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

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

Regeneration of Lasiurus scindicus from Tissue Culture

Embryogenic callus cultures were initiated from mature embryosof Lasiurus scindicus on Murashige and Skoog's medium supplementedwith 6 mg l^–1 2,4-Dichlorophenoxyacetic acid (2,4-D).These cultures were maintained on 2 mg l^–1 2,4-D. Plantletswere regenerated via somatic embryogenesis when the calli weretransferred onto hormone-free MS basal medium. Young plantswere successfully transplanted to pots and grown to maturityin a greenhouse. Grass, Lasiurus scindicus, Thar Desert, drought tolerant, somatic embryogenesis, plant regeneration     

Plant regeneration <Emphasis Type="Italic">via</Emphasis> somatic embryogenesis in <Emphasis Type="Italic">Pinus kesiya</Emphasis> (Rolye ex. Gord.) influenced by triacontanol

Embryogenic cultures were initiated and established for the first time in 3 different genotypes of Pinus kesiya using mature zygotic embryos and triacontanol. Mature zygotic embryos produced white-mucilaginous embryogenic callus when cultured on half strength MSG (Becwar et al. 1990) basal medium supplemented with 90 mM maltose, 2.0 g l⁻¹ Gellan gum, 9.0 M 2, 4-D and 10 g l⁻¹triacontanol. On subculture of such embryogenic callus on the maintenance medium (II) containing 2.0 M 2,4-D and 2.0 g l⁻¹ triacontanol induced cleavage polyembryogenesis with proembryos. The percentage of somatic embryogenesis was not similar in all the three genotypes. The highest percentage of somatic embryogenesis (88.5 %) was recorded in PK04 genotype. Somatic embryos were successfully germinated on half strength MSG basal medium without growth regulators. Somatic seedlings showed fast growth and a survival rate of 95%. This work for the first time reveals that triacontanol can be used as an effective growth regulator for inducing somatic embryogenesis in conifers.     

Effect of plant growth regulators on ovary culture of coconut (<Emphasis Type="Italic">Cocos nucifera</Emphasis> L.)

Coconut is a cross pollinating palm, propagated only by seeds. Tissue culture is the only vegetative propagation method available for coconut. Consistent callogenesis was obtained by culturing unfertilised ovaries at -4 stage in CRI 72 medium containing 100 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.1% activated charcoal. Callusing was improved by application of 9 μM thidiazuron (TDZ). Embryogenic calli were subcultured onto somatic embryogenesis induction medium containing 66 μM 2,4-D. Stunted growth was observed in the somatic embryos after subculture onto CRI 72 medium containing abscisic acid (ABA). Maturation of somatic embryos could be achieved in Y₃ medium without growth regulators. Conversion of somatic embryos was induced by adding gibberellic acid (GA₃) to conversion medium containing 5 μM 6-benzyladenine (BA) while 2-isopentyl adenine (2iP) increased the frequency of plant regeneration. A total of 83 plantlets was produced from 32 cultured ovaries.     

Somatic Embryogenesis and Plant Regeneration from Inflorescence Culture of Java Citronella: (Cymbopogon winterianus)

Embryogenic callus was induced from immature inflorescence segmentsof Java citronella (Cymbopogon winterianus) and maintained for2 years on Murashige and Skoog's medium supplemented with 2,4-D(l mg l^–1). The callus cells retained the original chromosomenumber of 2n = 20. The somatic embryos germinated into plantletson MS basal medium or medium with IAA, NAA, BAP or KN individually(l mg l^–1). The regenerated plantlets developed a goodroot system on full strength solid MS inorganics medium withIAA (1 mg l^–1). The regenerated plants were similar tothe donor plant in morphology and had the same chromosome number,but showed some variation in the essential oil content. Java citronella, Cymbopogon winterianus, somatic embryogenesis, regeneration, inflorescence culture     

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

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

Expression of gus in somatic embryo cultures of black spruce after microprojectile bombardment

Immature embryos (stage I) and cotyledonary somatic embryos(stage III) of black spruce [Picea mariana (Mill) B.S.P.] werebombarded with tungsten particles coated with a gene constructcontaining the fusion of gus:: nptll. GUS (ß-glucuronidase)activity was monitored histochemically with X-gluc giving ablue colour where transient gene expression was detected inthe bombarded tissues. A high transient expression of gus wasobserved in stage I embryo cultures 2 d after bombardment (202GUS foci per 300 mg tissue). GUS activity had substantiallydiminished in this material 14 d after bombardment, when grownin liquid LP maintenance medium containing BA (4.4µM),2,4-D (9µM) and 1% sucrose. However, when stage I embryoswere cultured on LP maturation medium containing BA (40 µM),IBA (1 µM), 3.4% sucrose and 0.8% agar, GUS activity after2 d was 335 GUS foci per 300 mg tissue, and the activity wasdetected until 30 d after bombardment. With stage III somaticembryos cultured on LP maintenance medium, 92% showed GUS activity2d after bombardment (16 GUS foci per embryo), and 31 % showedactivity 30 d after bombardment (4 GUS foci per embryo). GUSactivity was still evident in 12% of the embryos (2 GUS fociper embryo) 45 d after bombardment. Key words: Black spruce, gus = E. coli geneuid A encoding ß-glucuronidase, nptll = gene encoding neomycin phos-photransferase, somatic embryos     

Genotype, explant and position effects on organogenesis and somatic embryogenesis in eggplant (Solanum melongena L.)

The relative importance of genotype and explant, and their interactionsfor in vitro plant regeneration via both organogenesis and somaticembryogenesis in Solanum melongena (eggplant) has been studied.Hypocotyl, cotyledon and leaf explants of four commerciallygrown Indian cultivars, Pusa Purple Long, Long White Cluster,Pusa Kranti, and Pusa Purple Cluster were used in the study.A combination of benzyladen-ine (11.1 µM) and indoleaceticacid (2.9 µM) was found to be optimum for shoot regeneration.Naphthalene acetic acid induced embryogenesis in all the threeexplants; 32.2µM was optimum for hypocotyl explants while10.7µM yielded maximum number of somatic embryos fromcotyledon and leaf explants. Genotype, explant and genotype-explantinteraction had highly significant effects on both organogenesisand somatic embryogenesis with genotype exerting maximum effecton both these processes. Pusa Purple Long was found to be themost responsive genotype for regeneration of both adventitiousshoots and somatic embryos among the cultivars. Among the explants,hypocotyls yielded the maximum number of adventitious shootsfollowed by cotyledons and leaves. The embryogenic responseof leaves and cotyledons was, however, significantly higherthan that of hypocotyl explants. Significant differences formorphogenetic potential were also observed within a single explant(hypocotyl). There was a basipetal gradient for organogenesis(i.e. decrease in number of shoots from base to apex) whilethe terminal hypocotyl segments showed better embryogenic potentialthan the median segments. Key words: Solarium melongena, organogenesis, somatic embryogenesis, genotype, explant, position effect     

Somatic embryogenesis and plant regeneration from suspension cultures of Acanthopanax koreanum Nakai

High-frequency somatic embryogenesis was achieved from an embryogenic cell suspension culture of Acanthopanax koreanum Nakai. Stem segments were cultured on Murashige and Skoog (MS) medium containing auxins and cytokinins. Opaque and friable embryogenic callus formed on MS medium with 4.5 μm 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.0 μm kinetin or zeatin, but was highest on medium containing 4.5 μm 2,4-D alone. Embryogenic calli were transferred to MS liquid medium containing 4.5 μm 2,4-D and maintained by subculture at 2-week intervals. Initiation of somatic embryogenesis and development up to the globular stage from embryogenic cell clumps occurred in medium containing 0.45 μm 2,4-D, whereas maturation and germination of somatic embryos occurred in MS medium lacking 2,4-D. Cytokinin treatment suppressed the normal growth of embryos, but stimulated secondary somatic embryogenesis from the surfaces of primary embryos. Plants from somatic embryos were acclimatized in a greenhouse. Received: 14 January 1997 / Revision received: 17 June 1997 / Accepted: 5 July 1997     

Unfertilized ovary: a novel explant for coconut (<Emphasis Type="Italic">Cocos nucifera</Emphasis> L.) somatic embryogenesis

Unfertilized ovaries isolated from immature female flowers of coconut (Cocos nucifera L.) were tested as a source of explants for callogenesis and somatic embryogenesis. The correct developmental stage of ovary explants and suitable in vitro culture conditions for consistent callus production were identified. The concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) and activated charcoal was found to be critical for callogenesis. When cultured in a medium containing 100 μM 2,4-D and 0.1% activated charcoal, ovary explants gave rise to 41% callusing. Embryogenic calli were sub-cultured into somatic embryogenesis induction medium containing 5 μM abscisic acid, followed by plant regeneration medium (with 5 μM 6-benzylaminopurine). Many of the somatic embryos formed were complete with shoot and root poles and upon germination they gave rise to normal shoots. However, some abnormal developments were also observed. Flow cytometric analysis revealed that all the calli tested were diploid. Through histological studies, it was possible to study the sequence of the events that take place during somatic embryogenesis including orientation, polarization and elongation of the embryos.     

Direct Somatic Embryogenesis in Roots of Cichorium: Is Callose an Early Marker?

Direct somatic embryogenesis can be obtained from epidermaland cortical cells in roots from in vitro Cichorium plantlets.The first embryogenic cells are seen after six days of culturein darkness, at 35 °C, in a liquid medium supplemented withNAA (1 x 10^–7 M), 6-dimethylallyl-amino-purine (2·5x 10^–6 M), sucrose (0.03 M) and glutamine (1·7x 10^–3 M). Embryogenic cells undergo first a linear andthen a globular segmentation, with increasing cytoplasmic density.These cells and young embryoids show aniline blue fluorescence.SEM allows the same microglobular pattern to be seen on thesurface of young embryoids and on young microspores of Cichoriumused as controls. In this root system, callose deposition seemsto be an early marker in somatic embryogenesis. Somatic embryogenesis, callose, Cichorium     

Somatic Embryogenesis and Plant Regeneration from Freely-suspended Cells and Cell Groups of Panicum maximum Jacq

Embryogenic calluses derived from cultured immature embryosand young inflorescences of Panicum maximum Jacq. were placedin Murashige and Skoog's liquid medium supplemented with 1 mg1^–1 2, 4- dichlorophenoxyacetic acid (2, 4-D) and 2.5per cent coconut water, to initiate suspension cultures. Suspensionsconsisted of two types of cells: small, richly-cytoplasmic andoften starch-containing embryogenic cells, and large, vacuolatednon-embryogenic cells. A presumed sequence of developmentalstages from single embryogenic cells to globular and heart-shapedstages of embyrogenesis was observed in the suspension cultures.Plantlets were produced from the embryoids when the suspensionswere plated in an agar medium without any hormone or with only0.2 mg 1^–12, 4-D or naphthalene acetic acid. Embryogenicsuspension cultures derived from immature embryos as well asfrom inflorescence segments gave rise to plants which showedthe normal somatic chromosome number of 2n = 4x = 32. Panicum maximum Jacq., Guinea grass, embryogenesis, regeneration, suspension culture     

Plant regeneration of common ash (<Emphasis Type="Italic">Fraxinus excelsior</Emphasis> L.) by somatic embryogenesis

This is the first report on somatic embryogenesis in common ash (Fraxinus excelsior L.). Experiments on somatic embryogenesis induction were carried out on zygotic embryos at different phases of development and maturation. The embryo axes were isolated and cultured on media containing different plant growth regulators (PGRs). Embryogenic tissues were obtained from embryos collected at an incomplete maturation phase and cultured on a modified Murashige and Skoog medium containing 8.8 μM 2,4-dichlorophenoxyacetic acid and 4.4 μM benzyl-adenine (BA). Embryos isolated from seeds at an advanced stage of maturation showed only organogenetic phenomena. Embryogenic tissues were successfully subcultured and multiplied on medium containing a reduced concentration of PGRs. After their isolation, somatic embryos were induced to develop and mature by transfer to PGR-free medium and subsequent culture on medium containing 0.1 μM BA. Somatic embryos developed completely and also germinated spontaneously. Embryo germination and conversion were significantly improved when subjected to a period of storage at 4°C and transplant onto woody plant medium. Plantlets were successfully transferred to soil and acclimatized in a “misted” greenhouse.     

Plant regeneration through somatic embryogenesis in medicinally important <Emphasis Type="Italic">Centella asiatica</Emphasis> L.

Summary High-frequency somatic embryogenesis and plant regeneration was achieved on callus derived from leaf (petiole and lamina) and internode explants of Centella asiatica L. Growth regulators significantly influenced the frequency of somatic embryogenesis and plant regeneration. Calluses developed on Murashige and Skoog (MS) medium fortified with 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D) or 5.37 μM α-naphthaleneacetic acid (NAA), both with 2.32 μM kinetin (Kn), were superior for somatic embryogenesis. Callus developed on NAA and Kn-supplemented medium favored induction and maturation of embryos earlier compared to that on 2,4-D and Kn. Embryogenic callus transferred from NAA and Kn-supplemented medium to suspension cultures of half-strength MS medium with NAA (2.69 μM) and Kn (1.16 μM) developed a mean of 204.3 somatic embryos per 100 mg of callus. Embryogenic callus transferred from 2,4-D and Kn subsequently to suspension cultures of half-strength MS medium with 2,4-D (0.45 μM) and Kn (1.16 μM) developed a mean of 303.1 embryos per 100 mg of callus. Eighty-eight percent of the embryos underwent maturation and conversion to plantlets upon transfer to half-strength MS semisolid medium having 0.054 μM NAA with either 0.044 μM BA or 0.046 μM Kn. Embryo-derived plantlets established in field conditions displayed morphological characters identical to those of the parent plant.     

Somatic embryogenesis in cell suspension cultures of olive <Emphasis Type="Italic">Olea europaea</Emphasis> (L.) ‘Chetoui’

Somatic embryogenesis of olive Olea europaea (L.) ‘Chetoui’ was studied using cell suspension cultures initiated from mature leaf-derived calli. Calli were developed on half-strength MS medium supplemented with 10 μM NAA and 2.25 μM 2i-P in the dark. Different combinations of three plant growth regulators (2,4-D, NAA and zeatin) were tested to determine cell proliferation and somatic embryogenesis induction and differentiation. Embryogenic suspension cultures were established in olive-modified medium for embryogenesis (OMe) containing 2.5 μM 2,4-D and 2.5 μM zeatin. Pre-globular and globular embryos were induced from mature olive tissue in liquid medium. In addition, the nitrogen form as inorganic (reduced; (NH₄)₂SO₄ or oxidized; KNO₃) and organic (CH) was used separately or in combination to improve the cell growth and proliferation. The most effective growth rate and cell proliferation were obtained with the medium containing inorganic and organic nitrogen forms.     

Ontogenesis, Differentiation and Precocious Germination in Anther-derived Somatic Embryos of Grapevine (Vitis vinifera L.): Proembryogenesis

Histological steps of callogenesis and proembryogenesis in anthercultures ofVitis vinifera L. ‘Grenache noir’ aredescribed. Embryogenic calli were obtained on Nitsch and Nitschmedium supplemented with 1mgl^-12,4-dichlorophenoxyacetic acid(2,4-D) and 0.25mgl^-1benzylaminopurine (BAP). Calli were initiatedfrom anther connective cells only and no division of microsporesoccurred. The embryos were hence of somatic origin. Proembryosdeveloped either directly (i.e. without intervening callus)from the endothecium, or indirectly from the connective-derivedcallus. In both cases, proembryos originated from single cells.They developed from starchy differentiated cells of a predeterminedtype. The polarity of the somatic proembryo was establishedfrom the first divisions and it was marked by precocious developmentof an easily recognizable suspensor. Other analogies with thedevelopment of the zygote are also emphasised. Vitis vinifera L.; grapevine; somatic embryogenesis; proembryogenesis; histology     

Comparison of in vitro regeneration pathways in <Emphasis Type="Italic">Punica granatum</Emphasis> L.

When cotyledonary explants, excised from in vitro germinated seedlings, of pomegranate (Punica granatum L.) were incubated on solid Murashige and Skoog (1962) medium supplemented with 21 μM naptheleneacetic acid (NAA) and 9 μM 6-benzyladenine (BA), 80% of explants developed callus. A high frequency of shoot organogensis was obtained when explants were incubated on MS medium supplemented with 8 μM BA, 6 μM NAA, and 6 μM giberrellic acid (GA₃). However, adding 24 μM silver nitrate (AgNO₃) to this medium markedly enhanced shoot regeneration frequency (63%) and mean number of shoots per explant (11.26) and length of shoots (2.22 cm). Highest frequency of in vitro rooting, mean number of roots/shoot (4.32), and mean root length (2.71 cm) were obtained when regenerated shoots were transferred to half-strength MS medium supplemented with 0.02% activated charcoal. Well-rooted plantlets were acclimatized, and then transferred to soil medium. Moreover, when zygotic embryos of P. granatum, excised from seeds collected at 16 weeks following full bloom, were incubated on MS medium containing 30 g l⁻¹ sucrose, 15% coconut water, 21 μM NAA, and 9 μM BA, they developed the highest frequency of embryogenic callus, clumps with globular embryos, and mean number of both globular and heart-shaped embryos per callus clump. Subjecting zygotic embryo explants to six-week dark incubation period was essential for embryogenic callus induction, and these were subsequently transferred to 16 h photoperiod for further growth and development of somatic embryos. Germination of somatic embryos was observed when these were transferred to MS medium was supplemented with 60 g l⁻¹ sucrose.     

Somatic embryogenesis and two embryo specific proteins (38 and 33 kD) in <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

In the present study, the regeneration pathway, especially the different events of somatic embryogenesis (SE) have been studied morphologically and biochemically in Catharanthus roseus. Firstly, the calluses were induced from different explant sources (hypocotyl, epicotyl and root) by using various auxins. Embryogenic and non-embryogenic calluses were identified based on their morphology, colour and dry weight. Embryogenic callus was later cultivated on MS added with 0.45 μM 2,4-D, 6.62 μM BAP and 1.44 μM GA3 for obtaining various developmental stages of embryos. Different stages of embryos have been assayed for the establishment of marker based embryogenesis, particularly on embryo specific proteins whose presence or absence will ensure a rapid and efficient production of embryos that has a special application to clonal biotechnology. Two embryo specific proteins (38 and 33 kD) have been identified for the first time in C. roseus during torpedo stage of embryogenesis. Besides, multiple shoot formation from in vitro raised emblings was also attempted to examine the role of BAP and kinetin for shoot proliferation. The shoots were rooted with 5.37 μM NAA and 5.71 μM IAA before transplantation.     

Embryos and Plantlets from Cultured Anthers of Hybrid Grapevines

Embryos and plantlets were produced in large numbers from callusformed by cultured anthers of hybrid grapevines (Vitis viniferax Vitis rupestris). Anthers of Vitis vinifera produced smallamounts of callus or failed to grow in vitro. For embryo formationanthers containing uninucleate microspores were chilled (4 °C)for 72 h before culture with Nitsch medium containing 2, 4-D(5µM) and benzyladenine (1 µM). Highest yields ofembryos were with anthers cultured in darkness. For productionof normal plantlets embryos required chilling (4 °C) for2 weeks. Unchilled embryos produced mainly abnormal plantlets.Chilling was effective in promoting plantlet growth when appliedat any stage of embryogeny. In grapes ability to produce plantlets from cultured anthersis a genetically-determined trait and maleness, as distinctfrom hermaphroditism, may be a predisposing factor. Callus derivedfrom anthers contained both haploid and diploid cells but allplantlets produced so far are diploid. The genetic constitutionof plantlets, whether they are diploids of somatic origin ordiploids from spontaneously doubled haploid cells, is not yetknown and is being determined by standard genetic methods.     

Ploidy stability of somatic embryogenesis-derived <Emphasis Type="Italic">Passiflora cincinnata</Emphasis> Mast. plants as assessed by flow cytometry

In this study, flow cytometric analysis was used to evaluate the genetic stability of Passiflora cincinnata Mast. plants regenerated via primary and secondary somatic embryogenesis. Embryogenic calli obtained from culturing zygotic embryos on Murashige and Skoog (MS) medium containing 18.1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4 μM benzyladenine (BA) were transferred to differentiation medium. Torpedo and cotyledonary embryos were obtained. These primary embryos were maintained on differentiation medium to generate secondary embryos. Conversion of primary and secondary embryos yielded 305 and 138 normal plants, respectively. Almost 90% of plantlets survived following acclimatization. Flow cytometric analysis revealed that seed-derived plants had on average 3.01 pg nuclear DNA (2C), and all plants, except for a single plant regenerated via primary embryogenesis, maintained their ploidy. This single plant contained more than twice the average DNA content: 6.21 pg (4C). Epidermal stomata of leaves of the tetraploid plant were larger but lower in density than those of diploid plants, indicating that stomatal characteristics are useful in distinguishing between diploid and tetraploid plants of passion fruit. In summary, the procedure we employed to regenerated P. cincinnata plants via somatic embryogenesis generated mostly genetically true-to-type plants.