Metabolism of glutathione and ascorbate in lingonberry cultivars during in vitro and ex vitro propagation

Lingonberry (Vaccinium vitis-idaea L. ssp. vitis-idaea Britton) cultivars Regal, Splendor, and Erntedank were obtained by conventional softwood cuttings (taken as a control), by in vitro shoot proliferation of node explants, and by adventitious shoot regeneration from excised leaves of micropropagated shoots. In the plants propagated in vitro, the total ascorbate content increased and its pool was more oxidized, the total glutathione content also increased but its pool became more reduced. The leaves of plants obtained from the in vitro culture showed significantly higher antioxidant enzyme activities except for dehydroascorbate reductase which was at a similar level in all plants. Total soluble phenolics, tannins, and flavonoids were enhanced in fruits of in vitro-propagated plants whereas in leaves, the levels of these metabolites (except flavonoids) were higher in ex vitro derived plants. The total radical scavenging capacity was enhanced in berries of the in vitro propagated plants. It is suggested that the active morphogenetic process, characterized by intensive formation and scavenging reactive oxygen species is reflected in the activities of antioxidant enzymes and metabolites. The reduction potential of glutathione is the most important parameter which determines patterns of growth and differentiation in the investigated plants.     

Growth and survival of Mycoplasma neurolyticum in liquid media

Hottle, G. A. (Naval Biological Laboratory, University of California, Berkeley), and D. N. Wright. Growth and survival of Mycoplasma neurolyticum in liquid media. J. Bacteriol. 91:1834-1839. 1966.-Maximal growth of Mycoplasma neurolyticum (between 10(8) and 10(9) colony-forming units per ml) was obtained after 3 days of incubation at 36 C in broth media containing 10% agamma horse serum. When whole horse serum was used in the medium, a complement-mediated inhibition was observed. This inhibition could only be detected when growth was followed by daily plate counts. Maximal growth was delayed for about 24 hr by the horse serum, and the inhibition was spontaneously reversed at the temperature of incubation. Penicillin G was also found to have a temporary inhibitory effect. This was detected with as little as 40 units per ml. Maximal growth was delayed until the 6th day of incubation, when 200 units per ml was present, and until the 16th day, when 1,000 units per ml was present. The survival of M. neurolyticum at undetectable levels in cultures during the incubation period presented an "eclipse" phenomenon which has not been explained. The recrudescence of growth in such cultures late in the incubation period illustrates the events which may occur when mycoplasmas are isolated from clinical material by prolonged incubation in the presence of inhibitors. Survival data showed that M. neurolyticum had greatest stability at pH 8.0, with reduced viability at pH 9.0, 7.0, 10.0, and 6.0, in that order The data on growth and stability suggest a close relationship between the species. of Mycoplasma studied and bacteria.     

A protocol for in vitro mass propagation of asiatic hybrids of lily through liquid stationary culture

Summary A simple, rapid and cost-effective in vitro scheme has been proposed for mass propagating two cultivars of Asiatic lily hybrids. An average of seven bulblets was formed after 17 d when 1×1 cm² bulb scale segments (explants) were cultured on Murashige and Skoog (MS) medium with 3% sucrose and 0.5 μM α-naphthaleneacetic acid (NAA). On MS medium containing 0.5 μM NAA and 6 or 9% sucrose, depending on the cultivar, large numbers of bulblets of increased size (3.5–5.0 cm in circumference) were formed under a 16/8 h photoperiod. A continuous system of mass propagation of bulblets was achieved through in vitro scale formation (secondary explants) on MS medium supplemented with 23 μM kinetin and 0.5 μM NAA, as well as scale proliferation on MS basal liquid stationary medium. Upon transplantation all bulblets sprouted, of which 40% flowered in the first season. Under ideal conditions, ca. 9.68×10⁵ bulblets can be produced from a single scale segment in 1 yr by following the systematic propagation steps proposed here.     

In vitro PPFD and media composition affect both in and ex vitro performance of Alstroemeria Butterfly-hybrids

The objective was to reduce in vitro production costs while retaining or improving plant quality, in particular the suitability for pot plant production. Plants were grown at photosynthetic photon flux densities (PPFD) of 0–40 μmol m^-2 s^-1 and sucrose concentrations of 3–7% during the multiplication phase and the effects of sucrose, BA, and NAA during root formation were investigated. Ex vitro growth were tested in both experiments. A small reduction in the rhizome multiplication rate was found with increasing PPFD and sucrose concentration. Increasing sucrose concentration reduced the number of aerial shoots. Aerial shoots were etiolated when cultured in darkness and their number increased with increasing PPFD at 3% sucrose, whereas PPFD did not affect the number of aerial shoots at 5 or 7% sucrose. During the multiplication phase a synergistic promoting effect of PPFD and sucrose was observed on root formation. Root formation after transfer to rooting medium was affected by sucrose and PPFD during the multiplication phase. PPFD did not influence root formation after propagation on 7% sucrose, whereas on 3 or 5 % sucrose root formation was gradually inhibited when PPFD was decreased below 17 μmol m^-2 s^-1. The formation of thick roots was promoted by propagation in light, and not influenced by sucrose concentration. Ex vitro growth was not affected by in vitro conditions, except for 7% sucrose during the multiplication phase that reduced flowering. Root formation on rooting medium was reduced by BA and promoted both by NAA and high levels of sucrose. The root inhibiting effect of BA could not completely be overcome by simultaneous application of NAA and high sucrose concentrations. Thick roots were only produced in the presence of NAA, and not affected by sucrose treatment. Ex vitro flowering was negatively influenced by the presence of BA during root formation and by high levels of sucrose if BA was absent in the rooting medium. High sucrose levels and NAA could partially compensate for the negative effect of BA on flowering. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro propagation of endangered Lippia filifolia mart. and schauer ex schauer

Summary This work describes an efficient micropropagation protocol of Lippia filifolia. Nodal segments cultivation in MS medium supplemented with 6-benzylaminopurine (4.5 μM)/α-naphthaleneacetic acid (NAA; 54nM) induced multiple shoots (in average 27 shoots per explant). Elongated shoots were rooted with NAA (0.11 μM) and they maintained ploidy level of the in vitro produced explants. The basic chromosome number were 2n=2x=24. Regenerated rooted shoots were successfully acclimatized under shading house conditions. This is the first report involving the establishment of a protocol for shoot multiplication and rooting for endangered L. filifolia, contributing for germplasm preservation of this species.     

Meta-topolin and liquid medium mediated enhanced micropropagation via ex vitro rooting in Vanilla planifolia Jacks. ex Andrews

Plant Cell, Tissue and Organ Culture (PCTOC) - An advanced micropropagation protocol has been developed for the global spice crop Vanilla planifolia using meta-topolin [mT, 6-(3-hydroxybenzylamino)...     

The Role of Abscisic Acid in Acclimation of Plants Cultivated in vitro to ex vitro Conditions

The content of endogenous free abscisic acid (ABA) in the shoots of in vitro cultivated tobacco (Nicotiana tabacum L. cv. White Burley) and its changes during ex vitro acclimation of these plants to the greenhouse or growth chamber were estimated. The content of free ABA significantly increased at the 1^st and/or 2^nd day after plant transfer from in vitro to ex vitro. The ABA content of plants covered with transparent foil to maintain higher relative humidity (RH), did not significantly differ from ABA content of plants cultivated under ambient RH. Transfer to fresh medium also transiently increased the content of endogenous ABA. The ABA content in plants, which had been acclimated for 1 week to ex vitro conditions, decreased to the content found in the in vitro plants. Acclimation to ex vitro conditions affected the stomata on adaxial and abaxial sides differently: stomata on the adaxial side were less open than those on the abaxial one. The exogenous application of 5 μM ABA increased transiently its endogenous concentration in shoots of in vitro plants more than ten fold, but after 1 week the concentration in the shoots decreased. This revised version was published online in July 2006 with corrections to the Cover Date.     

Assessment of liquid culture procedures for in vitro propagation of Rheum emodi

Micropropagation of an endangered Indian medicinal plant, Rheum emodi Wall., was achieved on Murashige and Skoog's medium using different liquid culture procedures. Liquid static (submerged, semi-submerged and with filter paper bridge) and shake (80 and 120 rpm) culture procedures were assessed for their effects on growth and multiplication rates. Best results were obtained using liquid shake cultures, which resulted in 50% reduction in medium requirement, 37.5% reduction in time and 1.5–2.2 fold increase in growth and multiplication rate. Liquid culture-raised plantlets facilitated easy transplantation and 90–95% survived transfer to potting mix in glasshouse.Abbreviations BA 6-benzyladenine - IBA indole-3-butyric acid     

In vitro propagation of Bambusa nutans Wall. ex Munro through axillary shoot proliferation

This communication describes for the first time an efficient and reproducible protocol for large-scale multiplication of Bambusa nutans. Nodal segments collected from field-grown clumps and cultured on Murashige and Skoog (MS) medium supplemented with 4.4 μM benzylaminopurine (BA) and 2.32 μM kinetin (Kin) gelled with 0.2% gelrite yielded 80% aseptic cultures with 100% bud-break. The in vitro-formed shoots obtained after bud-break were successfully multiplied in MS liquid medium supplemented with 13.2 μM BA, 2.32 μM Kin, and 0.98 μM indole-3-butyric acid (IBA). Sub-culturing of shoots every 3 weeks on fresh multiplication medium yielded a consistent proliferation rate of 3.5-fold. Shoot clusters containing three to five shoots were successfully rooted with 100% success on half-strength MS liquid medium supplemented with 9.8 μM IBA, 2.85 μM indole-3-acetic acid (IAA), 2.68 μM naphthaleneacetic acid (NAA), and 3% sucrose. Plantlets grown in vitro were acclimatized and subsequently transferred to the field. Inter-simple sequence repeat analysis has confirmed the genetic uniformity of the tissue-cultured plants up to 27 passages.     

An efficient in vitro method for mass propagation of Potentilia potanii wolf

Summary This study reports a method for high-frequency shoot organogenesis and plant establishment of Potentilla potaninii Wolf. Hypocotyl and cotyledon explants of P. potaninii were cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of benzyladenine (BA) and α-naphthaleneacetic acid (NAA) to induce adventitious shoot formation for micropropagation. The highest frequency of adventitious shoot regeneration was achieved from hypocotyl and cotyledon explants grown on MS medium supplemented with 5.0 mgl^−1 BA and 1.0 mgl^−1 NAA. The regenerated shoots rooted most efficiently on half-strength MS medium supplemented with 1.0 mgl^−1 NAA and 0.5 mgl^−1 indole-3-acetic acid or indole-3-butyric acid. The acclimatized plants with normal morphology and growth characters flowered and set seeds in the following year.     

Wave propagation in bone media

The composite nature of bone dictates the use of a model for bone which is transversely isotropic. We solve the associated sets of partial differential equations governing the dynamic elastic behavoor of a two-layered cylindrical-shaped bone. The solution is analyzed for long, short, and intermediate length waves. The special case of compact bone is treated for long and short wave lengths and a numerical example is worked out to determine the wave speeds (for short wave lengths) given a set of elastic constants, determined by ultrasonic methods, and the bone density, wave frequency, and radius.     

Wave propagation in discrete media

 We have considered infinite systems of nonlinear ODEs on the one-dimensional integer lattice which describes the activity in an excitatorily coupled network of excitable cells. For an ideal nonlinearity, we calculated the speed of propagation of an activity and derived the condition for its existence. We also studied the existence and stability of the traveling wave solution and gave, in the simplest case, its explicit expression. We established that some unstable traveling waves lead to propagation with an enlarging profile defined by a front velocity and a wake velocity. We generalized some results to inhomogeneous medium and network with long range connections. Received: 3 July 2000 / Revised version: 17 April 2001 / Published online: 7 December 2001     

In vitro propagation of Allium tuberosum Rottl. ex. Spreng. by shoot proliferation

Halved shoot bases of Allium tuberosum Rottl. ex Spreng. proliferated both axillary and adventitious shoots on B5 medium (1968) supplemented with either 6-benzylaminopurine (0.5 mg/l) or 1-naphthalene acetic acid (0.1 mg/l) and 2-isopentenyladenine (0.5 mg11). Ia vitro shoots proliferated further numerous shoots upon subculture to fresh medium, and these shoots rooted spontaneously. Plantlets were transplanted successfully to soil and retained the diploid condition of the parents.     

In vitro propagation of Allium tuberosum Rottl. ex. Spreng. by shoot proliferation

Seeds of trifoliate orange (Poncirus trifoliata (L.) Raf.) are sensitive to desiccation, and could not withstand reduction in moisture level below 20%, whereas the excised embryonic axes could be easily desiccated to moisture levels as low as 14% without much loss in viability. Axes could be successfully cryopreserved in liquid nitrogen (–196°C) for eight months. The viable embryonic axes exhibited good growth on modified Murashige and Skoog medium supplemented with 1-Naphthalene acetic acid (NAA) and 6-Benzylaminopurine (BAP). Growth of cryopreserved axes was promoted in the presence of charcoal in the medium allowing for plant recovery.Abbreviations NAA Napthaleneacetic acid - BAP 6-Benzylamino-purine - MS Murashige and Skoog (1962) - LN Liquid nitrogen     

Role of N-terminal familial mutations in prion protein fibrillization and prion amyloid propagation in vitro

A self-perpetuating conformational conversion of the prion protein (PrP) is believed to underlie pathology and transmission of prion diseases. Here we explore the effects of N-terminal pathogenic mutations (P102L, P105L, A117V) and the residue 129 polymorphism on amyloid fibril formation by the human PrP fragment 23-144, an in vitro conversion model that can reproduce certain characteristics of prion replication such as strains and species barriers. We find that these amino acid substitutions neither affect PrP23-144 amyloidogenicity nor introduce barriers to cross-seeding of soluble protein. However, the polymorphism strongly influences the conformation of the amyloid fibrils, as determined by infrared spectroscopy. Intriguingly, unlike conformational features governed by the critical amyloidogenic region of PrP23-144 (residues 138-139), the structural features distinguishing Met-129 and Val-129 PrP23-144 amyloid fibrils are not transmissible by cross-seeding. While based only on in vitro data, these findings provide fundamental insight into the mechanism of prion-based conformational transmission, indicating that only conformational features controlling seeding specificity (e.g. those in critical intermolecular contact sites of amyloid fibrils) are necessarily transmissible by cross-seeding; conformational traits in other parts of the PrP molecule may not be "heritable" from the amyloid template.     

Biomass plug development and propagation in porous media

Exopolymer-producing bacteria can be used to modify soil profiles for enhanced oil recovery or bioremediation. Understanding the mechanisms associated with biomass plug development and propagation is needed for successful application of this technology. These mechanisms were determined from packed-bed and micromodel experiments that simulate plugging in porous media. Leuconostoc mesenteroides was used, because production of dextran, a water-insoluble exopolymer, can be controlled by using different carbon sources. As dextran was produced, the pressure drop across the porous media increased and began to oscillate. Three pressure phases were identified under exopolymer-producing conditions: the exopolymer-induction phase, the plugging phase, and the plug-propagation phase. The exopolymer-induction phase extended from the time that exopolymer-producing conditions were induced until there was a measurable increase in pressure drop across the porous media. The plugging phase extended from the first increase in pressure drop until a maximum pressure drop was reached. Changes in pressure drop in these two phases were directly related to biomass distribution. Specifically, flow channels within the porous media filled with biomass creating a plugged region where convective flow occurred only in water channels within the biofilm. These water channels were more restrictive to flow causing the pressure drop to increase. At a maximum pressure drop across the porous media, the biomass yielded much like a Bingham plastic, and a flow channel was formed. This behavior marked the onset of the plug-propagation phase which was characterized by sequential development and breakthrough of biomass plugs. This development and breakthrough propagated the biomass plug in the direction of nutrient flow. The dominant mechanism associated with all three phases of plugging in porous media was exopolymer production; yield stress is an additional mechanism in the plug-propagation phase.     

An efficient in vitro method for mass propagation of a woody ornamentalIxora coccinea L.

Various factors that affect culture establishment, shoot growth, proliferation and rooting ofIxora coccinea L., a woody shrub, were studied. Stem cuttings (decapitated shoot, three nodes) were the most suitable explants for multiple-shoot proliferation, and when cultured on a woody plant medium (WPM) containing 2.5 M BA produced axillary shoots which branched repeatedly, yielding an average of 27 shoots per explant after 6 weeks in culture. Kinetin, 2-iP, zeatin and thidiazuron all induced multiple-shoot formation, but were less effective than BA. While the presence of IAA in the multiplication medium was detrimental to shoot proliferation, shoot growth was not affected by IAA. The production of large amounts of basal callus and vitrification of shoots were the major problems to be avoided in proliferating shoot cultures. Addition of TIBA to the multiplication medium markedly reduced basal callusing, while sealing the culture vessels with a fluorocarbon polymer (tetrafluoroethyleneperfluoroalkyl vinyl ether) film (Neoflon PFA film) almost completely eliminated vitrification. A reduction in the number of vitrified shoots was also achieved with AVG treatment. Following this protocol of using BA-supplemented WPM and Neoflon film, it would be possible to produce more than 100,000 plants from a single stem cutting in 1 year.Abbreviations AVG Aminoethoxyvinylglycine - BA N⁶-benzyladenine - BM basal medium - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2-tiP N⁶-(2-isopentenyl)adenine - KIN kinetin - MS Murashige and Skoog medium - NAA -naphthaleneacetic acid - SRM shoot regeneration medium - TDZ thidiazuron - TIBA 2,3,5-triiodobenzoic acid - WPM woody plant medium - ZEA zeatin