proved successful for the in vitro multiplication of somatic embryos regenerated at low frequencies from filament and callus cultures. The occurrence and rate of adventive embryogenesis did not depend on the origin of the primary embryos (zygotic and somatic), but did depend on the developmental stage. Primary embryos are capable of embryogenesis in each of the different phases of embryogenesis, though the rate is different. BA concentrations of 22–44 M increased the rate of adventive embryogenesis and accelerated the development of embryos. The highest proliferation rate (22–25x/5 weeks) was achieved at hormone concentrations of 44 M BA and 5.4 M NAA.Abbreviations BA benzyladenine - CH casein hydrolysate - CM coconut milk - 2,4-D dichlorophenoxyacetic acid - MS Murashige & Skoog medium - WPM woody plant medium - NAA 1-naphtaleneacetic acid     

Somatic embryogenesis from immature zygotic embryos ofGinkgo biloba

Immature zygotic embryos ofG. biloba were taken, at various developmental stages, from ovules harvested in November 1993. Zygotic embryos showing the beginning of the cotyledonary development cultured on modified Murashige & Tücker (1969) media proliferated intensely. In fact, 98.5% of the immature zygotic embryos produced embryogenic and undifferentiated tissues (calluses), in proportions varying depending on the hormonal composition of the induction media. After two weeks of culture, direct embryogenesis was observed on the hypertrophic cotyledons when benzyladenine 10 M was used as the sole plant growth regulator in the induction media. The addition of different concentrations of NAA (5–10–20 M) and of BA (5 M) to the induction media led to an indirect embryogenesis after two months, when the calluses were transferred to the development media without auxin. The highest frequency of embryogenic tissues (90–95%) and the highest number of somatic embryos per explant (9.6) were obtained with benzyladenine (10 M) as the sole exogenous growth factor. Some embryos isolated mechanically or in situ on the callus developed as far as the later cotyledonary stage.Abbreviations AUX Auxin - BA Benzyladenine - CYT Cytokinin - IZE Immature zygotic embryo - MT Murashige & Tücker (1969) medium - NAA Naphtaleneacetic acid     

Possible involvement of abscisic acid in the induction of secondary somatic embryogenesis on seed-coat-derived carrot somatic embryos

When seed coats (pericarps) were picked from 14-day-old carrot (Daucus carota) seedlings and cultured on agar plates, embryogenic cell clusters were produced very rapidly at a high frequency on the open side edge. Embryo induction progressed without auxin treatment; indeed treatment caused the formation of non-embryogenic callus. The embryogenic tissues (primary embryos) developed normally until the torpedo stage; however, after this a number of secondary somatic embryos were produced in the hypocotyl and root regions. Tertiary embryos were formed on some of the secondary embryos, but many developed into normal plantlets. The primary embryos contained significantly higher levels of abscisic acid (ABA) than the hypocotyl-derived normal and seed-coat-derived secondary embryos. Fluridone inhibited the induction of secondary embryogenesis, while exogenously supplied ABA induced not only tertiary embryogenesis on the seed-coat-derived secondary embryos, but also secondary embryos on the hypocotyl-derived normal somatic embryos. These results indicate that ABA is one of the important endogenous factors for the induction of secondary embryogenesis on carrot somatic embryos. Higher levels of indole-3-acetic acid (IAA) in primary embryos also suggest the presence of some concerted effect of ABA and IAA on the induction of secondary embryogenesis in primary embryos.     

Regeneration of fertile plants from excised immature zygotic embryos of Arabidopsis thaliana

Summary We present protocols for the regeneration of fertile plants from immature zygotic embryos of Arabidopsis thaliana ecotype Zürich either directly by multiple shoot formation from the apical region or indirectly by shoot induction from embryo derived calli. The regeneration efficiency by multiple shooting depended on the developmental stage of the cultured embryos and ranged from 15% for early heart shaped to 90% for early torpedo shaped and further developed embryos. 85% callus induction was achieved from embryos in the early torpedo shaped or a later stage of development. The efficiency of shoot induction from embryo derived calli varied between 25% and 75% in different experiments.Abbreviations BAP 6-benzylaminopurine - CIM callus inducing medium - 2,4-D 2,4-dichlorophenoxyacetic acid - Gln glutamine - IAA indole-3-acetic acid - Kin kinetin - NAA l-naphthaleneacetic acid - 2ip 2-isopentenylaminopurine - Pro proline - RIM root inducing medium - SIM shoot inducing medium     

Somatic embryogenesis and plants from immature zygotic embryos of the species Musa acuminata and Musa balbisiana

Callus was obtained from immature zygotic embryos of semminiferous species (diploids) of Musa sp. using a medium derived from that of Murashige and Skoog. Picloram (7.5 M) was added and the medium was solidified with gelrite (2 gl^–1). Differentiation of the first somatic embryos occurred after transfer of the callus in the presence of 7.5 M picloram or 5.3 M NAA. Somatic embryos germinated on the medium supplemented with 5.3 M NAA. Serial sections of zygotic and somatic embryos showed perfect homology in their structure (epidermis, cotyledonary slit, shoot apex and 3 root primordia). Embryonic callus was characterised by a large quantity of protein storage in the cytoplasm.Abbreviations BAP 6-Benzylaminopurine - NAA -Naphthaleneacetic acid - HPLC High performance liquid chromatography     

Plant regeneration from somatic embryos ofTaxus brevifolia

Summary Taxusbrevifolia is the source of paclitaxel (Taxol®), an anticancer drug. A method for regeneration ofTaxus brevifolia from immature zygotic embryos via somatic embryogenesis is described. Embryogenic callus tissues were obtained by culturing immature zygotic embryos on Lloyd and McCown medium (MCM) supplemented with 160 M 2,4-dichlorophenoxyacetic acid (2,4-D) + 5 M benzylaminopurine (BA) + 5 M naphthaleneacetic acid (NAA) for 4 weeks. Putative embryoids were obtained following transfer of cultures to MCM medium supplemented with 4 M BA + 5 M kinetin + 1 M NAA for 6 to 8 weeks. Conversion of embryos was obtained on MCM medium supplemented with 40 M abscisic acid (ABA) + 1% activated charcoal. Development of bipolar structures with recognizable shoot and root apices was observed in somatic embryos. Five percent of somatic embryos were regenerated into plantlets on half-strength growth regulator-free MCM medium.     

Sialidase of swine influenza A viruses: variation of the recognition specificities for sialyl linkages and for the molecular species of sialic acid with the year of isolation

The sialidase of swine influenza A viruses of N1 and N2 subtypes, isolated from 1930 to 1992, was studied for substrate specificity with ganglio-series, lacto-series type II and GM3 gangliosides containing Neu5Ac2-3Gal, Neu5Gc2-3Gal and Neu5Ac2-6Gal linkages. All viral sialidases tested showed that the activity for hydrolysing substrates with Neu5Ac2-3Gal was higher than the activities with Neu5Gc2-3Gal and Neu5Ac2-6Gal linkages. When GM1b, GM3 and sialylparagloboside were used as substrates, the earliest strain (A/Wisconsin/15/30 H1N1, isolated in 1930) showed the activity ratio of Neu5Ac2-6Gal to Neu5Ac2-3Gal to be 0.13:0.2, and the ratio Neu5Gc2-3Gal/Neu5Ac2-3Gal to be 0.19:0.37, while those strains isolated from 1978 to 1992 exhibited ratios of 0.29:0.58 for Neu5Ac2-6Gal/Neu5Ac2-3Gal and 0.51:0.76 for Neu5Gc2-3Gal/Neu5Ac2-3Gal. The above results indicate that the substrate specificities of sialidases from swine influenza A viruses towards sialyl linkages and the molecular species of sialic acid are related to the year of isolation, i.e. strains isolated after 1978 exhibited higher activity towards Neu5Ac2-6Gal and Neu5Gc2-3Gal linkages when compared with strains isolated in an earlier year, 1930.Abbreviation Neu5Ac 5-N-acetylneuraminic acid - Neu5Gc 5-N-glycolyneuraminic acid - Gal d-galactose - Glc d-glucose - Cer Ceramide - II³(Neu5Ac)Lac Neu5Ac2-3Gal1-4Glc - GM3(Neu5Ac2-3Gal) Neu5Ac2-3Gal1-4Glc1-Cer - GM3(Neu5Gc2-3Gal) Neu5Gc2-3Gal1-4Glc1-Cer - GM1b(Neu5Ac2-3Gal) Neu5Ac2-3Gal1-3GalNac1-4Gal1-4Glc1-Cer - GMlb(Neu5Gc2-3Gal) Neu5Gc2-3Gal1-3GalNAc1-4Gal1-4Glc1-Cer - IV³(Neu5Ac)nLc4Cer Neu5Ac2-3Gal1-3GlcNAc1-4Gal1-4Glc1-Cer - IV³(Neu5Gc)nLc4Cer Neu5Gc2-3Gal1-3GlcNAc1-4Gal1-4Glc1-Cer - IV⁶(Neu5Ac)nLc4Cer Neu5Ac2-6Gal1-3GlcNAc1-4Gal1-4Glc1-Cer - TDC taurodeoxycholate.     

Somatic embryogenesis and plant regeneration from immature embryos of saw palmetto,an important landscape and medicinal plant

Somatic embryogenesis and plant regeneration from immature zygotic embryos was achieved for saw palmetto (Serenoa repens (Bartr.) Small). Embryos, isolated from immature fruit of native-grown plants, were cultured on Murashige and Skoog medium plus 0.15% (w/v) activated charcoal and supplemented with 452 M 2,4-dichlorophenoxyacetic acid (2,4-D) and 14.7 M N⁶-(2-isopentenyl)adenine (2iP). Clusters of somatic embryos developed from all immature zygotic embryos 5 weeks after culture initiation. After 12 weeks, explants were transferred to the same medium with the amount of 2,4-D reduced to 90.4 M which resulted in somatic embryo proliferation. Somatic embryos were then transferred to the basal medium containing 0.9, 9 M thidiazuron (TDZ), or no growth regulator for conversion into plantlets. The 9 M TDZ treatment was ineffective for plant regeneration. However, 12% of the embryos subcultured on 0.9 M TDZ were able to produce complete plantlets. Shoot production was obtained from 35% of the embryos subcultured in the absence of growth regulators. Rooting (100%) was achieved when these shoots were transferred onto medium containing 22.2 M -naphthaleneacetic acid (NAA).     

Plant regeneration from embryo-derived callus of oil palm – the effect of exogenous polyamines

Regeneration in oil palm was achieved through somatic embryogenesis/organogenesis from embryo-derived callus. Callus was induced from mature embryos of the cross 281 (D)×18 (P) on modified MS medium supplemented with 2,4-D (113.12 M) and 2-iP (14.76 M). The embryogenic calluses obtained were transferred to Blaydes medium supplemented with 2,4-D (0.045 M) and one of the following growth regulators: TDZ (4.54 M), zeatin riboside (2.85 M), putrescine (1 mM) and spermine (100 M). Secondary somatic embryogenesis was found to occur in media supplemented with polyamines. The efficiency of formation of somatic embryos, secondary somatic embryos and shoot meristemoids were significantly higher in putrescine containing medium. Histological studies were also undertaken.     

Growth and morphogenesis of immature embryos of sweet potato (Ipomoea batatas L.) in vitro

Sweet potato (Ipomoea batatas L.) embryos excised from the fertilized ovules of 6- to 12-day-old capsules were cultured on MS medium supplemented with NAA, BA, GA separately and in combinations. GA was found essential for initial morphogenesis of globular and heart stages. Seedlings were recovered from late globular stage onwards but recovery was best from advanced embryo stages. Differentiated embryos produced multiple shoots on MS medium +1M NAA÷2M BA +0.5M GA.     

In vitro somatic embryogenesis ofDalbergia sissoo Roxb. —a multipurpose timber-yielding tree

Somatic embryogenesis was achieved in callus cultures dervied from 40-day-old semimature zygotic embryos ofDalbergia sissoo on semi-solid Murashige and Skoog (MS) salts and vitamins supplemented with 0.46–1.16 M kinetin, 6.78–9.04 M 2,4-dichlorophenoxy acetic acid (2,4-D) and 30 g/1 sucrose. Somatic embryos proliferated rapidly by secondary somatic embryogenesis after transfer to half-strength basal MS medium supplemented with 0.46-1.16 M kinetin and 6.78–9.04 M 2,4-D with 2% (w/v) sucrose. The light-green somatic embryos germinated on half-strength MS salts and vitamins supplemented with 0.5 mg/1 abscisic acid and 2% (w/v) sucrose. The developmental stages of somatic embryogenesis were studied by light and scanning electron microscopy.Abbreviations ABA Abscisic acid - BA 6-benzyladenine - Kn kinetin - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - MS Murashige and Skoog (1962) basal medium     

Proliferation and germination of somatic embryos from embryogenic suspension cultures in <Emphasis Type="Italic">Phœnix dactylifera</Emphasis>

The present study demonstrates a procedure for the rapid development of a high number of somatic embryos from embryogenic suspension culture. This method might be efficient for mass propagation of Phnix dactylifera L. Embryogenic callus placed in liquid medium with 10^–5M ABA yielded an average 72 embryos per 100ml of culture medium within 2months, while those placed on solid medium yielded an average of 33, 20 and 16 embryos per 100ml of culture medium respectively for 10^–7, 10^–6 and 10^–5 M ABA after 4months. The combination of 2,4-DIchlorophenoxyacetic acid (2,4-D) (4.5×10^–7M), glutamine (6.7×10^–4M), and ABA (10^–5M) (L8 liquid medium) showed a beneficial effect on somatic embryos production compared to 2,4-D and glutamine alone, while this combination significantly (p<0.05) increased the accumulation of storage proteins (144 and 138mgg^–1 DW respectively for Jihel and Bousthami noir cultivars) in somatic embryos. The somatic embryos which underwent maturation on medium containing only 4.5×10^–7M 2,4-D and 10^–5M ABA (L6 liquid medium) accumulated more sugars (292 and 265mgg^–1 DW respectively for Jihel and Bousthami noir) than those matured on any other liquid medium. Histological studies revealed that somatic embryos (developed in L6 and L8 liquid media) accumulated less reserve compounds (proteins and sugars) than zygotic embryos. The addition of activated charcoal (0.25 and 0.5gl^–1) and phytagel^® (2.5gl^–1) to the germination medium may be useful for enhancing the germination of Phnix dactyliferasomatic embryos.     

Factors influencing secondary somatic embryogenesis inMalus x domestica Borkh. (cv ‘Gloster 69’)

A procedure for regeneration of apple plants through secondary somatic embryogenesis (SSE) was developed in apple Gloster 69. Primary somatic embryos were produced from cotyledon-derived cultures of immature zygotic embryos. These somatic embryos were multiplied by secondary somatic embryogenesis (SSE) on media with different Plant Growth Regulator (PGR) combinations. The highest SSE rate (55.5%) was obtained with a combination of NAA (5.3 M), BAP (0.9 M) and KIN (0.9 M) or with TDZ alone (10 M). In addition, effects of explant source, somatic embryo size, type and concentrations of carbohydrates and gelling agents on SSE were investigated. The optimum SSE (>73%) was obtained by the culture of large size somatic embryos or cotyledon-like structures on medium containing a combination of NAA/BAP/KIN or TDZ (10 M) alone, maltose (175 mM) and Phytagel (2.8 g/1).     

Analysis of the genetic stability of <Emphasis Type="Italic">Eucalyptus globulus</Emphasis> Labill. somatic embryos by flow cytometry

Flow cytometry was used to measure the nuclear DNA content of Eucalyptus globulus Labill. somatic and zygotic embryos and leaves in order to determine if somatic embryogenesis induces DNA content and ploidy changes in this species. Mature zygotic embryos derived from open-pollination orchard families were collected from a location in the centre of Portugal. One group was kept for nuclear DNA content and ploidy analysis, and the other group was used for establishing embryogenic cultures. Mature zygotic embryos were grown on Murashige and Skoog (MS) medium supplemented with 3% (w/v) sucrose and 3 mg l^–1 -naphthaleneacetic acid (NAA) for 3 weeks and then transferred to MS medium without growth regulators. Globular somatic embryos from approximately 8-month-old embryogenic cultures were used in the assay. DNA ploidy levels and the nuclear DNA content of mature zygotic embryos, somatic embryos and leaves from the mother field tree were determined using flow cytometry combined with propidium iodide staining. Zygotic embryos had a nuclear DNA content of 1.32 pg/2C, somatic embryos had a nuclear DNA content of 1.39 pg/2C and leaves from the field tree had a nuclear DNA content of 1.40 pg/2C. The values estimated for the somatic embryos and mother plant did not differ statistically from each other (P0.05), but both differed from those of the zygotic embryos (P0.05). These results clearly indicate that no changes were induced during the embryogenic process. However, the differences found between the field plants and zygotic embryos did suggest that some aspects must be evaluated carefully, as propidium iodide fluorescence may potentially be influenced by the presence of secondary compounds (e.g. anthocyanins, tannins) in E. globulus somatic embryos and mature leaves. Therefore we believe that the somatic embryogenesis methodology used did not induce major genetic changes in the somatic embryos and that our primary goal of true-to-type propagation was assured.     

In vitro somatic embryogenesis and plant regeneration in Acacia arabica

In vitro somatic embryogenesis and subsequent plant regeneration was achieved in callus cultures derived from immature zygotic embryos of Acacia arabica on semi-solid Murashige and Skoog (MS) basal salts and vitamins supplemented with 8.88 MBA, 6.78 M2,4-D and 30 g l^–1 (w/v) sucrose. Somatic embryos proliferated rapidly by secondary somatic embryogenesis after transfer to MS medium supplemented with 6.66 M BA, 6.78 M 2,4-D. The maximum number of somatic embryos per callus was 72.6 after 8 weeks of culture on medium containing 6.66 M BA and 6.78 M 2,4-D. The isolated somatic embryos germinated on half-strength basal MS salts and vitamins supplemented with 0.04 M BA, 0.94 M ABA and 2% (w/v) sucrose. The embryo-derived plantlets were acclimatized in the greenhouse and subsequently showed normal growth.     

Steroid synthesizing cellular sites in the ovary of the domestic pigeon Columba livia (Gmelin): a histochemical study.

Synopsis The ovary of the domestic pigeon,Columba livia, has been assayed histochemically for the localization of ⁵-3-hydroxysteroid dehydrogenase (⁵-3-HSDH), 17-hydroxysteroid dehydrogenase (17-HSDH), 11-hydroxysteroid dehydrogenase (11-HSDH), glucose-6-phosphate dehydrogenase (G6P-DH) and NADH-diaphorase activities during different periods of the reproductive cycle. ⁵-3-HSDH, 17-HSDH, 11-HSDH, G6P-DH and NADH-diaphorase activity was found in the theca interna of growing, atretic and postovulatory follicles, the granulosa of ovulatory, atretic and postovulatory follicles, and interstitial gland cells during the pre-incubation and the laying periods. During the incubation and squab feeding periods only ⁵-3-HSDH, G6P-DH and NADH-diaphorase activities were observed in the above mentioned cells. The steroidogenic potential of atretic follicles depends upon the type of atresia a follicle undergoes.     

Expression of the alpha subunit of 7S nerve growth factor in the mouse submandibular gland

-NGF is an inactive serine protease that is associated in the mouse submandibular gland with a closely related serine protease, -NGF, and the neurotrophic factor, -NGF. The heterogeneity of purified -NGF has been examined by DEAE-cellulose chromatography and SDS polyacrylamide gel electrophoresis. A possible explanation for the observed heterogeneity is presented. Antibodies have been prepared against -NGF and purified by affinity chromatography so that they do not cross-react with -NGF. This antibody preparation recognizes two very similar proteins in male mouse submandibular gland RNA-directed cell-free translation mixtures. The expression of only one of these forms is regulated by testosterone. Oligonucleotide probes specific for each of the three NGF subunits have been prepared and used for Northern blot analysis of RNA from the mouse submandibular gland. The three subunits were found to be coordinately expressed and each were 30-fold more abundant in male than in female glands.Abbreviations used NGF nerve growth factor - -, -, and -NGF -, -, and -subunits of mouse 7S NGF - PBS phosphate buffered saline - DTT dithiothreitol - PPO 2,5-Diphenyloxazole - DMSO dimethylsulfoxide - HEPES N-2-Hydroxyethylpiperazine-N-2-ethanesulfonic acid - SSC 0.15M NaCl, 15 mM sodium citrate Supported by USPHS research grant NS19964. This paper is respectfully dedicated to Profs. Eric M. Shooter and Silvio Varon in recognition of their many contributions to our understanding of the structure and function of nerve growth factor.