Organogenesis of stem and leaf protoplasts of a haploid golden delicious apple clone (Malus Xdomestica Borkh.)

Summary Highly viable protoplasts were isolated in large numbers from in vitro-grown leaf and stem tissues of a haploid clone of the apple scion cultivar Golden Delicious (Malus Xdomestica Borkh.). Protoplasts from both sources divided rapidly to give microcallus, when cultured in a modified Kao and Michayluk-based medium. Following two successive subcultures for callusing, shoot buds were regenerated from such calli, on half-strength Murashige and Skoog medium with an increased concentration of group B vitamins and containing 5.0 mg.l^-1 6-benzyl-aminopurine and 0.1 mg.l^-1 l-naphthaleneacetic acid (for the leaf protoplast-derived calli) or 4-indole-3yl-butyric acid (for stem protoplast-derived calli). The mesophyll protoplast-derived shoots were enfeebled and vitrified, in time with their ultimate death. Conversely, for those shoots deriving from the stem protoplasts, in vitro propagation was successfully achieved. This is the first report on the successful isolation, culture and organogenesis from stem protoplasts of a woody plant genotype.Abbreviations BAP 6-benzylaminopurine - FPE final plating efficiency - IBA 4-indole-3yl-butyric acid - IPE initial plating efficiency - f wt fresh weight - KM Kao and Michayluk (1975) - MES 2-N-morpholino ethane sulfonic acid - MPE intermediate plating efficiency - MS Murashige and Skoog (1962) - NAA l-naphthaleneacetic acid - PVP-10 polyvinylpyrrolidone (Av MW 10,000)     

Activity of 1,2-benzisoxazole-3-one and indole-2,3-dione on plant regeneration in vitro and on cell elongation

We tested the morphogenetic and cell elongating activity of 1,2-benzisoxazole-3-one, a compound similar to 1,2-benzisoxazole-3-acetic acid but lacking the lateral carbon chain. For comparison, we tested also the activity of indole 2,3-dione, having the same indolic ring as indole 3-acetic acid but no lateral carbon chain. The tests were made on the regeneration of tomato (Lycopersicon esculentum Miller var. Alice) from cotyledons and on pea (Pisum sativum L. var. Alaska) stem elongation. We found that 1,2 benzisoxazole-3-one retains part of the high shoot inducing activity of 1,2-benzisoxazole-3-aceticacid, while indole-2,3-dione is inactive. Both compounds have no effect on root induction or cell elongation. It seems therefore that the activity of 1,2 benzisoxazole-3-acetic acid is partly related to the structure of its ring, and that also in this respect 1,2 benzisoxazole-3-acetic acid differs from other auxinlike compounds.Abbreviations BOA 1,2-benzisoxazole-3-acetic acid - BOO 1,2-benzisoxazole-3-one - IAA in-dole-3-acetic acid     

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.     

The isolation and culture of protoplasts from an embryogenic cell suspension culture of Picea glauca (moench) voss

Conditions were standardized for the isolation and culture of protoplasts from an embryogenic cell suspension culture of Picea glauca. A combination of 0.5% Cellulase R-10, 0.25% Macerozyme, 0.25% Driselase, 0.25% Rhozyme HP-150 with 0.5M mannitol and 5 mM CaCl₂.2H₂O produced an average of 4.5 × 10⁶ protoplasts per gram fresh weight of cells. Of the several protoplast culture media tested, von Arnold and Eriksson and Kao and Michayluk (KM8P) media best supported mitotic divisions of protoplasts. A density of 10⁵ protoplasts per ml and the addition of 5 mM glutamine to the culture medium was necessary to induce sustained divisions and microcallus formation. Microcalli grew into subculturable callus using a nurse culture technique.Abbreviations BAP benzylaminopurine - 2,4-D 2,4-dichlorophenoxy-acetic acid - FDA fluorescein diacetate NRCC No. 27937     

A new approach to direct somatic embryogenesis in Medicago

A highly efficient system for direct somatic embryogenesis is described. Leaf sections originating from young trifoliate leaves of Medicago falcata line 47/1–5 and Medicago sativa line No2/9R, directly produced embryos after cultivation in liquid B5IV induction medium. In comparison with indirect somatic embryogenesis the system omits the callus stage and thus allows shortening of the process of somatic embryogenesis in alfalfa by 35–40 days. It permits the avoidance of secondary changes occurring during the process of dedifferentiation. A modified B5/3H medium containing Polyethylene Glycol 6000 promoted embryo development from globular up to torpedo stage. It was clearly shown that 2.5% Polyethylene Glycol stimulated this process for both H. falcata 47/1–5 and M. sativa No 2/9R. Maturation of torpedo stage embryos was carried out on solidified or liquid abscisic acidcontaining medium. A 30M abscisic acid concentration was optimal in allowing one embryo to yield one plant. Somatic embryo conversion to plants and plant regeneration was performed on Murashige and Skoog medium. Regenerated plants showed a normal morphology.Abbreviations ABA Abscisic acid - B5 Medium of Gamborg et al.(1968) - COT Cotyledone stage embryos - 2,4-D 2,4-dichlorphenoxyacetic acid - FW Fresh weight - GA3 Gibberellin A3 - MS Medium of Murashige and Skoog (1962) - PEG Polyethylene Glycol - POLY Polyembryos     

Isolation,culture, and cell division in cotyledon protoplasts of cotton (Gossypium hirsutum and G. barbadense)

Protoplasts were isolated from cotyledons and foliage leaves of cotton (Gossypium hirsutum and G. barbadense). Cotyledon protoplasts were larger and responded to culture better than leaf protoplasts. Cotyledon derived protoplasts regenerated cell walls and formed microcolonies of 2–3 cells in G. hirsutum and 5–8 cells in G. barbadense. However, the microcolonies did not grow beyond this stage. Protoplast yield and viability, cell wall regeneration and cell division were influenced by several factors, e.g., genotype, age, tissue and growth condition of donor plant, enzyme mixture and concentration, preplasmolysis period, incubation period, and culture medium.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine - GA₃ gibberellic acid - p CPA p-chlorophenoxyacetic acid - MES 2[N-morpholino]ethanesulfonic acid     

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     

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     

Anther culture of papaya (Carica papaya L.)

Papaya (Carica papaya L.) anther containing microspores in tetrad to early-binucleate stages were successfully cultured on 1/2 strength MS salts and vitamins with full strength Na-Fe-EDTA supplemented with 2 mg/l NAA, 1 mg/l BA and 6% sucrose for callus initiation and formation. Highest frequencies of callus induction were obtained when anthers at the uninucleate stage were cultured in the dark. Haploid plantlets and pollen-derived embryoids were obtained from anthers cultured at the uninucleate stage on solidified MS medium containing 3% sucrose without any growth regulators under a low light intensity (1,500 lux). Large quantities of embryoids were obtained when the original embryoids were transferred to MS medium with 3% sucrose and no growth regulators. Cytology of root tips of embryoid-derived plants confirmed the haploid chromosome number of 9 indicating that the embryoids originated from pollen.Abbreviations MS Murashige and Skoog (1962) - MAA naphthaleneacetic acid - BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid     

Metabolism of valproic acid by hepatic microsomal cytochrome P-450

Incubation of valproic acid with rat liver microsomes led to the formation of 3-, 4- and 5-hydroxy-valproic acid. The latter two metabolites, which have been characterized previously from in vivo studies, may be regarded as products of fatty acid ω-1 and ω hydroxylation, respectively. 3-Hydroxy-valproic acid, however, had been thought to derive from the β-oxidation pathway in mitochondria. Conversion of valproic acid to all three metabolites in microsomes required NADPH (NADH was less effective), utilized molecular oxygen, was suppressed by inhibitors of cytochrome P-450 and was stimulated (notably at C-3 and C-4) by phenobarbital pretreatment of the rats. It is concluded that rat liver microsomal cytochrome P-450 catalyzes ω-2 hydroxylation of valproic acid, a reaction not detected previously with fatty acids in mammalian systems, and that the product, 3-hydroxyvalproic acid, should not be used to assess in vivo metabolism of valproate via the β-oxidation pathway.     

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     

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     

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     

Analysis of characters coded for by T-DNA using hybridization between tumorous and normal plant cells

In somatic hybrids between tumourous Nicotiana tabacum (B6S3) and normal mesophyll Atropa belladonna cells, the following traits, directly or indirectly connected with T-DNA gene expression and tumourous growth, were analysed: lysopine dehydrogenase activity (LpDH), shoot suppression, root suppression, ability to grow on media with D-lactose as a sole carbon source and resistance to 2-aminoethylcysteine, 5-bromodeoxyuridine and 5-methyltryptophan. Dominant (semidominant) expression was observed for all but one trait studied, e.g. shoot suppression which behaved as a recessive character.Abbreviations LS-H Linsmaier and Skoog (1965) hormone-free medium - NAA Naphtaleneacetic acid - BAP 6-Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - 5-BUdR 5-Bromodeoxyuridine - 2-AEC 2-Aminoethylcysteine - 5-MT 5-Methyltryptophan     

Antiauxin enhanced microshoot initiation and plant regeneration from epicotyl-originated thin-layer explants of sugarbeet (Beta vulgaris L.)

An in vitro method was developed for microshoot initiation from thin-layer explants prepared from the elongated epicotyls of sugarbeet (Beta vulgaris L.). Intact epicotyls of 14-day-old seedlings were excised from the hypocotyls above the cotyledons and allowed to elongate on De Greef and Jacobs (1979) medium supplemented with 0.2 mg/l 6-benzyladenine, 0.2 mg/l gibberellic acid and 0.1 mg/l indole-3-acetic acid in darkness. After a 21-day-incubation, the elongated epicotyls were halved to obtain apical and basal segments prior to removing the leaves and lateral buds. Subsequently, 5–8 mm long, 2–3 mm wide and 0.8–1.0 mm thick tangential sections were prepared longitudinally from the exterior parts of the halved epicotyls. These thin-layer explants were incubated on microshoot initiating media containing various growth regulators. The combination of 1.0 mg/l 6-benzyladenine and the antiauxin 2,3,5-triiodobenzoic acid (1.0 mg/l) resulted in maximum microshoot development (6.3±0.2 microshoots/thin-layer explant). The final efficiency of our tissue culture system was significantly increased by the NaCl (100 mg/l) initiated in vitro rooting of microshoot originated plantlets.Abbreviations AC activated charcoal - asdp apical segment derived plantlet - asTLE apical segment derived thin-layer explant - BA-6 benzyladenine - bsdp basal segment derived plantlet - bsTLE basal segment derived thin-layer explant - EEM1-4 epicotyl elongation media - GA₃ gibberellic acid - GM germinating medium - IAA indole-3-acetic acid - IBA indole-3-butyric acid - KN kinetin - MES morpholino-ethanesulfonic acid - MSI1-6 microshoot initiating media - NAA -naphthalene acetic acid - PG_oB De Greef and Jacobs (1979) medium - RM1-3 rooting media - SDM shoot developing medium - SE standard error - TIBA 2,3,5 triiodobenzoic acid - TLE thin-layer explant - ZEA zeatin     

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     

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     

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     

Phytochrome-mediated responses of cells and protoplasts of green calli obtained from the leaves of a CAM plant

Green callus obtained from leaves of the CAM-inducible plant Kalanchoe blossfeldiana cv. Montezuma has previously been shown to perform C₃-type photosynthesis under 16-h days and to shift to crassulacean acid metabolism (CAM) under 9-h days. The utilization of photoperiodic regimes (i.e. night interruptions by 30 min red light) established that CAM induction in the callus was under the control of phytochrome, as shown by measurements of CAM criteria: phosphoenolpyruvate carboxylase activity and malic acid pools. Short-term responsiveness of the callus cells to phytochrome modulations by monochromatic radiations was also established by the rapid changes observed in the diameter of the callus-derived protoplasts. These results provide further evidence that whole plant correlations are not necessary for phytochrome operativity.Abbreviations CAM crassulacean acid metabolism - PAL phenylalanine ammonia lyase (EC 4.3.1.5) - PAR photosynthetically active radiations - PEPC phosphoenolpyruvate carboxylase (EC 4.1.1. 31) - Rubisco ribulose 1,5 bisphosphate carboxylase (EC 4.1.1.39)     

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