Characterization of embryogenic cell lines of Picea abies in relation to their competence for maturation

Summary Embryogenic cell lines of Picea abies are categorized into three groups (polar, solar, and undeveloped) based on the organization of the somatic embryos within the tissue and the ability of the somatic embryos to proceed through a maturation process when treated with ABA. The polar and the solar types consist of somatic embryos with densely packed embryonic regions subtended by vacuolated suspensors. Both types of tissue regenerate mature somatic embryos when treated with ABA. Almost all mature somatic embryos develop further into shoots or plantlets. The undeveloped type consists of somatic embryos comprised of only a few loosely aggregated cells in their embryonic regions. Mature somatic embryos were not observed with this tissue type.Abbreviations ABA cis-trans abscisic acid - A1 polar type - A2 solar type - B undeveloped type - BA benzyladenine - 2,4-D 2,4 di-chlorophenoxyacetic acid - LP von Arnolds medium (1987)     

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.     

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     

Morphogenic and genetic stability in longterm embryogenic cultures and somatic embryos of Norway spruce (Picea abies {L.} Karst)

Embryogenic cultures were initiated from mature zygotic embryos of Picea abies. The somatic embryos in the embryogenic cultures were first stimulated to mature and then either to develop further into plantlets or to differentiate new embryogenic cultures. The procedure was repeated three times during two years. The ability to give rise to new embryogenic cultures or to develop into plantlets was similar for all somatic embryos irrespective of how long they had been cultured in vitro. The nuclear DNA content, measured in a flow cytometer, was estimated at 32 pg/G1 nuclei in seedings developed from zygotic embryos. Nuclei isolated from embryogenic cultures and from plantlets regenerated from somatic embryos had the same DNA content as those isolated from seedlings.Abbreviations N⁶-benzyladenine BA - 2,4-dichlorophenoxyacetic acid 2,4-D - abscisic acid ABA     

Secretion of Specific Extracellular Proteins by Somatic Embryos of Picea abies is Dependent on Embryo Morphology

Embryogenic cell lines ofPicea abieswere categorized into twogroups, A and B, based on the morphology of the somatic embryosand the ability of the somatic embryos to proceed through amaturation process when treated with ABA. Group A embryos hada distinct, densely-packed embryonic region whereas group Bembryos had loosely packed cells in their embryonic region.Embryo morphology was shown to be regulated by changes in theplant growth regulators in the culture medium. Treatment withN⁶-benzyladenine stimulated embryos to develop large embryonicregions. The morphology of somatic embryos and especially thatof the embryonic regions was correlated with the presence ofspecific extracellular proteins. Only somatic embryos with denselypacked cells in the embryonic regions secreted proteins withrelative molecular weights of 28, 66 and 85kD. The extracellularprotein of 28kD was isolated and the first 21 amino acids inthe N-terminus were identified. These showed 52–57% identitywith the N-terminal sequence conserved among members of a proteinfamily which includes zeamatin and which have been shown tobe involved in plant anti-fungal mechanisms. Immunological studiesof extracellular chitinases and zeamatin-like proteins, as wellas of activity of extracellular peroxidase, revealed a closecorrelation between the presence of specific chitinases andembryo morphology. Auxin; cytokinin; embryogenic cell lines; embryo morphology; extracellular proteins; Norway spruce; Picea abies; somatic embryos     

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.     

新疆天山雪莲体胚诱导与分化研究

以新疆天山雪莲的叶片为外植体,分别用不同配方培养基诱导愈伤组织,后进行体胚诱导和分化培养形成再生雪莲植株.结果表明,诱导愈伤组织的最适培养基为MS 2,4-D 0.5 mg/L BA 1.5 mg/L,诱导率可达到100%;愈伤组织转移至MS 2,4-D 0.5 mg/L BA 1.5 mg/L培养基进行继代培养,增殖后的愈伤组织转移到MS 2,4-D 0.2 mg/L的液体培养基后成功诱导出雪莲体胚,出胚率达40%;将体胚接至MS ABA 0.5 mg/L培养基后,结果分化生长出大量的再生雪莲幼苗.     

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     

Development of white spruce (Picea glauca (Moench.) Voss) somatic embryos during culture with abscisic acid and osmoticum, and their tolerance to drying and frozen storage

The limit of permeability of white spruce (Picea glauca [Moench.]Voss) somatic embryo cell walls to molecules was in the orderof 30 . Polyethylene glycols (PEGs) and dextrans of molecularweights greater than 1000 and 6000, respectively, produced anonpermeating (non-plasmolysing) water stress which improvedembryo development. Somatic embryos converted to plantlets atfrequencies of 76–84% following slow drying and storageat –20 C for 1 year, which was similar to the 77% recordedfor control somatic embryos slowly dried then germinated withoutfreezing or storage. Culture for 7–8 weeks with mediumcontaining abscisic acid, 3% sucrose, and 7.5% PEG 4000 yieldedsomatic embryos with five times the embryo storage lipid contentrecorded for zygotic embryos. During culture with PEG the moisturecontent of the somatic embryos decreased from 96% for immaturesuspension-cultured somatic embryos, to 47% for mature embryos.Somatic embryos cultured for 7–8 weeks survived rapiddrying to 5% moisture content, and converted to plantlets atfrequencies of 60–70%, but no somatic embryos survivedrapid drying when cultured for only 4 weeks; however, slow dryingdid induce desiccation tolerance in 3-week cultured somaticembryos. Abscisic acid was important to maintain embryos ina developmental state, but ABA alone did not induce desiccationtolerance. In order to induce desiccation tolerance a waterstress treatment was required. Tolerance of rapid drying coincidedwith moisture contents below 55%, which occurred after 5 weeksof culture in the presence of PEG 4000 and abscisic acid. Key words: Dextran, molecular weight, polyethylene glycol, triacylglycerol, water stress     

Somatic embryogenesis and plant regeneration from embryogenic callus of soybean,Glycine max L.

During induction of somatic embryogenesis from immature embryos of soybean, a smooth-shiny and a rough callus were obtained. The smooth-shiny type developed from callus derived from cotyledonary tissue and cultured on media containing 10 mg/l 2,4-D. The rough type was derived from immature embryos, cotyledons, hypocotyls and hypocotyl segments from germinated seedlings on a medium containing various growth regulators. Plants were obtained from the smooth-shiny type but rough callus did not differentiate into plants. The conditions for formation of both callus types and regeneration of mature soybean plants from the smooth-shiny type were defined.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - IBA indolebutyric acid - ABA abscicic acid - GA3 gibberellic acid - BA benzyladenine - B5 Gamborg et al. - LS Linsmaier & Skoog - MS Murashige & Skoog - P proline     

Regeneration of Cladrastis lutea (Fabaceae) via somatic embryogenesis

Summary Immature embryos from 5 Cladrastis lutea (Michx.) K. Koch (yellowwood) trees were initially cultured on modified Schenk and Hildebrandt medium (SH) containing either 4.5, 9.0, 13.5 or 23 M 2,4-D. One-third of the explants were transferred to SH medium supplemented with 25.0 M NAA after 2 and 3 weeks respectively. The remaining explants were incubated on the initial 2,4-D containing media for 6 weeks. Groups of somatic embryos formed directly only at the proximal end of cotyledons; only a few formed as single embryos. The greatest numbers were formed from zygotic embryos explanted from 6–8 weeks post-anthesis and initially cultured on medium containing 9 or 13 M 2,4-D. However, all treatments supported somatic embryogenesis. In the second year, explants were initially cultured on SH medium containing either 9.0, 13.5, or 23 M 2,4-D and then transferred to SH medium containing 4.0 M ABA after 2 or 3 weeks. ABA did not affect the development of somatic embryos. Six of 21 somatic embryos germinated on half-strength SH medium without growth regulators. Three entire plantlets were formed, but only one was established in soil.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - ABA abscisic acid - CRAF III chromium trioxide-acetic acidformalin     

Somatic Embryogenesis and Plant Regeneration from Cultured Leaf Explants of Zea mays

Young leaf segments of Zea mays L. seedlings were cultured onMurashige and Skoog's basal nutrient medium supplemented with2 mg l^–1 2, 4-D and sub-cultured on medium containing8 mg l^–1 2,4-D. Two types of callus tissues appeared—embryogenicand non-embryogenic. The embryogenic callus tissue producednumerous somatic embryos which on transfer to media containinglow amounts of 2,4-D or ABA produced plantlets. Callus tissuesexhibited embryogenic potential for more than 1 year. Zea mays L. cv. Ageti-76, Zea mays L. cv. N-L-D-Comp., maize, leaf, callus, somatic embryogenesis, regeneration     

Effects of various partial pressures of oxygen and carbon dioxide on different stages of somatic embryogenesis in Picea abies

Effects of various partial pressures of oxygen (5, 20 and 45 kPa) and carbon dioxide (0.03 and 6 kPa) on initiation, proliferation and maturation of somatic embryos in Picea abies were studied. The pO₂ had a significant effect on the initiation of embryogenic tissue from mature zygotic embryos. However, the effect of pO₂ was dependent on the strength of the basal medium. Low pO₂ stimulated the formation of embryogenic tissue when the zygotic embryos were incubated on full strength medium, but was inhibitory when half-strength medium was used. Proliferation of embryogenic tissue was stimulated by higher partial pressures of both CO₂ and O₂. The effect of the gas phase on maturation of somatic embryos varied between different cell lines. However, there was a general tendency for 5 kPa O₂ and 6 kPa CO₂ to stimulate maturation.Abbreviations ABA abscisic acid - BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - ET embryogenic tissue     

Comparison of NAA and 2,4-D induced somatic embryogenesis in Cassava

NAA and 2,4-D were compared for their ability to induce somatic embryogenesis in cassava (Manihot esculenta Crantz). In all seven cultivars tested, only 2,4-D had the capacity to induce primary somatic embryos from leaf explants, however, both NAA and 2,4-D were capable of inducing secondary somatic embryos. More secondary somatic embryos were formed in NAA than in 2,4-D medium. Furthermore, the maturation period for secondary somatic embryos was shorter in NAA medium than in 2,4-D medium. In some cultivars, repeated subculture of secondary somatic embryos in NAA medium resulted in a gradual shift from somatic embryogenesis to adventitious root formation. This shift could be stopped and reversed by subculture of the material in 2,4-D medium. In NAA medium the most secondary somatic embryos were formed when they were subcultured every 15 days whereas in 2,4-D a 20 day subculture interval was optimal. Subculture of secondary somatic embryos at a high inoculum density (>1.5 g jar⁻¹) in NAA medium did not result in the formation of secondary somatic embryos, whereas in 2,4-D it lead to the formation of globular secondary somatic embryos. With 2,4-D the newly induced secondary somatic embryos were connected vertically to the explant and with NAA medium horizontally. For all cultivars tested, desiccation stimulated normal germination of NAA-induced somatic embryos. However, the desiccated, secondary somatic embryos required a medium supplemented with BA for high frequency germination. The concentration of BA needed for high frequency germination was higher when the desiccated secondary somatic embryos were cultured in light instead of dark. In only one cultivar desiccation enhanced germination of 2,4-D induced secondary somatic embryos and in three other cultivars it stimulated only root formation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of plant growth regulators on somatic embryogenesis in leaf cultures of Coffea canephora

The effects of plant growth regulators on somatic embryogenesis were studied in leaf cultures of Coffea canephora. The maximum number of somatic embryos were obtained on media that contained only cytokinin as a plant growth regulator. All of the auxins tested (NAA, IBA, IAA and 2, 4-D) inhibited the formation of embryos. The optimal concentration of each cytokinin (2-iP, BA and kinetin) for somatic embryogenesis was 5 M. Under optimal conditions, each explant formed more than 100 embryoids with little callus and few adventitious roots. Embryoids were formed only at the cut edges of the leaf discs. Cytokinins were absorbed only at the cut edges of leaf discs that were in contact with the medium, and were not transported to other parts of the explant.Abbreviations 2-iP iso-pentenyladenine - BA benzyladenine - NAA 1-naphthaleneacetic acid - IBA indole-3-butyric acid - IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Control of Morphogenesis in Sorghum by 2,4-Dichlorophenoxyacetic Acid and Cytokinins

2, 4-Dichlorophenoxyacetic acid caused a shortening of rootsand shoots when mature seeds of Sorghum bicolor (L.) Moenchcv. BT3197 were germinated on Murashige and Skoog (MS) mediumthat contained 2,4-D. Shoot growth was restored with cytokinins.A callus formed at the nodal region, the further differentiationof which was determined by the ratio of 2,4-D and cytokininsin the initial culture medium. A high auxin to cytokinin ratiopromoted primarily root differentiation while a high cytokininto auxin ratio promoted multiple bud development. Isolated shootapical meristem with the subtending node produced embryogeniccallus at low cytokinin levels and green buds on high cytokininlevels when cultured in the presence of 2,4-D. It is concludedthat cells potentially capable of differentiation into roots,somatic embryos or axillary buds are present in the first nodalregion. Sorghum bicolor, organogenesis, embryogenesis, 2, 4-D: cytokinin ratios, tissue culture     

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 and plant regeneration from suspension cultures of Picea glauca (White spruce)

Hakman, I. and von Arnold, S. 1988. Somatic embryogenesis and plant regeneration from suspension cultures of Picea glauca (White spruce). - Physiol. Plant. 72: 579–587.
Plantlets were regenerated from long-term embryogenic cultures of Picea glauca (Moench) Voss. (White spruce). Embryogenic calli, initiated from immature zygotic embryos and maintained by monthly subculture for 16 months, were used to establish suspension cultures. Small somatic embryos were continuously produced in liquid culture medium containing auxin and cytokinin and the cultures showed a sustained regeneration capacity for >6 months. Somatic embryos propagated in the suspension cultures developed further into embryos bearing cotyledons, about 1 month after transfer to solidified medium containing abscisic acid. Electron microscopic examination revealed that storage nutrients, lipids, proteins and carbohydrates, accumulated in the somatic embryos during this treatment with abscisic acid (ABA). Upon subculture to medium lacking plant growth regulators such embryos could develop into small green plantlets.     

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     

白Pian体细胞胚悬浮培养的动力学研究

白(ＰｉｃｅａｍｅｙｅｒｉＲｅｈｄ.ｅｔＷｉｌｓ.)是我国特有的云杉属树种,在林业生产和环境绿化中均具有重要地位。其体细胞胚胎发生的研究,一方面可用于优良种质的大规模快速繁殖,为植树造林和园林绿化提供优质苗木;另一方面可作为遗传转化的再生系统,进行树种遗传...