Virus-induced gene silencing as a tool for functional analyses in the emerging model plant Aquilegia (columbine, Ranunculaceae)

Background  

There is considerable interest in rapid assays or screening systems for assigning gene function. However, analysis of gene function in the flowers of some species is restricted due to the difficulty of producing stably transformed transgenic plants. As a result, experimental approaches based on transient gene expression assays are frequently used. Biolistics has long been used for transient over-expression of genes of interest, but has not been exploited for gene silencing studies. Agrobacterium-infiltration has also been used, but the focus primarily has been on the transient transformation of leaf tissue.     

Virus-induced PpCHLH gene silencing in peach leaves (Prunus persica)

Summary

Compared with other model plants or crops, studies on the molecular biology of fruit trees have lagged behind due to technical difficulties in gene transformation and manipulation. Therefore, developing an efficient system for gene manipulation is of particular significance in fruit trees. Here, we report on a method for virus-induced gene silencing (VIGS) by syringe-infiltrating a tobacco rattle virus (TRV) vector containing a specific target gene sequence into peach (Prunus persica) leaves to analyse gene function. The target gene (PpCHLH) was a 4,445 bp sequence encoding the H subunit of magnesium chelatase and was first cloned as a cDNA. This gene (PpCHLH) is reported to be related to chlorophyll biosynthesis, and any loss of function leads to a decrease in chlorophyll content, with concomitant yellow or white colour changes in the leaves. To silence the PpCHLH gene, a 1:1 mixture of Agrobacterium tumefaciens strain GV3101 cultures containing pTRV1 or a pTRV2 vector construct with a 650 bp cDNA fragment of the PpCHLH gene was infiltrated into leaves of 4 – 5 week-old peach seedlings. After 15 d, the inoculated areas of the green leaves faded and finally turned yellow or white. Loss of PpCHLH gene function was confirmed by semi-quantitative RT-PCR, real-time qRT-PCR, and siRNA northern blot analysis. The virus-induced gene silencing (VIGS) technique developed here could be used for further molecular studies on fruit trees.     

Interrelationships between plant functional types and soil moisture heterogeneity for semiarid landscapes within the grassland/forest continuum: a unified conceptual model

In semiarid landscapes, the ratio of herbaceous to woody plant biomass is a major determinant of ecosystem properties. This ratio depends to a large extent on the amount and spatial distribution of soil moisture that is available to plants, and these variables, in turn, are determined primarily by climate and land use. Current conceptual models for determining the ratio of herbaceous to woody plant biomass in semiarid plant communities are based either on differences in soil moisture with depth (vertical heterogeneity) from one site to another (Walter's two-layer model) or on differences in soil moisture between canopy and intercanopy patches at the same site (horizontal heterogeneity) that result from disturbances associated with land use (Schlesinger et al.'s model of desertification). We developed a model that unifies these two perspectives by relaxing two assumptions of Walter's two-layer model. First, our model recognizes that soil moisture varies horizontally between canopy and intercanopy patches, not only due to land-use disturbance, a general assumption of the Schlesinger et al. model, but also due to the physical nature of the canopy itself. Second, while retaining the general assumption of Walter that woody plants obtain moisture from deeper soil layers than do herbaceous plants, our model recognizes the existence of two types of woody plants: those that extract a substantial proportion of their moisture from deeper layers and those that extract mainly from shallower layers. By modifying the two-layer hypothesis to include four soil compartments and distinguishing between shallow- and deeper-rooted woody species, our model integrates three key concepts in semiarid ecology: (1) the proportion of woody cover increases as moisture in the deeper soil layers increases (Walter's two-layer hypothesis for coexistence of herbaceous and woody plants); (2) land use practices that cause a reduction in herbaceous vegetation and compaction of intercanopy soils lead to a long-term increase in the proportion of woody plants (Schlesinger et al.'s concept, or more generally, that at a given site multiple variations in the proportions of herbaceous and woody plant biomass are possible); and (3) changes in the ratios of herbaceous to woody plant biomass exhibit complex behavior (changes can happen quickly and are not directly reversible without intensive management). This integration of concepts results because rather than assuming a simple, one-way dependence of plant functional types on soil moisture heterogeneity, our model assumes an interdependence between the two: soil moisture heterogeneity constrains the composition of the plant community, which in turn modifies soil moisture heterogeneity. The four-compartment model that we propose enables, for the first time, an integrated picture of both dimensions of soil moisture heterogeneity – horizontal and vertical – and of the interdependence between soil moisture heterogeneity and the proportions of the plant functional types that make up a given plant community. This unified conceptual model can be applied to provide insight into the individual and the combined effects of climate and land use on semiarid plant communities within the grassland/forest continuum, which vary in the proportions of canopy and intercanopy patches.     

Combining endophytic fungi and bacteria for the biocontrol of Radopholus similis (Cobb) Thorne and for effects on plant growth

Eight endophytic fungal and bacterial isolates with antagonistic activity against Radopholus similis were evaluated in vivo for their individual and combined effects on biocontrol of R. similis and on the growth of “Grand Naine” cultivar banana plantlets in the greenhouse. Penetration efficiency (PE) of R. similis was between 3 and 21% in 29 biological agents (BAs) treatments, less than the 29% of the nematode-alone control (p ≤ 0.0001); 24 of the BAs treatments did not differ from the PE of 5% for a nematicide control. Twenty nine BAs treatments exhibited antagonistic activity against nematodes which reduced final population levels between 18 and 93%, relative to those on nematode-alone control plants (p ≤ 0.0001), and 14 BAs treatments were statistically similar to the nematicide treatment (88% reduction). Twenty four BAs treatments had increments of plant root biomass ranging from 20 to 58%, greater than the control plants; 37% of the treatments with single and combined BAs inoculations had root length increments ranging from 29 to 54% compared with control and chemical treatment. The nematicide, Terbufos 10GR, did not affect plant growth.     

Locust bean gum (LBG) as a gelling agent for plant tissue culture media

Locust bean gum (LBG) is a natural hydrocolloid extracted from the seeds of carob tree (Ceratonia siliqua L.). This work describes the successful use of LBG as a gelling agent in combination with agar for shoot multiplication and rooting of carob tree and Iberian rose shoots. Its presence did not affect the multiplication rate of both species. The rooting frequency of carob shoots was even significantly increased in the presence of 5 g of LBG plus 4 g of agar to the medium compared to medium solidified with 9 g of agar. Iberian rose shoots rooting was not influenced by the addition of this gum to the rooting medium. Results obtained show that LBG can be used in combination with agar in culture medium as a gelling agent without negative effect on plant material and with the advantage of reduced medium costs.     

Study of long-distance movement of Plum pox virus (Sharka) as an alternative resistance-evaluation method in Prunus

During the breeding programs for Plum pox virus (PPV, Sharka) resistance in Prunus, the evaluation of the new releases through symptoms observation on leaves has been contradictory and represents one of the main handicaps in these programs. In order to increase the accuracy of this traditional evaluation method, we here analyze an alternative method based on the study of the ability of a genotype to allow the long-distance movement of the virus through its vascular vessels. Two different plant models have been assayed: (a) in Model I, the inoculation was performed in the ‘GF305’ rootstock with a later grafting of the genotype under evaluation and a scion of healthy control ‘GF305’, to evaluate the long-distance movement through the studied genotype from the rootstock to the scion (xylem transport), and (b) in Model II, the inoculation with ‘GF305’ diseased scions was performed by grafting these diseased scions onto the studied genotypes, which were grafted onto healthy ‘GF305’ peach seedlings, to evaluate the long-distance movement through the studied genotype from the scion to the rootstock (phloem transport). The results show that, regardless of the presence of symptoms, susceptible genotypes allowed the movement of the virus through their vascular vessels in both directions studied. However, the resistant apricot ‘Stark Early Orange’ did not allow this movement. We propose the study of the ability of a genotype to allow the long-distance movement of the virus as an alternative and more accurate method for the evaluation of PPV resistance. However, this protocol is much more tedious than the traditional one and could be used mainly in the evaluation of a reduced number of more interesting genotypes.     

Identification of host plant resistance to pepper leaf curl virus in chilli (Capsicum species)

Three hundred and seven genotypes belonging to four cultivated and one wild species of Capsicum were screened against pepper leaf curl virus (PepLCV) causing devastating leaf curl disease of chilli (Capsicum annuum). Initial screening was done under field conditions based on coefficient of infection (CI), disease reaction to each genotype was assigned. Subsequently, selfed progenies of eight symptom-less and highly resistant lines were challenged by viruliferous white fly under glasshouse conditions, out of which only three genotypes, viz. GKC-29, BS-35 and EC-497636 showed no symptom. Using scion and root stalk of susceptible genotype (Pusa Jwala), these three putative symptom-less genotypes were further challenged by grafting and alternate grafting. The resistant reactions of GKC-29, BS-35, EC-497636 were confirmed because even after 50 days of successful grafting/alternate grafting, no viral symptom appeared on all the grafted plants of these genotypes. When subjected to PCR amplification with degenerate primers deigned to detect gemnivirus like PepLCV, the three symptom-less genotypes did not show any amplification, suggesting that the resistant reaction in three identified symptom-less resistant sources was because of the absence of viral genome and they are not symptom-less carrier.     

Response of Dendranthema grandiflora (Ramat) to three plant growth regulators in container paint mix applications

The effectiveness of plant growth regulators (PGRs) when applied in a paint mix to the inside of containers was evaluated. Two kinds of paint (regular flat latex and SpinOut^®) were evaluated for their respective effectiveness with three separate plant growth regulators (paclobutrazol, uniconazole, and ancymidol) on the growth of Dendranthema grandiflora (Ramat) (cv. Fina and Pelee) plants. Chrysanthemums grown in containers were subjected to three different application methods: interior surfaces covered by a mixture of paint and plant growth regulator (PGR) and soil drench or spray treatments according to label recommendations. The effect of composted pine bark on the effectiveness of two methods of application (paint and drench) was also compared. Paint alone had no effect on growth and development. None of the plants given treatments with paint with or without PGR, showed any sign of phytotoxicity. Plants exposed to PGRs appeared greener and were shorter than control plants. Both the regular flat latex and SpinOut paints were effective carriers of PGRs, although plants grown in containers coated with PGR/SpinOut paint were taller than plants grown with latex/PGR-coated containers. Composted pine bark slightly reduced the effectiveness of ancymidol on plant growth. This reduction in effectiveness was smaller when ancymidol was carried by latex paint but was greater when carried by SpinOut paint. These results indicate that the paint/PGR method may not represent any real advantage over the drench method to overcome the effect of composted pine bark in the growing mixes on the effectiveness of ancymidol. Chemical names used: β-((4-chlorophenyl)methyl)--(1,1-dimethyl)-1H-1,2,4,-triazole-1-ethanol (paclobutrazol); (E)-(+)-(S)-1-(4chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)-pent-1-ene-3ol (uniconazole-P); -cyclopropyl-(p-methoxyphenyl)-5 pyrimidinemethanol (ancymidol).     

A plant resource and experiment management system based on the Golm Plant Database as a basic tool for omics research

Background  

For omics experiments, detailed characterisation of experimental material with respect to its genetic features, its cultivation history and its treatment history is a requirement for analyses by bioinformatics tools and for publication needs. Furthermore, meta-analysis of several experiments in systems biology based approaches make it necessary to store this information in a standardised manner, preferentially in relational databases. In the Golm Plant Database System, we devised a data management system based on a classical Laboratory Information Management System combined with web-based user interfaces for data entry and retrieval to collect this information in an academic environment.     

一种植物表达载体的构建及其在金柑上的瞬时表达

以植物表达载体pCambia1301的带内含子的GUS(iGUS)基因替换植物表达载体pBI121中的GUS基因,构建了以pBI121为基础载体并含有iGUS的植物表达载体pLZ13。农杆菌染色表明,pCamiba1301和pLZ13两载体中的iGUS基因均不会导致GUS染色反应。以金柑不同组织为材料,通过农杆菌介导开展瞬时表达研究,结果表明,pLZ13和pCambia1301两载体的iGUS在CaMV35S启动子调控下的表达没有差异,证明构建的pLZ13是一种同时适于瞬时表达和转基因研究的植物表达载体。     

ECV304 cells can be used as a general model,tool or target in the research of biomedicine and pharmacology

ECV304 was reported first in 1990 as a spontaneously-transformed and immortalized cell line derived from a Japanese HUVEC. Subsequently, many studies validated that the ECV304 is a permanent endothelial cell line. It has been used widely as an endothelial cell model and an useful research tool in biomedicine and pharmacology. However, several distinct differences exist between ECV304 and HUVEC. Some studies even pointed out that ECV304 is not of HUVEC origin. According to the research data including ours, this reportedly endothelial-derived permanent human cell line ECV304 may be dedifferentiated towards an epithelial phenotype. It is therefore not an appropriate cell line to study endothelial cell biology. But cultured ECV304 cells can still be used as a model, tool or target in the pathophysiological and pharmacological studies, depending on whether or not their functional expression or markers are suitable for the research work.     

Syrian pear (Pyrus syriaca) as a pollinator for European pear (Pyrus communis) cultivars

In Israel four European pear cultivars are grown: ‘Spadona’ is the main cultivar and ‘Coscia’, ‘Gentile’ and ‘Spadochina’ are its pollinators. However, molecular S-genotyping revealed that ‘Spadona’ is semi-compatible with its three pollinators. This explains, at least in part, the relatively low pear yield in Israel. The Syrian pear (Pyrus syriaca) grows wild in Israel and blooms intensively, overlapping the blooming of the cultivated European pears. Cross-fertilization between Syrian pear and ‘Spadona’ was shown to be efficient suggesting that Syrian pear might be a potent pollinator for ‘Spadona’. Twenty-six Syrian pear seedlings, from different sites in north-east Israel were S-genotyped identifying 11 that are fully compatible with the four European pear varieties cultivated in Israel. By this screening, 24 different S-RNases were cloned; ten of them are new, whereas the other fourteen had been identified previously. In addition, seedlings of two wild pear species were also S-genotyped. Two seedlings from Pyrus betulifolia and one from Pyrus korshinskii were found to be genetically compatible with the four European pear cultivars. From these seedlings four S-RNases were cloned, two are new, one had been cloned previously and one was identical to an S-RNase allele cloned from Syrian pear in this work.     

RhizoTubes as a new tool for high throughput imaging of plant root development and architecture: test,comparison with pot grown plants and validation

Background

In order to maintain high yields while saving water and preserving non-renewable resources and thus limiting the use of chemical fertilizer, it is crucial to select plants with more efficient root systems. This could be achieved through an optimization of both root architecture and root uptake ability and/or through the improvement of positive plant interactions with microorganisms in the rhizosphere. The development of devices suitable for high-throughput phenotyping of root structures remains a major bottleneck.

Results

Rhizotrons suitable for plant growth in controlled conditions and non-invasive image acquisition of plant shoot and root systems (RhizoTubes) are described. These RhizoTubes allow growing one to six plants simultaneously, having a maximum height of 1.1 m, up to 8 weeks, depending on plant species. Both shoot and root compartment can be imaged automatically and non-destructively throughout the experiment thanks to an imaging cabin (RhizoCab). RhizoCab contains robots and imaging equipment for obtaining high-resolution pictures of plant roots. Using this versatile experimental setup, we illustrate how some morphometric root traits can be determined for various species including model (Medicago truncatula), crops (Pisum sativum, Brassica napus, Vitis vinifera, Triticum aestivum) and weed (Vulpia myuros) species grown under non-limiting conditions or submitted to various abiotic and biotic constraints. The measurement of the root phenotypic traits using this system was compared to that obtained using “classic” growth conditions in pots.

Conclusions

This integrated system, to include 1200 Rhizotubes, will allow high-throughput phenotyping of plant shoots and roots under various abiotic and biotic environmental conditions. Our system allows an easy visualization or extraction of roots and measurement of root traits for high-throughput or kinetic analyses. The utility of this system for studying root system architecture will greatly facilitate the identification of genetic and environmental determinants of key root traits involved in crop responses to stresses, including interactions with soil microorganisms.

     

Effects of explant type, culture media and growth regulators on callus induction and plant regeneration of Chinese jiaotou (Allium chinense)

To establish an efficient protocol of shoot regeneration from callus, effects of explant type, culture media and plant growth regulators on callus induction and shoot regeneration of Chinese jiaotou (Allium chinense) were evaluated. The results showed that basal plate was the best explant for callus induction (47.5%) when cultured on B5 medium supplemented with 0.1 mg l⁻¹ 6-benzylaminopurine (BA) and 1.0 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D), and B5 was the best medium to induce callus formation with 49.3% of the explants forming callus. The highest callus induction (65.2%) was achieved culturing basal plate on B5 medium supplemented with 0.1 mg l⁻¹ BA and 1.0 mg l⁻¹ 2,4-D after 8 weeks of culture. The best callus proliferation was observed on B5 medium with 1.5 mg l⁻¹ 2,4-D. Shoots regenerated at the highest frequency of 58.8% with 4.5 shoots when calli were cultured on B5 medium with 0.1 mg l⁻¹ BA and 1.0 mg l⁻¹ a-naphthaleneacetic acid (NAA). This protocol provides a basis for future studies on genetic improvement and could be applied to large-scale multiplication systems for commercial nurseries of Allium chinense.     

Efficient production of transgenic plants using the bar gene for herbicide resistance in sweetpotato

Efficient production of transgenic sweetpotato (Ipomoea batatas (L.) Lam.) plants using the bar gene for herbicide resistance was achieved through the use of embryogenic suspension cultures and Agrobacterium tumefaciens-mediated transformation. Cell aggregates from embryogenic suspension cultures of sweetpotato cv. Lizixiang were cocultivated with A. tumefaciens strain EHA 105 harboring a binary vector pCAMBIA3300 with the bar gene and uidA gene. Selection culture was conducted using 0.5 mg/l PPT. A total of 1431 plants were produced from the inoculated 870 cell aggregates via somatic embryogenesis. GUS assay and PCR analysis of the regenerated plants randomly sampled showed that 86.5% of the regenerated plants were transgenic plants. Stable integration of the bar gene into the genome of transgenic plants was confirmed by Southern blot analysis and transgene expression was demonstrated by Northern blot analysis. The copy number of integrated bar gene ranged from 1 to 3. Transgenic plants exhibited functional expression of the bar gene by in vivo assay for herbicide resistance. This study also provides a simple and efficient transformation system of sweetpotato based on the use of bar gene as a selectable marker gene, which can be combined with other agronomically important genes for the improvement of sweetpotato.     

Urban greenery as a habitat for parasitoids of the Pimplinae subfamily (Hymenoptera,Ichneumonidae)

Vegetation in green areas such as parks, gardens and allotment gardens provides suitable conditions for the development of parasitic insects, which can effectively reduce the population of plant pests feeding in this environment.The aim of the study was to determine the qualitative and quantitative structure of parasitoids of the Pimplinae subfamily (Hymenoptera, Ichneumonidae) inhabiting urban green areas and to determine the influence of anthropogenic pressure on the structures of these communities.The study was conducted between 2014 and 2016 at five sites in urban green areas in Poznań, which were exposed to low, medium or high anthropogenic pressure.In total 3096 samples were collected and 659 Pimplinae insects belonging to 51 species were caught. The largest population and the highest species diversity of the Pimplinae was noted at the Serbska (S) site, which was exposed to medium anthropogenic pressure. The smallest population and the lowest species diversity were observed at the Zoological Garden (ZG) site, which was exposed to heavy anthropogenic pressure.The research showed that the qualitative and quantitative structure of parasitoids of the Pimplinae subfamily depended on the degree of greenness in a particular area, its abundance in plant species, and the air pollution level.The research showed that the degree of anthropogenic pressure determined the similarity of the qualitative and quantitative structure of Pimplinae communities inhabiting urban green areas.The research showed that the species richness and the number of parasitoids of the Pimplinae subfamily in urban green areas were positively related with a well-developed and species-diverse plant cover with an abundance of shrub plants.Therefore, a floristically diverse urban environment may increase the species abundance of parasitoids of the Ichneumonidae family, including the Pimplinae subfamily, which may effectively regulate the number of pests feeding on plants in this environment.     

The riverine thruway hypothesis: rivers as a key mediator of gene flow for the aquatic paradoxical frog Pseudis tocantins (Anura,Hylidae)

Landscape Ecology - Rivers, landscape, and climate can alter patterns of gene flow and consequently, shape intraspecific genetic variation. While rivers are predicted to halt gene flow in...     

Effects of CO2, temperature and their interaction on the growth, development and yield of cauliflower (Brassica oleracea L. botrytis)

Stands of summer cauliflower were grown within polyethylene-covered tunnels along which a temperature gradient was imposed. Two tunnels were maintained at either normal or elevated CO₂ concentrations. At the last harvest (88 days from transplanting) no interaction between CO₂ and temperature on total biomass was detected. The total dry weight of plants grown at 531 μmol mol⁻¹ CO₂ was 34% greater than those grown at 328 μmol mol⁻¹ CO₂, whereas a 1 °C rise reduced dry weight by 6%. From serial harvests the radiation conversion coefficient was 2.01 g MJ⁻¹ and 1.42 g MJ⁻¹ at 531 μmol mol⁻¹ CO₂and 328 μmol mol⁻¹ CO₂, respectively, but was not greatly affected by differences in temperature. No effect of either CO₂ or temperature on the canopy light extinction coefficient was detected. The rate of progress towards curd initiation increased to a maximum at 15.5 °C, and declined thereafter. Provided the effect of temperature was accounted for, CO₂ enrichment did not affect the time of curd initiation. From serial harvests after curd initiation, the logarithm of curd weight or diameter were negative linear functions of mean temperature from initiation. Increases in curd weight and diameter at 531 compared with 328 μmol mol⁻¹ CO₂ were greater at warmer temperatures (27% at 13 °C compared with 47% at 15 °C, 57 days after initiation). Effects of CO₂ on curd diameter were less than those on curd dry weight because the curd dry matter content was greater at 531 compared with 328 μmol mol⁻¹ CO₂. Thus, the effects of elevated CO₂ concentrations on fresh weight based yield parameters of cauliflower were less than the increase in total dry matter production.