Improvement of regeneration ability in Phleum pratense L. in vitro culture by dicamba

Calli were induced from mature caryopses of timothy grass (Phleum pratense L.) on MS medium (Murashige and Skoog 1962) supplemented with 500 mg·dm⁻³ casein hydrolysate and 5 mg·dm⁻³ 2,4-D (2,4-dicholorophenoxyacetic acid) or 2 mg·dm⁻³ dicamba (3,6-dichloro-o-anisic acid). Twelve-week-old calli were passaged on media with reduced levels of auxins (2 mg·dm⁻³ 2,4-D or 1 mg·dm⁻³ dicamba). Tissues induced on medium with 2,4-D were transferred on medium with 2,4-D and on medium with dicamba; parallely calli initiated on medium with dicamba were passaged on medium with 2,4-D or dicamba. Calli from various media sequences were used to establish cell suspension cultures in media containing 2 mg·dm⁻³ 2,4-D or 1 mg·dm⁻³ dicamba. An assessment of regeneration ability of calli was made on MS medium containing 0.2 mg·dm⁻³ kinetin. Callus tissue induced and/or subcultured on any of the media with 2,4-D did not regenerate plants while dicamba added to the media was the effective stimulator of regenerability. In the presence of 2,4-D calli and suspensions produced a jelly-like extracellular matrix. In cell suspension this phenomenon was observed 4–5 days after each passage. The measurements of electric potential of calli, growing on MS medium with kinetin were performed. Non-regenerating callus areas had an electric potential close to 0 mV while parts of tissue with meristematic centres were characterized by lower values of electric potential.     

Structural diversity of Papaver somniferum L. cell surfaces in vitro depending on particular steps of plant regeneration and morphogenetic program

Culture of Papaver somniferum in vitro was used for a characterisation of cell surface structures and mode of cell adhesion and cell separation during cell differentiation and plant regeneration in somatic embryogenesis and shoot organogenesis. In early stages of somatic embryogenesis, cell type-specific and developmentally regulated change of cell morphogenesis was demonstrated. Cell wall of separated embryonic cells were self-covered with external tubular network, whereas morphogenetic co-ordination of adhered cells of somatic proembryos was supported by fine and fibrillar external cell wall continuum of peripheral cells, interconnecting also local sites of cell separation. Such type of cell contacts disappeared during histogenesis, when the protodermis formation took place. Tight cell adhesion of activated cells with polar cell wall thickening, and production of extent mucilage on the periphery were the crucial aspects of meristemoids. Fine amorphous layer covered developing shoot primordia, but we have not observed such comparable external fibrillar network. On the contrary intercellular separation of differentiated cells in regenerated organs, and accepting distinct developmental system of somatic embryogenesis and shoot organogenesis, cell adhesion in early stages and ultrastructural changes associated with tissue disorganisation, and the subsequent reorganisation into either embryos or shoots appear to be regulatory morphogenetical events of plant regeneration in vitro.     

Plant regeneration of mangosteen via nodular callus formation

Nodular callus was induced at a high frequency on young purple red, 5–15 mm long laminae taken from in vitro grown plants of mangosteen. The optimal medium was composed of Murashige and Skoog (MS) nutrients supplemented with 2.22 μM benzyladenine (BA), 2.25 μM thidiazuron (TDZ), 500 mg l^-1 polyvinylpyrrolidone (PVP 360 000) and 3% sucrose. A multiplication rate of two–three was obtained by subculture of the nodular callus at 3–4-week intervals. Plantlet regeneration from the nodules was achieved by transfer to woody plant medium (WPM) with 500 mg l^-1 PVP, 0.4 μM BA and 3% sucrose and overlaying with half strength liquid MS containing 0.32 μM naphthaleneacetic acid (NAA), 0.13 μM BA and 3% sucrose. Elongated shoots were rooted to 100% when wounded at the base of shoot, dipped in 4.4 mM indolebutyric acid (IBA) solution in the dark for 15 min and cultured on WPM supplemented with 1.11 μM BA, 0.25% activated charcoal, 34.5 μM phloroglucinol (PG) and 3% sucrose. This revised version was published online in June 2006 with corrections to the Cover Date.     

Simplified clonal multiplication of mulberry using liquid shake culture

Organogenesis was induced in callus derived from mature zygotic embryos of six families of loblolly pine (Pinus taeda L.) within 24 weeks of culture. Elongation of adventitious buds was achieved on TE medium supplemented with 0.5 mg l⁻¹ indole-3-butyric acid (IBA) and 2 mg l⁻¹ 6-benzyladenine (BA). The most suitable medium for root formation proved to be TE medium supplemented with 0.1 mg l⁻¹ IBA, 1 mg l⁻¹ BA, and 0.5 mg l⁻¹ gibberellic acid (GA₃). One hundred and sixty-nine regenerated plantlets were transferred to a perlite:peatmoss:vermiculite (1:1:1) soil mixture, and 98 plantlets survived in the field. This revised version was published online in June 2006 with corrections to the Cover Date.     

Production of transgenic tropical maize with cryIAb and cryIAc genes via microprojectile bombardment of immature embryos

To enhance the level of resistance to insects in tropical maize germplasm we have developed techniques to successfully transform elite tropical maize inbred based on the activity of specific cryI proteins against four major maize pests – corn earworm, fall armyworm, southwestern corn borer and sugarcane borer. Constructs containing cryIAb or cryIAc synthetic genes were used. To generate transgenic plants we have established methods for biolistic bombardment and the selection and regeneration of immature embryos and calli from the elite tropical lines CML72, CML216, CML323, CML327 and hybrids. Transgenic plants resistant to the herbicide Basta^TM contained the bands for the cry, bar and gus genes as detected by Southern blot analyses. A simple leaf bioassay presented varying levels of resistance to Southwestern corn borer of transgenic tropical maize carrying the cryIAc gene. Analyses of the progenies confirmed the sexual transmission of the introduced genes and their stable expression. Received: 25 September 1998 / Accepted: 27 October 1998     

Influence of genotype and gelling agents on in vitro regeneration by organogenesis in sunflower

Fourteen recombinant inbred lines of sunflower (Helianthus annuus L.) and their parents (PAC-2 and RHA-266) were tested for their organogenesis ability. Seeds were surface sterilized and germinated on hormone free half strength MS basal medium containing 10 g l^-1 sucrose solidified with five different gelling agents: Phytagar (Gibco laboratoires) 3 g l^-1, Phytagel (Sigma) 3 g l^-1, Agarose (Sigma) 5 g l^-1, Arcagel (Sigma) 4 g l^-1 and Agar-Agar (Fisher France) 7 g l^-1. Cotyledons from 2-day-old seedlings were split in half and the four explants of each seed were cultived in 55 mm diameter petri dishes containing 10 ml of MS medium supplemented with 50 μM KNO₃, 1 μM myo-inositol, 5 μM casein hydrolysate, 4.4 μM of BA and 5.4 μM of NAA solidified with the same gelling agents. The experimental design was a randomized complete block with 3 replications. A replicate for each genotype consisted of ten petri dishes containing four explants. The statistical analysis showed significant differences among genotypes and gelling agents. Of the fourteen recombinant inbred lines tested `C93' presented the highest values for all regeneration traits in the five different media and it was better than the best parent. Agarose and Agar-Agar were more better than other gelling agents for shoot induction. This revised version was published online in June 2006 with corrections to the Cover Date.     

Topography of cell traces studied by atomic force microscopy

Migrating adherent cells release material onto artificial substrates like glass and silicon while moving. Traces of mouse fibroblasts (L929) have been visualised by atomic force microscopy (AFM). “Non-contact” mode AFM in a liquid environment can extract topographic information from these traces. This dynamic mode allows the study of these soft structures without damage or compression. The AFM images show crossing and branching networks (with specific angles of branching), structured patches, nodular elements, linear elements with irregular height and other features. Fourier analysis of segment spacing in the strands is presented. These spatial features of fibroblast traces are strong indications that actin linked to structural proteins is involved in the formation of cell traces. We also give methods for trace preparation and undistorted imaging and discuss further perspectives. Received: 11 January 1999 / Revised version: 1 April 1999 / Accepted: 8 April 1999     

Direct electrochemical regeneration of NADH from NAD+ using cholesterol-modified gold amalgam electrode

The efficiency of the direct electrochemical regeneration of NADH from NAD+ was enhanced by applying a cholesterol-modified gold amalgam electrode. The modified electrode was prepared by immersing gold plate in mercury and casting few drops of cholesteryl oleate solution over the gold amalgam. Coenzymatically active NADH was formed from NAD+ directly at the cholesterol-modified gold amalgam electrode which is supposed to hinder the dimerization of the NAD radicals on its membrane surface. The direct electrochemical NAD+ reduction process was used favorably to drive an enzymatic reduction of pyruvate to d-lactate. d-Lactate of 18.2 mm was obtained from pyruvate of 25.3 mm at 21 h of total reaction time in the electrolysis of 50 cm3 solution with the electrode of 6 cm2area. The turnover number for NAD+ was estimated as 1400.     

Establishment of the invasive perennial herb Bunias orientalis L.: An experimental approach

A 2-year field experiment was performed to assess regeneration, establishment and growth of the invasive polycarpic perennial Brassicaceae Bunias orientalis L. from transplanted juveniles and propagules (seeds, fruits and root fragments). Further treatments were: different planting depths of the propagules, different root fragment sizes and a varied competition intensity by the matrix vegetation. In contrast to a high number of individuals established from planted juveniles (ca. 90 %), only relatively few propagules gave rise to seedlings or regenerating plants (0–16 %). However, upon emergence, all plants showed low mortality. Although the number of regenerated or recruited plants was affected by all treatments, only one treatment level (the use of very small root fragments of 1 cm length) completely failed in establishing plants. Plants experiencing low competition intensity attained high aboveground performance and reproductive output as well as reproductive success within two growth periods. Under high competition intensity recruitment, regeneration and growth were strongly negatively affected. The high morphological plasticity of rosette growth of B. orientalis seems to be advantageous for persistence in such situations. The results show that young B. orientalis plants are robust and can efficiently sequester and translate elevated resources into rapid growth and early reproductive output although the species is a rather long-lived iteroparous perennial. In addition, population foundation or regeneration can be based on both seeds and root fragments, even when buried under a thick soil layer. This seems to be a very efficient adaption to habitats which are subject to anthropogenic soil perturbations.     

Norway Maple (Acer platanoides) Invasion of a Natural Forest Stand: Understory Consequence and Regeneration Pattern

Norway maple (Acer platanoidesis) is invasive in a natural stand in suburban Ithaca, NY. To determine the understory pattern and consequences of a Norway maple invasion, I compared density and species richness under Norway maples and sugar maples (Acer saccharum). Mean sapling density was significantly lower (P<0.0027) under Norway maples (3.64/100 m²±1.6 SE) than under sugar maples (19.4/100 m²±4.4 SE). Mean sapling species richness was significantly lower (P<0.0018) under Norway maples (0.7/32 m²±0.18 SE) than under sugar maples (2.6/32 m²±0.48 SE). Likewise, Norway maple regeneration is more frequent under sugar maples than sugar maple regeneration: 57% of sugar maple plots had Norway maple saplings while 0% of Norway maple plots had sugar maple saplings. Two significant plot effects were found for presence–absence: Norway maple saplings grow under Norway maples with a significantly lower frequency (P<0.03) than under sugar maples; sugar maple saplings grow under Norway maples with a significantly lower frequency (P<0.000) than under sugar maples. Across the site, Norway maple saplings were the most abundant (29 saplings for 480 m²). The success of Norway maple regeneration and the reductions in total stem density beneath Norway maples is most likely the result of its strong competitive abilities, notably its high shade tolerance and abundant seed crops.