Vital DNA staining of agarose-embedded protoplasts and cell suspensions of Nicotiana plumbaginifolia

The DNA of agarose-embedded protoplasts of Nicotiana plumbaginifolia was stained with Hoechst 33342 by immersing microscope slides, coated with immobilized protoplasts, into Erlenmeyer flasks containing consecutively dye solution, pH-correcting washing solutions and culture medium. After staining, protoplasts regenerated cell walls, started to divide and proliferated to calli. The culture system with immobilized protoplasts permits rapid change of culture media and accurate control of experimental conditions. The staining technique offers the opportunity for continuous observation of chromosomal behaviour and cell dynamics in individual plant cells.The same staining procedure was successfully applied to DNA of plant cells in suspension. Flow cytometric analysis revealed a retarding effect of the dye on the cell cycle, but within hours the cells recovered and showed their normal growth characteristics as compared to the controls.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxy acetic acid - DAPI 4'6-diamidino-2-phenylindole - FDA fluorescein diacetate - LMT low melting temperature - MES 2(N-morpholino)ethanesulfonic acid - MS Murashige and Skoog-medium - NAA -naphthaleneacetic acid - PCV packed cell volume - Tris Tris(hydroxymethyl)amino methane     

The presence of concanavalin A and canatoxin in Canavalia ensiformis DC tissue culture

Isolated embryos, cotyledons and embryos plusa fragment of cotyledon from seeds of Canavalia ensiformis (jack bean) were cultured in vitro. Concanavalin A and canatoxin cross-reactive material were detected by double immunodiffusion tests. Canatoxin was detectable until 30 days in cultures of embryos, embryos plus cotyledons and hypocotyls. Concanavalin A was also present in all cultures being detected until 90 days in cultures treated with 6-benzylaminopurine. No concanavalin A was detected in root cultures. Concanavalin A was present in cell suspensions until 45 days of culture; the culture medium contained neither concanavalin A nor canatoxin. Tissue cultures thus can produce Con A and CNTX and will be an important research tool for studying the biosynthesis of such substances.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzylaminopurine - CNTX canatoxin - Con A concanavalin A - CRM cross-reactive material - DEAE-cellulose diethylaminoethyl-cellulose - IgG immunoglobulin G - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - Tris tris (hydroxymethyl) aminomethane     

Hyoscyamus muticus + Nicotiana tabacum fusion hybrids selected via auxotroph complementation

Protoplasts of the nicotinamide-deficient Hyoscyamus muticus cell line nic^– IVH2 and of the nitrate reductase-deficient Nicotiana tabacum cell line NR^– cnx 68 were induced to fuse. Selection for putative interspecific hybrid clones was via auxotroph complementation. Controls included tests for cross-feeding, cross inhibition, PEG-induced variation, culture-induced variation, reversion, viability, delayed selection. Protrotrophic cell lines were recovered exclusively from PEG-treated mixtures of both protoplast types. The putative hybrid clones grew independent of exogenously supplied auxins and cytokinins, and at a faster rate than either parent. The morphogenic potential of different clones varied from non-morphogenic to regeneration of fertile plants. Indirect evidence for the hybrid nature of the clones is provided from a) tight selection, b) hormone-independent growth, c) hybrid vigour, d) extreme morphological variation, e) isoenzyme bands from both parents, f) morphogenic potential. Definite proof for the hybrid nature was, however, provided from species-specific DNA hybridization. Chimerism could be excluded since only the large subunit of Hyoscyamus muticus ribulose bisphosphate carboxylase was found and since species-specific DNA hybridization identified clones which gave no Nicotiana tabacum signal.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - NAA Naphthaleneacetic acid - BAP 6-Benzylaminopurine - PEG Polyethyleneglykol - MES (2[N-morpholino]ethane sulfonic acid - Tris-HCl Tris(hydroxymethyl)-aminoethane-HCl % is given throughout in w/v - FMI Friedrich Miescher-Institut     

Marker proteins for embryogenic differentiation patterns in pea callus

Polypeptide pattern alterations during somatic embryogenesis were investigated using callus cultures of two Pisum sativum genotypes. Both genotypes show the formation of two different callus lines from the same explant after six to eight weeks in culture: a nodular yellowish callus line, which forms somatic embryoids in suspension cultures (e⁺) and a white compact callus line with no regenerative capacity (e^–). The cytosol proteins of the two different callus lines were separated in a semi-preparative two-dimensional system and the polypeptide patterns were compared. Two protein bands were found (P1: M_r=45000 D, pI=7.0–7.1; P2: M_r=7000 D, pI=<4.5), which were characteristic of the putatively embryogenic (e⁺) callus line in all tissues investigated (two genotypes × two explant sources). These proteins found in nodular (e⁺) pea cultures are very similar to two proteins found in Daucus carota suspension cultures preceding the formation of somatic embryos.Abbreviations BA 6-benzyl-aminopurine - Bistris 2-(bis(2-hydroxyethyl)imino)-2-(hydroxymethyl)-1.3-propanediol - PAGE polyacrylamide gel electrophoresis - pI isoelectric point - TCA trichloroacetic acid - Tris tris(hydroxymethyl)-aminomethane     

Plant regeneration from protoplasts of Dimorphotheca and Rudbeckia

Protoplasts were isolated from leaves, shoots, cotyledons, ray florets and callus cultures of Dimorphotheca aurantiaca (syn. D. sinuata) (Cape Marigold, Star of the Veldt) and Rudbeckia hirta, R. laciniata and R. purpurea; species of ornamental value. For Dimorphotheca, plants were regenerated from protoplasts of all sources apart from the ray floret, whilst for the Rudbeckia species, although protoplast division was induced in most cases, only leaf mesophyll protoplasts of R. hirta c.v. Marmalade gave plants. The establishment of plant regeneration for these ornamental species, from protoplasts, now provides a basis for their somatic hybridisation.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - NAA naphthaleneacetic acid - K kinetin - GA₃ gibberellic acid - MS Murashige and Skoog (1962) - f.wt. fresh weight     

Alkaloid production in cell suspension cultures of Thalictrum flavum and T. dipterocarpum

Both cell suspension cultures of Thalictrum flavum and T. dipterocarpum were found to produce berberine (0.3 and 0.4 g/l, respectively) as a main alkaloid. Berberine production in the latter was markedly stimulated by 1-naphthaleneacetic acid in combination with 6-benzylaminopurine, whereas it was rather suppressed by the same auxin in the former. T. flavum cultures accumulated berberine and columbamine in the cells without releasing them into medium. On the other hand, T. dipterocarpum cultures released berberine into medium during the logarithmic growth phase, but thereafter accumulated all the berberine synthesized in the cells.Abbreviations LS Linsmaier and Skoog - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - BA 6-benzylaminopurine - TFG a culture strain of T. flavum ssp. glaucum - TDP a culture strain of T. dipterocarpum     

Intergeneric protoplast fusion between Brassica carinata and Camelina sativa

Camelina sativa is a wild crucifer that is reported to be resistant to Alternaria blight. Polyethylene glycol mediated fusion was attempted between protoplasts from etiolated hypocotyls of Brassica carinata and mesophyll protoplasts of Camelina sativa. The mean frequency of heterokaryons was 6.8%. Three hybrid shoots were regenerated, each from a single fusionderived callus. These shoots failed to produce roots capable of withstanding transplantation. Confirmation of hybridity was obtained from the morphology of in vitro produced leaves, somatic chromosome number in leaf tips, and restriction fragment length polymorphism for a nuclear rDNA probe. Analysis for organelle constitution using RFLPs indicated that the hybrid contained chrloroplasts derived from the wild species and mitochondria from the cultivated Brassica species.Abbreviations 2,4-D 2,4-dichlorophenoxy-acetic acid - IAA Indole-3-acetic acid - NAA -Naphthaleneacetic acid - IBA Indole-3-butyric acid - GA₃ gibberellic acid - BAP 6-Benzylaminopurine - MS Murashige and Skoog (1962) basal medium     

Role of inhibitors in the induction of differentiation in callus cultures of Brassica,Datura and Nicotiana

The effect of various inhibitors on differentiation (shoot morphogenesis) in callus cultures of Brassica, Datura and Nicotiana has been investigated. Hormone medium without any inhibitor (control), resulted in 6% shoot formation. Addition of inhibitors such as actinomycin D, cordycepin, abscisic acid, trigonelline and theophylline greatly enhanced shoot formation. The results suggest that inhibitors play a regulatory role in the control of differentiation.Abbreviations ABA Abscisic acid - BA Benzyl adenine - Kn Kinetin - MS Murashige Skoog's - NAA 1-naphthalene acetic acid     

Plant regeneration from protoplasts of Felicia and Brachycome

Protoplasts were isolated from leaves of axenic shoot cultures of Felicia bergeriana (Kingfisher Daisy) and Brachycome iberidifolia (Swan River Daisy) and from callus cultures of Felicia. Plants were regenerated from all three sources and since both species are of ornamental value (blue flowered) the establishment of plant regeneration provides a basis for their incorporation in somatic hybridisation programmes involving important ornamentals such as Chrysanthemum.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - NAA naphthaleneacetic acid - KIN 6-furfurylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - MS Murashige and Skoog (1962) - FDA fluorescein diacetate - f. wt. fresh weight     

Induction of somatic embryogenesis in leaf-derived callus of Vicia narbonensis L.

A method for the induction of somatic embryogenesis in callus cultures, using explants from mature leaves of Vicia narbonensis L., is described. Callus developed on a solid medium (Murashige and Skoog 1962), which was supplemented with low concentrations of picloram and benzylaminopurine. Subsequent culture was carried out in different liquid media (culture length four months). The gradual reduction of auxin and cytokinin concentrations, and the addition of glutamine and pyridoxal·HCl were favourable. Somatic embryos appeared on solid media without phytohormones.Abbreviations MS Murashige and Skoog (1962) - 2,4 D dichlorophenoxy acetic acid - KIN 6-furfurylaminopurine - BAP 6-benzylaminopurine - picloram 4-amino-3,5,6-trichloropicolinic acid - NAA 1-naphthalene acetic acid - p-CPA parachlorophenoxy acetic acid - M1 - M7 media numbers (details in materials and methods)     

Plant regeneration in callus and suspension cultures of Brassica campestris cv. Yellow Sarson

Prolific shoot bud differentiation was induced in callus and suspension cultures of hypocotyl origin in Brassica campestris cv. Yellow Sarson on MS medium supplemented with K (13.9–23.2 M) or BA (13.3–22.1 M). Plantlets were obtained by rooting the in vitro differentiated shoots. Histological studies revealed a unique mode of meristemoid formation.Abbreviations MS Murashige and Skoog (1962) - 2,4-D 2,4-dichlorophenoxyacetic acid - BA Benzyladenine - IAA Indole-3-acetic acid - IBA Indolebutyric acid - K Kinetin - NAA Naphthalene acetic acid     

The animal ether phospholipid platelet-activating factor stimulates acidification of the incubation medium by cultured soybean cells

Addition of the animal ether phospholipid platelet-activating factor, 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine, (PAF) stimulates medium acidification in cultured soybean (Glycine max L.) cells. The pH of the medium after 8–10 hours is on the average one pH unit lower than in controls. With fusicoccin an average pH difference of 1.7 units is reached. Phospholipids, glycerol, 1-oleyl-2-acetyl-sn-glycerol, 1-0-hexadecyl-sn-glycerol, and triolein at the same concentrations as PAF had no stimulatory effect on medium acidification. The detergents CHAPS and deoxycholate lead to alkalinization of the medium whereas lysophosphatidylcholine (LPC), a detergent with structural similarity to PAF, shows no effect.Abbreviations CHAPS (3-((3-cholamylopropyl) dimethylamino)-1-propanesulfonate) - DOC deoxycholic acid - FC fusicoccin - LPC lysophosphatidylcholine - OAG 1-oleyl-2-acetyl-sn-glycerol - PAF platelet-activating factor = 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine - IAA indole-3-acetic acid     

In vitro plant regeneration from leaf explants of C3 and C3-C4-intermediate Flaveria species

Summary A procedure is described for the invitro regeneration of whole plants of Flaveria cronquistii (C₃ species) F. pubescens and F. chloraefolia (both C₃-C₄ intermediate species) using different concentrations of 6-benzylaminopurine and alpha-napnthalenic acid.Abbreviations BAP 6-benzylaminopurine - NAA alpha-naphthalenic acid - MS medium Murashige-Skoog-medium     

In vitro propagation through axillary bud multiplication in different mulberry genotypes

Axillary buds from 5 genotypes of mulberry belonging to 4 species were cultured on modified MS basal medium. A total of 30 media combinations were tried for all the genotypes. The response of axillary buds and the requirement for growth regulators varied with genotype. In Morus indica BAP (0.25–0.5 mg/l), and in M. alba and M. rotondifolia GA₃ (0.5–1.0 mg/l)were found to induce sprouting. Two genotypes of M. bombycis, namely Schimanochi and Mizusawa, developed healthy shoots on the incorporation of 2,4-D (0.5–1.0 mg/l) and BAP (0.5–2.0 mg/l), respectively. IBA (0.5 mg/l), along with cytokinin/auxin/gibberellin, had no effect on bud growth but helped root induction. Shoots developed from the axillary buds were further multiplied as nodal explants. MS basal medium supplemented with 0.5 mg/l IBA and LS vitamins was found best to produce healthy plantlets in all the genotypes. An average 89% survival was observed on transferring the plantlets to soil.Abbreviations MS Murashige and Skoog (1962) - LS Linsmaier and Skoog (1965) - IBA 3-indole-butyric acid - GA₃ Gibberellic acid - BAP 6-Benzylaminopurine - Kn Kinetin - 2,4-D 2,4-Dichlorophenoxyacetic acid     

Propagation of Indian Rhubarh (Rheum emodi Wall.) using shoot-tip and leaf explant culture

Shoot-tip explants of Rheum emodi Wall. (Polygonaceae) gave rise to multiple shoots when cultured on a Murashige and Skoog (1962) medium (MS) with 2.0 mg/l 6-benzylaminopurine (BAP) and 1.0 mg/l indole-3-butyric acid (IBA). Also, shoot buds developed from leaf explants using MS medium with 2.0 mg/l BAP and 0.25 to 1.0 mg/l indole-3-acetic acid (IAA) or IBA. Roots were induced when the resulting shoots were placed on MS medium with 1.0 mg/l IBA. Both regeneration procedures gave rise to healthy plantlets that were established in soil under glasshouse conditions at 80% frequency after hardening phase of two weeks. Regenerated plants showed a constant chromosome number of 2n=2x=22, same as the parent plant. The use of liquid shake cultures minimized the time and culture medium requirements for propagation. This procedure can be applied for the conservation and utilization of elite clones of R. emodi.Abbreviations BAP 6-benzylaminopurine - Dd H₂O Double glass distilled water - IAA indole-3-acetic acid - IBA indole-3-butyric acid - K Kinetin - MS Murashige and Skoog's (1962) medium - RH Relative humidity CIMAP Publication No. 876     

Plant regeneration from protoplasts of long-term callus cultures of Actinidia deliciosa var. deliciosa cv. Hayword (Kiwifruit)

Plant regeneration of Actinidia deliciosa var. deliciosa cv. Hayword was obtained from protoplasts isolated from petiole derived long-term callus cultures. Protoplasts were cultured in liquid medium over agarose gelled medium. Regenerated green callus, plated on solid medium, could develop shoots that rooted spontaneously in hormone-less medium. The plants obtained are growing fast in soil and present a normal phenotype.Abbreviations BAP benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - DTT dithiotreitol - IAA indole-3-acetic acid - IBA indole-3-butyric acid - Kin kinetin - MES 2-(N-morpholino) ethanesulphonic acid - MS Murashige and Skoog (1962) medium - NAA naphthalene-1-acetic acid - SH Schenk and Hildebrandt (1972) medium This Research was supported by JNICT and INIC     

Synthesis of [17,18-3H]trans-13-azaprostanoic acid. A labeled probe for the PGH2/TXA2 receptor

Because of its highly unstable nature, TXA₂, produced by platelet metabolism of arachidonic acid, does not lend itself to use as a receptor probe for its own receptor. As such, the stable TXA₂/PGH₂ antagonist, trans-13-azaprostanoic acid (trans-13-APA, 12b), was prepared as the [17,18 ³H] derivative ([³H] trans-13-APA, 12c) to study this receptor and to better evaluate the mechanism of action of these azaprostanoids. Tritiated trans-13-APA, 12c, was prepared in nearly theoretical specific activity (57 Ci/mmole) from (17z)-trans-13-azaprost-17-enoic acid (11b) by catalytic tritiation. The unsaturated 11b was prepared by condensation of cis-7-amino-3-heptene (8) with 2-(6-carboxyhexyl) cyclopentanone (9), NaBH₄ reduction, chromatography, and hydrolysis of the trans isomer so isolated. The olefins 11a and b were also of biochemical interest because of the unsaturation in the lower side chain. The presence of similar unsaturation in PGH₃ (4) and TXA₃ (3) renders these prostaglandins inactive as proaggregatory agents. Evaluation of the antiaggregatory activity of 11a and b indicated it to be about the same potency in inhibiting human platelet aggregation as the parent cis and trans-13-APAs, suggesting that introduction of a double bond at the 17 position in platelet prostaglandin antagonists is unlikely to result in enhanced antiplatelet activity.     

Studies on a drought resistant legume: The moth bean,Vigna aconitifolia (Jacq) marechal. II. morphogenetic studies

Plantlets regenerated from shoot apices, cotyledons and callus cultures in Moth bean, Vigna aconitifolia (JACQ) Marechal, a drought resistant legume and pulse crop, were rooted and transferred to soil. Explants for these studies were derived from seedlings pre-conditioned by germination of seeds on B5BA and WMB (control).Abbreviations MS Murashige and Skoog (1962) - B5 B5 basal medium (Gamborg et al 1968) - B5BA B5 basal medium containing BA (2.25 mg/l) - WMB Modified White's medium (Mascarenhas et al 1976) - BA 6-benzyladenine - IAA indole-3-acetic acid - NAA 1-napthaleneaceticacid - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indolebutyric acid - 2iP N(^–2 isopentyl) adenine - CM coconut milk NCL Communication No. 3375     

Production of chrysanthemic acid and pyrethrins by tissue cultures ofChrysanthemum cinerariaefolium

Tissue cultures ofChrysanthemum cinerariaefolium were established, and then used to study the production of pyrethrin insecticides, and their precursor chrysanthemic acid. Callus cultures and root-differentiated cultures did not contain pyrethrins whereas shoot differentiated callus was found to produce the pyrethrins. Chrysanthemic acid was isolated by extraction from callus cultures, and feeding¹⁴C-labelled chrysanthemic acid to a cell suspension ofC. cinerariaefolium established that the acid accumulates largely as a glucoside ester.Abbreviations MS Murashige and Skoog (1962) medium - 2,4-D 2,4-dichlorophenoxyacetic acid - 1AA Indoleacetic acid - BAP 6-Benzylaminopurine - GC-MS Gas chromatography - mass spectrometry     

Comparative shoot regeneration responses of diploid brassicas and their synthetic amphidiploid products

Comparative shoot regeneration responses of three diploid Brassica species, B. campestris (AA), B. nigra (BB) and B. oleracea (CC) and their synthetic amphidiploid combinations have been investigated. The study indicates that A genome has an inhibitory effect on regeneration as evident from significantly low responses of B. juncea (AABB) and B. napus (AACC) combinations, in comparison with regeneration response of B. nigra and B. oleracea. This inhibition may arise from genomic interactions or from the B. campestris cytoplasm interacting negatively with the alien genome. Significant cytoplasmic influence on regenerability has been observed in B. carinata (BBCC) synthesised from reciprocal crosses of B. nigra and B. oleracea.Abbreviations MS Murashige and Skoog (1962) - IAA Indole-3-acetic acid - NAA -napthaleneacetic acid - BA 6-Benzylaminopurine