Fast-track applications: The potential for direct delivery of proteins and nucleic acids to plant cells for the discovery of gene function

In animal systems, several methods exist for the direct delivery of nucleic acids and proteins into cells for functional analysis. Until recently, these methods have not been applied to plant systems. Now, however, several preliminary reports suggest that both nucleic acids and proteins can also be delivered into plant cells by very simple, direct application. This promises to open the way for high-throughput screening for gene function in a range of plant species.     

Cell type-specific characterization of nuclear DNA contents within complex tissues and organs

Background  

Eukaryotic organisms are defined by the presence of a nucleus, which encloses the chromosomal DNA, and is characterized by its DNA content (C-value). Complex eukaryotic organisms contain organs and tissues that comprise interspersions of different cell types, within which polysomaty, endoreduplication, and cell cycle arrest is frequently observed. Little is known about the distribution of C-values across different cell types within these organs and tissues.     

Review of methodologies and a protocol for the <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of wheat

Since the first report of wheat transformation by Agrobacterium tumefaciens in 1997, various factors that influence T-DNA delivery and regeneration in tissue culture have been further investigated and modified. This paper reviews the current methodology literature describing Agrobacterium transformation of wheat and provides a complete protocol that we have developed and used to produce over one hundred transgenic lines in both spring and winter wheat varieties.     

Host Specialization not Detected Among Isolates of the EC-1 Lineage of <Emphasis Type="Italic">Phytophthora infestans</Emphasis> Attacking Wild and Cultivated Potatoes in Peru

To determine whether populations of Phytophthora infestans attacking wild and cultivated potatoes in the highlands of Peru are specialized on their hosts of origin, we characterized isolates using several neutral markers, metalaxyl resistance and for aggressiveness in a detached leaf assay. One hundred and fifty-three isolates were collected from the northern and central highlands of Peru from different potato cultivars (both modern and native cultivars) and from different species of wild, tuber-bearing potatoes. All the isolates analyzed belonged to one of four clonal lineages that had been described previously in Peru: EC-1, US-1, PE-3 and PE-7. The EC-1 lineage (n = 133) was dominant and present in similar frequencies on wild and cultivated potatoes. PE-3 (n = 14) was found primarily on cultivated potatoes, with only one isolate coming from a wild host. US-1 (n = 2) and PE-7 (n = 4) were rare; all but one (PE-7) occurred on wild potatoes. Isolates from the EC-1 lineage from modern cultivars were compared in three separate detached leaf inoculation assays with EC-1 isolates from the wild potato species S. sogarandinum, S. bill-hookerii or S. huancabambense, respectively. No significant interactions between isolate type (from wild or cultivated potato) and host type (wild or cultivated) were measured for any assay. It appears that the pathogen genotypes in the EC-1 lineage indiscriminately attack both wild and cultivated tuber-bearing solanaceous hosts in Peru, and breeders should be able to select for resistance using the common EC-1 lineage.     

Germination of <Emphasis Type="Italic">Plasmodiophora brassicae</Emphasis> resting spores stimulated by a non-host plant

Plant-induced germination of Plasmodiophora brassicae resting spores was studied in a laboratory experiment. Spore reaction was analysed in nutrient solution with exudates from growing roots of different plant species – one host plant (Brassica rapa var. pekinensis) and four non-host plants (Lolium perenne, Allium porrum, Secale cereale and Trifolium pratense) – and in controls with distilled water and nutrient solution. It was found that root exudates from L. perenne stimulated spore germination more than exudates from the other plants, including those from the host plant. The effect could not be explained by differences in the nutritional composition of the solutions due to differential uptake of the plant species, or by differences in root activity, measured as exudation of soluble sugars. This is the first time such a separation of factors has been done in analysing the influence of plants on P. brassicae germination. Although stimulation of P. brassicae resting spore germination is not restricted to the presence of host plants, it seems to vary depending on the plant species.     

Preliminary results on detection of maize treated with electrons for seed dressing and according decontamination and infestation

Saatgut und Getreide kann mit niederenergetischen Elektronen (<300 kev) oder=" hochenergetischen=" elektronen=" (1–10 mev)=" wirksam=" behandelt=" werden,=" um=" mikroorganismen=" und=" insekten=" abzutöten.=" in=" dieser=" vorläufigen=" studie=" wurde=" mais=" mit=" niederenergetischen=" (125 kev)=" und=" hochenergetischen=" elektronen=" (10 mev)=" behandelt.=" um=" diese=" elektronenbehandlung=" nachzuweisen,=" wurden=" verschiedene=" verfahren=" eingesetzt:=" photostimulierte=" lumineszenz=" (psl),=" thermolumineszenz=" (tl)=" und=" dna-kometentest.=" für=" diese=" drei=" nachweismethoden=" existieren=" bereits=" europäische=" normen=" und=" sie=" sind=" als=">Allgemeine Codex Methoden zum Nachweis bestrahlter Lebensmittel etabliert. Die Ergebnisse zeigen, dass PSL und TL geeignete Verfahren sind, um sowohl eine Behandlung von Mais mit niederenergetischen als auch mit hochenergetischen Elektronen zu erkennen. Der DNA-Kometentest erwies sich als weniger geeignet: die Behandlung mit niederenergetischen Elektronen konnte—wie erwartet—nicht nachgewiesen werden. Die Behandlung mit hochenergetischen Elektronen konnte bei einer Maissorte erkannt werden, jedoch nicht bei einer anderen Sorte.     

Maceration of plant tissue by fungi is inhibited by recombinant antipectinase antibodies

Polyclonal antiserum from mice immunized with extracellular proteins from Rhizoctonia solani inhibited pectinase and cellulase activities in cell free culture supernatants of Rhizoctonia solani. Spleen mRNA from these mice was used to construct a cDNA library from which antipectinase ScFv antibodies were isolated using phage display techniques. Soluble ScFv antibodies produced by individual clones in Escherichia coli inhibited polygalacturonase in the culture supernatants of a range of fungal pathogens, including ascomycetes, basidiomycetes, and oomycetes. The soluble antibodies also inhibited maceration of potato tissue by these pathogens.     

Germination of <Emphasis Type="Italic">Gibberella zeae</Emphasis> ascospores as affected by age of spores after discharge and environmental factors

The effects of age of ascospores (0–18 days after discharge), photon flux density (0–494 mol m^–2 s^–1 PAR), temperature (4–30 °C), frost (–15 °C for 30 min), relative humidity (RH; 0–100%), pH (2.5–6.5) and dryness (0 and 53% RH for up to 40 min) on the germination of the ascospores of the mycotoxin-producing fungus Gibberella zeae (anamorph Fusarium graminearum) were studied. Freshly discharged ascospores germinated within 4 h at 20 °C and 100% RH. The rate of germination and the percentage of viable ascospores decreased over time after the spores were discharged from perithecia. The time course of ascospore germination was not significantly affected by photon flux density. The period of time required to obtain 50% germinated ascospores at 100% RH was 26.90 h at 4 °C, 10.40 h at 14 °C, 3.44 h at 20 °C and 3.31 h at 30 °C. There was no significant effect of frost on the percentage of viable ascospores. A small percentage (6.6 ± 3.8%) of the ascospores germinated at 53% RH. At RH 84% and 20 °C almost 100% of the freshly discharged ascospores germinated. The time course of ascospore germination was affected by pH. The maximum rate of ascospore germination was estimated to be at pH 3.76. Ascospores lost their ability to germinate following exposure to 0% RH almost instantaneously. No germinating spores were detected after an incubation period of 1 min at 0% RH. Incubating the ascospores at 53% RH decreased the percentage of viable spores from 93 to 6% within 10 min. The data demonstrate that age of spores, relative humidity, temperature and pH, but not photon flux density, are key factors in germination of G. zeae ascospores.     

Molecular characterization of <Emphasis Type="Italic">Pythium</Emphasis> group F isolates by ribosomal-and intermicrosatellite-DNA regions analysis

Pythium group F is a ubiquitous, though minor, pathogen in several soilless and soil cultures; investigations were carried out to analyze different regions of the DNA and better understand the nature of this group. Fourty-two isolates were obtained from a variety of plants (cucumber, lettuce, tomato) grown in soil or soilless cultures collected in various countries (Canada, Denmark, France, Norway, Sweden and United Kingdom). All Pythium group F isolates displayed amplified ITS1-5,8S-ITS2 ribosomal DNA region (rDNA) of similar length, whereas polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) revealed that, among the seven enzymes used, polymorphism was only identified with Hin6I. After cloning of ITS1-5,8S-ITS2 rDNA region from Pythium group F isolates that displayed restriction polymorphism patterns with Hin6I, comparisons of sequence and restriction mapping data showed a slight variation consisting in a single base change. Inter Simple Sequence Repeat (ISSR)-PCR method was also used to obtain data related to the entire genome and not only to a single DNA region. It identified repeated motifs in the genome of Pythium group F isolates. Two primers (CAC)₅ and (CCA)₅ detected polymorphism, and isolates were classified among 11 molecular clusters. The genetic diversity of this group was not correlated with the geographical locations or the host plants from which the isolates originated. Polymorphism of Pythium group F isolates pointed out by ISSR is discussed