An Insert for the Reorienting Gradient Rotor for Virus Extraction from Infected Plants

device made of nylon and which is inserted in the bowl of the reorienting gradient rotor is described. This insert acts as container for holding infected plant tissue for the purpose of separating virus by centrifugal force from intact and fresh plants, from frozen and thawed plants and from plant tissue disintegrated by mechanical means. The extracted fluid represents 80 to 90% of that present in the untreated plant tissue. Electron micrographs taken of concentrates of the viruses prepared by the centrifugation extraction procedure indicate that extraneous materials are reduced to low level.     

5. An Insert for the Reorienting Gradient Rotor for Virus Extraction from Infected Plants

ABSTRACT

A device made of nylon and which is inserted in the bowl of the reorienting gradient rotor is described. This insert acts as a container for holding infected plant tissue for the purpose of separating virus by centrifugal force from intact and fresh plants, from frozen and thawed plants and from plant tissue disintegrated by mechanical means. The extracted fluid represents 80 to 90% of that present in the untreated plant tissue. Electron micrographs taken of concentrates of the viruses prepared by the centrifugation extraction procedure indicate that extraneous materials are reduced to a low level.     

Ice-cap. A high-throughput method for capturing plant tissue samples for genotype analysis

High-throughput genotype screening is rapidly becoming a standard research tool in the post-genomic era. A major bottleneck currently exists, however, that limits the utility of this approach in the plant sciences. The rate-limiting step in current high-throughput pipelines is that tissue samples from living plants must be collected manually, one plant at a time. In this article I describe a novel method for harvesting tissue samples from living seedlings that eliminates this bottleneck. The method has been named Ice-Cap to reflect the fact that ice is used to capture the tissue samples. The planting of seeds, growth of seedlings, and harvesting of tissue are all performed in a 96-well format. I demonstrate the utility of this system by using tissue harvested by Ice-Cap to genotype a population of Arabidopsis seedlings that is segregating a previously characterized mutation. Because the harvesting of tissue is performed in a nondestructive manner, plants with the desired genotype can be transferred to soil and grown to maturity. I also show that Ice-Cap can be used to analyze genomic DNA from rice (Oryza sativa) seedlings. It is expected that this method will be applicable to high-throughput screening with many different plant species, making it a useful technology for performing marker assisted selection.     

The effect of genetic transformations for pest resistance on foliar solanidine-based glycoalkaloids of potato (Solatium tuberosuni)

Foliage of potato cv. Desiree was harvested from glasshouse‐cultivated plants of five experimental transgenic lines expressing three different insecticidal proteins (snowdrop lectin, Galanthus nivalis agglutinin (GNA); jackbean lectin, Concanavalin A (Con A), cowpea trypsin inhibitor; (CpTi)), tissue‐cultured control plants and standard control (non‐tissue cultured) plants. The foliage was subdivided into stems, upper, middle and lower leaves and analysed separately by HPLC for the solanidine‐based glycoalkaloids a‐solanine and a‐chaconine. The results demonstrate that one or more stages in the plant transformation process (i.e. insecticidal‐ and marker‐gene insertions, gene expression and tissue culture) resulted in a lower level of leaf glycoalkaloids than that found in either the tissue‐cultured controls or standard controls, based on the selected potato lines transformed for insecticidal protein expression. However, the distribution of glycoalkaloids throughout the plant foliage was unaffected by genetic transformation and tissue culture, with the highest glycoalkaloid levels being observed in the top third of the plant. The importance of investigating unexpected effects of genetic engineering on plant secondary metabolism is discussed from an ecological viewpoint.     

Plant tissue culture. Biotechnology. Milestones

Summary The progress in the development of the technologies of plant tissue and cell culture over the past four decades has been remarkable. This article covers my personal reflections on the various topics and is based on my involvement in the field during that period. There are three fundamental technologies which constitute most of what is referred to as plant in vitro technologies or tissue culture. The origin and some of the key persons involved in the development of each of these procedures will be discussed. The technology that is most common is growing plant tissue on gel-solidified nutrient media. That technology is being used in the most vital procedures, namely the regeneration of plants from cultured cells. The culture of plant cells in liquid suspension was developed very shortly after that, and has become a very effective technology for plant regeneration by somatic embryogenesis. The method of meristem culture arose out of a need for developing plants that were virus-free. In many species the technique is now being used to produce virus-free crop plants. Another important technology is the culture of anthers and microspores for producing haploid and homozygous plants. Included with plant tissue culture is the development of the plant protoplast and cell fusion technologies for the production of new plant hybrids. The final aspect of the development concerns the integration of tissue culture with molecular genetics, which has developed into the rapidly expanding field of biotechnology.     

Non-native plants rarely provide suitable habitat for native gall-inducing species

For insect herbivores, a critical niche requirement—possibly the critical niche requirement—is the presence of suitable host plants. Current research suggests that non-native plants are not as suitable as native plants for native herbivores, resulting in decreases in insect abundance and richness on non-native plants. Like herbivores, gall-forming insects engage in complex, species-specific interactions with host plants. Galls are plant tissue tumors (including bulbous or spindle-shaped protrusions on leaves, stems and other plant organs) that are induced by insects through physical or chemical damage (prompting plants to grow a protective tissue shell around the insect eggs and larvae). As such, we hypothesized that gall-inducing insect species richness would be higher on native than non-native plants. We also predicted higher gall-inducing insect species richness on woody than herbaceous plants. We used an extensive literature review in which we compiled gall host plant species by genus, and we assigned native or non-native (or mixed) status to each genus. We found that native plants host far more gall-inducing insect species than non-native plants; woody plants host more gall-inducing species than herbaceous plants; and native woody plants host the most gall-inducing species of all. Gall-inducing species generally are a very cryptic group, even for experts, and hence do not elicit the conservation efforts of more charismatic insects such as plant pollinators. Our results suggest that non-native plants, particularly non-native woody species, diminish suitable habitat for gall-inducing species in parallel with similar results found for other herbivores, such as Lepidopterans. Hence, the landscape-level replacement of native with non-native species, particularly woody ones, degrades taxonomically diverse gall-inducing species (and their inquilines and parasitoids), removing multiple layers of diversity from forest ecosystems.

     

Phytoseiid predator of whitefly feeds on plant tissue

Predatory mites of the family Phytoseiidae feed on herbivorous mites and insects but they also use a variety of non-prey food items, such as pollen and nectar. Plant tissue is another potential food source. We investigated whether plant feeding occurs in the two phytoseiids Euseius scutalis (Athias-Henriot) and Typhlodromips swirskii (Athias-Henriot), which are natural enemies of whiteflies. These predatory mites can suppress populations of Bemisia tabaci (Gennadius) on isolated plants and are candidates for biological control of this pest. Both species can be reared on a diet of pollen, but E. scutalis requires a leaf tissue substrate, suggesting that this species might feed on plant tissue. To test this hypothesis, we applied a systemic insecticide (aldicarb) to cucumber plants and assessed the survival of predatory mites on leaves from insecticide-treated plants and untreated plants, both in presence and absence of pollen. The survival of T. swirskii was not affected by the presence of systemic insecticide in the plant. However, the survival of E. scutalis on leaves from insecticide-treated plants was 10 times lower than on leaves from untreated plants. Since the two species showed similar sensitivity to the insecticide when applied in a slide-dip test, this suggests that E. scutalis ingested insecticide through feeding on the leaf tissue. Mortality on treated leaves was observed both in absence and presence of pollen, suggesting that plant feeding is indispensable for E. scutalis. The extent to which plants are used as food by E. scutalis requires further analysis.     

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day ^1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.     

In vitro culture: an epigenetic challenge for plants

In vitro plant cell and tissue culture techniques are the basis of many micropropagation and breeding programs for scientific research. Plant tissue culture (PTC) involves organogenesis and embryogenesis, and the outcome depends on the different conditions to which the tissue is exposed. PTC is a stressful environment—high relative humidity, low ventilation rate, high concentrations of plant growth regulators, and low light availability—for plants that need to rapidly change their molecular regulation in order to respond fast and efficiently during cell division and growth. For instance, somatic embryogenesis (SE), which plays an important role in plant multiplication, requires complex cellular, biochemical and molecular processes for embryo formation and development. New data has come out about a connection between plant morphogenesis and epigenetics. Epigenetics is a very sensitive regulatory mechanism, which in most of cases is affected by the environment. Although it is known that, under plant morphogenesis, the genome has little or no change, DNA methylation and histone modifications are very susceptible to those in vitro environmental conditions. In the present review, we highlight the most used in vitro systems such as organogenesis and SE in plants and discuss how epigenetics plays a pivotal role in the phenotype outcome. Furthermore, we discuss the big role that the small RNAs have during cell division and propagation and propose different challenges and opportunities to study epigenetics in plant cell tissue and organ cultures.     

Tissue analyses guidelines for diagnosing sulfur deficiency in white wheat

Summary Visual identification of S deficiency in white wheat is difficult since deficiency symptoms are nearly identical with those of N deficiency. In this study, S deficiency was best identified by determining the total N/S ratio rather than S concentration in vegetative tissue. Vegetative growth generally decreased from tillering to boot when the whole plant N/S ratio exceeded 17. The N/S ratio in S-sufficient plants declined gradually with age, implying that the critical N/S ratio may decline with advancing growth. Changes in stem: leaf ratio could have been responsible for the decline since the N/S ratio in stem tissue at heading was less than that of green leaf tissue.Sulphur concentration less reliably indicated S-deficiency, because differences in S levels between S-deficient and S-sufficient wheat, were often less than year-to-year variation of S concentration of plants sampled at the same growth stage. In addition, S concentration in whole plants declined sharply between tillering and heading. These factors make it difficult to designate a critical S level. Sulfur distribution among various plant organs suggests that critical S levels might best be obtained by utilizing green leaf tissue.Nitrogen concentration in S-sufficient wheat plants also decreased quite rapidly with growth, which indicates a similar difficulty for determining critical N percentages. Consequently, the most reliable distinction between N and S deficiency in wheat was accomplished by evaluation of the total N/S ratio in whole plant tissue.Contribution from the Agricultural Research Service, USDA, in cooperation with the Agricultural Experiment Station, Oregon State University. Technical Paper No.3953 of the latter.     

From phenotype to genotype: whole tissue profiling for plant breeding

Fourier transform infrared spectroscopy (FT-IR) was used to obtain ‘holistic’ metabolic fingerprints from a wide range of plants to differentiate species, population, single plant genotype, and chromosomal constitution differences. Sample preparation simply entailed the maceration of fresh leaves with water, and these samples were then dried and analysed by reflectance FT-IR where spectral acquisition was typically 10 s. All samples gave reproducible, characteristic biological infrared absorption spectra and these were analysed by chemometric methods. FT-IR is not biased to any particular chemical species and thus the whole tissue profiles produced measure the total biochemical makeup of the test sample; that is to say it represents a plant phenotype. We show that by simple cluster analysis these phenotypic measurements can be related to the genotypes of the plants and can reliably differentiate closely related individuals. We believe that this approach provides a valuable new tool for the rapid metabolomic profiling of plants, with applications to plant breeding and the assessment of substantial equivalency for genetically-modified plants.     

The effect of plant identity and the level of plant decay on molecular gut content analysis in a herbivorous soil insect

Plant roots represent an important food source for soil‐dwelling animals, but tracking herbivore food choices below‐ground is difficult. Here, we present an optimized PCR assay for the detection of plant DNA in the guts of invertebrates, using general plant primers targeting the trnT‐F chloroplast DNA region. Based on this assay, we assessed the influence of plant identity on the detectability of ingested plant DNA in Agriotes click beetle larvae. Six different plant species were fed to the insects, comprising a grass, a legume and four nonlegume forbs. Moreover, we examined whether it is possible to amplify DNA of decaying plants and if DNA of decayed plant food is detectable in the guts of the larvae. DNA of the ingested roots could be detected in the guts of the larvae for up to 72‐h post‐feeding, the maximum digestion time tested. When fed with living plants, DNA detection rates differed significantly between the plant species. This may be ascribed to differences in the amount of plant tissue consumed, root palatability, root morphology and/or secondary plant components. These findings indicate that plant identity can affect post‐feeding DNA detection success, which needs to be considered for the interpretation of molecularly derived feeding rates on plants. Amplification of plant DNA from decaying plants was possible as long as any tissue could be retrieved from the soil. The consumption of decaying plant tissue could also be verified by our assay, but the insects seemed to prefer fresh roots over decaying plant material.     

植物化学通讯研究进展

 生物的信息传递是生命科学中引人入胜的研究领域之一,生物种间种内和个体内都存在着物理和化学等各种信息交流方式。植物种间种内是否通过物理信号进行通讯交流还是一个未知数,但邻近的同种或异种植物通过化学物质为媒介的通讯关系确是客观存在的。最近,愈来愈多的研究证明：许多陆生植物种可以合成并释放特定的次生物质,这些次生物质可以通过空气和土壤两种载体进行信息传递,尤其是在植物受到侵袭和寄生条件下。茉莉酮酸甲酯、水杨酸甲酯和乙烯等挥发性次生物质被确证为以空气为媒介进行植物种间和种内通讯的化学信号分子。植物根分泌的黄酮和氢醌等分子也可以经土壤媒介传递信息。由于在自然条件下植物根系分泌物的收集和活性信号分子的俘获及鉴定技术还未能突破,这增加了以土壤为媒介的植物种间和种内化学通讯关系研究的难度。但不论如何,植物的化学通讯是植物种间和种内交流的主要方式,植物间的化学通讯关系的研究还处于突破的前夜,这方面的任一研究成果都会引起世界性的关注。因此,破译植物种间和种内化学通讯密码具有重要的学术价值。     

Beauveria bassiana (Balsamo) Vuillemin as an endophyte in tissue culture banana (Musa spp.)

Beauveria bassiana is considered a virulent pathogen against the banana weevil Cosmopolites sordidus. However, current field application techniques for effective control against this pest remain a limitation and an alternative method for effective field application needs to be investigated. Three screenhouse experiments were conducted to determine the ability of B. bassiana to form an endophytic relationship with tissue culture banana (Musa spp.) plants and to evaluate the plants for possible harmful effects resulting from this relationship. Three Ugandan strains of B. bassiana (G41, S204 and WA) were applied by dipping the roots and rhizome in a conidial suspension, by injecting a conidial suspension into the plant rhizome and by growing the plants in sterile soil mixed with B. bassiana-colonized rice substrate. Four weeks after inoculation, plant growth parameters were determined and plant tissue colonization assessed through re-isolation of B. bassiana. All B. bassiana strains were able to colonize banana plant roots, rhizomes and pseudostem bases. Dipping plants in a conidial suspension achieved the highest colonization with no negative effect on plant growth or survival. Beauveria bassiana strain G41 was the best colonizer (up to 68%, 79% and 41% in roots, rhizome and pseudostem base, respectively) when plants were dipped. This study demonstrated that, depending on strain and inoculation method, B. bassiana can form an endophytic relationship with tissue culture banana plants, causing no harmful effects and might provide an alternative method for biological control of C. sordidus.     

Opine synthesis in wild-type plant tissue

Opine production is associated with crown gall tissue, a neoplastic growth caused by infection of dicotyledonous plants with Agrobacterium tumefaciens. Recent publications have claimed that tissues of certain monocotyledonous plants can also be infected by Agrobacterium. Following infection, a part of the Agrobacterium Ti plasmid, T-DNA, is integrated into the chromosome of the infected plant. T-DNA, which codes for opine-synthesizing enzymes, is now used to add foreign genes to plants. A number of laboratories have used opine production in plant tissue, often after arginine feeding or preincubation as evidence for plant transformation by T-DNA vectors. In this report we provide microbiological, chromatographic, spectroscopic and chemical evidence indicating that opines can be formed in normal callus and plant tissue as a result of arginine metabolism. Therefore, researchers studying T-DNA should be aware of the capability of plant tissue to metabolize arginine to opines. Opine production following infection with T-DNA may not always be sufficient evidence to indicate transformation by the Agrobacterium Ti plasmid.     

Metabolic engineering of plants for alkaloid production.

Alkaloids purified from plants provide many pharmacologically active compounds, including leading chemotherapy drugs. As is generally true of secondary metabolites, overall productivity is low, making commercial production expensive. Alternative production methods remain impractical, leaving the plant as the best source for these valuable chemicals. Recently, significant progress in characterizing the biosynthetic pathways leading to various alkaloids has been made, and a number of relevant genes have been cloned. Metabolic engineering employing such genes provides a promising technology for improved productivity in plant cell cultures, plant tissue cultures, or intact plants. In exploring solutions though, metabolic engineers must be careful to recognize the limitations inherent in designing plant systems.     

Plant population growth and competition in a light gradient: a mathematical model of canopy partitioning

Can a difference in the heights at which plants place their leaves, a pattern we call canopy partitioning, make it possible for two competing plant species to coexist? To find out, we examine a model of clonal plants living in a nonseasonal environment that relates the dynamical behavior and competitive abilities of plant populations to the structural and functional features of the plants that form them. This examination emphasizes whole plant performance in the vertical light gradient caused by self-shading. This first of three related papers formulates a prototype single species Canopy Structure Model from biological first principles and shows how all plant properties work together to determine population persistence and equilibrium abundance. Population persistence is favored, and equilibrium abundance is increased, by high irradiance, high maximum photosynthesis rate, rapid saturation of the photosynthetic response to increased irradiance, low tissue respiration rate, small amounts of stem and root tissue necessary to support the needs of leaves, and low density of leaf, stem, and root tissues. In particular, equilibrium abundance decreases as mean leaf height increases because of the increased cost of manufacturing and maintaining stem tissue. All conclusions arise from this formulation by straightforward analysis. The argument concludes by stating this formulation's straightforward extension, called a Canopy Partitioning Model, to two competing species.     

Evidence for a senescence-associated gene induced by darkness

Tobacco (Nicotiana tabacum) plants transformed with a chimeric tobacco anionic peroxidase gene have previously been shown to synthesize high levels of peroxidase in all tissues throughout the plant. One of several distinguishable phenotypes of transformed plants is the rapid browning of pith tissue upon wounding. Pith tissue from plants expressing high levels of peroxidase browned within 24 hours of wounding, while tissue from control plants did not brown as late as 7 days after wounding. A correlation between peroxidase activity and wound-induced browning was observed, whereas no relationship between polyphenol oxidase activity and browning was found. The purified tobacco anionic peroxidase was subjected to kinetic analysis with substrates which resemble the precursors of lignin or polyphenolic acid. The purified enzyme was found to readily polymerize phenolic acids in the presence of H₂O₂ via a modified ping-pong mechanism. The percentage of lignin and lignin-related polymers in cell walls was nearly twofold greater in pith tissue isolated from peroxidase-overproducer plants compared to control plants. Lignin deposition in wounded pith tissue from control plants closely followed the induction of peroxidase activity. However, wound-induced lignification occurred 24 to 48 hours sooner in plants overexpressing the anionic peroxidase. This suggests that the availability of peroxidase rather than substrate may delay polyphenol deposition in wounded tissue.     

Field performance of transgenic potato plants compared with controls regenerated from tuber discs and shoot cuttings

Summary The objective of this study was to separate and determine effects on the field performance of transgenic potatoes that originate from the tissue culture process of transformation and from the genes inserted. The constructs introduced contained the reporter gene for betaglucuronidase (GUS) under the control of the patatin promoter (four different constructs) and the neomycin phosphotransferase gene under the control of the nopaline synthase promoter. Both genes might be expected to have a neutral effect on plant phenotype. The field performance of transgenic plants (70 independent transformants) was compared with non-transgenic plants regenerated from tuber discs by adventitious shoot formation and from shoot cultures established from tuber nodal cuttings. Plants from all three treatments were grown in a field trial from previously field-grown tubers, and plant performance was measured in terms of plant height at flowering, weight of tubers, number of tubers, weight of large tubers and number of large tubers. There was evidence of somaclonal variation among the transgenic plants; mean values for all characters were significantly lower and variances generally higher than from plants derived from nodal shoot cultures. A similar change in means and variances was observed for the non-transgenic tuber-disc regenerants when compared with shoot culture plants. Plant height, tuber weight and tuber number were, however, significantly lower in transgenic plants than in tuber-disc regenerants, suggesting an effect on plant performance either of the tissue culture process used for transformation or of the genes inserted. There were significant differences between constructs for all five plant characters. The construct with the smallest segment of patatin promoter and the lowest level of tuber specificity for GUS expression had the lowest values for all five characters. It is proposed that the nature of GUS expression is influencing plant performance. There was no indication that the NPTII gene, used widely in plant transformation, has any substantial effect on plant performance in the field.