Showing papers in "Plant Cell Reports in 1985"

Shoot multiplication from mature trees of Douglas-fir ( Pseudotsuga menziesii ) and sugar pine ( Pinus lambertiana )

Abstract: Opening of apical and axillary buds of mature Douglas-fir and sugar pine trees was obtained on a newly formulated basal medium (DCR) without growth regulators. Elongation of buds was observed on 1/2 strength DCR with 0.3% activated charcoal (DCR-1). In sugar pine, multiple shoots were obtained when explants on DCR with 0.5 mg/1 BAP for 5–6 weeks were transferred to DCR-1 medium. On subculture, axillary buds again developed when shoots were cultured on DCR with 0.2 mg/1 BAP for Douglas-fir and 0.5 mg/1 BAP for sugar pine. These buds were again elongated on DCR-1 medium. By subculturing 7–10 shoots of Douglas-fir and 2–3 shoots of sugar pine, over 100 shoots can be obtained in a year.

Influence of a loblolly pine (Pinus taeda L.). Culture medium and its components on growth and somatic embryogenesis of the wild carrot (Daucus carota L.).

Abstract: A new culture medium, originally designed and shown to grow cell suspensions from a variety of loblolly pine (Pinus taeda L.) explants, was used to study growth and somatic embryogenesis of the wild carrot (Daucus carota L.) in cell suspensions. The new loblolly pine medium (LM) differed from the standard wild carrot medium (WCM) in having very low Ca2+, very high Mg2+, and enrichment with PO inf4 sup3− and microelements. When WCM was altered to contain levels of Ca2+ or Ca2+ and Mg2+ equivalent to LM, it supported neither growth nor embryogenesis of the wild carrot. However, growth and embryogenesis in LM was superior to WCM. The phosphate level in WCM was found to be suboptimal.

A tissue culture technique for rapid clonal propagation and storage under minimal growth conditions of Musa (Banana and plantain).

Abstract: A tissue culture technique for rapid clonal propagation and storage under minimal growth conditions is presented in this paper Shoot-tip cultures of Musa cultivars (both banana and plantain) are induced by culturing small excised shoot apices on modified MS semisolid medium supplemented with various concentrations and combinations of auxins and cytokinins The effects of cytokinin concentration in the medium as well as the genotypic configuration of the cultivars on the rate of shoot-bud proliferation have been tested The established shoot-tip cultures grown on modified MS semisolid medium supplemented with IAA (018 mg/l) and BA (230 mg/l) have been successfully stored at 15°C with 1000 lux light intensity up to 13–17 months depending on the cultivar The cultivars tested in the present investigation seem to vary in their ability to withstand minimal growth temperature

Effect of the 1B/1R translocation on anther culture ability in wheat (Triticum aestivum L.).

Abstract: Using two varieties, their reciprocal hybrids, F8 lines and doubled haploids, results confirmed that three genetic components are involved in wheat anther culture ability, viz embryo induction frequency, regeneration ability and the frequency of albinism. In these experiments, no significant maternal effects were noticed. For embryo yields, transgressive lines were obtained from hybrids between distant genotypes. Regeneration of green plants depended upon two independent traits: regeneration ability and the frequency of albinos. F8 lines and two doubled haploids equaled the 50% regeneration rate of the hybrids, but they only regenerated green plants. Based upon cytological examination and gliadin patterns, it is suggested that genes favoring regeneration ability could be linked to the 1BL-1RS translocated chromosome from Aurora.

A simple culture method for Brassica hypototyl protoplasts.

Abstract: Hypocotyl protoplasts from oil rape, Brassica napus L cv Isuzu were cultured in the dark at 25°C in a modified Nitsch and Nitsch medium containing 13% sucrose, 5 g/l Ficoll, 05 mg/l BAP, 1 mg/l NAA and 05 mg/l 2–4 D Protoplasts floated on the surface of the medium and developed into microcolonies 05 mm in diameter in 4–6 weeks The microcolonies also remained on the surface of the medium Transfer to MS medium supplemented with 200 mg/l casein hydrolysate, 5mg/l BAP, 05 mg/l NAA and solidified with 06% agarose induced shoot regeneration in 3–4 weeks

Somatic embryogenesis and regeneration of plants in the bamboo Dendrocalamus strictus.

Abstract: Somatic embryogenesis leading to plant regeneration has been achieved in the bamboo, Dendrocalamus strictus, by culturing seeds (caryopses) on B5 basal medium supplemented with 2,4-dichlorophenoxyacetic acid. Callus cultures obtained from the embryonal end of the seeds differentiated chlorophyllous embryoids. On transfer to a germination medium (B5 liquid, sucrose, indolebutyric acid, and ∝ -naphthaleneacetic acid) 40% of the embryoids developed into plantlets. Further development of the plantlets occured on B5 liquid medium (half strength) + sucrose (1%) + IBA (5 × 10−7M) + NAA (10−7M).

Anthraquinones as phytoalexins in cell and tissue cultures of Cinchona spec.

Abstract: The addition of autoclaved mycelia of Aspergillus niger and the known phytopathogenic fungus Phytophtora cinnamomi to cultured cells of Cinchona ledgeriana Moens. caused a marked increase in the anthraquinone content of the plant cells. This finding in combination with the antimicrobial activity of the anthraquinones isolated from calli of Cinchona pubescens Vahl. led to the conclusion that anthraquinones are phytoalexins.

Cultivation of cell cultures of Berberis wilsonae in 20-l airlift bioreactors.

Abstract: Suspension cultures of Berberis wilsonae produce 4 berberine-type alkaloids: berberine, palmatine, columbamine and jatrorrhizine. In particular the formation of the phenolic alkaloids columbamine and jatrorrhizine and of berberine proves to be dependent on the concentration of dissolved oxygen. With higher aeration rates, berberine and jatrorrhizine yields can be increased considerably. Thus we reached an alkaloid yield of more than 3 g × 1−1 with 50% dissolved oxygen tension in the medium. As far as we know this is one of the best results in fermenting of alkaloid-producing cell cultures.

Gynogenesis in Beta vulgaris L.: From in vitro culture of unpollinated ovules to the production of doubled haploid plants in soil

Abstract: Our study concerns the different stages involved in the production of doubled haploid beet plants by in vitro gynogenesis. A histological study shows that the embryos obtained could come from the oosphere or the antipodals. Gynogenesis using male fertile plants gives nearly two haploid plants for 1000 ovules cultured: in this case, the problem is to avoid plating fertilized ovules. The gynogenetic plants obtained display an endopolyploidy phenomenon at the root meristem level while their shoot meristem remains haploid.

Electric field mediated stable transformation of carrot protoplasts with naked DNA

Abstract: We have developed an electroporation procedure for the transformation of carrot protoplasts with Ti-plasmid DNA from Agrobacterium tumefaciens. The uptake of pTiC58 into carrot protoplasts was mediated by high voltage electrical pulses at field strengths from 0.5 to 3.8 kV/cm. Protoplast regeneration, somatic embryogenesis and plantlet regeneration were unaffected by the electroporation conditions selected for DNA uptake. Uptake of plasmid pTiC58 resulted in hormone independent regeneration of carrot protoplasts. Transformed somatic embryos were detected in carrot cultures 45 days after electroporation. The transformed somatic embryos developed into teratomas which synthesized nopaline. Hybridization was obtained between a labeled T-DNA fragment from pTiC58 and DNA fragments from 4 month old teratomas regenerated from electro-transformed protoplasts. Based on the number of somatic embryos regenerated after electro-transformation, a frequency of 1.6×102 transformants/104 somatic embryos/μg pTiC58 DNA was obtained.

Effects of macronutrients on growth and rosmarinic acid formation in cell suspension cultures of Anchusa officinalis

Abstract: The influence of various macronutrients on growth and RA formation in cell suspension cultures of Anchusa officinalis has been investigated. Factors tested included sucrose concentration, alternate carbon sources, nitrate, phosphate and calcium concentration. The optimum concentration of sucrose was 3%. Fructose, glucose or their 1:1 mixture were also suitable carbon sources. The optimum concentrations of nitrate (15 mM), phosphate (3 mM) and calcium (0.25 mM) were, respectively, 3/5, 3x, and 1/4 those in normal B5 medium, when tested separately. These concentrations improved not only the yield of RA but also cell growth to a similar degree (10%-50%). Studies on the combined effects of these optimum macronutrient concentrations in B5 medium showed that RA production is inhibited by 2,4-D-containing revised medium but stimulated by NAA-containing revised medium.

Regeneration of plants from the culture of leaves and axillary buds in mulberry (Morus indica L.).

Abstract: Stem segments, axillary buds and leaves excised from established shoot cultures of Morus indica were soaked in MS liquid medium containing benzyladenine (0.5, 1, 2 mg/1) and were cultured subsequently on semi solid medium of the same composition. Numerous shoot buds differentiated from leaf and axillary buds but stem segments were unresponsive. The shoot buds on isolation and culture developed into plantlets. Callus tissues which developed at the base of the leaf explant upon subculture also differentiated numerous shoot buds.

In vitro vegetative propagation of Leucaena leucocephala (Lam.) de Wit.

Abstract: A method for in vitro clonal multiplication of Leucaena leucocephala cv K-8 is described. On MS with BAP (3×10−6M), at the optimum temperature of 30°C, shoots from seedling and adult trees multiplied at a rate of 6–7 fold every three weeks. The addition of adenine or glutamine reduced precocious leaf drop. All shoots rooted on MS with IAA (5×10−6M). Micropropagated plants have been successfully transferred to soil.

Late enzymes of vindoline biosynthesis. Acetyl-CoA: 17-O-deacetylvindoline 17-O-acetyl-transferase.

Abstract: From differentiated plants of Catharanthus roseus (L.) G. Don, a specific enzyme was isolated and named acetyl-CoA : 17-O-deacetylvindoline 17-O-acetyltransferase, acting on the biosynthetic formation of the Aspidosperma type alkaloid vindoline.

Somatic embryogenesis and plantlet regeneration in the soybean Glycine max.

Abstract: A tissue culture procedure for the regeneration of somatic embryos and plantlets from somatic cells of the soybean Glycine max is described. Bean pods of soybean cv. TGM119 were immersed in liquid nitrogen for 20 minutes. Young embryos were excised from the immature seeds and cultured to form calli. Calli grown from the young embryos were incubated in liquid culture for two weeks. The liquid suspension culture was filtered to obtain single cells. The soybean cells were cultured for one month in a liquid medium in hanging drop cultures for development into proembryoids. The proembryoids were maintained on a solid growth medium for 40 days. The resultant callus tissue was transferred into MS media containing selected combinations and concentrations of 2,4-Dichlorophenoxyacetic acid, Naphthaleneacetic acid, Kinetin, Benzyladenine and Indoleacetic acid. In the presence of Benzyladenine (0.2 mg/l) and Indoleacetic acid (0.01 mg/l), globular and heart shaped somatic embryos were formed on the surface of the calli. Calli containing somatic embryos were transferred into liquid medium and incubated under low light conditions. After six months further incubation, more than 1,000 plantlets and a large number of somatic embryoids at various developmental stages were obtained per flask.

Directed electrofusion between protoplasts with different responses in a mass fusion system.

Abstract: Protoplasts of Solanum brevidens (leaves) and Nicotiana rustica (suspensions) have been aligned and fused electrically between widely spaced electrodes, and the yield of 1:1 (binary) fusion products in chains of aligned protoplasts has been determined by light microscopy. Leaf protoplasts fuse more easily than protoplasts from suspension cultures (Tempelaar and Jones, 1985), thus electrical parameters and the ratio of leaf: suspension protoplasts can be varied to control the yield of binary and multifusion products. In experiments to determine optimum ratios for electrofusion, up to 60–70% of S. brevidens — N. rustica fusion products were binary at overall fusion frequencies of 40–50%. Fusions in samples of protoplasts with the same characteristics can also be controlled to direct the fusion process towards binary products. However, in this case, at least half of the binary products may be derived from self-fusions.

In vitro culture of Cucumis sativus L. V. Stabilizing effect of glycine on leaf protoplasts

Abstract: A method for leaf mesophyll protoplast isolation and plant regeneration of cucumber (Cucumis sativus L.) is described. Using an isolation solution complemented with 0.1 M glycine, 8.2·106 viable protoplasts were isolated from 1 g of fresh leaves. The effect of the growth substances indole-3-acetic acid, naphthalene acetic acid, 2,4,-dichlorophenoxy acetic acid, 6-benzylaminopurine, 2-isopentenyladenine and kinetin at concentrations from 0.5 to 5 mg·1−1 was studied using the multi-hanging drop technique. The optimal growth substance combination, namely 5 mg·1−1 naphthalene acetic acid and 3 mg·1−1 2-isopentenyladenine, together with agarose medium in a so-called bead culture resulted in a plating efficiency of 21%. Some of the colonies obtained regenerated to plantlets which developed to plants.

Effects of auxins and cytokinins on growth and rosmarinic acid formation in cell suspension cultures of Anchusa officinalis

Abstract: Cell suspension cultures of Anchusa officinalis required exogenous phytohormones for their normal growth. Cell lysis was observed at the third passage in a hormone-free medium. Using hormone — depleted cells, the effects of auxins (2,4-D, NAA, IAA and CFP) and cytokinins (BA, kinetin, and zeatin) on cell growth and RA production were investigated. All auxins tested could maintain growth and integrity of the cells whereas cytokinins alone could not, suggesting that this culture is auxindependent. Among the auxins tested, NAA had a pronounced effect on RA production. The total RA content obtained at optimum NAA concentration (0.25 mg/l) reached 1.7 g/l (12% of dry weight). The kinetics of growth and RA production suggested that the increase in final RA content was due to both an increase in the rate of RA synthesis and initiation of the period of synthesis in the exponential rather than the linear growth phase.

A method for the microinjection and culture of protoplasts at very low densities

Abstract: A method has been developed which allows the recovery of calli from a high proportion of individual, injected, mesophyll protoplasts of Nicotiana tabacum c.v. Xanthi. A small drop of low melting point agarose is used both to hold protoplasts during microinjection and for their subsequent culture in feeder dishes. The feeder dishes consist of "beads" of protoplasts at a high density set in agarose to "feed" the infected protoplasts across a liquid medium. The method has been used successfully both with normal protoplasts and protoplasts from which the vacuole has been removed.

Suspension culture of sugarbeet (beta vulgaris L.). induction and habituation of dedifferentiated and self-regenerating cell lines

Abstract: A systematic procedure is described to obtain habituated cell suspensions from different explant sources of sugarbeet (Beta vulgaris L.).

Infection of turnip and radish storage roots with Agrobacterium rhizogenes

Abstract: Within about 10 days after inoculation with Agrobacterium rhizogenes, the vascular bundles of storage root disks of turnip or radish developed small outgrowths with numerous root hairs. Thereafter, adventitious roots (hairy roots) emerged extensively from these outgrowths. The hairy roots which emerged fully supported the growth of host plants, though they lacked geotropism. An excised hairy root could be subcultured as an axenic root culture in the absence of phytohormones. Hairy root cultures with extensive lateral branches grew much more rapidly than those with few lateral branches or ordinary roots. Calli were induced from hairy root cultures in the presence of 2,4-D, and root proliferation from these calli occurred in the absence of 2,4-D. Both the primary hairy roots and the roots which grew from them synthesized agropine and mannopine.

Late enzymes of vindoline biosynthesis : S-Adenosyl-L-methionine: 11-O-demethyl-17-O-deacetylvindoline 11-O-methyltransferase and unspecific acetylesterase.

Abstract: From differentiated plants of Catharanthus roseus (L.) G. Don we have isolated a specific enzyme of the vindoline biosynthetic pathway catalysing the S-adenosylmethionine-dependent methylation of 11-O-demethyl-17-O-deacetyl-vindoline. The enzyme we named S-adenosyl-L-methionine : 11-O-demethyl-17-O-deacetylvindoline 11-O-methyltransferase. This transferase exhibits a high substrate specificity. Obviously the O-methylation at C-11 precedes the O-acetylation at the C-17 position during the biosynthesis of vindoline.

Induction of freezing tolerance in alfalfa cell suspension cultures

Abstract: The effects of ABA, 2,4-D, kinetin and cold exposure on the cold hardiness of Medicago sativa L. cell suspensions were investigated. Cultures treated with 5×10−5 M ABA at 2°C for 4 weeks in the absence of kinetin showed a 50% survival after freezing to −12.5°C, whereas cultures grown at 25°C under normal conditions tolerated freezing to only −3°C. The optimum ABA treatment of 5×10−5 M for 4 weeks was effective only in combination with cold exposure. Of six cell lines tested, all showed different degrees of induced cold hardiness. The results suggest that ABA alone cannot induce freezing tolerance on alfalfa cell suspension cultures and that the deletion of kinetin and combination of low temperature and ABA is critical for the induction of cold hardiness in alfalfa cell suspension cultures.

Correlation of the appearance of morphinan alkaloids and laticifer cells in germinating Papaver bracteatum seedlings.

Abstract: The course of alkaloid accumulation and laticifer cell appearance was compared in germinating P. bracteatum seedlings. Seedlings of various ages (0–14 days old) were analyzed for their dopamine, thebaine, morphinan alkaloid immunoreactivity, and benzophenanthridine alkaloid levels. Simultaneous electron microscopic studies revealed that seedlings were devoid of laticifer initials until day 3, where-upon their numbers increased with time. The appearance of appreciable amounts of thebaine only occurred after day 4 of germination. Conversely, dopamine was rapidly formed at the onset of germination and reached millimolar concentrations well before laticifer cells were detected. Benzophenanthridine alkaloid levels remained fairly constant over the period analyzed. These results support the theory that the presence of laticifer cells is necessary for the accumulation of morphinan but neither benzophenanthridine alkaloids nor their mutual precursor, dopamine.

Anther culture of papaya (Carica papaya L.).

Abstract: 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.

Partial purification and characterization of a NADPH dependent tetrahydroalstonine synthase from Catharanthus roseus cell suspension cultures.

Abstract: A new enzyme was discovered which specifically hydrogenates the iminium form of cathenamine at position 21 to yield the heteroyohimbine alkaloid tetrahydroalstonine. The enzyme was partially purified (35-fold) from Catharanthus roseus cell suspension cultures. It was shown to use exclusively NADPH as reductant, the pH optimum is at 6.6, the temperature optimum at 30°C, the half life of the soluble enzyme preparation is 26 min at 37°C, and the molecular weight is 81 000 ± 3%. Evidence is presented for the occurrence of two distinct and different cathenamine reductases, one reducing the iminium form of this central intermediate to give tetrahydroalstonine, the other one reducing cathenamine to yield ajmalicine. Tetrahydroalstonine synthase was present in cell suspension cultures of C. ovalis, C. roseus, Picralima nitida, Rhazya stricta, and Vinca herbacea.

S-adenosyl-L-methionine: (S)-scoulerine 9-O-methyltransferase, a highly stereo- and regio-specific enzyme in tetrahydroprotoberberine biosynthesis

Abstract: Suspension cultures of Berberis species are useful sources for the detection and isolation of a new enzyme which transfers the methyl group from S-adenosyl-L-methionine specifically to the 9-position of the (S)-enantiomer of scoulerine, producing (S)-tetrahydrocolumbamine. The enzyme was enriched 27-fold; it is not particle bound, has a pH optimum of 8.9, a molecular weight of 63 000 and shows a high degree of substrate specificity.

Electrofusion, a simple and reproducible technique in somatic hybridization of Nicotiana plumbaginifolia mutants.

Abstract: Electrofusion of protoplasts from two complementary nitrate reductase deficient mutants of Nicotiana plumbaginifolia has resulted in somatic hybrid lines. Mesophyll protoplasts isolated from the cofactor mutant CNX 20 and fluorescein diacetate stained protoplasts derived from a cell suspension culture of the NA 36 line, being defective in the apoenzyme, were used in the fusion experiments. In total, 594 lines were recovered which could proliferate on a selective medium with nitrate as the sole nitrogen source. This is including 141 putative hybrid lines which were obtained after transfer of 1048 heterokaryons with a micromanipulator one day after electrofusion. The hybrid character of some of the selected lines was confirmed by nitrate reductase activity measurements. Plants were grown from hybrid calli.

Brassica grown gall tumourigenesis and in vitro of transformed tissue

Abstract: A number of Brassica species and cultivars were tested and found to be highly susceptible to crown gall induction by both nopaline and octopine strains of Agrobacterium tumefaciens. Only B. napus did not form tumours when inoculated with octopine strains. Seedlings of very young plants were poor hosts but efficient infection occurred after 8–10 weeks of growth. Teratomas arising on tumours in planta were relatively frequent on induction with nopaline strains. Axenically cultured tumour calli of Brassicas were very active in opine synthase activity and stably maintained this transformed phenotype; however, transformed plants could not be regenerated. These results suggest that disarmed nopaline Ti plasmid vectors are well suited for the genetic engineering of this important crop family.

Effect of brassinosteroid on cell division and enlargement in cultured carrot (Daucus carota L.) cells.

Abstract: When added at 3.10−6 mM to auxin-starved suspension cultured carrot (Daucus carota L.) cells, the steroid hormone 24-epibrassinolide (BR) induces cell enlargement but not cell division. This is at variance from the effect of 2,4-D which, in the same experimental conditions, restores cell division without having any effect on cell enlargement. Furthermore, in the tested experimental conditions, the effect of BR is dominant over the effect of 2,4-D when the two hormones are simultaneously supplied to the auxin-starved culture.